. . . . ‘ , _ ‘ . .. :1» .q... .5: r. r . ‘ . 1.. .. :1 v.33? .‘ 3m. . . ,. : . swig... Ewgfiéswéfiw .. r aw, M31. 3 r.‘ 51.... :I. r I .1: .353. 3 a} ESlS fQO This is to certify that the dissertation entitled Comparative Mapping of the Chicken Genome presented by Steven P. Suchyta has been accepted towards fulfillment of the requirements for Phd . . Genetics degree in Date ;’ 2‘ 00 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Mlchlgan State Unlverslty PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE Sim c:ICIRC/DateDuo.p65-p. 15 COMPARATIVE MAPPING OF THE CHICKEN GENOME BY Steven P. Suchyta A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Genetics 2000 ABSTRACT COMPARATIVE MAPPING OF THE CHICKEN GENOME BY Steven P. Suchyta Comparative mapping has been performed between the chicken and human genomes in regions corresponding to human chromosomes 1, 4, and 9, along with several other smaller areas of conserved synteny. These regions were initially chosen because of their relevance to previously identified Marek's disease (MD) resistance quantitative trait loci (QTL) (Vallejo et a1. 1998, Yonash et a1. 1999). Segments of chicken orthologues of mapped human genes were PCR-amplified from parental DNA of the East Lansing Backcross (BC) reference population, and the two parental alleles were sequenced. Single nucleotide polymorphisms (SNP) differences were then used to design allele—specific PCR primers with which to genotype the mapping panel; 52 BC progeny. Inheritance data were analyzed and the map location of the chicken orthologues were determined. Statistical analysis, based on the theoretical treatment of Nadeau and Taylor (1984), was performed using the region specific comparative map data to derive an estimate of the genome-wide conservation of gene order between avian (chicken) and mammalian (human) genomes. The average length of a conserved segment was calculated to be 38 i 9 centimorgans (CM), approximately 1% of the present estimate of the total genome. This corresponds to a rate of .13 i 0.04 reciprocal translocations per million years of evolution, a rate substantially less than found for some intra-mammalian genomes, suggesting an unusual level of evolutionary stability exists among avian genomes. A significant portion of human chromosome 9 was shown to correspond to a portion of the chicken Z sex chromosome, thereby providing some insight into the evolution of ZW—type chromosomal sex determination in birds. In addition to the comparative map, the initial steps to building a physical map of the chicken genome were begun. Recently, through collaboration with the Texas A&M BAC Center, a 5—fold BAC library of the chicken genome has been generated. This is comprised of approximately 38,000 clones with an average insert size of 150 kb. The BAC library is composed of chromosomal DNA from a Jungle Fowl (JF) female parent of the reference population. Because of the relative marker density, MD QTL, and number and positions of conserved markers between humans and chickens, microchromsome E41 was chosen to begin the physical mapping project. The BAC library has been spotted on 20 nylon membrane filters and these were screened using radio-labeled probes derived from six markers on E41. Ten positive BAC clones have been identified from four of the six markers tested. TABLE OF CONTENTS List of Tables List of Figures Key to Abbreviations Chapter 1 Chapter 2 Chapter 3 Chapter 4 Appendix 1 Appendix 2 Appendix 3 References Introduction and Literature Review Comparative Mapping of the Chicken Genome Physical Mapping of Chicken Microchromosome E41 Summary Lack of Polymorphisms in Several Chicken Genes Primer Pairs to Sequenced Chicken Genes Comparative Mapping of the Chicken Genome Using the East Lansing Reference Population W vi viii 32 101 122 124 132 160 165 Chapter 2 1. Table l. 2. Table 2. 3. Table 3. 4. Table 4. 5. Table 5. 6. Table 6. 7. Table 7. 8. Table 8. Chapter 3 9. Table 1. Appendix 1 10. Table 1. Appendix 2 11. Table 1. Appendix 3 12. Table l. 13. Table 2. LIST OF TABLES Gallus gallus gene sequences and the percentage nt and protein identity with the corresponding human gene. Genes Mapped: Primer and PCR Information PCR conditions: Genes Mapped: Human Chromosome 1 Genes Mapped: Human Chromosome 9 Genes Mapped: Human Chromosome 4 Genes Mapped: Human Chromosomes 11, 12, and Others Genetic Lengths of conserved segments between chicken and humans Identification of BAC clones on chromosome E41. Non-polymorphic gene sequences: Primer Pairs to Sequenced Chicken Genes Primers used to amplify target regions in functional genes. Comparative location of chicken, human, and mouse genes. 'U'U'U'U'U .62-64 .65-83 .84 .85 .87 .88 .89 .90 .121 .130 .134 .161 .162 Chapter 1 1. Figure 1. Chapter 2 2. Figure 1. 3. Figure 2. 4. Figure 3. 5. Figure 4. 6. Figure 5. 7. Figure 6. 8. Figure 7. 9. Figure 8. 10. Figure 9. LIST OF FIGURES Markers on chicken chromosome E41. Syntenic groups mapped to human chromosome 1 and chicken chromosomes E54, 8, 1, E26, E04, and 3. Syntenic groups mapped to human chromosome 9 and chicken chromosomes Z, E18, 1, 2, and E41. Syntenic groups mapped to human chromosome 4 and chicken chromosomes E29, E38, and 4. Syntenic groups mapped to human chromosome 11 and chicken chromosomes 5, 1, E52, and E49. Syntenic groups mapped to human chromosome 12 and chicken chromosome 1. Syntenic groups mapped to human chromosome X and chicken chromosome 4. Syntenic groups mapped to human chromosome 2 and chicken chromosomes 3, 4, and 7. Syntenic groups mapped to human chromosome 15 and chicken chromosomes 5 and E29. Syntenic groups mapped to human chromosome 17 and chicken chromosomes E57, E21, E31, E59, and E16. 11. Figure 10. Curves illustrating expected Chapter 3 12. Figure 1. pBeloBacll large insert cloning vector. cumulative frequency distributions of segments containing two or more markers at different values of L. vi .31 .91 .92 .93 .94 .95 .96 .97 .98 .99 .100 .113 13. Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Figure 19. Figure Appendix 1 20. Figure Appendix 3 21. Figure Restriction enzyme testing for three of the gene markers, ABLl, AKl, and RING3L; run on a 1% agarose gel. Autoradiographs of the filters for plates 65-68 and 21-24. 3% Metaphor agarose gel of BAC clones 74/P21 and 23/J8 after PCR with the 6 sets of primers. 1% agarose gel of BAC clones 95/C11, 75/K22, 90/B4, and 71/I1. CHEF gels for the BAC clones tested in the study. 1% agarose gel of BAC clones tested in this study. Chromosome E41 (EL reference map) and BAC clones identified on the current study. Filter with White Leghorn (W) and Jungle Fowl (J) genomic digests. Mismatch Primer PCR Chicken B-Globin Gene vii .114 .115 .116 .117 .118 .119 .120 .131 .163 KEY TO ABBREVIAT I ONS AFLP: Amplified Fragment Length Polymorphism Amp: ampicillin BAC: Bacterial Artificial Chromosome BC: Backcross BLAST: Basic Local Alignment Search Tool c-chr: Chicken Chromosome cDNA: Complementary DNA CHEF: Clamped Homogeneous Electric Fields cm: Centimorgans DNA: Deoxyribonucleic Acid EL: East Lansing EST: Expressed Sequence Tagged Site FISH: Fluorescent in situ Hybridization Gb: Gigabase h-chr: Human Chromosome INRA: Institut National de la Recherche Agronomique JP: Jungle Fowl kb: Kilobase Mb: Megabase LCD: log10 of Odds MD: Marek’s Disease Mya: Million Years Ago NCBI: National Center for Biotechnology Information NOR: Nucleolar Organizer Region GMIM: Online Mendelian Inheritance in Man viii PASA: PCR Amplification of Specific Alleles PCR: Polymerase Chain Reaction PFGE: Pulse Field Gel Electrophoresis PPSCG: Primer Pairs to Sequenced Chicken Genes QTL: Quantitative Trait Loci RAPD: Random Amplified Polymorphic DNA RFLP: Restriction Fragment Length Polymorphism RB: Radiation Hybrid SNP: Single Nucleotide Polymorphism Tm: Melting Temperature UDEL: University of Delaware UMSTS: Universal Mammalian Sequence Tagged Sites WL: White Leghorn YAC: Yeast Artificial Chromosome ZOO-FISH: Interspecies Chromosome Painting ix Chapter 1: INTRODUCTION AND LITERATURE REVIEW Comparative Mapping: Terms and Techniques Before giving a definition of comparative mapping it will be useful to review the terms and techniques associated with the construction and utilization of comparative maps. Comparative maps rely on the placement of homologous genes on the genome maps of two or more species. An important factor to consider when analyzing homologous genes between different species is whether the genes are orthologous or paralogous. Orthologous genes are homologous genes in different species that are descended from the same gene in the last common ancestor of the two species. In contrast to this, paralogous genes are homologous genes that are not descended from the same ancestral gene. Paralogous genes arise through gene duplication prior to the existence of the last common ancestral species. Thus, paralogues may diverge and change location within the genome at times both before and after the time of the last common ancestor, whereas orthologues can only do so after that time point. Thus, selection of orthologous genes will provide the most accurate and useful comparative map. Two additional terms used to define the structure (similarities and differences) of a comparative map are conserved synteny and conserved segment. Originally, the term synteny was used to describe genes found on the same chromosome, regardless if they were genetically localized or not (Renwick, 1971). With the continued use of somatic cell hybrids, there was a need to classify genes found on the same chromosome, but that could not be linked through recombination analysis. The term conserved synteny is now used in comparative mapping to describe the situation when two or more genes are syntenic (reside on a single chromosome) in different species, regardless of gene order or non-contiguous interspersed segments. The determination of synteny is through some type of genome mapping, such as linkage analysis or radiation hybrid (RH) mapping. A conserved segment between two species is a chromosome interval (defined by two or more genes) that shares the same gene order and has no non-contiguous interspersed gene segment. A comparative map is constructed using established conserved segments and syntenies. Comparative maps are unique in that they rely on other types of genome maps for their construction. In order to maximize the information across species there has been an effort to produce homologous anchored reference loci. Two groups of reference loci have been developed, the comparative anchor tagged sequences (CATS) (Lyons et a1. 1997) and the Universal Mammalian Sequence Tagged Sites (UMSTS)(Venta et al. 1996). The CATS primer set was optimized for the cat and the UMSTS set was optimized for the canine. These markers were developed by designing PCR primers based on conserved exon sequences from many different mammalian species. The primers were designed through computer analysis of adjacent exonic sequences from over 20 mammalian species (Venta et al. 1996; Lyons et a1. 1997). The exon sequences can be used to verify the PCR product and the intron is a potential source for sequence or length polymorphisms to be used for genome mapping. Primer sets for over 500 genes are available and approximately 75% should be successfully amplified in any mammalian species (Lyons et a1. 1997). These tools will greatly aid in the construction of a reference comparative map that can be used across many mammalian species and there is now a comparative genome map between mice and humans based on 314 of the CATS anchor loci (Chen et a1. 1999). One of the most studied intra—mammalian comparative maps has been derived from the cat. Therefore, it will be useful to review its construction. The feline genome map was first developed using a rodent X cat somatic cell hybrid panel and fluorescent in situ hybridization (FISH) (O’Brien and Nash 1982; Yuhki and O’ Brien 1988; Lopez et a1. 1996; O’Brien et al. 1997). FISH mapping relies on fluorescently labeling a portion of the gene or marker of interest and hybridizing on metaphase chromosome spreads. Somatic cell hybrids can assign markers only to their respective chromosomes, and do not give information on gene order. An interspecies backcross (BC) population between the domestic cat and an Asian leopard cat has also been developed (Lyons et a1. 1994). The CATS or UMSTS reference loci could be mapped through linkage analysis of polymorphisms found in the PCR product (Lyons et al. 1997). There is a 4 cM limit to the resolution of this linkage map (Lyons et al. 1994). In order to develop a high—resolution gene map in the cat, a RH panel was developed (Murphy et al. 1999). For a complete review of RH mapping, see McCarthy (1996). RH panels are made by irradiating a donor cell line (in this case, derived from the cat) with a lethal dose of X—rays or y rays, the DNA fragments from the donor cell line are rescued by a recipient cell line (hamster cells were used for the feline RH panel). Using a selectable marker, the only post-fusion cells that will grow are those containing donor DNA. The hybrid colonies are picked individually and DNA is extracted. Genes or other markers are screened in the panel usually through PCR. The retention pattern of the markers for each hybrid is compared to determine linkage, and from this data, the map distances can be calculated. High-resolution RH maps have been successfully constructed for humans (Gyapay et al. 1996; Stewart et al. 1997) and mouse (McCarthy et al. 1997). The CATS or UMSTS markers are PCR primers and can be used for RH mapping as can any other sequence tagged site (STS). The current feline-human comparative map was developed using the RH panel and FISH mapped genes placed on the feline genome (Lyons et al. 1997). An additional tool used to assess the amount of conservation between the two genomes on a broader scale was interspecies chromosome painting (ZOO-FISH) (Lyons et al. 1997). The ZOO-FISH procedure first uses special PCR conditions to amplify flow sorted metaphase chromosomes and the amplified chromosome is fluorescently labeled and used for in situ hybridization on metaphase chromosomal spreads from distantly related species (O’Brien, 1993; Weinberg and Stanyon 1995; Rettenberger et al. 1995; Solinas—Toldo et al. 1995; Fronicke et al. 1996; Goureau 1996). The ZOO-FISH method can give a direct assessment of the amount of genome conservation between two species through visualization of the labeled metaphase chromosomes. Unlike the feline genome map, the chicken genome map has been developed primarily through linkage analysis. There are currently three main reference families through which DNA-based sequence polymorphims have been placed: the Compton population (Bumstead and Palyga 1992), the East Lansing (EL) population (Crittenden et a1. 1993,) and the Wageningen population (Groenen et a1. 1998). A consensus map combining all three that contains 1889 markers (approximately 300 are genes) has been developed (Groenen et al. in press). The chicken-human comparative map data developed in this thesis was based on genes placed on the EL reference map, so a more detailed description of it will be useful. The EL population was constructed by first mating an inbred male UCD001 Red Jungle Fowl (JF) to an inbred UCD003 White Leghorn (WL) female and then 2 F1 male progeny were backcrossed to the WL line (Crittenden et al. 1993). This interspecies cross maximizes the potential for sequence polymorphism and each marker is biallelic in the BC population. Four hundred animals were produced in the BC from which the panel of 52 BC birds normally used for the mapping panel are derived. Comparative Mapping: Definition and Utilization Comparative gene mapping is the comparison of the chromosomal arrangement of orthologous genes in the genomes of two or more species. Comparative gene mapping has been an essential tool in the genetic analysis of many species and has given insight into the evolution of genome organization. Among mammals, much of the power of comparative mapping relates to the extensive mapping and sequence information now available for human genes. To date, over 7,000 known genes and over 16,000 expressed sequence tagged sites (ESTs) have been mapped on the human genome (Adams et al. 1995; Hudson et al. 1995; Schuler et al. 1996; DeLoukas et al. 1998, Online Mendelian Inheritance in Man, OMIM, http://www.ncbi.nlm.gov/omim/, 2000). The other model mammalian species, the mouse, now has over 7,000 genes mapped on its genome (Copeland et al. 1993; Adams et al. 1995; Dietrich et al. 1996; Marra et al. 1999; Van Etten et al. 1999). All of this data is readily available through National Center for Biotechnology Information (NCBI) Genbank databases. Through the use of a framework comparative map between a reference/model genome (e.g., mouse, human) and a genome of interest that has been less extensively studied, it is possible to infer the location of genes in the latter species that exist in the gaps between orthologous genes previously mapped in both species. Framework comparative maps between a number of related species (e.g., mammals, O'Brien et al. 1999) depend upon placing orthologous comparative anchor loci on two or more members of that group. Ideally, the same anchor loci are mapped in several member species, which allows integration of the respective maps and, potentially, an estimate of the pattern of chromosome rearrangements that explain the evolution of gene order within the species group. Comparative mapping has been applied to the genomes of a variety of mammalian species (O'Brien et al. 1999). The mouse presents a special case in the development of its comparative map. The mouse genome has been far more extensively mapped than that of any other mammal, excepting humans. Additional interest derives from the putative unusual qualities of mouse chromosomes in an evolutionary sense (reviewed in Graves 1996). There is now a high— resolution comparative map between the mouse and human genomes, which provides great insight into chromosomal rearrangements that have occurred during the evolution of the mouse (Copeland et al. 1993; Debry and Seldin 1996; Carver and Stubbs 1997). Although there exist large regions with a high degree of conservation between the two genomes this is the exception (e.g., on both species’ chromosome 1 there is a >10Mb region with conserved gene content, spacing, and order, Oakley et al. 1992). Most syntenic segments contain numerous rearrangements. As maps have improved, several syntenic segments initially thought to be conserved intact are not truly contiguous. One example is the q arm of human chromosome 5 which contains a large segment initially thought to be completely conserved with a region on mouse chromosome 11, but which now has been shown to be interspersed with orthologous genes from mouse chromosomes 13, 18, and 17 (Carver and Stubbs 1997). At least four rearrangements in mouse chromosome 11 would be needed to account for this (Watkins—Chow et al. 1997). Extensive analysis of the mouse and human Major Histocompatibility Complex regions and T-Cell Receptor loci reveal that many deletions, duplications, and inversions exist between the two species (Weiss et al. 1984; Hood et al. 1993; Koop et al. 1992, 1994; Amadou et al. 1995). One of the advantages of comparing the mouse and humans genomes is the large amount of sequence information available for both (Januzzi et al. 1992; Koop et al. 1992, 1994; Lamerdin et al. 1995, 1996; Oeltjen et al. 1997) These studies compared the sequences from a diverse set of genes and flanking regions of the two genomes. Overall, it appears there is a general conservation of exons, introns, and intergenic sequences. Exonic sequences in the T-Cell Receptor gene region have a 66-79% similarity, whereas intronic and intergenic sequences have approximately 66% similarity (Oeltjen et al. 1997). The mouse and human gene regions also had conservation in the sizes and order of the exons, introns, and intergenic areas (Januzzi et al.1992; Renucci et al. 1992; Koop et al. 1992, 1994; Lamerdin et al. 1995, 1996; Oeltjen et al. 1997). Thus, it appears the relative instability of the mouse genome is in the placement and order of genes on the chromosomes, while the sequence and organization of the genes themselves has remained stable. Thorough analysis of the comparative map between human and mouse gives rise to 180 conserved segments with lengths ranging from 1 to 10 cM (Copeland et al. 1993; Debry and Seldin 1996; O’Brien et al. 1999). Although fewer data points are available for other mammals, comparative mapping across a wide range of mammals reveals that the mouse genome is the exception (with its large number of rearrangements), relative to that of the human. In other words, the rearrangements observed between the mouse and human genome have occurred primarily in the evolutionary line to the mouse, not to the human, from the last common ancestor of both species (O'Brien et al. 1999). An example of this high degree of conservation can be found in the feline-human comparative map. Even though the initial construction of the feline-human comparative map relied on somatic cell hybrid panels and FISH mapping (O’Brien and Nash 1982, Yuhki and O’Brien 1988, Lopez et al. 1996) and contained only 105 homologous genes, it showed a considerable amount of conservation between the two species (O’Brien et al. 1997). Now there are approximately 500 homologous markers mapped on the feline map (Yuhki and O'Brien 1988; Lopez et al. 1996; O’Brien et al. 1997; O’Brien et al. 1999), covering all 19 feline chromosomes. Many of these genes were mapped on the feline high resolution RH map (Murphy et al. 1999; O’ Brien et al. 1999). There is extensive syntenic conservation with the human map across most of the chromosomes. Chromosome D1 in the feline is conserved completely with human chromosome 11 and there is complete X chromosome conservation. Comparative map data based on gene maps will have gaps unless there are thousands of homologous markers as in the human and mouse. In order to confirm the comparative map, ZOO—FISH analysis was performed using feline-human reciprocal hybridizations (O’Brien et al. 1997). ZOO-FISH painting physically covers 90% of the chromosomes. This allows for direct observation of the minimal number of translocation rearrangements between the two genomes, but the technique will miss translocation of small segments or internal rearrangements within a single chromosome. The ZOO-FISH method also confirms that the framework provided by the location of homologous markers on the genetic map is accurate. The majority of differences between the feline and human genomes appear to be the splitting and rejoining of chromosomes; with only two interspersed human chromosomal segments in the feline genome (O’Brien et al. 1997). The high resolution RH 10 map illustrated that there was also a high degree of gene order conservation for human chromosomes 12 and 22 with feline chromosomes B4 and D3 respectively (Murphy et al. 1999) Very large segments of conserved synteny with the human genome have also been reported in other mammals such as dogs (Priat et al. 1998; Murphy et al. 1999; Neff et a1. 1999,), cattle (Yoo et al. 1994; Hayes et al. 1995; Solinas-Tolda et al. 1995; Wienberg and Stanyon 1995; Chowdhary et al. 1996; Pirottin et al. 1999) and pigs (Rettenberger et al. 1995; Fronicke et al. 1996; Goureau et a1. 1996; Marklund et al. 1996; Rohrer et al. 1996; INRA, http://www.toulouse. inra.fr/lgc/pig/compare/compare.htm). Unlike the feline map, the canine, pig, and bovine maps primarily have employed genetic mapping to build the comparative maps. These comparative maps have been confirmed on a larger scale and gene order appears to be conserved as well. As high- resolution maps are eventually made of these species, smaller rearrangements will likely appear, as was observed with the human—mouse high-resolution map (Carver and Stubbs 1997). This will pose a problem when the comparative map is used to locate potential candidate genes. One of the uses of comparative maps is to find candidate genes based on the assumption of common inheritance of a complete interval flanked by two syntenic framework markers. An example exists on bovine chromosome 2, which was shown to contain the gene for muscular hypertrophy ll (Charlier et a1. 1995; Dunner et al. 1997). This region shares conserved synteny with human chromosome 2, and there are several potential candidate genes in this area (Sonstengard et al. 1997b). Refinement of the comparative map in this region in cattle revealed several cases of complex gene shuffling throughout (Sonstegard et al. 1998). Rearrangements in gene order may cause the initially identified candidate genes to be reevaluated. This may result in considerably more effort than anticipated in gene identification, as was the case with muscular hypertrophy. However, it should be noted that the gene responsible for muscular hypertrophy, myostatin, was identified through a comparative approach (Grobet et al. 1997). Comparative maps built using anchor loci will be a valuable tool in identifying potential candidate genes, but small rearrangements in gene order show that dense comparative maps will often be required to make confident predictions. At the moment, maps with this level of resolution are lacking for vertebrate species outside of mouse, rat, and human. In general, the wider the evolutionary difference between two species, the greater is the desired resolution of comparative maps used to infer candidate genes for traits. Overall, it appears there is a great deal of genome conservation between mammalian species. Compared to the mouse and human genomes that can be divided into 180 conserved segments (O'Brien et a1. 1999)(when gene order is 12 considered there are over 200 segments, Eppig and Nadeau 1995; Debry and Seldin 1996), all of the other species studied have a much higher level of conservation. Human and feline maps are divided into 32 conserved segments (O’Brien et al. 1997; O’Brien et al. 1999), human and bovine maps have 50 conserved segments (Rettenberger et al. 1995; Fronivke et al. 1996; Goureau et al. 1996; O'Brien et al. 1999), and human and porcine maps can be divided into 47 segments (Marklund et al. 1996; Rohrer et al. 1996; O’Brien et al. 1999). These do not take into consideration small changes that affect gene order, but it is clear the genome organization is very similar among a variety of mammalian species. Chicken comparative mapping: One of the most important non—mammalian species is the chicken. It is of great importance as an agricultural commodity and as a research tool. At first glance, it appeared that building a comparative map between chicken and any mammalian species might be difficult. The last common ancestor between avian and mammalian lines lived approximately 300-350 million years ago (Mya), so there have been 600—700 million years of separate evolutionary history (along both lines) for chromosomal rearrangements to occur between the chicken and, for example, the human genome. Applying the formula of Paterson et al. (1996) (based on 13 comparative plant genome maps and early data from mouse and human genomes) leads one to calculate the size of a segment of the genome with a 50% probability of not being rearranged between chicken and human to be about 1.7 cM. As the chicken genome is about 3500 cM, this would be equivalent to roughly 2000 chromosomal rearrangements between the two genomes. Several studies, including those described in this thesis, have demonstrated that this is a gross over- estimate. Avian chromosomes have been conserved over a long period of time. Analysis of karyotypes of over 800 species of birds has shown that avian chromosome morphology (banding pattern) and number have been highly conserved for 150 million years (Rodionov 1996). This is similar to the case in turtles (Bickman 1981) and salamanders (Maxson and Wilson 1975), where chromosomes have remained relatively constant (at the cytogenetic level of analysis) for over 200 million years in some cases. The typical avian genome is comprised of eight to ten macrochromosomes and between 30 to 34 microchromosomes. The distinction is arbitrarily based on the size of the chromosome; there is no clear quantitative cut-off defining the boundaries between macro and microchromosomes. Generally macrochromosomes are between 2.5 to 6 um in length and the microchromosomes are less than 2.5 um long during mitosis (reviewed in Rodionov 1996, 1997). Avian macrochromosomes are probably generally homologous to turtle macrochromosomes (Takagi and Sasaki l4 1974; Stock and Mengden 1975), so there may be a similar evolutionary mechanism involved. The conservation of chromosomes over this long period may be due to a selection for high genomic homeostasis or a strong stabilizing selection for the ancestral chromosome number and morphology (Bickman 1981; Rodionov 1996). The stability of avian chromosomes should greatly increase the effectiveness of comparative mapping between mammals and chickens by reducing the amount of change that has occurred since the last common ancestor. In addition, the formula derived by Paterson et al. (1996) was heavily weighted by a few comparisons (e.g., mouse/human) in which high levels of genome rearrangement have occurred. Although there are not enough data to make a definitive estimate among birds, recent broad analysis of mammalian genomes (O'Brien et al. 1999) suggests that genomes are often highly stable over long evolutionary time, but that particular lineages (e.g., the rodent lineage) go through periods of unusually rapid rearrangement. Fortunately, as will be described below, such bursts of chromosomal rearrangement may have been relatively rare in the lineages leading to both the chicken and human from their last common ancestor. Using the data available at that time, Burt (1997) calculated that approximately one-third of the syntenic genes (genes on the same chromosome in this case) from the last common ancestor between human and chickens now have conserved synteny between the species. As discussed earlier, 15 there has been a high rate of chromosomal rearrangement in the mouse compared to other mammalian species, and only 40% of these original syntenic relationships remain between humans and mouse (Bengtsson et al. 1993). Only 18% of the original syntenic relationships remain for chickens and mice. The low percentage of conserved syntenies between chicken and mouse is heavily influenced by the high rate of rearrangements found in rodent species. The divergence time is approximately 70 million years between human and mouse (Graves 1996) and 300 million years between mammals and birds (Kumar and Hedges 1998). Considering the difference in divergence time between the species, it is interesting to note that the number of rearrangements predicted between the human and mouse genomes was similar to those predicted between the human and chicken genome. There has been recent further progress into the construction of a chicken-mammalian comparative map. One successful approach used by our group and others has been to map chicken genes with known sequence information (Klein et al. 1996; Smith et al. 1997; Fridolfsson et al. 1998; Groenen et al. 1999). FISH analysis, Restriction Fragment Length Polymorphisms (RFLP), and polymorphic intergenic microsatellite sequences are common methods used for the chromosomal placement of chicken genes (Klein et a1. 1996; Smith et a1. 1997; Fridolfsson et al. 1998; Groenen et al. 1999). FISH mapping using the gene of interest as a fluorescent probe allows for visualization of the 16 chromosomal placement of the gene. RFLP analysis uses the gene as a probe to identify a polymorphism and to genetically map the gene through linkage analysis in a reference population. PCR primers are designed to cross polymorphic intergenic microsatellite sequences in order to genetically map the gene through linkage analysis. A technique successfully used by our group has been the use of PCR amplification of specific alleles (PASA) to genotype sequence polymorphisms identified in the EL reference map population. Using available sequence information from a gene, primers are designed to cross a less conserved region such as an intron or 3' untranslated region (UTR), and sequence information is obtained from both parental lines of the EL population (WL, JF). If a polymorphism is found, segregation of the JF allele in the BC mapping animals is determined through preferential amplification of the JP allele. A more detailed description of this technique is described in Chapter 2 of this thesis. Although a few genes have been successfully amplified using the CATS and UMSTS set of primers (Smith et al. 1997), we have experienced a relatively high failure rate and now rely almost entirely on chicken genes with known sequence information. This initial work has shown that a robust chicken mammalian comparative map could be made. Several large regions with conserved synteny and regions with conserved segments were found. Some of the conserved regions extend over 50 cM on the chicken genetic map (Smith et al. 1997; 17 Groenen et al. 1999). For the purposes of this thesis, the focus was placed on the chicken-human comparative map. The mouse genome, as was discussed earlier, appears to be relatively unstable, which could limit its usefulness in a comparative map. Additionally, the human genome has by far the most comprehensive genome map. Although many regions of the chicken—human comparative map were added to in this thesis, we focused on a few select regions rather than seeking broad coverage. Since the comparative map of human chromosome 1 was the most complete, an attempt was made to fill in some of the gaps to identify the extent of the conservation. Our initial work had identified a large region conserved between human chromosome 4 and chicken chromosome 4, and an attempt was made to extend the chicken-human chromosome 4 map. Initial work by our group and others had identified a large region of conservation between human chromosome 9 and the Z chicken sex chromosome (Smith et al. 1997; Fridolfsonn et al. 1998; Nanda et al. 1999). The comparative map of the Z chromosome was extended in the hopes of elucidating some of the dynamics of the evolution of the avian sex chromosomes. By focusing on relatively few regions we hoped to get good coverage of these chromosomes, in an attempt to get a general idea of the number of chicken segments that would cover a human chromosome. One of the goals of the work in this thesis was to add to the number of conserved segments between chickens and humans. The approach taken was to try to saturate the 18 coverage over entire human chromosomes using those chicken orthologues that had already been sequenced. The starting points were the conserved groups found in our initial work in Smith et al. (1997). Additionally, the statistical approach of Nadeau and Taylor (1984) used in the early stages of the mouse—human comparative map was used to make a genome-wide estimate of the total level of conservation of gene order between the avian and mammalian genomes. Statistical approach: In the early 19805, far less map data existed for both the mouse and human genomes. In order to analyze the amount of genomic conservation between the two species, Nadeau and Taylor (1984) derived a method to estimate overall genome conservation from a limited data set of gene segment comparisons. They estimated the average length of a conserved segment between mouse and human genomes to be 8.1 i 1.6 cM. This was based on 13 known conserved linkage groups (containing two or more genes) and 54 mapped single homologous markers. In 1993, Copeland et a1. came to the same estimated conserved segment length of approximately 8 cM. This was based on over 140 conserved linkage groups, nearly covering both genomes. Thus, it appears that the Nadeau and Taylor (1984) model generated an accurate prediction of average genome conservation, despite the relatively poor level of map coverage at that time. In 19 comparing the human genome to those of most non-rodent mammals, direct observation techniques (chromosome banding patterns, ZOO-FISH), are most often used to estimate average conserved segment length (or estimated number of rearrangements), since there typically exist relatively few changes (O'Brien et al. 1999). However, until recently little effort has gone into comparative genome mapping between more distantly related species (e.g., birds and mammals) due to the greater challenge in identifying an adequate collection of orthologues and initial estimates that conserved segment lengths would be small (e.g., Paterson et al. 1996). Recently, Burt et al. (1999) looked at all of the available gene data on the chicken reference maps. By analyzing the total number of conserved segments between humans, mice and chickens, Burt et al. (1999) concluded that the organization the human genome is closer to that of the chicken genome than to the mouse genome. The work in this thesis will help to substantiate these findings as well as adding to the overall chicken—human comparative map. Physical mapping: Physical mapping is the construction of a genome map using large insert clones (e.g., Bacterial Artificial Chromosomes: BACs, Yeast Artificial Chromosomes: YACs) to ascertain the physical size of the chromosomes. 20 Additionally, these clones will serve as a source for a great deal of the sequence information in the genome. Physical mapping using large insert clone libraries has been applied successfully to a wide range of genomes (e.g., Hardy et a1. 1986; Burmeister et al. 1988; Martin et al. 1993; Bent et al. 1994; Song et al. 1995; Van Houten et al. 1996; Mcdermid et al. 1996; Yoshimura et al. 1996; Lauer et al. 1997; DeLoukas et al. 1998). Initially, limits on resources available and the state of technology in general led most investigators to take a regional map-building approach focused on a single large genome segment (e.g., major histocompatibility locus, Abderrhim et a1. 1994; Totaro et al. 1996) or chromosome (Chang et al. 1994; Kunz et al. 1994; Moir et al. 1994; Nagata et al. 1995; Smith et al. 1995; Soeda et al. 1995; Nagaraja et al. 1997). Cohen et al. (1993) were among the first to attempt the physical mapping of a large genome (human) all at once. This was based on fingerprint analysis of large human YAC clones. Fingerprinting is based on the analysis of banding patterns of large insert clones after cutting them with restriction enzymes. The digested clones are run on high—resolution polyacrylimide sequencing gels. The analysis is done with a computer and looks for common and overlapping bands (Zhang and Tao 1997; Chang et al. 1999; Tao et al. 1999). This approach, however, is complicated by the tendency of YAC inserts to rearrange and other difficulties in handling and mapping YAC clones. BAC clone inserts are generally smaller 21 (typically 100-300 kb) than observed in YAC libraries (up to about 1 Mb, on average), which means that many more clones must be analyzed to generate a complete map. However, BAC inserts are much more stable, and BAC DNA is comparatively easy to purify and fingerprint. Whole genome maps based on extensive BAC clone analysis have begun to appear (Marra et al. 1999; M020 et al. 1999). Recently, two chicken BAC clone libraries have been constructed at the Texas A&M BAC Center (Crooijmans et al. personal communication). The Crooijmans library consists of approximately 50,000 clones with an average insert size of 130 kb (about 5X coverage of the genome). The BAC library described in this thesis presently consists of about 38,000 clones with an average insert size of about 150 kb (ca. 5X coverage). Insert DNA fragments were derived from partial digestion with Mpg; and cloned into the ggmfi; site of pBeloBacll. Plans are underway to expand this library to about 80,000 clones including inserts derived by partial HindIII and Eppfi; digests. Our BAC library has been constructed using DNA from a female of the inbred UCD001 JF line of chickens. Use of DNA from a UCD001 bird allows the possibility that dominant markers (e.g., AFLP and RAPD) previously identified in UCD001 birds may be applied in BAC analysis, if necessary. 22 Physical mapping: thesis focus Originally, avian microchromosomes were considered genetically inert elements (Newcomer 1957; Ohno 1961; reviewed in Bitgood and Somes 1990). With continued study of the avian karyotype, it was found that there was a relatively constant number of these elements in most bird genomes. This led to the understanding that they were genuine chromosomes (Schmid 1962; Krishan 1964; Clement 1971). Additional studies showed that microchromosomes replicate, contain centromeres, and form meiotic bivalents (Kaelbling and Fechheimer 1983a, 1983b; Hutchison 1987; Bitgood and Shoffner 1990). Further study into their structure and recombination properties seem to indicate that microchromosomes may have some unique qualities. There have been many studies on the composition of the microchromosomes. Data regarding the distribution of non— coding sequences in the chicken genome are of several types. C—banding studies have shown that heterochromatin is found on certain microchromosomes (Stefos and Arrighi 1974; Bulatova et a1. 1977; Pollock and Fechheimer 1981; Belterman and De Boer 1984; Schmid and Guttenbach 1988; Rodionov et al. 1989), and clones showing a high proportion of repeated sequences have been isolated from microchromosomes (Matzke et al. 1992; Fillon et al. 1998). Therefore, there are non- coding regions found on microchromosomes. Additionally, 23 genetic markers based on non—coding repeat sequences such as microsatellites have been placed on microchromosomes (Cheng et al. 1995; Crooijamans et al. 1996). Primmer et al. (1997) demonstrated that, while microchromosomes contain microsatellite and other non—gene sequences, they appear to contain fewer than would be expected based on the genome content as a whole. They used Primed In Situ Labeling (Koch et al. 1989) with the (CA)10 ndcrosatellite on metaphase chicken chromosomes for this estimate. Initial studies on chicken microchromosomes showed (by differential staining) that several microchromosomes are comprised of GC—rich R blocks (Rodionov 1985; Rodionov et al. 1989). FISH with probes enriched for CpG islands (CGIs) indicated that CGIs are enriched on chicken microchromosomes (McQueen et al. 1996). Increased acetylation of the amino— terminus of histone H4 is strongly correlated with the presence of genes (Turner 1993; Wade et a1. 1997) Immunofluorescence with acetylated Histone H4 on metaphase spreads of chicken chromosomes, showed that the microchromosomes are enriched for acetylated Histone H4 (Mcqueen et a1. 1998). Additionally, McQueen et a1. (1998) demonstrated that microchromosomes replicate early in S phase, which is also associated with transcriptionally active DNA. By analyzing cosmids whose genomic origin was known, CGIs were approximately six times denser on microchromosomes (McQueen et a1. 1998) McQueen et al. (1998) predicts that approximately 75% of chicken genes are located 24 on microchromosomes. Clark et al. (1999) sequenced 18 cosmids with known chromosomal origin and found an increase in gene density on microchromosome based cosmids, but their data was inconclusive for CGIs due to the small sample size. At present (Groenen et al. in press), there does not appear to be an unusually high density of genes located on microchromosomes, but since the choice of genes to map has not been random and since little is known of the physical length of microchromosomal DNA, this may not refute the McQueen et al. (1998) conclusion. Analysis of BACs comprising the physical map of a microchromosome on a sequence level should give some insight into its gene density as well. Recombination rates on microchromosomes are also of interest. It was initially thought that crossover density in microchromosomes was less than macrochromosomes (Tegeldstrom and Ryttmann 1981; Slizinski 1964; Birshtein 1987), however, the opposite is now believed to occur. It is generally believed that chromosomes must have at least one or more cross—over events each (Carpenter 1994; Dutrillaux 1986; Kaback 1996) to insure proper meiotic segregation, and several studies have suggested that the microchromosomes also have about one chiasma per pair (Rahn and Solari 1986; Hutchinson 1987; Rodionov et al. 1992a, 1992b; Myakoshina and Rodionov 1994). Due to the small size of microchromosomes, if they indeed have at least one chiasma per meiosis, this would lead to unusually high recombination 25 frequencies per Mb of DNA. The macrochromosomes average about one crossover event per 30Mb (Rahn and Solari 1986; Rodionov et al. 1992a, 1992b; more recently, Groenen et al. in press, estimate the full length of the genome at 3800 cM, equivalent to 1 cM z 32 Mb for a 1.2 Gb genome), and it has been estimated that microchromosomes should have one crossover event every 11—12Mb (Rodionov et al. 1992a). Thus, the ratio of genetic length to physical distance of microchromosomes should be about 3X that of macrochromosomes. The present consensus map (Groenen et al. in press) contains several linkage groups of length substantially below 100 cM (equivalent to one cross-over per chromosome per meiosis), but it is not known how completely any of these linkage groups covers the full length of DNA within the putative microchromosome they represent. In one case, chromosome 16, two small linkage groups are known to be on the same microchromosome separated by a recombination hot spot which is located at the nucleolar organizer region. Nor is the actual physical length of DNA represented by any particular linkage group/microchromosome known. Building a contiguous physical map across a microchromosome might shed some light on this question. The large-scale project of building a genome-wide, BAC- based physical map of the chicken genome will be done through collaboration with the Texas A&M BAC center. The BAC research described in this thesis includes some preliminary characterization of the library and a test case use of the 26 library for regional physical mapping of linkage group E41. E41 has been identified as a microchromosome through FISH analysis (Sazanov, personal communication). Because of the small size of the microchromosomes (estimated at 1-10 Mb), it should be feasible to begin to construct a local physical map with relatively few (10-100) BAC inserts. Some regional physical maps have been based on enriched libraries constructed with DNA from a single chromosome or chromosomal region (using flow sorting, microdissection, or somatic cell hybrid—based procedures). For the most part, these resources are not available, at present, for the chicken. The alternative approach of screening a full genome library with markers previously localized to the genetic linkage group in question has been employed (Figure l, markers on E41). Restriction enzyme digestion patterns (fingerprints) of BAC inserts and cross-hybridization can be used to identify overlapping clones and build local clusters (called contigs) of such overlapping clones that contain the marker/gene used in screening the library. Given the present density of genetic markers in the chicken map (~2000 markers spanning 3,800 cM, Groenen et al. in press), rarely will it be the case that the contig containing one such marker will overlap with that containing the nearest available marker on the map. Gaps need to be filled either by increasing the density of useful genetic markers in the E41 genetic map and/or expanding contigs by "chromosome walking". Chromosome walking involves generation of new hybridization 27 probes from the ends of existing contigs (or isolated clones), followed by use of such probes to rescreen the BAC library. Each such "step" should extend the contig in question by about the length of a typical BAC insert (ca. 100-200 kb). The process can then be repeated to (slowly) fill in existing gaps. (Unless at least two genetic markers have already been placed relative to one another within a given contig, one must walk from both ends because the orientation of the contig to the genetic map is unknown.) In general, chromosome walking is too laborious for large- scale physical mapping, and it is mainly used to fill known gaps. Therefore, we have chosen to focus on a relatively densely mapped microchromosome to minimize the need for walking. The E41 test case will help to estimate the viability of such strategies for the chicken genome and our BAC library. As noted above, it is most reasonable to choose a microchromosome with dense marker coverage as a test case for regional physical map building using BACs. Linkage group E41 has 21 markers covering approximately 70cM (Figure 1). This includes 7 genes and 13 microsatellite and AFLP markers, which are the types of markers most easily mapped to BACs. The decision to use microchromosome E41 was also based on the location of a Marek’s Disease (MD) resistance Quantitative Trait Locus (QTL) on E41. MD is lymphproliferative disease that continues to be a significant health and financial problem for the poultry 28 industry (Purchase 1985). There is a continuing effort in the research community to improve the genetics of chickens to help combat this disease. One such approach has been to identify QTLs responsible for MD resistance, with the ultimate goal of finding the actual genes. Vallejo et al. (1998) and Yonash et al. (1999) did a genome wide scan for MD QTL, where a thorough description of the methods and results of the MD QTL analysis can be found. The E41 MD QTL specifically relates to differences in MDV viremia between similarly infected line 6 (resistant) and line 7 (susceptible) birds. Although actually locating the gene encoding this QTL is out of the scope of this research project, making a start on the E41 physical map might speed progress by others towards this ultimate goal. As will be described in Chapter 2, comparative mapping places several orthologues of known E41 genes to the end of human chromosome 9q. Detailed sequence analysis of this region in the human genome may also assist in suggesting candidate genes for this QTL—encoding chicken gene. Lines 6 and 7 were also shown to segregate MD QTL alleles found on chicken chromosomes four and eight, which was a factor in our choice to enhance the comparative chicken—human genome map covering these regions. This thesis describes the construction of a chicken- human comparative genome map over several selected regions. Statistical analysis of the resulting data has been used to estimate the average conserved segment length between the 29 human and chicken genomes. Microchromosome E41, which is an integral part of the comparative map for human chromosome 9, was the starting point for a preliminary analysis of physical clones from a newly constructed BAC library. 30 Figure 1. Markers on chicken microchromosome E41. OcM 70 cM I I I I I I I I I I I I I I I I I I I GSN RING3L R0820 L7a ABL1 AK1 AMBP ADL202 MCW335 ADL293 ADL149 MCW151 ACW117 ACW452 HUJ2 ADL199 ”3342 mcwsso 0039L1 ACW81 ACW118 Figure 1. Markers on chicken microchromosome E41. Included are gene markers GSN, RING3L, L7a, ABLl, AK1, CD39L1, and AMBP, the rest of the markers are either microsatellite (10) or AFLP markers (4). 31 Chapter 2: Comparative Mapping of the Chicken Genome INTRODUCTION Recent work in our lab and others has shown that a robust avian-mammalian comparative map can be made (Smith et al. 1997; Groenen et al. 1999; O’Brien et al. 1999). Several large regions with conserved synteny and regions with conserved segments have been found. For the work in Chapter 2, the focus was placed on the human—chicken comparative map. The mouse genome appears to be relatively unstable (reviewed in Graves 1996; Carver and Stubbs 1997; O’Brien et al. 1999), which could limit its usefulness in an avian comparative map. Additionally, the human genome has by far the most comprehensive genome map. Although many regions of the chicken-human comparative map were added to in Chapter 2, we focused on a few select regions rather than seeking broad coverage. Since the comparative map of human chromosome (h—chr) 1 was the most complete, and an attempt was made to fill in some of the gaps to identify the extent of the conservation. Our initial work had identified a large region conserved between h-chr 4 and chicken chromosome (c— chr) 4, and an attempt was made to extend the chicken—human chromosome 4 map. Initial work by our group and others had identified a large region of conservation between h—chr 9 and the chicken Z sex chromosome (Smith et al. 1997; Fridolfsonn et al. 1998; Nanda et a1. 1999). Two autosomal 32 sex—determining genes have recently been mapped to h—chr 9 and the c-chr Z (Nanda et al. 1999; Smith et al. 1999). In order to provide insight into the evolution of ZW—type chromosomal sex determination in birds, an effort was made to increase the comparative map between the chicken Z sex chromosome and human chromosomes 9. Additionally, c-chr 8, c—chr 4, and c—chr E41, which show a large degree of conservation with h-chr 1, h-chr 4, and h-chr 9 respectively, contain QTL for Marek’s disease resistance in the chicken (Vallejo et al. 1999; Yonash et al. 1999). A comparative map in these areas may assist in identifying potential candidate genes for MD resistance. By focusing on relatively few regions, we hoped to get good coverage of these chromosomes. This was done in order to get a general idea of the number of chicken segments that would cover a human chromosome. In the early 1980s, far less map data existed for both the mouse and human genomes. In order to analyze the amount of genomic conservation between the two species, Nadeau and Taylor (1984) derived a method to estimate overall genome conservation from a limited set of gene segment comparisons. When compared to data generated from a high-resolution human-mouse comparative map, their model generated an accurate prediction of average genome conservation (Copeland et al. 1993). Statistical analysis based on the work of Nadeau and Taylor (1984) was performed on the region specific comparative map data to derive an estimate of the 33 genome-wide conservation of gene order between chicken and humans. 34 MATERIALS AND METHODS Determination of Orthologues: Chicken cDNA sequences obtained either from National Center for Biological Information's Genbank database (NCBI: http://ncbi.nih.nlm.gov) or the University of Delaware (UDEL) cDNA library (Burnside and Morgan, http://udgenome. ags/chickenest/chick.htm) were compared to human gene sequences using the Basic Local Alignment Search Tool as provided by the NCBI web site (BLAST: http://www.ncbi.nlm. nih.gov/BLAST). Four main factors were used in determining the human orthologue to the chicken sequence. These are functional similarity, nucleotide (nt) sequence similarity, protein sequence similarity, and common chromosomal linkage relationships. Levels of nt identity were determined using the blastn program within BLAST and protein identity using the blastx program. Table 1 lists the Gallus gallus sequence and the percentage of nt and protein identities with the corresponding human genes. The comparison is made over the entire cDNA sequence. The nt identities range from 61%-94% and the protein identities range from 51%—99%. When there were multiple human genes that had high nucleotide and protein similarities, it was possible to distinguish the best candidate for the orthologue. For example, chicken skeletal muscle alpha-actinin cDNA (accession: X13874) has a nt identity of 80% and a predicted protein identity of 80% 35 with the human gene ACTNB. The two proteins also have a similar function. ACTN2 has a nt identity of 83% and a protein identity of 95% (Table l). ACTN2 is linked to ADPRT on ch-chr 3 and ACTN2 and ADPRT are closely linked on h—chr 1 (Figure 1), whereas ACTN3 is located on h—chr 11. Thus, both sequence homology and linkage relationship supports the conclusion that the X13874 sequence is orthologous to human ACTN2. If there are two copies of the gene in humans, the nt identity was naturally very high for both copies with the respective chicken gene. This was the case with splicing factor arginine/serine-rich 2 (SFSRZ). There is a copy of SFSRZ on h-chr 4 and another on h-chr 17. SFSRZ maps to linkage group E31 in chickens (Figure 9), along with two additional chromosome 17 syntenic loci, FAS and H338. Therefore, the SFSRZ found on E31 is mostly likely orthologous to the human gene on chromosome 17. All of the factors were taken into consideration when assigning chicken loci. In some cases, the Unigene (Unigene: http://ncbi.nlm. nih.gov/UniGene) and Online Mendelian Inheritance in Man (OMIM: http://ncbi.nlm.nih.gov/omim) databases within Genbank were used to identify human genes in regions of interest followed by a search for an orthologous chicken cDNA in Genbank or the UDEL database. The Genbank searches were performed by using the human gene sequence and running a BLAST search against the Gallus gallus sequences in the database. The UDEL cDNA database has been BLASTed against 36 the entire Genbank database and positive genes are listed along with the corresponding percentage positive nts. The orthologous chicken sequence information was used to construct polymerase chain reaction (PCR) primers used to clone and sequence chicken genomic DNA from parental DNAs of our map population. For the Abelson murine leukemia viral oncogene homolog 1 (ABLl) chicken orthologue, primers from the Universal Mammalian Sequence Tagged Site (UMSTS) set (Venta et al. 1996) were used. Primers for the chicken gamma—carboxylglutamic acid protein, matrix (MGP) gene were from the Primer Pairs to Sequenced Chicken Genes (PPSCG) panel (appendix 2). PCR Primer Design: Where possible, PCR primers were chosen to amplify a large fraction of the 3' untranslated region (UTR) of the chicken gene of interest. When it was necessary to amplify predominantly coding regions, only PCR products larger than the predicted cDNA size were analyzed further, since these presumably include intron regions which are more likely to be polymorphic. Occasionally, when the available 3' UTR sequence was small, primers were designed to cover as much of the 3’ UTR as possible and some coding region as well, in the hope that an intron would be included. One problem that arose during the amplification of the gene products was that of product size. A nucleotide 37 polymorphism between the WL and JF parents is needed to genetically map the gene. The 3’UTR was chosen as the region to amplify in most of the genes. A nucleotide difference in a non—coding region may not have as great an effect as a difference would in a coding region and 3’UTRs should be less conserved over evolutionary time. Additionally, there are also fewer introns in 3’ UTR. The product size between cDNA sequence and PCR products designed from the cDNA will more likely be the same. PCR products designed from within coding regions where there is no information about intron size or location can be problematic due to very large product size. An additional problem is that primers could be designed across intron boundaries. This can lead to the PCR product being too large to be cloned efficiently under normal conditions or no product at all. Both of the above problems were encountered where no 3' UTRs were available and primers were designed to cover coding regions. This was the case with the UDEL cDNA library where there are only partial cDNA sequences, occasionally the 3’UTR is just small in some genes, and in the PPSCG set of primers, which are designed within the coding region of the gene (Appendix 2). These large products led to a decrease in our success rate when these were the sources of our gene sequences. PCR primers were designed with the PrimerSelecttn PCR Primer & Probe Design program within the Lasergene Biocomputing Software (DNASTAR Inc., Madison, WI) suite of 38 programs. Criteria used in the design included: similarity of melting temperature (Tm) between the two primers, predicted absence of primer dimers, and absence of hairpins. An attempt was made to keep primer size from 18 to 24 nt in length with around 50% GC content. In the hope that these primers could be used in multiplex PCR, the predicted Tm were all kept in the 55—6OTIrange. All primers were purchased from the Michigan State University MacroMolecular Structure Facility. All PCR and primer information is contained in Table 2. Cloning and analysis of PCR products PCR was performed using the conditions described in Table 3. The entire PCR reaction was run on 1% low melting temperature (LMT) agarose. When a single band was observed, the WL and JF bands were extracted and cloned into the TOPO- TA“‘(Invitrogen Corporation, Carlsbad, CA) cloning vector using the Low—Melt Agarose Method for purification of PCR products as per the manufacturer’s recommendations. Transformation into the One—ShotTM Chemically Competent cells (Invitrogen Corporation, Carlsbad, CA) was done according to the manufacturer’s recommendations. The cells were plated on LB plus 50 ug/ml ampicillin (AMP) with 40 pl 40 mg/ml X—GAL per plate and incubated atl3fC for 16 to 18 h. An additional test was performed on white colonies prior to sequencing to ensure they contained the product of 39 interest. Colonies were picked into 240 pl of LB plus AMP in individual wells of a flat bottomed 96 well plate (Cell Wells“, Corning Glass Works, Corning, NY). The plate was incubated at 37°C for 8—12 h. Four ul of the cells were placed into individual wells of a 96 well thin walled PCR plate (Thermowellm, Model M, Corning Glass Works, Corning, NY), covered by a drop of mineral oil, and heated to 94%: for five minutes to lyse the cells. The appropriate PCR mixture (23 ul) was added to the lysed cells and PCR was performed under the same conditions as for genomic PCR. The PCR reactions were then run on a 1% agarose gel to determine if they contained the same size insert as expected. Plasmid DNA purification was done using the QiaprepCm miniprep kit protocol (Qiagen Inc., Valencia, CA). Concentrations of purified plasmid DNAs were determined by fluorimetry (TKO Mini Fluorometer, Hoefer Scientific Instruments, San Francisco, CA). Three individual clones from both JF and WL genomic templates were sequenced using SP6 or M13 reverse primers and the T7 primer, using ABI 377 automated sequencers at either the Michigan State University Sequencing Facility or at the U.S.D.A. Avian Disease and Oncology Laboratory. Three clones were sequenced to insure that observed polymorphisms were unlikely to arise from PCR or sequencing errors. 40 Sequence analysis and genetic mapping: BLAST analysis between cloned PCR products and chicken sequence data previously found in Genbank confirmed that the correct gene had been cloned. Greater than or equal to 99% identity in the known coding regions and 3’UTR was considered positive. Intron sequences were sometimes found in the cloned product that, of course, were absent from the earlier cDNA sequences, but the identity of introns could be confirmed by the presence of consensus intron boundary sequences (Keller and Noon 1984 Mariman et al. 1984). In order to control for sequencing errors, the alignment of the sequences from the cloned plasmids was performed using the Seqmancm Sequence Assembly and Contig Management program within the Lasergene Biocomputing Software (DNASTAR Inc., Madison, WI) suite of programs. The alignment of the sequences into contigs makes it possible to distinguish true SNPs and sequence differences due to errors in sequencing or PCR induced sequence errors. Alignment of successfully sequenced plasmid clones was done under the manufacturer’s recommended parameters for contig assembly (Seqmanw, DNASTAR Inc., Madison, WI). Alignment of the sequences into contigs allowed for the identification and placement of any SNP between WL and JP. 41 For mapping in the reference BC population, polymerase amplification of specific alleles (PASA) primers were designed based on the polymorphic nt alteration (usually a SNP) such that only the JF allele successfully amplified. PASA primers were designed to minimize the possibility of hairpin or dimer formation. If there were multiple JF vs. WL polymorphisms, the one giving rise to the predicted optimal allele-specific primer (ASP) was used. Either the forward or the reverse primer from the original PCR amplification was chosen as the other primer, based on best fit with the ASP. ASP were generally designed with the JF-specific nt at the 3’ end and an additional mismatch to both the WL and JF sequence, three nt from the 3' end. As demonstrated by Okimoto and Dodgson (1996), the additional mismatch provides increased specificity and accuracy in genotyping. Occasionally, additional changes were made to adjust the Tm or to avoid predicted hairpins and/or dimer formation. In one case, TNNTZ, there were multiple SNP available, and two opposing ASP were found to be necessary for genotype analysis (Table 2). All the PASA PCR primer information is provided in Table 2. PASA PCR genotyping was performed in duplicate on the 52 animals of the reference BC population (Crittenden et a1. 1993). PCR products were run on 1% or 2% agarose gels and absence or presence of the JF allele was determined (Figure 1, Appendix 3). Segregation data were analyzed using MAPMANAGER version 2.6.5 (Manly, K., Roswell Park Institute, Buffalo, NY). The correct map positions were 42 determined using the following criteria: within the strain distribution patterns, the position with the least number of crossovers and with minimal double recombinants that generated the highest possible log10 of odds (LOD) score. In order to be considered linked to other markers the LOD score had to be greater the 3.0. 43 RESULTS In order to generate a preliminary View of chromosomal evolution between birds and mammals, we chose to focus on a representative subset of the vertebrate genome, those genes contained on h-chr 1,4 and 9. These regions initially were targeted due to the fact that preliminary evidence suggested that they may contain QTL-encoding genes for resistance/susceptibility to Marek's Disease Virus as mapped by Vallejo et al. (1998) and Yonash et al. (1999). Subsequently we chose to map as many chicken orthologues as possible of the human genes already known to map to these regions. We believe that these observations can be extrapolated to derive conclusions about the overall comparative chicken-human genome map. Figure 1 is a graphical representation of the comparative map of h-chr 1 and the corresponding segments of c-chr (or linkage groups, where a specific c-chr has yet to be identified). Table 4 lists the genes mapped in this study that provide comparative map coverage of h-chr 1. The source of the chicken cDNA sequences is also listed for each chicken gene (either Genbank or UDEL). As outlined in Materials and Methods, chicken gene sequence information was used to design PCR primers for amplification, cloning, and sequence analysis of selected gene segments from parental DNAs of the East Lansing reference mapping family (Crittenden et al. 1993). When sequence polymorphism was observed between the WL(UCD003) and JF(UCD001) alleles, PCR— based assays were developed with which to genotype the standard reference gene mapping panel, thereby locating the chicken orthologue on the East Lansing reference map (http://poultry.mph.msu.edu) and the consensus chicken gene map (Groenen et al., in press). The map position on the EL reference map is listed in Table 4 along with the human physical map position from OMIM (OMIM: http://ncbi.nlm.nih. gov/OMIM). The human genetic map information from Unigene (Unigene: http://ncbi.nlm.nih.gov/UniGene) tends to be more accurate than the physical map information (chromosomal placement is more precise). Because of this, there are a few discrepancies between the tables and figures. This was done when the physical map position covered a large range, such as XPA, the physical position is 9q22.3-q31, but the genetic map information more accurately places XPA near 9q22. Bold and underlined genes were mapped in the current study. Six segments of the chicken genome provide almost complete coverage of h-chr 1, with a few gaps not covered by corresponding chicken segments. Four chicken genome segments contain three or more genes whose orthologues map to h-chr 1. Two of these are linkage groups E54 (telomeric end of 1p) and E04 (1q31-q32.1). It is likely, but not certain that these linkage groups correspond to chicken microchromosomes. An internal segment of c-chr 3 appears to correspond to the telomeric end of 1q. C-chr 8 shows conservation to both the p and q arms of h—chr 1. RPL5 has only been mapped on the 45 Compton reference population (Compton and Palyga 1992) and its precise location among the other markers is not known. Between these two conserved segments are two genes on h—chr 1 that map to a segment of c-chr 1 (HSD3B) and to E26 (MCLl), respectively. HSDBB is in a region of h-chr 1 for which we have no nearby marker information and MCLl is the only gene mapped to E26. Thus, further comparative mapping will be required to ascertain whether these two associations are part of large conserved segments, derive from small translocations (e.g., transposon—mediated rearrangements), or result from mistaken assignment of orthology. However, that the largest h-chr (approximately 300cM) appears to correspond to as few as 4—8 chicken genome segments is noteworthy, as is the fact that relative gene order is almost completely conserved (i.e., lack of evidence for inversions). Figure 2 shows the location of chicken orthologues of genes on h—chr 9, with further information provided in Table 5. Conventions and methods used are as described above for Figure 1 and Table 4. In addition, one of these genes, the ABLl proto-oncogene, was amplified using UMSTS primers (Venta et al. 1996; Smith et al. 1997). Figure 2 demonstrates that much of h-chr 9 derives from segments that correspond to the chicken Z chromosome and the probable microchromosome E41, the latter corresponding to the telomeric end of human 9q. However, the chicken Z chromosome segment also contains at least four genes that do 46 not map to h-chr 9, and the human segment in question contains a single gene (ALDHl) which maps to the E18 linkage group. Again, further comparative map data will be required to elucidate the relevance of these single gene homologies. In addition, the h—chr 9—chicken Z chromosome segment exhibits two internal alterations in gene segment order (the TPM2 gene and the CTSL to XPA segment). These could be due to inversions (intrachromosomal) within one large conserved segment or to independent translocations (interchromosomal) between the same pair of ancestral chromosomes. The independent origin of the avian sex chromosomes as opposed to their mammalian counterparts has been noted previously by others (Fridolffson et a1. 1998; Nanda et al. 1999), and in some cases, rearrangements appear more common on sex chromosomes than autosomes. However, this trend is most striking on the sex chromosome that is mostly non—coding, i.e., the avian W and mammalian Y chromosomes. E41 is a microchromosome (Sazanov, personal communication). All seven genes mapped have the same gene order as on h—chr 9. It appears that most small linkage groups have been well conserved, for example E54 and E04 (Figure 1), although this is not always the case (E29, Figure 3; E52, Figure 4). Table 6 and Figure 3 show the positions of chicken orthologues of genes on h—chr 4. A large section of c-chr 4 is conserved with the q arm of h-chr 4. Assuming that EDNRA, SPPl, ALB—GC, PPAT, and NFKBI are placed accurately, there appear to have been at least two inversions or three 47 independent translocation events in either the avian or mammalian line since the last common ancestral genome. The FGFR3 gene at the distal end of h-chr 4p is also on c—chr 4, but this gene is quite distant from the segment previously described and is separated from it by at least two genes that map elsewhere in the chicken, so the synteny of FGFR3 and the segment is likely to be fortuitous. Unfortunately, we have not been able to map chicken orthologues of genes at the most telomeric end of h-chr 4q. In the early stages of this study and in the course of trying to extend or define conserved segments described above, several other genes were added to the overall chicken-human comparative map. These are summarized in Table 7 and Figures 4 through 9. Although we did not add more than one or two new genes to each of the relevant chromosomes or linkage groups, in several cases, our observations extended conserved segments observed by other laboratories (Fridolfsson et a1. 1998, Nanda et al. 1999, Groenen et al. 1999). Rate of Chromosomal Evolution: Nadeau and Taylor (1984) calculated the expected lengths of conserved segments between the human and mouse genomes using thirteen homologous segments known at that time. As noted previously, the Nadeau and Taylor predictions in 1984 turned out to be surprisingly robust. Thus, we applied the Nadeau and Taylor theory to 19 conserved 48 segments between humans and chicken (Table 8). Table 8 lists the chromosomal location of the chicken genes and the corresponding location on the human genome. The majority of the conserved segments were found or added to in this study. Additional groups (such as DNECL—CKB and CRYB—IGVPS—MIFLZ) were found by searching the chicken genome database in Arkdb—CHICK (http://www.ri.bbsrc.ak.uk/chickmap) for gene clusters that formed conserved segments with the human genome. The mean of the expected segment lengths (mean m = 67) is transformed to account for segments lacking identified genes and conserved segments with single markers. The mean length would be biased toward longer segments since only those with two or more genes are included. The complete mathematical transformation is discussed in Nadeau and Taylor (1984). Their final equation is: E(x’) = (sz + 3L)/(LD+1) where E(x’) is the mean of the transformed lengths (67.4), and D is the total number of mapped homologous loci (~150 consensus map) (Groenen et al. in press) divided by the genome size (3,800 cM, Groenen et al. in press). The mean length of conserved segments between humans and chicken (using the data from Table 8) is 38 i 9 cM. The rate of chromosomal evolution between humans and chickens can also be calculated based on the model of Nadeau and Taylor (1984). This first step is to calculate the number of disruptions that have accumulated during the 49 evolutionary divergence of chickens and humans. The formula of Nadeau and Taylor (1984) is: R = (G/L) - N0 R is the number of disruptions, G is the genome length and 1% is the total number of haploid chromosomes in the last common ancestor. The true.Nois not known; therefore, the lower haploid number of the compared species (23) was used (O'Brien et a1. 1999). (Reasonable values of Rh have little effect on our final conclusions.) Using the value of L as 38, R = 77 i 24. The average rate of reciprocal disruption is R divided by twice the estimated time to the last common ancestor (300 myr, Kumar and Hedges 1998) to account for disruptions in both species or about 0.13 i 0.04 disruptions per myr. 50 Discussion Comparative map: One of the goals of this project was to test whether it would be feasible to build an avian—mammalian comparative genome map. Our initial results and those of others (Klein et al. 1996; Smith et al. 1997; Fridolfsson et a1. 1998; Groenen et al. 1999) showed that there were surprisingly large conserved segments between the human and chicken genomes. While a complete comparative map for these two species was beyond the scope of the present project, a more limited analysis focusing on human chromosomes 1, 4 and 9 was performed. Our results suggest that there will typically be between four to eight chicken segments per human chromosome, so the long-term goal of a complete comparative map between chicken and mammalian genomes is feasible. Two preliminary genome-wide comparative maps, based on some of the data reported herein plus that available from other labs, have recently been described (Burt et a1. 1999; Groenen et al. in press). There is now general agreement that the chicken genome can be even more closely aligned with the human genome than can that of the mouse (Burt et al. 1997; Burt et a1. 1999; O’Brien et al. 1999; Groenen et al. in press). The level of similarity between the human and chicken genomes is especially remarkable, given the fact that the 51 former contains almost three times as much DNA as the latter. As can be seen in Figures 1 and 2, as yet there is no evidence for large, chromosome-sized segments of human DNA that contain no obvious chicken orthologues. If this is confirmed in more detailed comparative maps, one must conclude that the "excess" human DNA is mostly interspersed. Indeed, based on anecdotal evidence, it was observed long ago that chicken gene families tended to be more closely packed, and have smaller introns and fewer pseudogenes than their mammalian counterparts (Dodgson et a1. 1979). Thus, it seems likely that a very large number of small deletions from the mammalian genome and/or insertions into the chicken genome have occurred during their separate evolution without significantly affecting the larger scale gene order. Thus, while at the level of DNA sequence the smallest evolutionarily conserved segments between the human and chicken genomes are likely to be rather small (probably on the order of a typical exon or about 1 kb), at the level of gene order, the average conserved segment appears to be 30— 40 cM (ca. 10 Mb of chicken DNA and 30 Mb of human DNA). Thus, the mechanisms by which small deletion/insertion events occur (replication errors, transposable elements, unequal recombination, etc.) must be very distinct from large scale chromosomal rearrangements. A similar situation exists for several plant genome comparisons, for example, corn vs. rice (Gale and Devos 1998). 52 Microchromosomes: One problem in assembling maps of the chicken genome has been the fact that chicken microchromosomes are not cytologically distinct (other than chromosome 16 which contains the NOR). However, with improved genetic maps (Groenen et al. in press) and preliminary fluorescent in situ hybridization experiments (Fillon et al. 1998), there has been some progress in categorizing microchromosomes. Identification of 16 chicken microchromosomes by molecular markers using two-color fluorescence in situ hybridization (FISH). Fillon et al. (1998) confirm that most of the undefined linkage groups in the EL reference map correspond to microchromosomes. Many presumptive microchromosomes, e.g., E41, appear to be conserved as a single block in the human genome. However, most of them do not contain enough cross—mapped genes to be confident of this conclusion. On the face of it, it is not surprising that microchromosomal segments survive intact, given that many of them may not be much larger than the average conserved segment length of 38 cM. On the other hand, microchromosomes have been proposed to be rich in both genes and recombination events compared to the autosomes (Rodionov 1996, 1997; Primmer et al. 1997; Sazanov et al. 1996; Fillon 1998). It remains unclear as to how one might reconcile differential gene density between micro and macrochromosomes with a high level of conservation of gene order with the human genome, where, to the best of 53 our knowledge, no such gene density distinction exists. Perhaps the density of internal insertion/deletion events discussed above (which generally appear to have little effect on gene order), may have been substantially different in genome segments which are microchromosomal in chickens vs. macrochromosomal. Microchromosome E41 is of special interest and will be discussed further in chapter 3. It contains a suggestive QTL for MDV viremia levels (marker ADL0149 has a LOD = 2.5 with the QTL; Vallejo et al. 1998; Yonash et al. 1999). The Major Histocompatibility Complex (MHC, called the B complex in chickens) of genes on chromosome 16 is known to play an important role in MD infection and severity of disease (Bacon 1987). The Ring3-Like gene, which has been mapped to E41, is found near the qter end of h-chr 9 in band 34. Ring3 is a gene in the MHC class II region on chromosome 6, but there has been a second similar copy mapped to 9q34 (Thorpe et al. 1996). Based on its high protein and nucleotide similarity and its conserved linkage, it is highly probable that RINGBL is on E41 and it was so designated in Figure 2. Several other MHC—related genes have also been mapped near RING3L on h-chr 9q, including Proteasome Subunit, Beta—Type, 7, PSMB7; Pre-B-Cell Leukemia Transcription Factor 3, PBX3; and Homolog of Drosophila Notch 1, NOTCHl. It seems likely that a similar group of the chicken orthologues of these genes will be found on E41, and they could serve as potential candidate genes for the MDV viremia—encoding QTL 54 allele(s). This is a preliminary, but illustrative, example of how the comparative human—chicken genome map can aid in the search for genes encoding chicken traits of interest. Relevance of the Nadeau and Taylor Model to the Chicken- Human Comparative Map: The original estimate of mouse vs. human average conserved segment length made by Nadeau and Taylor (1984) was 8.1 cM. Copeland et a1. (1993) later calculated the average to be 8.8 cM, and O'Brien et a1. (1999) estimates 8.1 cM in a review of several published reports. Thus, at least in the case of mouse vs. human, the model appears very robust. Still, there are many assumptions made in the model that need consideration. The first is that synteny between two markers in both species is presumptive evidence for iconserved linkage. Evidence from many species (reviewed in Nadeau and Sankoff 1998 and O'Brien et al. 1999) generally supports this assumption, at least within mammals. The number of apparent conserved segments with several common markers, often in the same order (Figures 1-9; Burt et al. 1999; Groenen et al. in press), also supports the validity of the assumption when comparing chicken and human genomes, although probable exceptions (e.g., FGFR3, Figure 3) exist at low frequency. Second, the model assumes that chromosomal rearrangements fixed during evolution are randomly distributed throughout the genome. Although it is 55 well known that recombination rate is not uniform, this assumption is probably adequate for the calculation of mean conserved segment length at the level of resolution of presently available data. The model also assumes that orthologous markers are randomly distributed throughout the two genomes of interest. This assumption is important because the initial calculation of the expected value of r (r= the actual length in cM of the conserved segment, m: the expected value of r) is determined by calculating the expected range of a random sample taken from a uniform distribution. In this case, the random sample will be the mapped markers from the chicken map. An account is made for the bias toward long segments by assuming the frequency of segments containing two or more markers will follow a truncated Poisson distribution. A plot of the normalized cumulative distributions of the frequency of increasing adjusted segment sizes is illustrated in Figure 10. Included are curves for L = 5, 20, 30, 40, 56, and 75, as well as the cumulative distribution of the transformed segment lengths from this study. It appears that for the larger segment sizes the model fits quite well, (L > 50 cM) with the best fit around L = 40 GM, as calculated above. The smaller transformed segment lengths do not follow the same curve, tending to be smaller than would be expected. There could be several reasons for this, both technical and biological. Technical errors could include sampling error (less than complete coverage and non—random selection of some 56 markers), errors in assessment of orthology or errors in the genetic map itself. Non-random marker placement could lead to an increase in the number of segments relatively small in size. In the current study, an attempt was made to cover certain human chromosomes but not to focus on a small area of interest, but this may not be true for all markers used in the analysis. In an attempt to increase the number of markers and to increase the density of the comparative map in a certain chromosomal area containing a gene of interest (such as a QTL), genes mapped by others may have focused on a narrow chromosomal region. Although the limited sequence analysis of many chicken gene family members could create possible mistakes in assigning orthologous genes, most gene family members which show high sequence homology tend to be closely linked in the genome, in which case such an error would have no impact on the comparative map. Mapping errors are more likely in the chicken map, most of which is based on only 52 meioses. These would be most likely to alter the internal gene order within a conserved gene segment, thereby leading to a mistaken estimate of an inversion event. If a gene has been erroneously included as part of a conserved segment, this would lead to overestimation in the size of the conserved segment. There are also possible biological explanations for the higher than expected proportion of short segments. First, it has been proposed that both recombination rate and gene 57 density on microchromosomes are abnormally high (Rodionov 1996, 1997; Primmer et al. 1997; reviewed in Fillon 1998). Although neither of these assertions has yet been proven by physical genome mapping or sequencing, either or both phenomena could contribute to the biphasic distribution seen in Figure 10. Second, chromosome rearrangements presumably involve multiple mechanisms, for example, intrachromosomal inversions, interchromosomal translocations, movement of internal segments via flanking transposable elements, etc. It seems unlikely that these different mechanisms would produce similar spectra of segment sizes. The effect of diversity in recombinational mechanism may be more apparent in the distant comparison of avian vs. mammalian genomes than it was in comparing mouse and human genomes. Estimated Rate of Autosome Evolution: Application of the Nadeau and Taylor (1984) model led us to estimate the average chromosomal evolution rate that separates the chicken and human genomes to be 0.13 i 0.04 disruptions per myr. It has become increasingly clear that chromosomal evolution rate varies considerably in different evolutionary lines ranging from about 0.01 to >2.0 disruptions per myr (e.g., Bickham 1981; Nadeau and Taylor 1984; Paterson et al. 1996; O'Brien et al. 1999). It should be pointed out that the low end of this range (in turtle species, Bickham 1981) was based on karyotypic analysis of 58 banded chromosomes only and is likely an underestimate. Our estimate of 0.13 disruption/myr is similar to the estimates of O'Brien et al. (1999) for the most stable mammalian genomes (e.g., human, feline) relative to the common ancestral mammalian genome. This suggests that a similar rate of chromosomal evolution has been maintained in the lines leading to both the human and chicken genome from their last common ancestor. As noted by Rodionov (1996), karyotype analysis suggests a high level of genome stability within birds in general and thus, by extrapolation, within the line leading to modern chickens from the common mammalian-avian ancestor. Our comparative genetic mapping results confirm this conclusion. Sex Chromosome Evolution: In birds, the heterogametic sex is the female (ZW)and the homogametic sex is the male (ZZ). Very little is known about ZW sex determination in birds. Figure 2 demonstrates that a surprising number of chicken orthologues of genes on h—chr 9 were mapped to the Z chromosome. Previously, a few chromosome 9 genes had been mapped to the Z chromosome by our group and others (Smith et al. 1997; Fridolsson et al. 1998; Nanda et al. 1999), but the extent of conservation was unknown. The current theory of mammalian and avian sex chromosome evolution maintains that the respective sex chromosomes evolved independently from different autosomes 59 within the two evolutionary lines (Ohno 1966; Watson et al. 1991; Reed and Graves 1993; reviewed in Marin and Baker 1998). The genes mapped on the Z chromosome and chromosome 4 appear to fit this model (Figures 2 and 6). As is expected, sex—controlling genes are found on avian sex chromosomes and sex reversal has been reported for different triploid arrangements in chickens (reviewed in Thorne and Sheldon 1992). The sex-determining gene SRY has been mapped in humans to the human Y chromosome (Sinclair et al. 1990). Sex reversal phenotypes can arise from chromosomal abnormalities on several autosomes as well as on the sex chromosomes in mammals (reviewed in Wachel 1987; reviewed in Reed and Graves 1993). One case of particular interest is XY chromosomal males that have a female phenotype and which exhibit a 9pter deletion (Raymond et al. 1998; Fleijter et al. 1998; Guioli et al. 1998). The phenotypes associated with this abnormality range from ambiguous genitalia to complete gonadal dysgenesis. The human genes DMRTl and DMRTZ have been mapped to the minimal region contained in the deletion (Raymond et al. 1998, 1999). These genes were isolated due to their homology to the male regulatory genes doublesex in Drosophila and mab-3 in Caenorhabditis elegans. Genetic analysis in the humans has shown that DMRTl and/or DMRTZ may operate in a dose— dependent fashion in the male sex-determination pathway (Raymond et al. 1999). Recently the chicken gene DMRTl 60 has been mapped through FISH to the chicken Z chromosome at the p21 position (Nanda et al. 1999) Additionally, chickens have been shown to have gonadal specific expression of DMRTl (as does the mouse) (Smith et a1. 1999). Two genes in the 9pter region (VLDLR and TYRPl) were mapped to the Z chromosome (Figure 2). The DMRTl and DMRTZ genes lie within the microsatellite markers D98129 and D9Sl43 on the pter region of h-chr9 segment (the interval is 1.9cM) (Raymond et a1. 1998, 1999; Fleijter et al. 1998; Guioli et a1. 1998). VLDLR is near the p telomere of chromosome 9 within the interval defined by D98129 and D9Sl43 and TYRPl is about 25 cM down from VLDLR. The farthest VLDLR could be from DMRTl and DMRTZ in humans would be 4.2 cM. Based on the formula from Nadeau and Taylor (1984) for calculating the probability of linkage based on the estimated mean conserved length (Probability = e”“, where x = 4.2 cM and L = 37.5 cM), there is a 90% probability that these loci are this closely linked to VLDLR on the Z chromosome. Therefore, it appears that this entire ancient sex-determining region has remained as a conserved segment between humans and birds. 61 Table 1. Gallus gallus sequence: Genbank Human Nucleotide Protein accession loci: identities": identities": or UDEL cDNA #: collapsin response mediator U17277 CRMP1 79% 97% protein CRMP—62 PR264 X62446 SFRSZ 83% 99% endothelin type A receptor AF040634 EDNRA 87% 80% trans Golgi network protease 268093 PACE 84% 81% furin Caspase—1 AF031351 CASP1 61% 49% villin J03781 VIL 84% 71% NF-kappaB p50 precursor M86930 NFKB1 85% 71% preproalbumin X60688 ALB 91% 61% n-calpain-1 large subunit 038028 CAPN1 71% 80% poly(ADP-ribose) X52690 ADPRT 79% 79% polymerase tyrosine kinase M35195 FGFRS 82% 82% alpha-tubulin V00388 TUBAL1 85% 98% stem cell factor D13516 MGF 90% 51% homogenin AF042795 GSN 83% 79% ABL proto-oncogene U66284 ABL1 87% 98% aldehyde dehydrogenase X58869 ALDH 81 % 91 % 62 Table 1. Cont. Gallus gallus sequence: Genbank Human loci: nucleotide protein accession identities": identities": or UDEL cDNA #: tyrosinase-related protein-1 AF003631 TYRP1 82% 82% precursor skeletal muscle alpha-actinin X13874 ACTN2 83% 95% axonin-1 X63101 TAX1 82% 75% glutamine synthetase $45408 GLUL 79% 88% troponin T form I M10013 TNNT2 83% 77% prostaglandin G/H synthase M64990 PTGS2 81% 82% xpacch 031896 XPA 81% 72% cytosolic phospholipase A2 010329 PLAZG4 80% 83% Iysyl hydroxylase M59183 PLOD 80% 77% trkB X74109 NTRK2 85% 77% pepsinogen 000215 CTSE 87% 62% smooth-muscle alpha- K02446 TPM2 87% 95% tropomyosin RPK-2 014460 TGFBR1 85% 92% glutamine M60069 PPAT 80% 83% phosphoribosylpyrophosphat e amidotransferase VLDUvitellogenin receptor X80207 VLDLR 83% 83% matrix GLA protein Y13903 MGP 71% 61% 63 Table 1. Cont. Gallus gallus sequence: Genbnk Human nucleotide protein accession loci: identities": identities“: or UDEL cDNA #. UDEL cDNA pk0033.h4 RINGSL 83% 81% UDEL cDNA pk0061.c12 JAK1 79% 89% UDEL cDNA pk0012.d1 UBE2A 89% 99% UDEL cDNA pk0006.b2 CTSL 79% 71 % UDEL cDNA pk0031.e6 MCL1 83% 61 % UDEL cDNA pk0049.16 GC 85% 66% Table 1. GaIIus gallus gene sequences and the percentage nt and protein identity with the corresponding human gene. *Percentage nucleotide identity obtained through a blastn comparison. ** Percentage protein identity obtained through a blastx comparison. Table 2. Genes Mappgd: Primer and PCR Information Jgnus Kinase 1 JAK1 product size: 8006p annealing temperature: 59°C upper primer: lower primer: 5' TCG AAA AAG TGA ACT 5' GAT TCG CTC CAC GCA CCT GAC AAC 3' TTC TI" 3' JF specific - product size: 140bp PASA ' annealing temperature: 57°C primer: use with lower primer 5' TGG ACA AAT ACT TCG GCT ACA 3' ubiguitin- product size: ~1kb Coniugating Enyme E2A (QQEZA) annealing temperature: 59°C upper primer: lower primer. 5' ATC CAA ATA AGC CAC 5' CAA CAA TCA CGC CAA CTA CTG 3' CTC T 3' JF specific - product size: 250bp PASA annealing temperature: 57°C primer: use with upper primer 5' TTC TGC CCC CTT ACT AAA C 3' 65 Table 2. Cont. Gamma- Carboxyglutamic Acid ProteinI Matrix (MGP) JF specific - PASA product size: >2kb annealing temperature: 59°C upper primer: lower primer. 5' TGC GTG CTC TCA TCG 5' CTC CTC CCA AAA TAG TCC T 3' TGC CTG TAA 3' product size: 170bp annealing temperature: 57°C primer. use with lower primer 5' CAT AGA CAG ATA TIT AAG ATA CCA 3' Trgponin T 2 (MNTZ) JF specific- PASA product size: 500bp annealing temperature: 59°C upper primer. lower primer: 5‘ AAC GGA GCG GGA GAA 5' ATG TGG GGG TGT GAA GAA AAA 3' GGA GAT GAG AAT 3' product size: 80bp annealing temperature: 57°C upper primer. lower primer. 5' GGC TCT GCT GCC TCC 5' GCT GAG CAC CTG CCA ACG 3' CCC ACC ACA 3' Table 2. Cont. Vgg Low Density Lipoprotein Receptgr (\_ILOR) JF specific- PASA product size: 900bp annealing temperature: 59°C upper primer. lower primer: 5' GCT TGG GCT GTT CTT 5' TAT CAT CCC CGT CCT ATC T 3' AAG TGT AAA AC 3' product size: 360bp annealing temperature: 57°C primer: use with upper primer 5' AAA GTC ACT TGG CAG GTC TTC G 3' legplin (GSN) JF specific - PASA product size: ~1.5kb annealing temperature: 59°C upper primer: lower primer: 5' GGA GCT CGC CCA GTA 5' GGG CAT CTT TTC CAG GTT TC 3' CAA TCC ATA CA 3' product size: 21 Obp annealing tmperature: 57°C primer: use with lower primer 5' AAG CTT CCT GTC ATC ACC ACT A 3' 67 Table 2. Cont. Ring3-Like Gene (RING3L) JF specific- PASA Collapgin Rgpponse Mediator Progin 1 (CRMP1) JF specific - PASA product size: ~1kb annealing temperature: 59°C upper primer: lower primer. 5' TAG TTA TGT TCC AGG 5' CAT CAG TTT GCT CGT TTC TTG 3' TGG CCT TTC TAC 3' product size: 220bp annealing temperature: 57°C primer. use with lower primer 5' ATC TCT CCA GCT CTG AAA AAC GAT 3' product size: 2kb annealing temperature: 59°C upper primer: lower primer: 5' AAT CAC CAT CGC AAC 5' CCC CGC AGG ACA CAA ACC AA 3' GCA GTG AGT 3' product size: 300bp annealing temperature: 59°C primer: use with lower primer 5' TTG CTG CTC CAT GCT TTT ACC AGT 3' 68 Table 2. Cont. Tgnsforming Growth product size: 500bp Factor-Beta Receptor, T313 1 (TGFBR1) annealing temperature: 52°C upper primer. lower primer. 5' CAG AGT GGC GTG TTA 5' TCC CCA CTA CTG AGA AGG TI 3' AAT GAG GTC 3' JF specific- PASA product size: 80bp annealing temperature: 51°C primer: use with lower primer 5' TGT TGG AGT ATG CTT TGC GAG 3' §plicing FactorI product size: 500bp ArginineISerine-Rich. 2 (§FR§2) annealing temperature: 59°C upper primer: lower primer. 5' CTA CGG GAG CAG 066 5' TGG AGA CAG 'l'l'A CG 3' ACG AGG ACT TTG ACT 3' JF specific- PASA product size: 180bp annealing temperature: 57°C primer. use with upper primer 5' GCT AAG GCT GCT GGG GAG AG 3' 69 Table 2. Cont. Tyrosinase-Related Protein 1 (TYRP1) JF specific- PASA product size: 335bp annealing temperature: 59°C upper primer: lower primer: 5' AAT ACA ACA 5' TGC CAT CTC TTC ATA CGA TGG TGC CTT CA 3' TCT 3' product size: 250bp annealing temperature: 57°C primer: use with upper primer 5' GAA GAC TAG AAG AGC AAA CAC 3' En o helin Re or TIE A (EDNRA) JF specific - PASA product size: ~1kb annealing temperature: 59°C upper primer: lower primer. 5' TAC CAC AAT CTT CTT 5' GGC ACT GGC ACC CGA CTG 3' ATT TTG ACC TT 3' product size: 150bp annealing temperature: 57°C primer: use with lower primer 5' AA CCC ATC AGA AAA ATC TAT TAT 3' 70 Table 2. Cont. Paired Basic Amino Acid Cleaving Enyme (PACE) JF specific- PASA product size: 400bp annealing temperature: 59°C upper primer. lower primer: 5' GGA GGG CCC TTC GGA 5' CCA GTC AGG GTC G 3' GCA ACA CCA ACA AG 3' product size: 200bp annealing temperature: 57°C primer: use with upper primer 5' GAG GGG AGC CCA GAA TGA CG 3' Troppmypsjn 2 (TPM2) JF specific - PASA product size ~1.5kb annealing temperature: 59°C upper primer: lower primer: 5' TGA ACC GCC GCA TCC 5' GCG CTC CAG AG 3' CTC TCC CTC AAG 3' product size: 150bp annealing temperature: 57°C primer: use with upper primer 5' GGA TGG TGA CTC CAT CAG AAG 3' 71 Table 2. Cont. Aldehyde Dehydrigengse 1 (ALOH1) JF specific - PASA product size: 1kb annealing temperature: 59°C upper primer: lower primer: 5' CTT AGC AGC AGC AGT 5' AAG GCC ATA TTC TTT TA 3' TCC CAG TT 3' product size: 250bp annealing temperature: 57°C primer. use with lower primer 5' TCA GGG TAT ACT GCT ATC AC 3' Fibroblast Growth Factor Receptor 3 (EGFRQ) JF specific - PASA product size: 450bp annealing temperature: 59°C upper primer. lower primer: 5' CCG CTT GGT GAG GGC 5' GCC CTG AGG TGT TTT 3' TAT TCC CGC AAG 1T 3' product size: 150bp annealing temperature: 55°C upper primer. 5' TTT TCT CAT AAG TTT ACA ATC ACG 3' 72 Table 2. Cont. Xeroderma PigmentosumI Complemtption Group A (XPA) JF specific - PASA product szie: 550bp annealing temperature: 59°C upper primer: lower primer: 5' CAT GAA TAC GGA CCA 5' GAA ACC TCC CTC GAA GAA AAT 3' CAT CAA GT 3' product size: 200bp annealing temperature: 55°C primer. use with upper primer 5' GGT AAA CTT CCC TCC AG 3' Cpthegin L (CTSL) JF specific - PASA product size: 450bp annealing temperature: 60°c upper primer. lower primer: 5' TGA TGA ATG GCT ATA 5' AGC CCA GCA AAC ACA AGA 3' AGA GCC ACA C 3' product size: 200bp annealing temperature: 57°C primer. use with upper primer 5' GAG GTA CTG AAT TTT ACT AAT CG 3' 73 Table 2. Cont. Prostaglandin- Endogroxide Synthpsp 2 (PTGSZ) JF specific - PASA product size: 1.3kb annealing temperature: 60°C upper primer lower primer. 5' GGT TGC CCT AGA TTC 5' AGT TCC CCA GCT CTT TA 3' GAG TTT AT 3' product size: 400bp annealing temperature: 57°C primer: use with lower primer 5' AAT TGG GAT GCT CTA CTA A 3' TubulinI Alpha-Like, 1 (TUBAL1 ) JF specific - PASA product size: 695bp annealing temperature: 60°C upper primer: lower primer. 5' ACT GCG CTT CGA TGG 5' CGG GGG TGG GGC TCT GA 3' GGT GGG GGA TAA 3' product size: 350bp annealing temperature: 57°C primer: use with upper primer 5' GAT GCC CAC CTT GAA ACC ACT T 3' 74 Table 2. Cont. Abelson Murine Leukemia Viral Oncogene Hpmolog 1 (ABL1) JF specific - PASA Phpgpholippge A2I §roup IV (PLAZQ) JF specific - PASA product size: 600bp annealing temperature: 60°C upper primer: lower primer: 5' GAG GAC ACC ATG GAG 5' GTG GAT GAA GAA GTG GA 3' GTT CTT CTT CTC 3' product size: 400bp annealing temperature: 55°C primer: use with upper primer 5' AAT TAT TAG GTA AGT GAT AAA TAG CG 3' product size: 625bp annealing temperature: 60°C upper primer. lower primer. 5' GCA AGG CCA AGT GAT 5' AGT TGT GCA CAG TCC AGT C 3' CCC TTT ATT TCA 3' product size: 78bp annealing temperature: 55°C primer: use with lower primer 5' GCT TCA AGA AAC TGA TTC TTT T 3' 75 Table 2. Cont. Caspase 1I Apoptosis- product size: 450bp Related Cysteine Pro 9 e ASP1 JF specific - PASA annealing temperature: 60°C upper primer. lower primer. 5' GCC AGC GCC ATC TTC 5' GCC CTT CGC ATT G 3' TCA TCT CCT CTA 3' product size: 400 bp annealing temperature: 57°C primer. use with lower primer 5' GCC CAG GCC CAA AGA CAC TCA A 3' Villin (ll-IL) JF specific - PASA product size: 755bp annealing temperature: 60°C upper primer: lower primer. 5' CTG CAG CGG GGA TGA 5' AGG GCA AGT GCG TGA GA 3' TGG CAA GGC AGA GC 3' product size: 200bp annealing temperature: 57°C primer: use with lower primer 5' TGA TGT GAC C'l'l' GTC 006 CC 3' 76 Table 2. Cont. Transiently-Expressed product size: 600bp Axonal Glycoprotein (IAX1) JF specific - PASA annealing temperature: 60°C upper primer: lower primer: 5' CTG AAG GGA GGA AGA 5' GCA TGG CAG AAG AA CA 3' CTG ATA CAA ACA 3' product size: 200bp annealing temperature: 57°C primer. use with lower primer 5' CTC TAA GGA GCG ATG GCA C 3' Agtininll Alphp 2 (ACTN2) JF specific - PASA product size: >1 kb annealing temperature: 60°C upper primer: lower primer. 5' AGA GAA ACA GCA GAT GGA CAG ACA ACC ACA GAC ACG 3' TAA AAC CAA CA 3' product size: 132bp annealing temperature: 57°C primer. use with upper primer 5' CTG CAA GTAA AGG GGG C 3' 77 Table 2. Cont. ADP- Ribosyltransferase (ADPRT) JF specific - PASA product size: >2kb annealing temperature: 60°C upper primer: lower primer: 5' AGT CAG CGT TAC AAG 5' GTT TCA GCA GGT CCA TTA 3' ACT TCA GAT T 3' product size: 200bp annealing temperature: 57°C primer: use with lower primer 5' GCT TGA AAT GTT AGG ACT CCA 3' Calgin 1I (CAPN1) JF specific - PASA product size: 800bp annealing temperature: 60°C upper primer lower primer: 5' ACC ATG TAC GCC TAA 5' CCA GGC CAA CCC CAG AGC 3' GGC ATA CCC AGA C 3' product size: 232bp annealing temperature: 57°C primer: use with upper primer 5' CTG TTG AAA GTA AAT GTC CAG G 3' 78 Table 2. Cont. Albumin (ALB) JF specific - PASA product size: 1kb annealing temperature: 60°C upper primer: lower primer: 5' CAT GGC GAG GCA GAC 5' GGG CTT GCG TTT TTC C 3' AAT GAG GTT G 3' product size: 78bp annealing temperature: 57°C primer: use with upper primer 5' GTA CTC CCA AGG CAG GCT 3' Lfiyl Hyproxylase (PLQD) ' JF specific - PASA product size: 800bp annealing temperature: 60°C upper primer: lower primer: 5' CCG CAG TTT AAG GGG 5' GCA GTG GCG AGC ATT CAT 3' GGC AGA GGA 3' product size: 220bp annealing temperature: 57°C primer. use with lower primer 5' CTC TGA GGG CTC TTT GCG T 3' 79 Table 2. Cont. Cathegin E (CTSE) Jf specific - PASA product size: >2kb annealing temperature: 60°C upper primer: lower primer: 5' ACC CCT GCT GAA CAC 5' AGG CCT CTT GCT CCT GGA CAT 3' GCT CTG AAA AAC 3' product size: 350bp annealing temperature: 57°C primer. use with upper primer 5' CCG GTG TCG AAG ACC ACT GC 3' QIutamlne Synthetase (GLUL) JF specific - PASA product size: 600bp annealing temperature: 60°C upper primer: lower primer. 5' GTG CTC CCC GTA CCC 5' GAG ATC GCC TGA CTA AAC TTC 3' CTT CCA ATG A 3' product size: 250bp annealing temperature: 57°C primer: use with lower primer 5' CCG ACT TCC CCT TAT TTG AT 3' 80 Table 2. Cont. Nuclear Factor Kpppp- product size: 800bp 8 P105 Subunit (NFKB1) JF specific - PASA annealing temperature: 60°C upper primer: lower primer: 5' CGT GTG ACA GCG 5' TGA AGG GAA CAG GCG TAG AGA C 3' CCA GAA ACC ATC 3' product size: 300bp annealing temperature: 57°C primer: use with upper primer 5' AGG AAG TGA GGT TGA GGA TTT 3' W Com onent i minD Singing Protpin (GC) JF specific - PASA product size: >2kb annealing temperature: 60°C upper primer. lower primer. 5' GTA GCA ACT CAC GCC 5' GAT GGG CAG GGA GAA CAC C 3' AAG GGG AGT C 3' product size: 450bp annealing temperature: 57°C primer: use with lower primer 5' AAT GAA GAG CTT ACC ACA CAC GCA 3' 81 Table 2. Cont. Npurotrophic Tyrosine KinaseI Receptpr. Tyg 2 (NTRK2) JF specific - PASA product size: upper primer: lower primer: 5' GAT GTC TGG AGC 5' TTT AAT GGA GTT CTG GGA GTT GTA 3' CAG CGG CAG TTG 3' product size: 170bp annealing temperature: 57°C primer. use with lower primer 5' GGA TGT TGG CTA CGG GAA CCT AAT 3' Mast Cell h fee or product size: >2kb (MGF) annealing temperature: 59°C upper primer: lower primer. 5' ATG GCA TGT TTA GCT 5' TGC CTC TTT GTT TTT GAT A 3' ACT GTT ACT GCT 3' JF specific - PASA product size: 220bp annealing temperature: 57°C primer: use with upper primer 5' CTA TGT TAA CAG AGT GTA GTG 3' 82 Table 2. Cont. Myeloid Cell Leukemia 1 (MCL1) JF specific - PASA product size: 129bp annealing temperature: 60°C upper primer: lower primer: 5' TCG GAA ACT CAC 5' GCA ACA AAG GCA GCC GAA CAC C 3' CCA AAT G 3' product size: 90bp annealing temperature: 57°C primer: use with lower primer 5' GTG TGA GGT GGC TGC TGA C 3' Phosphoribgylpyroph osphate Amidotransferase (PPAI) JF specific - PASA product size: 992kb annealing temperature: 60°C upper primer: lower primer: 5' CTT GCC CTG AAT 5' AAG ATG GGG AAG GTG AGA TA 3' GAA AAA G 3' product size: 440bp annealing temperature: 57°C primer: use with lower primer 5' TTT TTC GCC TTC CAG ATT GC 3' 83 Table 3. PCR conditions: 25u| Reaction: PCR cycle: 10X PCR Buffer 94°C 2 min. 30 sec. 1.5mM MgCL2 94°C 30 sec. .2mM dNTPs 55°C-60°C 1 min. 30 sec. .2uM each primer 72°C 2 min. 1U Taq Polymerase cycle 30 times 30 ng genomic DNA 72°C 10 min.. (WL or JF) 4°C Table 4. Genes Mapped: Human Chromosome 1 Source‘: Region Chicken Map Human Map Amplified”: Position: Position Lvsyl Hydroxvlase; PLOQ Genbank 3' UTR Chromosome 1p36.3-p36.2 cDNA E54, 59.6 Janus Kinase 1;.I:AK1 U.Del. Within coding Chromosome 1p31.3 cDNA region 8, 0.0 Myeloid Cell Leukemia 1; MCL1 U.Del. Within coding Chromosome 1q21 cDNA region E26, 0.0 Phospholipase A2. Group IV: Genbank 3' End Chromosome 1q25 PLAZG4 cDNA including UTR 8, 50.5 Prostaqlandin-Endoperoxide Genbank 3' UTR Chromosome 1q25.2-q25.3 Synthase 2; PTGS2 cDNA 8, 50.5 Glutamine nthetase' GLUL Genbank 3' End Chromosome 1q31 cDNA including UTFi 8, 82.4 Cathepsin E; CTSE Genbank 3' End Chromosome 1q31 cDNA including UTR E04, 17.7 Troponin T2I Cardiac; TNNT2 Genbank 3' End Chromosome 1q32 cDNA including UTR E04, 15.7 Transientlv-Expressed Axonal Genbank 3' UTR Chromosome 1q32.1 glycoprotein; TAX1 cDNA E04, 9.6 ADP-Ribosyltransferase; Genbank Within coding Chromosome 1q42 ADPRT cDNA region 3, 75.6 Actinin.flpha 2; ACTN2 Genbank 3' End Chromosome 1q42-q43 cDNA including UTR 3, 132.2 Table 4. Genes mapped that are orthologous to genes on human chromosome 1. *Source: cDNA source for the chicken genes; Genbank: Genbank database at N.C.B.I., U.Del.: University of Delaware cDNA library. **Region amplified: region of genomic DNA sequenced; 3’UTR: 3’ untranslated region, 3’ End including UTR: 3’ coding region and some or all of the 3’UTR, Within coding region: strictly coding region. 85 Table 5. Genes mapped that are orthologous to genes on human chromosome 9. *Source: cDNA or primer source for the chicken genes; Genbank: Genbank database at N.C.B.I., U.Del.: University of Delaware cDNA library, UMSTS: Universal Mammalian Sequence Tagged Sites. **Region amplified: region of genomic DNA sequenced; 3’UTR: 3’ untranslated region, 3’ End including UTR: 3’coding region and some or all of the 3’UTR, Within coding region: strictly coding region. 86 Table 5. Genes Mapgd: Human Chromosome 9 Very Low Density Lipoprotein Receptor; VLDLR ItLrosinase-Related Protein 1; TYRP1 Tropomyosin 2; TPM2 Aldehyde Dehydrogenase 1; ALOH1 Cathepsin L; CTSL Neurotrophic Tyrosine Kinase. Receptor. Type 2; NTRK2 Xenoderma Piqmentosu Group A Complentinq Protein: XPA Transformin Growth Factor-Bela Receptor. T e l' TGFBR1 mlson Murine Leukemia Viral Oncogene Homolog 1' ABL1 Gelsolin' GSN Ring3-Like Gene; RING3L Figure 5. m Genbank cDNA Genbank cDNA Genbank cDNA Genbank cDNA U. Del. CDNA Genbank cDNA Genbank cDNA Genbank cDNA UMSTS Genbank cDNA U.Del. cDNA Reqion Amplified: Within coding region 3' End including UTR Within coding region Within coding region Within coding region 3' End including UTR 3' End including UTR 3' UTR Within coding region Within coding region Within coding region Legend on facing page. 87 Chicken Map Lem Chromosome 2, 92.3 Chromsome 2, 102.3 Chromsome 2, 5.8 Chromosome E18, 10.0 Chromosome 2, 113.1 Chromosome 2, 115.5 Chromosome 2, 175.1 Chromosome 2, 153.8 Chromosome E41, 30.8 Chromosome E41, 16.0 Chromosome E41, 13.5 Human Map Position: 9p24 9p23 9p13.2-13.1 9q21 9q21-q22 9q221 9q22.3-q31 9q21-22 9q34.1 9q34 9q34 Table 6. Genes Mapped: Human Chromsome 4 Fibroblapt Growth Factor Receptor 3; FGFR3 Collapsin Response Mediator Protein 1° CRMP1 Phosmorposvlpvrophosphat e Amidotranlerase; PPAT Albumin: ALB Group-Specific Component (Vitamin D Bind_inq Protein); EQ Nuclear F_a_ptor Kappa-B P105 ubunit' NFKBI Endothelin Recgator. Type A: EDNRA Source‘: Genbank cDNA Genbank cDNA Genbank cDNA Genbank cDNA U.Del. cDNA Genbank cDNA Genbank cDNA 59.91% Amplified“: Within coding region Within coding region 3' UTR 3' End including UTR Within coding region 3' UTR 3' UTR Chicken Map Posmon: Chromosome 4, 3.8 Chromosome E38, 0.0 Chromosome 4, 173.4 Chromosome 4, 132.3 Chromosome 4,132.3 Chromosome 4, 165.7 Chromosome 4, 108.7 Human Map 4p16.3 4p15-16.1 4q12-13 4q11-q13 4q12 4q23-q24 4q27-28 Table 6. Genes mapped that are orthologous to genes on human chromosome 4. genes; *Source: University of Delaware cDNA library. region of genomic DNA sequenced: End including UTR: all of the 3’UTR, Within coding region: region, 3’ region. 3 I 88 3’UTR: 3! cDNA source for the chicken Genbank: Genbank database at N.C.B.I., U.Del.: **Region amplified: untranslated coding region and some or strictly coding Table 7. Genes Mapped: Human Chromosomes 11I 12I and Others Source‘: Region Chicken Map Human Map Amplified": Position: Position: Calpain 1; CAPN1 Genbank 3' UTR Chromosome 5, Chr.11 cDNA 768 Caspase 1I Apoptosis- Genbank 5' UTR Chromosome 11q22.2-q22.3 Related Cysteine cDNA E52, 43.1 Protease: CASP1 Gamma-Carboxyglutamic Genbank Within coding Chromosome 1, 12p12.3-13.1 Acid Protein, Matrix; MGP cDNA region'“ 151.8 first Cell Growth Factor; Genbank Within coding Chromosome 1, 12q22 MGF cDNA lemon 1432 TubulinI Alpha-Like, 1; Genbank 3' End including Chromosome Chr.12 TUBAL1 cDNA UTR E22, 20.4 Ubiguitin-Coniugating U. Del. Within coding Chromosome 4, Xq24-25 Enzyme E2A; UBE2A cDNA region 81.0 Villin; VIL Genbank 3' End including Chromosome 7, 2q35-q36 cDNA UTR 731 flared Basic Amino Acid Genbank 3' UTR Chromosome 15q25-26 Cleavinq Enzyme; PACE cDNA E29, 6.3 Spicing Factor, Genbank Within coding Chromsome E31, 17q24 Arqinine/Serine-Rich, 2; cDNA region 0.0 SFRS2 Table 7. 12, X, 2, 15, and 17. *Source: Genes mapped that are orthologous to genes on human chromosome 11, C DNA source for the chicken genes; Genbank: Genbank database at N.C.B.I., region, 3’ region. U.Del.: **Region amplified: 3’UTR: 3' untranslated region, 5' UTR: 5' End including UTR: 3’ all of the 3'UTR, Within coding region: Pairs to Sequenced Chicken Genes set. 89 University of Delaware cDNA library. region of genomic DNA sequenced: untranslated coding region and some or strictly coding ***Within coding region: primers from the Primer Table 8. Gene Combination Length of Segment, CM Chromosome r" m“ chicken human SFRS, H338, and FAS 9.8 19.6 E31 17q PACE, lGF1 R, and 82M 98.1 196.2 E29 15q SPPl, ALB, GC, and PPAT 35.3 58.8 4 4q RPL37A, VIL, C028, and EEF1 B 44.3 73.8 7 2q CDC2L1, AGRN, ENOL. PLOD, 71.2 106.8 E54 1p and SLCZA1 JAK1 and GGTB3 27.4 82.2 8 1p PLA2G4, PTGSZ, and GLUL 31.8 63.6 8 1q TAX1, TNNT2, and CTSE 8 16 E04 1q ADPRT, TGFBZ, and ACTN2 65.1 130.2 3 1 q RING3L, GSN, L7a, ABL1, AK1, 80.9 107.9 E41 9q 0039, and AMBP VLDLR and TYRP1 7.7 23.1 2 9p CTSL and NTRK2 2.1 6.3 2 9q ALDOB and XPA 26.6 79.8 2 9q GAPD and LDHB 17.3 51.9 1 12p PGK1 and UBE2A 9.6 28.8 4 X WNT11 and FUCTIV 29.6 88.8 1 Ho MPR1, PLN, ME1, and GSTA2 80.2 133.7 3 6q-6p DNECL and CKB 1.9 5.7 5 14q CRYB, IGVPS, and MIFL2 4 8 E18 22q Table 8. Genetic lengths of conserved segments between chickens and humans. *r: genetic distance between outermost markers in the group based on the EL reference map. **m: expected value of the length of the conserved segment based on the treatment of Nadeau and Taylor (1984). Mean of r=34.3, standard deviation (SD)=30.3, mean of m=67.4, 80:52.1. 90 E54 E04 Part Ch 3 Figure 1. Syntenic groups mapped to human chromosome 1 and chicken chromosomes E54, 8, 1, E26, E04, and 3. The human physical map is compared to the chicken genetic map. Genes mapped in the current study are in bold and underlined. Dotted lines on the ends of c-chr represent the ends of the conserved segment. If all genes currently on the map of a c- chr are found on the same syntenic group, these are bordered by closed ends. AGRN’and ENOl are found on the pter end of h-chr 1. RPL5 is mapped on chromosome 8 on the Compton chicken genetic map (Compton and Palyga, 1992) 91 Figure 2. ATP5A12 TPM2 (15") /// GHpr) / VLDLR\ LYRFLt _ ChrZ GGTBZ —CHD-Z (Sq) IREB1 —-—CHRN83 . (89) cm E18 % ALDH CTSL / NTRK2 , ; ALDOB Part Chr2 EBA "”’/”,,,»»”” ‘ / AMBP 0039L ‘ E41 AK1 ABL '/ E// m/ Figure 2. Syntenic groups mapped to human chromosome 9 and chicken chromosomes Z, E18, 1, 2, and E41. The human physical map is compared to the chicken genetic map. Genes mapped in the current study are in bold and underlined. Dotted lines on the ends of c-chr represent the ends of the conserved segment. If all genes currently on the map of a 92 Figure 3. Part ChEZ Ch E38 Part CH4 Figure 3. Syntenic groups mapped to human chromosome 4 and chicken chromosomes E29, E38, and 4. The human physical map is compared to the chicken genetic map. Genes mapped in the current study are in bold and underlined. Dotted lines on the ends of c-chr represent the ends of the conserved segment. If all genes currently on the map of a c-chr are found on the same syntenic group, these are bordered by closed ends. 93 Figure 4. Part of /TH— _ CH5 HBB / WNT11 Part 0' .// /; I CH1 r FUCTIV / i Part of // M _ 552 } APOA1 \ \ EPOC1 \ \ ETSi E49 Figure 4. Syntenic groups mapped to human chromosome 11 and chicken chromosomes 5, 1, E52, and E49. The human physical map is compared to the chicken genetic map. Genes mapped in the current study are in bold and underlined. Dotted lines on the ends of c-chr represent the ends of the conserved segment. If all genes currently on the map of a c—chr are found on the same syntenic group, these are bordered by closed ends. 94 Figure 5. \ Part of Chr 1 \ GAPD NAGA (22q11) lGF1 i Figure 5. Syntenic groups mapped to human chromosome 12 and chicken chromosome 1. The human physical map is compared to the chicken genetic map. Genes mapped in the current study are in bold and underlined. Dotted lines on the ends of c- chr represent the ends of the conserved segment. If all genes currently on the map of a c-chr are found on the same syntenic group, these are bordered by closed ends. 95 Figure 6. Figure 6. Syntenic groups mapped to human chromosome X and chicken chromosome 4. The human physical map is compared to the chicken genetic map. Genes mapped in the current study are in bold and underlined. Dotted lines on the ends of c- chr represent the ends of the conserved segment. If all genes currently on the map of a c-chr are found on the same syntenic group, these are bordered by closed ends. 96 Part of CH 3 Part of Part of CH 7 COL3A1 (COM) 2q31 EEFlBZ 0028 Figure 7. Syntenic groups mapped to human chromosome 2 and chicken chromosomes 3, 4, and 7. The human physical map is compared to the chicken genetic map. Genes mapped in the current study are in bold and underlined. Dotted lines on the ends of c-chr represent the ends of the conserved segment. If all genes currently on the map of a c-chr are found on the same syntenic group, these are bordered by closed ends. Figure 8. ”w“-.. we I l Part of CH 5 i . i i __ __ 82M E29 IGFR‘i //////////’PACE Figure 8. Syntenic groups mapped to human chromosome 15 and chicken chromosomes 5 and E29. The human physical map is compared to the chicken genetic map. Genes mapped in the current study are in bold and underlined. Dotted lines on the ends of c-chr represent the ends of the conserved segment. If all genes currently on the map of a c—chr are found on the same syntenic group, these are bordered by closed ends. 98 Figure 9. -4fl£MA1 - COL1 A1 l I '\\ Part of 515 ARHGDIA \ Figure 9. Syntenic groups mapped to human chromosome 17 and chicken chromosomes E57, E21, E31, E59, and 816. The human physical map is compared to the chicken genetic map. Genes mapped in the current study are in bold and underlined. Dotted lines on the ends of c-chr represent the ends of the conserved segment. If all genes currently on the map of a c-chr are found on the same syntenic group. these are bordered bv closed ends. Figure 10. Cumulative frequency o. —— ..... on . o (o-l ° O. O ‘tl 0 Nut . o I I 1 T 0 50 100 150 200 Segment length, cM Figure 10. Curves illustrating expected cumulative frequency distributions of segments containing two or more markers at different values of L. The circles represent the cumulative distribution of adjusted segment lengths used in this study. 100 Chapter 3: Physical Mapping of Chicken Microchromosome E41 Introduction: The typical avian karyotype is composed of 8 macrochromosomes, plus the Z and W sex chromosomes, and around 30 microchromosomes. Although the microchromosomes vary in size, they are not large enough, nor do they have a banding pattern distinct enough to distinguish between them. Thus, the term "microchromosome" is somewhat arbitrary. There are many questions concerning avian microchromosomal physical and genetic structure. It has been hypothesized that microchromosomes may have an increased rate of recombination compared to macrochromosomes since it is thought that at least one chiasmata is required per microchromosome, regardless of size, to ensure proper meiotic segregation (Rodionov et al. 1992; Rodionov 1998). Microchromosomes have reduced levels of non-coding sequences such as microsatellites and initial studies suggest they may also be gene-rich (Sazanov et al. 1996; Rodionov et al. 1996; Primmer et al. 1997; McQueen et a1. 1996, 1998, Clark et al. 1999). All of these theories remain unproven in the absence of a detailed physical map of any avian chromosome. Recently, through collaboration with the Texas A&M Bacterial Artificial Chromosome (BAC) Center, a 5—fold BAC library of the chicken genome has been generated through the insertion of partial BamHI DNA fragments into pBeloBacll 101 (Figure 1). This is comprised of approximately 38,000 clones with an average insert size of 150 kb. The DNA source used is a UCDOOl female Red Jungle Fowl. This is the same line as the non-recurrent parent of the East Lansing (EL) Reference Backcross family (Crittenden et a1. 1993) which allows for identification of dominant JF markers such as AFLP (Knorr et a1. 1999) within the library. BAC libraries have been used extensively in the generation of physical contig maps (Marra et al. 1999; M020 et al. 1999), and we have begun to develop such a map for the chicken genome in a continuing collaboration with the BAC Center. As a test of the feasibility of such an approach, we have made initial steps into the generation of a contig map for the E41 microchromosome which are described below. E41 is one of the most densely mapped microchromosomes (Groenen et al. in press) and several known genes are among the mapped markers (Smith et al. 1997; Chapter 2 of this thesis). Interestingly, all of the genes mapped on E41 are syntenic with telomeric portion of the q arm of human chromosome 9 (Chapter 2, Figure 2). The overall map of E41 contains 20 markers across approximately 70 cM (Chapter 1, Figure 1). Additionally, a quantitative trait locus (QTL) for a differential response in viremia to Marek’s Disease Virus in line 6 and 7 chickens has been mapped to E41 (Vallejo et a1. 1998; Yonash et al. 1999). These factors led to the decision to begin testing the newly constructed BAC library using genetic markers on E41. Our long—term goal is 102 to construct a complete physical contig across E41, which will allow for, among other things, comparison of its physical and genetic sizes. 103 Materials and Methods: BAC library screening: Six markers were chosen from linkage group E41 to screen the BAC library: RING3L (Ring3—Like Gene), AK1 (Adenylate Kinase 1), L7a (Ribosomal Protein L7a), ABL1 (Abelson proto-oncogene 1), GSN (Gelsolin), and microsatellite marker ROSOOZO. Our group had mapped RING3L, AK1, ABL1, GSN, and L7a, so the primers for these markers were available and had been tested. We wanted to test microsatellite markers for probing the BAC library and ROSOOZO is positioned between GSN and L7a on the genetic map (Chapter 1, Figure 1). All primer and PCR information including ROSOOZO are available on the chicken genome mapping web site (http://poultry.mph.msu.edu/). PCR products from the markers were cloned into the TOPO-TAtm cloning vector (Invitrogen Corporation, Carlsbad Ca.). Plasmid isolation of positive clones was done using the Qiaprepcm miniprep kit protocol (Qiagen Inc., Valencia, CA). pBeloBacll contains A cos and LACZ gene sequences, therefore insert DNA to be used as a probe must first be extracted from any vector that contains these sequences (such as TOPO—TA“). Several restriction enzyme combinations based on the TOPO-TAcm vector-cloning site were tested to ;produce the largest useful insert (Figure 2). Insert DNA was 104 isolated using the Qiaex II“ gel purification kit protocol (Qiagen Inc., Valencia, CA). The BAC library has been spotted in duplicate onto Hybond—N+ (Amersham Pharmacia Biotech, Piscataway, NJ) nylon membrane filters. We employed a 30,000 clone sublibrary spotted on 20 filters. Prior to hybridization, the filters were prehybridized with 0.263 M Nagumh, 2% SDS, 1% BSA, 1 mM EDTA, and 200 ug/ml denatured salmon sperm DNA (HYB solution). Ten filters were prehybridized with 20 mls of HYB solution. Prehybridization was carried out at 65°C for 16 to 18 h with constant rotation. Approximately 25 ng of the purified fragments were radiolabeled with [32P]—dCTP using the Prime—It IIm Primer Labeling Kit (Stratagene Cloning Systems, La Jolle, CA). All six denatured labeled probe reactions were added to the filters along with 10 m1 fresh HYB solution and hybridization was carried out for 48 h at 65°C. Following hybridization, the filters were washed four times with 0.5x SSC and 0.1% SDS, 0.5-1 h each, at 65°C with gentle agitation. Autoradiography was carried out using Kodak Bio- tm Max (Eastman Kodak Company, Rochester, NY) film exposed at -70°C for 48 h. BAC DNA purification: Several protocols for isolating BAC DNA were tested including one from the PACBAC Resource Center at the Roswell Park Cancer Center Institute, Buffalo, NY 105 (http://bacpac.med.buffalo.edu/framebpmini.htm), the PSICLONEcm BAC DNA Kit (Princeton Separations, Adelphia, NJ), and the protocol for BAC Clone Analysis from the Texas A&M BAC Center (http://hbz.tamu.edu/bacindex.html). All of the procedures are similar, except that the PSICLONEcm procedure uses a filter column. In our lab, the protocol from the Texas A&M BAC Center produced the greatest amount of high quality BAC mini-prep DNA. All further analysis was performed using DNA isolated using that procedure. BAC Insert Size Analysis 0 Miniprep BAC DNA (1—2 #91 was digested overnight at 37 C. Digested DNA was run in 1% agarose on a CHEF—DRCm II, Pulsed Field Electrophoresis (PFGE) System (Bio—Rad Laboratories, Richmond, CA) with a 5 5 initial pulse time, 15 5 final pulse time, 6 V/cm, for 16 h. 1X TAE buffer was continuously circulated over the gel and cooled to 14°C: using the Model 1000 Mini Chiller (Bio-Rad Laboratories, Richmond, CA). 106 Results and Discussion: Figure 3 demonstrates autoradiographic exposures of two of the filters after hybridization. There were several strong positive as well as many weakly positive signals throughout. The double spotting helps distinguish between background spots and likely positive signals. Since the probes are all single copy PCR based markers, the putative positive clones were confirmed by PCR. Miniprep BAC DNA from strong positives and weak positives were used as the template in PCR reactions with all six primer pairs for the respective markers. The six PCR reactions were run on 3% Metaphor agarose gels along with a positive control templated by JF genomic DNA. Figure 4 shows a Metaphor gel with two of the positively identified markers. BAC 74/P21 amplified with ABL1 primers is in lane 7 (JF genomic DNA positive control with ABL1 primers is in lane 8) and BAC 23/J8 with GSN’primers is in lane 10 (positive control is in lane 16). As is evident from the PCR reactions with BAC 74/P21 (lanes 3, 5, and 9), there was a problem with contamination, possibly from E. coli chromosomal DNA, which led to faint bands showing up in multiple lanes. This was a common problem and the PCR reactions were performed several times to confirm the identification. Positive identification was only given when there was at least one test PCR reaction with no background. Even with the occasional background 107 problem, after several trials it was clear which clones were positive for the markers. Figure 5 is a 1% agarose gel (additional trials were occasionally run on 1% agarose) with 4 putative positive BACs amplified with the six different primer sets (no JF genomic was run on these gels). On this gel there are no background bands and BAC 75/K22 is positive for ABL1 (lane 12) and BAC 90/B4 is positive for AK1 (lane 19). BAC 95/C11 (lanes 2-7) and BAC 71/Il (lanes 20-25) are negative for the six primers tested. This is a clear example of two positives and two negative clones without background. Through this approach, we were able to identify 10 positive BACs representing four of the markers (Table 1). All of the positively identified BACs initially had strong positive signals on the filters, suggesting that the weakly hybridizing spots were due to background hybridization. There are several possible reasons for the failure to isolate BACs corresponding to two of the markers tested. In this preliminary screen no effort was made to insure that all probes were of similar specific activity, so if a probe happened to be of low specific activity, it might have been obscured by the background of a more radioactive probe. Another possibility is that these markers are underrepresented in the BAC library. The sample screened was theoretically about 4X in coverage, but our lab and others have often detected only one (or no) positives to a particular probe. Regions of the genome very rich or very poor in BamHI sites could have been lost or depleted in the 108 library construction process. Microsatellite—based probes such as ROSOOZO may be particularly problematic, especially when the original clone is not available, but only the PCR— amplified region. Amplified microsatellite fragments are often designed to be fairly small (for high resolution of alleles on sequencing gels) and, by definition, they contain repetitive DNA sequences that could hybridize widely in the genome. (The actual simple sequence repeat is often found embedded in other repetitive sequences, as well.) We are presently screening the BAC library again with RING3L and ROSOOZO, to eliminate the likelihood of low quality probes and will attempt to use poly d(GT)—d(CA) as a competitor to minimize background repeat hybridization. Once the BAC library is expanded with HindIII and EppR; partial digest inserts, we will also screen this more representative library. Twenty—eight BACs that gave weak and strong positives on the filters were tested in the above manner. Although only 10 were confirmed as positives, all 28 were digested with Npp; (1U per ug) (New England Biolabs, Beverly, MA) in order to test the average BAC insert size (Figure 6). There are several points to note. Gel 1 contains twenty—eight BAC clones isolated using the PSICLONEcm or Roswell method of BAC DNA isolation. This gel exhibits considerable smearing and several BACs do not show up at all. Gel 2 contains the same BACs (except 74/P21) isolated using the Texas A&M BAC Center protocol, and there appears to be less shearing and all BACs 109 were successfully detected. The 7.4kb band seen in most of the lanes is the pBeloBacll vector. The average insert size is approximately 150 kb, consistent with previous estimates (H. Zhang, personal communication), with several larger BACs over 200kb. Lanes 11, 13, 15, and 17 of Gel 2 all contain a unique band that is smaller than the vector. These four BACs are 28/C12, 25/D13, 90/B4, and 42/N21, the four positive for AK1. These four also share additional larger bands. These shared fragments, especially the common, unusually small Npp; fragment, suggest that the four BAC inserts overlap, as might be expected, since they were positive for the same probe. Together the four clones form an initial contig in the AK1 gene region. As expected, it appears that the BAC clones for GSN'share common bands, as do the BAC clones for ABL1 (Figure 6). This suggests that all or most BAC clones isolated and confirmed by PCR do indeed contain the gene of interest and not some partially homologous sequence from elsewhere in the genome. In order to confirm the overlapping nature of the BACs, a HindIII fingerprint digest was performed on each BAC DNA. The HindIII recognition site is AAGCTT, and it would be expected to produce more bands than a Npp; digest. (In 50% GC, random sequence DNA there is about one HindIII site per 4 kb of DNA or around 40 in a 160 kb insert, whereas there would be one Npp; site per 65 kb or 2—3 per 160 kb insert.) Figure 7 shows the HindIII digested BAC clones run on a 1% agarose gel (not PFGE). The first four lanes are the BACs 110 positive for AK1, lanes 6 through 8 are the ABL1 clones, and lanes 9 and 10 are the GSN clones. Although, as expected, there are many bands in each lane, it is clear there are common bands among the putative overlapping clones. As noted above, Npp; (recognition site: GCGGCCGC) would be expected to cut random sequence, 50%-GC DNA approximately every 65 kb. However, it cuts most eukaryotic DNA much less frequently, since CpG dinucleotides are unusually rare and the NotI recognition site contains two CpG sequences. In an initial test of the BAC library by the Texas A&M BAC center, 56 random BAC clones were digested with Nppl (unpublished results). These BACs were cut on average 1 to 2 times, and rarely three or more (averaging about one Nppl cut per 100 kb). The E41 BACs isolated in the current study appear to be cut significantly more frequently by NotI, usually three or more cleavages per insert (Figure 6). This may reflect that these BACs all were isolated on the basis on the gene they contain, and gene sequences, especially promoters, are known to be comparatively rich in CC and especially in CpG dinucleotides (so-called CpG "islands", McQueen et al. 1996, 1998). Another possibility is that since these BACs derive from E41, a microchromosome, and since microchromosomes are known to be GC—rich and rich in CpG dinucleotides (McQueen et al. 1996, 1998), these sequence biases are reflected in the resulting BACs. Obviously considerably more work will need to be done to confirm this speculation. 111 We have isolated ten BAC clones from our UCD001 JF BAC library. This is an important first step in our long-term goal of building a physical map of the chicken genome. Along with a whole genome approach, we will continue to focus on the microchromosome E41. Figure 8 is a graphical representation of the EL genetic map alongside the BAC clones isolated to date. On—going experiments are aimed at reducing the gaps, especially between AK1 and ABL1, by chromosome walking experiments, as well as screening the library with the rest of the available E41 gene and microsatellite markers. Improved hybridization screening methods and/or PCR—based screening may be required for some of these markers. In addition, the project can benefit from on-going efforts to expand our BAC library and from the use of another chicken BAC library that is now available (Crooijmans and Groenen, personal communication). 112 Figure 1. 17* *" 596 SBI i NOiI B H NO“ OSN IOXP lacZ/ Eco RI par C arB r fl CM \ Sna Bl pBeloBAC 11 7.4 kb Xho I parA my Xba [ rep E Eco RV Eco RV Figure l. pBeloBacll large insert cloning vector. B: BamHl cloning site, H: HindIII cloning site. 113 Figure 2. ABL1 AK1 RINGBL Figure 2. Restriction enzyme testing for three of the gene markers, ABL1, AK1, and RING3L; run on a 1% agarose gel. From right to left the enzyme combinations for each are EcorI, NotI and KpnI, NotI and SpeI. The first lane is a 100bp lambda ladder. In this case any of the three enzyme combinations extracted the entire insert from the TOPO-TA vector for ABL1 and RING3L. The AK1 insert must have an internal KpnI site, since there are two bands in that column. In the case of AK1, either EcorI or NotI and SpeI would be used for the large preparation of the insert. 114 Figure 3. Filter for plates 65—68 Figure 3. Autoradiographs of the filters for plates 65-68 and 21—24. Lines point to the positive signals (in duplicate) for 67/P10 (ABL1 probe) and 23/J18 (GSN probe) respectively. 115 Figure 4. 74/P21 23/J8 Figure 4. 3% Metaphor agarose gel of BAC clones 74/P21 and 23/J8 after PCR with the 6 sets of primers. Lane 1 and 18: 100bp lambda ladder. Lanes 2 and 17: 1kb lambda ladder. The order of primers for 74/P21: GSN, R0520, L7a, RING3L,-ABL, JF genomic DNA with ABL1, and AK1. The order of primers for 23/J8: GSN, R0320, L7A, RING3L, ABL, JF genomic DNA with GSN. 116 Figure 5. 95/C11 75/K22 911/34 71/I1 Figure 5. 1% agarose gel of BAC clones 95/C11, 75/K22, 90/B4, and 71/Il. Lanes 1 and 25: 100bp lambda ladder. The order of primers for all: GSN, R0520, L7a, RING3L, ABL1, and AK1. 75/K22 is positive for ABL1 and 90/B4 is positive for AK1. 95/C11 and 71/I1 gave no amplified product for all six primer sets. 117 III Gel 2 Figure 6. CHEF gels for the BAC clones tested in the study. CHEF gel conditions: 1% agarose, 5 second initial pulse, 15 second final pulse, 6 Volts/cm, 16 hours, 15C. End lanes on both gels MidRange PFG Marker I (New England BioLabs, Beverly, MA). The Midrange Marker ranges from 15kb to 291kb. Positive clone from the present study on Gel 1: Lane 5— 74/P21. Positive clones from the present study on Gel 2: Lane 3- 23/J18, Lane 7— 22/I10, Lane 9— 98/F13, Lane 11- 28/C12, Lane 13— 25/013, Lane 15— 90/B4, Lane17— 42/N21, Lane 18— 75/K22, Lane 19- 67/P10 118 Figure 7. Figure 7. 1% agarose gel of BAC clones tested in this study. All BACs were digested with HindII at BTC for 16 hours. Positive clones: Lane 1: 90/B4, Lane 2: 25/013, Lane 3: 42/N21, Lane 6: 75/K22, Lane 7: 67/P10, Lane 8: 74/P21, Lane 9: 23/J18, Lane 22/110, and Lane 11: 98/F13. The additional lanes are from BAC clones that gave a weakly positive signal on the filters. 119 Figure 8. Chr £41 Backcross Stats, 95: Lilit «T RING3L 12 O o (H-MCDOSSS 4.0 22/110 1-4- GSN U5 23/J18 5.8 ”-0- 18059929 15.4 98/F13 "Il- L70 D 15.4 74/P21 75/K22 l +4 II pABL ' l I 67/P10 .1] «Ir-- ADLOZSS 25/913 13 l 2e4n2 lepri ' 90/34 3.3 BBQ/21 z 1 «4»ADL0149 ’ «A. HUJOOOZ 10.9 LADL0199 C039 iAMBP LBACBQBIOO T 1.9 2.1 [i Figure 8. Chromosome E41 (EL reference map) and BAC clones identified in the current study. 120 "5'! I52“- . Table 1. Marker: BAC ID: Insert Size: Gelsolin: GSN 22/110 160kb Gelsolin: GSN 23/310 150kb Ribosomal Protein L7a: L7a 98/F13 140kb Abelson Murine Leukemia 74/P21 200kb Viral Oncogene Homolog 1: ABL1 Abelson Murine Leukemia 75/K22 llokb Viral Oncogene Homolog 1: ABL1 Abelson Murine Leukemia 67/P10 140kb Viral Oncogene Homolog 1: ABL1 Adenylate Kinase 1: AK1 25/013 250kb Adenylate Kinase 1: AK1 28/C12 200kb Adenylate Kinase 1; AK1 90/B4 200kb Adenylate Kinase 1; AK1 42/N21 100kb Table 1. Identification of BAC clones on chromosome E41. BAC ID: Plate location of BAC clone. Insert size: approximate insert size based on NotI digest of the clone. 121 SUMMARY The work in this thesis began the process of building a comparative map between avians (chickens) and mammals (humans). The comparative map data provided coverage for most of human chromosome 1. human chromosome 9. and human chromosome 4. while other regions were also added to. The pews-“ml regional comparative map data was used to produce an estimate on the mean conserved lengths of segments between humans and chickens (38 :9 cM) and to estimate the rate of chromosomal evolution between humans and chickens (0.13 i 0.04). This is a rate considerably less than for humans and mouse. but comparable to other intra-mammalian comparisons (e.g., cat. cattle. pig). The comparative map will be an invaluable tool for identification of potential candidate genes. The comparative map data for human chromosome 9. provides some insight into chicken ZW—type sex chromosome evolution. Two genes mapped in this thesis. YTKFU and 142238 suggest that an ancient sex—determining region has been conserved between mammals and birds. The comparative map for chicken chromosome E41, gave rise to MHC type genes in an area where a QTL for Marek's disease lies. MHC genes are known to play a role in Marek‘s disease susceptibility and severity of disease. RUAEUZ is a MHC gene and was mapped in this thesis to microchromosome E41. £73652 on human n2 chromosome 9. is closely linked to several additional MHC— related genes. Due to the amount of conservation between microchromosome B41 and human chromosome 9. there is a high probability that this group of MHC-related genes are also on E41. This illustrates that the comparative map can already be used to identify potential candidate genes. Additionally. the building of a regional physical map on microchromosome E41 was begun. Six markers from E41 were 5. 3:3 ”-0": fl. tested. two based on genes mapped in this thesis. A total of 10 BACs were isolated covering 4 of the markers tested. This a was an initial screening of our newly constructed BAC library and the BACs isolated had an average insert size of 150 kb. These BACs were isolated from microchromosome E41 and appear to have an unusually high GC content. It has been suggested that microchromosomes are gene and GC-rich. Although this was a preliminary test. analysis of several isolated BAC clones. appear to support this theory. The regional physical map will eventually lead to a better understanding of the mechanisms and make-up of microchromosomes. Markers generated in the bu11ding of the r _ comparative map were used and will continued to be used to build a genome-wide physical map. The ultimate goal of the physical map will be to align the genetic and physical maps. and to provide sequence data for the chicken genome. 123 APPENDIX 1: Lack of Polymorphisms in Several Chicken Genes Introduction: One of the reasons the East Lansing (EL) reference map has been successful is the genetic diversity between the two inbred lines used to produce the Backcross (BC) mapping population. Previous studies have shown that there is approximately a 1% difference between UCDOOl Jungle Fowl (JF) and UCDOO3 White Leghorn (WL) (Okimoto and Dodgson 1996; Okimoto et al. 1997). By using two inbred lines, selected to be as different from one another as possible, Crittenden et al. (1993) hoped to optimize one's ability to identify sequence polymorphisms and to insure that all markers were strictly bi-allelic. Furthermore, a BC mating design was used to facilitate mapping of dominant fingerprint-type markers from the JP genome. Despite the average 1% sequence difference observed between the UCD001 and UCDOO3 genomes (Okimoto and Dodgson 1996; Okimoto et al. 1997), sequenced blocks longer than 1 kb have been observed with no detectable polymorphisms. As part of the comparative map generation described elsewhere in this thesis, we have identified several other long stretches of DNA, both coding and non—coding, that were not polymorphic. Genes that could be important in filling in gaps on the comparative map were analyzed in detail through sequencing and Restriction Length Fragment Polymorphism 124 (RFLP) analysis between WL and JF. Although RFLP analysis only samples a small percentage of the flanking genome (those which contain the restriction sites for which we probed), it can efficiently sample large regions of DNA that flank a cloned gene of interest. Our data confirm that near certain genes, sequence conservation between UCDOOl and UCDOO3 appears to extend across relatively large regions of DNA . 125 Materials and Methods: Amplification and sequence analysis of gene fragments: These techniques are described in detail in Chapter 2 of this thesis. RFLP analysis: Five ug of both WL and JF genomic DNA were digested with 10 different six—base cutters. Six—base cutters were used because the fragment size should be large enough to extend out of the gene itself. The genomic digests were run on 1% agarose gel at 30 volts for 16 to 18 hours. The DNA was then transferred to Zetabind“ nylon filters (CUNO, Life Sciences Division, Meridan, CT) by the method of Southern (Southern 1975). Insert DNAs of the cloned 3’UTR or coding regions were used as probes. 25 ng of the purified insert was labeled with [32P]—dCTP, using the Prime-It IIm Labeling Kit (Stratagene, La Jolla, CA). The filters were pre-hybridized overnight with constant rotation at 65°C in 10 ml of 0.263 M Nagfiwk, 1% SDS, 1% BSA, 1mM EDTA. Hybridization was carried out at 65°C overnight with constant rotation. Filters were washed three times at 65°C in 0.1 X SSC, 0.1% SDS, for 30 min each with gentle agitation. Autoradiography was carried out at —70°C for 48 h, using Kodak Bio-Max"m (Eastman Kodak Company, Rochester, NY) film. 126 Results and Discussion: Table 1 lists the genes for which sequence analysis failed to detect polymorphisms. The bulk of the sequence data is from 3'UTR and introns. A 302 bp fragment from the Gelsolin gene. containing protein coding sequence and about 100bp of 3’UTR had no polymorphisms between WL and JF. A larger fragment containing over 1 kb of intron yielded four polymorphisms. and the gene was subsequently mapped to linkage group E41 on the EL reference map. The majority of the sequenced regions cover over 800 bp of non-protein coding sequence. The genes IRFZ. 720V. and KIT. map to 4q12. 4q35.1, and 9q31. respectively. on the human genome. Placement of these genes on the chicken map would fill in gaps in the chicken- human comparative map. RFLP analysis was performed using probes for these three genes in an attempt to identify polymorphisms that could be used to map the genes. However. no RFLP were detected for any of the genes upon surveying 10 enzymes with 6 bp restriction sites. Figure 1 shows the autoradiography for the AUUFRFLP analysis. Our results (Table 1) suggest that the observed sequence diversity between UCDOOl and UCDOO3 is not randomly distributed across the genome. For example. based on 1% sequence diversity. randomly distributed. the predicted probability of a 800 bp non—polymorphic region would be 127 .8!“ 0.03% (the Poisson 0 term = e7). Although it is difficult to assess statistical significance because our choice of genes and respective regions within genes to be sequenced was not random. it seems unlikely on the face of it that we would have obtained the results of Table 1 on this basis. The RFLP analysis samples fewer base pairs of sequence (12 bp per enzyme tested or 120 bp per observable fragment generated). but it does detect large insertion/deletion events over many kb of flanking DNA. Our limited RFLP results suggest that those genes lacking sequence polymorphism within the gene may be closely related. if not identical. in UC0001 and UCDOOB. Based on our limited results to date and those of previous members of this lab (Okimoto and Dodgson 1996: Okimoto s¢'aJL 1997: Levin. unpublished results). we conclude that the UC0001 and UCDOO3 genomes show substantial linkage disequilibrium. The most likely explanation is that the UC0001 genome is not purely of Red Jungle Fowl origin. Inadvertent contamination of the line may have occurred by modern chickens (most likely. White Leghorn). This may have occurred in the early stages of developing the UC0001 line. Wild JP are difficult to breed in captivity. so WL traits/genes that were initially rare in the flock may have been highly selected. Furthermore, the inbreeding process itself and the likely narrow origins of the UC0001 and UCDOO3 lines would tend to promote linkage disequilibrium. The high level of interfertility observed between UC0001 and 128 ”“71 Irr- UCDOO3 (Crittenden at a]. 1993) also suggests that the two genomes have very few major chromosomal rearrangements with respect to one another. and that they are likely more similar to one another than might otherwise have been expected. Thus. the observed non—random distribution of polymorphism between the two lines is not surprising. 129 I“? I‘. Table 1. Non—polymorphic gene sequences: Gene: Genbank Region(s) accession: sequenced: CYSTEINE- AND GLYCINE- X73831 RICH PROTEIN 1: CSRPl "300bp 3'UTR PARATHYROID HORMONE- X52131 ~800bp 3'UTR LIKE HORMONE: PTHLH CONTACTIN 1; CNTNl ANTI—MULLERIAN X14877 ~600bp 3'UTR U6l754 ~800bp 3'UTR HORMONE: AMH NATRIURETIC PEPTIDE U Del. cDNA ”1kb coding region PRECURSOR A: NPPA (700bp: 1 intron) GELSOLIN: GSN AF042795 300bp coding region (100bp: 1 intron) PHOSPHODIESTERASE 6C, L29233 CGMP-SPECIFIC. CONE. ALPHA PRIME: PDE6C ~800bp 3'UTR. 800bp coding region (500bp: 1 intron) INTERFERON REGULATORY X95478 ~1kb 3'UTP FACTOR 2; IRF2 THIORODEXIN: TXN 303882 ~2kb coding region (1.5 kb: 3 introns) ~1.5kb 3'UTR. 1kb coding region V—KIT HARDY-ZUCKERMAN D13225 4 FELINE SARCOMA VIRAL ONCOGENE HOMOLOG: KIT (700bp: 2 introns) Table 1. Non—polymorphic sequence data for genes listed. U.Del. cDNA: cDNA sequence from the University of Delaware cDNA library (Burnside and Morgan. http://udgenome.agS/ chickenest/chick.htm). Genbank accessions are from the National Center for Biotechnolgy Information Genbank database.3'UTR: 3' untranslated region. B0 Figure 1. 1?. Ba BsH BSE D Ea K S H P UJUJUJUJUJUJUJUJUJUJ Figure 1. Filter with White Leghorn (W) and Jungle Fowl (J) genomic digests. Filter was probed a dctP32 labeled KIT 1.5kb 3'UTR fragment. Restriction enzymes: E: Ecorl, Ba: BamHl, BsH: Bsle, BsE= BspEl, D: Dral, Ea: Eagl, K: Kpnl, S: Sspl, H: Hindlll, and P: Pstl. 131 APPENDIX 2: Primer Pairs to Sequenced Chicken Genes A set of 300 PCR primers pairs, designed to amplify previously sequenced chicken genes has been developed. The cDNA sequences for these genes were taken from the National Center for Biological Information Genbank database. Primer pairs were designed using the PrimerSelectcm PCR Primer & Probe Design program within the Lasergene Biocomputing Software (DNASTAR Inc., Madison, WI) Primers were optimized to have similar melting temperatures (1;) and to minimize any propensity to contain hairpin loops or to generate primer-dimers during amplification. Among other possible uses, this collection primarily is designed to be used in Reverse-Transcription PCR (RT-PCR). RT-PCR is most successful when the primers are within the coding region, away from the 3’ end. The 3’end of genes may include untranslated sequences, which may produce secondary structures that can interfere with primer annealing. Therefore, all of the primers in this set amplify from within the coding region of the gene. The sequences of the 300 primer pairs are listed in Table 1, along with the gene name, locus symbol, and the RT-PCR product size. The majority of chicken gene sequences now available are from cDNA clones, so cDNA sequences were used for all genes within the panel to maintain consistency. The region amplified by any given primer pair may include one or more introns which could interfere with successful amplification 132 from genomic DNA templates. However, at least several of the primer pairs may also be successful using chromosomal DNA templates, especially if conditions are optimized for long PCR product amplification (Cheng et al. 1994; Barnes, 1994). As an example, the primers for Matrix GLA protein (MGP, Table 1) have been used to amplify Jungle Fowl and While Leghorn genomic DNA. The RT-PCR product size is 298bp (Table 1) and the genomic product is over 2kb, due to intervening sequences. The genomic product was confirmed by sequence analysis to be MGP. MGP was mapped on the East Lansing Reference map to chromosome 1, position 151.8. These primers, and subsequent gene primer panels yet to be synthesized, are being provided free of charge to interested users as part of the USDA—CSREES National Animal Genome Research Program Poultry Coordination effort. They are designed to be useful in analyzing transcription levels by RT-PCR, generating probe DNAs for microarrays, and cloning and sequencing portions of candidate genes (either from cDNA or genomic DNA) in hopes of locating a useful polymorphism for genetic linkage analysis (such as demonstrated for the MGP gene above). 133 Table 1. Primer Pairs to Sequenced Chicken Genes Locus Gene Name: Genbank Product Primer 1: Primer 2: Symbol: l0: size: AANAT Arylalkylamine N- 1781379 546 GCAGGGCC GCATGGCCC Acetyltransferase CCCGCAAC CGCACCTC TC ACAC Acetyl-CoA 2170499 645 GCGGGCAC TCATCATCC Carboxylase GGCAGGTT ACGTCCCCA CTCATT TCAGTT ACTN1 Actinin, Alpha-1 517084 622 CAAGGAGG GAAGGCGG GGCTGCTG GCCGGTTGC CTGTGGTG TCA ACVR2A Activin A Receptor 505347 553 ACAAGGTT AATGCTGGT 2A GCTGGCTG GCCTCGC‘I'I' GATGACA CTCTG ADHF Alcohol 2326999 582 AGGCTATG ATCACGGTG Dehydrogenase F GGGCTGCT CGAATGCTI’ GTCA TTG ADPRT ADP- 1638784 709 GACATGGC GGCCCCGA Ribosyltransferase CCTGAACT CCCCACTGC CCTTIGAT AGTR1 Angiotensin 1763531 589 CTGGCTCC GGGCAAGC Receptor1 TTGCTGGT GTATAT'ITI’C GTGG TGGTG AK1 Adenylate Kinase 222785 656 GATGGCAA CTCGCGAG 1 ACTCCTGG GGTAGCCGT GGGTGGTG CAATG AKT1 Serine-Threonine 2745888 603 CCGGACGG ACAAAGTGC Protein Kinase, TATTATGCT GTGGAAATC Oncogene AKT1 ATGAA TAATCT APE Aminopeptidase 2766186 592 GCCGCCCC TGCAGCCCC Ey GCAGCCAT TCCTTGAAC TG ACATCT APH Aminopeptidase H 1850771 576 GCCCGTCA TAGAACTGC CCAACCAG ACCGGTGTC AAGAACTC ATAGGA ASCL4 Achaete-Scute 1905985 282 GTGGCCCG GGAACAGG Complex GCGCAACG GCGAGGCG (Drosophila) AACG GAGGAATA Homolog-Like 4 134 Table 1. Cont. A Homolog 5 TCAGGCT ATAATGGT VR Basic 16683 Gene 1 50 Protein 22 AGTGGAC T TCAGGCGT Division Division 127799 TGGTG 1841295 1T 135 Table 1 . Cont. Locus: Gene Name: ID# Size: Primer 1: Primer 2: CDHSB Cadherin 68 867998 556 TCGGTTCC CAATG‘ITTC CCCAGAGC CCGGTCAA AC GAGl TTT CENPC Centromeric Protein C 2749772 520 AATCGCAC ATCCTCCC CATCATCAC TTGGCATC CTTCTCC ACCCTTCT CFRA CPR-Associated 2737970 587 GGTTGCCA CCTGCCCG Protein TTGCCTGT TGGTAAAG CA TCC CHOR Chordin 2826738 594 GCCGAGCC CTGCGGCG GTGTGCGT GGCGGTAA TTCA TGGTG CHRND Cholinergic Receptor, 211060 665 GGCGGTGT AGCCCGTC Nicotinlc, Delta CTGTGTCC CTGCCCCT CAACTG ACTCA CHRNG Cholinergic Receptor, 211061 628 CCAGCCCC TTCCCCATC Nicotinlc, Gamma GCACATAA CCC'I‘I'GCA CTCATCC TCACTTA CL C—Type Lectin 1142649 709 GCGGCTGT ACGGCGCC GGTTCTGG GGTTTGAT GGTCCTT GTTCC CNBP Cellular Nucleic Acid 2232216 402 GGCCGTGG GCCGCAGC Binding Protein TCGTGGGA GATAGCAG TGAG TTGAC CNP2 Cyclic Nucleotide 2760607 625 AGGCCGGC CCCCGTTT Phosphodiesterase 2 CAGGTGTT GTTGGTGC CTTG TCTGTGTA COCHSB Cochlear 5b2 2293561 522 TGGGCTTC GGCCACTA 2 ATCTTCTCA TACCAGCTT G CTTCTA COL1A2 Collagen, Type 1, 2587064 150 GAGACAAA AGTCAGCC Alpha 2 GGGCCACA GCT'ITAGAT GGGAGAA GGAT COL6A1 Collagen, Type 6, 576463 678 TACTTCCG TT'TGTCGC Alpha-1 Chain CTGTGACC CCTTCATTC GCTTCCT CTTGGTA B6 Table 1. Cont. Type 9, 1040 Chain 5 1 143827 Beta-A4 Kinase 1 55 L Beta 1 1455 906770 1 1A Against 374774 137 16 TGGACTG TGTGTTG TCCAAGA TGCAGGA Table 1. Cont. EPHAS Receptor Tyrosine Acid 1145 Growth 10 TTGTG 1827499 Receptor 1857 ranscription Factor AACCAGA 1 TCACGAG and Brain 71808 TCCTCT TTCACT T 3802 Line-Derived Factor 138 Table 1. Cont. 1009246 Family 1932736 3 1839476 1A 685 A TTGAAC Deacetylase 687 Growth 1419543 TCTG TGCATG Group TGCCTTCT Group 1160514 14 Box 7 146 A2 1 5799 AGCCCT 139 Table 1. Cont. Phosphatase the Endoplasmic HPER1 1 7-Beta- 1 944048 7B 7-Beta 4 Shock Protein of Apoptosis 55 1490877 Protease 1, Receptor 2 Channel ATGGCTTC 140 Table 1. Cont. Alpha 1 Tyrosine 1816447 Alpha 1 1 907288 Acitvated. Conductance, M, Alpha 1 Beta 2-Like ange Receptor Homeodomain Proteoglycan) 141 TGATGG Table 1. Cont 16723 Family Family Family , bZlP ranscription Factor 1 1065994 ransporter 3 1 GLA Protein MMP115 15-kDa 1655466 Matrix 142 Table 1. Cont. Locus: Gene Name: 104: Size: Primer 1: Primer 2: MP M-Protein 222832 533 ACGGGAAG AAATATTGC CTAACCATA CCTCCTCAT AAAACTG CCACAC MSTN Myostatin 2623569 526 CATGCCACA TCAGCGGG ACCGAGAC TAGCGACA GATTAT ACAT MUARP1 Mu-Adaptin-Related 1929344 773 CGGGAGGG CTCCCCGC Protein GCGGCACTT CGGCTCCC CGTC ACTCCA MYBPC3 Myosin Binding 1110448 586 GTGGTGGCT GCCGGGAC Protein C, Cardiac GGGAACAAA ATGCCAATA CTGAG GA MYF6 Myogenic Factor 6 222834 617 AACCGGCTC AGGCCGAC CTAT'ITC‘ITC GACTCCAC TACTI' CAT MYLK Myosin-Light- 992992 671 AGAAGCCCC GGGAGTAG Ploypeptide Kinase CTGCAGAGA CTGCT‘ITI’G ATGG GAGGAGT NEL Nel Gene 1483183 514 CACGC'ITTG GGGCTTCT CCTTCTCCT CCACAACT CT CTTI’CATA NEU Neuropilin 10600870 525 AGCCCCATC CCAGCAGG ATTTACTCG CACAGTAC CAGAA AGGACAA NFKB2 Nuclear Factor 755083 411 CGCCCTTGC CGCCGTTC Kappa-B, Subunit 2 ACCTCGCCA ACATCCGC TCATCC ACCCTTCC NKH1 Hyperglycinemia, 222820 763 GCCGCGGC GGAGCTGC Isolated Nonketotic, ACGATGACT CCAGGACA Type1 ACA NKH2 Hyperglycinemia, 222867 521 CATGGAGG GGGGCCCC Isolated Non ketotic, GCAGAGCA ACCGATGT Type 2 GCAGAACT CAGC NPPA Natriuretic Peptide 2170460 303 CAGCCCAG GCCGAAGC Precursor A CAGAGCCAA AGCCAGAA CC TC 143 Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: NRTRA Neurturin Receptor 2213804 627 GGGCTGGC GCAGGAGC Alpha CGAAGGAG ACCAGGGC AAGAGTT GAGATAGT OAZ Omithine 2317775 577 CCCTGCAGC TGGGGACA Decarboxylase GGATACTCA AGGGGATG Antizyme AC C OPOML Opioid-Binding 2897596 411 GATGGCCG GCCAGCCG Protein, Cell CACTCCTCC CTTGTCGT Adhesion Molecule- TCTT C'ITT Like PAD Peptidylarginine 2897752 580 GCTGGGCC TGCCCGCA Deiminase GCATCCTCA CCCGCTCC TTGG TC PARA Paranemin 2828800 (679 AGCGCCTG CAGCCCCT GAGTAGCAT CCTCGGTG CTTTG AACT PAX6 Paired Box 6 2576236 (660 CCCAGGGC GATGGGGA GATCGGAG TGTGGCTG GTAGTAAG GGAGTGTT PAX7 Paired Box 7 2576238 510 GCGCCCACT CTGCGGCG GCCCAACCA CTGCTTCCT CATC CTTCAAA P02 Protocadherin 2 2196557 557 GCTGTACCC AACCACCC CCTCCCGAA CGCACGGC CTCCAC ATCAACAT PG Pepsinogen 2760810 I646 GCACCCCAC GCCCCGAC CGCAGGACT TGCGC‘ITT TCACT GGATG PHOX Paired-Related 222850 382 CCCGGCCG TGGGTCTI' Homeobox GAGCTTGTT GGAGCTGG GGAGTC GCGAGGTA Pl3K Phosphoinositide 3- 2245505 571 TAAAGGCCG CATTGCTTG Kinase Catalytic GAAGGGTG CTCTGGCT Subunit CTAA TGATT POU1F1 Pou Domain, Class 1, 2842418 122 AGGAAGCG TTCTCAAGA Transcription Factor CAGAACCAC TTAAGCCC 1 CATA CTCAGC 144 h I” Table 1. Cont. 2A Subunfi 1 Receptor Protein Tyrosine Tyrosine 17097 14S Table 1. Cont. -Associated 1 Protein AATAC ATGCTG Protein Binding Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: SCYC1 Lymphotactin 2827881 205 CTCCACGCC TGATACCAT Precursor ACAGTTCTC TTGCAGTG ATAA SERC1 C-Serrate1 1236280 685 GCAACACTG AAGCACTG GCCCCGATA GGCACCGT AATACC TCTGG SF 1 Steroidogenic Factor 2541859 576 AACCCCGCC CTGCGCCG 1 GCCCTGACA CCACTGCT CCT GACA SIAT8 Sialyltransferase 8 1763266 594 CCCTCGGC GGCCCGTC GTCTTCGTC CCCGTCTT CTCTG CATTG SMP1 Smooth Muscle Prot. 2198741 547 CAGGCTCCG GTACGGGG Phosphatase Type 1- GGGCTGGC CTTGGGGC Binding Subunit ACTC TCTGAATG SNF2L2 Sucrose 996019 783 GGCCAAACC CAGGCGTC Nonfermenting, CCAGATATG TATC'ITTCT Yeast, Homolog-Like AGTGTC ‘TI'TGGTC 2 $00 Superoxide 1142717 322 AAGGCCGT TGCAGTGT Dismutase GTGCGTGAT GGTCCGGT GAAGG AAGAGAAA SOX2 SRY-Box 2 849043 469 AATGGCCCA CTGCGAGC GGAGAACC TGGTCATG CGAAGAT GAGTTGTA SRC1 Neural SRC 2582523 454 ACCCCGTTA GAACAGGC Interacting Protein CTTCCGCAG AGGTCTTG CATCTT AGGCAGTC STX1B Syntaxin1B 2564017 502 GGGCCTCAA CACCCCAA CCGCTCCTC GAACAACG ACGAAAAT SULT Sulfotransferase 2687359 513 “TTTGAAGCC TCCCAGGG AGAAGTGAT ‘TTTGATTCT GATGTC C'ITI'TAG TAD Thymocyte Activation 2665789 546 GCACGCCGT CAGGGATG and Developmental TCAGAAGTA TGGTGAGC Protein AGATG AGAGGTA 147 Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: TBP1 TATA-Binding Protein 1183016 667 ACAGCTTG ACATCACAG 1 CCGCCCTA CTGCCCACC CG AT TCRG T-Cell Receptor 2707426 489 GGCACCGT TAAATCCCAG Gamma Chain Vg1- GAGAAGAA TAGGCACAG Jgg TG TAGTA TENP Transiently Express in 2599571 568 CACCAGG TGAGCCCGC Neural Precursors GAGGCAG CCCAATGTG AAAGCAAG AAC TC TFAP2 Transcription Factor 2289947 561 GGTCTTCG AAGCCGTGC AP-2 GCGGGGT GAGATGAGG GGTGA ‘TTGAAG TFT T Brachyury 2529385 695 TCGGCGC CGCCGGGGT CCACTGGA GATGGTGCT TGAAGG G‘ITACT TGM2 Transglutaminase 2 2148921 736 GCCGCTAC AGCGCTTGC CGCCTGAC CACCCATCG ACTG TATCC THRBz Thyroid Hormone (63822 82 ATGGACAT ATGGCGACT Receptor Beta 2 GGCCCTG GCACTTGAG AATC AAAA TlMP2 Tissue lnhibitorof 2352472 291 TCGGCGAA CCGCTGGTT Metalloproteinase 2 GGAGGTG GAGGCTCTT GATT CTTCT TJP Tight Junction Protein 464148 1614 TCGCCATG CTGGTCGCC GCCGTGCT CCGGCTGCT GTGCTTCC GTAGGT TMP E3- Putative 2425049 568 ACGCCAAG CCACGGCAG 16 Transmembrane GAGCCGG AGGCGGTAA Protein E3-16 AGGATGT ATAAAG TNNC1 Troponin C, Slow 222844 414 AGGCGGC TGTTT'ITGTC GGTTGAGC GCCATCTTTC AGTTG ATCA TNNT Troponin T (variant) 2921774 548 GCCTTGAT CAGCGCCCG TGACAGCC CCAACCTT ACTTT 148 Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: TOM1B Tom1B Protein, v-Myb 1915893 530 GACGGATC AAGGTGACG - Target Gene CTTGCGG GGGTGGAG GGTGAG AATGGA TOP1 Topoisomerase1 1786131 765 AAAATGGG GTTGGCACG CATGTTGA GTTATAGGA AGAGACG AAGGAT TOPZB Topoismerase (DNA) 2463528 549 GTGAATGC ATCTACGTA ll beta CGCTGACA ACTGCGAAA ATAAG TCCAT TRP1 Tyrosinase-Related 2828811 667 TGGCCCAT GGATGGGA Protein 1 ACGCTTC‘IT CCGCCTTC CAAC AGT TSC22 Transforming Growth 1722682 352 TGTAGACC CGGAGGAT Factor Beta GGCGGCAA GGCGGGGA Stimulated Clone 22 TGGAT ACC TSHB Thyrotropin Beta- 2660744 297 CGTGGAGA TGTGGCTT Subunit AGCGGGAG GGTGCAGT TGTG AGTTTGTC TYR Tyrosinase 1655468 687 CCGCCCTG TGGGCTGA GGATGGAG GTAAATTAG AT GGTTGGT UBA52 Ubiquitin/Ribosomal 1763014 289 TTACGGGG TTCAGCAC Protein Fusion AAGACCAT GGCAAGTT Product CAC TA UBP41 Ubiquitin Specific 2736063 522 CCGCGGGC GGTGGTGC Protease 41 CAATGCTG CCGAGTGG AC TTAGAGAC VDR Vitamin 0 Receptor 2245698 676 GCTGAAGC TCCGGCTT GCTGCGTG GGGTGACA GACATTGG TCGCTGAC VEGF Vascular Endothelial 2897813 447 CTGGCGGC CCGGCCTT Growth Factor GCTGCTCT TCTTGCGC ATCTGC TTTCTC‘IT VMO1 Vitelline Membrane 487905 499 ACTCATCCT GAGCGCTG Outer Layer Protein 1 GCTCTTCTT TATCATCAC TTTCTA GA 149 Table 1. Cont. Group A 1 Finger Protein 5 399186 Protein Gene Product) 150 Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: AKR Homeodomain Protein 857681 617 TCGGGCAAA GGTGGGGT AKR CGGAGGAG TGGAGGAG ACG GAGTGTTA BF1 Brain Factor1 1546781 716 CGCCGCGG AGGGCATG CCGAAGAAG GGGTGGCT AGGAC GGGGTAGG BF2 Brain factor2 1546783 859 CGTCGGCG AAGAGGGC CTGGCTGAA GGAGTGGG GAGA GGTGGTAG CAMZAB Calcium/Calmodulin- 3668370 968 CTGCAACCG TCACGCCG Dependent Kinase 2, CTTCACCGA TCATTCTTC Alpha-B GGAGTA TTGTTGC CAM2B Calcium/Calmodulin- 3668372 1041 GACGGGCG GTGAGCCC Dependent Kinase 2, GAGAGCTGT CGGGTCGC Beta TTGAGGA AGATTTTC CBX1 Chromobox Protein1 3649782 343 GAGGAGGA CCCCGCTG GGAGTATGT GAATCTGT GGTGGAG GG CBX2 Chromobox Protein 2 3649784 277 AAACAGATG CATGAATG GTGCGAAAA CCAAGTTA GAAAAT GTCGTT CBX3 Chromobox Protein 3 3649786 1179 CTTCGCCCG GGCTGCTG CCGCTCCAA CGGGGGCT CAT CTACG CCNC CyclinC 1118027 612 TCCAGGCTT TAGCCATCT TAGGTGAAC CTlTCCTCT ATCTTA CATCAA CDA Cytidine Deaminase 3746538 789 GGGCTGCA AGGGGACC GGCTGGGA GGCTGGGG CACG ATGG C06 Putatitive Calcium- 3341750 502 TGACTGCAC GTGGGCCA Activated Potassium AGAAGCGA AAGGAAAG Channel Regulatory GGAGAG TGAAGAG Subunit CRE82 Cyclic AMP Response 3757574 574 TT'lTI'ATGCA GAAACGGG Element-Binding CTGCCCCTG CCTGGAAC Protein 2 GATGT TGGAACTA 151 Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: OCT Dopacnrome 3510493 881 CCCGGGCA TCTTGGCC Tautomerase GAGGCACA Tl’CGTrGG GTTC AGCAGTC E2F1 E2F Transcn'tpion 944827 756 CCGGCAGA GCGGCGAC Factor1 GGGGCAAA AGGCTCAC GT G ECH Erythroid Cell-Derived 1037159 1022 CCAGCTCAG GGGCCAGC CNC Family CGCGTTCAG AGGAGGGT Transcritpion Factor TC CTTT EDNRA Endothelin Receptor, 2961104 995 CCTTGTATT TCTGCCGG Type A TGCGAGTTT GATCTCTTT CTTCAC CATTAT EK10 Eph-Related Tyrosine 312201 689 GCGGCCCG TCCGTCCA Kinase 10 GGGACG'ITC GCCGCAGC AAATC GAGTTCTT EK6 Eph-Related Tyrosine 312901 814 AACGGGGAT GCCGGGCC Kinase 6 GGGGAGTG GTGTTGGT GATGG CTGA EK7 Eph-Related Tyrosine 3122058 870 GTGGGTGG CCTCCACG Kinase 7 GCTTCTTCT GCTTTAATC CTGC ACATCTT EK8 Eph-Related Tyrosine 312216 826 AGCAGGAG GTGGCAAC Kinase 8 GCGCAGCA CGATACCC AATACAGT TTCCTCAA EPH9 Ephrin Receptor 9 758788 756 AAGTAAGTG TGTGGGCA TCCGGGATG GGGCAGAG ATAAGG AAG ER81 ER81 Protein 3869359 1253 CCGCGTGG AGTAAGGG GAGAAACTG GCGCTGGT TAATGAG TGTCTGG ETS1 Erythroblastosis Virus 63382 776 ACCCCCAGC GGCAGGGC E2 Oncogene AGCAAGGAA GGCGGGGT , ATGATG AGT EYK Eyk Proto-Oncogene 438522 761 GGGAGAGG ACGTCGGT GGGAGTTCG CGGTCAGC GGTCAGT AGGTTCAG ”2 Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: FGFR2 Fiborblast Growth 63085 722 GGGCGCCC CATGGAGG Factor Receptor 2 TATTGGACA CGATCAGG CA AAGACC FKH1 Forkhead 1 3341440 717 CCGGGCTTC GCTGCCGG AGCGTGGA GAACGCCA CAACATC TCTGACA FOS V-Fos F BJ Murine 62891 485 CTCGGTCGC GCGGCGCC Osteosarcoma Viral CCCCTCCCA TCGGTCATI' Oncogne Homolog; GAAC AGC FYN Fyn Oncogene 62861 748 TC‘lTl'TTGA GGGCCTCC Related to Src, F gr, GGCGCTTTA TAGACACC Yes TGACT ACAG 622P1 ' Thyroid Autoantigen, 3374508 1123 GGGGCGGG AGTGTTCC 70-KD ACAGCTTGA GGCGGGCG ”‘lTl'I'CT ATGTAT GATA1 Gate-Binding Protein 212628 640 GGCTCCCCC GGGGGCGC 1 ACTCCGTTC CGCT'ITTI’A C CC GATA2 Gate-Binding Protein 3650486 357 GTGCGGTTG ACGGGGGC 2 GGGGCGGT AGAAGGGT GTGG GGGAGGAA GATA4 Gate-Binding Protein 511479 743 GCCCGTGTC TGGGGCGC 4 ACCTCGCTT AT‘lTCCTCA CTCCTT GTGGTC GATA5 Gate-Binding Protein 511481 735 TGGACGGC GAGCGCCA 5 CGGACACTI' GGGCACAC TGAGAGC CACGAGTC GATA6 Gate-Binding Protein 511483 655 TCCGCGCCC TGGTGGTG 6 AGCTCTCCC GTGGTGTG GTCTAC GCAGTTGG GJ83 Gap Junction Protein, 3746661 615 TTCCGTATC CTCATGGTT Beta-3 ATGATCCTG GGGGTGGT GTTGTG GTTTCTG HLXB9 Homeo Box Gene 3777536 686 CCGCGCAC CGCCTCCC H89 CGACAGCC GCCGCCTT CCTCTC TCTCC 153 Table 1. Cont. Box B1 AAGGTA Harvey Rat \firal 121 16 TGG 524050 2 Transformation 1017830 Target 2221 7 Oncogene Protein 1 TCCCCC'TT Enhancer- Factor 1 T ACCTGATG Factor AC TT Protein TCGCCCCTC Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: MAX Max Protein 414723 770 GGGGGCTG CAGGGCG TGGTGATG TTGTGGT GGACTCTC GGGCTCG TT M|M1 Myeloid Protein 1 212341 878 GCAGGCGC GGAGGGC TACAGATCT AGTGAGG TATITAC GGTGAG MYB Myeloblastosis Viral 558575 772 CACTCCGC ACACGCA Oncogene CTGCTATC TTCAGTTT CTA CTTCTTA NEURO Neurogenic 3094019 665 GCGCGGCC CGGCGGG D Differentiation CCAAGAAG GTAGTGC AAGAAGAT ATGGTGA AGG NFIA Nuclear Factor VA 63661 954 CCTGCAAG GAAGGCG CCCGAAAG AGGGACT AGAAAATA GCTGAAA CC NFlC Nuclear Factor NC 63677 1029 ACGAGGAG GGATGGC CGGGCGGT CGTGTGG GAAGGA GGGAAAT AGG NFKB1 Nuclear Factor Kappa- 2130627 845 GACGACGG CGCACCC B, Subunit 1 CGCGGCTC CGCTGTC AACCA CTGTCCA TTC NFM Nuclear Transcription 296511 646 GCAGCGGC CAGCGGG Factor M GGCGGCAA GCGAGGA GAAGC AGCGAGC AG NFYB Nuclear Transcription 63690 439 CCACGACG GTTCCCC Factor Y, Beta GATGCTTCT CAATTCC CAGTTAG CTTTTCTC C NOG Noggin, Mouse, 3695028 525 AGCACCCG AGCACTT Homolog of GACCCTAT GCACTCC CTTTGACC GCGATGA TGG NURR1 Nuclear Receptor— 683561 262 GGAGGGCC CCGTGGG Related 1 CTGCAAAAT GCCAGCA GAAGAG GAGGT OCT6 Octamer Bindirg 3172416 1641 CCGCGAGG CCTCGAA 155 Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: OKRT Otokeratin 3746659 924 GTCCCAG GTCCCC GGGTCAG GTGTTTC CCAGGTG CCAGCA GTG PEA3 Polyomavirus 3869361 508 AGCGCCC CGTI'CCC Enhancer Activator 3 CATGTCTG CTGCCAC AGC CTTCTG PITX1 Paired-Like 3236449 548 ACGACCC ATAGGGA Homeodomain GGCGAAG CAAGCG Transcription Factor 1 AAGAAGAA GGCGAG GC GACAT PITX2 Paired-Like 3335642 782 CGCCTGG GCCGAG Homeodomain GAGCCGG TTGAGGG Transcription Factor 2 GAATAATA AGGGGTT AG GC POMC Proopiomelanocortin 3869132 313 CAGCAGC GCCTTCC GGAGGGC TCTTCCT ACAAAA CCTCTTC TTC POU2F1 Pou Domain, Class 2, 212466 1103 GCAGGGG GTAATGC Transcription Factor1 CAGCAGG GGCTGCT GTCTCC GCTGCTG TTT PRH Proline Rich 297086 608 GGCGTCG CCTTCCG Homeobox GCGTCCCT CCTCCTC CTGTA CTTTTTG GTG PS1 Processed 63334 440 ACGCGGC CCATGTC Pseudogene Related CGGCAAAA AGCCCTT to the Ras Oncogene CCAC CGTAGAG Superfamily TCC REL Oncogene Rel 63922 829 AAGGGGC TTGCCTT ATGCG'ITT TTTGCTT CAG TGTTACC ATA REM1 Rem 1 Protein 529655 322 GCTGCGC CCCGTTG CCTGAAGT CCATCCA GCT GGTC SDHB Succinate 3851611 637 ATGTGGGC AGCGGC Dehydrogenase CTATGGTA TGCCTTC Complex, Subunit B, CTTGATGC TC‘ITI’GT Iron Sulfur Protein 156 Tal El 521 Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: SlM1 Single-Minded, 1173853 186 CGGACTAG GCTGGTG Drosophila, Homolog GCGGGAGA CGGCTGG of, 1 AAGAAAAC AGTGG SLUG Neural Crest 495237 678 TCATACCG CTACGCA Transcription Factor CAGCCAGA GCAGCCA GAT GA‘l'i'C SOX1 Sry-Box1 2947024 848 GCTGGGCG CCCCCGT CCGAGTGG GCTGGCG AAGGTGAT CTCTGGT AGT SOX11 Sry-Box 11 2982741 691 GCCTGGGC CTGCCGG AAGCGGTG CCGACGA GAAAATG GGTGGAG ATG SOX3 Sry-Box 3 2947026 539 CGGGGCCG TCTGCGA ATTGGAAG GTGCGAG C GTGATGG SOX9 Sry-Box 9 2982739 735 CATCTCCC CCGGCGG CCAACGCC CGTGGCT ATCTTCAA GTAGTAG GAG SPl1 Spleen Focus Forming 2369862 709 CCTCATTCC CCCCCTT Virus Proviral CCCTCCCT CCCATCA Integration Oncogene CTG CCTCA T T Brachyury 2529385 755 TCGGCGCC CACCGGA CACTGGAT GAGCCAC GAAGG GCAGGAA CT TAL1 T-Cell Acute 62844 657 GCCACGAG GCCCCTI' Lymphocytic CGAGCCCG TGGTTTC Leukemia 1 ACAGC CTTCCTC CTC TBX2 T-Box Transcription 3236441 379 CGGGTGAG GGTAGGC Factor2 CGGCCTGG GGTGACG ACAAGAA GCGATGA AGT TBX3 T-Box Transcription 3236443 303 ATAAAAGA CGTGCTT Factor 3 GGCACGGA GTCGGAG GATGGT ATGTTG TBX4 T-Box Transcription 3236445 499 AAGCAGGC AGGTCGC Factor 4 AGGAGGAT TGTCGTC GTTT ACTTC 157 Table 1. Cont. Locus: Gene Name: ID#: Size: Primer 1: Primer 2: TBX5 T-Box Transcription 3236447 445 ACGAGGTG GAGGTAACA Factor 5 GGGACGGA GCGATGAAG GATG GCAGTC TBX6L T-BoxTranscription 1806623 539 GCCCCAGCT CATGGCTGC Factor 6L CCCTTGTCG TGTTCTGCT CTGA TBXT T-Box Transcription 1806621 601 TCCCCTTTG GTGGGGAG Factor T GCAGAGATT CCTGTGGAG CA AGTG TCF15 Transcription Factor 3413459 569 GGCCGGGT CGGGGCGG 15 CCCCACTGC GTCTCCAAC TGCTC ACG TCF4 Transcription Factor 63356 800 ATCACCATC ACATCCGGC 4 GCCGCTTAC CGAGTTCTI' AGG TGA TEAD1B Tea Domain Family 1256008 926 GGGAGGGG TGCGCTGCT Member 18 CGGGAAGAT GTATI’GACT GG GCTGAC TFAPZB Transcription Factor 3309576 816 GGTACGGC GTGAGGGC AP2 Beta GGCCAGAT GGCGCAGAT GTCC AGC THRB Thyroid Hormone 63820 455 TGGCATGGC CGGGGTCAT Receptor, Beta AACAGATTT AGCGAACT UBP46 Ubiquitin Specific 3800759 860 CAGAGATAC TCTCGGGGC Protease 46 GCCCCACG TTTCTGCTG CTTTGTT TTCTTG UBP52 Ubiquitin Specific 3800761 760 TTCGGGGCT CTTGGGGAT Protease 52 GCACACGTC GGGCAGGG GGATAG AGAGGTC UBP66 Ubiquitin Specific 3800763 1108 ATGCCGGG GGGCCGGG Protease 66 CTCCCTGCT TACATGCGT GGTCT GAGGAT WH1 Winged Helix Protein 1766072 631 GACGGGGC CGTAGCGAA 1 GAAATACAG GCCGGGCA CGAGGAC GGAAGG 158 Table 1. Locus: Cont. Gene Name: ID#: Size: Primer 1: Primer 2: WH2 Winged Helix Protein 2 1 766074 788 AACCCGCCGC CCCCAAGGAC GCTGCACG CCGCGCTG TAACC WH3 Winged Helix Protein 3 1 766076 636 GCTGCCGCTG CCGCTGGACG AG AGAGCGGC GGGGTGCG GGTAGG Wl'1 Wilms Tumor 987062 655 GAGCGCTTI'C ACCGTCCACTT CT GGGGCGTT TTTCATTI'G TCTCACT YRK Yes Related Kinase 63895 695 GCAGGCGCAC AGCAGCATCA CAG TGCCCGGC TTCAGCGT CTTCACT ZFP161 Zinc Finger Protein 161 1399186 726 ATTGGGGAAC CTAACGATACC GCAGGAAC CGCAGACA AAA H9 Comparative Mapping of the Chicken Genome Using the East Lansing Reference Population EUGENE I. SMl'I'I-I)”1 LESLIE A. LYONS,‘t HANS H. CHENG,’ and STEVEN P. SUCHYTA' ‘USDA, Agricultural Resmrch Service, Avian Disease and Oncology laboratory, 3606 East Mount Hope Road, East Lansing, Michigan 48823, and *National Cancer Institute, laboratory of Viral Carcinogenesis, Frederick Cancer Research and Development Center, Frederick, Maryland 21702 ABSTRACT The annotation of known genes on linkage maps provides an informative framework for synteny mapping. In comparative gene mapping, con- served synteny is broadly defined as groups of two or morelinkedmarkersthatarealsolinkedintwoormore species. Although many anonymous markers have been placed on the chicken genome map, locating known genes will augment the number of conserved syntenic groups and consolidate linkage groups. In this report, 21 additional genes have been assigned to linkage groups or chromosomes; five syntenic groups were identified. Ultimately,co conserved syntenic groups may help to pinpoint important quantitative trait loci. (Key words: synteny, polymerase chain reaction, comparative mapping, linkage, genes) INTRODUCTION In the assembly of linkage maps, functional genes (Type I) (0’ Brien, 1993) and anonymous polymorphic DNA markers (Type II), have served as markers in mapping the chicken genome (Burt et al., 1995; Cheng et al., 1995; Crooijmans et al., 1996). Because chickens diverged from mammals about 300 million yr ago (Hedges, 1994), Type I markers have also revealed conserved linkage associations among other species. In view of the considerable amount of research associated with expresed genes, they are especially informative candidates for conserved synteny mapping. Using anchor lod, syntenic comparisons may provide clues to the location and orientation of orthologous genes. Functionalgenesarealsousefulasprobesinfluorescent in situ hybridizations (FISH), in the physical mapping of geneaandintheassignmentoflinkagegroupsto specific chromosomes. Currently, about 41 linkage groups and more than 617locihavebeenplacedonthe£astlansing(EL) reference map; 101 loci represent known genes. An objective of this research is to annotate the genetic map of the chicken to facilitate marker-assisted selection of economically important traits. This therefore, extends earlier mapping data (Smith et al., 1996). Winn-MAW“, 1996. AmptedforpubliationlamraryBS! 1997. l‘l‘owhomcorresponderueahould beaddruaed. Mambmgvlncwm MNW. ‘KindlyprovldedbyKManley, RoswellParkCancerlnstimte, 743 160 1997 Poultry Science 76:743—747 MATERIALS AND METHODS Candidate Genes and PCR Primers Chicken genes were selected based on the availability of sequencesin the GenBank database. GenBankaccession numbers and sequences were obtained through the Entrez2 retrieval system at the National Center for Biological Information. Primers were selected using the OLIGO3 primer analysis program To determine con- served synteny, priority was given to cognate genes that were mapped in the human or mouse. Exon-based primers (18—mer) that amplified across introns were selectedbecausethereisagreaterlikelihood thatbase substitutions would be found in introns rather than in the more conserved exons. Alternatively, primers were based on sequences in the 3’ untranslated region (UTR) of complementary DNA (cDNA). Two chicken genes were mapped based on primer sequences from nonavian sources. Linings Analysts Segregation data on inheritance of the Jungle Fowl 0F) alleleofSZrnalemeioseswasenteredintotheELdatabase usingMAP MANAGER4 version 2.6. Genes with the least number of crossovers between adjacent loci and minimal double recombinants were located in strain distribution patterns. Only genes with logm of odds (LOD) scores greaterthan3.0weremnsideredtobeltnkedtoother markersMarkersweredepositedintheI-ZLgenome 744 SMITH ET AL. TABLE 1. Primers used to amplify target regions in functional genes Accession Gene Forward/ reverse 5'-3’ number References rcccacn'rcrrcrcrr/ ’ Nicotinic acetylcholine receptor CCCI'I'CAAAACCI‘CCATC X83739 Gammon-Hernandez et al.. 1995 TGATGGAAACGATACCI‘C/ Retinoblastoma oncogene TGGC'ITCAATCAGTAACG X7228 Boehmelt et al., 1994 AMACAACAGC‘ITCACCA/ G protein coupled purinoreceptor CGAGTCAAAACAGACATC 1.06109 Kaplan et al.. 1993 AGAGGTACACCAGCCAGT/ Hydroxy steroid dehydrogenase TGCTCAATGAC’ITGGGTA 043762 Nakabayshi et al., 1995 TCCCAAAGCACTGCTCAT/ N-acetyl galactoaaminidase GGTCACTGGGAACACTCC 1.18754 Davis et al.. 1993 TCAATGTGGCCGAATGTG/ Beta-globin AGCATCCCCAAAAGGAGGT V00409 Dolan et al., 1983 GTGCTGCTATGTCACCI‘G/ W‘mglessprelated MMTV int site ATGGCAAAGAT'ITI‘GGAA 031901 Tanda et al., 1995 GAGGACACCATGGAGGTGGA/ Abelson viral oncogene homologI - CTGGATGAAGAAGTTCI'I‘CI'I‘C‘I‘C M14752 Shtivelman et al., 1986 ATGGTCGCAGACCI‘GCCG/ Alpha miaoglobulinl AGTAGAAC’I'I'G‘ITGCCG‘ITGCC X54818 Vetr and Gebhard, 1990 GAGCCACAGCGACAAGAC/ Glucose transporter 1 TGCTCAATCTATCGGCTI' 1.07300 Wagstaff et al., 1995 CGTTGTCI‘CCGTITCTCT/ CelldivisioncycleZproteinkinase CGCTGTCTCCGTITC’I‘CI‘ U16344 Uetal.,1995 GTITI‘GCTGGAAGGAACT/ EnolaaeA GCI'CCAAACACTGAAGAA 037900 Tanaka et al., 1995 TGCI'AATGTGAGGAAAAT/ Glutatl'iione-S-tnnsferue TAAAAAGGGAGGGAAGAG 1.15387 Liu et al., 1993 TGCITC'ITI'GAACCTGAGAG/ Vimentin CTGTCCI’CI'I‘CGAGTGAGTG 102759 Zehner et al., 1987 CAAAATCCAAAACATCTA/ (GM? phosphodiestensealpha ‘lTlTl‘CTCGACAGTATGC 1.29233 Scripts-Rowland and Green. 1994 CCGCAG'IC‘ICATCGTCAG/ Bela Bl crystalline ATCTCCCCACGCATGTI‘G U09951 Duncan et al.. 1995 GCI'TGCAGCC'I'ITAGGAG/ Beta-Z-ntkroglobulin TAAGCCGAGCI‘GGGA'ITA Z4892] Riegert et al.. 1996 AAAGCTGCCAGGAAGCTG/ Osteopontin . GGCCTCATCCTCAATGAG U01844 Rafidl et al.. 1994 GTATCAGGATCAACTCGG/ Ryanodine receptor 3 CCCTCTGATC'ITCAAG‘IT X95267 Ottini et al., 1996 TAGTGCI'ITI'I‘GGI‘ATGG/ Riboaomal Protein 137a GAAATGCI‘AATGTCTCCA 014167 Madrida et al.. 1993 GAGGACCCTATACCITITGA/ Ski-novel overexpreaoed N A‘I‘GI'I'ITGI'I'CTI'CCAGCAT $78406 Givol et al., 1995 1Deriiotesprirnersrile-livedfriornnonaviansequences. database (Chick GBASE5) and will be integrated into a miifiedmapwithmarkersfromtheComptonreference population (Burt et al., 1995, Crittenden et al., 1995). RESULTS AND DISCUSSION TheI'FxWhiteleghornMLIbackcrosstEL reference population (Crittenden et al., 1993) and methodsusedtodeterminethesegregationoftheIF- specific allele were previously described (Smith et al., 1996). Briefly, introns or 3’ UTR were amplified using PCRSequenceanalysesofclonedPCRproductsfrom theIFandWLparentsofthereferencepopulationwere conducted to identify base substitutions in either parent. Wherinudeoddesubstitufioriswaefoundsegregation of the nonredundant IF allele was typed through Snap: / / www.pou1try.mph.rnsu.edu/ . 161 preferential amplification oftheIFallelefromDNAof BC progeny of the EL family. References to nucleotide sequences and primers used toamplifyintronsor3’UTRofcandidategenesare listed in Table 1. The initial PCR products were between 250 and 650 bp in size. Products for hydroxysteroid dehydrogenase (HSDSB) and B-crystalline (CRYBBI ). however,had2and11bpdifferencesbetweenWLand IF, respectively. After electrophoretic separation, the differences in size enabled detection of the IF allele. For the other genes, base substitutions were found and primersmismatchedattheB’ter-minuswithrespectto the WL allele were designed for preferential amplifica- tionoftheIFallele.Althmghtransitionsoccurredmore frequently, mismatched primers based on transversions were preferred because they are less prone to false pruning. Typelmndidategenesthatweremappedarelisted in Table 2. Their location and the position of other genes MAPPING KNOWN CHJCKEN GENES 745 TABLE 2. Comparative location of chicken, human, and mouse genesI Gene Symbol Chicken Human Mouse Lysoson'ial glyooprotern' LAMP] Chi 13q.34 8 oncogene RBI Chi 13q14.3 14 G protein puririoreceptor PZYS Chi 13q14.3 NM Globin HBB Ch1 11p15 7 Winglas WNT11 Chl NM 7 Abelson viral oncogene homologue ABL1 E41 9q34 2 Adaiylate kinase 1' AK1 E41 9q34 2 Alpha microglobulin AMBP E41 9q32 4 AldolaseB' ALDOB F1 9q22.3 4 Iron raponse element‘ [KEEP E2 9 k 15 4 Cell division cycle 2 protein kinase CDC 2L1 £54 1p36 4 Enolase A ENOI F54 1136 4 Glucose transporter 1 SLQAI UL 1p35 4 Ryanodine receptor 3 RYIB 507 15114 2 Beta-Z-miaoglobulin 32M UL 15q21 2 Hydroxy steroid dehydrogenase HSD3B Ch] 1p13 3 ' NAGA Chl 22q13 NM V tin VIM Ch2 10p13 2 Gluhthione-Stransfeme GSTA2 C113 6p12 9 Osteopontin SPPI CM 4q11 5 Creatine ldnase B' CKB ChS 14q32 12 Ribosomal proteimLS’lA L37u Ch7 NM NM Vitellogenin 2' 1762 018 NM NM Phosphodisterase P054 £11 5q31 18 Beta crystalline CRYBBI E18 2:111 11 Ski novel overexprrssed N SNON 36 NM NM Apo ' Al' APOA] B49 11:18.3 9 Acetylcholine receptor EZ 8p11 NM ‘Genes reported earlier (Smith et al., 1996) are marked with an asterisk. UL, unlinked; NM, not mapped. on the EL map enabled us to identify five novel conserved groups. OnChromosome (Ch) LLAMPI, the retinoblastoma susceptibility gene (RBI), and a G- protein coupled r (PZYS) gene, exhibited conserved synteny with humanCh 13. We note thatin human,P2Y5isinintron 17 of RBI (Webbetal., 1996). Apparently, the RBI-P2Y5 linkage has remained intact throughout evolutirm (LOD score 15) Humanorthologstothewntfamilyhavenotbeen reported, butthedtickenorthologoftheDrosophila segmentpolaritygenewingless, Writ-11, islinkedwithfi- globinonChl;HBBandet-11arelinkedonmouse Ch7.lnchicken,Wnt-11andHBBareabmt15cMapart, whermstheyareabouthMapartinmouse.A representationofPCRproductder-ivedfrom tial amplification of the IF allele of H38 among 15 BC progenyisshowninfigurel. of the Abelson viral oncogene (ABL1), adenylate kinase 1 (AK1), and alpha microglobulin- bikunin precursor (AMBP) comprising 28.4 cM on chickmlinkagegroupfllaresynterucinhumanCh9 andmnththneandhmnhowws,ABLl andAlCl areabouthM . Z-Linked aldolaseBiALDOB)andestheironresponse elernentbindingproteinmtfiBP) es,purportedtobe acytosolicisoformofacorfitase,($aitohetal.,1993).are linkedtowithirilscM'l'lwsegenesarealsolinkedin 4. 162 Glucose transporter 1 (SLCZAI). p58 ein associated with cell division cycle 2 (CDC2:L1), and enolase (ENOI) are syntenic in hum and mouse. The latter two gens are provisionally placed in E54 (they are 26.9 cM apart, LOD score 2.5). In this context, we also note that CDCZLI and ENOI are telomeric on human Ch 1p. Ofthe38pairsofchickenautosoms, about30pairs are classified as microchromosomal. Recently, CpG islands(CGI)werefoundtobehighlyconcentratedon microchromosomes and in situ hybridizations with a CG] probe suggested that microchromosomal euchroma- tin '5 gene-rich (McQueen et al., 1996). Although B« 2-microglobulin (82M) is, unlinked on the EL map, it wasshownbyFlSHtobemiaocluomosomalmiegertet al., 1996). Theribosomalproteingeneas7a),wasalsomapped byFlSHtochickenmacrochromosome7mandaetaL, 1996). Although 137a was originally mapped to linkage groupEOZ, E02,it probablyrepresentsCh7. TheFISl-l mappingsupportstheconsemusmapbecausetheupper portionofEOZisCh7(BumsuadandCheng,unpub- lished data) '1'heothergeneslistedinTable2havenot,atthis point, been assodated with a syntenic group, but we note that vitellogenin and ostepontin are major componentsofavianeggyolkandshellmbrane, mpecfively. 746 SMITH ET AL. MISMATCH PRIMER PCR Chicken B-Globin Gene Backcross Progeny JF1W4 5 6 7 8910111213141516171819 JJWJJWJJ FIGURE 1. Preferential amplification“ of the 189 bp polymerase rupecfiv lackingDNA. Molecularsizeuurker,Misamultipleo{1flbp With the relatively high incidence of polymorphisms in vertebrate gnomes, selective PCR amplification of less conserved regions and prefaential amplifimtion of specific alleles provides a convenient and efficient approach to mapping cloned genes. Moreover, PCR requires little DNA and is amenable to largescale testing, whereas restriction fragment polymorphisms require time-consuming Southern blot hybridizations that are fraught with techniml difficulties Linkage mapping and FISH will collaterally charac- terize the numerous chicken microchromosomes that constitute about 25% of the chicken genome (McQueen et al., 1996). In the case of L371 reported above, marker linkagesupportedthel-‘ISHassignmenttoCh7. Apartfromthosediscussedhere,19otherconserved syntenic groups have been found (Burt et al., 1996). Althoughtherepertoireofclonedchickengenssis limited, additional synteny may be revealed using comparative anchor tagged site primers (CATS) that are based on consa'ved exon of mammalian species. In this context, we have successfully used non 163 JWWWJWJJJW—TM chainreaction(PCR)productolthelungleFowlalleleofthe threspecttotheS’ terminusoftheWhite alleleand ' WAmmquluwlemmelmma ely.;anl.anes4to18representhprogury-Trepresenls aliquotofa B-globingeneusing theinitialreverseprimerfiable1).Thefii-st avian-based primers to amplify chicken ABL1 and AMBP. Ultimately, a marker-rich map annotated with respect to orthologous mammalian will inform poultry geneticists on loci associated with economically important traits. ACKNOWLEDGMENTS We thank Cecyl Fischer and Barbara Okimoto for their skilled technical assistance. Jerry Dodgson provided constructive comments. Funding was provided, in part, through a USDA~ARS Cooperative Agreement 58-3635-1-106 with ' tural Research Service and Michigan State University. Stephen O’ Brien generously provided an extensive set of comparative anchor tagged site primers. Patrick Vents also provided primers for ABL1. REFERENCES Boehmelt, G., E. Ulrich, R. Kurzbauer, G. Mellitzer, A Bird, andMZehnke,1994.Structureandexpressionofthe MAPPING KNOWN CHICKEN GENES 747 chicken retinoblastoma gene. Cell Growth Differ. 5: 221-230. Burt, D. W., N. Bumstead, J. J. Bitgood, F. A. Ponce deLeon, and L. B. Crittenden, 1995. Chicken genome mapping a new era in avian genetics. Trends Genet. 11:190-194. Burt, D. W., C. T. Jones, D. R. Morrice, and l. R. Paton, 1996. Mapping the chicken genome—An aid to comparative studies. Page 105 in: XXVth International Conference on Animal Genetics. (Abstr.) Cheng, H. H., I. Levin, R L. Vallejo, H. Khatib, J. B. Dodgson, L. B. Crittenden, and J. Hillel, 1995. Development of a genetic map of the chicken with markers of high utility. Poultry Sci. 74:1855—1874. Clemencia-Hemandez, M., L. Erkrnan, L. Matter-Sadzmski, T. Roztocil, M. Ballivet, and J. M. Matter, 1995. Characteriza- tion of the nicotinic acetylcholine receptor 63 gene. J. Biol. Chem. 270:3224—3233. Crittenden, L. B., L. Provencher, L. Santangelo, I. Levin, H. Ablanalp, R. Briles, W. E. Briles, and J. Dodgson, 1993. Characterization of a Red Jungle Fowl Backcross reference population for molecular mapping of the chicken genome. Poultry Sci. 72:334—348. Crittenden. L B., J. Bitgood, and D. Burt, 1995. Genetic Nomenclature Guide Trends Genet. 11(Suppl.):33—34. Crooijmans, R. P., P.A.M. van Oers, J. A. Strijk, J. J. van der Poel, and M.A.M. Groenen, 1996. Preliminary linkage map of the chicken (Gallus domesticus) genome based on microsatellite markers. Poultry Sci. 75:746—754. Davis, M. 0., D. J. Hata, D. Smith, and J. C. Walker, 1993. Cloning and sequence of a chicken alpha-N- acetylgalactosaminidase gene. Biochim. Biophys. Acta 1216:296—298. . Dolan, M., J. B. Dodgson. and J. D. Engel, 1983. Analysis of the adult chicken B-globin gene. J. Biol. Chem. 2583983—3990. Duncan, M. K., H. J. Roth, M. Thompson, M. Kantarow, and J. Piatigorsky, 1995. Chicken 881 crystalline: gene sequence and evidence for functional conservation of promoter activity between chicken and mouse. Biochim. Biophys. Acta 1261:68—76. Givol, 1., P. L. Boyer, and S. H. Hughes, 1995. Isolation and characterization of the chicken c-sno gene. Gene 156: 271-276. Hedges, S. B., 1994. Molecular evidence for the origin of birds. Proc. Natl. Acad. Sci. USA. 91:2621-2624. Kaplan, M., D. 1. Smith, and R. S. Sundick. 1993. Identification of a G—protein coupled receptor induced in activated T cells. J. Immunol. 151:628-638. Li, H., J. Grenet, M. Valentine, J. M. Lahti, and V. Kidd, 1995. Structure and expression of chicken protein kinase PITSLRE-encoding genes. Gene 153237-242. Liu, L-F., S.-H. Wu, and M. F. Tam, 1993. Nucleotide sequences of class alpha glutathione S-transferases from chicken liver. Biochim. Biophys. Acta 1216:332-334. Machida, M., S. Toku, N. Kenmochi. and T. Tanaka, 1993. The structure of the gene encoding chicken ribosomal protein L37. Eur. J. Biochern 213:77—80. McQueen, H. A., J. Fantes, S. H. Cross, V. C. Clark, A. L. Archibald, and A. P. Bird, 1996. CpG islands of chicken are concentrated on microchromosomes. Nature Genet. 12: 321-323. 164 Nanda, N., T. Tanaka, and M. Schmid, 1996. The intron- containing ribosomal protein-encoding genes L5, L70 and 1.37:: are unlinked in chicken. Gene 170:159-164. Nakabayashi, 0., O. Nomura, K. Nishimori, and 5. Mizuno, 1995. The cDNA cloning and transient expression of a chicken gene encoding a 3B-hydroxysteroid de- hydrogenase unique to major Steroidogenic tissues. Gene 162:261-265. O’Brien, 5. J., J. E. Womack, L. A. Lyons, K J. Moore, N. A. Jenkins, and N. G. Copeland, 1993. Anchored reference Loci for comparative mapping genome mapping in mammals. Nature Genet. 3:103-112. Ottini, L., G. Marziali, A. Conti, A. Charlesworth, and V. Sorrentino, 1996. Alpha and B isoforms of ryanodine receptor from chicken skeletal muscle are the homologues of mammalian RYRl and RYR3. Biochem. J. 315207-216. Rafidi, K., I. Simkina, E. Johnson, M. A. Moore, and L. C. Gerstenfeld, 1994. Characterization of the chicken osteopont‘m—encoding gene. Gene 140:163—169. Riegert, P., R. A. Andersen, N. Bumstead, C. Dohring, M. Dominguez-Steglich, J. Engberg, J. Salomonsen, M. Schmid, J. Schwager, K Skjodt, and J. Kaufman, 1996. The chicken BZ-microglobulin gene is located on a non-major histocompatibility complex microchromosome: A small, G+C-rich gene with X and Y boxes in the promoter. Proc. Natl. Acad. Sci. USA 93:1242-1248. Saitoh, Y., A. Ogawa, T. Hori, R. Kunita, and R. Kunita, 1993. Identification and localization of two genes on the chicken 2 chromosome: implication of evolutionary conservation of the Z chromosome among avian species. Chromosome Res. 1:239—251. Sample-Rowland, S. L., and D. A. Green, 1994. Molecular characterization of the alpha-subunit of cone photorecep- tor cGMP phosphodiesterase in normal and rd chicken. Exp. Eye Res. 59:365-372. Smith, E. J., H. H. Cheng, and R L. Vallejo, 1996. Mapping functional chicken genes: an alternative approach. Poultry Sci. 75:642-647. Shtivelman, E., B. Lifshitz, R P. Gale, B. A. Roe, and E. Canaani, 1986. Alternative splicing of RNAs transcribed from the human ab! gene and from the bcr-abl fused gene. Cell 47:277—284. Tanaka, M., K. Maeda, and K. Nakashima, 1995. Chicken alpha-enolase but not B—enolase has a sec-dependent tyrosine-phophorylation site cDNA cloning and nucleo- tide sequence analysis. J. Biochem 117554-559. Tanda, N., Y. Kawakami, T. Saito, S. Noji, and T. Nohno, 1995. Cloning and characterization of Wnt-4 and Wnt-ll cDNAs from chicken embryo. DNA Seq. 52277—281. Vetr, H., and W. Gebhard, 1990. Structure of the human alpha- l-microglobulin-bikunin gene. Hope-Seyler 371:1185—1196. Wagstaff, P., H. Y. Kang, D. Mylott, P. J. Robbins, and M. White, 1995. Characterization of the avian GLUTl glucose transporter: Differential regulation of GLUTl and GLUT3 in chicken embryo fibroblasts. Mol. Biol. Cell 6:1575—1589. Webb, T. E, M. G. Kaplan, and E. A. Barnard, 1996. Identification of GM as a P2Y purinoreceptor: P2Y5. Biochim. Biophys. Res. Commun 219:105-110. Zehner, Z., Y. Li, B. A. Roe, B. M. Paterson, and C. M. Sax, 1987. The chicken vimentin gene. J. Biol. Chem. 262: 8112-8120. M References Abderrahim, H., J.L. Sambucy, F. Iris, P. Ougen, A. Billaut, I.M. Chumakov, J. Dausset, D. Cohen, and D. LePaslier, 1994. Cloning the Human Major Histocompatibility Complex in YACs. Genomics 23:520-527. Adams, M.D., A.R. Kerlavge, R.D. Fleischmann, R.A. Fuldner, C.J. Bult, N.H. Lee, E.F. Kirkness, k.G. Weinstock, J.D. Gocayne, 0. White, 1995. Initial Assessment of Human Gene Diversity and Expression Patterns based Upon 83 Million Nucleotides of cDNA Sequence. Nature (Suppl. 65478) 37, 3— 174. Amadou, C., M.T. Ribouchon, M.G. Mattei, N.A. Jenkins, D.J. Gilbert, N.G. Copeland, P.Avoustin, and P. Pontarotti, 1995. Localization of New Genes and Markers to the Distal Part of the Human Major Histocompatibility Complex (MHC) Region and Comparison with the Mouse: New Insights into the Evolution of Mammalian Genomes. Genomics 26:9-20. Bacon, L.D., 1987. Influence of the Major Histocompatibility Complex on Disease Resistance and Productivity. Poultry Sci. 66:802-811. Barnes, W, 1994. PCR Amplification of up to 35kb DNA With High Fidelity and High Yield Prom Lambda Bacteriophage Templates. Proc. Natl. Acad. Sci. 91:2216-2220. Belterman, R.H.R, and L.E.M. De Boer, 1984. A Karyological Study of 55 Species of Birds, Including Karyotypes of 39 Species New to Cytology. Genetica 65:39-82. Bengtsson, B.O., K. Klinga Levan, and G. Levan, 1993. Measuring Genome Reorganization from Synteny Data. Cyto. Cell Genet. 64:198-200. Bent, A.F., B.N. Kunkel, D. Dahlbeck, K.L. Brown, R. Scmid, J. Hiraudel, J. Leung, and B.J. Staskawicz, 1994. RPSZ of Arabidopsis Thaliana: A Leucine-Rich Repeat Class of Plant Disease Genes. Science 265:1856-1860. Bickham, J.W., 1981. Two—Hundred-Million-Year-Old- Chromosomes: Deceleration of the Rate of Karyotypic Evolution in Birds. Science 212:1291-1293. Bird, A.P., 1987. CpG Islands as Gene Markers in the Vertebrate Nucleus. Trends Genet. 3:342. Birshtein, V.Ya., 1987. Tsitogenetic and Molecular Aspects of Evolution of Vertebrates. Nauka, Moscow. 165 ‘73??? E. Bitgood, J.J., and R.G. Somes, 1990. Linkage Relationship and Gene Mapping. Poultry Breeding and Genetics, pp.469-405; Elsevier, Amsterdam. Bitgood, J.J., and R.N. Schoffner, 1990. Cytology and Cytogenetics, Poultry Breeding and Genetics. Elesevier, Amsterdam. Bulatova, N.Sh., 1977. Structure and Evolution of Avian Chromosomes, Cytogenetics of Hybrids, Mutations, and Evolution of the Karyotype, pp. 248—259. Novosibirsk, Nauka. Bumstead, N., and J. Palyga, 1992. A Preliminary Linkage Map of the Chicken Genome. Genomics 13:690-697. Burmeister, M.A, P. Monaco, E.F. Gillard, G.B. van Ommen, N.A. Affara, M.A. Fergusn—Smith, L.M. Kunkel, and H. Lehrach, 1988. A lO—Megabase Physical Map of Human Xp21, Including Duchene Muscular Dystrophy Gene. Genomics 2:189. Burt, D.W., N. Bumstead, J.J. Bitgood, F.A. Ponce DeLeon, and L.B. Crittenden, 1995. Chicken Genome Mapping: A New Era in Avian Genetics. Trends Genet. 11:190-194. Burt, D.W., 1997. Comparative Mapping with the Chicken - Clues to Our Ancestral Vertebrate Genome. Avian Molecular Cytogenetics Symposiumm Leicester, England. Abstract. Burt, D.W., N. Bumstead, T. Burke, R. Fries, M. Groenen, M. Tixier-Boichard, and A. Vignal, 1997. Current Status of Poultry Genome Mapping — June 1997. In: Proceedings of the 12th AVIAGEN Symposium: Current Problems in Avian Genetics, Prague, Czech Republic, pp. 33—45. Burt, D.W., C. Bruley, I.C. Dunn, C.T. Jones, A. Rmage, A.S. Law, D.R. Morrice, I.R. Paton, J. Smith, D. Windsor, A. Sazanov, R. Fries, and D. Waddington, 1999. The Dynamics of Chromosome Evolution in Birds and Mammals. Nature 402:411— 413. Carpenter, A.T.C, 1994. Chiasma Function. Cell 77:959-962. Carver, E.A., and L. Stubbs, 1997. Zooming in on the Human— Mouse Comparative Map: Genome Conservation Re-examined on a High-Resolution Scale. Genome Res. 7:1123—1137. Chang, Y.-L., Q. Tao, J. Wang, C. Scheuring, K. Meksem, and H.-B. Zhang, 1999. A Large Scale Plant Transformation- and Genome Sequence-Ready Physical Map of the Arabidopsis thaliana Genome. Proceedings of the Plant and Animal Genome VII Conference, p. 37 (abstract). 166 Chang, E., J. Luna, J. Giacalone, D. Uyar, G.A. Silverman, and U. Francke, 1994. Regional Localization of 56 New Human ' Chromosome lB—Specific Yeast Artificial Chromosomes. Cytogenet. and Cell Genet. 65:136—139. Charlier, C., W. Coppieters, F. Farnir, L. Grobet, P.L. Leroy, C. Michaux, M. Mni, A. Schwers, P. Vanmanshoven, and R. Hanset, 1995. The mh Causing Double—Muscling in Cattle Maps to Bovine Chromosome 2. Mamm. Genome 6:788-792. Chen, Z.-Q., J.A. Lautenberger, L.A. Lyons, L. McKenzie, and S.J. O’Brien, 1999. A Human Genome Map of Comparative Anchor Tagged Sequences. J. Hered. 90:477-484. Cheng, S., C. Fockler, W. Barnes, and R. Higuchi, 1994. Effective Amplification of Long Targets From Cloned Inserts and Human Genomic DNA. Proc. Natl. Acad. Sci. 91:5695-5699. Cheng, H.H., R.L. Vallejo, H. Khatib, J.B. Dodgson, L.B. Crittenden, and J. Hillel, 1995. Development of a Genetic Map of the Chicken with Markers of High Utility. Poultry Sci. 74:1855-1874. Chowdharry, B.P., L. Fronicke, I. Gustavsson, and H. Scherthan, 1996. Comparative Analysis of the Cattle and Human Genomes: Detection of ZOO—FISH and Gene-Mapping Based Chromosomal Homologies. Mamm. Genome 7:297—300. Clark, M.S., Edwards, Y.J.K., Y.J.K. Edwards, S.E. Meek, S. Smith, Y. Umrania, S. Warner, G. Williams, G. Elgar, 1999. Sequence Scanning Chicken Cosmids: A Methodology for Genome Screening. Gene 227:223—230. Clement, W.M., 1971. DNA Replication Patterns in the Chromosomes of the Domestic Fowl. Cytologia 8:168—172. Cohen, D., I. Chumakov, and J. Weissenbach, 1993. A First- Generation Physical Map of the Human Genome. Nature 366:698- 701. Copeland, N.G., N.A. Jenkins, D.J. Gilbert, J.T. Eppig, L.J. Maltais, W.F. Dietrich, A. Weaver, S.E. Lincoln, and R.G. Steen, 1993. A Genetic Linkage Map of the Mouse: Current Applications and Future Prospects. Science 262:57-66. Crittenden, L.B., L. Provencher, I. Santangelo, H. Levin, H. Abplanalp. R.W. Briles, W.E. Briles, and J.B. Dodgson, 1993. Characterization of a Red Jungle Fowl by White Leghorn Backcross Reference Population for Molecular Mapping of the Chicken Genome. Poultry Sci. 72:334-348. Crooijmans, R.P.M.A., J.J. van der Poel, and M.A.M. Groenen, 1994. Functional Genes Mapped on the Chicken Genome. Anim. Genet. 26:73-78. 167 Crooijmans, R.P.M.A., P.A.M. Van Oers, J.A. Strijk, J.J. Van Der Poel, and M.A.M. Groenen, 1996. Preliminary Linkage Map ' of the Chicken (Gallus domesticus) Genome Based on Microsatellite Markers: 77 New Markers Mapped. Poultry Sci. 75:746-754. Debry, R.W., and M.F. Seldin, 1996. Human/Mouse Homology Relationships. Genomics 33:337-351. Deloukas, P., G.D. Schuler, G. Gyapay, E.M Beasley, C. Soderlund, P. Rodriguez-Tome, L. Hui, T.C. Matise, K.B. McKusick, J.S. Beckmann, S. Bentolila, M.-T. Bihoreau, B.B. Birren, J. Browne, A. Butler, A.B. Castle, N. Chiannilkulchai, C. Clee, P.J.R. Day, A. Dehejia, T. Dibling, N. Drouot, S. Duprat, C. Fizames, S. Fox, S. Gelling, L. Green, P. Harrison, R. Hocking, E. Holloway, S. Hunt, S. Keil, P. Lejnzaad, C. Loiis-Dit-Sully, J. Ma, A. Mendis, J. Miller, J. Morissette, D. Mesulet, H.C. Nusbaum, A. Peck,S. rozen, D. Simon, D.K. Slonim, R. Staples, L.D. Stein, E.A. Stewart, M.A. Suchard, T. Thangarajah, N. Vega- Czarny, C. Webber, X. Wu, J. Hudson, C. Auffray, N. Nomura, J.M. Sikela, M.H. Polymeropoulos, M.R. James, E.S. Lander, T.J. Hudson, R.M. Myers, D.R. Cox, J. Weissenbachm M.S. Boguski, and D.R. Bentlry, 1998. A Physical Map of 30,000 Human Genes. Science 282:744-746. Dietrich, W.F., J. Miller, R. Steen, M.A. Merchant, D. Damron-Boles, Z. Husain, R. Dredge, M.J. Daly, K.A. Ingalls, and T.J. O’Connor, 1996. A Comprehensive Genetic Map of the Mouse Genome. Nature 380:149-153. Dodgson, J.B., J. Strommer, and J.D. Engel, 1979. Isolation of the Beta-Globin Gene and a Linked Embryonic Beta-Like Globin Gene From a Chicken DNA Recombinant Library. Cell 17:879—887. Dunner, S., C. Charlier, F. Farnir, B. Brouwers, J. Canon, and M. Georges. 1997. Towards Interbreed IBD Fine Mapping of the mh Locus: Double—Muscling in the Asturiana de los Valles Breed Involves the Same Locus as in the Belgian Blue Cattle Breed. Mamm. Genome 8:430-435. Dutrillaux, B., 1986. Le Role des Chromosomes dans L'Evolution; une Nouvelle Interpretation. Ann. Genet. 29:69- 75. Eppig, J.T., and J.H. Nadeau, 1995. Comparative Maps: The Mammalian Jigsaw Puzzle. Curr. Opin. Genet. Dev. 5:709—716. Fillon, V., 1998. The Chicken as a Model to Study Microchromsomes in Birds: A Review. Genet. Sel. Evol., 30:209-219 168 Fillon, V., M. Morisson, R. Zoorob, C. Auffray, M. Douaire, J. Gellin, and A. Vignal, 1998. Identification of 16 Chicken' Microchromosomes by Molecular Markers Using Two-Colour Fluorscence in situ Hybridization. Chromosome Res. 6:307- 313. Flejter, W.L., J. Fergestad, J. Gorski, T. Varvill, and S. Chandrasekharappa, 1998. A gene Involved in XY Sex Reversal is Located on Chromosome 9, Distal to Marker D9Sl779. Am. J. Hum. Genet. 63:794-802. Fridolfsson, A.-K., H. Cheng, N.G. Copeland, N.A. Jenkins, H.—C., Liu, T. Raudsepp, T. Woodage, B. Chowdhary, J. Halverson, and H. Ellegren, 1998. Evolution of the Avian Sex Chromosomes from and Ancestral Pair of Autosomes. Proc. Nat. Acad. Sci. 95:8147—8152. Fronicke, L., B.P. Chowdhary, H. Scherthan, and I. Gustavsson, 1996. A Comparative Map of the Porcine and and Human Genomes Demonstrates ZOO-FISH and Gene Mapping-Based Chromosomal Homologies. Mamm. Genome 7:285. Gale, M.D., and K.M. Devos, 1998. Comparative Genetics in the Grasses. Proc. Natl. Acad. Sci. 95:1971-1974. Goureau, A., M. Yerle, A. Schmitz, J. Riquet, D. Milan, P. Pinton G. Frelat, and J. Gellin, 1996. Human and Porcine Correspondence of Chromosome Segments Using Bidirectional Chromosome Painting. Genomics 36:252-262. Graves, J.A., 1996. Mammals That Break the Rules: Genetics of Marsupials and Monotremes. Annu. Rev. Genet. 30:233-260. Grobet, L., L.J.R. Martin, D. Poncelet, D. Pirottin, B. Brouwers, J. Riquet, A. Schoeberlein, S. Dunner, F. Menissier, J. Massabanda, R. Fries, R. Hanset, and M. Georges, 1997. A Deletion in the Bovine Myostatin Gene Causes the Double-Muscled Phenotype in Cattle. Nature Genetics 17:71—74. Groenen, M.A.M., and R.P.M.A. Crooijmans, Personal Communication. Department of Animal Breeding, Wageningen Institute of Animal Breeding, Wageningen Agricultural University, P.O. Box 338, 6700 AH Wageningen, The Netherlands. Groenen, M.A.M., R.P.M.A. Crooijmans, A. Veenendaal, H.H. Cheng, M. Siwek, and J.J. van der Poel, 1998. A Comprehensive Microsatellite Linkage Map of the Chicken Genome. Genome Res. 7:1162-1168. 169 Groenen, M.A.M., R.P.M.A. Crooijmans, R.J.M. Dijkhof, R. Acar, and J.J. van der Poel, 1999. Extending the Chicken— Human Comparative Map by Placing 15 Genes on the Chicken Linkage Map. Anim. Genet. 30:418—422. Groenen, M.A.M, H.H. Cheng, N. Bumstead, B. Benkel, E. Briles, D.W. Burt, T. Burke, L.B. Crittenden, J. Dodgson, J. Hillel, S. Lamont, F.A. Ponce de Leon, H. Takahashi, and A. Vignal, 2000. A Consensus Linkage Map of the Chicken Genome. Anim. Genet., In Press. Guioli, S., K. Schmitt, R. Critcher, M. Bouzyk, N.K. Spurr, T. Ogata, J.J. Hoo, L. Pinsky, G. Gimelli, L. Pasztor, and P.N. Goodfellow, 1998. Molecular Analysis of 9p Deletions Associated with XY Reversal: Refining the Localization of a sex-Determining Gene to the Tip of the Chromosome. Am. J. Hum. Genet. 63:905-908. Gyapay, G., K. Schmitt, C. Fizames, H. Jones, N. Vega- Czarny, D. Spillett, D. Muselet, J.-.F Dib, C. Auffray, J. Morissette, J. Weissenbach, and P.N. Goodfellow, 1996. A Radiation Hybrid Map of the Huma Genome. Hum. Mol. Genet. 5:339-346. Hardy, D.A., J.I. Bell, E.O. Long, T.Liindsten, and H.O. McDevitt, 1996. Mapping of the Class II Region of the Human Major Histocompatibility Complex by Pulsed Field Gel Electrophoresis. Nature 3323:453. Hayes, H., 1995. Chromosome Painting With Human Chromosome- Specific DNA Libraries Reveals the Extent and Distribution of Conserved Segments in Bovine Chromosomes. Cytogenet. Cell Genet. 71:168. Hood, L., B.P. Koop, L. Rowen, and K. Wang, 1993. Human and Mouse T-Cell-Receptor Loci: The Importance of Comparative Large—Scale DNA Sequence Analyses. Cold Spring Harbor Symp. Quant. Biol. 58:339-348. Hudson, T.J., L.D. Stein, S.S. Gerety, J. Ma, A.B. Castle, J. Silva, D.K. Slonim, R. Baptista, L. Kruglyak, S.H. Xu, A.M.E. Colbert, C. Rosenberg, M.P. Reeve-Daly, S. Rozen, L. Hui, X. Wu, C. Vestergaard, K.M. Wilson, J.S. Bae, S. Maitra, S. Ganiatsas, C.A. Evans, M.M. DeAngelis, K.A. Ingalls, R.W. Nahf, L.T.Jr. Horton, M.O. Anderson, A.J. Collymore, W. Ye, V. Kouyoumjian, I.S. Zemsteva, J. Tam, R. Devine, D.F. Courtney, M.T. Renaud, H. Nguyen, T.J. O’Connor, C. Fizames, S. Faure, G. Gyapay, C. Dib, J. Morissette, J.B. Orlin, B.W. Birren, N. Goodman, J. Weissenbach, T.L. Hawkins, S. Foote, D.C. Page, and E.S. Lander, 1995. An STS-based Map of the Human Genome. Science 270:1945-1954. 170 Hutchison, N., 1987. Lampbrush Chromosomes of the Chicken, Gallus domesticus. J. Cell Biol. 105:1493—1500. Januzzi, J.L., N. Arzolan, A. O’Connell, K. Aalto-Setala, and J.L. Breslow, 1992. Characterization of the Mouse Apolipoprotein Apoa—l/Apoc-3 Gene Locus: Genomic, mRNA, and Protein Sequence Comparisons to Other Species. Genomics 14:1081-1088. Kaback, D.B., 1996. Chromosome-Size Dependent Control of Meiotic Recombination in Humans. Nat. Genet. 13:20-21. Kaebling, M. and N.S. Fechheimer, 1983a. Synaptonemal Complexes and the Chromosome Complement of Domestic Fowl, Gallus Domesticus. Cytogenet. Cell Genet., 35:87-92. Kaebling M., and F.S. Fechheimer, 1983b. Synaptonemal Analysis of Chromosome Rearrangements in Domestic Fowl, Gallus Domesticus. Cytogenet. Cell Genet. 36:567—572. Keller, B.B., and W.A. Noon, 1984. Intron Splicing: a Conserved Internal Signal in Introns of Animals Pre-mRNAs. Proc. Natl. Acad. Sci. 81:7417—7420. Klein, S., D.R. Morrice, H. Sang, L.B. Crittenden, and D.W. Burt, 1996. Genetic and Physical Mapping of the Chicken IGFl Gene to Chromosome 1 and Conservation of Synteny with Other Vertebrate Genomes. J. Hered. 87:10-14. Knorr, C., H.H. Cheng, and J.B. Dodgson, 1999. Application of AFLP Markers to Genome Mapping in Poultry. Anim. Genet. 30:28-35. Koch, J.B., S. Kolvraa, K.B. Peterson, N. Gregersen, and L. Bolund, 1989. Oligonucleotide-Priming Methods for the Chromosome—Specific labelling of Alpha Satellite DNA in situ. Chromosoma 98:259-265. Koop, B.F., R.K. Wilson, K. Wang, B. Vernooij, D. Zallwer, C.L. Kuo, D. Seto, M. Toda, and L. Hood, 1992. Organization, Structure, and Function of 95Kb of DNA Spanning the Murine T-Cell-Receptor C ALpha/C Delta Region. Genomics 13:1209- 1230. Koop, B.F., L. Rowen, K. Wang, C.L. Kuo, D. Seto, J.A. Lenstra, S. Howard, W. Shan, P. Deshpande, and L. Hood, 1994. The Human T-cell Receptor TCRAC/TCRDC (C a/C 5) Region: Organization, Sequence, and Evolution of 97.6Kb of DNA. Genomics 19:478-493. Krishan, A., 1964. Microchromosomes in the Spermatogenesis of the Domestic Turkey. Exp. Cell Res. 33:1-7. 171 Kumar, S., and S.B. Hedges, 1998. A Molecular Timescale for Vertebrate Evolution. Nature 392:917-920. ' Kunz, J., S.W. Scherer, I. Klawitz, S. Soder, Y.Z. Du, N. Speich, M. Kaiff-Suske, H.H. Heng, L.C. Tsui, and K.H. Grzeschik, 1994. Regional Localization of 725 Human Chromosome 7—Specific Yeast Artificial Chromosome Clones. Genomics 22:439—448. Lamerdin, J.E., M.A. Montgomery, S.A. Stilwagon, L.K. Scheodecker, R.S. Tebbs, K.W. Brookman, L.H. Thompsom, and A.V. Carrano, 1995. Genomic Sequence Comparison of the Human and Mouse XRCCl DNA Repair Gene Regions. Genomics, 25:547- 554 Lamerdin, J.E., S.A. Stilwagon, M.H. Ramirez, L. Stubbs, and A.V. Carrano, 1996. Sequence Analysis of the ERCC2 Gene Regions in Human, Mouse, and Hamster Reveals Three Linked Genes. Genomics 34:399-409. Larson, P., G. Gunderson, R. Lopez, and H. Prydz, 1992. CpG Islands as Gene Markers in the Human Genome. Genomics 13:1095. Lauer, P., N.C. Meyer, C.E. Prass, S.M. Starnes, R.K. Wolff, and A. Gnirke, 1997. Clone—Contig and STS Maps of the Hereditary Hemochromatosis Region on Human Chromosome 6p21.3—p22. Genome Res. 7:457-470. Lopez, J.V., S. Cevario, and S.J. O'Brien, 1996. Complete Nucletide Sequences of the Domestic Cat (Felis catus) Mitochondrial Genome and a Transposed mtDNA Tandem Repeat (Numt) in the Nuclear Genome. Genomics 33:229-246. Lyons, L.A., M. Menotti-Raymond, and S.J. O’Brien, 1994. Comparative Genomics: The Next Generation. Anim. Biotechnol. 5:103-111. Lyons, L.A., T.F. Laughlin, N.G. Copeland, N.A. Jenkins, J.E. Womack, and S.J. O’Brien, 1997. Comparative Anchor Tagged Sequences (CATS) for Integrative Mapping of Mammalina Genomes. Nat. Genet. 15:47—56. Manly, K.F., 1993. A Macintosh Program for Storage and Analysis of Experimental Genetic Mapping Data. Mamm. Genome 4:303-313. Mariman, E.C., P.T. Sillekens, R.J. vanBeekReinders, and W.J. vanVenrooij, 1984. A Model for the Excision of Introns 1 and 2 from Adenoviral Major Late Pre-Messenger RNAs. J. Mol. Biol. 178:47-62. 172 Marklund, L., M. Moller-Johansson, B. Hoyheim, W. Davies, M. Fredholm, R.K. Juneja, P. Mariani, W. Coppieters, H. ' Ellegren, and L. Andersonn, 1996. A Comprehensive Linkage Map of the Pig Based on a Wild Pig-Large White Intercross. Anim. Genet. 27:255—269. Marra, M., T. Kucaba, M. sekhon, L. Hillier, R. Martienssen, A. Chinwalla, J. Crockett, J. Fedele, H. Grover, C. Gund, W.R. McCombie, K. McDonald, J. McPherson, N. Mudd, L. Parnell, J. Schein, R. Seim, P. Shelby, R. Waterson, and R. Wilson, 1999. Nat. Genet. 22:265-270. Marin, I., and E.S. Baker, 1998. The Evolutionary Dynamics of Sex Determination. Science 281:1990-1994. Martin, G.B., S.H. Brommonschenkel, J. Chunwongse, A. Frary, M.W. Ganal, R. Spivey, T. Wu, E.D. Earle, and S.D. Tanksley, 1993. Map—Based Cloning of Protein Kinase Gene Conferring Disease Resistance in Tomato. Science 262:1432-1436. Matzke, A.J.M, F. Varga, P. Gruendler, I. Unfried, H. Berger, B. Mayr, and M.A. Matzke, 1992. Characterization pf a New Repetitive Sequence That is Enriched on Microchromosomes of Turkey. Chromosoma 102:9-14. Maxson, L.R., and A.C. Wilson, 1979. Rates of Molecular and Chromosomal in Salamanders. Evolution 33:734-740. McCarthy, L.C., 1996. Whole Genome Radiation Hybrid Mapping. Trends Genet. 12:491-493. Mccarthy, L.C., J. Terrett, M.E. Davis, C.J. Knights, A.L. Smith, R. Critcher, K. Schmitt, J. Hudson, N.K. Spurr, and P.N. Goodfellow, 1997. A First-Generation Whole-Genome Radiation Hybrid Map Spanning the Mouse Genome. Genome Res. 7:1153—1161. McDermid, H.E., K.E. McTaggart, M.A. Riazi, T.J. Hudson, M.L. Budarf, E.S. Emanuel, and C.J. Bell, 1996. Long-Range Mapping and Construction of a YAC Contig Within the Cat Eye Syndrome Critical Region. Genome Res. 6:1149—1159. McQueen, H.A., J. Fantes, S.H. Cross, V.H. Clark, A.L. Archibald, and A.L. Bird, 1996. CpG Islands of Chickens are Concentrated on Microchromosomes. Nat. Genet. 12:321—324. McQueen, H.A., G. Siriaco, and A.P. Bird, 1998. Chicken Microchromosomes are Hyperacetylated, Early Replicating, and Gene Rich. Genome Res. 8:621-630. 173 Moir, D.T., T.E. Dorman, J.C. Day, N.S. Ma, M.T. Wang, and J.I. Mao, 1994. Toward a Physical Map of Human Chromosome 10: Isolation of 183 YACs Representing 80 Loci and Regional Assignment of 94 YACs by Fluorescence in situ Hybridiztion. Genomics 22:1-22. M020, T., K. Dewar, P. Dunn, J.R. Ecker, S. Fischer, S. Kloska, H. Lehrach, M. Marra, R. Martienssen, S. Meier- Ewert, and T. Altmann, 1999. A Complete BAC-Based Physical Map of the Aribidopsis thaliana genome. Nat. Genet. 22:271— 275. Murphy, W.J., M. Menotti-Raymond, L.A. Lyons, M.A. Thompson, and S.J. O'Brien, 1999. Development of a Feline Whole Genome Radiation Hybrid Panel and Comparative Mapping of Human Chromosome 12 and 22 Loci. Genomics 57:1. Myakoshina, Y.A., and A.V. Rodionov, 1994. Meiotic Lampbrush Chromosomes in Turkey Meleagris gallopavo (Galliformes:Meleagridea). Genetika 30:649-656. Ezra: Nadeau, J.H., and B.A. Taylor, 1984. Lengths of Chromsomal Segments Conserved Since Divergence of Man and Mouse. Proc. Nat. Acad. Sci. 81:814-818. Nadeau J.H., and D. Sankoff, 1998. Counting on Comparative Maps. Trends Genet. 14:495-501. Nagaraja, R., S. MacMillan, J. Kere, C. Jones, S. Griffin, M. Schmatz, J. Terrell, M. Shomaker, C. Jermak, C. Hott, M. Masisi, S. Mumm, A. Srivastava, G. Pilia, T. Featherstone, R. Mazzarella, S. Kesterson, B. Mccauley, B. Railey, F. Burough, V. Nowotny, M. D’Urso, D. States, B. Brownstein, and D. Schlessinger, 1997. X Chromosome Map at 75-kb STS Resolution, Revealing Extremes of Recombination and GC Content. Genome Res. 7:210-222. Nagata, T., E.H. Weiss, K. Abe, K. Kitagawa, A. Ando, Y. yara-Kikuti, M.P. Seldin, K. Ozato, H. Inoko, and M. Taketo, 1995. Physical Mapping of the Retinoid X Receptor in Mouse and Human. Immunogenetics 41:83-90. Nanda, I., Z. Shan, M. Schart, D.W. Burt, M. Koehler, H. Nothwang, F. Grutzner, I.R. Paton, D. Windsor, I. Dunn, W. Grutzner, P. Staeheli, S. Mizuno, T. Haaf, and M. Schmid, 1999. 300 Million Years of Conserved Synteny, Between Chicken Z and Human Chromosome 9. Nat. Genet. 21:258-259. Neff, M.W., K.W. Broman, C.S. Mellersh, K. Ray, G.M. Acland, G.D. Aguirre, J.S. Ziegle, E.A. Ostrander, and J. Rine, 1999. A Second-Generation Genetic Linkage Map of the Domestic Dog, Canis familiaris. Genetics 151:803. Newcomer, E.H., 1957. The Mitotic Chromsomes of the Domestic Fowl. J. Hered. 48:227—234. PM Oakley, R.J., M.L. Watson, and M.F. Seldin, 1992. Construction of a Physical Map on Mouse and Human Chromosome' 1: Comparison of 13Mb of Mouse and lle of Human DNA. Hum. Mol. Genet., 1:613-620. O’Brien, S.J., and W.G. Nash, 1982. Genetic Mapping in Mammals: Chromosome Map of the Domestic Cat. Science 216:257-265. O’Brien, S.J., 1993. Comparative Biology: The Genomics Generation. Curr. Biol. 3:395-397. O’Brien, S.J., J. Wienberg, and L.A. Lyons, 1997. Comparative Genomics: Lessons From Cats. Trends Genet. 13:393-399. 3 O’Brien, S.J., M. Menotti-Raymond, W.J. Murphy, W.G. Nash, J. Wienberg, R. Stanyon, N.G. Copeland, N.A. Jenkins, J.E. Womack, and J.A.M. Graves, 1999. The Promise of Comparative Genomics in Mammals. Science 286:458—464. t_‘-' "77.1) I7 Li. L Oeltjen, J.C., T.M. Malley, D.M. Muzney, W. Miller, R.A. Gibbs, and J.W. Belmont, 1997. Large—Scale Sequence Analysis of the Human and Murine Bruton’s Tyrosine Kinase Loci Reveals Conserved Regulatory Domains. Genome Res. 7:315-329. Ohno, S., 1966. Sex Chromosomes and Sex-Linked Genes. Springer-Verlag Berlin, Heidelberg, and New York. Okimoto, R., and J.B. Dodgson, 1996. Improved PCR Amplification of Multiple Specific Alleles (PAMSA) Using Internally Mismatched Primers. Biotechniques 21:20-22,24,26. Okimoto, R., H.H. Cheng, and J.B. Dodgson, 1997. Characteriztion of CR1 Repeat Random PCR Markers for Mapping the Chicken Genome. Anim. Genet. 28:139-145. Paterson, A.H., T.—H., Lan K.P. Resichmann, C. Chang, Y.-R., Lin, M.D. Burow, S.P. Kowalski, C.S. Katsar, T.A. DelMonte, K.A. Feldman, K.F. Schertz, and J.F. Wendel, 1996. Toward a Unified Genetic Map of Higher Plants, Transcending the Monocot-Dicot Divergence. Nat. Genet. 14:380—382. Pirottin, D., D. Poncelet, L. Grobet, L.J. Royo, B. Brouwers, J. Masabanda, H. Takeda, R. Fries, Y. Sugimoto, J.E. Womack, S. Dunner, amd M. Georges, 1999. High— Resolution, Human-Bovine Comparative Mapping Based on a Closed YAC Contig Spanning the Bovine MH Locus. Mamm. Genome 10:289-293. Pollock, D.L., and N.S. Fechheimer, 1981. Variable C-Banding Patterns and a Proposed C-Band Karyotype in Gallus domesticus. Genetica 54:273-279. 175 Priat C., C. Hitte, F. Vignaux, C. Renier, Z. Jiang, S. Jouquand, A. Cheron, C. Andre, and F. Galibert, 1998. A Whole-Genome Radiation Hybrid Map of the Dog Genome. Genomics 54:361. Primmer, C.R., T. Raudsepp, B.P. Chowdhary, A.P. Moller, and H. Ellegren, 1997. Low Frequency of Microsatellite in the Avain Genome. Genome Res. 7:471—482. Purchase, H.G., 1985. Clinical Disease and its Economic Impact. In Marek's Disease, Scientific Basis and Methods of Control (ed. L.N. Payne) pp. 17-42. Martinus Nkjhoff Publishing, Boston. Rahn, M.I., and A.J. Solari, 1986. Recombination Nodules in the Oocytes of the Chicken, Gallus domesticus. Cytogenet. Cell Genet. 43:187-193. Raymond, C.S., C.E. Shamu, M.M. Shen, K.J. Seifert, B. Hirsch, J. Hodgkin, and D. Zarkower, 1998. Evidence for Evolutionary Conservation of Sex-Determing Genes. Nature 391:691-695. Raymond, C.S., E.D. Parker, J.R. Kettlewell, L.G. Brown, D.C. Page, K. Kusz, J. Jaruzelska, Y. Reinberg, W.L. Flejter, V.J. Bardwell, B. Hirsch, and D. Zarkower, 1998. A Region of Human Chromosome 9q Required for Testis Development Contains Two Genes Related to Known Sexual Regulators. Hum. Mol. Genet. 8:989-996. Reed, J.A., and K.C. Graves, 1993. Chapter 10. In Sex Chromosomes and Sex—Determining Genes. Harwood Academic Publishers, Switzerland. Renwick, J.H., 1971. 4th International Congress of Human Genetics. Paris, France. Renucci, A., V. Zappavigna, J. Zakany, J.C. Izpisua- Belmonte, K. Burki, and D. Duboule, 1992. Comparison of the Mouse and Human HOX—4 Complexes Defines Conserved Sequences Involved in the Regulation of HOX-4.4. EMBO J. 11:1459-1468. Rettenberger, G., C. Klett, U. Zechner, J. Kunz, W. Vogel, and H. Hameister, 1995. Visualization of the Conservation of Synteny Between Humans and Pigs by Heterologous Chromosomal Painting. Genomics 26:372-378. Rodionov, A.V., 1985. Genetic Activity of DNA from G and R Blocks of of Human Mitotic Chromosomes. Genetika 21:2057— 2065. PM Rodionov, A.V., L.A. Chelysheva, E.V. Kropotova, and E.R. Gaginskaya, 1989. Heterochromatic Chromosome Regions of Chickens and Japanese Quail in Mitosis and at the Lampbrush Stage. Tsitologia 31:867-873. Rodionov, A.V., Y.U. Myakoshina, L.A. Chelysheva, and E.P. Gaginskaya, 1992a. Chiasmata on Lampbrush Chromosomes of Gallus gallus domesticus. Cytogenetic Investigations of Recombination Frequency and Linkage Group Length. Genetika 28:53-63. Rodionov A.V., L.A. Chelysheva, I.V. Solovei, and Y.U. Myakoshina, 1992b. Chiasma Distribution in the Lampbrush Chromosomes of the Chicken Gallus gallus domesticus: Hot Spots of Recombination and Their Possible Role in the Proper Dysjunction of Homologous Chromosomes at the First Meiotic Division. Genetika 28:151-160. Rodionov, A.V., 1996. Micro versus Macro: A review of Structure and Functions of Avian Micro and Macrochromosomes. Russian J. Genet. 5:517-527. Rodionov, A.V., 1997. Evolution of Avian Chromosomes and Linkage Groups. Russian J. Genet. 6:605-617. Rohrer, G.A., L.J. Alexander, Z. Hu, T.P. Smith, J.W. Keele, and C.W. Beattie, 1996. A Comprehensive Map of the Porcine Genome. Genome Res. 6:371-391. Sazanov, A., Department of Animal Breeding, Technical University of Munich, Alte Akademie 12, 85350 Freising- Weihenstephan. Sazanov, A., L.A. Alekseevich, A.L. Sazanova, and A.F. Smirnov, 1996. Mapping the Chicken Genome: Problems and Perspectives. Genetika 32:869-878. Schmid, W., 1962. Replication Patterns of the Heterochromosomes in Gallus Domesticus. Cytogenetics 1:344- 352. Schmid, M., and M. Guttenbach, 1988. Evolutionary Diversity of Reverse (R) Fluorescent Chromosome Bands in Vertebrates. Chromosoma 97:101-114. 177 Schuler, G.D., M.S. Boguski, E.A. Stewart, L.D. Stein, G. Gyapay, K. Rice, R.E. White, P. Rodriguez, A. Aggarwal, E. Bajorek, S. Bentolila, B.W. Birren, A. Butler, A.B. Castle, N. Chiannikulchai, A. Chu, C. Clee, S. Cowles, P.J.R. Day, T. Dibling, N. Drouot, I. Dunham, S. Duprat, C. East, C. Edwards, J.B. Fan, N. Fang, C. Fizames, C. Garrett, L. Green, D. Hadley, M. Harris, P. Harrison, S, Brady, A. Hicks, E. Holloway, I. Hui, S. Hussein, C. Louis—Dit-Sully, J. Ma, A. MacGilvery, C. Mader, A. Maratukulam, T.C. Matise, K.B. McKusick, J. Morissette, A. Mungall, D. Muselet, H.C. Nusbaum, D.C. Page, A. Peck, S. Perkins, M. Piercy, F. Qin, J. Quackenbush, S. Ranby, T. Reif, S. Rozen, C. Sanders, X. She, J. Silva, D.K. Sloinim, C. Soderlund, W.L., Sun, P. Taber, T. Thangarajah, N. Vega-Czarny, D. Vollrath, S. Voyticky, T. Wilmer, X. Wu, M.D. Adams, C. Auffray, N.A.R. Walter, R. Brandon, A. Dehjia, P.N. Goodfellow, R. Houlgatte, J.R. Hudson Jr., S.E. Ide, K.R. Iorio, W.Y. Lee, N. Seki, T. Nagase, K. Schmitt, R. Berry, K. Swanson, R. Torres, J.C. Venter, J.M. Sikela, J.S. Beckmann, P. Deloukas, E.S. Lander, and T.J. Hudson, 1996. A Gene Map of the Human Genome, Science 274:540-546. Sinclair, A.H., J.W. Foster, J.A. Spencer, D.C. Page, M. Palmer, P.N. Goodfellow, and J.A.M. Graves, 1990. Sequences Homologous to ZFY, a Candidate Human Sex-Determining Gene, are Autosomal in Marsupials. Nature 336:780—783. Slizynski, B.M., 1964. Cytological Observations on a Duck Hybrid: Anas clypeata X Anas penelope. Genet. Res. Camb. 5:441-447. Smith, T.P., G.A. Rohrer, L.J. Alexander, D.L. Troyer, K.R. Kirby-Dobbels, M.A. Janzen, D.L. Cornwell, C.F. Louis, L.B. Schook, and C.W. Beattie, 1995. Directed Integration of the Physical and Genetic Linkage Maps of Swine Chromsome 7 Reveals that the SLA Spans the Centromere. Genome Res. 5:259-271. Smith, B.J., L.A. Lyons, H.H. Cheng, and S.P. Suchyta, 1997. Comparative Mapping of the Chicken Genome Using the East Lansing Reference Population. Poultry Sci. 76:743—747. Smith, C.A., P.J. McClive, P.S. Western, K.J. Reed, and A.H. Sinclair, 1999. Conservation of a Sex-Determining Gene. Nature 402:601-602. Soeda, B., D.X. Hou, K. Osoegawa, Y. Atsuchi, T. Yamagata, T. Shimokawa, H. Kishida, S. Okano, and I. Chumakov, 1995. Cosmind Assembly and Anchoring to Human Chromosome 21. Genomics 25:73—84. Solinas—Toldo, S., C. Lengauer, and R. Fries, 1995. Comparative Genome Map of Human and Cattle. Genomics 27:486- 496. 178 Song, W.Y., G.L. Wang, L.L. Chen, H.S. Kim, Y.P. Pi, T. Holsen, J. Gordnee, B. Wang, W.X. Zhai, L.H. Zhu, C. Faouquet, and P. Ronald, 1995. A Receptor Kinas-Like Protein Encoded by the Rice Disease Resistance Gene, Xa21. Science 270:1804-1806. Sonstegard, T.S., N.L. Lopez—Corrales, S.M. Kappes, C.W. Beattie, and T.P. Smith, 1997. Comparative Mapping of the Bovine and Human Chromosome 2 Identifies Segments of Conserved Synteny and Increases Informative Marker Density Near the Bovine mh Locus. Mamm. Genome 8:751-755. Sonstegard, T.S., S.M. Kappes, J.W. Keele, and T.P.L. Smith, 1998. Refinement of the Bovine Chromosome 2 Linkage Map Near the mh Locus Reveals Rearrangements Between the Bovine and Human Genomes. Anim. Genet. 29:341-347. Southern, E.M.. 1975. Detection of Specific Sequences Among DNA Fragments Seperated by Gel Electrophoresis. J. Mol. Biol. 98:503. Stallings, R.L., N.A. Doggett, D. Callen, S. Apostolou, L.Z. Chen, J.K. Nancarrow, S.A. Whitmore, P. Harris, H. Michison, and M. Breuning, 1992. Evaluation of a Cosmid Contig Physical Map of Human Chromosome 16. Genomics 13:1031-1039. Stefos, A.D., and F.E. Arrighi, 1974. Repetitive DNA of Gallus domesticus and Its Cytological Localization. Exp. Cell. Res. 83:9-14. Stewart, E.A., K.B. McKusick, A. Aggarwal, E. Bajorek, S. Brady, A. Chu, N. Fang, D. Hadley, M. Harris, S. Hussain, R. Lee, A. Maratukulam, K. O’Connor, S. Perkins, M. Piercy, F. Qin, T. Reif, C. Sanders, X. She, W.L. Sun, P. Tabar, S. Voticky, S. Cowles, J.B. Fan, D.R. Cox, et al., 1997. An STS-Based Radiation Hybrid Map of the Human Genome. Genome Res. 7:422-433. Stock, A.D., and G.A. Mengden, 1975. Chromosome Banding Pattern Conservatism in Birds and Nonhomology of Chromosome Banding Patterns Between Birds, Turtles, Snakes, and Amphibians. Chromosoma 50:69-77. Takagi, N. and M. Sasaki, 1974. A Phylogenetic Study of Bird Karyotypes. Chromosoma 46:91-120. Tao, Q., Y.—L. Chang, J. Wang, H. Chen, M.N. Islam-Faridi, C. Scheuring, B. Wang, D.M. Stelly, and H.-B. Zhang, 1999. A Large-Scale Sequence-Ready Physical Map of the Rice Genome. Proceedings of the Plant and Animal Genome IV Conference, p. 101 (abstract). 179 Tegelstrom, H., and H. Ryttman, 1981. Chromosomes in Birds (Aves): Evolutionary Implications of Macro- and Microchromosome Numbers and Lengths. Hereditas 94:225—233. Thorne, M. H., and B.L. Sheldon, 1992. Triploid Intersex and Chimeric Chickens: Usefel Models for Studies of Avian Sex Determination. Chapter 15. in Sex Chromosomes and Sex- Determining Genes. Harwood Academic Publishers, Switzerland. Totaro, A., J.M. Rommens, A. Grifa, C. Lunardi, M. Carella, J.J. Huizenga, A. Roetto, C. Camaschella, G. DeSandre, and P. Gasparini, 1996. Hereditary Hemochromatosis: Generation of a Transcription Map within a Refined and Extended Map of the HLA Class I Region. Genomics 31:319-326. Turner, B.M., 1993. Decoding the Nucleosome. Cell 75:5-8. Vallejo, R.L., H. Liu, R.L. Witter, M.A.M. Groenen, J. Hillel, and H.H. Cheng, 1998. Genetic Mapping of Quantitative Trait Loci to Marek’s Disease Virus Induced Tumors in.F} Intercross Chickens. Genetics 148:349—360. Van Etten, W.J, R.G. Steen, H. Nguyen, A.B. Castle, D.K. Slonim, B. Ge, C. Nusbaum, G.D. Schuler, E.S. Lander, and T.J. Hudson, 1999. Radiation Hybrid Map of the Mouse Genome. Nat. Genet. 22:384-387. Van Houten, W., N. Kurata, Y. Umehara, T. Sasaki, and Y. Minobe, 1996. Generation of a YAC Comtig Encompassing the Extra Glume Gene, eg, in Rice. Genomics 39:1072—1076. Venta, P.J., J.A. Brouillette, V. Yuzbasiyan-Gurkan, and G.J. Brewer, 1996. Gene-Specific Universal Mammalian Sequence—Tagged Sites: Application to the Canine Genome. Biochem. Genet. 34:321-341. Wachtel, 8.8., 1987. Evolutionary Mechanisms in Sex Determination. CRC Press Inc., Florida. Wade, P.A., D. Pruss, and A.P. Wolfe, 1997. Histone Acetylation: Chromatin in Action. Trends Biochem. Sci. 4:128-132. Watkins—Chow, D.E., M.S. Buckwalter, M.M. Newhouse, A.C. Lossie, M.L. Brinkmeier, and S.A. Camper, 1997. Genetic Mapping of 21 Genes on Mouse Chromosome 11 Reveals Disruptions in Linkage Conseervation with Human Chromosome 5. Genomics 40:114-122. Watson, J.M., J.A. Spencer, A.D. Riggs, and J.A. Marshall Graves, 1991. Sex Chromosome Evolution: Platypus Gene Mapping Suggests that Part of the Human X Chromosome was Originally Autosomal. Proc. Nat. Acad. Sci., 88:11256—11260. 180 Wienberg, J., and R. Stanyon, 1995. Curr. Opin. Genet. Dev. 5:792-797. Weiss, E.H., L. Golden, K. Fahrner, A.L. Mellor, J.J. Devlin, H, Bullman, H. Tiddens, H. Bid, and R.A. Flavell, 1984. Organization and Evolution of the Class I Gene Family in the Major Histocompatibility Complex of the C57BL/10 Mouse. Nature 310:650-655. Yonash, N., L.D. Bacon, R.L. Witter, and H.H. Cheng, 1999. High Resolution Mapping and Identification of New Quantitative Trait Loci (QTL) Affecting Susceptibility to Marek’s Disease. Anim. Genet. 30:126-135. Yoo, J., R.T. Stone, S.M. Kappes, and C.W. Beattie, 1994. Linkage Analysis of Bovine Interleukin Receptor Types I and II (IL-1R I, II). Mamm. Genome 5:820-821. Yoshida, K., M.P. Strathman, C.A. Mayeda, C.H. Martin, and M.J. Palazzolo, 1993. A Simple and Efficient Method for Constructing High Resolution Physical Maps. Nucleic Acids Res. 21:3553-3562. Yoshimura, S., Y. Umehara, N. Kurata, Y. Nagamura, T. Sasaki, Y. Minobe, and N. Iwata, 1996. Identification of a YAC Clone Carrying the Xa—l Allele, a Bacterial Blight Resistance Gene in Rice. Theor. Appl. Genet. 93:117-122. Yuhki, N., and S.J. O’Brien, 1988. Molecular Characterization and Genetic Mapping of Class I and II MHC Genes of the Domestic Cat. Imunnogenetics 27:414—425. Zhang, H.—B., and Q. Tao, 1997. A Simple, Economic and Universal Kit for Rapidly Fingerprinting Cloned DNA. Invention No.: TAMUS#1228 (International Patent in Pending). 181 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII (HI(HIWWI[llzllljlillflllflilllflllllll