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IGAN SYATE U {Lug/HI(NIHllllilllllifllllliil 93 01421 6588 This is to certify that the dissertation entitled MOLECULAR GENETIC LINKAGE MAP IN ASPARAGUS presented by Chunxiao Jiang has been accepted towards fulfillment of the requirements for Ph.D. Horticulture degree in \ 7 . 1/' M a jor professor Date #{q/?é MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 LEBR'ARY Michigan $tate * University PLACE ll RETURN 30X to romavothb chockwtflom your "cord. TO AVOID FINES Mum on or baton duo duo. DATE DUE DATE DUE DATE DUE Molecular Genetic Linkage Map in Asparagus By Chunxiao Jiang A DISSERTATION Submitted to Michigan State University in partial fulfilhnent of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture Plant Breeding and Genetics 1 996 ABSTRACI' Molecular Genetic Linkage map in Asparagus By Chunxiao Jiang Two linkage maps of asparagus (Asparagus officinalis L.) were constructed using a double pseudotestcross mapping strategy with restriction fragment length polymorphisms (RFLPs) , random amplified polymorphic DNAs (RAPDs) , and allozyme markers in a population generated from crossing two heterozygous parents. TWO sets of data were formed according to the inheritance patterns of the markers and analyzed as a backcross population. The maternal MW25 map has a total of 163 markers placed in 13 linkage groups covering 1280.9 cM with an average and maximum distance between adjacent markers of 7.9 and 28.5 cM, respectively. The paternal A19 map has 183 markers covering 1324 .1 cM in 9 linkage groups with an average and maximum distance between two adjacent markers of 7.7 and 29.3 cM, respectively. Six multiallelic RFLP markers (markers segregating in the pattern a/c x b/c) and eight markers segregating in the pattern a/b x a/b (HZ loci) , including four RAPD and four RFLP polymorphisms, were comnon to both maps and were used as locus bridges to align homologous groups between the two maps. In this case, RFLP markers were more efficient than RAPD markers . Nine linkage groups in the MW25 map were identified as being homologous to six groups in the A19 map. In two cases, two or more bridge loci were in corrmon, thus, the orientation was also determined; in the other 4 cases, only one locus was common in the two groups and the orientation is unknown. MDH, four RFLP and 13 RAPD markers were assigned to chromosome L5 that has the sex locus M. Bulk segregant analysis (BSA), RAPD and sequence characterized amplified region (SCAR) techniques were used to map the sex determinate locus M. A total of 760 arbitrary decanter oligonucleotide primers were used for RAPD analysis, of which one, produced two RAPD band linked with the M locus . RAPD markers OC15—98 and OC15-30, amplified by the same primer, were both found closely linked to sex locus M at a distance of 1.6 cM. RAPD fragment OC15-98 was cloned and the sequence used to design 24 —mer oligonucleotide primers. This pair of SCAR primers amplified a single DNA band that was the same size as the RAPD fragment cloned. The SCAR marker remained dominant as the RAPD marker OC15-98 from which it was derived . DEDICATION To my wife Jie Zhang and daughter Anna Jiang iv ACKNOWLEDGMENTS My sincere thanks to Dr. K.C. Sink for his support and help. Thanks are also due to my committee members Drs. A.Iezzoni, J.Kelly and D.Douches for their valuable discussions and support. Also my thanks to Mark Lewis, Roger May, Sergei Krasnyanski, Terry Ball for their help and.assistance. Special thanks also go to Professor Dewei Zhu for giving me this opportunity to study at MSU. Finally, but not limited, my thanks to my wife Jie Zhang and daughter Anna Jiang for their patients and support. TABLES OF CONTENTS Page LIST OF TABLES ........................................ ix LIST OF FIGURES ....................................... X SECTION 1. Literature Review .............................. 1 1. General introduction ................................... 2 1.1 Original Background ................................ 2 1.2 Growth Condit1ons .................................. 3 1. 3 Botanical Characteristics ......................... 5 1. 4.Asparagus Breeding Goals ........................... 7 1.5 Breeding Methodology ............................... 9 2. Genetic Linkage Mapping ............................... 12 2.1 Historical Background ............................. 12 2.2 Isozyme markers ................................... 15 2.3 RFLP markers ...................................... 16 2.4 RAPD markers ...................................... 21 2.5 Advantages of Mblecular Markers ................... 26 3. Constructing a Linkage Map with DNA.Markers ........... 27 3.1 Choice of Mapping Population ...................... 28 3. 2 Population Size ................................... 33 3. 3 Likage.Analysis ................................... 34 4. Genetic Mapping of Simply Inherited Trait ............. 37 5. Genetic Mapping of Quant1tative Trait Loci (QTLs) ..... 43 Literature Cited ...................................... 46 SECTION'Z.(llfliflflflDIUUXDPWE)FTTE’MOLECULAR.LINKAGE MAP OF ASPARAGUS ......................................... 60 Abstract ................................................. 61 Introduction ............................................. 63 Material and Methods ..................................... 66 Plant Material ......................................... 66 Isozyme.Analysis ....................................... 66 DNA Isolation .......................................... 67 RFLP Assay ............................................. 68 RAPD assay ............................................. 68 Primer Pre-screening ................................... 68 Loci NOmenclature ...................................... 69 S egation and Linkage Analysis ....................... 69 Resu ts and.Discussion ................................... 72 Literature Cited ......................................... 86 vi SECTION 3. RAPD AND SCAR MARKERS LINKED TO THE SEX DETERMINATION LOCUS M IN ASPARAGUS ....................... 90 Abstract ................................................. 91 Introduction ............................................. 92 Material and.Methods ..................................... 94 Plant Material ......................................... 94 DNA Isolation ......................................... 95 PCR .Analysis .......................................... 96 Sequence Characterized.Amplified Region (SCAR) ......... 97 Linkage Analysis ....................................... 98 Results and Discussion ................................... 98 Literature Cited ........................................ 114 vii LIST OF TABLE Page Table 1.Sequence of 24-mer oligonucleotide primers for SCAR locus derived from.RAPD marker OC15-98 linked to maleness of asparagus ........................ 100 viii LIST OF FIGURES SECTION 2 Fig. 2. 1 Primer pre— screening results. "BC" type RAPD polymorphism produced by primer OP13 was inherited from paternal parent A19 (A); primer 219 was inherited from maternal parent MW25 (B); and "HZ" type lyrrorphism inherited from both parents (C). From eft to right: 123 bp DNA ladder, MW25, A19, and 6 individuals in the mapping population. Polymorphic bands are indicated by arrows ............ Fig. 2. 2 Segregation pattern of RAPD markers in the mapping population. In both panels, the first lane is the 123 bp DNA ladder; the second and third are MW25 and A19, respectively. The other lanes are individuals in the ma ping population. RAPD assay with primer P13 P) and primer 0219 (B) . Segregat 1ng RAPD markers are indicated by arrows ................. Fig. 2. 3 Genetic linkage maps of asparagus parents A19 and MW25. Solid black bars and open bars indicate linkage groups in A19 and MW25 maps, respectively. Markers with "O" are RAPD markers while "R" indicates RFLP markers. The loci heterozygous in both parents that serve as locus bridges end with "HZ". Markers with " -" were normall coded data, while those with " " are inverse y coded data. Vertical hatched bars to each linkage group indicate uncertain locations of the RAPD HZ loci. Markers enclosed in parentheses have been located to corresponding intervals with LCD < 3.0. The numbers at the end of the names are the 1/10 of the size of the DNA fragments in base pairs .............. SECTION 3 én3 1 RAPD markers OC15- 30 and OC15- 98 linked to ess of asparagus. From left to right, lane 1 is 123- -bp DNA molecular weight ladder, lanes 2 and 3 are parent MW25 (female) and A19 (male), respectively. Lanes 4- 13 are female (F) and male (M) individuals of the map ing population. Segregating RAPD markers are in icated by arrow ...... Page 78 80 83 . . .105 Fig. 3.2 RAPD marker 0AA02-38 linked to femaleness of asparagus. From left to right, lane 1 is 123-bp DNA molecular weight ladder, lanes 2 and 3 are parents MW25 (F) and A19 (M), respectively. Lanes 4-17 are female (F) and male (M) individuals of the mapping population. Segregating RAPD marker is indicated by arrow ................................. Fig.3.3 Southern hybridization of cloned OC15-98 marker to PCR product amplified by primer PC15. From left to right, lane 1 is 123—bp DNA molecular weight ladder, lanes 2 and 3 are MW25 (F) and A19 (M), respectively, lane 4 is ,1 DNA digested b HindIII and lanes 5—8 are female (F) and male (M individuals of the mapping population. Segregating RAPD marker is indicated by arrow. (A) RAPD marker OC15-98 (arrow) linked to maleness of asparagus, and (B) Southern hybridization of the RAPD fragment OC15—98 to individuals shown in panel A ..... Fig.3.4 Amplification of genomic DNAs using SCAR primers (arrow). Left to right, lane 1 is 123-bp DNA molecular weight ladder and lane 2 and 3 are parents MW25 (F) and A19 (M), respectively. Lanes 4-16 are individual males (M) and females (F) of the mapping population ............................... Fig . 3 . 5 Diagram of recombination- facilitated RAPD marker-assisted selection for sex identification using dominant RAPD markers OC15-98/OC15-30. CC = markers OC15~98/OC15-30, which linked with sex locus M at a coupling phase. . . Page . . .107 . . .109 . . .110 . . .113 APPENDIX Table 1. Segregation ratio, X2 value of RAPD loci from A19 parent .................................... 116 Table 2. Segregation ratio, X2 value of RAPD loci from A19 parent .................................... 120 Section 1 Literature Review 1. General Introduction: 1 . 1 Origin of Asparagus The genus Asparagus belongs to the Liliacea family which is comprised of 150-300 herbaceous and woody perennial species (Baily et al. 1942; Lawrence 1982) . In the genus Asparagus, only Asparagus officinalis L. is cultivated for food. It is European-Siberian continental plant related to the east mediterranean vegetation. As a crop it is native to the orient and to the eastern parts of the Mediterranean gene center (Reuther 1984; Peirce 1987) . Asparagus officinalis L. is considered one of the oldest garden vegetables. The Greeks introduced it into their motherland from the eastern nations and at a later date, the Romans adapted its culture from the Greeks (Luzny 1979) . As early as the Imperial Roman time, asparagus was considered as a popular delicious vegetable, and it was said that the Roman troops introduced it to central Europe. The first description of asparagus as a vegetable is documented in a French monastery in the eleventh century. The first beds of it were established in Germany in 1567 in the vicinity of Stuttgart . Before the seventeenth century, its growth was limited mainly to the gardens of castles and monasteries (Luzny 1979) . Roman writers set down instructions on how to grow asparagus, which was prized as a food crop. These early writings indicated a preference for the forms of asparagus grown from seed collected from the wild form. Obviously, since asparagus plants are found growing wild, 3 birds have readily dispersed asparagus seed. Wild growing A. officinalis plants are to be found throughout the United States and Canada (Nonnecke 1989) . Activities in asparagus breeding first took place in France and Germany. According to Henslow (1911) , until the seventeenth century, ancient herbalists directed more attention to the medicinal properties of asparagus than to its use as a vegetable. By 1776, asparagus was growing in every colony along the Atlantic coast (Gleason and Cronquist 1963) . During the following century, it became widely distributed in North America. Today, it is grown almost everywhere in the United States. Michigan is the third largest asparagus producer following California and Washington. In Michigan, asparagus is grown on sandy soils which are often not suitable for other vegetable crops. 1 . 2 Growth Conditions As a perennial herbaceous species, asparagus has specific growing requirements that must be taken into account . Asparagus has a wide geographic compatibility spectrum reflecting very wide ranging environments including the sea shore, the desert, southern and northern latitudes (20° to 55° N) and high elevations . These diverse environmental conditions indicate wide adaptability. The best annual regrowth of asparagus is predicted on a mean temperature range of 16° to 24°C for spear and fern production (Ellison et al. 1959). Where fall killing frost occurs, the asparagus plant goes into 4 a resting period, which is essential for regrowth and ongoing spear production. Where the temperature does not create frost conditions, asparagus should be dried down by withholding irrigation to permit an induced rest period equivalent to a killing frost. If this rest period does not occur, as may happen at lower elevations, especially in the southeast, asparagus production quickly diminishes and becomes uneconomic. In the tropics, asparagus plantings are good for one or at best two annual spear harvests because of the difficulty of inducing the needed rest period. During the active growing season, asparagus responds to rainfall or to irrigation (Nonnecke 1989) . Asparagus is a long term crop, requiring careful soil preparation that will insure a long, vigorous, productive cycle of growth. Once asparagus is planted, it becomes deep rooted, especially with an open soil, and it may be in place from fifteen to twenty years. Although asparagus has been considered to be tolerant of both alkaline and acidic conditions, the main requirement is that the pH level be such that the movement of nutrients is not inhibited (Hartung 1987) . Careful soil analysis is necessary for asparagus production. In preparing the planting bed, all factors must be brought to a level that will provide good maintenance for asparagus growth. Ideally, prior preparation of the planting bed should include the incorporation of organic matter either by ploughing under long-standing alfalfa or other legume crops 5 or incorporating all growth debris in the year immediately preceding the planting year (Ellison 1986) . 1.3 Botanical Characteristics Asparagus officinalis is a long—lived monocot, herbaceous perennial, and is dioecious. It is winter hardy and considered a cool season crop (Ellison 1986) . The plant is grown for its fleshy shoots (spears) , which appear after a prolonged winter rest period. As in other monocotyledonous species, the crown of the plant is the critical growth center. The plant consists of underground stems (rhizomes), fleshy roots, and fibrous roots (Pierce 1987) . The fleshy roots serve as storage organs and the fibrous roots as absorption organs . The fibrous roots die after each year's growth. Likewise, the fleshy roots die after providing nourishment for the next generation of spears (Shelton et al. 1978). If not cut for food, the shoots that give rise to the spears each year eventually become the ferns of the asparagus plant. As the spear ages, lignin accumulates in the pericycle region to provide support for the ferns. The ferns of the asparagus plant are cladophylls, which perform as leaves but are actually modified stems (Flory 1932) . The true leaves are scale—like structures that form at the tip of the spear and down the stem. As the plant grows, the scales give rise to cladophylls, which in turn give rise to the flowers (Pierce 1987) . Structurally, a cross section of a spear shows five anatomical regions: the epidermis, cortex, pericyclic 6 fibers, ground parenchyme and vascular bundles (Pierce 1987). Asparagus officinalis L. is a dioecious species which.produces male and female florets on separate staminate and pistillate plants. The normal ratio of staminate to pistillate plants is 1:1 (Bracale et al. 1991). Sometimes, single plants will produce perfect flowers. According to Bracale et al. (1991), this is the result of a modifier gene or mutation in male plants which induces the andromonoecious state. Andromonoecious plants occur at about 2% in most populations (Wricke 1979). The male flowers are slender, bell-shaped and greenish white in color. Each flower has an aborted ovary and well developed anther bearing orange pollen. Female flowers are smaller than male flower and contain vestigial, functionless anthers and a well developed ovary, style and three feather-like stigmas (Flory 1932). Asparagus flowers are insect pollinated, and.pollen does not move with the wind.until the plant is dead and dried out (Flory 1932). The flowers are individual and form one to six small seeds which are nearly round, flattened on one side with a hard black coat. The seeds are primarily endosperm tissue with a small elongated embryo. As the seed germinates, a radical appears first, followed by the primary stem (Pierce 1987; annecke 1989). 1 . 4 Asparagus Breeding Goals Asparagus has an average harvest period of 10 years and also demands a great amount of financial and labor input before any economic return is realized. Therefore, improvement of cultivars by breeding has high priority. Important breeding aims are all-male hybrids, high yield and quality characterized by an increased number of spears of large diameter per plant, uniformity among the spears, low level of fiber in the spears, high resistance to disease, salt tolerance, and climatic adaptability (Reuther 1984) . Recently, fusarium is considered the most limiting factor in asparagus production (Mace et al. 1981) . Fusarium oxysporium (Schlecht) f . sp. asparagi Cohen (FOA) is the causal agent of the wilt and root rot disease and Fusarium monilifonme Sheld. emend. Snyd. & Hans (FM) is the causal agent of stem and crown rot (Hartung 1987) . Because of fusarium, asparagus plantings decline to uneconomical levels as soon as 5—6 years due to the reduction in plant vigor and loss of crowns. Replanting in fields where asparagus was grown previously results in losses of up to 50% of the new plants within the first year. This problem is described as the asparagus decline and replant problem (Hanna 1947; Hartung 1987) . Because PFOA and PM are transmitted through soil, the use of fungicides and fumigation for long term control is limited. So, breeding for disease resistant varieties is the most effective way for control of this disease. Takatori and Southern (1978) in California and 8 Ellison (1986) in New Jersey made attempts to select for asparagus resistance to fusarium, but to date no such cultivar has been developed. However, new lines introduced from Rutgers such as Jersey Giant have considerable tolerance to this disease. Ellison (1986) attempted to use exotic germplasm in breeding for fusarium resistance. He collected numerous seed samples of A. acutifolins in the wild from Greece, Italy and Spain and seed of wild A. maritimus from Yugoslavia. Unfortunately, all of those accessions were found to be susceptible to fusarium. Only ornamental species such as A. springerii and A. lumosus have been found to be highly resistant to fusarium, but neither of them can hybridize sexually with A. officinalis (Ellison 1986) . There are many differences in the growth of female and male plants. Male plants give rise to more spears and are consequently higher yielding than the female. Female spears generally are heavier individually but fewer in number than in male plants (Ellison and Scheer 1959) . Also, male plants do not produce seedling weeds which compete with the established crowns in nutrition and may favor disease epidemic. For these reasons, breeding efforts are concentrated on developing all- male cultivars that will produce higher, more stable yields of spears (Ellison 1986; Reuther 1984) . 1 . 5 Breeding Methodology As a dioecious plant, asparagus has its specific breeding methodology. Since higher yield is correlated with staminate plants, there have been efforts in asparagus breeding to produce all-male varieties (Loptien 1979; Franken 1970) . In conventional breeding methods, this aim can be reached by selfing hermaphroditic flowers in an andromonoecious plant which are cytogenetically male. Such selfing produces inbred lines consisting of two M alleles-the supermales (M4) . The supermale plant can also be produced through anther culture (Colby et al. 1988) . Pollination of female plants with the supermale plants results in all-male F1 hybrids. To identify the supermale plants, individual male or supermale plants are crossed to female plants, then the progeny is grown and the sex phenotypes are determined by checking the mature flowers . This whole process takes at least 4-5 years (Reuther 1984). After the identification of the supermale (MM) genotype, combining ability for yield and quality must be evaluated. If a supermale plant with a set of genes for yield and other valuable characters has been selected, limiting factors for a large scale F1 hybrid seed production are inbreeding depression of the supermale parent and possible difficulties in vegetative propagation by conventional methods . Therefore, cloning of parent plants by tissue culture is important for setting up a seed production field (Nonnecke 1987) . Since asparagus is a cross-pollinated plant, it has 10 high degree of natural heterozygosity. In many cases it would be of considerable interest to produce completely homozygous plants by obtaining haploids and subsequently doubling the chromosomes (Franken 1970) . F1 hybrids could be produced from homozygous parents with demonstrated combining ability. In asparagus, haploids have been recovered in two ways: First, by selection of polyembryonic seeds, and second, by anther culture (Corriols-thevenin 1979) . After treatment with colchicine, fertile homozygous plants are obtained. Genetic diversity among asparagus cultivars is relatively low. Since asparagus originated in the eastern part of Mediterranean region and it was cultivated by the Greeks more than 2000 years ago, and introduced by the Roman legions to central Europe, the native forms of asparagus presently found there, may have originated from those cultivated types that reverted to wild forms (Luzny 1979) . For example, according to Luzny (1979) , the population "Gewore Hollandse" developed in the eighteenth century in the Netherlands has been largely cultivated in other countries and has been called in England "Purple Dutch" which gave rise to the "Argenteuil" varieties in France in the nineteenth century (Flory 1932) . Later on, these varieties were introduce into America to give rise to the Washington varieties as the populations "Mary Washington" and "Martha Washington" originated from a breeding program for rust resistance (Norton 1913) . Selections from "Purple Dutch" have also been made in England and Germany 11 (Flory 1932) . As a result, the population "Purple Dutch" may be regarded as the form from which all the modern types have been derived at least in the Netherlands, France, German, England, and the United States. In addition, most strains of asparagus developed in the United States and Canada since 1930, have been selections of Martha or Mary Washington (Ellison 1986) . However, there is still considerable variation in these varieties (Robbines & Jones 1925; 1926; Ellison & Schermerhorn 1958; Bannerot et al. 1969; Geoffriau 1992) . The improvement of asparagus cultivars has been achieved by cross- pollination of selected high-yielding male and female plants of the same variety or of different varieties with subsequent selections among the progeny. F1 hybrids were tested for their combining ability in order to improve or to maintain heterosis (Ellison 1986) . By using a polycross system, optimum combinations of female and male genotypes could be identified. A great number of cultivars have been selected in the past according to the different climatic and soil conditions of asparagus growing areas and local market demands . The old cultivars are dioecious, consisting of female and male plants. Several selections and crosses with other varieties have been developed with improved yield and quality from the American cultivars Mary Washington and Martha Washington and University of California types. Mary Washington, introduced in 1910, is a cross by J .B. Norton, Concord, Massachusetts of Mary (a giant female seedling selected in a bed of Reading Giant from 12 England) and Washington (Flory 1932; Norton 1913) . This improved variety provided rust resistance, good spear and growth habits, and purplish-tinged spearhead with tight, pointed buds . The branching of spears does not begin until well after market length is obtained. The Martha Washington is a cross of Martha and Washington. These two cultivars are the main cultivars in asparagus production and the main breeding material in the United States and Canada. Because of the occurrence of fusarium and the absence of the resistant genes in most current cultivars, University of California types, such as UC 157 and UC72 are replacing the Washington types and introducing tolerance to Fusarium oxysporium (Reuther 1984) . As a result of the inbreeding of andromonoecious lines and of anther culture, pure male Fl hybrids have been introduced into asparagus cultivation. Recently, Jersey Centennial and Jersey Giant play a great role in commercial asparagus production (Nonnecke 1989). 2 . Genetic Linkage Mapping 2 . 1 Historical Background Plant breeding is both an art and a science dealing with a wide range of traits, including both quantitative and qualitative traits (Stuber 1992) . Most agronomic traits of economic importance, such as grain or forage yield and protein or oil content, are considered to be quantitative, even though some traits are simply inherited (Lander and Botstein 1989) . 13 Although numerous investigations have been conducted on the inheritance of quantitative traits (primarily using classical biometrical methods), the plant breeder typically has little information on the number of loci involved in the expression of traits, the chromosomal locations of these loci and the relative size of the contribution of individual loci to trait expression (Comstock 1978) . For marker-based procedures to be effective in the manipulation and improvement of quantitative and qualitative traits, the genetic basis underlying quantitative and qualitative trait variation must be better understood (Stuber 1989c) . Marker—based techniques should assist scientists in the development of a better understanding of these underlying bases as well as providing plant breeders and geneticists with a powerful approach for mapping and manipulating individual loci associated with the expression of these traits (Shoemaker 1994) . The ability to identify specific quantitative trait loci (QTL) should lead to more powerful means for investigating epistasis, pleiotropy, and the genetic bases of heterosis. Effective use of mapped genetic markers should allow significant advances in plant improvement and selection strategies (Kelly 1995). The basic concept of associating markers with quantitative and qualitative traits is not of recent origin and was first proposed by Sax in 1923 . He originally explored the association between a qualitative character, a marker, and 14 a quantitative character for indirect selection, using seed coat color and seed size in cormon bean (Phaseolus vulgaris) . Mather and Jinks (1971) summarized several cases where simply inherited markers were associated with continuously inherited variation. For example Rasmusson (1935) demonstrated linkage of flowering time in peas with a simply inherited gene for flower color. Emerson and Schaller (1955) found morphological markers closely linked to a genomic segment affecting yield in barley. Extensive work in Drosophila demonstrated the effects of individual chromosomes on quantitative traits (Mather and Harrison 1949) . Thoday (1961) reiterated the possibility of using genetic markers for the selection of quantitative characters through the development of specific lines and segregating populations. He also suggested the use of a cross between homozygous lines differing in quantitative value as a general means to follow the effect of different genotypes on the expression of the quantitative character. These and other studies provided the framework of theory and observation on which the area of plant breeding is presently based. Since that time, a number of scientists have contributed to the general concepts and theory of using mapped genetic markers for identifying, locating and manipulating QTLs . Some of the major contributors are Mather and Jinks (1971) , McMillan and Robertson (1974) , Tanksley et al. (1982) , Lander and Botstein (1989) and Stuber (1992) . The concept is based upon using the marker locus as a point of reference for the chromosomal 15 segment in the marker' 8 vicinity and permitting that segment to be followed through appropriate genetic manipulation. The frequently used markers at that time were morphological and isozymes . 2.2 Isozyme Markers Isozymes constitute a group of multiple molecular forms of the same enzyme that exist in a species as a result of more than one gene encoding for the same enzyme (Moss 1982) . They display the same catalytic activity, however they can have different kinetic properties and be separated by biochemical procedures. This means that isozymes of the same group differ in their amino acid sequence which may result in a different secondary, tertiary or quartenary protein structure. The number of isozymes for a particular enzyme can be related to the number of subcelluar compartments where the same catalytic reaction takes place (Gottlieb 1982) . The basic assumption made when using isozyme analysis is that differences in mobility along an electric field are the result of differences at the level of the DNA segment that encodes for such enzymes . Thus, if the banding patterns of two individuals differ, it is generally assumed that such differences have a genetic basis (Murphy et al. 1990) . The genetic control of isozymes occurs by several genes, which can be alleles at the same locus or be located in different loci. Isozymes encoded by allelic genes are also defined as allozymes. The main advantage of an 16 isozyme is its co—dominant characteristics, ie. heterozygote and homozygote genotypes at a locus can be easily distinguished. This characteristic allows direct estimation of genetic parameters such as genotype frequencies, gene frequencies and genetic diversity and heterozygosity. The main limitations are the number of loci that can be resolved and analyzed is limited, especially when the investigation requires a more ample coverage of the genome, such as the case of genetic mapping . 2 .3 RFLP Markers A major breakthrough occurred when it was realized that genetic maps could be constructed by using pieces of chromosomal DNA as direct markers for the segregation pattern of chromosome segements (Botstein et al. 1980) . The first such DNA markers to be utilized were fragments produced by restriction enzyme digestion (Botstein et al. 1980) . It was soon found that restriction fragments from a given chromosomal locus often varied in size (or length) in different individuals of the same or different species and these differences were designated restriction fragment length polymorphisms or RFLPs (Botstein et al. 1980) . RFLPs have their origin in base sequence changes or in DNA rearrangements, and are naturally occurring, simply inherited, Mendelian characters . Thus, they can be used to construct genetic maps (Kochert 1994) . 17 RFLP occurs because the DNA of distinct individuals differ in the presence or absence of specific 4 to 8 base pair sites recognized by restriction enzymes. Alternatively, the DNA sequence of two genotypes can differ as a result of insertions, deletions or other rearrangements that alter the relative distance between.any two restriction sites. The RFLP detection technique involves several steps. Initially, the DNA of the individuals that one wishes to analyze is extracted and subjected to digestion with a particular enzyme. The DNA. sequence is cut into a large number of fragments. Differences in the DNA.sequence between individuals (DNA polymorphisms) results in different populations of fragments, which.are size fractionated by agarose gel electrophoresis. Due to the large number of fragments produced in eukaryotic genomes, a smear of fragments is observed on the gel. Discrete fragments cannot be discerned and therefore differences in length of the fragments between.individuals cannot be visualized directly on the gel. To detect RFLPs, the fragments separated by' electrophoresis are transferred and immobilized by capillary or vacuum.transfer onto nylon or nitrocellulose membranes in a process called "Southern Blotting" (Southern 1975) . The identification of a polymorphic fragment between individuals is done by'tnmmidizing small cloned DNA fragments, called labeled probes, to homologous sequences of the fragments immobilized onto the membrane . Probe labeling usually involves the incorporation of nucleotides containing radioactive 18 phosphorous (32F) or sulfur (358) isotopes. Following probe hybridization, the membrane is exposed to an X-ray film in a process called autoradiography, resulting in dark bands of spots on the film that constitute the genetic marker data. These bands result from.the sensitivity of the film to beta particles emitted by the radioactivity incorporated to the probe . The probe hybridi zes exclusively to fragments containing homologous sequences , and only where the probe hybridizes, a signal is obtained on the fihm..Alternatively, non—radioactive detection systems use modified nucleotide containing groups such as digoxigenin or biotin, that are later recognized by antibody—enzyme conjugates. In the chemiluminescence system, for example, the antibody recognizes the digoxigenin group on the hybridized probe , and the conjugated enzyme, typically an alkaline phosphatase, catalyzes a chemiluminescent reaction involving a substrate that , when degraded by the enzyme, emits light photons . These photons play the same role as beta particles in sensitizing the filma If two individuals display fragments that differ in length following restriction digestion, such fragments will migrate to different positions on the gel and also be immobilized at different positions on the membrane. Upon probe hybridization and detection, bands in distinct positions will be observed on the X-ray film, characterizing a so called RFLP. If the Mendelian segregation and inheritance is 19 confirmed by analyzing parents and progeny, the particular RFLP is a defined locus and can be used as a genetic marker. The membrane prepared at the beginning of the procedure can be reused several times for hybridization with different probes, provided that the previous probe is adequate 1y removed and the immobilized DNA is not degraded. Each probe typically provides one polymorphic marker, and occasionally detects more than one . The DNA clones to be used as probes can be obtained in different ways, the most common being: (1) by reverse transcription of messenger RNA of the organism under study, producing a complementary DNA (cDNA) library of clones corresponding to expressed sequences of the genome; (2) random genomic DNA sequences, in the form of a genomic library that includes both expressed and non-expressed sequences; (3) from known clones of specific genes, either by PCR amplification or synthetic oligonucleotide construction. Typically, once the library of fragments to be used as probes is obtained, a necessary step before any analysis can be carried out is the selection of clones to be used as probes. In this screening procedure, the objective is to select those clones that contain exclusively single or low copy sequences, that is, that do not contain repetitive sequences present in the genoie. Clones containing such repetitive DNA, will hybridize to all the fragments immobilized on the membrane that contain such repetitive elements . After autoradiography, this pattern 20 of hybridization will show large numbers of bands or continuous smears that cannot be interpreted or followed as genetic markers . On the other hand, probes containing moderately repetitive elements become very useful for the objective of obtaining unique fingerprints of genomes. Libraries of cDNA fragments are intrinsically made up of a large majority of low single copy clones. Genomic libraries are not, however they can be constructed in specific useful probes with similar amounts of detectable polymorphism. RFLPs present the main advantage of potentially covering the whole genome of interest , depending on the type of library used to obtain the probes. The use of RFLP markers substantially increases the probability of finding significant associations between markers and genetic loci of breeding interest. RFLP markers typically display co—dominant inheritance, and therefore a larger amount of genetic information is obtained per locus when compared to dominant markers. Co-dominant markers allow the analysis of intralocus interactions between alleles at genetic loci of interest as well as epistatic interactions between alleles at different loci . Finally, as with any other DNA based markers, RFLPs share the advantage of the high stability of DNA which can be extracted from any tissue, at any stage of development and can be reused over long periods of time. The main limitations for this technique is that it is labor intensive and time consuming, Another limitation is that a library of useful 21 probes is needed. The use of RFLP's requires personnel with skills in recombinant DNA technology, and in the management of radioactive material safely. 2 .4 RAPD Markers Williams et al. (1990) suggested application of the widely utilized polymerase chain reaction (PCR) to develop random amplified polymorphic DNA (RAPD) markers. RAPDs are created using primers of 10-17 bases with an arbitrary sequence and the target region is anonymous. When two sites complementary to the primer sequence are adjacent (less than 4000 base pairs apart) and in an inverted orientation, the amplification between these sites takes place following the same procedure as in a standard PCR. As a result of the large quantity of DNA produced, the amplified fragment can be directly visualized as a band on an agarose gel by ethidium bromide staining. Alternatively, high resolution polyacrylamide gels, coupled to isotope incorporation during PCR and autoradiography or silver staining can be used to increase the sensitivity of the method. Typically each primer directs the synthesis of several DNA fragments simultaneously in different regions of the genore, so that several bands of different sizes are observed on the gel. Welsh and McClelland (1991) suggested the use of multiplexing pairs of arbitrary primers to increase the number of detectable polymorphisms. The probability with which a RAPD site occurs in a genome of a certain size can be estimated, 22 such that a linear relationship is expected between the sequence corplexity of the genome and the number of bands observed. Experimental data have shown that the number of bands is relatively independent of genome complexity. In other words, the number of amplified bands from a simpler genore such as bacteria, is similar to that amplified from more corplex genome such as soybean (Williams et al. 1990) . These results indicate that the final products of the RAPD reaction depend more on the corpetition between different RAPD sites than on the number of sites, which imply that not all the fragments amplified are the result of a perfect match between the primer and the template DNA. A more specific corplementarity at the 3' end of the primer, where the extension begins seems to be more critical than the 5' end (Williams et al. 1990) . RAPD polymorphism results from changes that affect amplification of the DNA located between the two priming sites . These changes include most probably single base substitution as well as deletions or insertions that either change the priming sequence or the size of the amplified DNA (Williams et al. 1990) . Martin et al. (1991) assessed nearly isogeneic tomato lines using 144 random primers and were able to distinguish between them. In sore cases, RAPD markers produce single band patterns; but in other cases, a multiple band pattern can be generated. RAPD markers usually behave as dominant genetic markers. Dominance in this case does not refer to the classical meaning in terms of intralocus 23 interaction between alleles, rather purely from the standpoint of relationship between genotype and phenotype . When a RAPD band is observed on the gel, it is not possible to distinguish whether that fragment is derived from the amplification from one or two allelic dosages. That is, in diploid homozygous individual AA for a RAPD locus, amplification is carried out from two copies of the RAPD allele A. In a heterozygous individual Aa for the same locus, the allele A is amplified and the allele a is not. The means of detecting RAPD fragments does not have sufficient quantitative sensitivity to discriminate between these two cases, and a band of identical intensity will be observed on the gel in both cases. So, the hoiozygous recessive genotype aa is identified as the absence of the band (null genotype) and the dominant horozygous and heterozygous genotypes have the same molecular phenotype, ie. presence of a band. This behavior is equivalent to that of a morphological dominant marker. The RAPD assay detects only one allele at each locus. The absence of the band corresponds to all the other genotypes represented by undetectable alleles at that locus that cannot be amplified. The basic technological difference between the RFLP and RAPD assays is that RFLP is based on DNA hybridization, while RAPD is based on DNA amplification. This difference results in a number of practical advantages of RAPD that can be summarized in two attributes: simplicity and speed. Data acquisition is several orders of magnitude faster. For example 24 RAPD analysis was four to six times more technically efficient than RFLPs when mapping polymorphisms linked to disease resistance, and 10 times more efficient in terms of time and labor (Paran et al. 1991) . This is basically the result of the detection procedure which is done directly by visualizing the bands on the gel , eliminating all the steps involving Southern blotting , probe preparation and autoradiography . The RAPD technique does not require the prior development of a library of probes. The same set of oligonucleotide primers can be used for any organism. Because it is not based on DNA hybridization , it does not require the handling of any type of radioactive isotope . Another important advantage is that minimal amounts of DNA are necessary for the genotypic analysis of an individual. For each RFLP data point, micrograrm quantities of DNA are needed. RAPD requires only nanogram amounts of DNA, i.e. three orders of magnitude less. This substantially facilitates and accelerates DNA extraction procedures. The cost of the RAPD technique is lower than RFLP in terms of cost per data point. If one include the cost of probe library development , the cost for RAPDs would be much lower. The main limiting feature of RAPD markers is their low genetic information content per locus, since only one allele is amplified, while all other alleles are detected as nulls. This limitation is comonly treated as dominant behavior of RAPD markers as described previously. As with any 25 other type of marker, RAPDs are not an.exception in that some of the.bands Observed.on.the gel are easily and unambiguously interpreted, while others are less interpretable or repeatable. Ambiguity in the interpretation may result from: (1) low discriminatory power between distinct sites of a specific primer as a result of its nucleotide sequence; (2) competition between distinct amplification sites for substrate and enzyme, such that the occurrence of particular sites interfere or out compete the amplification of others, in a process equivalent to an epistatic interaction between marker loci; (3) problems related to the standardization of amplification conditions. Laboratory to laboratory variation, as a result of differences in thermal profiles of different PCR cyclers has been observed (Penner et al. 1993 ; Wang et al. 1993) . It has also been suggested that RAPD markers should be used with caution for phylogenetic analysis, fingerprinting and paternity determination, because corpetition between different fragments may lead to ambiguous results (Heun and Helentjaris 1993; Thormamm and Osborn 1992; Thormann et al. 1993; Riedy et al. 1992). Due to the sensitivity of the PCR protocol the optimization and standardization of reaction corponents and amplification conditions represent an important step in the application of the RAPD technique. 26 2.5 Advantages of Molecular Markers The relative advantage of molecular markers, such as isozymes, RFLPs, and RAPDs over morphological markers for most genetic and breeding applications were discussed by Tanksley et al. (1989) and Stuber (1987; 1989a; 1989b; 1989c; 1992) and are summarized as follows: (1) For molecular markers, genotypes usually can be determined at whole plant, tissue, or cellular levels. For most morphological trait markers, genotypes generally can be ascertained only at the whole plant level, and, frequently, the mature plant is required; (2) For many plant species, several naturally occurring alleles are available at most molecular marker loci. Thus, natural variation in existing populations can be used without the need to construct special genetic stocks, as may be required for many morphological traits; (3) The majority of molecular markers appear to be phenotype—neutral , whereas morphological markers frequently cause major alterations in the phenotype which are often undesirable in breeding population; (4) Unfavorable epistatic interactions frequently occur among loci encoding morphological-marker traits and limit the number of segregating markers that can be tolerated or unequivocally scored in a single segregating population. Most molecular markers appear to be free of epistatic effects (Kelly 1995) , thus, the number of loci that can be monitored in a single population is theoretically unlimited. 27 3. Constructing a Linkage Map with Molecular Markers The most critical decision in constructing a linkage map with DNA markers is the mapping population. This is determined by the plant to work with, the goal of the mapping project (Young 1994) , and the availability of inbred lines (Weeden 1994) . The goal of mapping may be to generate a framework map to provide a set of mapped loci for the future (Vallejos et al. 1992) , or instead, identify and orient DNA markers closely linked with a target gene for eventual marker-based cloning (Martin et al. 1993) . Perhaps the goal is mapping quantitative trait loci (QTL) , or the monitoring of several disease resistance loci in the process of pyramiding them into a single background (Kelly 1995) . These goals will have a critical influence on which parents are chosen and the size of the population. Sufficient DNA sequence polymorphism between parents must be present . This point cannot be overemphasized, for in the absence of DNA polymorphism, segregation analysis and linkage mapping are impossible (Stuber 1992) . Naturally, outcrossing species, such as maize, tend to have high levels of DNA polymorphism and virtually any cross that does not involve related individuals will provide sufficient polymorphisms for mapping (Helentj aris et al. 1986) . However, in naturally inbreeding species, levels of DNA sequence variation are generally lower and finding suitable DNA polymorphisms may be more challenging (Miller and Tanksley 1990) . Sometimes, mapping of inbreeding species requires that parents be as distantly related as possible. 28 This can often be estimated based on geographical, morphological, or isozyme diversity ( Miller and Tanksley 1990) . Lines without common ancestry, or the use of interspecific crosses have been frequently used to optimize the genotypic analysis (Bonierbale et al. 1988; Tanksley et al. 1982) . Recently, however, molecular markers based on highly polymorphic simple sequence repeat sites have eliminated the problem of lack of polymorphism in selfing species (Morgante et al. 1994) . And also, in the context of quantitative trait mapping, selection of parents becomes only a function of the contrasting phenotypes of the parents to evaluate the effects of allelic substitutions at loci that control traits of interest. 3.1 Choice of Mapping Population: Once suitable parents have been chosen, the type of genetic population to use for linkage mapping must be considered. For plant species in which inbred lines are easily produced and in which large populations can conveniently be handled, either the F2 produced from crossing two lines or the backcross of the F1 to one of the parents provides an appropriate mapping population (Weeden et al. 1994) . A polymorphic marker between the two inbred lines will have genotype AA in one line and aa in the other. The F1 will all be heterozygous Aa. In an F2 produced by selfing the F1 generation, the marker will segregate 1 (AA) : 2 (Aa) : 1 (aa) for co—dominant 29 markers and 3 (Aa) : 1 (aa) for a dominant one. In a backcross to the horozygous recessive line, both these markers will segregate 1 (AA) : 1 (aa) . Alternatively, one can use a population of recombinant inbred lines (RIL) derived from an F3 through successive rounds of selfing (Burr et al. 1988; Reiter et al. 1992) or a population of doubled.haploid lines produced by anther culture of F1 individuals (Ferreira 1993) . Both kinds of populations are analogous to a backcross in that a 1 (AA) : 1(aa) segregation ratio is expected. Although more corplex to analyze, an F2 provides almost twice as much information as a backcross, because markers are segregating in both the male and female gametic populations generating the F2 (Lander et al. 1987) . Recombinant inbred lines also are useful for generating linkage maps (Burr et al. 1988; Haley et al. 1994c) . For plant species for which inbred lines are not available, such as woody and dioecious plants, the above 'mapping' population cannot be constructed. easily' through traditional plant breeding. weeden et al. (1994) developed a strategy for apple genome mapping. They used trees that are highly heterozygous as an F1. The approach they employed was developed by the following reasoning: Selfing of the "F1" plant is generally impossible as a result of strong incompatibility barriers or inbreeding depression, but researchers working with conifers have demonstrated that genetic analysis can be performed on single trees by examining segregation of heterozygous loci in megagametophytic tissue 30 (O'Malley et al. 1979; Tulsieram et al. 1992) . Woody and other plants may not have megagametophyte large enough to be easily isolated or sampled. However, in a controlled cross, the contribution of the pollinator could be identified, and the segregation of heterozygous loci from the "F1" plant should be able to be detected by means of pseudo—testcross. Indeed, the other parent also was highly heterozygous , and the progeny derived from the cross also represent a segregating set of the second parent's gametes. Thus, the progeny of any cross between different varieties can be thought of as a double pseudo-testcross, and segregation of the heterozygous loci in both genomes can be analyzed simultaneously (Weeden 1994) . The F1 generation of such crosses is genetically heterogeneous and segregates for marker genotypes and phenotypic traits. An F1 in the forest tree breeding concept is actually genetically closer to an F2 or backcross generation in an annual self fertilizing crop. Genetic markers segregate in different Mendelian ratios depending upon the genotypes of the parents and the mating configuration (Ritter et al. 1990) . For example, if both parents are heterozygous for the same alleles, a 1:2:1 genotypic ratio for co-dominant markers and 3:1 for dominant ones is expected. If the mating involves three or four alleles, as is often the case, a 1:1:1:1 ratio is expected for co-dominant markers, however a more simple 1:1 ratio is expected, if only one dominant allele is detectable out of the four alleles in the mating configuration. Although 31 less information per locus is acquired with dominant markers, a.more simple and.straightforward analysis of a 1:1 ratio can be performed following essentially the same analysis used in a backcross or testcross mating in annual crops. RAPD markers are particularly advantageous in this case, because they are highly polymorphic and detect only one allele (presence of the amplified band) at a locus, while all the other alleles are detected as null. So essentially, the meiotic segregation of the two alleles (presence and.absence) at a RAPD locus in one parent of the cross can be followed in exactly the same fashion as in the analysis of haploid megagametophyte, as long as the other parent has a RAPD null genotype at the same locus. Because the analysis of RAPD markers is quick and simple, it is possible to prescreen large numbers of oligonucleotide primers and select only those that amplify markers segregating in the informative 1 : 1 configuration. This screening is typically done by analyzing the two parents and a sample of six or more F1 progeny individuals in search of RAPD bands that are present in one of the parents, absent in the other and polymorphic, i .e. present and absent in the progeny sample. After genotyping all individuals for the preselected markers, two independent marker data sets are generated, one for each.parent. These will in turn result in two independent genetic linkage maps by analyzing the co— segregation of markers. Markers for which only one allele is detected have 32 been called single dose polymorphism or single dose restriction fragments when derived from RFLP analysis (Gebhart et al. 1989; Ritter et al. 1990; Da Silva et al. 1993; Carlson et al. 1991; Sobral and Honeycutt 1993) . They have been efficiently used to map polyploid species such as sugar cane (Wu et al. 1992; Sobral and Honeycutt 1993) . The term "pseudo—testcross" (Weeden 1994) was suggested for this strategy because the 1:1 testcross mating configuration of the markers is not known a priori as in a conventional testcross where the tester is homozygous recessive for the locus of interest. Rather, the configuration is inferred a posteriori after analyzing the parental origin and genetic segregation of the marker in the progeny of a cross between highly heterozygous parents with no prior genetic information. When this inference is done for both parents involved in the cross, the term "two—way pseudo—testcross" or "double pseudo- testcross" is more appropriately used (Grattapaglia and Sederoff 1994) . This conceptually simple strategy co'tbined with the polymorphism detection power of the RAPD assay, has allowed the construction of the linkage maps in Eucalypsus grandis and E. urophylla (Grattapaglia and Sederoff 1994) , apple (Lawson et al. 1992) , sugar cane (Sobral and Honeycutt 1993), alfalfa (Echt et al. 1993) and Vitis (Lodhi et al. 1995) . The major advantage of the pseudo—testcross mapping strategy combined with RAPD markers is that it is a general 33 strategy for the construction of genetic linkage maps in outbred plants as well as in any highly heterozygous living organism. It can be immediately applied to these species without any prior genetic information. The only requirement is sexual reproduction between two individuals that results in the generation of a progeny set large enough to allow the reliable estimation of recombination frequencies between segregating markers. Its efficiency will be directly proportional to the level of genetic heterozygosity of the species under study, which is a function of the mating system and the genetic diversity between the parents . In interspecific crosses, practically every arbitrary RAPD primer detects at least one informative pseudo-testcross marker and frequently several. At the intraspecific level, the frequency is corparatively less, however, still high enough to make this strategy extremely efficient (Carlson et al. 1991 ; Grattapaglia et al. 1994) . Finally, the possibility of constructing linkage maps using only two-generation pedigrees is of great relevance to plant breeding because it allows the use of existing populations. 3.2 Population Size: Resolution of a map and the ability to determine marker order is largely dependent on population size. (Mathers 1938; Young 1994) . Clearly, population size may be limited technically by how many seeds are available or by the amount of DNA samples 34 that can be reasonably prepared. Whenever possible, the larger the mapping population the better. Populations of less than 50 individuals probably provide too little mapping resolution to be useful (Mathers 1938) . Moreover, if the goal of high resolution mapping in specific genomic regions or mapping QTL of minor effect, ITILlCh larger populations will be required (Young 1994) . For example, Messeguer et al. (1991) examined over 1000 F2 plants to construct a high resolution map around the Mi gene of tomato and Stuber et al. (1987) analyze of over 1800 maize F28 to find QI‘L controlling as little as 1% of the variation in yield components. 3.3 Linkage Analysis As a segregating population is produced and a particular genetic mode of inheritance is expected, the mapping population is genotyped for a large number of markers. These markers had originally been selected because they are polymorphic between the parents . A matrix of data is generated where typically the rows correspond to the markers and the columns to the progeny individuals. A first statistical test (X2) is applied to each marker to test the null hypothesis of the expected segregation ratio, 1:1, 1:2:1, or 3:1. Significant XZ can indicate segregation distortions that have a biological basis (eg. lethal) or be merely the result of a small sample of individuals genotyped. Subsequently, markers are then submitted to a test of independence of segregation, 35 or linkage. Basically, this test is performed on all possible pairwise combinations of markers. Rejection of the null hypothesis-—two markers segregating independently--is an indication of genetic linkage between them. The next step involves the estimation of genetic map distance, which is not physical distance but reflects the frequency of genetic recombination events (crossover) per meiosis per generation. This parameter is expressed in % recombination or translated into CentiMorgan units by using different mapping functions that take into consideration double crossovers and genetic interference (eg. Haldane, Kosambi) . In the simplest case, a backcross, genetic distance is the proportion between the observed number of recombinant genotypes and the total number of individuals genotyped. The correlation between physical distance on the chromosome and the frequency of recombination between any two markers may seem intuitively high and linear, it need not to be so. There are regions of small physical distance where there is a high probability of genetic recombination, the so called "recombination hot spots" (Tanksley et al. 1992) . On the other hand, there are very long stretches of DNA where genetic recombination is suppressed due to the presence of heterochromatin, or around the centromere or in teloreric regions and therefore the genetic distance is small . When all the markers are grouped in linkage groups and all two-point recombination distances are estimated, a genetic map can be constructed by hand in a classical procedure called 36 two—point mapping, the relative position of the markers is determined and gradually a gene order is obtained. This procedure becomes tedious when.hundreds of markers need to be ordered. Also, each two-by—two distance has an associated error, which is a fUnction of its magnitude and the sample size. This complicates the correct estimation of the relative position of the markers and becomes increasingly' more difficult with closer markers. To mutigate these problems, maximum likelihood multipoint estimates of recombination frequency are typically used. A.growing number of software packages have been developed to perform those calculations. The most widely-used genetic mapping software is MAPMAKER (Lander et al. 1987). Mapmaker is based on the concept of the LOD score, the "log of the odds—ratio"..A.LOD score indicates the log (10) of the ratio between the odds of one hypothesis (for example, linkage between two loci) versus an alternative hypothesis (no linkage in this example) (Morton 1955; Staub 1995; Risch 1992). In plants, MAPMAKER.has become a virtual standard for constructing genetic linkage maps. More recently, a new computer software package called.JoinMap (Stam 1993) was developed. It is used to merge horologous linkage groups of two or more populations. In essence, JoinMap pools the segregation data of all the mapping populations, recalculates the pairwise recombination frequencies and LCD scores, and then synthesizes a consensus linkage map using a best—fitting arrangement. JoinMap is very useful when two or 37 more populations and/or more than one data type is available. It can also be used to identify horologous linkage groups produced with the (double-) pseudo-testcross mapping strategy (Hammet et al. 1994; Lewis and Sink 1995) . In recent years, molecular markers have allowed a significant advance in the ability to generate linkage maps quickly. Maps of molecular markers were reported for loblolly pine (Grattapaglia et al. 1991) , white spruce (Tulsieram et al. 1992) , Arabidopsis (Reiter et al. 1992) , apple (Lawson et al. 1995; Hammat et al. 1994; Weeden 1994), slash pine (Nelson et al. 1993; Kubisiak 1995) , Eucalyplis grandis and E. urophylla (Grattapaglia 1994; 1995) , sunflower (Gentzbittel et al. 1995), citrus (Cai et al. 1994); lettuce (Robbines et al. 1994; Kesseli 1994) , torato (Tanksley 1992) , potato (Freyre and Douches 1994; Tanksley et al. 1992) , alfalfa (Yu et al. 1993; Echt et al. 1994) , barley (Kleinhofs et al. 1994; Huen et al. 1991) , celery (Yang et al. 1995) , soybean (Morgante, 1994; Shoemaker et al. 1995) , carrot (Schulz et al. 1994) , wheat (Eastwood et al. 1994) , maize (Heun et al. 1993) , cucumber (Kennard et al. 1994) , sorghum (Pereira et al. 1994; Xu et al. 1994; Pammi et al. 1994), pepper (Lefebvre et al. 1995) and Vitis (Lodhi et al. 1995) . 4 . Genetic Mapping of Simply Inherited Traits The association between molecular markers and traits that display simple Mendelian inheritance have been established in 38 several crop species. Isozymes, RFLPs and RAPDs have been used to map genomic regions significantly associated with traits such as disease and pest resistance. From a theoretical view, gene mapping of such traits is quite simple, as more or less discrete phenotypic classes for the traits can be observed. The traditional mapping scheme involves crossing of a susceptible line with a resistant one. The F1 can be either susceptible, resistant or intermediate, depending on the pattern of interallelic interaction (recessive, dominant or incomplete dominant) . The F1 is either selfed or backcrossed to one of the parents to form a segregating population. This population is subject to an inoculum under controlled conditions and the phenotypes are evaluated. When the phenotypes segregate according to a particular genetic model and associated Mendelian expectations, the resistance trait is treated as another marker. When only a few genes of large effects are involved, discrete phenotypic classes can still be determined. Typically, subjective systems of disease severity scores are used and associations between markers and resistance can be found. A classical example of this approach was described in tomato when a significant association was established between an isozyme marker locus (Alkaline Phosphatase 1) and a locus that controls resistance to nematode Meloidogyne incognita. These two loci were found to be linked at a 1 cM distance (Medina-Filho 1980) . Several tomato breeding programs have 39 used this information to select nematode resistant plants indirectly based on the presence of a particular isozyme allele (Tanksley 1983a; b). Another example of strong association is between the locus that confers resistance to BYMV (Bean Yellow Mosaic Virus) and an allele at the isozyme locus, PGM (phospho-glucomrutase) in pea (Weeden et al. 1984) . These two loci are 2 cM apart. In both cases, the indirect selection of the isozyme allele avoids or reduces the manipulation of the pathogen and facilitates the selection of resistant individuals. Genetic linkage between isozymes and simply inherited traits has also been detected in apple (Malus) for resistance to aphids, downy mildew and several self incorpatibility alleles (Chavreau and Laurens 1987; Manganaris and Alston 1987) . In barley, an association has been detected between an isozyme locus and a major gene controlling differentiation of shoots in callus cultures (Koratsuda et al. 1993) . The use of RFLP and RAPD techniques has significantly increased the number of associations detected between markers and traits of economical interest, both with simple and corplex inheritance. The large number of markers allows a more corplete sampling of the genore for significant linkages. RFLPs have been used to locate major genes involved in disease resistance in tomato (Young et al. 1988; Klein-Lankhorst et al. 1991a; Sarfatti et al. 1989) , species of Brassica (Landry et al. 1992; Ferreira 1993), rice (Yu et al. 1991), soybean 40 (Muehlbauer et al. 1991; Diers et al. 1992) and maize (Bentolila et al. 1991). Mutant lines have been used for the detection of an RFLP closely linked to the phl gene that controls chromosome pairing in wheat (Gill and Gill 1991). A (Hg insensitive dwarfing gene and growing habit were mapped with RFLP markers in rye (secale cereale) (Plaschke et al. 1993) . Since their introduction, RAPD markers have been extensively used for the purpose of mapping resistance genes in tomato (Klein-Lankhorst et al. 1991a; b; Martin et al. 1991), bean (Miklas et al. 1993; Haley et al. 1993), lettuce (Michelmore et al. 1991; Paran et al. 1991), Beta vulgaris (Uphoff and Wricke 1992) and Pinus lambertiana (Devey et al. 1993) . Most of the studies where disease resistance loci were mapped, relied on a strategy involving near isogeneic lines (NIL) derived from sequential backcross breeding. Gene mapping using NIL is based on the principle by which a gene of interest is introduced in a cultivated line from a donor line. .After several generations of backcrossing, the genome of the selected progenies becomes more and more similar to that of the recurrent parent with the exception of the DNA segments that contain the introgressed. gene of interest. These segments, if genetically polymorphic at the DNA level in relation to the recurrent genome, can.be used as a target to determine if a particular marker is located near the gene of interest . Near isogenic lines have been used to identify two 41 RFLP probes very closely linked (0.4 cM) from the Tim-2a gene in tomato that confers resistance to Tomato Mosaic Virus (TMV) (Young et al. 1988) . Similarly, a set of NIL derived from the cross of lines susceptible and resistant to Pyricularia orizae was used to identify two RFLP markers associated with the resistant genes (Yu et al. 1991) . One of the markers, RFLP clone RF64, is 2.8 cM from a resistance locus and can potentially be used to select for resistant genotypes. That same marker is also strongly associated with a photoperiod sensitivity gene (Mackill et al. 1993) . NIL were also used to map the gene conferring resistance to Fusarium oxysporum and Cladosporium fulvum in torato (Sarfatti et al. 1989; Van der Beek et al. 1992) and Phytophthora megasperma in soybean (Diers et al. 1992). There are, however, two major limitations in using such an approach to identify linkages of breeding interest . The first one has to do with the creation of the NIL. In the vast majority of plants this is impracticle, especially in allogamous plants with a long generation span such as trees. When possible, it is still a time consuming process because more generations of backcrossing are necessary. The second limitation is that frequently many DNA segments of the donor genore are inadvertently introduced into the recurrent line (Young and Tanksley 1989) . This results in the identification of polymorphic markers between the NILs that in fact are not linked to any gene of interest. An alternative procedure which 42 is also restricted to some species, involves the use of dihaploid lines derived from microspore culture of a single F1 individual . Such an approach was used to identify DNA markers linked to loci for resistance to Leptoshaeria maculans and Albugo candida in colza (Brassica napus) (Ferreira 1993) . A powerful alternative technique to target genomic regions associated to the inherited traits involves the analysis of pools of genome that are fixed at the locus of interest. This strategy was originally proposed by Amhein et al. (1985) for the identification of RFLP in linkage disequilibrium with loci controlling diseases in humans as a result of natural selection. Markers linked to the locus of interest were identified based on their linkage disequilibrium with the disease trait in relation to the rest of the population. This idea was later adopted by Michelmore et al. (1991) for the rapid identification of markers in specific genomic regions using a segregating population, a technique termed bulked segregatant analysis (BSA). RAPD analysis was used to corpare two groups of pooled DNA from individuals of a segregation population originating from a single cross from parents that differed by only a single discrete character. According to Maisonneuve et al. (1994) , BSA analysis use a large number of segregating individuals in each pool with the objective of minimizing or eliminating variation not associated with the specific trait of interest. Haley et al. (1994a; b) used the BSA methodology to identify several RAPD 43 markers linked to a disease resistance gene in common bean and Mulcahy et al. (1992) and Hormaza et al. (1994) using the same approach, found several markers for sex determination in Silene latifolia and Pistacia versa, respectively, both of which are dioecious species. 5. Genetic Mapping of Quantitative Trait Loci (QI'Ls) Most heritable traits of economic importance are the result of the joint action of several genes with unequal effects and variably influenced by the environment. These traits are called polygenic, quantitative or complex ones. The resulting phenotypes display a continuous variation instead of discrete phenotypic classes. Productivity, plant size and precocity are typical quantitatively inherited traits. For the vast majority of complex traits, very little information is available on their genetic architecture, which includes the number, relative chromosome location, magnitude of effect and possible interactions of the genetic loci controlling the expression of the trait. The classical OIL mapping strategy is based on pedigrees traditionally used for the construction of linkage maps . Two inbred lines, genetically divergent and preferentially extreme in the quantitative trait of interest are identified. These lines, homozygous for alternative alleles at the (file and polymorphic for a number of molecular markers, are crossed and a segregating population is generated 44 from the F1. Segregating populations can be of different kinds: F2 intercross, backcross, recombinant inbred lines (RIL) or“ dihaploid lines. This type of pedigree where phenotypically extreme inbred lines are initially crossed is considered ideal because it maximizes linkage disequilibrium between markers and QTLS. The power to detect a OH. is a function of the magnitude of its effect on the quantitative trait, the heritability' of the trait, the size of the segregating population and the genetic map distance (recombination frequency between marker and QTL. Evidently the larger the effect, the heritability and the sample size, and the closer is the marker, the more powerful will be the efficiency of detection. QTL mapping experiments will therefore typically involve the evaluation of a few hundred individuals for the quantitative traits of interest and their genotyping for several dozens of molecular markers selected for being evenly spaced (10 to 30 cM) throughout the genome. A search is then carried out for significant associations between segregating markers and trait values. various statistical methods can.be applied.to QTL analysis. A. t test of difference between.means is the most simple one, and.it is generally' considered. to Ibe rdbust especially' for trait distributions departing from normality, or in cases where the genotypic classes involve a mixture of distributions due to recombination between marker and QTL. In a population where markers segregate 1:1, all the individuals can be classified 45 in either one of two classes depending on their marker genotype, For each marker, a mean and a variance can be calculated for each genotypic class. A hypothesis test is constructed where the null hypothesis of no difference between the mean trait value of the two classes is tested using a t test. The software package MapMaker—QTL (Lander et al. 1987) implements this type of QI'L analysis. The output is given in numerical form by LOD scores at each map interval or uses a specified portion of interval. Alternatively, these data can be presented graphically as LOD score profile scans for each linkage group where peaks indicate the presence and location of a GIL. Functions for the analysis of F2 intercross, backcross and RIL are available. In the F2 intercross model, options are available to fit different models of gene action at each QT'L so that additive and dominant effects can be estimated. In the backcross situation, however, only the additive effect of an allelic substitution can be estimated. 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Section 2 Confined RAPD and RFLP Molecular linkage W of Asparagus 61 Abstract Two linkage maps of asparagus (Asparagus officinalis L.) were constructed using a pseudo-testcross mapping strategy with restriction fragment length polymorphisms (RFLPs) , random amplified polymorphic DNAs (RAPDs) , and allozyme markers in a population generated from crossing two heterozygous parents . Two sets of data were formed according to the inheritance patterns of the markers and analyzed as a backcross population. The maternal MW25 map has a total of 163 markers placed in 13 linkage groups covering 1280 .9 cM with an average and maximum distance between adjacent markers of 7.9 and 28 .5 cM, respectively. The paternal A19 map has 183 markers covering 1324 .1 cM in 9 linkage groups with an average and maximum distance between two adjacent markers of 7.7 and 29.3 cM, respectively. Six multiallelic RFLP markers (markers segregating in the pattern a/c x b/c) and eight markers segregating in the pattern a/b x a/b (HZ loci) , including four RAPD and four RFLP polymorphisms, were common to both maps and were used as locus bridges to align horologous groups between the two maps. In this case, RFLP markers were more efficient than RAPD markers . Nine linkage groups in the MW25 map were identified as being horologous to six groups in the A19 map. In two cases, two or more bridge loci were in cormon, thus, the orientation was also determined; in the other 4 cases, only one locus was cormon in the two groups and the orientation is unknown. Mdh, four RFLP and 14 RAPD markers were assigned to chrorosome L5 that has the sex locus M. 62 Key' words: asparagus, 'molecular 'markers, double pseudo- testcross, sex, bridge loci 63 Introduction Asparagus belongs to the lily family and is an important high value vegetable crop worldwide. As a dioecious species, asparagus provides a challenge for genetic and breeding research. Dioecious sex expression is controlled by a major gene (M) which is located on the L5 chrorosome (Loptien 1979) . 'Ihe naturally occurring male type is heterozygous Mm, while the female type is horozygous (mm) . So-called supermales, which can be produced from the selfing of a perfect flower on an androronoecious plant or derived from anther culture, are homozygous dominant (MM) . 'Ihese supermale plants are very useful for the production of all-male populations which are highly productive . ‘Ihus, a genetic linkage map would be very useful for the early determination of sex type, identification of quantitative trait loci (QI'Ls) , and for molecular tagging of disease resistance traits. Restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs) are the most widely used molecular markers for map construction and gene tagging. RFLPs are generated using low copy nuclear DNA sequences as hybridization probes on Southern blots (Botstein et al . 1980) . RAPDs are detected by the polymerase chain reaction (PCR) using arbitrary primers, which are much simpler to assay than RFLPs, and can detect polymorphisms in both low copy and repetitive DNA sequences (Williams et al. 1990) . RFLP markers are codominant and it is possible to detect many 64 alleles at a locus, such as the a/b x a/b, a/c x b/c crosses; while most RAPD markers are dominant and can only detect two alleles at a locus (presence or absence of the band). 'Ihus, RAPD markers provide less genetic information than RFLP markers in F2 populations. Together with other molecular markers, RFLP and RAPD markers have been used to develop genetic linkage maps for many agricultural important plant species, including lettuce (Robbins et al. 1994; Kesseli et al. 1994), tomato (Tanksley et al. 1992), potato (Freyre and Douches 1994) , barley (Kleinhofs et al. 1994) , soybean (Morgantee et al. 1994; Shoemaker et al. 1995) , wheat (Eastwood et al. 1994) and maize (Heun et al. 1993). Allozyme markers have been used to assess genetic variation among asparagus populations and cultivars (Geoffriau et al. 1992; Brettin and Sink 1992) and to identify some doubled haploid lines derived from anther culture (Colby and Peirce 1988) . Recently, Restivo et al. (1995) constructed a preliminary low density genetic map (43 markers in 12 linkage groups) of asparagus on the basis of RFLP and allozyme markers derived from backcrosses of doubled haploid individuals. More recently, an RFLP linkage map containing 81 RFLP markers was constructed by Lewis and Sink (1995) using the double pseudo- testcross mapping strategy. Two linkage maps were constructed initially, one for each heterozygous parent, and the loci of the configuration a/b x a/b (HZ loci) and a/c x b/c were used as locus bridges to align seven homologous linkage groups 65 between the two maps . This asparagus map is presently not saturated sufficiently for marker assisted selection (MAS) for QI‘L analysis . Most genetic linkage maps of plants are readily constructed from segregating populations derived fron crosses of inbred lines. Such populations are not available for some plant species or not easy to obtain. In conifers, haploid megagametophytes have been used for the analysis of linkage in gametes and the construction of genetic linkage maps (Conkle et al. 1981; Grattapaglia and Sederoff 1994; Tulsieram et al. 1992; Nelson et al. 1993). For plants that do not have megagametophytes large enough to be used for sampling and DNA isolation, double pseudo-testcrosses may be used for map construction (Grattapaglia and Sederoff 1994; Echt et al. 1993; Hemmat et al. 1994; Weeden 1994) . In the double pseudo-testcross approach, genetic analysis is conducted on both parents in a controlled cross by keeping track of which loci are heterozygous in each parent . Two maps are generated in an F1 population produced by crossing the two non- inbred lines and utilizing the apparent high level of heterozygosity of the parents in this outcrossing species. In such a cross of heterozygous lines, many single-dose polymorphic markers are heterozygous in one parent, null in the other and therefore segregate 1:1 in their progeny. The term "double pseudo- testcross" (Weeden 1994) was used for this strategy, because the genotype of the markers in each parent is not known before 66 the marker is scored. This is in contrast to the standard testcross configuration where the tester is homozygous recessive in the loci analyzed. In the double pseudo-testcross strategy, the two parents serve for each other as a tester. Outcrossing species generally exhibit high levels of genetic polymorphism and individuals in these species have a high level of heterozygosity. In this paper, we report the development of asparagus genomic maps based on segregating RFLP, RAPD, and isozyme markers in a double pseudo-testcross population derived from non- inbred parents . One map was constructed for each parent and the two maps were aligned where possible to provide an integrated map . Material and Methods Plant Material The genetic analysis was performed using a mapping population of 63 plants produced from a controlled cross between two heterozygous clones, A19 (male, Mm) and MW25 (female,mn) . A19 is a parent used in the asparagus breeding program in Michigan State University, and MW25 is a seedling selected for genetic studies based on a moderate degree of allozymes diversity. These parents appeared to have many corplementing loci to provide useful information in mapping (Brettin and Sink 1992) . 67 Isozyme analysis Fern and newly emerged spears of asparagus plants grown in the greenhouse or field were used for the analysis of aconitase (ACO) , isocitrate dehydrogenase (IDH) , malate dehydrogenase (MDH) , peroxidase (PRX) , shikimate dehydrogenase (SKDH) and triosephosphate isomerase (TPI) . Isozyme analyses were conducted according to standard protocols modified for asparagus (Brettin and Sink 1992) . All enzymes were extracted with 0.1 M potassium phosphate, 0.005 M EDTA, pH 7.5 extraction buffer. Electrophoresis was conducted on 12% horizontal starch gels by using six standard buffer systems (Brettin and Sink 1992) for resolving the different allozymes. Other enzyme systems, alcohol dehydrogenase (ADH) , esterase (EST), glucose-6-phosphate dehydrogenase (G6PDH) , glutamate dehydrogenase (GDH) , nitrite reductase (NIR) and phosphoglucomutase (PGM) , which have not been described previously in asparagus were resolved on horizontal gels . The protocols for the new enzyme systems were as described in Geoffriau et al. (1992) for ADH and PGM, Soltis et al. (1983) for EST, Vallejos (1983) for GDH and NIR and Conkle et al. (1982) for G6PDH. mm Isolation Total genome DNA was isolated from fresh fern tissue using the protocol of Doyle and Doyle (1990) with some modifications. Cold (-20°C) ethanol (2.5 volumes of the DNA solution) was 68 used in place of isopropanol to precipitate the DNA. The sample was dissolved in TE buffer (10 mM Tris-HCl (pH 7.4) , 1mM EDTA) , and adding 5 M NaCl and pre—warmed 2x CI‘AB buffer. The mixture was incubated at 60°C for 30 min with frequent agitation. Subsequently, the mixture was treated with an equal volume of chloroform: isoarmyl alcohol (24:1) , and the DNA was precipitated with 2.5 volumes of cold ethanol. Finally, the dried precipitate was dissolved in TE buffer. DNA concentration was measured by fluorometer, gel electrophoresis and/ or by comparison with fluorescence on an ethidium bromide-stained agarose plate. RFLP Assay Methods used for the RFLP analysis are as described by Lewis and Sink (1995) . RAPD Assay Random 10-bp primers (kits A through AL) were obtained from Operon Technologies Inc. (Alameda, CA). Amplification reactions were carried out according to Haley et al. (1994) : reaction mixture (25 ul) included 25 ng of genome template DNA, 25 ng of primer, 200 mM of each dNI'Ps (dATP, dTTP, dGTP and dCI'P) , 5mM MgCl2 and 2.5 units of Ampli Taq Stoffel DNA polymerase (Perkin-Elmer Cetus) in reaction buffer. The PCR cycling profile used was 3 cycles of 1 min/94°C, 1 min/35°C, 2 min/72°C, 34 cycles Of 15 S/ 94°C, 15 S/ 40° C, 75S/ 72 °C, 69 and 1 cycle of 7 min/ 72°C. The reaction was run in a Perkin- Elmer Cetus System 9600. RAPD products were analyzed after electrophoresis in 2.0% agarose gels (BRL) for 5 h at 100V in 1x TBE buffer (Sambrook et al. 1989) . The gels were stained in 1mg/ml ethidium bromide solution for 30 min and photographed on a transilluminator using either Polaroid film 667 or video imaging system Gel Print 20001 and saved for corputer analysis and scoring. Primer Pre-screening: The two parents and six randomly chosen mapping progeny were used for the initial screening of a total of 760 10-bp random primers.Distinct RAPD fragments that were polymorphic (presence vs absence) between the two parents (BC loci) and segregated in the progeny (Fig. 1A and B) were detected and were subsequently analyzed for the entire mapping population . These markers will be referred to as "BC" loci. Primers that generated RAPDs heterozygous in both parents (HZ loci) and segregated in the progeny (Fig. 1C) were also detected and analyzed in the mapping population. These markers will be referred to as HZ loci. Using the six individual progeny, the probability of missing a polymorphic marker useful for mapping (1:1 segregation ratio) is about 9.4%. Loci Nanenclature The identification of each marker on the maps included the marker type ("0" for RAPD markers, "R" for RFLP markers) , and 70 the primer or probe used to generate the polymorphism and the type of the data ("-" for the natural uninverted data, " . " for inverted or recoded data) followed by the size of the marker in 1/10 base pairs. Segregation and linkage analysis Segregating RAPD markers were scored for presence or absence of the appropriate band. Three separate data sets were kept according to the parental origin of the markers: markers present in male (A19) parent and absent in female (MW25) parent (Fig 2A) , markers present in MW25 and absent in A19 parent (Fig. 2B) or markers present and heterozygous in both A19 and MW25 parents (Fig. 1C) . In the first two cases, the bands are expected to segregate 1 :1 band presence to band absence in the pseudotestcross, while in the third case, the expected segregation ratio is 3 :1 present to absent. Markers that were present in A19 (heterozygous) and absent (null) in MW25 were mapped in the A19 parent , and markers that were present in MW25 (heterozygous) and absent in A19 (null) were mapped in the MW25 parent . These data sets were analyzed as a backcross. Polymorphic fragments were scored as "H" (heterozygous) for present, "A" for absent, and "-" for missing data in the whole population. The markers were tested for eipected 1:1 or 3:1, 1:2:1 using Chi-square test. All marker fragments that deviated significantly from the expected ratio at p= 0.01 (but not at p= 0.05,) were used for linkage 71 analysis (Appendix). Prior to linkage analysis, the data for each marker were recoded or "inverted" ("H" for absent, "AF for present) and merged with the normally coded markers to detect repulsion phase linkage (Grattapaglia and Sederoff 1994) . RAPD markers together with the published RFLP markers (Lewis and Sink 1995) were jointly analyzed with MAPMAKER Version 3.0 (Lander et al. 1987) . The MAPMAKER backcross model assumes that all markers are in the coupling phase and consequently does not recognize linkages for markers in repulsion. The repulsion phase linkages can be detected by analyzing recoded data (1 .e. , presence recoded to absence, and 'vice versa) together with.the unrecoded.data set. Analysis of the combined data yielded twice the expected number of linkage groups , corresponding to the two hotologous chromosomes within each parent. The two homologous groups contained inversely coded markers in the same exact locus order. .A. LOD score of 4.0 and maximum distance p=0.30 were set as default linkage thresholds for grouping markers . The preliminary framework of the linkage orders was established with the order and/ or matrix correlation method and the "ripples" command was used to 'verify' the order; Markers 'were retained. within. the framework only if the LOD value for ripples was > 3.0. All remaining markers were assigned to intervals within the LOD 3.0 framework using the "try" command. Map distances in cM were calculated using the Kosambi mapping function (Kosambi 72 1944) . Error detection was used to find possible miss-scoring or errors . The potential errors were re-checked and were treated as missing data if they were difficult to confirm. Loci with alleles segregating from both parents were represented in both maps, which provided a set of comon loci that served as a locus bridge to align horologous linkage groups . JoinMap was used to identify linkages between the BC and HZ markers. A linkage LOD score of 4.0 and a map LOD of 0.5 were used for the JoinMap analysis. Finally, corputer program DRAWMAP (Van Ooijen 1994) was used to draw the map with sore final modifications in CorelDraw ver. 3.0. Results and Discussion For the enzyme systems examined, only MDH, SKDH, ACO, PGVI and IDH had sufficient activity to be scored. MDH, SKDH and A00 exhibited polymorphisms. In prescreening the 760 10-base arbitrary primers, from three to 18 fragments were generated by each primer and 30% of the primers identified at least one polymorphism. About 14% of the primers did not produce any band and 16% generated more than one polymorphic band pattern. A total of 483 marker loci were identified in the mapping population including 382 RAPDs, three allozymes, sex (the single morphological trait), and 87 RFLP markers. Chi- square analysis of the fitness of the segregation of markers 73 indicated that 6.3% of RAPD markers (Appendix) and 5% of RFLPs deviated significantly from the expected Mendelian ratio. This frequency of segregation distortion was similar to that reported for some of other plant species, such as pine (Kubisiak et al. 1995) , Eucalypus grandis and E. urophyllas (Grattapaglia and Sederoff 1994) , sorghum (Pereira et al. 1993); but lower than that for carrot (Schulz et al. 1994) and celery (Huestis et al. 1993) . According to Schulz et al. (1994) , the distorted segregation ratio may be caused by gametic or zygotic selection or poorly scorable bands in small mapping populations . Markers with distorted segregation ratios (deviated significantly at p=0.01 but not at p=0.05) were included in the linkage analysis, because according to Schulz et al. (1994) , the gametic or zygotic selection will have no effect on the estimation of the recombination value of two marker loci as long as at least one marker shows codominance. Of the 346 markers identified, 183 were inherited from A19 and 163 from MW25. The markers in the A19 map were assigned into 9 linkage groups covering 1324.1 cM (Fig. 3). The MW25 map was constructed with 179 markers which formed 13 linkage groups covering 1280.9 cM (Fig. 3) . Thirty—one HZ loci were found segregating from both parents . The HZ loci were used to identify homologous linkage groups between the two maps, eight of them were mapped in both maps (the other 23 HZ markers were unlinked), including 4 RFLP and 4 RAPD polymorphisms . Six RFLP markers which segregated in the 74 pattern a/c x b/c from both parents were also used as locus bridges. For some linkage groups (groups II and VI) , two or more locus bridges were found. Some groups, such as I, III, IV and V, contained only one locus bridge and the relative orientation of these groups could not be determined. Five relatively small linkage groups in the MW25 map were horologous to two single large groups A19-II and A19-VI in the A19 map (Fig. 3) . The number of linkage groups of MW25 map was reduced to 10 (2n=2x=20) when more than one linkage group of this map showed homology to a single group in the A19 map. There are 23 and 10 unlinked loci in the A19 and MW25 map, respect ive ly . Two maps were produced using molecular markers and the double pseudo—testcross mapping strategy. This mapping strategy can be used in any type of molecular markers. Because the primary type of segregation was 1:1 band presence to band absence in the mapping population, the dominant nature of RAPD markers, which is a significant disadvantage when mapping using an F2 population, is not a limitation. In cases where both parents are heterozygous , RAPD markers are less informative than codominant markers. In this situation, the fragment-absent phenotype (horozygous recessive) is still useful for the mapping, but it occurs in only one-fourth of the progeny. These RAPD markers are useful for identifying hotologous linkage groups between the two maps . In asparagus, four out of 22 (18%) such markers were used to join the 75 horologous linkage groups in the A19 and MW25 maps. RFLP markers, which are codominant and segregated 1:2:1 in the HZ loci and 1:1:1:1 in a/c x b/c loci, were more informative than RAPDs; hence, more useful for the identification of homologous groups between the two maps. Four HZ type RFLP and six a/c x b/c type locus bridges were found in six pairs of linkage groups from the two maps. The two maps cover similar distances. In both maps, the markers are not distributed evenly . The average map distance between adjacent markers is 7.7 and 7.9 cM for A19 and MW25 map, respectively. The largest gap in the MW25 map is 28.5 cM between OV14-40 and OT02-92 on linkage group MW-C. In the A19 map, the largest gap is 29.3 cM between OAC12.66 and RF06-34 on linkage group A19—V. Clusters of markers are also present in several groups in both of the two maps. For the A19 map, the biggest cluster is on groups A19-VI. With the MW25 map, the cluster region located on MW-VI which is homologous to the cluster on group A19-VI of the A19 map. These high density regions may correspond to centromeric regions or in sore cases, teloreric regions (Tanksley et al. 1992) . Clustering of markers may be the result of inhibited crossover of chromosoral sequences and also it could be due to the presence of heterochromatin in the centromeric region (Lodhi et al. 1995; Tanksley et al. 1992) . Asparagus chrorosomes were karyotyped as metacentric and submetacentric and satellites exist in some of the ten chrorosore pairs 76 (Loptien 1979) . Additional effort would help to join the molecular linkage map to the chromosoral karyotype, and the present maps may be found to reflect the identity of the centromeric regions. Clusters were also found at the end of linkage groups, such as A19—V and A19-IV, these high density marker regions may correspond to telomeres. Recombinational hot spots on a chromosome may also result in the uneven distribution of markers on the linkage map (Lindahl 1991) . All the 81 RFLP markers, 7 bridged horologous linkage groups in Lewis and Sink's map (1995) were integrated into the current map. Six unmapped markers, three for A19 parent and 3 for MW25 parent were mapped. Only Mdh loci and one RFLP marker, RIP-34, and sex locus M were shared by the Italian map (Restivo et al. 1995) and our map. Data inversion or recoding was used to detect repulsion linkage phase. Because of the joint analysis of naturally and inversely coded data, two duplicate maps were formed for each parent . Similar linkage groups were identified and only the copy containing more naturally-coded markers was retained for further analysis. The merging of the natural and recoded data also decreased the number of unlinked markers and led to the merging of small linkage groups into larger ones, leading to a decrease in the overall number of linkage groups. One allozyme, four RFLP and 14 RAPD markers were assigned to the A19-V which carries the sex-determination locus (Loptien 1979). RAPD markers OC15-98 and OC15-30, 77 generated by the same primer, were found closely (1.6 cM) linked with the M allele of the sex locus. Thus, male genotypes (MM and Min) could be identified by scoring OC15-98/ OC15-30 .However, because both OC15-98 and OC15-30 are dominant RAPD markers, the genotypes NM, Mm cannot be distinguished. The genore size of asparagus is approximately 1.79 x 109 bp/C (Galli et al. 1988) . For the A19 and MW25 maps, 1 cM presently corresponds to 1351.8 kbp and 1397.4 kbp, respectively. Corpared with ca. 750 kbp/CM for the high density torato map, which is used for genome walking and gene cloning (Flavell et al. 1993) , both of the present asparagus maps appear to be only moderately saturated. When compared with the maps constructed by Restivo et al (1995) , which contained 43 markers in 12 linkage groups and Lewis and Sink' s 81-marker map (1995) , the present map is more saturated. 78 Fig.2.1 Primer pre-screening results. "BC" type RAPD polymorphism produced by primer OP13 was inherited from paternal parent A19 (A); primer OZ19 was inherited from maternal parent MW25 (B); and "HZ" type polymorphism inherited from both parents (C). From left to right: 123 bp DNA ladder, MW25, A19, and 6 individuals in the mapping population. Polymorphic bands are indicated by arrows . 6L 80 Fig.2.2 Segregation pattern of RAPD markers in the mapping pulation. In both panels, the first lane is the 123 bp DNA adder; the second and third are MW25 and A19, respectively. The other lanes are individuals in the mapping population. RAPD assay with primer OP13 (A) and primer 0219 (B). Segregating RAPD markers are indicated by arrows . 8 Moo 5o 223m >3 0:. . . anm D.Dinvt m 30mg? lllv 82 z/{Nm >3 wanna Illnv 83 Fig. 2.3 Genetic linkage maps of asparagus parents A19 and MW25. Solid black bars re resent linkage groups identified in A19 and the open bars indicate linkage groups of MW25 . 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O O > w 00.0301 020.001 0.80.001 0800.00. 0209.001 0000.001 0.30.81 0000.21 0.65.001 0<00.001 050.001 02.5..001 0>00...T 0......001 0):.0001 Oontfil 02.30.01 0000-301 0 055.001. 0)00.-001 9.6.0.001 02000.31 0260.001 030-001 $30 > O U 10>0.0 0o 0.50.00 10x0?00 10020.00 10>:.0..o 00.0. .00 \0...o.00 1050.50 1050.00 /0>moo.u0 1000. .00 100.600.: 100.000 w 10.00.00 10.30.00 .102.0..00 0>00000 R01 .wma. 1020.900 10.00.00 19.60.00 100.000 10>0.0.00 1080.00 10000.05 010100.00 10x. 0.00 1005.00 1050.8 10200.0. 10))0000 10:00.0. 16‘3.UN fij”§.50 10.60.00 C 86 LITERATURE CITED Botstein, D., White, R. L. Skolnick, M. and Davis, R. W. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am. J. Hum. Genet. 32:314—332. Brettin, T. S. and Sink, K. C. 1992. Allozyme variation and genetics in asparagus. J. Heredity, 75. 383— 386. Colby, L. W. and Peirce, L. C. 1988. Using an isozyme marker to identify doubled haploids from anther culture of asparagus. 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RAPD linkage mapping in a long leaf pine x slash pine F1 family. Theor. Appl. Genet. 90: 1119- 1127. Lander, E.S., Green, P., Abrahamson, I., Barlow, A., Daly, M.J., Lincoln, S.L., and Newsbers, L. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental populations. Genomics, 1: 182- 195. Lewis, M. and Sink K.C. 1995. RFLP linkage map of Asparagus. Genome (submitted). Lindahl, K.P. 1991. His and.hers recombinational hot spots. Trends Genet. 7:273-276. Lodhi, M.A., Daly, M.J., Ye, G.N., Weeden, N.F., and Reisch, B.I. 1995. A molecular marker based linkage map of Vitis. Genome, 38:786-794. 88 Loptien, H. 1979. Identification of the sex chromosome pair in asparagus (Asparagus officinalis L.) . Z. Pflanzenzuc tg. 82:162-173. Morgante, M., Rafalski, A., Biddle, P., Tingey, S., and Olivieri, M. 1994 . Genetic mapping and variability of seven soybean simple sequence repeat loci. Genome, 37: 763—769. Nelson, C.D., Nance, W.C., Doudrick, R.L. 1993. A partial genetic linka e map of slash pine (Pinus elliottii Engelm. var. elliottii) based on random amplified polymorphic DNAs. Theor. Appl. Genet. 87:145-151. Pereira, M.G., Lee, M., Bramel-Cox, P., Woodman, W., Doebley, J ., and Whitkus, R. 1994. Construction of an RFLP map 1n sorghum and comparative mapping in maize. Genome, 37:236-243 . Restivo, F.M., Tassi, F., Biffi, R., Falavigna, A., Caporali, E., Carboni, A., Doldi, M.L., Spara, A., and Marzianl, G.P. 1995. Linkage arrangement of RFLP loci in progenies from crosses between doubled haploid Asparagus officinalis L. clones. Theor. Appl. Genet. 90:124-128. Robbins, M.A., Witsenboer, H., Michelmore, R.W., Laliberte, J.F., and Fortin, MG. 1994. Genetic mapping of turnip mosaic virus resistance in Lactuca sativa. Theor. Appl. Genet. 89:583-589. Sambrook, J., Fritch, E.F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, New York, Cold Spring Harbor Press. Schulz, B., Westphal, L., and Wricke, G. 1994. Linkage groups of isozymes, RFLP and RAPD markers in carrot (Daucus carota L.sativus). Euphytica, 74:67-76. Shoemaker, R.C., and Specht, J.E. 1995. Integrating of the soybean molecular and classical genetic linkage groups. Crop Sc1. 35:436-446. Soltis, D.E., Hanfter, C.H., Darrow, D.C., and Gastony, G.J. 1983. Starch gel electrophoresis of ferns: A compilation of buffers, gel and electrode buffers, and staining schedules. .Amer. Fern J. 73:9-27. Tanksley, S.D., Ganal, M.W., Prince, J.P., de Vicente, M.C., Bonierbale, M.W., Broun.P., Fulton, T.M., Giovannoni, J.J., Martin, G.B., Messeguer, R., Miller, J.C., Paterson, A.H., Pineda, 0., Roder, M.S., Wing, R.A., Wu, W. and Young, N.D. 1992. High density molecular linkage maps of the tomato and potato. Genetics, 132:1141-1160. 89 Tulsieram, L.K., Glaubitz, J.C., Kiss, G., and Carlson, J.E. 1992. Single tree genetic linkage mapping in conifers using haploid DNA from megagametophytes. Blo/Technology, 10: 190— 194. Vallejos, C.E., Sakiyama, N.S., and Chase, C.D. 1992. A molecular-based linkage map of Phaseolus vulgaris L. Genetics, 131:733-740. Vallejos, C.E. 1983. Enzyme activity staining, P.481-512. In: Tanksley, S.D. and Orton, T.J., (eds.) . Isozymes in plant genetics and breeding, Part A. Elsevier, Amsterdam. Van Ooijen, J. W. 1994. DrawMap: a computer program for drawing genetic linkage maps. J. Hered. 85:66. Weeden, N. F. 1994. Approaches to mapping in horticultural crops, In P.M. Gresshoss (ed.) Plant Genome Analysis, pp57- 68, CRS press. Williams, J., Kubelik, A., Livak, K., Raflski, J., and Tingey, S. 1990 . DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nuc. Acids Res. 18:6531-6535. :rgq, m- r K Secfion 3 RAPD and SCAR Markers Linked to the Sex Determination locus M in Asparagus 9O 91 Abstract Bulk segregant analysis (BSA), random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) techniques were used to map the sex determination locus M and linked markers in dioecious Asparagus officinalis L. . Two asparagus clones, A19 (male,Mm) and MW25 (female,mm), and 63 progeny were used for the study. Two DNA bulks were made, one male and one female, by pooling equal amounts of DNA from such individuals in the population. A total of 760 arbitrary decamer oligonucleotide primers were used for RAPD analysis, of which two, OPC15 and OPAAO2 , produced RAPD bands linked with the sex gene. RAPD markers OC15-98 and OC15—30, which were amplified by the same primer, were both found closely linked to maleness (M_) at a distance of 1.6 cM, while marker 0AA02-38 was linked to the female genotype (mm) at a distance of 11.5 cM. Amplified RAPD fragment OC15-98 was cloned and sequenced. The sequence was used to design flanking 24-mer oligonucleotide primers. This pair of SCAR primers amplified a single DNA band with the same size as the RAPD fragment cloned. The SCAR marker remained dominant as RAPD marker OC15- 98 from which it is derived. Key words: Asparagus, RAPD, SCAR, sex determination : - m .Li'v'!"’ 92 Introduction Asparagus (Asparagus officinalis L.) is a dioecious perennial plant which is native to Europe and Eastern Asia. As a dioecious plant, asparagus provides a great challenge for genetic and physiological studies on sex determination and expression. Male plants have higher yields and vigor than females and males do not create "asparagus weed problem" by producing seeds which may germinate and grow in the field (Yeager and Scott 1938) . Thus, an all-male population is one of the major goals of asparagus breeding . According to Loptien (1979) , sex determination is controlled by a single dominant gene M which is located on the L5 chromosome. The male genotypes are dominant (M_) , while the female is homozygous recessive (mu). Normal crosses between males and females give populations that typically segregate 1:1 male to female. However, there are so-called supermales (le) that when crossed with females, yield all male populations. Supermales can be produced by self ing the rarely occurring hermorphroditic flowers of andromonoecious plants or by anther culture. Presently, in order to distinguish male and female plants, the seedlings or tissue culture—derived plants must be grown to flowering which generally takes 2-3 years. In addition, to identify supermale plants, testcrosses must be performed by crossing to a female. This whole process generally takes about 5—6 years (Reuther 1984) . Franken (1970) in a study on the inheritance of 93 andromonoecy in asparagus suggested that male and female plants possess modifying genes which may influence pistil development . The modifying genes do not modify the female plants, since the plants have the major gene and the flower already has fully developed immature pistils. In males, however, depending on the presence or absence of the inhibitor, modifying genes become active, even in some cases producing functioning pistils (Bracale et al. 1991) . The presence of these modifying factors complicate the study of sex determination. Bracale et al. (1991) searched for protein messages specifically expressed in reproductive structures by 2D-electrophoresis of existing and newly synthesized polypeptide or of in vitro translation products of poly (A)+ RNA from male and female flowers and by isolating specific monoclonal antibodies against sex specific floral antigens. A set of floral specific polypeptides which are not present in the phylloclads was found. These floral specific polypeptides are mainly associated with reproductive organs . The polypeptide patterns are remarkably similar in male and female flowers at the premeiotic stage; thus, in agreement with indistinguishable floral appearance at this stage of development. Differences, as expected, were observed in post meiotic male and female flowers either at the level of existing, newly synthesized and poly (A)+ RNA in vitro translated product . Bracale et al. (1991) and Restivo (1995) found 94 isozyme marker malate dehydrogenase (MDH) linked with the sex factor on chromosome 5 at a distance of about 20 cM. In addition, an RFLP marker was reported by Restivo (1995) linked with the sex locus at 6 . 9 cM. Obviously, both markers are too far away from the sex gene (s) to be used for the sex identification . Molecular markers and the bulk segregant analysis (BSA) strategy (Michelmore et al. 1991) have been widely used in disease resistant gene tagging. Michelmore et al. (1991) used BSA for the rapid identification of markers linked to the gene(s) for resistance to downy mildew in lettuce. Haley et al. (1994a, b) described the finding of RAPD markers linked to disease resistant genes in common bean. Mulcahy et al. (1992) and Hormaza et al. (1994) found several markers for sex determination in Silene latifolia and Pistacia versa, respectively, both of which are dioecious species. In this paper, we describe the associations found between the sex locus with RAPD markers . A SCAR (sequence characterized amplified region) marker was developed from a RAPD marker closely linked to sex locus. Material and methods Plant material The plant materials used in this study were the mapping population produced from the cross between A19 (male,Mn) and MW25 (female,mn) parents. A19 is a plant used in the asparagus 95 breeding program at Michigan State University, and MW25 is a seedling selected from a Mary Washington population. The mapping population has 63 individuals, including 32 males and 31 females. DNA isolation Total genomic DNA was isolated from 1-2 gram fresh fern tissue using the protocol of Doyle and Doyle (1990) with some modification: Cold (—20°C) ethanol (2.5 volumes of the DNA solution) was used in place of isopropanol to precipitate the DNA. The DNA was dried and dissolved in TE buffer (10m Tris— HCl, 1 mM EDTA, pH 7.4) , and adding 5 M NaCl and pre-warmed 2x CI‘AB buffer. The mixture was incubated at 60°C for 30 min with frequent agitation. Subsequently, the mixture was treated with an equal volume of chloroform: isoamyl alcohol (24:1) , and the DNA was precipitated with 2.5 volumes of cold ethanol. Finally, the dried precipitate was dissolved in TE buffer. DNA concentration was measured by f luorometer, gel electrophoresis and/ or by comparison with the fluorescence on an ethidium bromide stained agarose plate. Two bulks of DNA. samples were made by pooling an identical amount of DNA from 10 individuals: one for male and one for female. All DNA samples were diluted to approximately 10 ng/ul in distilled water prior to use as template for PCR. 96 PCR analysis A total of 760 random 10—bp primers (Kits OPA through OPAL) obtained from Operon Technologies Inc. (Alameda, CA) were used to screen for DNA markers linked with sex locus. For each primer, DNAs from the two parents, six individuals in the mapping population and two sex bulks were used for polymerase chain reaction (PCR) . The amplification reactions were carried out according to Haley et al. (1994a): 25 ul reaction mixture included 25 ng of genome template DNA, 25 ng of primer, 200 mM of each dNTPS (dATP, dCI‘P, dTTP, dGI'P), 5 mM MgCl2 and 2.5 units of Ampli Taq Stoffel DNA polymerase (Perkin-Elmer Cetus) in reaction buffer. The PCR cycling profile used consisted of the following steps: 3 cycles of 1 min/94°C, 1 min/35°C, 2 min/72°C; 34 cycles of 10 S/94°C, 20S/40°C, 2 min/72°C; 1 cycle of 5 min/ 72°C. The reaction was run in a Perkin-Elmer Cetus 9600 PCR system. The PCR reaction products were analyzed after electrophoresis in 2.0% agarose (BRL) gels for 5 h at 100V in 1x TBE buffer (Sambrook et al. 1989) . The gels were stained with 1mg/ml ethidium bromide solution for 30 min and photographed on a transilluminator using either Polaroid film 667 or video imaging system Gel Print 20001 and saved for computer analysis and scoring. DNA markers present in one parent and the corresponding bulk of male or female and absent in the alternate parent and sex bulk were recognized as a potential 97 sex linked marker. Subsequently, PCR analysis was conducted using these primers on DNAs from the other plants in the mapping population. Sequence Characterized Amplified Region (SCAR) The RAPD fragment, found linked to sex determination, was cloned using the methods of Maniatis et al. (1989): the DNA fragment was excised from an ethidium bromide stained agarose gel, and the DNA was purified by the "Pre-A-Gene" kit (Bio- rad) . The purified DNA was reamplified using the same primer that identified the RAPD polymorphism. Filling and phosphorylation of the fragment termini were performed using the Klenow fragment of E. coli and T4 DNA kinase according to Maniatis et al. (1989) . After purification, it was blunt-end ligated into Sma I site of pUCl9 plasmid as described by Maniatis et al. (1989). Subsequently, competent cells of E. coli strain 10HB (BRL) were transformed by electroporation. The cells were then spread on LB medium containing ampicillin (50ug/ml) , IP‘IG (0.95 ug/ml) , and X-gal (40ug/ml) , and incubated at 37°C overnight . Transformants with inserts were identified as white colonies. Plasmid minipreps were prepared from overnight cultures using the Bio—Rad (Hercules, CA) Prep- A—Gene DNA MiniPrep kit. The identities of the cloned products were verified by hybridization of the cloned fragment to Southern blots of individuals that segregated for that particular RAPD. Double—strand sequencing was done by the —_..—2_ I. at] - all . ‘_ L_._._ -_. _=A —- — \f.‘ 98 dideoxynucleotide chain termination method using primer M13 and -21M13 . Two oligonucleotides were designed to be used as SCAR primers. Each primer contained the original 10 bases of the RAPD primer plus the first 14 bases from each end. Amplification of genomic DNA with SCAR primers was done using the protocol of: 1 cycle of 94°C/4 min, 30 cycle of 94°C/ 1 min, 67°C/1 min, 72°C/2 min; 1 cycle of 72 °C/7 min. The amplification products were resolved by electrophoresis in a 2.0% agarose (BRL) gel. Linkage Analysis RAPD and SCAR markers which were assumed linked with the sex locus were analyzed for fitness to Mendelian segregation ratio by Chi-square. Marker order and map distances were estimated using MAPMAKER version 3.0 b (Lander 1987) , LOD score of 4.0 was used. Map distance was calculated using Kosambi map function (Kosambi 1944). Results and Discussion The three RAPD markers found linked to the sex determinate locus were RAPD markers OC15-30 and OC15-98 which were amplified by primer OC15(5' GACGGATCAG 3') and OAA02—38 amplified by primer OAAOZ (5'GAGACCAGAC3 ' )1 . OC15-30 and OC15- 98 were present only in the bulked male sample, male parent and individual males (Fig.1) , while OAA02-38 was present in 99 the female bulk, female parent and individual females and absent in males (Fig. 2) . Scoring the mapping population with each of these three markers indicated that OC15-30 and OC15-98 were linked to maleness at 1.6 cM while femaleness and marker OAA02—38 was linked at 11.5 cM. The recombination between the sex gene(s) and the OC15-98/OC15—30 and OAA02—38 loci indicates that the two " sex" homologous chromosomes in asparagus can pair and undergo crossing over, in agreement with the results of Bracale et al. (1991) . This and the viability of the MM genotype suggest that dioecy in asparagus may have been derived relatively recently (Dellaporta and Calderon-Urrea 1993; Charlesworth 1991) . RAPD fragment OC15-98 linked to maleness was cloned and verified by Southern hybridization to be associated with individuals that segregated for the particular RAPD (Fig. 3) . The hybridization pattern was identical to the segregating RAPD indicating that the cloned fragment was derived from the OC15-98 . The cloned amplification products were sequenced for about 600 bp from each end. The sequences at the two ends of the cloned RAPD products did not reveal inverted repeats longer than the 10 bases that exactly matched the primer sequences. Two specific 24 —mer primers, coded SCC15-1 and SCC15—2 and corresponding to each end of the insert, were synthesized (Table 1). When subsequently used as primers, SCC15-1 and SCC15-2 amplified a 980bp fragment in male 100 SCC15-2 and corresponding to each end of the insert, were synthesized (Table 1). When subsequently used as primers, SCC15-1 and SCC15-2 amplified a 980bp fragment in male individuals but not in females (Fig. 4). Table 1.Sequence of 24-mer oligonucleotide primers for SCAR locus derived from RAPD marker OC15-98 linked to sex locus M Primer Sequence* ii- 3 SC C 1 5-1 GACGGATCAGATCCAGTTGATAAA r") ' SCCIS-Z GACGGATCAGGAMTAGAAGCAAC l *The underlined sequence represents the sequence of the progenitor RAPD primer Both RAPD and SCAR analysis of the 63-plant mapping population indicated that the observed polymorphism with the two techniques corresponded to the same locus. For the SCAR marker, amplification occurred with both parents 2' 603C annealing temperature (data not shown) , but when the annealing temperature was increased to 67’=c, only one band was amplified in males and none in females (Fig.4), Thus, this SCAR was scored as a dominant marker. Digesting the SCAR fragment produced at the 609C annealing temperature with four endonucleases (.E'aeIII, MboI, RsaI, Altf) failed to produce a small fragment length polymorphism. This result is different 101 enzymes tried. SCAR marker are sometimes advantageous over RAPD markers because they detect only a single locus and their amplification is less sensitive to reaction conditions. Since only one PCR-product is generated, post-amplification electrophoresis could be eliminated because the amplified DNA can be visualized either by staining with ethidium bromide or by measuring DNA concentration in the reaction mixture using an ELISA reader or other rapid scanning devices capable of measuring absorbence at 260 nm (Weeden 1994) . In addition to its use as genetic markers, SCAR's will be useful in physical mapping. SCAR' s will bridge the gap between the ability to obtain molecular markers linked to genes of interest in a short time and the use of these markers in a map-based cloning approach (Paran and Michelmore 1993) . In asparagus , the linked marker could be used for screening genomic libraries for physical mapping of the M region and to obtain overlapping clones in the process of chromosome walking to clone the M gene. Genetic evidence indicates that sex determination in asparagus is controlled by a single gene located on chromosome pair L5. Thus, markers OC15-98, OC15-30 and SCC15 may be linked to the M allele in the Min plants, in another word, these markers linked with sex locus in a coupling phase. These markers could be used to discriminate genetic males (Mm) from female (mm) plants. RAPD marker OAA02-38 was linked with the 102 "femaleness" of the plants in the mapping population. Marker OAA02-38 segregated as a "recessive marker" which is present in the homozygous recessive genotype (female, mmu and.absent in heterozygous individuals (male, Mm). This association cannot be explained by the hypothesis of repulsion phase linkage, because recessive allele m is present in both male (Mm) and female (mm) plants. Marker OAA02-38 may be associated with the modifier gene (Franken 1944; Bracale et al. 1991) . To prove this hypothesis, genetic linkage analysis 'with. a population containing all the three genotypes, MM, Mm and mm, should.be conducted. Marker—assisted selection (MAS) proposed. by Melchinger (1990), could.greatly facilitate sex.gene tagging in asparagus, since direct selection for the sex phenotype is time consuming;.According'to'Tanksley'(1983), the application of MAS requires tight linkage (less than 5 cM) between the marker and.gene of interest or the use of two loosely linked markers flanking the targeted locus. During the gene tagging process, coupling—phase as opposed to repulsion-phase markers are usually sought. However, in asparagus sex gene mapping, the genotypes Mm and MM cannot be distinguished with the coupling phase marker. Haley et al. (1994a) showed that selection against a repulsion-phase RAPD, as opposed to selection.for a couplingephase RAPD in.disease resistant gene tagging in common bean is more useful, since the genotypes homozygous and heterozygous resistant can be distinguished. 103 Furthermore, it was indicated that even a loosely linked (7. 1 cM) repulsion—phase marker provided greater selection efficiency than a tightly linked (1.9 cM) coupling-phase marker. In order to identify a repulsion phase linkage marker (marker linked with recessive m allele at the sex locus), a population containing the three genotypes, MM, Mm and mm should be used. This kind of population can be derived from the selfing of perfect flower on andromonoecious plant or from the cross of supermale and female and backcross the F1 male to the female. To do this, three parents-- supermale, F1 male and female-— and three bulks each from one genotype are needed. These dominant coupling phase linked markers can be converted into a repulsion phase marker by the method of recombination—faciliated RAPD using a recombinant line from the original mapping population as parent (Miklas et al. 1996; Kelly 1995) . First, the female (mm) plant with the dominant OC15-98/OC15-30 markers (recombinant individual) in the mapping population is crossed to a male or supermale plant lacking the dominant markers (can be individuals outside the mapping population or from another gene pool) . Then one male progeny is selected and crossed to a female plant either without or with these markers (Fig. 5) . If a plant without markes was used as female parent, all the male progenies should not have marker OC15-98/OC15-30 and all the females should have these markers . Thus, selection against the marker 104 would be practiced to identify the male (Mm) individuals. If a plant with markers was used as female parent, both male and female progenies in the population should have the markers and, thus, supermale (MM) individuals can be identified by selecting against these markers. This recombination- facilitated marker-assisted selection can be used in asparagus sex identification to overcome gene pool specificity of the dominant RAPD markers and improve selection efficiency. RAPD markers OC15—98/OC15—30 and SCAR marker SCC15 can be used in the sex identification at the early stage of asparagus growth . 105 Fig.3.1 RAPD markers OC15—30 and OC15-98 linked to maleness of asparagus. From left to right, lane 1 is 123-bp DNA molecular weight ladder, lanes 2 and 3 are parent MW25 (female) and A19 (male), respectively. Lanes 4-13 are female (F) and male (M) individuals of the mapping population. Segregating RAPD markers are indicated by arrows . 106 107 Fig. 3.2 RAPD marker OAA02-38 linked to femaleness of asparagus. From left to right, lane 1 is 123—b DNA molecular we1ght ladder, lanes 2 and 3 are parents MW25 F) and A19 (M), respectively. Lanes 4-17 are female (F) and male (M) ind1viduals of the mapping population. Segregating RAPD marker is indicated by arrow. 108 p>>ON® m 109 Fig.3 .3 Southern hybridization of cloned OC15-98 marker to PCR roduct amplified by primer PC15. From left to right, lane 1 1s l23-bp DNA molecular weight ladder, lanes 2 and 3 are MW25 (F) and A19 (M), respectively, lane 4 is A DNA digested by HindIII and lanes 5-8 are female (F) and male (M) individuals of the mapping population. Segregating RAPD marker is indicated by arrow. (A) RAPD marker OC15-98 (arrow) linked to maleness of asparagus, and (B) Southern hybridization of the RAPD fragment OC15-98 to individuals shown in panel A. HO 111 Fig. 3.4 Amplification of genomic DNAs using SCAR primers (arrow). Left to right, lane 1 is 123-bp DNA molecular weight ladder and lane 2 and 3 are parents MW25 (F) and A19 (M), respectively. Lanes 4—16 are individual males (M) and females (F) of the mapping population. 112 E8 >3 2— m m Z3 m 113 Ca. 3 ca 7.. cc 2. X C: CA. _: cc _: cc Z 28.25.3215: Amide—.513 E ,/ >V / cc _: :0 Z 0Q _: on 2. x x on _: 00 3 00 _: On 3 OO 3 on E Co 3 On. 3 on Z CC 3 cc _: Ch. _: >:c<< 8 ding Zia 233v >=o€ 8 :22: 9:62:30 237$ Em. m. USES: 188393239790z:epaa EU quroe-mmm_m8a mianro: 3.. max 59:50.33: :35. 95:38: 7200 3918a 00 _ mom. OOH—:2.me OO _ mom. £30: :18:— 5 occczsm promo <33 Z 525. 114 LITERATURE CITED Bracale, M. , Caporali,E. , Galli,M.G. ,Longo, C, Marziani- longo,G. , Rossi,G. , Spada,A. , Soave, C. , Falav1gna,A. , Raffaldi, F. ,Maestri,E. ,RestivoF.M. , and Tassi, F. 1991 .Sex determination and differentiation in Asparagus officinalis L. Plant Science 80:67-77. Charlesworth B. 1991. The evolution of sex chromosomes. Science, 251:1030-1033. Del-Castillo I. , Cohen—Salmon M. , and Petit C. 1993. Molecular analysis of inherited diseases by positional cloning isolation and characterization of the gene responsible for the X chromosome—linked Kallmann symdrome. Methods Mol. Cell Biol 4:87-92. Dellaporta, S.L. and Calderon-Urrea, A. 1993. Sex determination of flowring plants. The plant cell, 5:1241- 1251. Doyle,J.J. , and Doyle,J.L. 1990. Isolation of plant DNA from fresh tissue. Focus, 12:13-15. Franken, A. A. 1970. Sex characteristics and inheritance of sex in asparagus Euphytica, 19:277-287. Haley,S. D. , Afanador,L.K. , and Kelly,J.D. 1994a Selection for monogenic resistance traits with coupling- and repulsion- phase RAPD markers. Crop Sci. 34:1061-1066. Haley,S.D. , Afanador,L.K. and Kell ,J.D. 1994b. Identification and application of a random ampli ied pol rphism DNA marker for the I gene (Potyvirus resistance in common bean. Phytopathology, 84 :157-160 . Hormaza,J. I. , Dollo,L. , Polito,V.S. 1994. Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis. Theor. Appl. Genet. 89:9—13. Kelly,J.D. 1995. Use of random amplified polymorphic DNA markers in breeding for major gene resistance to plant pathogens. HortScience, 30:467—465. Kosambi,D.D. 1944. The estimation of map distances from recombination values. Ann. Eugen. 12:172-175. Iander,E. S. , Green, P. , Abrahamson, I . , Barlow,A. , Daly,M.J. , Lincoln,S.E. , and Newburg,L. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental populations. Genomics, 1:182-195 . 115 Loptien, H. 1979. Identification of the sex chromosome air in asparagus (Asparagus officinalis L.) . Z. Pflanzenzuct 82:162-173. Melchinger, A. E. 1990. Use of molecular markers in breeding for oligogenic disease resistance. Plant Breeding 104. 1- 19. Michelmore, R. W., Paran, I. and Kesseli, R. V. 1991. Identification of markers linked to disease- resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by us1ng segregation populations. Proc. Natl. Acad. Sci. USA 88: 9828- 9832. Miklas,P.N. , Afanador,L. , and Kelly, J.D. 1996. Recombination- facilitated RAPD marker-assisted selection for disease resistance in common bean. Crop Sci. 36: Mulcahy D. L. W,eeden N. F. ,Kesseli, R. “Carroll S. B. 1992. DNA probes fro the Y- chromosorie of Silene latifolia, a dioecious angiosperm. Sex Plant Reprod. 5. 85- 88. Paran I. and Michelmore R. W. 1993. Development of reliable PCR— based markers linked to downy mildew resistance genes in lettuce. Theor. Appl. Genet. 85: 985- 993. Restivo, F.M. , Tassi, F. ,Biffi,R. , Falavigna,A. ,. Caporali,E. , Carboni, A. Doldi, M. L. Spara, A. and Marziani, G. P. 1995. Linkage arrangement of RFLP loci in progenies from crosses between doubled haploid Asparagus officinalis L. clones. Theor. Appl. Genet. 90: 124—128. Reuther G. 1984. Handbook of plant cell culture Vol. 2, pp. 211-239. Sharp R. S., Evanse, D. A. Ammirato, P. V. Yamada,Y. (eds. ) MacMillan Publishing Co. New York. Tanksley,S. D. 1983. Molecular markers in plant breeding. Plant Mol. Bio. Report 1 (1): 3- 8. Weeden, N. F. 1994. Approaches to mapping in horticultural crops, In: P. M. Gresshoff (ed.) Plant Genome analysis, pp57- 68. CRS Press. Yeager, A. F. and Scott, H. 1938. Studies of mature asparagus lantings with special reference to sex, survival and rooting its. Proc. Amer. Soc. Hort. Sci. 36: 513- 514. 116 Appendix 1. Segregation ratios and X 2 values of RAPD loci isozymic loci, the M locus from A19 parent Loci Obs. ratio X2 0A05-l 11 28 : 35 0.78 0A08-70 31 :29 0.07 0A08-148 28 : 29 0.02 0B06-350 34 : 28 0.58 0808-120 24 : 37 0.80 OBIS-42 29: 31 0.06 0B17-40 32: 28 0.27 0318-62 34: 29 0.40 0C11-37 27: 35 1.03 0Cll-68 36: 26 1.61 0C11-76 26: 36 1.61 0C14-62 24: 35 2.05 0C15-30 31 : 32 0.02 0C15-95 31 : 32 0.02 0C15-98 31 : 32 0.02 0D10-37 35 : 26 1.40 01312-74 31 : 32 0.02 0F04-68 36 : 27 1.29 0F13-30 27 : 34 0.80 0003-52 32 : 31 0.20 0003-80 27 : 36 1.29 0003-98 43 : 20 8.67“ OGlO-36 32 : 25 0.86 0010-80 25 : 32 0.86 0011-74 30: 32 0.14 01102-60 29 : 33 0.25 01103-68 29 : 33 0.25 01107-49 34 : 28 0.58 01109-92 29 : 32 0.14 01119-70 33 : 29 0.25 01119-46 31 : 31 0.00 0DD1-l48 30 : 31 0.02 0116-98 31 :31 0.00 OL07-46 35 : 28 0.63 0LO8-70 24 : 27 0.18 0L08-49 31 : 32 0.02 0L08-48 31 : 32 0.02 0L20-74 31 : 32 0.02 0107-86 22 : 33 2.20 M- 31 : 32 0.00 0K06-50 31 : 29 0.07 01(14-74 39 : 20 5.60* 0K20-62 38 : 23 5.59“ OKS89-46 31 : 32 0.02 0L10-43 35 :28 0.77 Appendix 1 (cont) 0M12-75 0M12-62 0KS13-98 0M17-86 0N04- 1 30 0N01-98 0N07-60 0N10-86 0Nl4-74 0Nl4-120 0012-40 0020-98 0020-92 0020-58 0020-35 0P08-86 0P08-26 0P12—37 0P12—39 0P12-40 0P1 3-40 0P1 3-46 0P15-110 0P15-49 0P17-45 SKD- 0005-86 0016-55 0R08-60 0R1 3-61 0R13-35 0R20-50 0303-37 0S05-52 0S05-36 0S18-80 0S18-49 0819-74 0106-98 0T08-1 10 0120-90 0U03-52 0U05-55 0U05-80 0U16-73 0U20-68 0V08-50 0V18-50 0W20-48 0X09-80 33 33 29 33 29 43 29 33 35 21 33 31 31 32 31 27 33 33 :30 27: 27: 32: :29 :34 26: 28: 32: 25: 36 34 29 32 30 30 36 :30 30: 28: 30: 30 34 32 :33 35: 28: 29: 29: 29: 36: 38: 35: 30: 25 33 34 30 30 27 25 28 33 :20 :29 :29 34: 28 :26 27: 34: 32: 26: 36 29 30 37 :42 28: 27: 33 32 :26 :32 :32 :28 :30 34: 30: :35 29: 38: :29 :29 32: 34: 26 31 26 23 24 29 0.14 1.29 0.80 0.14 0.25 0.60 0.62 0.06 0.06 1.98 0.14 0.02 0.58 0.06 0.58 1.29 0.60 0.60 0.02 0.02 1.29 2.68" 0.78 0.14 8.67“ 0.00 0.06 0.80 1.33 1.28 0.80 0.06 1.33 7.01 * 0.40 0.40 0.83 0.02 0.02 0.26 0.02 1.03 0.02 1.23 0.16 3.75* 0.25 0.25 1.47 0.80 117 Appendix 1 (cont) 0X19-60 0Y04-80 0Y04-90 0Y05-48 0Y08-62 0Y08-92 0Y 1 3-86 0Z08-1 1 0 0Z20-32 0AA01-42 0AA02-20 0AA02-38 0AA06- 1 7 0AA06-25 0AA 1 1-32 0AA 12- 1 0 0AA 14-42 0AA 1 5-48 0ABO 1-20 0ABO4-70 OABl 1-28 0AB1 7-67 0ABl 8-45 DAB 1 8-48 DAB 1 8-67 OAB 19-86 0AC04-53 0AC04-66 0AC05-48 0AC06-86 0AC08-61 OAC 12-64 0AC12-66 0AC1 5-57 OAC 19-52 0AD05-71 0AD06-72 0AD10-73 0AD12- l 8 0AE03-58 0AE04-74 0AE05- 12 0AE08-63 OAE 10-80 OAE14-1 2 0AE17-70 0AEZO-60 0AF 02-90 0AF04-90 OAF 06-48 41 37 33 31 31 34 23 33 30 32 36 34 32 35 31 29 20 32 34 35 34 25 31 25 29 :21 32: :21 :28 28: 32: 28: 24: 30 33 30 35 34 :29 :29 26: 35 :28 :40 :30 32: 30 :30 :28 32: 31 :26 :27 :28 29: 28: 29: 31 34 33 :27 :27 28: :22 27: 28: 30: 30: :41 :28 28: 29: :26 :25 :27 29: :29 26: 32 34 33 31 31 31 32 31 35 :31 30: 29: 30: :34 :31 28: 24: 30 34 32 34 35 118 4.59* 0.06 4.40" 0.40 0.40 0.06 0.78 1.40 0.06 0.06 1.30 1.03 4.59" 0.14 0.06 0.00 0.26 0.02 1.61 0.80 0.26 0.06 0.60 0.26 1.03 0.27 0.26 0.96 0.80 0.40 0.02 0.02 7.00"“ 0.26 0.14 0.15 1.07 1.67 0.80 0.06 0.30 1.33 0.00 0.00 0.80 0.06 1.37 0.06 0.80 1.50 119 Appendix 1 (cont) 0AF06-49 27 : 34 1.03 0AF1 1-49 26 : 35 1.26 0AF15-55 25 : 35 1.67 0AGOl-55 24 : 28 0.31 0AGO3-30 30 : 33 0.14 0AGO4—52 32 : 28 0.26 0AGO9-61 23 : 32 1.47 0AGlO-86 34 : 25 2.05 0AGl3-40 32 : 27 0.42 0AGlS-12 27:30 0.16 0AH01-86 26 : 32 0.62 0A1-113-60 28 : 33 0.40 0A1-118-12 25 : 31 0.64 0AH20-63 31 : 30 0.02 0AKO4-48 25 : 36 1.98 0AK05-43 32 : 27 0.42 MDH- 29 : 30 0.02 *,** Indicate significant deviation from expected 1 :1 segregation ratio at the 5% and 1% levels, respectively. 120 Appendix 2. Segregation ratios and X2 of RAPD loci from MW25 parent Loci Obs. ratio X2 OA05-55 32 : 31 0.02 0A09-60 29 : 30 0.02 0A09-86 31 :28 0.15 OA17-148 28 : 31 0.15 OB10—1 11 25:34 1.37 OBIS-18 29 : 30 0.02 OBIS-63 21 :36 4.38" OB18-80 34 : 29 0.20 OC01-49 30: 33 0.14 _‘. OD05-41 29 : 30 0.02 . 01313-86 26: 31 0.16 '. OF16-88 29 : 32 0.15 ,1 OFl6—26 36 : 25 1.98 11 0003-98 29 : 34 0.39 1 0610-98 32 : 22 1.85 ' 0611-104 28:29 0.02 01108-68 29 : 34 0.15 01118-92 33 : 28 0.46 01118-74 38 : 25 440* 01118-104 31 :30 0.02 OKl7-38 26 : 34 1.06 01(17-98 29 : 31 0.06 01(18-80 33 : 27 0.60 OL04-90 27 : 35 1.03 OL07-55 31 :32 0.02 OL07-46 39 : 20 5.01“ OL08-74 30 : 30 0.00 OL08-62 30 : 33 0.14 OL09-86 29 : 34 0.20 OL10-49 26 : 37 1.92 OL11-90 28 : 34 0.53 OL13-38 28: 34 0.53 OL19—30 31 : 32 0.02 OM03-37 33 : 29 0.25 ONOl-123 22: 36 3.38” ON13-73 33 : 29 0.25 ON14-135 25: 35 1.67 0003-35 27: 32 0.42 0019-35 26: 37 1.92 OP02-74 31 : 27 0.27 OP08-38 29: 28 0.02 OP08—75 27: 24 0.15 OP12—85 31 : 29 0.02 OP12-38 34 : 29 0.20 OP13-68 36 : 26 1.61 0P15-110 32 :23 1.47 Appedix 2 (cont) 0P17-92 0Q12-55 0R01-45 0R1 3-95 0S05-50 0805-85 0809-98 0S16-104 0T02-96 0T06-63 0T07-35 0U10-35 0U16-40 0V01-37 0V01-75 0V14-40 0W02-50 0W02-55 0W04-32 0W04-86 OX03-49 0X08-63 0X08-73 0X09-92 0X13-60 0X18—55 0Y08-55 0Y16-30 0Y17-61 0Y20-53 0Z06-60 0Z08-50 0Z12-18 OZ 1 9-48 OZ 1 9-68 0AA01-73 0AA01-84 0AA03-56 0AA07-37 0AA07-98 OABO 1 -64 0AB02-35 0ABl 1-52 0AB 16-42 0AB 19-49 0AC05-28 0AC15-100 0AC17-27 0AD12-80 0AD19—68 30: 37: 34: 30: 33 22 31 24: 32: 38: 36: :35 23 29: 27: 29: 27: 33 22: 29: 26: 32: .27: 29: 26: 29: 38: :29 :24 31 36 30: 20: 35: 22: 17: 26: 32: 25: 26: 37: 26: 35: 29: 40: 32: 29 24 27 31 :29 26: 30: 27: 30: :41 30: 34: 27: :30 35 33 33 27 31 29 31 39 30 19 22 33 35 33 36 :29 41 32 35 31 35 34 34 34 23 31 40 27 34 19 35 31 37 34 21 35 25 33 21 26 0.02 4.39* 0.80 0.02 0.26 1.33 0.13 0.60 0.16 4.59“ 0.02 0.20 0.28 0.02 3.57" 0.06 3.95“ 4.03“ 2.48 0.26 1.03 0.26 1.13 0.26 4.59* 0.15 1.33 0.02 1.03 0.20 1.26 0.20 3.68” 0.13 2.40 0.02 6.67“ 0.79 2.48 0.11 1.13 0.02 2.32 1.26 4.41“ 1.33 1.67 0.26 6.50” 0.62 121 WT' Appendix 2 (cont) 0AD19-52 0AE03-49 0AE06-42 0AE09-74 0AE10—72 0AE1 1-48 0AE12-45 0AE1 3-33 0AE13-41 0AE17-35 0AF02-45 0AF04-60 0AF06-104 0AF1 1-45 0AF1 5-46 0AFl7-1 23 0AGl 1-3 7 0A0 1 3-36 OAG 15-55 CAI-10 1 ~92 0AH09-1 12 0AH10-100 0AH19-123 0AH20-55 0AK04-42 0AK08-104 33 29 33 30 34 33 31 :26 :31 30: 37: 28: 32 25 35 :30 29: 34 :26 29: 26: 30: :28 24: 29: 28: 30: 28: 37: 27 36 30 35 32 33 29 34 23 :28 32: 29: 26 29 :27 26: 30 32:28 24:35 32:25 122 0.83 0.13 0.06 2.32 0.57 0.14 0.40 0.29 0.07 1.61 0.00 0.58 2.05 0.15 0.41 0.00 0.26 3.27“ 0.41 0.62 0.00 0.28 0.28 0.27 2.05 0.85 *,"‘* Indicate significant deviation from expected segregation ratio 1: 1 at the 5% and 1% levels, respectively. -4 .A"UJ|L.3' I l‘iv.‘ We.“ \ MICHIGAN STATE UNIV. 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