Novel porcine repetitive elements

BACKGROUND: Repetitive elements comprise ~45% of mammalian genomes and are increasingly known to impact genomic function by contributing to the genomic architecture, by direct regulation of gene expression and by affecting genomic size, diversity and evolution. The ubiquity and increasingly understood importance of repetitive elements contribute to the need to identify and annotate them. We set out to identify previously uncharacterized repetitive DNA in the porcine genome. Once found, we characterized the prevalence of these repeats in other mammals. RESULTS: We discovered 27 repetitive elements in 220 BACs covering 1% of the porcine genome (Comparative Vertebrate Sequencing Initiative; CVSI). These repeats varied in length from 55 to 1059 nucleotides. To estimate copy numbers, we went to an independent source of data, the BAC-end sequences (Wellcome Trust Sanger Institute), covering approximately 15% of the porcine genome. Copy numbers in BAC-ends were less than one hundred for 6 repeat elements, between 100 and 1000 for 16 and between 1,000 and 10,000 for 5. Several of the repeat elements were found in the bovine genome and we have identified two with orthologous sites, indicating that these elements were present in their common ancestor. None of the repeat elements were found in primate, rodent or dog genomes. We were unable to identify any of the replication machinery common to active transposable elements in these newly identified repeats. CONCLUSION: The presence of both orthologous and non-orthologous sites indicates that some sites existed prior to speciation and some were generated later. The identification of low to moderate copy number repetitive DNA that is specific to artiodactyls will be critical in the assembly of livestock genomes and studies of comparative genomics

SNP discovery in swine by reduced representation and high throughput pyrosequencing

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Approximately 5 million sequence reads were collected and assembled into contigs having an overall observed depth of 7.65-fold coverage. The approximate minor allele frequency was estimated from the number of observations of the alternate alleles. The average coverage at the SNPs was 12.6-fold. This approach identified 115,572 SNPs in 47,830 contigs. Comparison to partial swine genome draft sequence indicated 49,879 SNP (43%) and 22,045 contigs (46%) mapped to a position on a sequenced pig chromosome and the distribution was essentially random. A sample of 176 putative SNPs was examined and 168 (95.5%) were confirmed to have segregating alleles; the correlation of the observed minor allele frequency (MAF) to that predicted from the sequence data was 0.58.</p> <p>Conclusion</p> <p>The process was an efficient means to identify a large number of porcine SNP having high validation rate to be used in an ongoing international collaboration to produce a highly parallel genotyping assay for swine. By using a conservative approach, a robust group of SNPs were detected with greater confidence and relatively high MAF that should be suitable for genotyping in a wide variety of commercial populations.</p

Characterization of the aldo-keto reductase 1C gene cluster on pig chromosome 10: possible associations with reproductive traits

BACKGROUND: The rate of pubertal development and weaning to estrus interval are correlated and affect reproductive efficiency of swine. Quantitative trait loci (QTL) for age of puberty, nipple number and ovulation rate have been identified in Meishan crosses on pig chromosome 10q (SSC10) near the telomere, which is homologous to human chromosome 10p15 and contains an aldo-keto reductase (AKR) gene cluster with at least six family members. AKRs are tissue-specific hydroxysteroid dehydrogenases that interconvert weak steroid hormones to their more potent counterparts and regulate processes involved in development, homeostasis and reproduction. Because of their location in the swine genome and their implication in reproductive physiology, this gene cluster was characterized and evaluated for effects on reproductive traits in swine. RESULTS: Screening the porcine CHORI-242 BAC library with a full-length AKR1C4 cDNA identified 7 positive clones and sample sequencing of 5 BAC clones revealed 5 distinct AKR1C genes (AKR1CL2 and AKR1C1 through 4), which mapped to 126–128 cM on SSC10. Using the IMpRH(7000rad )and IMNpRH2(12000rad )radiation hybrid panels, these 5 genes mapped between microsatellite markers SWR67 and SW2067. Comparison of sequence data with the porcine BAC fingerprint map show that the cluster of genes resides in a 300 kb region. Twelve SNPs were genotyped in gilts observed for age at first estrus and ovulation rate from the F8 and F10 generations of one-quarter Meishan descendants of the USMARC resource population. Age at puberty, nipple number and ovulation rate data were analyzed for association with genotypes by MTDFREML using an animal model. One SNP, a phenylalanine to isoleucine substitution in AKR1C2, was associated with age of puberty (p = 0.07) and possibly ovulation rate (p = 0.102). Two SNP in AKR1C4 were significantly associated with nipple number (p ≤ 0.03) and another possibly associated with age at puberty (p = 0.09). CONCLUSION: AKR1C genotypes were associated with nipple number as well as possible effects on age at puberty and ovulation rate. The estimated effects of AKR1C genotypes on these traits suggest that the SNPs are in incomplete linkage disequilibrium with the causal mutations that affect reproductive traits in swine. Further investigations are necessary to identify these mutations and understand how these AKR1C genes affect these important reproductive traits. The nucleotide sequence data reported have been submitted to GenBank and assigned accession numbers [GenBank:DQ474064–DQ474068, GenBank:DQ494488–DQ494490 and GenBank:DQ487182–DQ487184]

Evaluation of genotype quality parameters for \u3ci\u3eSowPro90\u3c/i\u3e, a new genotyping array for swine

Understanding early predictors of sow fertility has the potential to improve genomic predictions. A custom SNP array (SowPro90 produced by Affymetrix) was developed to include genetic variants overlapping quantitative trait loci for age at puberty, one of the earliest indicators of sow fertility, as well as variants related to innate and adaptive immunity. The polymorphisms included in the custom genotyping array were identified using multiple genomic approaches including deep genomic and transcriptomic sequencing and genome-wide associations. Animals from research and commercial populations (n = 2,586) were genotyped for 103,476 SNPs included in SowPro90. To assess the quality of data generated, genotype concordance was evaluated between the SowPro90 and Porcine SNP60 BeadArray using a subset of common SNP (n = 44,708) and animals (n = 277). The mean genotype concordance rate per SNP was 98.4%. Differences in distribution of data quality were observed between the platforms indicating the need for platform specific thresholds for quality parameters. The optimal thresholds for SowPro90 (≥97% SNP and ≥93% sample call rate) were obtained by analyzing the data quality distribution and genotype concordance per SNP across platforms. At ≥97% SNP call rate, there were 42,151 SNPs (94.3%) retained with a mean genotype concordance of 98.6% across platforms. Similarly, ≥94% SNPs and ≥85% sample call rates were established as thresholds for Porcine SNP60 BeadArray. At ≥94% SNPs call rate, there were 41,043 SNPs (91.8%) retained with a mean genotype concordance of 98.6% across platforms. Final evaluation of SowPro90 array content (n = 103,476) at ≥97% SNPs and ≥93% sample call rates allowed retention of 89,040 SNPs (86%) for downstream analysis. The findings and strategy for quality control could be helpful in identifying consistent, high-quality genotypes for genomic evaluations, especially when integrating genotype data from different platforms

Association of Porcine Heparanase and Hyaluronidase 1 and 2 with Reproductive and Production Traits in a Landrace–Duroc–Yorkshire Population

The ovary and placenta are dynamic structures requiring constant modification both structurally and through cell–cell communication capabilities. The extracellular matrix and basement membranes are primarily composed of a milieu of glycosaminoglycans, including heparan sulfate and hyaluronan. Heparanase (HPSE) and hyaluronidases (HYAL) are responsible for degrading heparan sulfate and hyaluronan, respectively. Therefore, the objective of this study was to evaluate the relationship of SNPs distinct to HPSE, HYAL1, and HYAL2 with measurements of reproduction and production traits in swine. Single trait associations were performed on a Landrace–Duroc–Yorkshire population using SNPs discovered and identified in HPSE, HYAL1, and HYAL2. Analyses were conducted on an extended pedigree and SNPs were found to be associated with reproductive and production traits. Prior to multiple-testing corrections, SNPs within HPSE were weakly associated (P < 0.03) having additive effects with age at puberty (−2.5 ± 1.08 days), ovulation rate (0.5 ± 0.24 corpora lutea), and number of piglets born alive (0.9 ± 0.44 piglets). A HYAL1 and two HYAL2 SNP were nominally associated (P ≤ 0.0063) with number of piglets born alive after multiple-testing corrections (effects between 1.02 and 1.44 piglets), while one of the same HYAL2 markers maintained a modest association (P = 0.0043) having a dominant effect with number of piglets weaned (1.2 ± 0.41 piglets) after multiple-testing correction. Functionally, HPSE and HYAL1 and 2 have been shown to participate in events related to ovarian and placental activity. SNPs from these studies could potentially assist with understanding genetic components underlying sow lifetime productivity as measured by piglet survivability based on number born alive and number weaned, thereby contributing to a greater number of pigs/sow/year

Genomic regions associated with kyphosis in swine

<p>Abstract</p> <p>Background</p> <p>A back curvature defect similar to kyphosis in humans has been observed in swine herds. The defect ranges from mild to severe curvature of the thoracic vertebrate in split carcasses and has an estimated heritability of 0.3. The objective of this study was to identify genomic regions that affect this trait.</p> <p>Results</p> <p>Single nucleotide polymorphism (SNP) associations performed with 198 SNPs and microsatellite markers in a Duroc-Landrace-Yorkshire resource population (U.S. Meat Animal Research Center, USMARC resource population) of swine provided regions of association with this trait on 15 chromosomes. Positional candidate genes, especially those involved in human skeletal development pathways, were selected for SNP identification. SNPs in 16 candidate genes were genotyped in an F2 population (n = 371) and the USMARC resource herd (n = 1,257) with kyphosis scores. SNPs in <it>KCNN2 </it>on SSC2, <it>RYR1 </it>and <it>PLOD1 </it>on SSC6 and <it>MYST4 </it>on SSC14 were significantly associated with kyphosis in the resource population of swine (<it>P </it>≤ 0.05). SNPs in <it>CER1 </it>and <it>CDH7 </it>on SSC1, <it>PSMA5 </it>on SSC4, <it>HOXC6 </it>and <it>HOXC8 </it>on SSC5, <it>ADAMTS18 </it>on SSC6 and <it>SOX9 </it>on SSC12 were significantly associated with the kyphosis trait in the F2 population of swine (<it>P </it>≤ 0.05).</p> <p>Conclusions</p> <p>These data suggest that this kyphosis trait may be affected by several loci and that these may differ by population. Carcass value could be improved by effectively removing this undesirable trait from pig populations.</p

MicroRNA transcriptome profiles during swine skeletal muscle development

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Twelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus) and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate.</p> <p>Conclusion</p> <p>These data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.</p

Detection of transcriptional difference of porcine imprinted genes using different microarray platforms

BACKGROUND: Presently, multiple options exist for conducting gene expression profiling studies in swine. In order to determine the performance of some of the existing microarrays, Affymetrix Porcine, Affymetrix Human U133+2.0, and the U.S. Pig Genome Coordination Program spotted glass oligonucleotide microarrays were compared for their reproducibility, coverage, platform independent and dependent sensitivity using fibroblast cell lines derived from control and parthenogenic porcine embryos. RESULTS: Array group correlations between technical replicates demonstrated comparable reproducibility in both Affymetrix arrays. Glass oligonucleotide arrays showed greater variability and, in addition, approximately 10% of probes had to be discarded due to slide printing defects. Probe level analysis of Affymetrix Human arrays revealed significant variability within probe sets due to the effects of cross-species hybridization. Affymetrix Porcine arrays identified the greatest number of differentially expressed genes amongst probes common to all arrays, a measure of platform sensitivity. Affymetrix Porcine arrays also identified the greatest number of differentially expressed known imprinted genes using all probes on each array, an ad hoc measure of realistic performance for this particular experiment. CONCLUSION: We conclude that of the platforms currently available and tested, the Affymetrix Porcine array is the most sensitive and reproducible microarray for swine genomic studies

Synaptogyrin-2 influences replication of Porcine circovirus 2

Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association, ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (\u3e24 hpi) and supernatant (\u3e48hpi)(P \u3c 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load