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This is to certify that the

thesis entitled

DEVELOPMENT OF NEW GENETIC MARKERS IN THE PIG
USING TWO APPROACHES:caMpARATIVE> MAPPING AND
REPRESENTATIONAL DIFFERENCE ANALYSIS

presented by
Charles R. Farber

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6/01 cJCIRC/DaleDue.p65-p.15

 

DEVELOPMENT OF NEW GENETIC MARKERS IN THE PIG USING Two
APPROACHES: COMPARATIVE MAPPING AND REPRESENTATIONAL
DIFFERENCE ANALYSIS
By

Charles R. Farber

A THESIS
Submitted to Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Animal Science

2000

ABSTRACT

DEVELOPMENT OF NEW GENETIC MARKERS IN THE PIG USING TWO
APPROACHES: COMPARATIVE MAPPING AND REPRESENTATIONAL
DIFFERENCE ANALYSIS
By
Charles R. Farber

The development of pig genome maps has rapidly progressed in recent years. This is
largely due to the development of genomemapping resources such as somatic cell hybrid
panels, radiation hybrid panels and a number of reference populations. The objective of
this study was to further increase the marker density on pig genome maps using
comparative mapping information and the novel technique of representational difference
analysis (RDA). Information on conservation of synteny between the human and pig was
used to generate markers surrounding the evolutionary breakpoint on human chromosome
(HSA) 12, which is syntenic with pig chromosomes (SSC) 5 and 14. Seven gene markers
were mapped using cytogenetic, radiation hybrid and genetic linkage mapping
techniques. The recently described technique of RDA was also implemented to isolate
genetic markers. RDA was performed using two pig populations divergent in genotype at
the RYRl locus and in phenotype for pork production traits. Eighteen markers were
isolated using RDA, and most flank putative quantitative trait loci (QTL) for performance
traits. Two of the isolated markers were homologous to the IRF6 and KCNBZ genes,
which were subsequently characterized. The markers developed in this study further
refine the human-pig comparative map and provide resources necessary for a better

understanding of QTL for economically important pig production traits.

This thesis is dedicated to my parents who have always been there with love, support and
guidance.

iii

ACKNOWLEDGMENTS

I have to acknowledge a number of people who have contributed to my project. First,
I would like to thank my major professor, Dr. Cathy Ernst, who was always there with
friendship and guidance. She always went the extra mile for me and I will be forever
grateful. I would also like to thank the members of my guidance committee (Drs. Ron
Bates, Gretchen Hill and Pat Venta) for their valuable insight and guidance regarding my
research, thesis and graduate program.

I also owe a special thanks to Nancy Raney. Her guidance and support in the lab
made long days go by fast and her patience and understanding were critical to the
completion of my research. I would also like to thank fellow lab members Melvin Pagan,
Stephanie Wesolowski and Chris Wilkinson for all of the help that each provided. The
interactions we shared will be greatly missed.

I would like to thank Drs. Patty Weber, Paul Coussens, Rob Templeman and Jianbo
Yao for providing technical assistance in the areas of lab protocols and statistical
analysis. Also I have to thank Drs. Daryl Kuhlers and Matt Doumit for providing DNA
and cells used in my project.

I am also indebted to a number of fellow graduate students and faculty. These groups
of great people have provided me with assistance and opportunities that I would not have
had otherwise.

I would also like to thank Amy Guzik for critically reviewing portions of my thesis.
In addition her special friendship has encouraged me to strive for perfection not only in

my work but also as a person.

iv

Lastly, I would be remiss if I did not acknowledge Dr. Gordon Jones. His
contribution to this work was indirect, however, without his vision and belief in me none

of this work would have happened.

TABLE OF CONTENTS

LIST OF TABLES ..................................................................................................... ix
LIST OF FIGURES .................................................................................................... x
LIST OF ABBREVIATIONS .................................................................................... xii
CHAPTER ONE
LITERATURE REVIEW ............................................................................................. 1
I. Introduction .............................................................................................................. 1
II. History and Current Status of Pig Genome Maps ...................................................... 2
A. Cytogenetic Maps ............................................................................................... 2
B. Radiation Hybrid Maps ....................................................................................... 6
C. Genetic Linkage Maps ........................................................................................ 7
III. Pig Comparative Maps ........................................................................................... 10
A. Comparative Map of HSA12 ............................................................................. 15
IV. Representational Difference Analysis ..................................................................... 16
A. Principles of RDA ........................................................................................... 16
B. Applications of RDA ....................................................................................... 18
C. RDA in the Development of Genetic Markers .................................................. 18
V. Summary ............................................................................................................... 22
CHAPTER TWO

COMPARATIVE MAPPING OF SEVEN GENES F LAN KING THE HUMAN

CHROMOSOME 12 EVOLUTIONARY BREAKPOINT IN THE PIG ................. 23
I. ABSTRACT ........................................................................................................... 23
11. INTRODUCTION .................................................................................................. 24
III. MATERIALS AND METHODS ............................................................................ 27

vi

A. Amplification of HSA12 Genes ........................................................................ 27

B. STS Confirmation ............................................................................................. 28
C. Cytogenetic Mapping ........................................................................................ 28
D. Radiation Hybrid Mapping ............................................................................... 29
E. Polymorphism Identification ............................................................................. 30
F. Linkage Analysis and Map Construction ........................................................... 31
IV. RESULTS .............................................................................................................. 32
A. Identification of Pig STS for HSA12 Genes ...................................................... 32
B. Cytogenetic Mapping ........................................................................................ 32
C. Radiation Hybrid Mapping ................................................................................ 32
D. Polymorphism Identification ............................................................................. 33
E. Linkage Analysis .............................................................................................. 34
V. DISSCUSSION ...................................................................................................... 35
VI. CHAPTER TWO TABLES AND FIGURES ......................................................... 38
CHAPTER THREE

IDENTIFICATION OF GENETIC MARKERS BETWEEN TWO PIG
POPULATIONS USING REPRESENTATIONAL DIFFERENCE ANALYSIS 50

I.

II.

III.

IV.

ABSTRACT .......................................................................................................... 50
INTRODUCTION .................................................................................................. 5 1
MATERIALS AND METHODS ............................................................................ 54
A. DNA Isolation and RYRl Genotyping .............................................................. 55
B. Representational Difference Analysis ................................................................ 55
C. Cloning and Sequencing of RDA Products ........................................................ 57
D. Sequence Analysis and Identification of RDA Sequence-Tagged Sites .............. 58
E. Radiation Hybrid Mapping ................................................................................ 59
F. Polymorphism Identification ............................................................................. 59
G. Statistical Analysis ........................................................................................... 60
H. Linkage Analysis and Map Construction ........................................................... 61
RESULTS .............................................................................................................. 61
A. RDA and STS Identification ............................................................................. 61
B. Polymorphism Identification ............................................................................. 62
C. STS Mapping .................................................................................................... 62
D. Allele Frequencies ............................................................................................ 62

vii

V. DISCUSSION ........................................................................................................ 63
VI. CHAPTER THREE TABLES AND FIGURES ...................................................... 70
CHAPTER FOUR

CHARACTERIZATION OF THE PORCINE [RF 6 AND KCNB2 GENES:
CDNA CLONING, EXPRESSION ANALYSIS AND CHROMOSOMAL

LOCALIZATION ....................................................................................................... 92
1. ABSTRACT ........................................................................................................... 92
11. INTRODUCTION .................................................................................................. 93
III. MATERIALS AND METHODS ............................................................................ 95
A. IRF6 and KCNB2 cDNA Isolation .................................................................... 95
B. RT-PCR ............................................................................................................ 97
C. Northern Blot Analysis ..................................................................................... 97
D. Polymorphism Identification ............................................................................. 98
E. Cytogenetic Mapping ........................................................................................ 99
F. Radiation Hybrid Mapping ............................................................................... .99
G. Linkage Analysis and Map Construction ......................................................... 100
IV. RESULTS ............................................................................................................ 101
A. IRF 6 and KCNB2 cDNA Isolation .................................................................. 101
B. Expression Analysis ........................................................................................ 102
C. Polymorphism Identification ........................................................................... 102
D. Cytogenetic, RH and Genetic Linkage Mapping ............................................. 102
V. DISCUSSION ...................................................................................................... 103
VI. CHAPTER FOUR FIGURES ............................................................................... 107
CHAPTER FIVE
SUMMARY AND RECOMMENDATIONS FOR FUTURE RESEARCH ........... 130
REFERENCES .......................................................................................................... 135

viii

LIST OF TABLES

CHAPTER ONE

Table 1.1. Summary of correspondence between human and porcine
chromosomal segments .................................................................................................. 11

CHAPTER TWO

Table 2.1. Pig sequence tagged sites developed for human chromosome
12 genes ........................................................................................................................ 39

Table 2.2. Summary of cytogenetic, radiation hybrid (RH) and genetic

linkage localizations of HSA12 genes in the pig ............................................................ 40
CHAPTER THREE

Table 3.1. Summary of RepeatMasker results for Bam HI and Bgl II inserts ................. 71
Table 3.2. Summary of RDA STS developed ............................................................... 72
Table 3.3. Summary of RH and genetic linkage mapping results ................................. .73
Table 3.4. Summary of SNP haplotypes present in RDA markers ................................ 74

Table 3.5. Summary of RDA marker allele frequency differences
between select and control lines ..................................................................................... 75

ix

LIST OF FIGURES

CHAPTER TWO

Figure 2.1. Agarose gels illustrating polymorphisms in pig STS .................................. 42
Figure 2.2. DNA sequence illustrating the use of the pool-and-sequence

strategy to identify a SNP in TCFl ................................................................................ 44
Figure 2.3. Diagram illustrating the cytogenetic and genetic linkage maps

of SSCS and SSC14 and the comparative map location of genes on HSA12 ................... 46
Figure 2.4. Diagrams illustrating the cytogenetic and RH maps of

SSCS and SSC14 ........................................................................................................... 48
CHAPTER THREE

Figure 3.1. Diagram of the RDA process ..................................................................... 76
Figure 3.2. RDA marker consensus sequences ............................................................. 78
Figure 3.3. Identification of a 15 bp length polymorphism in MSURDA42 .................. 79
Figure 3.4. Diagrams of the pig cytogenetic, RH and genetic linkage maps

for pig chromosomes containing RDA markers .............................................................. 81
CHAPTER FOUR

Figure 4.1. Alignment of porcine, human, mouse and sheep IRF6
nucleotide sequence ..................................................................................................... 110

Figure 4.2. Alignment of the porcine, human, mouse and sheep
IRF6 amino acid sequence ........................................................................................... 114

Figure 4.3. Alignment of porcine, human, canine and rat KCNBZ nucleotide

sequence ...................................................................................................................... 116
Figure 4.4. Results of RT-PCR analysis for IRF 6 and KCNBZ ................................... 122
Figure 4.5. IRF 6 and KCNB2 northern blot analysis .................................................. 124

Figure 4.6. Agarose gel image of KCNBZ RH mapping results obtained
using the IMpRH panel ................................................................................................ 126

Figure 4.7. Diagram of the pig cytogenetic, RH and genetic linkage maps for
SSC4 and SSC9 ........................................................................................................... 128

xi

LIST OF ABBREVIATIONS

ASP: Chromosome arm specific paint

BAC: Bacterial artificial chromosome

BLAST: Basic Local Alignment Search Tool

bp: Base pair

CATS: Comparative anchored tagged-Sites

cDNA: Complementary DNA

cM: Centi-Morgan

COL2A1: Collagen type II alpha 1

cR: Centi-Ray

DISC-PCR: Direct in Situ single-copy polymerase chain reaction
DNA: Deoxyribonucleic Acid

DUSP6: Dual specificity MAP kinase phosphatase

EDTA: Ethylenediaminetetraacetic acid

EST: Expressed sequence tag

FISH: Fluorescence in situ hybridization

GB4: GeneBridge 4

GDRDA: Genetically-directed representational difference analysis
GPP: Grandparent pool

HSA: Human chromosome

IFN: Interferon

IMpRH: [NRA-University of Minnesota porcine radiation hybrid panel

xii

INRA: Institut National de la Recherche Agronomique

IRF : Interferon regulatory factor

IRF6: Interferon regulatory factor 6

KCNB2: Potassium voltage-gated channel, Shah-related subfamily, member 2
LMA: Loin muscle area

LINE: Long interspersed element

LOD: Logarithm of the odds

LTR: Long terminal repeat

MAS: Marker assisted selection

MGF: Mast cell growth factor

mRNA: Messenger ribonucleic acid

NCBI: National Center for Biotechnology Information

nt: Nucleotide

PAGE: Polyacrylamide gel electrophoresis

PAH: Phenylalanine hydroxylase

PCR: Polymerase chain reaction

PDRDA: Phenotype-directed representational difference analysis
PiGMaP: International Pig Gene Mapping Project

pIRS-PCR: Porcine interspersed repetitive sequence-polymerase chain reaction
PLA2: Phospholipase A2

PSS: Porcine stress syndrome

PXN: Paxillin

QTL: Quantitative trait locus

xiii

RACE: Rapid amplification of cDNA ends

RDA: Representational difference analysis

RFLP: Restriction fragment length polymorphism
RH: Radiation hybrid

RNA: Ribonucleic acid

RT-PCR: Reverse-transcription-polymerase chain reaction
RYRl: Skeletal muscle ryanodine receptor 1

SCHP: Somatic cell hybrid panel

SINE: Short interspersed nucleotide elements

SNP: Single nucleotide polymorphism

SSC: Pig chromosome

SSCP: Single-stranded conformational polymorphism
STS: Sequence tagged site

TCF l: Hepatic transcription factor 1

TOASTS: Traced orthologus amplified sequence tags
UM-STS: Universal mammalian sequence-tagged site
USDA: United States Department of Agriculture

UTR: Untranslated region

xiv

CHAPTER 1

LITERATURE REVIEW

Introduction

Genome mapping in livestock Species has rapidly progressed in recent years as high-
resolution maps have been published for cattle (Barendse et al. 1997; Kappes et al. 1997),
sheep (de Gortari et al. 1998) and pigs (Archibald et al. 1995; Rohrer et a1. 1996). As of
1999, the number of mapped loci was 2850 for cattle, 1774 for pigs and approximately
1000 for sheep (as reviewed by, Kappes, 1999). The availability of high-density genome
maps has facilitated the elucidation of genomic regions harboring quantitative trait loci
(QTL) in livestock (Andersson et a1. 1994; Stone et al. 1999). Upon isolation of a QTL,
the subsequent step is gene identification. However, analysis based on segregation of
marker alleles with favorable phenotypes in a large resource population provides only
gross estimations of QTL location. This results in the identification of a genomic region
containing many genes. In this context, there is a need for both genome maps with a high
density of gene loci and for the evaluation of novel techniques designed to generate
genetic markers targeted to a small region of the genome. Comparative mapping has
proven highly effective for producing high density gene maps. In addition, the recently
described technique of representational difference analysis (RDA) has been used to target
genetic markers to precise locations of a genome (Lisitsyn et al. 1993). The purpose of
this literature review is to provide an historical and current perspective on pig genome
mapping efforts and to outline the utility of RDA for the generation of targeted genetic

markers.

History and Current Status of Pig Genome Maps

A. Cytogenetic Maps

The cytogenetic map of the pig has been developed through the use of physical
mapping techniques, which assign markers to specific genomic locations (Ruddle et al.
1986). The cytogenetic map contains information regarding both the part of the genome
conserved among mammalian species (i.e., coding sequences) and anonymous DNA
markers (largely microsatellites). Since a significant portion (36%) of the most recent
cytogenetic map of the pig is comprised of genes, it makes the cytogenetic map a very
useful tool for comparative studies between the pig and other mammalian Species (Yerle
et al. 1997). A dense cytogenetic map with a high percentage of mapped coding
sequences will also assist in the cloning of genes that potentially control economically
important traits, through the positional candidate approach (Collins, 1995).

Echard et a1. (1992) published a compilation of early gene mapping activities in the
pig. This map consisted of 84 loci, including linkage and physical assignments, covering
17 chromosomes. Yerle et al. (1997) published the most recent cytogenetic map of the
pig containing 436 physical assignments, thus demonstrating the rapid progress made in
the development of the pig cytogenetic map. O’Brien et al. (1993b) identified marker
loci as belonging to one of two types. Type I loci refer to known genes or coding
sequences and Type II loci are anonymous DNA markers, most commonly microsatellite
loci. Of the 436 cytogenetically mapped loci on the pig cytogenetic map, 160 represented
Type I loci.

Two techniques have been used for developing the pig gene map, in Situ hybridization

(or recently fluorescence in situ hybridization, FISH) and somatic cell genetics. In situ

hybridization was the first technique used extensively to physically map cloned pig DNA
segments containing genes or microsatellites. This was demonstrated by the assignment
of the major histocompatability complex to chromosome 7 (Rabin et al. 1985; Echard et
al. 1986). In contrast, the use of FISH to map porcine genes dramatically increased the
precision of assignments. Chowdhary et al. (1994) used FISH to precisely localize genes
for glucose phosphate isomerase (GPI), calcium release channel (CRC), hormone-
sensitive lipase (LIPE) and growth hormone (GH). This study demonstrated the utility of
FISH to refine previous assignments made with traditional in situ hybridization that
utilized radioactive labeling.

The integration of the physical and genetic linkage maps has also been accomplished
by FISH. Ellegren et al. (1994a) used FISH along with linkage data to anchor the pig
physical and linkage maps with 18 cosmid-derived genetic markers. Their results
indicated the first assignment of loci with both in Situ and genetic data for chromosomes
2p, 3, 4, 10, 12q, and 16. In a similar experiment, Alexander et al. (1996) physically
assigned 68 porcine cosmid and bacteriophage lambda clones containing polymorphic
microsatellites using FISH, providing additional anchor loci between the physical and
genetic linkage maps.

Numerous gene markers have also been assigned using FISH. Localizations for genes
such as immunoglobulin lambda (IGL) to pig chromosome (SSC) 14q17-q21
(Rettenberger et al. 1995b), immunoglobulin kappa (IGKC) to SSC3q12-ql4
(Rettenberger et al. 1995a), choline acetyltransferase (CHAT) to SSCl4q25-q27
(Murakami et al. 1995) and cytoplasmic inhibitor of NF-KB (IKBA) to SSC7q15-q21

(Musilova et al. 1996) have all been accomplished using FISH.

Recently, a modification of traditional in situ hybridization was developed. This
technique, termed direct in situ single~copy polymerase chain reaction (DISC-PCR), uses
the PCR to exponentially amplify DNA fragments directly on metaphase chromosome
spreads. The use of DISC-PCR was authenticated by physically mapping five porcine
microsatellites to the long arm of SSCl (Troyer et al. 1994).

To date, three porcine somatic cell hybrid panels (SCHP) have been developed. Two
of these allow only gross chromosomal assignment of genetic markers (Rettenberger et
al. 1994; Zijlstra et al. 1996). In contrast, the SCHP constructed by Yerle et al. (1996)
allows for regional localization of genetic markers on pig chromosomes. Rettenberger et
al. (1994) developed a partially informative porcine SCHP. This panel was constructed
by filsing porcine lymphocytes and fibroblasts with three different permanent rodent cell
lines, establishing 21 stable somatic cell hybrid lines. Confirmation of the assignments of
transition protein 2 (TNP2) and protamine 1 (PRMl) to chromosome 3 and polyubiquitin
(UBC) to chromosome 14, were determined using this SCHP.

In comparison, Zijlstra et al. (1996) developed a SCHP consisting of 15 independent
pig-hamster and 6 independent pig-mouse cell lines. Fluorescence painting, GTG-
banding of metaphase chromosomes and screening with twelve microsatellite markers
was used for characterization of this panel.

The most extensively used SCHP for cytogenetic mapping in the pig was constructed
by Yerle et a1 (1996). This panel of 27 pig-rodent somatic cell hybrids (19 pig-hamster
and 8 pig-mouse) was cytogenetically characterized by hybridizing porcine SINE (short
interspersed nucleotide elements) and pIRS-PCR (porcine interspersed repetitive

sequence—PCR) probes to metaphase chromosomes. Additionally, molecular verification

was conducted by amplification of six previously mapped microsatellites and one gene
using the PCR. This panel has advantages over previous SCHP because it is the only one
capable of making regional localizations (Yerle et al. 1996). In addition, it has been
useful for the integration of the cytogenetic and genetic linkage maps. One hundred and
thirty-three microsatellites were physically anchored using this panel, which led to the
assignment of the “R” linkage group to SSC6 (Robic et al. 1996; Robic et al. 1997).
Refinement of the human-pig comparative map has also been demonstrated using the
SCHP. Jargensen et al. (1997) mapped 22 expressed sequence tags (ESTS) isolated from
a porcine small intestine cDNA library. Additionally, the physical mapping of human
ESTS (Lahbib-Mansais et al. 1999), porcine ESTS (Fridolfsson et al. 1997; Wintero et al.
1998) and porcine Type I loci (Rettenberger et al. 1996) using the SCHP have also
contributed to the advancement of the human-pig comparative map. AS an illustration to
the power of this technique, a SCHP was used to map 40% of the loci on the latest
cytogenetic map (Yerle et al. 1997).

The accumulation of cytogenetic mapping results has recently been published in two
cytogenetic maps (Yerle et al. 1995; Yerle et al. 1997). The first version of this map
contained 154 loci (Yerle et al. 1995) in contrast to 436 loci localized on the most recent
map. Of these 436 assignments, 160 (36%) represent known genes and the remaining
276 (64%) are Type II loci. This map is especially useful for pig gene mapping because
it integrates the genetic and cytogenetic maps, with 95% of the Type II and 36% of the
Type I loci being located on both (Yerle et al. 1997). These maps are invaluable for

current and future comparative mapping studies, and the production of higher resolution

cytogenetic maps will aid in the identification of genes controlling variation at QTL
(Archibald et al. 1994).
B. Radiation Hybrid Maps

The technology of generating radiation hybrid (RH) panels using irradiation and
fusion gene transfer was first developed by Goss and Harris in 1975. Briefly, x-rays are
used to break the genome into numerous fragments. These broken fragments are
recovered in rodent cells and approximately 100-200 hybrid clones are analyzed for the
presence or absence of Specific DNA markers. The farther apart two markers, the higher
the probability of breakage between them. This information can then be used to estimate
frequency of breakage, or distance between the markers, similar to meiotic mapping (Cox
et al. 1990). However, RH mapping is different from meiotic mapping because only
Single-copy DNA is analyzed. Thus, this differs from linkage mapping, which relies on
the analysis of diploids possessing differences in one of the copies of DNA. RH mapping
can utilize both polymorphic and nonpolymorphic markers (Cox et al. 1990).

To date, the construction of one porcine-hamster RH panel has been reported (Yerle et
a1. 1998). This panel was developed at the INRA in France and was further characterized
at the University of Minnesota (Hawken et al. 1999) so it is referred to as the INRA-
Minnesota porcine Radiation Hybrid (IMpRH) panel. It was constructed by fusing
porcine lymphocytes or fibroblasts, after irradiation with 6,000-7,000 rads, to hamster
Wg3hC 12 cells. The panel was cytogenetically characterized by hybridization with
porcine SINE and primed in situ labeling (PRINS) probes to identify pig chromosomal
fragments. Thirty-two markers, chosen based on chromosomal location, were used for

molecular verification of the panel. These characterizations resulted in the reduction of

the number of hybrids used for the final panel from 154 to 118. Thirty-four hybrids were
eliminated because they had retained little or no porcine DNA.

The IMpRH panel was extensively verified by the production of the first-generation
porcine whole-genome radiation hybrid map (Hawken et al. 1999). The map was
developed with analysis of 903 markers, of which 22 were ESTS and 49 were genes. RH
data was obtained for 757 markers placed in 128 linkage groups constructed with a lod
score 2 4.8. Fifiy-nine of the markers were unlinked. The average retention frequency
for the markers used for map construction was 29.3%. Additionally, the size of each
chromosome was divided by the number of cR (centi-rays) per chromosome to give the
RH map a genome wide ratio of approximately 70 kb/cR7ooo, and a theoretical resolution
of 145 kb.

A radiation hybrid map of the region containing the redemenent napole (RN) gene was
developed using the IMpRH panel (Robic et al. 1999). Ten microsatellites and eight
genes were mapped within an interval of 55 cM to test the resolution of the IMpRH
panel. These data indicted the cR-moo IMpRH panel was sufficient to develop high
resolution maps of small genomic intervals and they also further refine the pig-human-
mouse comparative map of this region (Robic et al. 1999). RH maps will be an essential
resource to the pig genome mapping community, allowing for estimates of the location
and order of genetic markers in a small genomic region (Cox et al. 1990; Yerle et al.
1998)

C. Genetic Maps
Andresen et al. (1970a) published some of the first studies demonstrating linkage

between two markers in the pig. In this study, linkage was demonstrated between the H

blood group and the locus for 6-phosphogluconate dehydrogenase (6-PGD). In a similar
subsequent study, linkage was described for the H blood group and the locus for
phosphohexose isomerase (PHI) (Andresen et al. 1970b). The culmination of these two
studies represented the first case of an autosomal linkage group involving three loci in
farm animals (Andresen et al. 1970b).

Since these early reports, the pig genetic linkage map has progressed rapidly. To date,
three primary maps have been published; the Nordic map (Ellegren et al. 1994b;
Marklund et a1. 1996), the United States Department of Agriculture (USDA) map (Rohrer
et al. 1994; Rohrer et al. 1996) and the International Pig Gene Mapping Project
(PiGMaP) consortium map (Archibald et al. 1995).

The Nordic map was first published in 1994 (Ellegren et al. 1994b), and subsequently
a comprehensive Nordic map containing markers from all three maps was published in
1996 (Marklund et al. 1996). The Nordic map was constructed by typing 128 markers in
pigs from a cross between the European Wild Boar and the Large White. In construction
of this map, it was determined that the rate of genetic recombination was much lower in
the pig as compared with humans. The sex-averaged genetic length of the pig genome
was estimated to be approximately 1873 cM as compared to the estimate for the human
map of 3800-4000 cM (Ellegren et al. 1994b).

The comprehensive Nordic map increased the total number of mapped loci to 236,
incorporating new markers from both the USDA and PiGMaP linkage maps located in
areas poorly covered in the first Nordic map. The sex-averaged genetic. length of this
map was estimated to be 2300 cM, with linkage groups assigned to all 18 autosomes and

the sex chromosomes. The authors also noted a significant difference in male and female

recombination rate, with the average ratio of female to male recombination estimated at
1.4:1 (Marklund et al. 1996).

In 1994, Rohrer et al. reported the most extensive genetic linkage map for any
livestock species at that time. The map was constructed using the USDA reference
families. These families were developed using a mating regime of backcrossing White
Composite (WC) boars with eight F1 WC sows. This map contained 376 microsatellite
loci and seven RF LP loci. These markers were assigned to 24 linkage groups covering
1997 cM of the pig genome with an average marker interval of 5.5 CM. However, since
all chromosomes were not completely covered, the entire porcine genome was estimated
to be approximately 2300 cM (Rohrer et al. 1994).

The highest resolution genetic map constructed to date was reported by Rohrer et al. in
1996. This map was constructed by genotyping the USDA reference families for the
majority of publicly available microsatellite markers. One thousand forty-two loci
spanning 2286.2 cM, with an average marker interval of 2.23 cM, in 19 linkage groups
were genotyped. Linkage groups were anchored to chromosomes using 123 informative
loci assigned cytogenetically by FISH. This high-density map has been the basis for the
identification of QTL in swine and for a better genetic and physiological understanding
of polygenic inheritance (Rohrer et al. 1996).

The European PiGMaP consortium has reported one version of their genetic linkage
map of the pig (Archibald et al. 1995). The map was constructed using 10 F2 families
with approximately 10 full-sibs in each family (Archibald, 1994). These families are
subsets of larger pedigrees produced for QTL studies in laboratories around Europe.

Families were developed from crosses between Wild Boar and Large White, Pietrain and

Large White or Meishan and Large White. Two hundred and thirty-nine markers were
genotyped across the reference families. Eighty-one of the markers corresponded to
Type I loci. This map further integrated the physical and genetic linkage maps because
69 of the markers were present on both. The sex-averaged genetic map of the pig was
estimated to be approximately 1800 cM. Since this map contributed both Type I and
Type II loci, it is useful for both comparative mapping and QTL studies (Archibald et al.
1995)

Pig Comparative Maps

It is generally agreed that eutherian mammals diverged approximately 70 million
years ago sharing between them a high level of genome conservation (Comparative
Genome Organization; First International Workshop, 1996). This conservation of coding
sequences between related species forms the basis of comparative genome mapping in
mammals (Edwards, 1994). Comparative mapping utilizes information from species
possessing marker dense genetic maps (primarily human and mouse). This information
allows for the construction of genetic maps containing a high density of Type I loci in
marker poor species including livestock species. Such high resolution gene maps then
aid in the identification of positional candidate genes located in genomic regions
containing QTL (O’Brien et al. 1999).

Many techniques and strategies have been used in developing the comparative map
between the human and pig. Early studies used chromosomal painting (Zoo-FISH)
techniques to establish genomic benchmarks. These studies allowed for the direct
visualization of segment-to-segment conservation between the two genomes.

Rettenberger et al. (1995c) hybridized labeled human chromosome-specific DNA

10

libraries on porcine metaphase spreads to visualize conserved synteny. This analysis
revealed 47 conserved chromosomal segments between the human and pig karyotypes.
In contrast, Copeland et al. (1993) estimated that the human and mouse genomes shared
approximately 150 conserved syntenic segments. These results indicate that there is three
times more conservation of synteny between humans and pigs than between humans and
mice. In comparison, F rOnicke et al. (1996) also probed porcine metaphase spreads with
labeled human chromosome-specific DNA libraries and confirmed the observation of 47
conserved human-pig chromosomal segments. The most comprehensive study to date
was conducted by Goureau et al. (1996). In contrast to previous studies, these
researchers used a bidirectional approach, hybridizing both human probes to porcine
metaphase chromosomes and porcine probes to human metaphase spreads. These
experiments allowed for more precise localization of syntenic regions. They also
revealed new homologies, estimating 37 conserved syntenic groups between humans and
pigs (Table 1.1).

Table 1.1. Summary of correspondence between human and porcine chromosomal

 

 

segments”

Segment Pig chr. % size Type of Human chr. % size

No. segment pig hybridizationb segmentc human
genome genome

1a 1p 3.5 <— 6q 3.4

lb 1q12-q14 0.7 <— 18

1c 1q12-q22 2.5 <— 15

1d 1q23-q24 0.7 <— 14

1e 1q24-q2.12 2.6 <— 9

2a 2p 2.4 <——> 11p-q13 2.6

2d 2q11-q21 1.5 <——> 19p 0.8

2c 2q22-q28 2.1 <——> 5q14-qter 3 .2

3a 3p14-p11 1.4 <——> 16p 1.2

3b 3q13-qter 3 <—-> 2p-q21 .2 4.5

4a 4p-q14 3 .4 (— 8q 3.1

4b 4q14-qter 2.6 <— 1

 

11

 

Table 1.1. Continued.

 

 

Segment Pig chr. % size Type of Human chr. % size
No. segment pig hybridization segment human
genome genome
5a 5pter-p14 0.6 <---> 22q12-qter 0.6
5b 5p14-q24 4.1 <——> 12pter- 3.7
q24.1
6a 6p 2 €——> 16q 1 7
6b 6q11-q21 0.8 <——> 19q 1.1
6c 6q22-q26 l <——> 1pter-p3 1 2.5
6q31-qter 2
6d 6q27-q31 1.2 t-—> 18q11-q12 0.6
7a 7p-q 14 2.7 <--9 6p 2.]
7b 7q 1 3-q22 l .9 <——> 15q24-qter 0.9
70 7q21-q25 1.9 <——> 14q11-q13 0.6
14q22-qter 1 .4
8a 8 6 <—-—> 4p-q31.3 5.1
9a 9p 2.7 <— 11q14-qter 1.8
9b 9q 3 <— 7pter-p15.2
7q11-q31.2
9c 9q23-qter 1 <— 1q31-qter
10a 10p 2.9 <- 1q41
10b 10q12-qter 1,8 ‘- 10p 1,5
11a 11 4 <—-—> 13q 2.6
12a 12 3.5 <--> 17 2.7
13a 13 7.6 <--> 3 6.5
13b 13q46-qter 0.9 <— 21 1.5
143 14q11-q16 1.7 <——> 8p 1.5
14b 14q21-q22 0.9 t-—> 12q22-qter 1.3
14c 14q21-q23 1.3 <——> 22q11-q13.1 0.9
14d 14q23-qter 2.9 <——> 10q > 2.9
153 15ql3-q14 0.6 <——> 2q 4.8
15q22-qter 2.4
16a 16 3 <- 5p-q13
17a 17 2.4 <-—9 20q 1.1
18a 18 2.3 <——> 7p15.2-p12 0.8
7q31.3-qter 1.4

 

a Table adapted from Goureau et al. (1996).
b Arrows indicate unidirectional painting with human probes on pig chromosomes (<——) or

bidirectional painting (<——>).
° Data in italics were not obtained by FISH but were deduced from other results.

In addition to providing comparative mapping benchmarks, Zoo-FISH has also

been used to develop the comparative map between the pig and other mammalian species.

12

Zoo-FISH was used to determine conservation of synteny between humans, pigs and
horses (Chaudhary et al. 1998). In this study, microdissected arm specific paints (ASPS)
for human chromosomes 2, 5, 6, 16, and 19 were used as probes on pig and horse
chromosomes. These results established links integrating the human, pig and horse
comparative map. Hybridization analysis with microdissected ASPs also provided more
refined and accurate comparative perspectives than whole chromosome specific paints.
Pinton et al. (2000) localized 113 anchor loci in pigs using Zoo-FISH, hybridizing
bacterial artificial chromosomes (BACs) containing genes already mapped in humans and
goats to pig chromosomes. Taking gene order into account, the authors identified 84, 106
and 70 conserved segments between the autosomes of humans and pigs, humans and
goats, and pigs and goats, respectively. Similar to the study by Chaudhary et al. (1998),
these results establish the human, pig and goat comparative map.

The development of PCR primer pairs for the amplification of Type I loci across
diverse mammalian species has proven to be a valuable comparative mapping resource.
Three projects have been reported using this approach. Venta et al. (1996) developed 86
gene specific universal mammalian sequence-tagged sites (UM-STS) primers. The
authors proposed this primer set to be valuable for the development of genetic maps in a
number of mammalian species. Lyons et al. (1997) designed 410 comparative anchored
tagged-sites (CATS) PCR primer pairs to amplify reference loci in a diverse set of
mammals. To increase levels of polymorphism for each marker, PCR primers were
designed to span intronic regions. Three hundred and eighteen primer pairs were
optimized in the domestic cat and a screen of 20 mammals from 11 orders revealed 35-

52% of the total PCR primers yielded a single product in each sample. J iang et al. (1998)

13

constructed a panel of 225 PCR primer pairs corresponding to 146 functional genes,
which were termed traced orthologus amplified sequence tags (TOASTS). Out of 225
PCR primer pairs, 182 (80.9%) produced a single PCR product when tested in pig
genomic DNA.

Recently, mapping of both human and pig ESTS has allowed for the rapid progression
of the human-pig comparative map. Several strategies have been devised to utilize the
abundance of available human and pig ESTS (as reviewed by Gellin et al. 2000). Lahbib-
Mansais et al. (1999) described the mapping of human ESTS in the pig. Out of 10,000
Généthon human EST primer pairs, approximately 600 amplified in the pig. They
reported mapping of 65 human ESTS in the pig using a SCHP. This study added a
significant number of Type I loci to the pig cytogenetic map and also further refined the
human-pig comparative map.

In addition to human ESTS, many pig specific ESTS have been mapped. F ridolfsson
et al. (1997) described the linkage and physical mapping of pig ESTS isolated from a
porcine small intestine cDNA library. This study clarified the occurrence of an inversion
event in a region of conserved synteny between SSC6q and human chromosome (HSA)
1p. Also, they showed that SSC14q does not contain a region homologous to HSAl and
that the homology between SSC17 and HSA20 includes the p-arm of HSA20. Using
ESTS from the same small intestine cDNA library, Jorgensen et al. (1997) mapped 22
ESTS using both linkage analysis and physical mapping techniques. Wintera et al. (1998)
reported mapping 33 Type I loci using pig ESTS providing comparative data for 13 pig

autosomes and the X chromosome. In addition, several European and US laboratories are

14

in the process of constructing pig specific cDNA libraries for the isolation and
subsequent mapping of ESTS (as reviewed by Gellin et al. 2000).
A. Comparative Map of HSA12

The bidirectional heterologous chromosome painting experiments conducted by
Goureau et a1. (1996) demonstrated that human chromosome (HSA) 12 is conserved on
SSC5 and 14. Precisely, SSC5p14-q24 is homologous to HSA12pter-q24.1 and
SSC14q21-q22 shares homology with HSA12q22-qter, with the evolutionary breakpoint
within the interval of HSA12q22-q24. 1. Unidirectional Zoo-FISH experiments (FrOnicke
et al. 1996; Rettenberger et al. 1995c) also established the conservation of HSA12 on
SSC5 and 14. Additionally, many linkage and physical assignments have localized
HSA12 genes to SSC5 and 14 (Johansson et al. 1995; Rohrer et al. 1997; Fridolfsson et
al. 1997; Wintem et al. 1998; Lahbib-Mansais et al. 1999). Whole chromosomal painting
demonstrated the presence of four syntenic groups on SSC14. However, hybridization of
gene-containing BACS expanded the conservation on SSC14 to 6 different human
chromosomal fragments, including a previously unreported segment of HSA12 on SSC14
(Pinton et al. 2000). In a separate study, Lahbib-Mansais et al. (1999) mapped an
anonymous EST from HSA12q21.33-q22 to SSC5q25 further extending knowledge of
HSA12 conservation through SSC5q25. Intrachromosomal rearrangements have been
documented for SSC5 genes relative to gene order on HSA12. Rohrer et al. (1997)
mapped 11 HSA12 genes to SSC5 and showed extensive gene order rearrangements.
Four of the genes mapped in this study were located in the region of HSA12q12-q13

(DAGKl, HOXC@, RARG and LALBA). In the pig, three of the four

15

 

 

(DAGKI , HOXC@ and RARG) mapped near each other on SSCSp. However, LALBA
mapped to SSC5q22-q23. In addition, IGF 1 and IFNG were located within HSA12q22-
q24, but when mapped in the pig, IFNG localized near the centromere on SSCSp while
IGF l was near the telomere on SSCSq. These data suggest multiple SSC5
rearrangements, however, a much higher gene density will be needed to precisely

determine the number and locations of such rearrangements.

Representational Difference Analysis

A. Principles of RDA

Representational difference analysis (RDA) was developed to isolate polymorphic
DNA fragments existing between two genomes. RDA is based on the principle of
traditional subtractive hybridization methods. However, traditional methods have proven
inefficient for the analysis of eukaryotic genomes due to the increase in sequence
complexity relative to prokaryotes. Additionally, RDA is unique when compared to other
hybridization-based genome analysis techniques because it combines subtractive
hybridization with the polymerase chain reaction (PCR). This combination allows for a
reduction in sequence complexity leading to efficient subtractive hybridizations. RDA
also provides enrichment of target sequences. Single-copy sequences can be enriched by
1010 relative to non-target sequences (Lisitsyn et a1. 1993 and Lisitsyn et al. 1995).

As described by Lisitsyn et al. (1993), there are two steps in the process of RDA,
representation and kinetic enrichment. RDA involves the analysis of two different

genomes denoted as the “tester” and “driver” samples. The tester and driver genomes

16

are identical except for the presence of target or unique fragments resulting from
polymorphisms that are to be isolated.

Representation involves digesting both tester and driver with a particular restriction
endonuclease. Oligonucleotide adaptors are ligated to the 5’ ends of the restriction
fragments, and DNA polymerase is subsequently used to fill in the complement of the
Oligonucleotide adaptors on the 3’ ends of the restriction fragments. This allows the
original oligonucleotides to be used as primers for the PCR. The PCR is subsequently
used to preferentially amplify only the small restriction fragments (average size of 0.6
kb). The amplified fragments are referred to as “amplicons”. Through representation the
sequence complexity is reduced to approximately 2-15% of the total genome. Thus, the
entire genome can be scanned using an array of restriction endonucleases.

During the kinetic enrichment step, the Oligonucleotide adaptors are removed from
tester and driver amplicons and a new set of Oligonucleotide adaptors are ligated to the 5’
ends of tester amplicons. Tester and driver amplicons are mixed (with driver in excess),
melted and allowed to re-associate. Unique tester sequences re-anneal in the presence of
competing driver fragments. These tester homoduplexes are selectively amplified using
the PCR and single-stranded tester and driver fragments are digested with single-stranded
DNA specific mung-bean nuclease. RDA final difference products are isolated after two
or three rounds of representation and kinetic enrichment, and subsequently cloned for
further analysis. RDA is designed to produce cloned restriction fragments that can be
amplified by the PCR from tester but not driver. Thus, probes can be produced to detect
RFLPs between tester and driver samples (Lisitsyn et al. 1993; Lisitsyn et al. 1994a).

RDA allows geneticists to isolate genetic markers without prior knowledge of the locus

17

(i.e., DNA sequence, genetic map location, etc.). These markers lead to higher resolution
genetic maps and ultimately positional cloning of genes influencing quantitative traits
(Lisitsyn et al. 1993).
B. Applications of RDA

Lisitsyn et a1. (1995) reviewed the use of RDA for three applications; 1) the detection
of genetic lesions in cancer, 2) the discovery of new pathogens and 3) the isolation of
polymorphic markers linked to a trait. One of the most recent applications of RDA has
been for analysis of cDNA. This technique has been used to analyze differential gene
expression in various tissue types and cell lines (Moses et al. 1999; Melia et al. 1998;
Iwama et al. 1998; Hubank and Schatz, 1994). It has also been used successfully for the
isolation of rare transcripts (O’Neill and Sinclair, 1997) and the identification of human
transcripts from a defined chromosomal region using somatic cell hybrids (Groot and van
Oost, 1998). Welford et al. (1998) coupled the use of cDNA-RDA with microarray
technology allowing for the analysis of a large set of enriched differentially expressed
genes from primary tumor cell lines that differed phenotypically. Results of these studies
indicate that cDNA-RDA has the potential to make major contributions to the
characterization of metabolic events at the molecular level (Hubank and Schatz, 1994).
C. RDA in the Development of Genetic Markers
There have been many variations of the original RDA protocol for the elucidation of
polymorphic genetic markers existing between two genetically distinct populations. One
variation of RDA as defined by Lisitsyn et al. (1994b) is the use of genetically-directed
representational difference analysis (GDRDA) to generate markers linked to a specific

gene locus. GDRDA involves the use of either inbred congenic lines or two generation

18

crosses to produce tester and driver amplicons differing only in the region flanking a
selected locus. GDRDA was validated by targeting three polymorphisms within a 1 cM
interval flanking the mouse nude locus (Lisitsyn et al. 1994b). GDRDA also proved
effective in isolating seven loci within a 10 cM interval surrounding the jcpk locus on
mouse chromosome 10 (Baldocchi et al. 1996). In this study, genomic subtractions
involved DNA from the C57BL/6 inbred line and its partially congenic partner B6-
jcpk/jcpk. Toyota et al. (1996) used GDRDA to isolate four polymorphic markers around
the DINccl locus in mice using the inbred lines AC1 and BUF. This was accomplished
by generating genomic DNA pools homozygous for ACI alleles at the DINch locus, but
containing different levels of ACI and BUF alleles for loci flanking DINccl . The pools
were used as driver and DNA from the ACI inbred line was used as tester.

In chickens, RDA was used to target marker loci to chromosome 16 (Wain et a1.
1998). In this study, the authors used one bird homozygous for alleles of inbred line N at
three chromosome 16 loci (the major histocompatability complex [MHC], the nucleolar
organizer region [NOR] and the pr-Y complex loci) for the tester. The driver contained
DNA from sixteen of the tester bird’s siblings that were homozygous for inbred line 151
alleles at these three loci. Sixty-two polymorphisms were identified using RDA and ten
loci were mapped to chromosome 16. GDRDA is therefore capable of targeting genetic
markers in the vicinity of a gene without prior knowledge of the gene’s location or
function.

Phenotype-directed representational difference analysis (PDRDA) has also been
effective in isolating polymorphisms surrounding loci controlling variation in a specific

quantitative trait. Toyota et al. (1998) used RDA to isolate polymorphisms linked to the

19

thymus enlargement loci (T en] and T en2) in rats. Out of 28 loci isolated, eight were
linked to T enI and one was linked to Ten2. These results demonstrated the ability of
RDA to isolate flanking polymorphisms for multiple loci when animals divergent for a
particular quantitative phenotype are analyzed. Xu et al. (1996) used a Mus domesticus
/Mus spretus mouse congenic line selected for retention of Mus spretus DNA around the
pearl locus by selecting for the recessive pearl phenotype, creating a highly polymorphic
region flanking this locus. Conducting two series of RDA experiments using the
congenic line as tester in one and as driver in the other, four loci linked to the pearl locus
were isolated. PDRDA can thus be used to isolate genetic markers linked to genes
controlling the variation of a quantitative trait.

Toyota et al. (1996) constructed a rat genetic map by the use of RDA. This study
analyzed DNA from the ACI and BUF inbred lines leading to the isolation of 131
polymorphic markers. For 125 of the markers, the size differential between ACI and
BUF alleles made these markers suitable for high-throughput genotyping by performing
dot blot analysis with amplicons derived from ACI X BUF F2 individuals.

Another variation of RDA was preformed by Ushijima et al. (1998). In this
experiment, amplicons were prepared from ACI and BUF inbred lines using primers
complementary to rat BI repetitive sequences. Probes were produced that detected either
the presence or absence of fragments. Through dot blot analysis of F2 progeny, markers
could be mapped rapidly and inexpensively. Forty-eight markers were developed, all
suitable for high-throughput genotyping. In a very similar study, Yoshida et al. (1999)
used arbitrary primers to produce amplicons from two inbred lines. In total, 47 markers

suitable for large-scale genotyping were developed. These studies suggest that RDA can

20

be used for the creation of large sets of polymorphic loci suitable for rapid and
inexpensive genotyping. These markers could be used to greatly simplify QTL analysis
(Toyota et al., 1996).

Recently, Everts et al. (2000) reported the use of RDA to isolate genetic markers in
moderately inbred dogs. Thirty—nine tester specific fragments were determined to be
unique difference products. These markers were mapped using the dog radiation hybrid
panel, RHDF5000. These results suggest that RDA is suitable to generate DNA markers
within lines of pedigree dogs (Everts et al. 2000).

Two recent studies have reported the use of RDA for pig genomic DNA. Pe’rez—Pe’rez
et al. (1998) used RDA to isolate pig male-specific DNA fragments. In this study, DNA
from an Iberian male pig and a Landrace female pig was used as tester and driver,
respectively. Four genomic clones were isolated that only hybridized to male
genomic DNA as determined by Southern analysis. Three of the isolated fragments
exhibited high homology to pig male-specific repetitive sequences. In another report,
RDA was used to identify sequences associated with postweaning multisystemic wasting
syndrome (PMWS) in pigs (Bratanich et al. 2000). For the analysis, DNA from PMWS-
affected pigs was used as tester and DNA from normal pigs was used as driver. After
three rounds of RDA, nine fragments were cloned. Six of the RDA fragments were
homologous to pig centromeric repeats, one was a microsatellite and two fragments
displayed no homology to sequences in the GenBank database. The authors hypothesized
that PMWS triggered the amplification of genomic regions containing repetitive DNA

sequences. However, based on our own observations using RDA in the pig, it is more

21

likely that the identification of repetitive sequences was a consequence of the RDA

technique and had no relationship to PMWS.

Summary

Genome maps provide the necessary framework for a complete understanding of
genome function. Without precisely defined genome maps, the elucidation of gene
function on a global level will be impossible. Genome maps also provide valuable links
between studies identifying QTL and large-scale gene expression analysis. Thus,
continued research in the areas of comparative mapping and identification of genetic
markers flanking QTL is critical. For the present study, three research objectives were
developed.

Objective 1: Map HSA12 genes to increase the density of gene loci on SSC5 and 14 and
further refine the comparative map of the evolutionary breakpoint on HSA12.

Objective 2: Generate genetic markers existing between pigs phenotypically divergent
for carcass and pork quality characteristics using representational difference analysis
(RDA).

Objective 3: Characterize potential candidate genes for production traits in pigs isolated

using RDA.

22

CHAPTER 2

COMPARATIVE MAPPING OF SEVEN GENES F LANKING THE

HUMAN CHROMSOME 12 EVOLUTIONARY BREAKPOINT IN THE PIG

Abstract

Previous reports have indicated that genes located on human chromosome 12
(HSA12) are conserved on pig chromosomes 5 and 14 (SSC5 and SSC14), with
HSA12q22-q24.1 harboring the evolutionary breakpoint between these chromosomes.
The objective of this study was to map pig sequence-tagged sites (STS) for HSA12 genes
in order to refine the comparative map of the region surrounding this breakpoint. STS
were identified for collagen type II alpha 1 (COL2A1), dual specificity MAP kinase
phosphatase (DUSP6), mast cell growth factor (MGF), phenylalanine hydroxylase
(PAH), phospholipase A2 (PLA2), paxillin (PXN), and hepatic transcription factor 1
(TCFI). Oligonucleotide primers used for amplification of each STS were designed from
pig sequence or distributed as part of the comparative anchor tagged sequences (CATS)
(Lyons et al. 1997) or universal mammalian sequence-tagged sites (UM-STS) (Venta et
al. 1996) projects. Gene identity for each STS was confirmed by sequencing of PCR
products. Four of the pig STS were physically mapped using a pig-rodent somatic cell
hybrid panel. COL2A1, MGF and PAH were mapped to SSC5 (risk of error less than
0.1%) and TCF 1 was mapped to SSC14 (risk of error less than 0.5%). COL2A1
localized to SSC5q12-q25, PAH localized to SSC5q25, MGF localized to SSC5q25 and

TCFl localized to SSC14. Radiation hybrid (RH) mapping was performed for six of the

23

genes using the INRA-Minnesota porcine radiation hybrid (IMpRH) panel. Two-point
analysis of the RH data showed significant linkage between DUSP6, MGF and PAH and
markers on SSC5 (LOD>l3.0). PLA2, PXN and TCF] showed significant linkage with
SSC14 markers (LOD>7.0). For genetic linkage mapping, single—stranded
conformatiOnal polymorphisms (SSCP) or restriction fragment length polymorphisms
(RFLP) were identified for COL2A1, DUSP6, PAH, PLA2 and TCFl. The PiGMaP
reference families were subsequently genotyped for each identified polymorphism and
linkage analysis was performed using the two-point option of CRI-MAP 2.4. COL2A1,
PAH and DUSP6 showed significant linkage (LOD>4.0) with markers on SSC5. PLA2
and TCF 1 showed significant linkage (LOD>10.0) with markers on SSC14. These
results further refine the comparative map of the evolutionary breakpoint region shared

between HSA12 and SSC5 and 14.

Introduction

It is generally agreed that eutherian mammals diverged approximately 70 million
years ago, sharing between them a high level of genome conservation (Comparative
Genome Organization; First International Workshop, 1996). The conservation of coding
sequences between related Species forms the basis of comparative genome mapping in
mammals. Comparative mapping utilizes information from species (such as human and
mouse) possessing marker dense genome maps. This information allows for the
construction of gene dense genome maps in the pig, which aid in the identification of

positional candidate genes located in genomic regions containing QTL.

24

Cytogenetic, radiation hybrid and genetic linkage mapping techniques can be used to
anchor specific genes to regions of conserved synteny and also to elucidate gene order.
O’Brien et al. (1993b) proposed a list of expressed genes (termed Type I markers) which
could be mapped in diverse species and serve as anchor loci for the alignment of
conserved genomic regions. These Type I markers were referred to as comparative
anchor tagged sequences (CATS) and Oligonucleotide primers for use in the PCR were
designed and distributed to researchers mapping genes in various species (Lyons et al.
1997). A similar set of Oligonucleotide primers for amplifying Type I markers was
designed by Venta et al. (1996). This set of markers was referred to as universal
mammalian sequence-tagged sites (UM-STS). These primer sets have been valuable
resources for comparative mapping efforts.

The use of somatic cell genetics in the pig has greatly facilitated the mapping of Type
I loci (Rettenberger et al. 1994; Zijlstra et al. 1996; Yerle et al. 1996). A somatic cell
hybrid panel consisting of 27 pig-rodent somatic cell hybrids was constructed providing a
resource for regional localization of genetic markers (Yerle et al.1996; Robic et al. 1996).
Complementing the establishment of various EST projects worldwide, this panel has been
used to physically assign over 100 known and anonymous ESTS (Fridolfsson et al. 1997,
Jargensen et al. 1997; Wintera et al. 1998). Recently, collaboration between the Institut
National de la Recherche Agronomique (INRA, Toulouse, France) and the University of
Minnesota led to the construction of a 7000 rad radiation hybrid panel (IMpRH) (Yerle et
al. 1998). The IMpRH panel consists of 118 pig-hamster hybrids and was used for the

development of a first generation radiation hybrid map of the porcine genome (Hawken

25

et al. 1999). The IMpRH panel will be an invaluable resource for fine mapping and
determining gene order within small regions of the pig genome.

Chromosomal segments with conserved synteny between human and pig have been
broadly defined using bidirectional chromosomal painting (Goureau et al. 1996). In these
experiments, conservation of synteny was observed between human chromosome (HSA)
12 and pig chromosomes (SSC) 5 and 14. Most of HSA12 is conserved on SSC5, with
the only exception being the distal portion of the q arm, which is homologous with two
distinct segments of SSC 14 (Pinton et al. 2000). However, chromosomal painting could
not resolve the precise location of the evolutionary breakpoint on HSA12.

In light of the information provided by chromosomal painting experiments, genes
surrounding the HSA12 breakpoint have been mapped in the pig genome. Insulin like
growth factor 1 (IGFl) and interferon gamma (IFNG) lie within HSA12q22—q24.1 and
both have been mapped to SSC5 (Rohrer et al. 1997). In addition, various EST mapping
projects have assigned genes residing near or in the breakpoint region to SSC5 and 14
(Fridolfsson et al. 1997; Lahbib-Mansais et al. 1999).

Although several HSA12 genes and ESTS have been mapped in the pig, there is still
not a clear picture of the precise location of the evolutionary breakpoint. The refinement
of this region will be critical to correctly identify HSA12 genes residing within QTL
located on either SSC5 or SSC14. Therefore, the objective of this study was to further
develop the comparative map in the region flanking the evolutionary breakpoint on

HSA12.

26

Materials and Methods

A. Amplification of HSA12 Genes

The HSA12 genes collagen type II alpha 1 (COL2A1), mast cell growth factor (MGF),
phenylalanine hydroxylase (PAH) and hepatic transcription factor 1 (TCFl) were
originally amplified using CATS primers. Phospholipase A2 (PLA2) was amplified
using UM-STS primers and dual specificity MAP kinase phosphatase (DUSP6) and
paxillin (PXN) were amplified using heterologous primers designed from available
mammalian genomic sequence information in GenBank. However, a number of primer
pairs did not amplify in an efficient or repeatable manner. CATS primers for PAH and
MGF amplified multiple background bands in addition to the correct PCR product, which
interfered with polymorphism identification. To circumvent this problem, the products
were gel purified in 1% agarose gels using the QIAquick Gel Purification kit (Qiagen,
Valencia, CA). The fragments were then cloned into the pGEM-T-Easy vector
(Promega, Madison, WI). Plasmid DNA was isolated with the QIAprep Spin Miniprep
kit (Qiagen, Valencia, CA) and digested with EcoR 1 (20,000 U/ml, New England
Biolabs, Beverly, MA) to confirm the presence of an insert. One plasmid for each STS,
containing an insert of the correct size, was sequenced on an ABI 373 automated DNA
sequencer using the ABI Prism dye terminator cycle sequencing ready reaction kit
(Applied Biosystems, Foster City, CA). A comparison to sequences in the GenBank
database confirmed the isolation of pig PAH and MGF. This sequence was used to
design pig specific Oligonucleotide primers for efficient amplification of these STS.
Heterologous primers designed for DUSP6 and PXN also tended to generate background

PCR products in addition to the correct product. To improve the efficiency of

27

amplification of these STS, the original PCR products were sequenced and pig-specific
primers were designed for use in subsequent analyses.

The PCR was performed for each STS using 25 ng genomic DNA in 10 [.11 reactions
containing 1 X PCR buffer (Promega, Madison, WI), 1.5 ’or 2.0 mM MgCl2, 200 “M each
dNTP, 0.5 or 2.0 uM each primer and 0.5 U Taq DNA polymerase (Promega, Madison,
WI). The PCR profiles included an initial denaturation of 3 min at 94°C followed by 30
cycles of 94°C for 1 min, 55-62°C for 1 min, 72°C for 1 min and a final extension of
72°C for 10 min.

B. STS Confirmation

Pig PCR products were visualized on 1% agarose gels containing 0.4 1.1ng ethidium
bromide. Following optimization of PCR conditions to obtain a single amplification
product, fragments were sequenced using an ABI 373 automated DNA sequencer using
the ABI Prism dye terminator cycle sequencing ready reaction kit (Applied Biosystems,
Foster City, CA). Identities of pig STS were confirmed by comparison with sequences in
the GenBank database.

C. Cytogenetic Mapping

Physical mapping of COL2A1, MGF, PAH and TCFl was accomplished using a pig-
rodent somatic cell hybrid panel (Yerle et al. 1996; Robic et a1. 1996). Analysis of 27
pig-rodent hybrids by the PCR allowed for chromosomal localizations. PAH and TCFl
amplified both mouse and hamster products, but were discemable from the pig products
due to differences in Size. Although no hamster product was detected for COL2A1, a
mouse product of the same size as the pig product was detected. The pig and mouse

COL2A1 products were distinguished by digestion with the restriction enzyme Dpn II

28

which cleaved the mouse but not the pig product. PCR products were not obtained from
mouse or hamster genomic DNA for MGF. PCR products were visualized on 1%
agarose gels containing 0.4 ug/ml ethidium bromide, and regional assignments were
determined as described by Chevalet et al. (1997) by submitting data through the INRA
web site (http://www.toulouse.inra.fr/lchpig/hybridhtm).
D. Radiation Hybrid Mapping

RH mapping of DUSP6, MGF, PAH, PLA2, PXN and TCF l was preformed using the
IMpRH panel (Yerle et al. 1998). Each primer pair was tested in hamster DNA to ensure
the absence of amplification or differences in product sizes between pig and hamster.
TCFl was the only primer pair to amplify hamster DNA. However, a large size
difference between pig and hamster products was observed allowing a clear distinction to
be made between the two products. Optimized PCR profiles for each STS were used to
amplify the 118 pig-rodent hybrids of the IMpRH panel. The PCR was conducted in 96-
well plates utilizing the same PCR reaction components as previously described with the
exception that 12.5 ng of DNA for each hybrid was used instead of 25 ng. Products from
the PCR were visualized on 3% agarose gels containing 0.4 11ng ethidium bromide.
Gels were destained in 1 X TBE buffer (89 mM Tris base, 89 mM Borate and 2 mM
EDTA; pH 8.0) for 1-2 hrs to minimize background fluorescence. Amplification of each
hybrid was scored as either positive (1), negative (0) or ambiguous (‘2). Preliminary two-
point and multipoint analysis of RH data was performed using the IMpRH server
mapping tool as outlined by Milan et a1. (2000) (http://imprh.toulouse. inra.fr/). This data

was subsequently used to determine the approximate order for markers on SSC5 and 14.

29

E. Polymorphism Identification

A DNA pooling strategy was used to identify potential RF LP in each STS (Sun et al.
1998). DNA from F0 individuals of the PiGMaP reference families was used to create a
grandparent pool (GPP). The pool was constructed by aliqouting equal amounts of DNA
from each individual. For the analysis, each STS was amplified from the GPP. The PCR
products were digested with a panel of restriction endonucleases with 4-base recognition
sites. DNA fragments were electrophoresed on 2% agarose gels containing 0.4 ug/ml
ethidium bromide. Digested fragments showing a banding pattern consistent with the
presence of two possible alleles in the GPP were further evaluated using PiGMaP
individuals.

STS not containing an observable RF LP were evaluated using SSCP analysis. DNA
from the GPP and 12 individual pigs was amplified and PCR products were heated to
98°C for 5 min in a denaturing loading buffer (95% formamide, 0.05% xylene cyanol,
0.05% bromophenol blue). The Bio-Rad D-Code Mutation Detection System (Bio-Rad
Laboratories, Hercules, CA) was used for SSCP analysis. Denatured DNA fragments
were electrophoresed on 8% native polyacrylamide gels with 5% glycerol at 4°C. Each
gel was run at a constant wattage, which ranged from 25-40 W depending on equipment
conditions, for 6-12 hrs. Gels were stained in a 1 ug/ml ethidium bromide solution for 3
min and destained in deionized water for 3 min with subsequent documentation
performed using a Bio-Rad Gel Doc 2000 Image Analysis System (Bio-Rad Laboratories,
Hercules, CA). Following optimization of conditions to detect an SSCP in a specific

STS, the SSCP was used to genotype individuals from informative PiGMaP families.

30

No RFLP was detected in TCFI so this STS was used to evaluate the use of a pool-
and-sequence strategy to identify single nucleotide polymorphisms (SNPs). TCF 1 was
amplified from the GPP. The resulting PCR product was purified using a Microcon 100
concentrator (Millipore, Bedford, MA) and sequenced using an ABI 373 automated DNA
sequencer using the ABI Prism dye terminator cycle sequencing ready reaction kit
(Applied Biosystems, Foster City, CA). The sequence was then analyzed for ambiguous
base pair sites that resulted from the presence of two different bases in the GPP. Using
this approach, a SNP was identified and subsequent genotyping was performed using
SSCP analysis to detect the two SNP alleles. Due to its large size, the TCF 1 fragment
was digested with Dpn II (10,000 U/ml, New England Biolabs, Beverly, MA) to obtain
smaller fragments. SSCP analysis of these fragments was used as described above to
confirm the SNP in TCFl and individuals from informative PiGMaP families were
genotyped.

F. Linkage Analysis and Map Construction

For STS with an identified polymorphism, linkage analysis and map construction was
performed using CRIMAP 2.4 (Green et al. 1990). The two—point option was used to
obtain preliminary pairwise comparisons between markers. The “all” and “flips” options
using genotypic data for previously reported markers (Archibald et al. 1995; Rohrer et al.
1996) were used to obtain the most likely marker order. In addition, linear order of
markers was determined using the “fixed” option. Linkage maps were drawn using the
CorelDraw 8 software package and aligned next to chromosome ideograms based on the

standard porcine karyotype as described by Gustavsson (1988).

31

Results

A. Identification of Pig STS for HSA12 Genes

In the present study, PCR profiles were optimized for the amplification of COL2A1,
DUSP6, MGF, PAH, PLA2, PXN and TCF]. Sequencing of each product after the PCR
confirmed amplification of the correct loci. The optimized PCR conditions were used to
amplify these STS for cytogenetic mapping, radiation hybrid mapping and polymorphism
identification. PCR product sizes, primer sequences and PCR conditions for each STS
are listed in Table 2.1.
B. Cytogenetic Mapping

The COL2A1, MGF, PAH and TCF 1 STS were cytogenetically mapped using a
somatic cell hybrid panel (Yerle et al 1996; Robic et al. 1996). Regional assignments
were made for COL2A1, MGF, and PAH. COL2A1 localized to SSC5q12-q25, and
MGF and PAH localized to SSC5q25. TCFl was mapped to SSC14 but it was not
regionally assigned due to the limited informativeness of the panel for SSC14. The
cytogenetic assignments for these genes are summarized in Table 2.2 and shown in
Figure 2.3.
C. Radiation Hybrid Mapping

Using the IMpRH panel, five HSA12 genes were placed on the porcine RH map.
Chromosomal localizations on the RH map with the most significantly linked marker for
each STS are presented in Table 2.2. Three of the genes mapped to SSC5 including
DUSP6 and MGF, which were both significantly linked to SW378 (LOD=13.8 and LOD
13.47, respectively). PAH also mapped to SSC5 exhibiting significant linkage with IGF 1

(LOD=15.47). The remaining three genes mapped to SSC14. PLA2 showed Significant

32

linkage with SW295 (LOD=8.05), PXN was linked to SW1321 (LOD= 11.81) and TCFl
was linked to SW1527 (LOD=7.64). Updated maps including these markers are shown
in Figure 2.4.
D. Polymorphism Identification

In order to identify polymorphisms for use in genetic linkage mapping, each STS was
first screened for the presence of PCR—RFLPS. STS were amplified from the GPP and
PCR products were subsequently digested with a panel of restriction enzymes having four
base recognition sequences. Three of the seven STS produced a banding pattern
consistent with the possible presence of two alleles in the GPP. Genotyping of F 0
individuals from the PiGMaP reference families confirmed the presence of a PCR-RFLP
in each of the three genes. A Rsa I PCR-RFLP was identified in COL2A1 (Figure 2.1 A).
Autosomal Mendelian segregation of the COL2A1 alleles was observed in 69 pigs from 3
three-generation PiGMaP families. DUSP6 contained a Msp I PCR-RFLP and DUSP6
alleles displayed autosomal Mendelian segregation in 20 pigs from 1 three-generation
family. A Dpn II PCR-RFLP (Figure 2.1 B) was identified in PLA2 and autosomal
Mendelian segregation of PLA2 alleles was observed in 98 pigs from 3 three-generation
PiGMaP families.

PCR-RFLPS were not observed for any of the remaining STS. However, SSCP
analysis revealed different single strand conformations among PiGMaP family
individuals for PAH (Figure 2.1 C). Autosomal Mendelian segregation of PAH alleles
was observed for 31 pigs from 1 three-generation PiGMaP family. A SNP in TCFl was
identified by using the pool-and-sequence method (Figure 2.2). To confirm this

polymorphism, SSCP analysis was performed. The TCFl fragment was too large to use

33

directly so smaller fragments suitable for SSCP analysis were obtained by digestion with
Dpn II to obtain a 425 bp fragment and a 175 bp fragment containing the SNP. SSCP
analysis of the Dpn II digested TCF 1 STS identified two SSCP alleles and sequencing of
each allele confirmed that they were the result of the SNP (Figure 2.1 D). Autosomal
Mendelian segregation of TCFl alleles was observed for 59 pigs from 3 three-generation
families.
E. Linkage Analysis

Two-point analysis of marker data revealed significant associations between markers
identified in this study and previously reported markers on SSC5 and 14 (Archibald et a1.
1995; Rohrer et al. 1996). PLA2 was linked to 15 markers on SSC14 with the most
significant linkage to the microsatellite SW295 (LOD=14.15, 0:0.00). TCF 1 was linked
to three SSC14 markers, with the most significant linkage to S0058 (LOD=10.96,
0=0.00). COL2A1, DUSP6 and PAH were all linked to markers on SSC5. COL2A1
exhibited linkage with three SSC5 markers, the most significant to S0018 (LOD=4.49,
0:0.17). PAH was also most significantly linked to S0018 (LOD=4.58, 0:005) and
DUSP6 showed most significant linkage to SW1954 (LOD=5.12, 0=0.00).

Figures 2.3 and 2.4 illustrates cytogenetic, RH and genetic linkage maps for SSC5 and
SSC 14 and the comparative location of genes on HSA12. For the genes mapped in this
study, marker order on the linkage map of SSC5 was PAH-(7.8 cM)-COL2A1-(13.3 cM)-

DUSP6 and the order on the SSC14 linkage map was PLA2—(1.7 cM)-TCF1.

34

Discussion

Seven genes flanking the human-pig evolutionary breakpoint on HSA12 were mapped
to SSC5 and 14. Six of the seven genes are located within the interval of HSA12q22-
q24.1, which harbors the breakpoint. Therefore, localizations determined in this study
significantly increase the gene density in this region or the pig-human comparative map.

To assess gene order rearrangements between the human and pig, human gene order
was determined by searching current human genome map databases. Cytogenetic
information was not useful because of the relative close proximity of the selected genes
within the human genome. A search of STS mapped on GeneMap’99 (Deloukas et al.
1998) revealed that all genes under investigation, with the exception of PLA2, were
mapped on the GeneBridge 4 (GB4) RH panel (Gyapay et al. 1996). A BLAST search of
human PLA2 mRNA sequence against the high-throughput genome sequence (htgs)
database revealed one clone, RP11-144BZ (GenBank accession # AC073930), containing
sequence homologous to PLA2. Using the electronic PCR (ePCR) (Schuler et al. 1998)
application available on the NCBI website (http://www.ncbi.nlm.gov/genome/
sts/epcr.cgi), STS were discovered within this clone. None of the STS exhibited
homology to PLA2. However, an STS located on the clone near the sequence
homologous to PLA2 had been localized on the GB4 RH map. This allowed for an
estimation of PLA2 position. Interestingly, a STS for PXN was also located on this clone
illustrating the close proximity shared between PLA2 and PXN. In addition, TCF 1 was
located just downstream of these genes on the GB4 RH map. Despite the high resolution

of GeneMap’99, it is still difficult to confidently assign order for PXN, PLA2 and TCFl.

35

However, based on the RH data available, the most likely order in the human genome is
PXN-PLAZ-TCF 1.

By using GB4 RH data for each gene, the apparent order for these markers in the
human genome is COL2A1-MGF-DUSP6-PAH-PXN-PLA2-TCF1. Data obtained in
this study indicate that this gene order is conserved in the pig. The three genes that
mapped to SSC14 (PXN, PLA2 and TCF 1) are located within a very small region of the
human genome. Genetic linkage data was obtained for PLA2 and TCF]. This data
indicated the relative position of these two markers on SSC14 as PLA2 - (1.7 cM) -
TCFI. Cytogenetic mapping of TCFl confirmed the location of this gene on SSC14, but
the resolution of the SCHP did not allow a more precise regional assignment to be made.
RH mapping data was obtained for all three genes and it further confirmed gene order
conservation between HSA12 and SSC14 with the approximate RH map order of PXN-
PLA2-TCF 1.

Despite extensive conservation of synteny between HSA12 and SSC5, considerable
intrachromosomal rearrangements have occurred during mammalian evolution
(Johansson et a1. 1995; Rohrer et al. 1997). Genes mapped in this study also add
evidence for rearrangements on SSC5. Using the SCHP, both MGF and PAH mapped to
the single cytogenetic band of SSC5q25 and COL2A1 localized to SSC5q12-q25.
Additionally, linkage data placed PAH, COL2A1 and DUSP6 near each other with an
order of PAH-(7.7 cM)-COL2Al-(l3.3 cM)-DUSP6. These data are in contrast to
human gene order in which PAH, MGF and DUSP6 are localized to HSA12q22-q24.1,
but COL2A1 resides on HSA12ql2-ql3.2. Previous reports localized interferon gamma

(IFNG) to SSC5p12-q1 1. However, in the human genome, IFNG resides on

36

HSA12q24.1 (Rohrer et al. 1997). These data suggest an early inversion event
subsequently followed by similar smaller inversions or translocations leading to extensive
gene order rearrangements within this syntenic block.

Information obtained in this study indicates that PAH and most likely PXN flank the
breakpoint on HSA12. One gene, D-amino acid oxidase (DAO) falls between PAH and
PXN on the GB4 map at position 427.31 cR3ooo. In the pig, DAO maps to SSC14 (Yerle
et al. 1997) indicating that the possible location of the breakpoint is between PAH and
DAO. With this level of resolution, genes in the HSA12 breakpoint region can be
assigned with a fairly high level of confidence to either SSC5 or SSC14.

The human-pig comparative map has progressed rapidly over recent years. With the
advent of technologies such as radiation hybrid mapping which allows for high resolution
mapping of nonpolymorphic markers, this progress will certainly continue. However,
some regions of the comparative map still contain relatively few markers. Focused
efforts must be initiated to increase the density of markers in these genomic regions. In
addition, it will be critical to precisely define all human chromosomal breakpoints. Such
knowledge will allow researchers to more accurately identify potential positional or
functional candidate genes responsible for the genetic variation observed in economically

important traits.

37

Chapter 2

Tables and Figures

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40

Figure 2.1. Agarose gels illustrating polymorphisms in pig STS. Panel A, Rsa I PCR-
RFLP in COL2A1. Panel B, Dpn II PCR-RFLP in PLA2. Panel C, a SSCP for PAH.
Panel D, a SSCP for TCF 1. For panels A and B lanes are labeled as 100, 100 bp ladder; U,
undigested sample; AA, homozygous genotype; AB, heterozygous genotype; BB,
homozygous genotype. For panel C lanes are labeled as AA, homozygous genotype; AB,
heterozygous genotype; BB, homozygous genotype. For panel D lanes are labeled as U,
undenatured sample; AA, homozygous genotype; AB, heterozygous genotype; BB,
homozygous genotype

41

   

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Figure 2.2. DNA sequence illustrating the use of the pool-and-sequence strategy to
identify a SNP in TCF 1. Panel A shows the presence of an ambiguous base pair site in
TCF 1 sequence obtained from a pooled DNA sample containing equal amounts of DNA
from the PiGMaP family F0 individuals. Panel B, the sequence of an individual
homozygous for the G allele. Panel C, the sequence of an individual homozygous for the
T allele. Figure is presented in color.

43

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CHAPTER 3

IDENTIFICATION OF GENETIC MARKERS BETWEEN TWO PIG

POPULATIONS USING REPRESENTATIONAL DIFFERENCE ANALYSIS

Abstract

Representational difference analysis (RDA) was preformed using genomic DNA from
Landrace pigs selected for increased loin muscle area. In addition, pigs used for the
analysis were divergent in genotype at the skeletal muscle ryanodine receptor (RYRl)
locus and in phenotype for various carcass and pork quality traits. Two RDA
experiments were performed using Bam HI and Bgl 11. Difference products from both
experiments were cloned. After plasmid isolation, fourteen Bam HI and 37 Bgl II inserts
were sequenced. A homology search between each RDA sequence and the non-
redundant database of GenBank revealed that fragments MSURDA79 and MSURDAI 11
were homologous to the interferon regulatory factor 6 (IRF 6) gene and the potassium
voltage-gated channel, Shah-related subfamily, member 2 (KCNB2) gene, respectively.
In addition, a high percentage of difference products were homologous to porcine
repetitive DNA sequences. The RepeatMasker web-based server (Smit and Green,
RepeatMasker at http://ftp.genome.washington.edu/ RM/RepeatMasker.html) was used to
characterize the repetitive sequences from both experiments. It was observed that Bam
HI fragments contained a higher proportion of repeat sequences (58.46%) then Bgl II
fragments (20.89%). Oligonucleotide primers were designed to amplify each RDA

difference product using the polymerase chain reaction (PCR). Primers were designed to

50

flank the repetitive portion and sequence-tagged sites (STS) were developed for a total of
18 RDA fragments (2 Barn HI and 16 Bgl II). Each STS was mapped using the IN RA-
Minnesota porcine Radiation Hybrid (IMpRH) panel. In addition, polymorphisms were
identified and used to localize nine RDA markers on the pig genetic map. STS were
localized throughout the genome. However, clustering of RDA markers was observed on
pig chromosomes (SSC) 11 and 14. Additionally, many of the markers mapped to
regions containing previously reported quantitative trait loci (QTL) for growth and
carcass traits including regions of SSC4, 6 and 7. The development of RDA markers will
increase the resolution of the pig genome maps. In addition, RDA provides a means to
increase marker density in areas of known QTL and to isolate potential candidate genes

for economically important traits in pigs.

Introduction

The development of high-resolution pig genetic maps has recently been reported
(Archibald et al. 1995; Rohrer et a1. 1996; Marklund et al. 1996). These maps have
provided the framework for the identification of a number of quantitative trait loci (QTL)
for various production traits in the pig (Andersson et al. 1994; Andersson-Eklund et al.
1998; Rohrer et al. 1998a; Rohrer et al. 1998b; de Koning et al. 1999). The ultimate goal
of QTL identification is the isolation of the gene(s) responsible for the observed
phenotypic variation. Traditional QTL analysis is dependent on marker-phenotype co-
segregation in large resource populations, which leads to the isolation of genomic regions
containing many genes. Also, polygenic traits rely on the concordant expression of a

large number of genes. Thus, the QTL for a given trait may reside on several

51

chromosomes spanning a significant portion of the pig genome. In order to positionally
clone the genes at QTL, there is a need to evaluate novel techniques such as
representational difference analysis (RDA), which have the potential to generate genetic
markers flanking these QTL.

In 1993, Lisitsyn et al. described the process of RDA. The RDA technique is based on
the principle of subtractive hybridization coupled with the use of the PCR. This allows
for the efficient subtraction of complex eukaryotic genomes. In RDA, DNA from two
distinct populations (referred to as tester and driver) is used. First, both tester and driver
are digested with a restriction enzyme, Oligonucleotide adaptors are ligated to the 5’ ends
of the restriction fragments and the PCR is used to produce amplicons. Tester and driver
amplicons contain different sets of restriction fragments due to the presence of restriction
fragment length polymorphisms (RF LP). Next, subtractive hybridization is performed
with driver present in excess and the PCR is used to selectively amplify testerztester
homoduplexes. After the second or third round of hybridization, followed by PCR
amplification of unique sequences, these fragments are cloned for further analysis.

The RDA process was first used to isolate genetic lesions leading to the formation of
cancerous cell types. Subsequently, RDA has also been used for the discovery of new
pathogens and the isolation of “targeted” genetic markers (Lisitsyn et al. 1995). A
modification of the original protocol, genetically-directed RDA (GDRDA), uses two
populations that are congenic except for a narrow interval flanking a particular locus.
The GDRDA technique has been used to identify genetic markers within small intervals
flanking various loci in mice (Lisitsyn et al. 1994; Baldocchi et al. 1996) and to target

markers to chicken chromosome 16 (Wain et al. 1998).

52

A similar RDA modification, termed phenotype-directed RDA (PDRDA), has been
used to isolate genetic differences in two populations arising from selection for particular
QTL alleles. PDRDA differs from GDRDA in that highly inbred animals are not
required. Thus, PDRDA holds great promise for use in moderately inbred or outbred
(most livestock) populations. In theory, individuals divergent phenotypically should also
be divergent genetically at loci influencing the quantitative trait under selection. PDRDA
has been used successfully in mice divergent for the thymus enlargement phenotype to
isolate nine genetic markers flanking two loci known to have a large effect on the
phenotype (Toyota et al. 1998).

In pigs, a point mutation in the skeletal muscle ryanodine receptor 1 (RYRl) gene has
been associated with porcine stress syndrome (PSS) (Fuji et al. 1991). It has also been
highly associated with increases in total percent lean, a trait of significant economic
importance to pork producers (Lundstrom et al. 1995). Despite this association, evidence
strongly indicates that the RYRl mutation is not likely to be the causative factor for
improvements in lean growth, and this allele is not a prerequisite for leanness and
muscularity since many elite breeding lines do not possess the mutation (O’Brien et al.,
1993a). Therefore, if specific loci controlling lean growth could be identified, selection
could be placed directly on these loci thus separating the positive and negative
phenotypes associated with the RYRl mutation.

Various selection experiments have been used in animal populations to study the
effect of selecting for particular traits. Afier many generations of selection, it is likely
that the frequency of favorable QTL alleles will have increased. Thus, these populations

provide an ideal model for the identification of QTL (Rathje et al. 1997; Ollivier et al.

53

1997). In addition, they also possess genetic differences at loci controlling traits of
interest, which can be rapidly isolated using RDA.

We have performed RDA using DNA from pigs divergent at the RYRl locus and also
differing in phenotype for production traits as a result of selection for loin muscle area
(LMA) over five generations. The markers isolated in this study are potentially linked to

QTL and can be used in subsequent studies to further fine map these genomic regions.

Materials and Methods

A. DNA isolation and RYRl Genotyping.

Selection for increased LMA in Landrace pigs for five generations was performed by
Dr. Daryl Kuhlers at Auburn University (Kuhlers et al. 1998). The select and control
lines contained 8 boars and 16 sows. Approximately 10 ml of blood was collected in
sterile vacuum tubes containing EDTA (Vacutainer, Becton Dickinson, Franklin Lakes,
NJ) from 21 pigs in the select and 23 pigs in the control line. DNA from each blood
sample was extracted using standard procedures (Sambrook et al. 1989). A fragment of
the RYRl locus containing the causative mutation for PS8 was PCR amplified using the
following primer pair: forward 5’-TCCAG'I'I‘GCACAGGTCCT ACCA-3 ’ and reverse
5’-TTCACCGGAGTGGAGTC TCTGAGT—3’ (O’Brien et al. 1993a). Amplification
was performed in a 10 ul reaction consisting of 1 X PCR buffer (Promega, Madison, WI),
2.0 mM MgC12, 200 uM each dNTP, 0.5 uM each primer and 0.5 U T aq DNA
polymerase (Promega, Madison, WI). The PCR profile included an initial denaturation
of 3 min at 94°C followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1

min and a final extension of 72°C for 10 min. Subsequently, each sample was digested

54

with 5 U BsiHKA 1 (10,000 U/ml, New England Biolabs, Beverly, MA). Reactions were
incubated in a 65°C water bath for 3-6 hrs. After digestion, DNA fragments were
subjected to gel electrophoresis on 3% agarose gels containing 0.4 11ng ethidium
bromide at 100 V for approximately 1.5 hrs. Gels were documented using the Bio-Rad
Gel Doc Image Analysis System (Bio-Rad Laboratories, Hercules, CA).

B. Representational Difference Analysis (RDA)

RDA was performed using DNA from one select line pig as tester and a pool of DNA
from five control line pigs as driver. Ultrasound measurements for the tester pig were
1.48 cm of backfat measured at the 10th rib and 39.75 cm2 LMA. Average backfat and
LMA measurements for the five driver pigs were 2.87 cm and 25.71 cmz, respectively. In
addition, the tester pig was homozygous mutant “rm” and driver pigs were homozygous
normal “NN” at the RYRl locus. The same tester and driver samples were utilized for
two RDA experiments with Bam HI and Bgl 11 (10,000 U/ml, New England Biolabs,
Beverly, MA). A diagram 'of the RDA procedure is presented in Figure 3.1. The Bam HI
experiment was performed precisely as outlined by Lisitsyn (1993). For the Bgl II
experiment, modifications described by Romani et a1. (1999) were implemented. The
modified procedure consisted of digesting 1 pg of genomic DNA from each pig with 200
U of Bgl 11. Each sample was subsequently purified using Microcon 100 concentrators
(Millipore, Bedford, MA), and eluted in 10 ul of TE (lOmM Tris-HCl, 1 mM EDTA; pH
8.0). Following purification, 2 111 of DNA (200 ng) was electrophoresed on a 1% agarose
gel containing 0.4 ug/ml ethidium bromide to insure complete digestion.

Ligation of Oligonucleotide adaptors (Oligonucleotide sequences published by Lisitsyn

et al. 1993) was preformed in 30 pl reactions containing 0.8 ug of digested DNA, 450

55

pmoles of each Oligonucleotide (RBg112 and RBg124) and 1 X ligation buffer (New
England Biolabs, Beverly, MA). Oligonucleotides were melted and allowed to re-anneal
in a thermal cycler using the following program: 55°C for 30 seconds, decreasing 1°C
per cycle for 40 cycles. Once the reaction had reached 15°C, 400 U of T4 DNA ligase
(400,000 U/ml, New England Biolabs, Beverly, MA) was added. The reaction was
incubated at 15°C overnight and subsequently diluted to 800 pl with TB.

One hUndred pl of each driver sample was pooled. Two amplification reactions for
the tester and 16 for the driver were prepared in 0.5 ml PCR tubes containing 5 X PCR
buffer (335 mM Tris-HCl, 20 mM MgClz, 80 mM ammonium sulfate, 50 mM B-
mercaptoethanol, 100 pg/ml BSA), 4 mM of each dNTP, 40 ng of ligated DNA and 240
pmoles RBg124. Once the reactions had reached 72°C in a thermal cycler, 15 U of T aq
DNA polymerase was added. The PCR profile included an initial denaturation of 5 min
at 72°C followed by 20 cycles of 95°C for 1 min, 70°C for 3 min, and a final extension of
72°C for 10 min.

All amplicons were digested with Bgl II and purified with Microcon 100
concentrators. Tester amplicons (0.8 pg) were subsequently ligated to JBgllZ and
JBg124 as previously described. Next, 200 ng of the tester/adaptor ligation was mixed
with 40 pg of driver amplicon, phenol chloroform extracted and precipitated with
isopropanol. The pellet was resuspended in 4 pl of 3 X EE buffer (30 mM EEPS, 3 mM
EDTA; pH 5 .5), vortexed and overlaid with mineral oil. The sample was denatured in a
thermal cycler for 4 min at 98°C. After denaturation, 1.5 pl of 5 M NaCl was added and

hybridization was conducted at 67°C for 40 hrs.

56

The sample was then diluted in 390 pl of TE and 8 pl of tRNA solution (5 mg/ml)
(Sigma, St. Louis, MO). The 3’ recessed ends of each amplicon fragment were filled in
by setting up a PCR reaction identical to the reaction used for amplicon production, with
40 pl of the hybridization reaction. After the 5 min incubation with T aq DNA
polymerase at 72°C, 600 pmoles of JBgl 24 was added. The following PCR program was
used to amplify tester homoduplexes: 5 min at 72°C followed by 10 cycles of 95°C for 1
min, 70°C for 3 min, and a final extension of 72°C for 10 min. Following amplification,
each sample was purified using Microcon 100 concentrators and single stranded DNA
was digested using 20 U of Mung-Bean nuclease (10,000 U/ml, New England Biolabs,
Beverly, MA) for 30 min at 30°C. The digest was diluted to 200 pl with 50 mM Tris-
HCl (pH 7.5) and heat inactivated at 98°C for 5 min.

The first round RDA product was digested as before with Bgl II and purified with a
Microcon 100 concentrator. Oligonucleotides NBg112 and NBg124 were ligated to first
round product as described above. For the second round of RDA, 100 ng of the first
round product/adaptor ligation was hybridized with 40 pg of digested driver. The
remaining protocol for the second round subtractive hybridization/selective amplification
was conducted as described for the first round.

C. Cloning and Sequencing of RDA Products

Difference products obtained from Bam HI and Bgl II RDA experiments were cloned
into pGEM-3Z (Promega, Madison, WI) and pGEM-T-Easy vectors (Promega, Madison,
WI), respectively. Recombinant vectors were transformed in DHSOL competent cells
(Life Technologies, Rockville, MD) according to the manufacturer’s protocol. Cultures

were plated on LB + ampicillin plates and incubated at 37°C overnight. Single colonies

57

 

were picked, 3 ml of LB + ampicillin medium was inoculated and cultures were
incubated with shaking at 37°C overnight. Plasmid DNA was isolated with the QIAprep
spin miniprep kit (Qiagen, Valencia, CA) and digested with either Bam HI (Bam HI
RDA) or EcoR I (Bgl II RDA) to confirm the presence of an insert. Fourteen of the Bam
HI and 37 of the Bgl II RDA inserts were sequenced on an ABI 373 automated DNA
sequencer using the ABI Prism dye terminator cycle sequencing ready reaction kit
(Applied Biosystems, Foster City, CA).

D. Sequence Analysis and Identification of RDA Sequence-Tagged Sites (STS)

The BLAST search tool (http://www.ncbi.nlm.nih.gov.BLAST/) was used to identify
sequences homologous to RDA difference products in the non-redundant database of
GenBank. Subsequently, RDA difference products were screened for known repetitive
DNA elements using the RepeatMasker web-based server (Smit and Green,
RepeatMasker at http://fip.genome.washington.edu/ RM/RepeatMasker.html).
Oligonucleotide primers to amplify individual RDA fragments were designed to avoid
repetitive sequences and to produce PCR products approximately 200 bp in length. Each
primer set was optimized using pig genomic DNA in 10 pl PCR reactions consisting of l E
X PCR buffer (Promega, Madison, WI), 2.0 mM MgCl2, 200 pM each dNTP, 0.5 pM L.

each primer and 0.5 U Taq DNA polymerase (Promega, Madison, WI). The PCR profiles

 

included an initial denaturation of 3 min at 94°C followed by 30 cycles of 94°C for 1 ..
min, 55-57°C for 1 min, 72°C for 1 min and a final extension of 72°C for 10 min.
Nomenclature used to distinguish each RDA STS included the MSURDA prefix with the

insert number attached. This number corresponded to the number given to the original

58

insert. The Bam HI inserts were numbered 1-70 and the Bgl II inserts were numbered 75-
115.
E. Radiation Hybrid Mapping

Radiation hybrid (RH) mapping of STS was preformed using the IMpRH panel (Yerle
et al. 1998). Amplification in hamster DNA was not observed for any RDA STS.
Optimized PCR profiles were used to amplify the 1 18 pig-rodent hybrids of the IMpRH
panel. The PCR was conducted in 96 well plates utilizing the same PCR reaction as
previously described except that, 12.5 ng of DNA was used for each hybrid instead of 25
ng. Products from the PCR were visualized on 3% agarose gels containing 0.4 pg/ml
ethidium bromide. Gels were destained in 1 X TBE buffer (89 mM Tris base, 89 mM
Borate and 2 mM EDTA; pH 8.0) for 1-2 hrs to minimize background fluorescence.
Amplification of each hybrid was scored as either positive (1), negative (0) or ambiguous
(?). Preliminary two point and multipoint analysis of RH data was performed using the
IMpRH server mapping tool as outlined by Milan et al. (2000) (http://imprh.toulouse.
inra.fr/). This data was subsequently used to determine the approximate order for each
RDA marker relative to previously reported markers (Hawken et al. 1999).
F. Polymorphism Identification

To identify polymorphisms, single-stranded conformational polymorphism (SSCP)
analysis was performed for each STS. The STS were amplified from both a DNA pool
consisting of DNA from all pigs in the Landrace selection experiment and twelve
individual Landrace pigs. The PCR products were heated to 98°C for 5 min in a
denaturing loading buffer (95% formamide, 0.05% xylene cyanol, 0.05% bromophenol

blue). The Bio-Rad D-Code Mutation Detection System (Bio-Rad Laboratories,

59

 

Hercules, CA) was used for SSCP analysis. Denatured DNA fragments were
electrophoresed on 8% native polyacrylamide gels with 5% glycerol at 4°C. Each gel
was run at a constant wattage, which ranged from 25-40 W depending on equipment
conditions, for 6-12 hrs. Gels were stained in a 1 mg/ml ethidium bromide solution for 3
min and destained in deionized water for 3 min with subsequent documentation using a
Bio-Rad Gel Doc 2000 Image Analysis System.

The markers MSURDA42 and MSURDA91 did not contain a discemable SSCP.
However, it was observed that MSURDA42 possessed single-stranded patterns consistent
with the presence of two PCR products of different lengths. This insertion/deletion
polymorphism was confirmed by polyacrylamide gel electrophoresis (PAGE).
MSURDA91 displayed a SSCP pattern too complex to be easily genotyped. Two
individuals possessing different SSCP conformations were sequenced and sequence
analysis revealed the presence of a single-nucleotide polymorphism in a Nla III
recognition site. A restriction fragment length polymorphism (RFLP) assay was
subsequently developed to genotype MSURDA91.

To fithher characterize polymorphisms observed in each marker, alleles were
sequenced. This was accomplished by amplifying each marker from individuals either
homozygous or heterozygous for SSCP alleles and sequencing the PCR products on an
ABI 373 automated DNA sequencer.

G. Statistical Analysis

Pigs from both the select and control lines were genotyped for each RDA marker. The

PROC FREQ function of SAS (1998) was used and a Fisher’s exact test was performed

to determine differences in allele frequencies between the two lines.

60

H. Linkage Analysis and Map Construction

Markers with an identified polymorphism were used to genotype pigs from the
PiGMaP reference families. Linkage analysis was performed using CRIMAP 2.4 (Green
et al. 1990). The two-point option was used to obtain preliminary pairwise comparisons
between markers. The “all” and “flips” options using genotypic data for previously
reported markers (Archibald et al. 1995; Rohrer et al. 1996) were used to obtain the most
likely marker order. In addition, a linear map of markers was determined using the
“fixed” option. Linkage maps were drawn using the CorelDraw 8 software package and
aligned next to chromosome ideograms based on the standard porcine karyotype as

described by Gustavsson (1988).

Results

A. RDA and STS Identification

Two and three rounds of RDA were performed for Bgl II and Bam HI RDA fragments,
respectively. The final round for each experiment contained multiple difference products
visible on an agarose gel. These fragments were cloned and sequence analysis revealed
that all Bam HI inserts were unique and only one (MSURDA108) Bgl II insert was
present in two copies. A BLAST search indicated that RDA fragments either had no
significant homology match, were homologous to porcine repetitive elements or in the
case of MSURDA79 and MSURDAl 11 were homologous to known human genes. The
RepeatMasker web-based server was used to screen each RDA fragment for repetitive

DNA elements. Table 3.] summarizes the RepeatMasker results. Oligonucleotide primer

61

 

sets were designed and a total of 18 STS were developed. Table 3.2 summarizes primer
sequences, fragment sizes and PCR conditions for each STS.
B. Polymorphism Identification

Polymorphisms including six SSCP, one insertion/deletion and one RFLP were
identified in eight STS (Table 3.3). Marker alleles were sequenced to identify the
specific SNPs leading to different SSCP conformations. The sequence analysis results
are presented in Table 3.4 and Figure 3.2. A 15 bp insertion/deletion polymorphism
identified in MSURDA42 is shown in Figure 3.3.
C. STS Mapping

A total of 18 RDA markers were localized to the porcine RH map and 9 (50%) were
placed on the genetic linkage map. Table 3.3 lists the RH and genetic linkage map
information, including chromosomal locations and most significantly linked markers with
corresponding LOD scores. MSURDA79 and MSURDA] 11 were found to be
homologous to interferon regulatory factor 6 (IRF6) and potassium voltage-gated
channel, Shab related subfamily, member 2 (KCNB2), respectively. Map results for
these two markers will be presented in Chapter 4. Figure 3.4 illustrate the pig
cytogenetic, linkage and RH maps showing the location of RDA markers relative to
previously reported markers (Archibald et al. 1995; Rohrer et al. 1996; Hawken et al.
1999).
D. Allele Frequencies

To determine if the isolated RDA markers were segregating in the Landrace selection
experiment, allele frequency differences between select and control lines were analyzed.

Genotypes were determined for individuals in both the select and control lines for all

62

 

markers. Table 3.5 summarizes the allele frequency data and the results of the Fisher’s

exact test.

Discussion

A large number of unique RDA difference products were obtained using both Bam HI
and Bgl II. Sequencing of fragments from both experiments revealed that a portion of the
fragments contained porcine repetitive DNA elements. However, the frequency of
repetitive DNA elements was much higher in Bam HI fragments. Since the same DNA
samples were used in both experiments, the difference in frequency could potentially be
caused by differences in the location of recognition sequences for each enzyme in the
porcine genome. It is possible that the Bam HI recognition site is more frequently
associated with porcine repetitive DNA elements than the Bgl II recognition site. This
apparent association has been observed in the construction of porcine bacterial artificial
chromosome (BAC) libraries in which Bam HI produced BAC inserts with a high
frequency of repetitive DNA elements flanking the ends (Dr. T.P.L. Smith, personal
communication). In addition, we have analyzed two porcine-specific repeat elements that
were homologous to a number of our Bam HI inserts. One sequence, a 1258 bp pig
gender-neutral repetitive DNA element (GenBank accession #X16513), contained a total
of four Bam HI sites with two sites clustered in each of two 200 bp regions (Akamatsu et
al. 1989). The other sequence was a porcine short interspersed element (SIN E)
approximately 300 bp in length containing two Bam HI sites approximately 200 bp apart
(Frengen et al. 1991). Both elements are present in the pig genome in hundreds of

thousands of copies and digestion with Bam HI would create an abundance of restriction

63

fragments approximately 200 bp in length. These fragments would be easily amplified in
the RDA process. It is also interesting to note that Bgl 11 sites are not present in either of
these repetitive DNA elements. To alleviate the high frequency of repetitive DNA
elements in future experiments, either different restriction enzymes could be used or
normalized tester samples could be produced. Normalization could be achieved by
isolating repetitive inserts afier the first round of RDA, then adding the inserts at a high
concentration to the final subtractive hybridization.

Polymorphisms were identified in eight of the 16 markers (50%) reported in this
chapter. Due to the high level of polymorphism observed for RDA fragments, SSCP
analysis provided a rapid and inexpensive technique for polymorphism identification. All
of the SSCPs used for mapping were reproducible and easily scored. In addition, 48
samples were analyzed simultaneously, enabling each marker to be mapped quickly.

Eight RDA markers with an identified polymorphism contained a total of 20 SNPs
(not including the insertion/deletion polymorphism in MSURDA42) with a range of 1 to
5 SNPs per marker. This resulted in a SNP frequency of approximately 1 SNP per 91.4
bp. However, this estimation is likely biased given the fact that only markers with a
SSCP were sequenced. Markers without a detectable SSCP would likely have a much
lower (possibly zero) SNP frequency.

Four of the identified SNPs were found within restriction enzyme recognition
sequences. This will allow for the implementation of RFLP assays for four of the six
SSCP markers. The polymorphic recognition sites in these four markers were a th I
site in MSURDA7, a My I site in MSURDA84, an Aci I site in MSURDA95 and an Alu I

site in MSURDA99. These RFLP assays are currently under development and evaluation

64

 

in our laboratory. After assay development, these markers along with MSURDA91 will
be suitable for high-throughput genotyping and can be evaluated in large resource
populations.

RDA markers were localized to 11 pig chromosomes. Each chromosome contained
one marker with the exception of SSCll and SSC14, which had three and four markers,
respectively. Markers MSURDA78, MSURDA89 and MSURDA95 appear to be
randomly distributed across the RH map of SSC] 1. However, three of the four markers
(MSURDA82, MSURDA84 and MSURDA91) on SSC14 fall within an approximately
33.8 cM interval on the genetic map. In recent studies, suggestive QTL have been '
identified for product yield, backfat and loin muscle area on SSCll and 14 (Rohrer et al.
1998a; Rohrer et al. 1998b). In addition, suggestive QTL for length of small intestine
and average backfat have been identified in the region containing the SSC14 RDA
markers (Knott et al. 1998). The clustering of RDA markers in these two regions of the
genome could potentially be the result of random sampling. However, it is also possible
that QTL exist on these chromosomes for traits segregating in the Landrace population.

The first QTL reported in pigs was localized to SSC4. This genomic region
significantly affected growth (birth to 70 kg), length of small intestine, average backfat
and abdominal backfat percentage (Andersson et al. 1994). As a result of its large effect
on fatness traits, this QTL has also been confirmed in a number of other studies
(Andersson-Eklund et al. 1998;Walling et al. 1998; Knott et al. 1998; Marklund et a1.
1999; Walling et al. 2000). Interestingly, MSURDA86 is located within the interval

containing the most likely QTL position, between markers $0001 and S0107. It is

65

 

probable that this putative QTL is segregating and under selection in the Landrace
population and it is possible that MSURDA86 is linked to this QTL.

Significant QTL having large effects on backfat, intramuscular fat and muscling traits
have been identified on SSC6 and 7. Various QTL on SSC6 have been reported for
backfat, intramuscular fat and loin muscle area (Ovilo et al. 2000; de Koning et al. 1999)
including a paternally expressed backfat QTL (de Koning et al. 2000). The RDA marker
MSURDA42 localized to SSC6 just flanking the most probable position of these QTL.
Similarly, MSURDA7 flanks significant QTL on SSC7 having large effects on loin
muscle area and backfat (Rohrer et al. 1998a; Rohrer et al. 1998b; de Koning et al. 1999;
de Koning et al. 2000; Rattink et al. 2000).

These genomic regions on SSC4, 6 and 7 appear to have major effects on fat
deposition and muscling traits. In addition, a number of RDA markers flank QTL with
significant and suggestive levels of significance for numerous other traits. Marker
MSURDA] 10 is located on SSC8 in the region of a QTL for carcass length (Andersson-
Eklund et al. 1998). Additionally, MSURDA106 is located in a suggestive QTL for tenth
rib backfat (Rohrer et al. 1998a), and a suggestive QTL for loin muscle area and average
daily gain was reported on SSC3 (Andersson-Eklund et al. 1998; Casas-Carrillo et al.
1997) corresponding to the location of MSURDA96.

Most RDA markers in this study were determined to localize in close proximity to
previously reported QTL for growth, carcass and pork quality traits. Significant QTL
affecting number of corpora lutea on SSC8 and ovulation rate on SSC8 and SSC13 have
also been reported (Rathje et al. 1997; Wilkie et al. 1999). Markers MSURDAI 10

(SSC8) and MSURDA106 (SSC13) flank these QTL. Similarly, a QTL for gestation

66

length has recently been described on SSC9 overlapping the region containing
MSURDA93 (Wilkie et al. 1999).

The majority of RDA markers generated in this study mapped to regions containing or
flanking previously reported QTL for various traits. It is interesting to note that the QTL
mentioned above were identified in different populations and have varying effects on the
phenotypes involved. It is not likely that all of these QTL are segregating in the
Landrace population. Also, in QTL studies, only those QTL contributing a large
percentage of F2 variance are identified. RDA is capable of generating markers flanking
QTL regardless of magnitude, which might explain why RDA markers were isolated on
several chromosomes or in regions not located near previously identified QTL.

In addition to 16 anonymous DNA markers, two genes were isolated. The
identification of IRF 6 and KCNB2 make this the first report of the isolation of coding
sequences with genomic RDA. Given the fact that only 3-5% of the pig genome is
comprised of genes, it seems unlikely that the identification of two genes in this study
was random. It is more plausible that selection in the Landrace population led to
segregation of alleles at these loci. The cDNA cloning, expression analysis and
chromosomal localization of IRF6 and KCNB2 will be presented in Chapter 4. In
addition, the roles of these genes as potential candidate genes for production traits will be
discussed.

Allele frequencies for each marker were determined in the select and control lines.
This was done to determine if selection had significantly changed the allele frequencies
between the select and control lines. It must be noted that the levels of significance

presented are overestimated since genetic drift was not accounted for in the analysis

67

using Fisher’s exact test. The analysis revealed that the allele frequencies for five of the
eight RDA markers did significantly differ between the select and control lines. These
data suggest that alleles for at least a portion of the isolated markers are segregating in the
select and control lines. It also should be noted that different QTL with differing
magnitudes of effect might be under differing levels of selection pressure. This could
explain why three of the markers were not divergent between the two lines. However,
this preliminary data does suggest that all markers generated in this study should be
tested in larger populations for potential associations with various economically
important traits.

Interestingly, the frequencies of the RYRl alleles are significantly divergent between
the select and control lines. It has been demonstrated that pigs heterozygous at the RYRl
locus are heavier muscled and leaner (Lundstrom et al. 1995). While most would agree
that the difference in lean meat content is not a direct result of the RYRl locus, it appears
from these data that the RYRl locus is under selection. Marker MSURDA42 is located
only 13.6 cM (on the sex-averaged genetic map) from the RYRl locus. Its isolation may
be the result of physical linkage with the RYRl locus or due to selection for QTL in the
vicinity of MSURDA42. However, alleles for MSURDA42 were not found to be
significantly different between the select and control lines suggesting that selection
pressure on this locus differed from selection for RYRl alleles. It has been hypothesized
by MacLennan and Phillips (1992) that the RYRl mutation leading to a down regulated
calcium channel could have a direct affect on muscling and leanness. This is because a
malfunctioning calcium channel could possibly allow the muscle fibers to maintain a

small but constant level of contraction, which would lead to fiber hypertrophy and the

68

energy utilized in the process could contribute to less fat deposition. Thus, the RYRl
locus may be under selection in this population as a result of its direct affect on carcass
traits or due to linkage disequilibrium with other loci.

In this study, 18 markers were isolated from pigs divergent both in genotype and
phenotype using RDA. Markers isolated in this study increase the marker density on
both the pig genetic and RH maps. In addition, most of the identified markers were in
close proximity to known putative QTL reported in previous studies. These markers can
now be tested in large resource populations. If strong associations are found between
RDA markers and various phenotypes, these markers could potentially be used in marker

assisted selection programs.

69

Chapter 3

Tables and Figures

70

Table 3.1. Summary of RepeatMasker results for Bam HI and Bgl II inserts.

 

 

 

 

RDA experiment

Bam HI Bgl II
Number of inserts sequenced 14 37
Total amount of filtered sequence (bp) 4,442 10,216
GC content (%) 48.46 44.92
Frequency of SINE sequences (%)a 31.04 10.70
Frequency of LINE sequences (%)b 0.00 1.41
Frequency of LTR sequences (%)° 0.00 1.23
Frequency of satellite repeat elements (%) 26.90 0.00
Frequency of simple repeat elements (%) 0.52 3.29
Total amount of repetitive sequences (%) 58.46 20.89

 

a

short interspersed elements (SINE).
b long interspersed elements (LINE).
° long terminal repeats (LTR).

71

Table 3.2. Summary of RDA STS developed.

 

 

Size PCR conditions
Marker Primer sequences (bp) (TA/MgCIZ/Primer)al
MSURDA7 F-5’GTCAGAAAGCGAACAGG 3’ 201 55 / 1.5 / 5
R-5’CCCAGTGCTCCCAAGAC 3 ’
MSURDA42 F-S’GCAGTCACCAGCTACCG 3’ 171 55 / 1.5 / 5
R-5 ’AGGGAAGGTTTCAGCAAG 3 ’
MSURDA78 F-5’CTGCAGCAATGACACACTG 3’ 123 56/ 1.5 / 5
R5 ’AAGGAGCCATCTTTGTTGACT 3 ’
MSURDA79 F-S’GCTGCCACTGTCAAAGGAT 3’ 237 55 / 1.5 / 5
R-5 ’GATGGTCCAGCGAAATGAAG 3 ’
MSURDA81 F-S’AAAGCAAAGGCCATCATCCA 3’ 226 57 / 1.5 / 5
R-5’GCAGCCCACCCGTCAG 3’
MSURDA82 F-S’GACACCCTGCGTATGAGC 3’ 172 55 / 1.5 / 5
R-5 ’CCAGGCAATACATCTGACCA 3 ’
MSURDA84 F-S’GGGCAGCCTGTCTCCTGTA 3’ 195 55 / 1.5 / 5
R-S ’CCATAAGATGTGCGGTGAGC 3 ’
MSURDA86 F-5’AGCTCCCACTTGGTCTTCT 3’ 200 57/ 1.5 / 5
R-5 ’AAGGTAGGGACAAATTGGTG 3 ’
MSURDA89 F-5’CAATCCTTGTGGCTAAGACCC 3’ 192 57/ 1.5 / 5
R-S’GCAAGCCCAGTGAACAGAAT 3’
MSURDA91 F-S’GCCAACACCTCCACCCTTAG 3’ 208 57 / 1.5 / 5
R-S ’GGACACTGCCATCTTGCTTC 3 ’
MSURDA93 F-5’GAAACCTGCTGTGCC 3’ 135 57 / 1.5 / 5
R-S ’ACCTTCTTGTAAGCCAGTAA 3 ’
MSURDA95 F-5’CATGATGACATTCCCAAAT 3’ 172 55 / 1.5 / 5
R-S ’ACTATGAGACTTTCGCACAG 3 ’
MSURDA96 F-5’GCTCCCAAACAACATACTTC 3’ 166 55 / 1.5 / 5
R-5’TTTACACTGCCGTGGAAC 3’
MSURDA99 F-5’CCATCACAGTCCTATTTCTT 3’ 182 55 / 1.5 / 5
R-5 ’GCAAATAACTCTGTGTGCTA 3 ’
MSURDA106 F-S’ACTGCAGGAGGAAGGAGA 3’ 147 57 / 1.5 / 5
R-S’GGGTGCAGTTATTTGATTTG 3’
MSURDA108 F-S’ACAGGCCATGCCTTTGAACA 3’ 150 56/ 1.5 / 5
R-5 ’TGACCTAGAAGAACCACCTC 3 ’
MSURDAl 10 F-S’T'ITGGTGTGTTCATGGGAGG 3’ 197 56/ 1.5 / 5
R-5’TGGGAGAGCTGCGTAGGAAT 3’
MSURDAl 11 F-5’TGAGGCGGAGTTACAACGAA 3’ 209 57 / 1.5 / 5

R-5 ’CACCTCCTCCATCTCTATCT 3 ’

 

a TA, PCR annealing temperature in °C; MgClz, concentration in mM; Primer,
concentration in pM.

72

Table 3.3. Summary of RH and genetic linkage mapping results.

 

b

 

Marker RH mappinga Type of polymorphism Genetic linkage
mappingc
MSURDA7 SSC7 SSCP SSC7
(SW1959, 11.81) (S0334, 28.60)
MSURDA42 SSC6 INS/DEL SSC6
(SW1473, 6.81) (sw122, 13.11)
MSURDA78 SSCll
(SW1460, 8.23)
MSURDA81 SSC5
(ACOZ, 6.57)
MSURDA82 SSC14 SSCP SSC14
(SW1032, 5.88) (80058, 9.92)
MSURDA84 SSC14 SSCP SSC14
(SW761, 16.33) (SW761, 28.17)
MSURDA86 SSC4
(SW45, 25.24)
MSURDA89 SSCll
(SW903, 6.98)
MSURDA91 SSC14 RFLP SSC14
(SW361, 19.56) (30007, 16.08)
MSURDA93 SSC9
(SW2093, 7.31)
MSURDA95 SSCll SSCP SSCll
(SW435, 13.41) (S0230, 11.57)
MSURDA96 SSC3
(SWR2069, 6.10)
MSURDA99 SSC16 SSCP SSC16
(SW1645, 20.26) (SW419, 15.68)
MSURDA106 SSC13 SSCP SSCI3
(80282, 6.07) (80219, 12.58)
MSURDA108 SSC14
(SW857, 11.42)
MSURDAI 10 SSC8

(80098, 10.63)

 

a Radiation hybrid mapping data presented as chromosomal location with most
significantly linked marker and LOD score in parentheses.

b SSCP, single-stranded conformational polymorphism; INS/DEL, insertion/deletion
polymorphism; RFLP, restriction fragment length polymorphism.

° Genetic linkage mapping data presented as chromosomal location with most
significantly linked marker and LOD score in parentheses.

73

Table 3.4. Summary of SNP haplotypes present in RDA markers.

 

 

 

SNP haplotype
Marker Number of SNPs
SSCP genotypea Haplotypeb

MSURDA7 2 AA T-G

AB N-N

BB C-A
MSURDA82 1 AA A

AB N

BB G
MSURDA84 4 AA C-G-T—T

AB N-N-N-N

BB T—A-A-G
MSURDA91c 4 AA C-T-C-T

BB T-C-T-C
MSURDA95 2 AA A-G

AB N-N

BB G-T
MSURDA99 5 AA C-A-T-T-A

AB N-A-T-T-A

BB T-A-T-T-A

BC N-N—N-N-N

AC C-N-N-N-N

CC C-G-C-A-T
MSURDA106 1 AA T

AB N

BB A

 

a Genotypes obtained using SSCP analysis.

b Haplotype of all SNPs detected by DNA sequencing. A, adenine; G, guanine; C,
cytosine; T, thymine; N, not distinguishable.

° SSCP identified was too complex to score. SSCP genotypes were arbitrarily assigned
and only two individuals possessing two distinct SSCP conformations were sequenced
which led to the identification of a Nla III RFLP.

74

Table 3.5. Summary of RDA marker allele frequency differences between select and

control lines.

 

Allele Frequencies

 

 

 

Marker F isher’sd
Selecta Controlb Difference° exact test

RYRl N= 0.57 N: 0.83 0.26 *
n= 0.43 n= 0.17

MSURDA7 A: 1.0 A= 0.95 0.05 NS
8: 0.0 B: 0.05

MSURDA42 A: 0.76 A= 0.80 0.04 NS
B= 0.24 B= 0.20

MSURDA82 A: 0.12 A: 0.32 0.20 *
B: 0.88 B= 0.68

MSURDA84 A= 0.29 A: 0.30 0.01 NS
B= 0.71 B= 0.70

MSURDA91 A= 0.05 A= 0.22 0.17 *
B= 0.95 B= 0.78

MSURDA95 A: 0.10 A= 0.0 0.10 *
B: 0.90 B= 1.0

MSURDA99 B: 1.0 B= 0.86 0.13 "‘
C= 0 0 C= 0.13

MSURDA106 A: 0.05 A= 0.52 0.47 **
B= 0.95 B= 0.48

a n=21.
b n=23.

° Absolute difference in allele frequencies between the select and control lines.

d * P< 0.05, ** P< 0.01.

75

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76

Figure 3.2. RDA marker consensus sequences. Polymorphic sites are indicated by
parentheses ( ) with each of the two possible bases listed.

77

MSURDA7 Consensus sequence

TTGTCAGAAAGCGAACAGGTGGAAAATCACAATAAATCAAACATGTAGGTGTTGCCCCCAAG
TGGCATGAAAGTAGCTTAGGTTCCAACAATTGCTTTCCATTAATGGCA(T/C)CTTAAAAAT
GAGATGGAGGTGGTG(G/A)TTTAAAAATGACATCAGGAGTTCCCGCTGTGGTGCAACAAGA
TGGGCAGTGTCTTGGGAGCACTGGGGA

MSURDA82 Consensus sequence

CCTTGACACCCTGCGTATGAGCCATAGAGAGTGGTAGGATAGAGTGTCTCACTCCTCAGGTC
TTCCTGGATGACACGAGAGGAGTCCCTCTGGTCTAGCCTGGACACTCTTCTTCAGGTCCTGT
GAAGGACATGCC(A/G)TCTTCTGACCCTCTCTTATTGGTCAGATGTATTGCCTGGA

MSURDA84 Consensus sequence

TTGGGCAGCCTGTCTCCTGTATTACC(CIT)ACCCACGAAAAGCCCCTCGCCCCGCAGCACC
GTGACCAGGAATAATGGGGCCCCAGGTTGTAACACAGGGGTTCGGGATATGACATGTGTGCT
G(G/A)TGGG(T/A)GTCTCATTTCAGAATATGTCCCGCT(T/G)ACAGCAAGAAGCCCGTC
TCTAGGAAGCTCACCGCACATCTTATGGA

MSURDA91 Consensus sequence

GCCAACACCTCCACCCTTAGGCCCACGAGGGTTTCCCGTTTGAGCCAAAACCCGGGGACTCC
TTGTCTTGCCCAT(T/C)CTGTCTGTTCTCCCTTCTCACAGTGAATCACTGGCCTCACATTC
ACTCAAATACACAAAATATACAAACTGCACAGACTGGTTTCCAAATTCAAAGAGTGGTTCCA
TCTTCTGAAGCAAGATGGCAGTG

MSURDA95 Consensus sequence

TTCATGATGACATTCCCAAATACATATTTATAGAAAGGAAAGCCAGTGATCC(A/G)ACTTA
CAACCAG(G/T)TTTGATATAAATGACCCATGGATTTTATACATGGAGCTTTATGCTCTATG
CTATTAAGATGGGTTATAGCTGAGGGCAAAAACAGCTGCTGTGCGAAAGTCTCATAGTA

MSURDA99 Consensus sequence
CCATCACAGTCCTATTTCTTTTTTTCTCTGAAATAAACCTCAAATCCAGG(T/C)TTTTTGT
TATGTTTTTGCATTCCCACTGTCACCAGCCCAGTCCA(A/G)GTCACCTCAG(T/C)TTCTC
AT(T/A)TGGCTTCCTGAGAAC(A/T)ACAAATGTCCTACTTCAATATAGTCTCTGTTTAGC
ACACAGAGTTATTTGGCA

MSURDA106 Consensus sequence

ACCATAAAAAGACTCCGGCCACAAAGTCAGTTAAGCATGTTT(T/A)CTGCCCTTTGGGAAG
AAGCGAG

78

TAG‘ICCCI‘GT‘GCGSPGQPCCCACCTCCC’I‘GCC'IGG ICCCA CTCA'IG TCC'P'IG CPGAA ACCI’ICIII '1’
40 l 0

m ,le lll llllll l...

 

Figure 3.3. Identification of a 15 bp length polymorphism in MSURDA42. Underlined
sequence indicates the 15 bp insertion in the A allele. Arrow indicates location of the 15
hp deletion in the B allele. AA and BB, homozygous genotypes; AB, heterozygous
genotype. Figure is presented in color.

79

Figure 3.4. Diagrams of the pig cytogenetic, RH and genetic linkage maps for pig
chromosomes containing RDA markers. RDA markers are underlined and shown in
bold face type. Maps show the position of RDA markers relative to previously
mapped markers (Archibald et al. 1995; Rohrer et al. 1996, Yerle et al. 1999; Hawken
et al. 1999). The sex-averaged linkage map position is represented in cM (centi-
Morgans) and RH map position is in cR (centi-Rays). Chromosome ideograms were
drawn to represent the standard porcine karyotype described by Gustavsson (1988).
Panel A, SSC3; Panel B, SSC4; Panel C, SSC5; Panel D, SSC6; Panel B, SSC7; Panel
F, SSC8; Panel G, SSC9; Panel H, SSC] 1; Panel 1, SSC13; Panel J, SSC14; Panel K,
SSC16.

80

 

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91

CHAPTER 4
CHARACTERIZATION OF THE PORCINE IRF 6 AND KCNB2 GENES: CDNA

CLONING, EXPRESSION ANALYSIS AND CHROMOSOMAL LOCALIZATION

Abstract

Representational difference analysis (RDA) was used to isolate genetic markers from
pigs differing in loin muscle area. A BLAST search revealed that RDA fragments
MSURDA79 and MSURDAl 11 were homologous to the interferon regulatory factor 6
(IRF 6) gene and the potassium voltage-gated channel, Shab-related subfamily, member 2
(KCNB2) gene. IRF 6 is a member of the interferon regulatory factor family of
transcription factors, which regulate the function of multiple interferon genes in a host of
molecular mechanisms. KCNB2 is one of two members of the Shab-related subfamily of
potassium channels, which are known to aid in the regulation of the contractile status of
smooth muscle cells. IRF6 and KCNB2 clones were isolated from pig intestinal and
cerebellum cDNA using PCR primers designed from RDA fragments and human IRF6
and KCNB2 mRNA sequence and 3’ rapid amplification of cDNA ends (RACE). RT-
PCR analysis revealed the presence of both IRF 6 and KCNB2 transcripts in pig aorta,
cerebellum, duodenum, ileum, jejunum, liver, skeletal muscle and uterus and IRF6
transcripts in kidney. Northern blot analysis confirmed expression of 2.5- and 2.0-kb
IRF 6 transcripts in pig uterus, kidney and ileum. KCNB2 transcripts of 6.0-, 2.8- and
1.6-kb were expressed in pig aorta, uterus, cerebellum, kidney, liver and skeletal muscle
and the expression of the 6.0-kb transcript was observed in pig ileum. [RF 6 has been

localized to human chromosome (HSA) lq32.3-q4l. This region of HSAl is conserved

92

on pig chromosome (SSC) 9q23-qter. Physical mapping using a pig-rodent somatic cell
hybrid panel placed IRF6 in the interval of SSC9q1 l-q26 (risk of error less than 0.1%).
Radiation hybrid (RH) mapping using the INRA-Minnesota RH panel (IMpRH) showed
significant linkage between IRF 6 and two markers on SSC9, SW749 (LOD=14.72) and
SW1651 (LOD=14.43). To further confirm this localization, a single-stranded
conformational polymorphism (SSCP) was identified in IRF 6. The PiGMaP reference
families were genotyped and linkage analysis was conducted using the two-point option
of CRI-MAP 2.4. IRF6 exhibited significant linkage with SW749 on SSC9 (LOD=5.42,
0:0.00). Human KCNB2 has been localized to HSA8q12-q13. This region of HSA8 is
conserved on SSC4p15-q16. Physical mapping placed KCNB2 in the interval of
SSC4q15-q16 (risk of error less than 0.1%). Radiation hybrid (RH) mapping showed
significant linkage between KCNB2 and four markers on SSC4 with the most significant
linkage to SW839 (LOD=9.00). Using a pool-and-sequence strategy, a single nucleotide
polymorphism (SNP) was identified in KCNB2 and a genotyping assay is currently being
developed. Further analysis of porcine [RF 6 and KCNB2 as candidate genes for traits of

economic importance is warranted.

Introduction

The development of high-resolution pig genetic maps has recently been reported
(Archibald et al. 1995; Rohrer et al. 1996; Marklund et al. 1996). These maps have
provided the framework for the identification of a number of QTL for various production
traits in the pig (Andersson et al. 1994; Andersson-Eklund et al. 1997; Rohrer et al.

1998a; Rohrer et al. 1998b; de Koning et al. 1999). The ultimate goal of QTL

93

identification is the iSolation of the gene(s) responsible for the observed phenotypic
variation. However, QTL studies provide only gross estimations of QTL size and
location and do not produce the resolution needed for positional cloning. Therefore,
there is a need to evaluate novel techniques such as representational difference analysis
(RDA), which have the potential to identify gene sequences located in QTL regions.

Interferons (1F Ns) are a family of multi-functional cytokines that were discovered as
mediators of cellular resistance against viral infection and later shown to play a diverse
role in the immune response to pathogens and various other cellular processes. There are
two classifications of IFNs, type I (IFNOt and IFNB) and type II (IFNy). Type I
interferons are produced by virus-infected cells and primarily fimction as a cell’s first
response to a viral infection, whereas IF NY functions to engage in the appropriate
immune response and pathogen clearance (Stark et al. 1998). The interferon regulatory
factor (IRF) family of transcription factors function to regulate the activity of both type I
and type 11 IF Ns. The IRF family contains ten members all sharing a conserved 115
amino acid DNA binding domain (Mamane et al. 1999). IRF6 is one of the most recent
additions to the family and its specific function is unknown.

Potassium (K+) channels determine the contractile status of smooth muscle by
controlling the influx of Ca2+ through voltage-gated Ca2+ channels. Two types of K+
channels have been identified in smooth muscle, the voltage-gated “delayed rectifier”
channels that are insensitive to intracellular Ca2+ and the voltage-activated or Ca2+-
activated channels. The Shab related subfamily of voltage-gated K+ channels received its
name due to their shared homology with the Shab family of Drosophila melanogaster

voltage-gated K+ channels. The Shab related subfamily has two members, KCNBI and

94

KCNB2. KCNB2 expression has been identified in all regions of the canine
gastrointestinal tract, brain, artery, uterus and vein and most likely functions as one of the
components of the delayed rectifier system in many smooth muscles (Schmalz et al.
1998)

We have previously applied the technique of RDA to isolate genetic differences
between two pig populations differing in genotype at the RYRl locus and also differing
in phenotype for various traits (see Chapter 3). Two of the RDA difference products
were determined to be IRF6 and KCNB2. The purpose of this study is to provide the
preliminary characterization for both of these genes in the pig. This will provide the
information needed to evaluate each gene as a potential candidate gene for economically

important pork production traits.

Materials and Methods

A. IRF6 and KCNB2 cDNA Isolation

IRF6 coding sequence (nucleotide (nt) position 1...1070) was amplified from pig
intestinal cDNA using the PCR (IRF6-F, 5’-AGAGACAGG GAAGTACCAGG-3’;
IRF6-R, 5’-GCTGCCACTGTCAAAGGAT-3’). In addition, 3’ rapid amplification of
cDNA ends (RACE) (Life Technologies, Rockville, MD) using two pig specific PCR
primers (MSURDA79-IRF6, 5’-AGGACAGATAGAGAAGCAGCC-3’; IRF6-GSP2-3,
5’-ACGATCCTITGACAGTGGCA-3’) was used to amplify the remaining 3’ end,
according to the manufacturer’s protocol.

A similar approach was taken to clone KCNB2 cDNA. A cDNA fragment of KCNB2

(nt position 586. . .241 1) was amplified from pig cerebellum cDNA using the PCR

95

(MSURDAl l l-F, 5 ’-TGAGGCGGAGTTACAACGAA-3 ’; KCNBZ-R, 5 ’-
TTCAGTGAAGCTCCTCCTC-3’). The remaining 3’ end of KCNB2 was amplified
using 3’RACE with pig specific primers (KCNB2-GSPl-3’, 5’-TTCCGCCACATTGT
CAGGGA-3 ’; KCNB2-GSP2-3 ’ ,5 ’-CTGACGCCTCGGTT1"C CCAA-3 ’).

The PCR for each IRF6 and KCNB2 cDNA fragment was performed using
approximately 25 ng of cDNA in 10 pl reactions containing 1 X PCR buffer (Promega,
Madison, WI), 1.5 mM MgC12, 200 pM each dNTP, 0.5 or 2.0 pM each primer and 0.5 U
T aq DNA polymerase (Promega, Madison, WI). The PCR profiles included an initial
denaturation of 3 min at 94°C followed by 30 cycles of 94°C for 1 min, 55°C for 1 min,
72°C for 1 min and a final extension of 72°C for 10 min.

All of the amplified cDNA products were cloned into the plasmid vector pGEM-T-
Easy (Promega, Madison, WI). Recombinant vectors were transformed into DHSOL
competent cells (Life Technologies, Rockville, MD) according to the manufacturer’s
protocol. Cultures were plated on LB + ampicillin plates and incubated at 37°C
overnight. Single colonies were picked, 3 ml of LB + ampicillin medium was inoculated
and cultures were incubated with shaking at 37°C overnight. Plasmid DNA was isolated
with the QIAprep spin miniprep kit (Qiagen, Valencia, CA) and digested with EcoR I to
confirm the presence of an insert. One plasmid with an insert of the correct size for each
fragment was sequenced on an ABI 373 automated DNA sequencer using the ABI Prism
dye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City,
CA). Bi-directional sequence was obtained using primer walking. Overlapping
sequences were aligned using BioNavigator (http://www.bionavigator.com) and sequence

comparisons were determined using the Biology Workbench (http://workbench.sdsc.edu).

96

B. RT-PCR

Total RNA was extracted from various pig tissues with the Qiagen RN easy kit
(Qiagen, Valencia, CA) according to the manufacturer’s protocol. The mRN A was
reverse transcribed using the SuperScript II Reverse Transcriptase (Life Technologies,
Rockville, MD) according to the manufacturer’s protocol. IRF 6 and KCNBZ were
amplified (primer sequences: IRF 6, MSURDA79-IRF 6, 5’-AGGACAGATAGAGAAG
CAGCC-3’ and MSURDA79-F, 5’-GCTGCCACTGTCAAAGGAT-3’; KCNB2,
MSURDA] 1 l-F, 5’-TGAGGCGGAGTTACAACGAA-3’ and MSURDAl 1 l-R, 5’-
CACCTCCTCCATCTCTATCT-3;) using previously described PCR conditions. The
IRF 6 primers spanned intron 6 of IRF 6 in genomic DNA. Amplification of IRF6 and
KCNB2 in cDNA resulted in the amplification of a 195 bp fragment of IRF 6 and a 209
bp fragment of KCNB2. Products from the PCR were visualized on 2% agarose gels
containing 0.4 pg/ml ethidium bromide. The IRF6 and KCNB2 RT-PCR products were
cloned and sequenced to confirm correct amplification.
C. Northern Blot Analysis

Eight pg of total RNA was denatured (24 mM HEPES, 6 mM sodium acetate, 1.2 mM
EDTA, 50% deionized formamide, 2.2 M formaldehyde) and fractionated in a 1.2 %
agarose gel containing 2.2 M formaldehyde in 20 mM phosphate buffer, pH 7.0. RNA
was transferred to Hybond N+ nylon membranes (Amersham, Piscataway, NJ) using a
VacuGene XL vacuum blotter (Amersham Pharmacia Biotech, Uppsala , Sweden) in 20
X SSC (1 X SSC is 0.15 M NaCl and 0.015 M sodium citrate) and membranes were UV
crosslinked in a GS Gene Linker UV chamber (Bio-Rad Laboratories, Hercules, CA).

Prehybridization was carried out at 65°C for 2 hrs in 6 X SSC, 5 X Denhardt’s solution,

97

0.5 % SDS and 100 pg/ml salmon sperm DNA. Hybridization to 32P-labeled IRF6 and
KCNB2 probes (labeled to specific activities of 1.5-3.0x109 cpm/pg) was performed at
65°C overnight. Blots were rinsed twice in 2 X SSC/0.1 % SDS at room temperature for
5 min, twice in 0.2 X SSC/0.1 % SDS at 42°C for 5 min and twice in 0.1 % SSC/0.1%
SDS at 65°C for 5 min. Blots were exposed to Kodak X-Omat X-ray film in the presence
of intensifying screens at -70°C for 1-4 days.
D. Polymorphism Identification

The sequence-tagged sites (STS) derived from the IRF 6 and KCNB2 RDA products
(MSURDA79 and MSURDA] l 1) were subjected to single-stranded conformational
polymorphism (SSCP) analysis. RDA was performed on DNA samples from Landrace
pigs that were either unselected or selected for increased loin muscle area. Details
regarding the RDA experiment are reported in Chapter 3. The IRF 6 and KCNB2 STS
were amplified from both a DNA pool consisting of DNA from all pigs in the Landrace
selection experiment and twelve individual Landrace pigs. The PCR profile included an
initial denaturation of 3 min at 94°C followed by 30 cycles of 94°C for 1 min, 55°C for l
min, 72°C for 1 min and a final extension of 72°C for 10 min. The PCR products were
heated to 98°C for 5 min in a denaturing loading buffer (95% formamide, 0.05% xylene
cyanol, 0.05% bromophenol blue). The Bio-Rad D-Code Mutation Detection System
(Bio-Rad Laboratories, Hercules, CA) was used for SSCP analysis. Denatured DNA
fragments were electrophoresed on 8% native polyacrylamide gels with 5% glycerol at
4°C. Each gel was run at a constant wattage, which ranged from 25-40 W depending on
equipment conditions, for 6-12 hrs. Gels were stained in a 1 pg/ml ethidium bromide

solution for 3 min and destained in deionized water for 3 min with subsequent

98

documentation using a Bio-Rad Gel Doc 2000 Image Analysis System (Bio-Rad
Laboratories, Hercules, CA).

KCNB2 was screened for single nucleotide polymorphisms using a pool-and—sequence
strategy. An approximately 1500 bp fragment of KCNB2 was amplified (KCNB2-PW]-
F, 5 ’-TGAGGCGGAGTTACAACGAA-3 ’; KCNBZ-R, 5 ’-TTCAGTGAAGCT
CCTCCTC-3’) using the PCR from a DNA pool of the PiGMaP reference family
grandparents (GPP) and one grandparent individual. Each amplified product was gel
purified using the Qiagen gel purification kit (Qiagen, Valencia, CA). The purified DNA
product was sequenced on an ABI 377 automated DNA sequencer (Applied Biosystems,
Foster City, CA).

E. Cytogenetic Mapping

Physical mapping of IRF 6 and KCNB2 was accomplished using a pig-rodent somatic
cell hybrid panel (Yerle et al. 1996; Robic et al. 1996). IRF6 (MSURDA79 primers, F-
5 ’-GCTGCCACTGTCAAAGGAT-3 ’ and R-S ’-GATGGTCCAGCGAAATGAAG-3 ’)
and KCNB2 (MSURDAI 11 primers, F-5’-TGAGGCGGAGTTACAACGAA-3’ and R-
5—’CACCTCCTCCATCTCTATCT-3’) amplification of the 27 pig-rodent hybrids by the
PCR allowed for chromosomal localizations. PCR products were visualized on 1%
agarose gels containing 0.4 pg/ml ethidium bromide and regional assignments were
determined as described by Chevalet et al. (1997) (http://www.toulouse.inra.fr/lgc/pig/
hybrid.htm).
F. Radiation Hybrid Mapping

Radiation hybrid (RH) mapping of IRF6 and KCNB2 was preformed using the INRA-

Minnesota porcine radiation hybrid (IMpRH) panel (Yerle et al. 1998). IRF6

99

(MSURDA79 primers, F-5-’GCTGCCACTGTCAAAGGAT-3’ and .R-5’-GATGGTCC
AGCGAAATGAAG-3’) and KCNB2 were amplified (KCNB2-PW1—F, 5’-TGA
GGCGGAGTTACAACGAA-3’; KCNB2-PW2-R, 5’-CACCT CCTCCATCTCTATCT-
3 ’) from the 118 pig-rodent hybrids of the IMpRH panel. Products from the PCR were
visualized on 3% agarose gels containing 0.4 pg/ml ethidium bromide. Gels were
destained in 1 X TBE buffer (89 mM Tris base, 89 mM Borate and 2 mM EDTA) for 1-2
hrs to minimize background fluorescence. Amplification of each hybrid was scored as
either positive (1), negative (0) or ambiguous (?). Preliminary, two-point and multipoint
analysis of RH data was performed using the IMpRH server mapping tool as outlined by
Milan et al. (2000) (http://imprh.toulouse. inra.frl). This data was subsequently used to
determine the approximate marker order for IRF6 and KCNB2 relative to previously
reported markers (Hawken et al. 1999).
G. Linkage Analysis and Map Construction

The IRF6 SSCP was used to genotype the PiGMaP reference families. Linkage
analysis was performed using CRIMAP 2.4 (Green et al. 1990). The two-point option
was used to obtain preliminary pairwise comparisons between markers. The “all” and
“flips” options using genotypic data for previously reported markers (Archibald et al. ;

1995; Rohrer et al. 1996) were used to obtain the most likely marker order. A linear map

 

of markers was determined using the “fixed” option. The linkage map was drawn using
the CorelDraw 8 software package and aligned next to chromosome ideograms based on

the standard porcine karyotype as described by Gustavsson (1988).

100

Results

A. IRF6 and KCNBZ cDNA Isolation

The full length coding sequence and a portion of the 3’ untranslated region (UTR) was
isolated for pig IRF6 (Figure 4.1). The cDNA consisted of a 1404 nt coding region and a
401 nt 3’ UTR. The 3’ UTR contained a poly T15 tract 17 nt downstream of the stop
codon (UAA). In addition, a polyadenylation signal (AAAUAA) 19 nt upstream of the
poly A tail was observed. (This sequence differs from the consensus AAUAAA
polyadenylation sequence seen in the 3’ UTR of most mRNAs.) A comparison with
IRF6 sequences from other species indicated a high level of sequence conservation.
Porcine IRF6 nucleotide sequence was 92%, 91% and 90% homologous to human, mouse
and sheep IRF6, respectively. Additionally, the deduced amino acid sequence of IRF 6
was highly conserved (Figure 4.2). Porcine and human IRF6 shared 100% homology
across all 467 amino acid residues. Sheep and mouse IRF 6 amino acid sequences were
98% and 97% identical to porcine IRF6. Porcine IRF 6 also shares a high level of
homology with the evolutionarily distant Xenopus xIRF6 with 76% of the amino acid
residues conserved between them.

An 1836 nt sequence of the porcine KCNB2 coding region was cloned along with 366
nt of the 3’UTR (Figure 4.3). A consensus poly A signal was not observed in the 3’UTR,
however, a 17 nt poly A tract (with one G interruption) was identified. The isolated
coding region spanned from nt position 586. . .2422(2419. . .2422 corresponded to the stop
codon) based on human KCNB2 cDNA sequence. This sequence was 87% homologous

to human and 86% homologous to canine and rat KCNB2.

101

B. Expression Analysis

RT-PCR was performed for IRF 6 and KCNB2 to determine if expression could be
detected in various pig tissues. Expression of IRF6 was observed in pig aorta,
cerebellum, duodenum, ileum, kidney, jejunum, liver, skeletal muscle and uterus (Figure
4.4A). KCNB2 transcripts were identified in pig aorta, cerebellum, duodenum, ileum,
jejunum, liver, skeletal muscle and uterus (Figure 4.4B). Northern blot analysis revealed
the expression of 2.5- and 2.0-kb IRF 6 transcripts in pig uterus, kidney and ileum (Figure
4.5A). KCNB2 transcripts of 6.0-, 2.8- and 1.6-kb were observed in pig aorta, uterus,
cerebellum, kidney, liver and skeletal muscle. Only the 6.0-kb transcript was observed in
pig ileum (Figure 4.5B).
C. Polymorphism Identification

The RDA fragments for both IRF6 and KCNB2 were subjected to SSCP analysis.
This led to the identification of a SSCP in IRF 6. No SSCP was observed in the 209 bp
RDA fragment for KCNB2. However, a pool-and-sequence strategy was applied to a
1500 bp fragment of the KCNB2 gene and a G to A transition was identified at nt 1404.
This SNP resides in a T spR I restriction site and digestion with this enzyme was used to
confirm the presence of the SNP in individual samples. However, T spR I digestion was
inefficient so it could not be used to discern all genotypes. In order to efficiently
genotype the PiGMaP families for genetic linkage mapping, an alternative genotyping
assay is currently under development.
D. Cytogenetic, RH and Genetic Linkage Mapping

Physical mapping placed IRF6 in the interval of SSC9q11-q26 (risk of error less than

0.1%) and KCNB2 in the interval of SSC4q1 5-q16 (risk of error less than 0.1%). Both

102

genes were also successfully mapped using the IMpRH panel. PCR amplification of
KCNB2 in the 118 pig-hamster hybrids is shown in Figure 4.6. Similar results were
obtained for IRF 6 (data not shown). IRF 6 mapped to SSC9 and was significantly linked
to SW749 (LOD=14.72) and SW1651 (LOD=14.43). KCNB2 mapped to SSC4 and was
significantly linked to SW839 (LOD=9.00), SW1475 (LOD=8.32), SW724 (LOD=7.23)
and SW1073 (LOD=5.53). In addition, linkage analysis revealed a significant association
between IRF6 and two SSC9 markers on the pig genetic map, SW749 (LOD=5.42,
0=0.00) and S0568 (LOD=3.91, 0:0.00). Figure 4.7 illustrates the pig cytogenetic,
genetic linkage and RH maps showing the location of IRF6 and KCNB2 relative to
previously reported markers (Archibald et al. 1995; Rohrer et al. 1996; Yerle et al. 1997;

Hawken et al. 1999).

Discussion

In this study, the complete coding region of pig IRF 6 was cloned and sequenced.
Homology searches with available sequences in the GenBank database revealed a high
level of nucleotide and amino acid sequence conservation across various mammalian
species. A 1844 nt fragment of the KCNB2 gene was also cloned and it too displayed
significant nucleotide sequence conservation with KCNB2 from other species. Currently,
we are isolating the remaining 586 nt from the 5’ end of KCNB2 so that the complete
coding sequence will be available for this gene. The availability of full-length cDNA
sequences for both IRF6 and KCNB2 will allow these genes to be extensively screened
for SNPs. SNPs found in cDNAs will provide the basis for functional studies and the

determination of genotype-phenotype associations.

103

RT-PCR and northern blot analysis was used to characterize the expression profiles of
IRF 6 and KCNB2 in various pig tissues. IRF6 has only recently been identified so there
are no reports in the literature regarding the location or timing of IRF 6 expression.
Therefore, this study represents the first report of an IRF6 expression profile in any
mammalian species. RT-PCR identified the presence of IRF 6 transcripts in all tissues
investigated including skeletal muscle tissue. In order to determine if IRF6 was
expressed by myogenic cells, RT-PCR was performed on RNA derived from pig
myogenic satellite cells and myotubes. No IRF 6 product was detected in satellite cells or
myotubes suggesting that expression is derived from non-myogenic cell types in skeletal
muscle tissue. Northern blots confirmed a relatively high abundance of IRF6 mRNA in
pig uterus, kidney and ileum. The expression of KCNB2 has been documented in various
smooth muscle tissues in the dog (Schmalz et al. 1998). Our results for the pig are in
agreement with the canine results because we also detected KCNB2 expression in smooth
muscle tissues. In addition, we observed KCNB2 expression in skeletal muscle, liver and
kidney. This could be the result of relatively high expression of KCNB2 in the smooth
muscle cell population present in these tissues (i.e., vascular tissue) or it could be due to
expression in other cell types. Further investigation of purified cell types will be needed
to develop a detailed expression profile for both IRF 6 and KCNB2.

IRF6 and KCNB2 have both been mapped in the human genome. IRF 6 is located on
HSA1q32.3-q4l and comparative information indicates that this region is conserved on
SSC9q23-qter (Goureau et al. 1996). Cytogenetic, RH and genetic linkage mapping
confirmed the localization of pig IRF6 to the distal q arm of SSC9. KCNB2 has been

mapped in the human genome to SSC8q12-q13, a region conserved on SSC4p1 5-q16

104

(Goureau et al. 1996; Lahbib-Mansais et al. 1999). We confirmed this region of
conserved synteny by cytogenetic and RH mapping of KCNB2.

Interestingly, putative QTL have been reported on both SSC4 and SSC9. IRF 6 is
located in the most probable position of a QTL affecting gestation length in a Meishan X
Yorkshire population (Wilkie et al. 1999). Wilkie et al. observed that individuals
inheriting both QTL alleles from the Meishan had a gestation length of 3.04 days longer.
Similarly, KCNB2 is located within a QTL region on SSC4 reported to have large effects
on backfat in a Large White X Wild Boar population (Andersson et al. 1994). This QTL
explained 18 % of the F2 variance in fatness traits in this population and, due to its large
effect, the QTL has been confirmed in multiple studies (Andersson-Eklund et al. 1998;
Walling et al. 1998; Knott et al. 1998; Marklund et al. 1999; Walling et al. 2000). In our
RDA experiments, RDA markers flanking both genes were isolated (see Chapter 3)
further supporting the possibility that IRF 6 and KCNB2 are excellent positional
candidate genes for QTL involved in carcass and reproductive traits.

The potential functional role that IRF6 and KCNB2 play in various production traits is
unclear. Although members of the IRF family that have been characterized are known to
regulate interferon genes, the primary function of IRF6 is unknown. Interferon plays a
role in numerous cellular processes, many of which impact pig performance. KCNB2
may also play a significant role in pig growth. The influx of Ca2+ is a major regulator of
the release of growth hormone (GH). Voltage-gated Ca2+ and K+ channels are the
primary controls for Ca2+ influx and are regulated by GH-releasing hormone (GHRH)
(Chen et al. 2000). More research is needed to identify the specific functions of IRF6 and

KCNB2 in the pig.

105

In this study, cDNA sequences, expression profiles and chromosomal localizations
were determined for porcine IRF 6 and KCNB2. This information will be essential to
begin understanding the role of these genes in molecular processes. The data presented
suggest that IRF 6 and KCNB2 could potentially be associated with traits of economic

importance in pork production, and their fithher investigation is warranted.

106

Chapter 4

Figures

107

Figure 4.1. Alignment of porcine, human, mouse and sheep IRF6 nucleotide sequence.
Alignment was prepared using the ClustalW program on the Biology Workbench web-
based server (http://workbench.sdsc.edu).

108

 

 

momanm
mcam:
mummp
205mm

mOHOHsm
mcams
mammp
ZOCMm

mOHOHDm
mcam:
mdmmp
Zocmm

momoHsm
mcam:
mdmmv
zocmm

monanm
magma
mvmmp
ZOCmm

Hme
mem
mem
mem

mem
mem
mem
mem

mem
mem
HWMm
mem

mem
Hme
HNMm
mem

mem
mem
mem
mem

H HH NH wH AH mH mH 4H
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109

wOHOHsm
magma
mrmmfi
305mm

mOHoHsm
mcams
mummp
305mm

monanm
magma
mUmmp
ZOCmm

mOHOHsm
magma
mummp
Zocmm

mOHOHsm
mcam:
msmmv
305mm

mem
Hme
Hme
mem

mem
mem
mem
mem

mem
mem
mem
mem

wam
mem
mem
HWMm

mem
mem
mem
mem

AOH AHH AMH AwH apH awH bmH ¢QH

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110

monoHsm
scam:
msmmw
zocmm

monoHsm
mcam:
mjmmv
ZOCmm

mOHanm
mcam:
mammv
ZOCmm

wOHOHsm
mcaws
mammv
306mm

mOHOHsm
mcam:
mqmmp
Zocmm

mem
Hme
mem
mem

mem
mem
mem
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HWmm
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mem
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111

womowsm
mcam:
mammp
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womnwsm
mcams
mammp
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mOHoHsm
mcam:
mrmmw
305mm

mem
mem
Hme
mem

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mem
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mem

mem
mem
mem
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112

Figure 4.2 Alignment of the porcine, human, mouse and sheep deduced IRF6 amino acid
sequence. Alignment was prepared using the ClustalW program on the Biology
Workbench web-based server (http://workbench.sdsc.edu).

113

momanm
mcam:
mummp
zocmm

momoHsm
mcam:
mammv
Zocmm

mOHOHsm
mcam:
mummv
305mm

mOHOHDm
magma
mammp
305mm

wonnwsm
mcams
msmmv
305mm

mOHOHsm
mcam:
mammw
zocmm

mem
mem
mem
mem

Hme
mem
wam
mem

mem
mem
mem
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mem
mem
mem
mem

mem
mem
Hme
Hme

HWWm
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Hme

 

H HH NH wH aH mH mH QH
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.............. ............................<....................Um.....m.........
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...................... ................m.........................O..............0
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...... ................................................>......O...>.

114

Figure 4.3. Alignment of porcine, human, canine and rat KCNB2 nucleotide sequence.
Alignment was prepared using the ClustalW program on the Biology Workbench web-
based server (http://workbench.sdsc.edu).

115

momowsm
mcam:
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wonnwsm
mcaw:
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mOHOHDm
mcam:
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mcams
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monowsm
chm:
nmzwsm
won

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116

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monowsm
magma
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monnwsm
mcam:
omsHsm
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monowsm
mcam:
owsHsm
won

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117

mononm
Imam:
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mom

wonanm
mean:
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monoHsm
Icams
nmsHsm
won

monanm
mcams
0m3Hsm
wmn

mOHOHsm
mcam:
nmbHsm
won

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118

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magma
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won

mOHanm
mcam:
OmsHsm
wow

wonowsm
mcaw:
Omszm
won

mOHOHbm
:cam:
nmsHsm
won

wonoHsm
mcams
nmsHsm
wow

xOZwN
x0zwm
x0zww
x0zmm

x0zwm
WOZwN
x0zwm
w0zwm

WOZwN
m0zwm
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W0zmm

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w0zwm
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119

mouoHsm

mcam:

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wmd

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mcams

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wmm

momowsm

mcam:

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won

m0sz
x0zwm
m0zwm
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x0zwm
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120

Figure 4.4. Results of RT-PCR analysis for IRF 6 and KCNB2. Panel A, the 195 bp band
represents amplification of IRF6. Lane 1, 100 bp ladder; Lane 2, Skeletal muscle; Lane 3,
Myogenic satellite cells; Lane 4, Myotubes; Lane 5, Cerebellum; Lane 6, Aorta; Lane 7,
Uterus; Lane 8, Liver; Lane 9, Duodenum; Lane 10, Jejunum; Lane 11, Ileum; Lane 12,
Kidney. Panel B, the 209 bp band represents amplification of KCNB2. Lane 1, 100 bp
ladder; Lane 2, Aorta; Lane 3, Uterus; Lane 4, Duodenum; Lane 5, Jejunum; Lane 6, Ileum;
Lane 7, 100 bp ladder; Lane 8, Skeletal muscle; Lane 9, Liver; Lane 10, Cerebellum.

121

123456789101112

195 bp

 

 

122

 

Figure 4.5. IRF6 and KCNB2 northern blot analysis. Panel A, IRF6 northern blot
showing the expression of 2.5- and 2.0-kb transcripts in various pig tissues. Panel
B, KCNB2 northern blot analysis showing the expression of 6.0-, 2.8- and 1.6-kb
transcripts in various pig tissues. Lane 1, Aorta; Lane 2, Uterus; Lane 3,
Cerebellum; Lane 4, Kidney; Lane 5, Liver; Lane 6, Skeletal muscle; Lane 7,
Ileum. ' -

123

 

 

A

2.5-kb
2.0-kb

 

6.0-kb

2.8-kb

1 .6-kb

 

124

Figure 4.6. Agarose gel image of KCNB2 RH mapping results obtained using the
IMpRH panel. Samples are numbered on the lefi side of the gel and arrows (1) indicate
positive hybrids. The negative control hamster (H) sample and positive control pig (P)
sample are labeled.

 

 

i 125

 

 

 

 

126

Figure 4.7. Diagram of the pig cytogenetic, RH and genetic linkage maps for SSC4 and
SSC9. Maps show the position of IRF 6 (Panel A) and KCNB2 (Panel B) relative to
previously mapped markers (Archibald et al. 1995; Rohrer et al. 1996, Yerle et al. 1997;
Hawken et a1. 1999). The sex-averaged linkage map position is represented in cM (centi-
Morgans) and the RH map position is in cR (centi-Rays).

 

127

 

 

 

 

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129

CHAPTER 5

SUMMARY AND RECOMENTDATIONS FOR FUTURE RESEARCH

Animal molecular genetics is a rapidly evolving field that promises to revolutionize
our perceptions of traditional animal breeding. Currently, new technologies and
resources are being developed at an astounding pace. Innovative genome mapping
technologies adapted from human molecular genetics have led to the development of
high-resolution genome maps in all major livestock species. These maps provide the
framework for QTL identification, implementation of marker assisted selection and
future genome sequencing efforts.

This study was designed to contribute new markers to the pig genome map. The
specific objectives included: 1) Map HSA12 genes to increase the density of gene loci on
SSC5 and 14 and further refine the comparative map of the evolutionary breakpoint on
HSA12; 2) Generate genetic markers existing between pigs phenotypically divergent for
carcass and pork quality characteristics using representational difference analysis (RDA);
and 3) Characterize potential candidate genes for production traits in pigs isolated using
RDA.

The genes collagen type II alpha 1 (COL2A1), dual specificity MAP kinase
phosphatase (DUSP6), mast cell growth factor (MGF), phenylalanine hydroxylase
(PAH), phospholipase A2 (PLA2), paxillin (PXN) and hepatic transcription factor 1
(TCF 1), all of which flank the HSA12 evolutionary breakpoint, were mapped. Pig

genome localizations were determined using the combination of cytogenetic, RH and

130

genetic linkage mapping techniques. COL2A1, DUSP6, MGF, and PAH mapped to
SSC5 and PLA2, PXN and TCFl mapped to SSC14. These results provide a high
density map of the breakpoint region on HSA12.

Intrachromosomal rearrangements were also identified for SSC5. However, the
number of HSA12 genes mapped on SSC5 is still too few to precisely characterize the
chromosomal restructuring that has occurred. Therefore, there is a need to map
additional HSA12 genes from the region syntenic with SSC5 to better understand the
complex evolutionary relationship between these two chromosomes. This detailed
understanding of SSC5 structure will be critical to correctly identify genes located at
potential QTL.

Of the genes mapped in this study, it appears that PAH and PXN flank the HSA12
breakpoint. The distance between these genes in the human is 64.8 CROWD) on
GeneMap’99. Since the GB4 RH panel has an approximate resolution of 208 kb/cR(3ooo),
this would equate to an approximate physical distance of 13.5 Mbp. This is still a
relatively large genomic region and further mapping of genes within this interval is
needed to confidently predict the precise location of the HSA12 breakpoint in the pig
genome.

Eighteen markers that mapped to 11 different pig chromosomes were isolated using
RDA. A number of the markers mapped to regions known to contain putative QTL for
pork production traits so they can potentially be used in studies designed to fine map
these regions. These markers should also be evaluated for their potential association with
various performance traits. Five of the isolated RDA markers contain RFLPs, providing

a rapid genotyping assay for use in large resource populations.

131

Not only should the markers developed in this study be further evaluated, but
additional RDA experiments implementing variations of the original RDA protocol such
as arbitrarily primed-RDA (Toyota et al. 1996) and repetitive sequence-RDA (U shijima
et al. 1998) should be pursued. These experiments have the potential to generate large
numbers of markers, which could be rapidly and inexpensively genotyped expediting the
process of QTL identification in large resource populations. In theory, a genetic map
could be constructed and genetic data obtained for each F 2 individual in a short period of
time. However, a larger number of RDA markers would be required compared to highly
polymorphic microsatellite markers due to lower levels of informativeness. Nonetheless,
the construction of a RDA genetic map would provide a more efficient set of markers for
genotyping.

RDA has the potential to generate large numbers of STS suitable for RH mapping.
RDA STS have an advantage over EST sequences for RH mapping. Since most RDA
markers are comprised of anonymous DNA sequence, only a small number will amplify
hamster DNA, whereas a much larger percentage of ESTS or type I marker STS amplify
hamster DNA products. To efficiently utilize RH mapping resources, primer sets suitable
for multiplexing could be developed, allowing the simultaneous localization of a number
of loci on the RH map. Markers that map within QTL regions could be further analyzed
for polymorphisms so that each could be placed on the pig genetic map. This would help
to facilitate the isolation of the gene(s) responsible for the genetic variation observed in
economically important traits.

In theory, RDA products should be specific to the tester sample due to the presence of

polymorphic restriction sites that yield fragments contained only in the tester and not the

132

driver. In this study, we were not able to confirm tester specificity due to a high level of
repetitive sequences present in the isolated RDA fragments. These repetitive sequences
made it impossible to use hybridization techniques to distinguish unique tester products.
It is possible that this problem could be avoided by producing normalized tester samples.
In addition, this study used closely related pigs from the fifth generation of a selection
experiment, whereas previous reported studies have used highly inbred populations.
Completely inbred populations are by definition assumed to be homozygous at all loci,
which makes them inherently more suited to RDA analysis than outbred populations.
However, due to the large number of generations required to create inbred populations,
there are very few such pig populations available. Nonetheless, the results of this study
demonstrate that novel markers can be generated from pig genomic DNA using RDA. It
may be possible to develop experimental populations from existing pig breeds or herds
that provide a resource for more efficient RDA analysis in the pig.

Two of the RDA markers generated were found to be homologous to the interferon
regulatory factor 6 (IRF6) gene and the potassium voltage-gated channel, Shab related
subfamily, member 2 (KCNB2) gene. These genes were subsequently characterized and
expression was identified in a number of pig tissues. In addition, IRF 6 was mapped to
the distal q arm of SSC9 and KCNBZ was mapped near the centromere on SSC4q. Both
of these genomic regions contain putative QTL. The region of SSC4 contains a highly
significant QTL for backfat traits (Andersson et al 1994) and the region of SSC9 contains
a significant QTL for gestation length (Wilkie et al. 1999). Interestingly, each gene was
mapped in close proximity with an additional RDA marker. Thus, IRF 6 and KCNBZ are

strong positional candidate genes for pig carcass and reproductive traits and they warrant

133

further investigation. SNPs were identified in each gene and these SNPs can be used in
genotype-phenotype association studies. In addition, further characterization of each
gene is needed to identify potential functional roles in important biological pathways.

In summary, a total of 25 new markers have been added to the pig genome maps as a
result of this project. This work has made a significant contribution to improving the
resolution of the pig-human comparative map. In addition, a number of novel
anonymous markers have been developed using the RDA technique. Together these
markers will impact future efforts to identify the specific genes controlling economically

important traits in the pig.

134

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