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.

BackgroundThe availability of a high-density SNP genotyping chip and a reference genome sequence of the pig (Sus scrofa) enabled the construction of a high-density linkage map. A high-density linkage map is an essential tool for further fine-mapping of quantitative trait loci (QTL) for a variety of traits in the pig and for a better understanding of mechanisms underlying genome evolution.ResultsFour different pig pedigrees were genotyped using the Illumina PorcineSNP60 BeadChip. Recombination maps for the autosomes were computed for each individual pedigree using a common set of markers. The resulting genetic maps comprised 38,599 SNPs, including 928 SNPs not positioned on a chromosome in the current assembly of the pig genome (build 10.2). The total genetic length varied according to the pedigree, from 1797 to 2149 cM. Female maps were longer than male maps, with a notable exception for SSC1 where male maps are characterized by a higher recombination rate than females in the region between 91–250 Mb. The recombination rates varied among chromosomes and along individual chromosomes, regions with high recombination rates tending to cluster close to the chromosome ends, irrespective of the position of the centromere. Correlations between main sequence features and recombination rates were investigated and significant correlations were obtained for all the studied motifs. Regions characterized by high recombination rates were enriched for specific GC-rich sequence motifs as compared to low recombinant regions. These correlations were higher in females than in males, and females were found to be more recombinant than males at regions where the GC content was greater than 0.4.ConclusionsThe analysis of the recombination rate along the pig genome highlighted that the regions exhibiting higher levels of recombination tend to cluster around the ends of the chromosomes irrespective of the location of the centromere. Major sex-differences in recombination were observed: females had a higher recombination rate within GC-rich regions and exhibited a stronger correlation between recombination rates and specific sequence features.

BackgroundTo enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP) discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock population.ResultsThe reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the validated markers were associated with rainbow trout transcripts.ConclusionThe use of reduced representation libraries and pyrosequencing technology proved to be an effective strategy for the discovery of a high number of putative SNPs in rainbow trout; however, modifications to the technique to decrease the false discovery rate resulting from the evolutionary recent genome duplication would be desirable.

Background: Relatively little information is available for sequence variation in the pig. We previously used a combination of short read (25 base pair) high-throughput sequencing and reduced genomic representation to discover > 60,000 single nucleotide polymorphisms (SNP) in cattle, but the current lack of complete genome sequence limits this approach in swine. Longer-read pyrosequencing-based technologies have the potential to overcome this limitation by providing sufficient flanking sequence information for assay design. Swine SNP were discovered in the present study using a reduced representation of 450 base pair (bp) porcine genomic fragments (approximately 4% of the swine genome) prepared from a pool of 26 animals relevant to current pork production, and a GS-FLX instrument producing 240 bp reads.

Background: MicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult.

We report the rotationally resolved, one-photon threshold photoelectron spectrum of the methyl radical, CH3, produced by supersonic-jet, flash pyrolysis. Only rotational transitions with ΔK=0, ±2 are observed and this result is shown to be consistent with photoionization selection rules in D3h symmetry. Assignment of the threshold photoelectron spectrum results in an adiabatic ionization potential of 79 349±3 cm−1.

The objectives of this study included: (1) identify the expression of miRNAs specific to bovine cumulus-oocyte complexes (COCs) during late oogenesis, (2) characterize the expression of candidate miRNAs as well as some miRNA processing genes, and (3) computationally identify and characterize the expression of target mRNAs for candidate miRNAs. Small RNAs in the 16-27 bp range were isolated from pooled COCs aspirated from 1- to 10-mm follicles of beef cattle ovaries and used to construct a cDNA library. A total 1798 putative miRNA sequences from the cDNA library of small RNA were compared to known miRNAs. Sixty-four miRNA clusters matched previously reported sequences in the miRBase database and 5 miRNA clusters had not been reported. TaqMan miRNA assays were used to confirm the expression of let-7b, let-7i, and miR-106a from independent collections of COCs. Real-time PCR assays were used to characterize expression of miRNA processing genes and target mRNAs (MYC and WEE1A) for the candidate miRNAs from independent collections of COCs. Expression data were analyzed using general linear model procedures for analysis of variance. The expression of let-7b and let-7i were not different between the cellular populations from various sized follicles. However, miR-106a expression was greater (P<0.01) in oocytes compared with COCs and granulosa cells. Furthermore, all the miRNA processing genes have greater expression (P<0.001) in oocytes compared with COCs and granulosa cells. The expression of potential target mRNAs for let-7 and let-7i (i.e., MYC), and miR-106a (i.e., WEE1A) were decreased (P<0.05) in oocytes compared with COCs and granulosa cells. These results demonstrate specific miRNAs within bovine COCs during late oogenesis and provide some evidence that miRNAs may play a role regulating maternal mRNAs in bovine oocytes.

Pork quality has a large impact on consumer preference and perception of eating quality. A genome-wide association was performed for pork quality traits [intramuscular fat (IMF)], slice shear force (SSF), color attributes, purge, cooking loss, and pH] from 531 to 1,237 records on barrows and gilts of a Landrace-Duroc-Yorkshire population using the Illumina PorcineSNP60 BeadChip. Associations were detected using MTDFREML for all traits. Intramuscular fat had the greatest number of SNP associations, followed by pH, purge, cooking loss, shear force, and color. Two regions contained associations for multiple traits; one on SSC1 at 255 Mb near calcineurin subunit B (PPP3R2) was associated with SSF, moisture loss, and pH, and one on SSC6 from 28 to 29.5 Mb for purge and IMF containing the candidate genes glucose-6-phosphate isomerase (GPI) and KCTD15. Some of the other compelling candidate genes in regions associated with meat quality include CEBPA, SNAI1, and FAM132A for IMF, CAPN1 for SSF, GLUL for pH, and PRKAG3 and ITGB1 with cooking loss.