Genetic variation at the tumour virus B locus in commercial and laboratory chicken populations assessed by a medium-throughput or a high-throughput assay.

The tumour virus B (TVB) locus encodes cellular receptors mediating infection by three subgroups of avian leukosis virus (B, D, and E). Three major alleles, TVB*S1, TVB*S3, and TVB*R, have been described. TVB*S1 encodes a cellular receptor mediating infection of subgroups B, D, and E. TVB*S3 encodes the receptor for two subgroups, B and D, and TVB*R encodes a dysfunctional receptor that does not permit infection by any of the subgroups, B, D, or E. Genetic diversity at the TVB locus of chickens was investigated in both layer and broiler commercial pure lines and laboratory lines. Genotyping assays were developed for both medium-throughput and high-throughput analysis. Of the 36 broiler lines sampled, 14 were fixed for the susceptible allele TVB*S1. Across all broiler lines, 83% of chickens were typed as TVB*S1/*S1, 3% as TVB*R/*R, and 14% as TVB*S1/*R. In the egg-layer lines, five of the 16 tested were fixed for TVB*S1/*S1. About 44% of egg-layers were typed as TVB*S1/*S1, 15% as TVB*R/*R, with the rest segregating for two or three of the alleles. In the laboratory chickens, 60% were fixed for TVB*S1/*S1, 6% for TVB*S3/*S3, 14% for TVB*R/*R, and the rest were heterozygotes (TVB*S1/*S3 or TVB*S1/*R). All commercial pure lines examined in this study carry the TVB*S1 allele that sustains the susceptibility to avian leukosis viruses B, D, and E. More importantly, the TVB*R allele was identified in multiple populations, thus upholding the opportunities for genetic improvement through selection.

Genetic diversity analysis using lowly polymorphic dominant markers: the example of AFLP in pigs.

DNA markers are commonly used for large-scale evaluation of genetic diversity in farm animals, as a component of the management of animal genetic resources. AFLP markers are useful for such studies as they can be generated relatively simply; however, challenges in analysis arise from their dominant scoring and the low level of polymorphism of some markers. This paper describes the results obtained with a set of AFLP markers in a study of 59 pig breeds. AFLP fingerprints were generated using four primer combinations (PC), yielding a total of 148 marker loci, and average harmonic mean of breed sample size was 37.3. The average proportion of monomorphic populations was 63% (range across loci: 3%-98%). The moment-based method of Hill and Weir (2004, Mol Ecol 13:895-908) was applied to estimate gene frequencies, gene diversity (F(ST)), and Reynolds genetic distances. A highly significant average F(ST) of 0.11 was estimated, together with highly significant PC effects on gene diversity. The variance of F(ST) across loci also significantly exceeded the variance expected under the hypothesis of AFLP neutrality, strongly suggesting the sensitivity of AFLP to selection or other forces. Moment estimates were compared to estimates derived from the square root estimation of gene frequency, as currently applied for dominant markers, and the biases incurred in the latter method were evaluated. The paper discusses the hypotheses underlying the moment estimations and various issues relating to the biallelic, dominant, and lowly polymorphic nature of this set of AFLP markers and to their use as compared to microsatellites for measuring genetic diversity.

Genetic diversity within and between European pig breeds using microsatellite markers.

An important prerequisite for a conservation programme is a comprehensive description of genetic diversity. The aim of this study was to use anonymous genetic markers to assess the between- and the within-population components of genetic diversity for European pig breeds at the scale of the whole continent using microsatellites. Fifty-eight European pig breeds and lines were analysed including local breeds, national varieties of international breeds and commercial lines. A sample of the Chinese Meishan breed was also included. Eleven additional breeds from a previous project were added for some analyses. Approximately 50 individuals per breed were genotyped for a maximum of 50 microsatellite loci. Substantial within-breed variability was observed, with the average expected heterozygosity and observed number of alleles per locus being 0.56 [range 0.43-0.68] and 4.5 respectively. Genotypic frequencies departed from Hardy-Weinberg expectations (P < 0.01) in 15 European populations, with an excess of homozygotes in 12 of them. The European breeds were on average genetically very distinct, with a Wright F(ST) index value of 0.21. The Neighbour-Joining tree drawn from the Reynolds distances among the breeds showed that the national varieties of major breeds and the commercial lines were mostly clustered around their breeds of reference (Duroc, Hampshire, Landrace, Large White and Piétrain). In contrast, local breeds, with the exception of the Iberian breeds, exhibited a star-like topology. The results are discussed in the light of various forces, which may have driven the recent evolution of European pig breeds. This study has consequences for the interpretation of biodiversity results and will be of importance for future conservation programmes.

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers.

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.

Development of a single nucleotide polymorphism map of porcine chromosome 2.

Single nucleotide polymorphism markers are developed on SSC2, predominantly on the p-arm. Several studies reported a quantitative trait loci (QTL) for backfat thickness in this region. Single nucleotide polymorphisms were identified by comparative re-sequencing of polymerase chain reaction (PCR) products from a panel of eight individuals. The panel consisted of five Large Whites (each from a different Dutch breeding company), a Meishan, a Pietrain and a Wild Boar. In total, 67 different PCR products were sequenced and 301 SNPs were identified in 32,429 bp of consensus sequence, an average of one SNP in every 108 bp. After correction for sample size, this polymorphism rate corresponds to a heterozygosity value of one SNP in every 357 bp. For 63% of the SNPs, there was variation among the five Large Whites, and these SNPs are relevant for linkage and association studies in commercial populations. Comparing the Whites with other breeds revealed higher variation rates with: (i) Meishan, 89%; (ii) Pietrain, 69%; (iii) Wild Boar, 70%. Because many of the experimental populations to identify QTL are based on crosses between these breeds, these SNPs are relevant for the fine mapping of the QTL identified within these crosses.

Improving the comparative map of SSC2p-q13 by sample sequencing of BAC clones.

To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.

A whole-genome scan for quantitative trait loci affecting teat number in pigs.

A whole-genome scan was conducted using 132 microsatellite markers to identify chromosomal regions that have an effect on teat number. For this purpose, an experimental cross between Chinese Meishan pigs and five commercial Dutch pig lines was used. Linkage analyses were performed using interval mapping by regression under line cross models including a test for imprinting effects. The whole-genome scan revealed highly significant evidence for three quantitative trait loci (QTL) affecting teat number, of which two were imprinted. Paternally expressed (i.e., maternally imprinted) QTL were found on chromosomes 2 and 12. A Mendelian expressed QTL was found on chromosome 10. The estimated additive effects showed that, for the QTL on chromosomes 10 and 12, the Meishan allele had a positive effect on teat number, but, for the QTL on chromosome 2, the Meishan allele had a negative effect on teat number. This study shows that imprinting may play an important role in the expression of teat number.

A high-resolution comparative RH map of porcine chromosome (SSC) 2.

A high-resolution comparative map was constructed for porcine Chromosome (SSC) 2, where a QTL for back fat thickness (BFT) is located. A radiation hybrid (RH) map containing 33 genes and 25 microsatellite markers was constructed for this chromosome with a 3000-rad porcine RH panel. In total, 16 genes from human Chromosome (HSA) 11p, HSA19p, and HSA5q were newly assigned to SSC2. One linkage group was observed at LOD 3.0, and five linkage groups at LOD 4.0. Comparison of the porcine RH map with homologous human gene orders identified four conserved segments between SSC2 and HSA11, HSA19, and HSA5. Concerning HSA11, a rearrangement of gene order is observed. The segment HSA11p15.4-q13 is inverted on SSC2 when compared with the distal tip of SSC2p, which is homologous to HSA11p15.5. The boundaries of the conserved segments between human and pig were defined more precisely. This high-resolution comparative map will be a valuable tool for further fine mapping of the QTL area.

Detection and characterization of quantitative trait loci for meat quality traits in pigs.

In an experimental cross between Meishan and Dutch Large White and Landrace lines, 785 F2 animals with carcass information and their parents were typed for molecular markers covering the entire porcine genome. Linkage was studied between these markers and eight meat quality traits. Quantitative trait locus analyses were performed using interval mapping by regression under two genetic models: 1) the line-cross approach, where the founder lines were assumed to be fixed for different QTL alleles and 2) a half-sib model where a unique allele substitution effect was fitted within each of the 38 half-sib families. The line-cross approach included tests for genomic imprinting and sex-specific QTL effects. In total, three genome-wide significant and 26 suggestive QTL were detected. The significant QTL on chromosomes 3, 4, and 13, affecting meat color, were only detected under the half-sib model. Failure of the line-cross approach to detect the meat color QTL suggests that the founder lines have similar allele frequencies for these QTL. This study provides information on new QTL affecting meat quality traits. It also shows the benefit of analyzing experimental data under different genetic and statistical models.

Fine mapping and imprinting analysis for fatness trait QTLs in pigs.

Quantitative trait loci (QTL) for fatness traits were reported recently in an experimental Meishan x Large White and Landrace F(2) cross. To further investigate the regions on pig Chr 2 (SSC2), SSC4, and SSC7, 25 additional markers from these regions were typed on 800 animals (619 F(2) animals, their F(1) parents, and F(0) grandfathers). Compared with the published maps, a modified order of markers was observed for SSC4 and SSC7. QTL analyses were performed both within the half-sib families as well as across families (line cross). Furthermore, a QTL model accounting for imprinting effects was tested. Information content could be increased considerably on all three chromosomes. Evidence for the backfat thickness QTL on SSC7 was increased, and the location could be reduced to a 33-cM confidence interval. The QTL for intramuscular fat on SSC4 could not be detected in this half-sib analysis, whereas under the line cross model a suggestive QTL on a different position on SSC4 was detected. For SSC2, in the half-sib analysis, a suggestive QTL for backfat thickness was detected with the best position at 26 cM. Imprinting analysis, however, revealed a genome-wise, significant, paternally expressed QTL on SSC2 with the best position at 63 cM. Our results suggest that this QTL is different from the previously reported paternally expressed QTL for muscle mass and fat deposition on the distal tip of SSC2p.

Genome-wide scan for body composition in pigs reveals important role of imprinting.

The role of imprinting in body composition was investigated in an experimental cross between Chinese Meishan pigs and commercial Dutch pigs. A whole-genome scan revealed significant evidence for five quantitative trait loci (QTL) affecting body composition, of which four were imprinted. Imprinting was tested with a statistical model that separated the expression of paternally and maternally inherited alleles. For back fat thickness, a paternally expressed QTL was found on Sus scrofa chromosome 2 (SSC2), and a Mendelian-expressed QTL was found on SSC7. In the same region of SSC7, a maternally expressed QTL affecting muscle depth was found. Chromosome 6 harbored a maternally expressed QTL on the short arm and a paternally expressed QTL on the long arm, both affecting intramuscular fat content. The individual QTL explained from 2% up to 10% of the phenotypic variance. The known homologies to human and mouse did not reveal positional candidate genes. This study demonstrates that testing for imprinting should become a standard procedure to unravel the genetic control of multifactorial traits.

The -323Ins10 polymorphism for factor VII is not associated with coronary atherosclerosis in symptomatic men. The REGRESS study group.

Elevated factor VII coagulant activity (FVII:C) has been associated with an increased risk of ischaemic heart disease, particularly for fatal events. Results of studies on the association between FVII:C and atherosclerosis are not consistent. FVII:C levels are influenced by several environmental factors and by genetic factors. One of the genetic factors is the -323Ins10 polymorphism in the promoter region of the factor VII gene, which is strongly related to FVII:C, and thus may be associated with ischaemic heart disease. We studied the association of this polymorphism with the severity and progression of atherosclerosis. In 511 male patients of the Regression Growth Evaluation Statin Study, the genotype for the -323Ins10 polymorphism was determined. The minimum obstruction diameter and the mean segment diameter were determined at baseline and after a 2-year follow-up period, and new lesion formation was assessed as well. Cardiovascular events were recorded. No relationship was observed between the -323Ins10 polymorphism and angiographic measures of disease progression, nor on the risk of new cardiovascular events. The results suggest that there is no association between the -323Ins10 polymorphism for factor VII and the severity or progression of coronary atherosclerosis in male patients with symptomatic coronary artery disease.

Detection of quantitative trait loci for backfat thickness and intramuscular fat content in pigs (Sus scrofa).

In an experimental cross between Meishan and Dutch Large White and Landrace lines, 619 F(2) animals and their parents were typed for molecular markers covering the entire porcine genome. Associations were studied between these markers and two fatness traits: intramuscular fat content and backfat thickness. Association analyses were performed using interval mapping by regression under two genetic models: (1) an outbred line-cross model where the founder lines were assumed to be fixed for different QTL alleles; and (2) a half-sib model where a unique allele substitution effect was fitted within each of the 19 half-sib families. Both approaches revealed for backfat thickness a highly significant QTL on chromosome 7 and suggestive evidence for a QTL at chromosome 2. Furthermore, suggestive QTL affecting backfat thickness were detected on chromosomes 1 and 6 under the line-cross model. For intramuscular fat content the line-cross approach showed suggestive evidence for QTL on chromosomes 2, 4, and 6, whereas the half-sib analysis showed suggestive linkage for chromosomes 4 and 7. The nature of the QTL effects and assumptions underlying both models could explain discrepancies between the findings under the two models. It is concluded that both approaches can complement each other in the analysis of data from outbred line crosses.