Identification of Novel Genetic Markers

in the Pig Using Representational Difference Analysis

C.R. Farber¹,N.E. Raney¹,K. Nadarajah², D.L. Kuhlers², and C.W. Ernst¹

¹Department of Animal Science, Michigan State University, East Lansing

²Animal and Dairy Sciences Department, Auburn University, Alabama

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) providing 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 studies is the identification of the gene(s) responsible for the phenotypic variation observed in a particular trait. Traditional QTL analysis, which relies on marker-phenotype co-segregation, leads to the isolation of large genomic regions. Additionally, polygenic traits rely on the concordant expression of multiple genes. Thus, QTL for a given trait may reside on several chromosomes spanning a significant portion of the pig genome potentially harboring hundreds of genes. Therefore, to increase QTL resolution, there is a need to evaluate novel techniques such as representational difference analysis (RDA; Litsitsyn et al., 1993; Romani et al., 1999), which have the potential to generate genetic markers flanking these QTL.

The RDA technique is based on restriction fragment length polymorphisms (RFLP) that exist between two DNA populations (referred to as tester and driver). In RDA the first step is to digest both tester and driver DNA samples with a specific restriction enzyme, ligate oligonucleotide adaptors to the ends of the restriction fragments and produce amplicons via the polymerase chain reaction (PCR). Subtractive hybridization is then performed with driver present in excess and the PCR is used to selectively amplify unique target sequences. After the second or third round of hybridization followed by PCR amplification, difference fragments are cloned for further analysis.

Various selection experiments have been used in pig populations to study the effects of selecting for particular traits. After many generations of selection, it is likely that the frequency of favorable QTL alleles will have increased. This provides an ideal model for the identification of QTL (Rathje et al., 1997; Ollivier et al., 1997) and the use of RDA to fine map QTL regions.

In our study we have isolated 16 anonymous DNA markers and two coding sequences (the interferon regulatory factor 6 (IRF6) and potassium voltage-gated channel, Shab related subfamily, member 2 (KCNB2) genes) using RDA. This was accomplished through the use of DNA from pigs divergent at the skeletal muscle ryanodine receptor 1 (RYR1) locus and also differing in phenotype for production traits as a result of selection for loin muscle area (LMA) over five generations (Kuhlers et al., 1998). The markers isolated in this study are potentially linked to QTL for production traits and can be used in subsequent studies to further fine map these genomic regions.

Materials and Methods

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 for 10^th rib backfat and 39.75 cm²forLMA. Average 10^th rib backfat and LMA measurements for the five driver pigs were 2.87 cm and 25.71 cm²_,respectively. In addition, the tester pig was homozygous mutant “nn” and driver pigs were homozygous normal “NN” at the RYR1 locus. The same tester and driver samples were utilized for two RDA experiments with the restriction enzymes Bam HI and Bgl II. 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 subsequently optimized to create a sequence-tagged site (STS) for each RDA difference product.

Mapping and allele frequency determinations for RDA STS

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 118 pig-rodent hybrids of the IMpRH panel. 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/). 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. In addition to SSCP markers, one RDA difference product was found to contain an insertion/deletion, one was found to contain an RFLP and one was found to contain a microsatellite. Markers with an identified polymorphism were used to genotype pigs from the PiGMaP reference families and linkage analysis was performed using CRIMAP 2.4 (Green et al., 1990). Additionally, 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.

Characterization of IRF6 and KCNB2

IRF6 and KCNB2 coding sequences were amplified from pig intestinal and cerebellum cDNA, respectively, using the PCR. Additionally, 3’ rapid amplification of cDNA ends (RACE) using pig specific PCR primers was used to amplify the remaining 3’ untranslated regions (UTR) of both IRF6 and KCNB2. To analyze the expression profiles of IRF6 and KCNB2 in various pig tissues, total RNA was extracted and the mRNA was reverse transcribed using an oligo d(T) primer. IRF6 and KCNB2 were amplified using the pig specific PCR primers. Amplification of IRF6 and KCNB2 in cDNA resulted in the amplification of a 195-bp and a 209-bp PCR fragment, respectively. The IRF6 and KCNB2 RT-PCR products were cloned and sequenced to confirm correct amplification. To further establish the nature of IRF6 and KCNB2 expression in pig tissues, northern blot analysis was preformed. Eight mg of total RNA was denatured and fractionated in a 1.2 % denaturing agarose gel. RNA was blotted onto nylon membranes and hybridized to ³²P-labeled IRF6 or KCNB2 probes. Blots were rinsed twice and exposed to X-ray film.

Results

Representational Difference Analysis (RDA) and characterization of RDA STS

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 subsequently cloned and sequenced. A BLAST search indicated that RDA fragments had no significant homology match or were identical to porcine repetitive elements. Exceptions to this were MSURDA79 and MSURDA111 which were found to be highly similar to human IRF6 and human KCNB2, respectively. A total of 18 RDA markers were localized to the porcine RH map and 10 were placed on the genetic linkage map. Table 1 lists the RH and genetic linkage map information, including chromosomal locations and most significantly linked markers with corresponding LOD scores. Figure 1 is a representative map illustrating the map locations of four RDA STS on pig chromosome 14. To determine if the RDA markers were segregating in the Landrace selection lines, allele frequency differences between select and control lines were analyzed. Genotypes were determined for individuals in both lines for all biallelic markers. Table 2 summarizes the allele frequency data and the results of the Fisher’s exact test.

Characterization of the pig IRF6 and KCNB2 genes

The full-length coding sequence and a portion of the 3’ UTR were isolated for pig IRF6. The cDNA consisted of a 1,404 nucleotide (nt) coding region and a 401 nt 3’ UTR. A comparison with IRF6 sequences from other species indicated a high level of sequence conservation. Pig IRF6 nucleotide sequence was 92%, 91% and 90% similar to human, mouse and sheep IRF6, respectively. Additionally, the deduced amino acid sequence of IRF6 was highly conserved. Pig and human IRF6 shared 100% similarity across all 467 amino acid residues. Sheep and mouse IRF6 amino acid sequences were 98% and 97% identical to pig IRF6. Pig IRF6 also shares a high level of similarity with the evolutionarily distant Xenopus laevis xIRF6 with 76% of the amino acid residues conserved between them. A 1,844 nt sequence of the pig KCNB2 coding region was cloned along with 366 nt of the 3’UTR. The isolated coding region spanned from nt position 586…2,430 (2,427…2,430 corresponding to the stop codon) based on human KCNB2 cDNA sequence. This sequence was 87% similar to human and 86% similar to canine and rat KCNB2.

Table 1. Summary of RH and genetic linkage mapping results

Marker	GenBank Acc. No.	RH mapping^a	Type^b	Genetic linkage mapping^a
MSURDA7	G67709	SSC7 (SW1959, 11.81)	SSCP	SSC7 (S0334, 28.60)
MSURDA42	G67710	SSC6 (SW1473, 6.81)	INS/DEL	SSC6 (SW122, 13.11)
MSURDA78	G67711	SSC11 (SW1460, 8.23)
MSURDA81	G67712	SSC5 (ACO2, 6.57)
MSURDA82	G67713	SSC14 (SW1032, 5.88)	SSCP	SSC14 (S0058, 9.92)
MSURDA84	G67714	SSC14 (SW761, 16.33)	SSCP	SSC14 (SW761, 28.17)
MSURDA86	G67715	SSC4 (SW45, 25.24)
MSURDA89	G67716	SSC11 (SW903, 6.98)
MSURDA91	G67717	SSC14 (SW361, 19.56)	RFLP	SSC14 (S0007, 16.08)
MSURDA93	G67718	SSC9 (SW2093, 7.31)
MSURDA95	G67719	SSC11 (SW435, 13.41)	SSCP	SSC11 (S0230, 11.57)
MSURDA96	G67720	SSC3 (SWR2069, 6.10)
MSURDA99	G67721	SSC16 (SW1645, 20.26)	SSCP	SSC16 (SW419, 15.68)
MSURDA106	G67722	SSC13 (S0282, 6.07)	SSCP	SSC13 (S0219, 12.58)
MSURDA108	G67723	SSC14 (SW857, 11.42)	MS	SSC14 (LPL, 16.84)
MSURDA110	G67724	SSC8 (S0098, 10.63)
IRF6	AF327368	SSC9 (SW749, 14.72)	SSCP	SSC9 (SW749, 5.42)
KCNB2	AF327369	SSC4 (SW839, 9.00)

^aRadiation hybrid mapping or genetic linkage 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; MS, microsatellite.

Table 2. Summary of RDA marker allele frequency differences between select and control lines.

Marker

Allele Frequencies

Fisher’s exact test^d

Select^a

Control^b

Difference^c

RYR1

A= 0.57

B= 0.43

A= 0.83

B= 0.17

0.26

MSURDA7

A= 1.0

B= 0.0

A= 0.95

B= 0.05

0.05

MSURDA42

A= 0.76

B= 0.24

A= 0.80

B= 0.20

0.04

MSURDA82

A= 0.12

B= 0.88

A= 0.32

B= 0.68

0.20

MSURDA84

A= 0.29

B= 0.71

A= 0.30

B= 0.70

0.01

MSURDA91

A= 0.05

B= 0.95

A= 0.22

B= 0.78

0.17

MSURDA95

A= 0.10

B= 0.90

A= 0.0

B= 1.0

0.10

MSURDA99

B= 1.0

C= 0.0

B= 0.86

C= 0.13

0.13

MSURDA106

A= 0.05

B= 0.95

A= 0.52

B= 0.48

0.47

^a n=21.

^b n=23.

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

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

Figure 1. Diagram of the pig cytogenetic, genetic linkage and RH maps for pig chromosome 14 (SSC14). RDA markers are underlined and shown in bold face type.

RT-PCR was performed for IRF6 and KCNB2 to determine if mRNA 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. KCNB2 transcripts were identified in pig aorta, cerebellum, duodenum, ileum, jejunum, liver, skeletal muscle and uterus. Northern blot analysis revealed the expression of 2.5- and 2.0-kb IRF6 transcripts in pig uterus, kidney and ileum. 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.

Discussion

The majority of RDA markers generated in this study mapped to regions containing or flanking previously reported QTL for various production traits. These QTL have been identified in different populations and have varying effects on the phenotypes involved. Therefore, it is not probable that all of these QTL are segregating in our Landrace selection experiment populations. Also, in QTL studies, only those QTL contributing a large percentage of the F₂ variance are identified. In contrast, RDA has the capacity to generate markers flanking QTL regardless of magnitude, which might explain why RDA markers were isolated on multiple chromosomes or in regions not located near previously identified QTL.

Allele frequencies for each marker were determined in both the select and control lines from the Landrace LMA selection experiment. This was done to examine 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 using Fisher’s exact test. However, the analysis indicated that the allele frequencies for five of the eight RDA markers significantly differed between the select and control lines. These data suggest that alleles for at least a portion of the markers isolated are segregating in this population and indicate that the markers generated in this study should be tested in larger populations for potential associations with various economically important traits.

In addition to 16 anonymous DNA markers, two genes were isolated. The identification of IRF6 and KCNB2 make this the first report of the isolation of coding sequences using the genomic RDA technique. 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 segregating alleles at each locus are affecting the phenotypes under selection in this population, resulting in their isolation using RDA.

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.

Acknowledgements

The authors would like to thank the PiGMaP consortium and the pig gene mapping group at the Institut National de la Recherche Agronomique (INRA) and the University of Minnesota for providing genome mapping tools. The technical assistance of Drs. Patty Weber, Paul Coussens and Jianbo Yao at Michigan State University was greatly appreciated. Thanks to Dr. Rob Templeman at Michigan State University for statistical advice. Funding was provided by a Michigan State University All University Research Initiation Grant (AURIG) and USDA Grant 99-33205-8150.

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