Abstract
BackgroundThe dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs). This study was conducted to discover several hundreds of thousands of porcine SNPs using next generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay.Methodology/Principal FindingsA total of 19 reduced representation libraries derived from four swine breeds (Duroc, Landrace, Large White, Pietrain) and a Wild Boar population and three restriction enzymes (AluI, HaeIII and MspI) were sequenced using Illumina's Genome Analyzer (GA). The SNP discovery effort resulted in the de novo identification of over 372K SNPs. More than 549K SNPs were used to design the Illumina Porcine 60K+SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs were included on the Beadchip. Results from genotyping the 158 individuals used for sequencing showed a high overall SNP call rate (97.5%). Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic yielding a SNP conversion success rate of 94%. The average minor allele frequency (MAF) for all scorable SNPs was 0.274.Conclusions/SignificanceOverall, the results of this study indicate the utility of using next generation sequencing technologies to identify large numbers of reliable SNPs. In addition, the validation of the PorcineSNP60 Beadchip demonstrated that the assay is an excellent tool that will likely be used in a variety of future studies in pigs.
Highlights
One of the main limitations to the dissection of economically important traits in livestock species has been the lack of a sufficient number of genetic markers for the development of high-density and high-throughput assays for association studies
Restriction enzymes producing blunt end fragments were chosen because this would result in DNA fragments that could be directly ligated to the adaptors used in the preparation of libraries for sequencing on the Illumina Genome Analyzer
The total number of generated sequences surpassed 370 million and after filtering based on the quality criteria described in the materials and methods, over 247 million reads remained for single nucleotide polymorphisms (SNPs) discovery using MAQ (Table 1)
Summary
One of the main limitations to the dissection of economically important traits in livestock species has been the lack of a sufficient number of genetic markers for the development of high-density and high-throughput assays for association studies. The genetic regulation of quantitative traits is complex and the identification of the genes that underlie genetic variation requires large numbers of genetic markers, such as microsatellites or SNPs. To date, many QTL (quantitative trait loci) have been localized to large chromosomal regions in several species of domestic animals, including the pig [1,2]. The dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs). This study was conducted to discover several hundreds of thousands of porcine SNPs using generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay
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