G. A. Rohrer

Design of a high density SNP genotyping assay in the pig using SNPs identified and characterized by next generation sequencing technology

Background 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 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 Findings A 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 Illuminas 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/Significance Overall, 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.

Interval mapping of growth in divergent swine cross

Abstract. A genomic scan of 18 swine autosomal chromosomes was constructed with 119 polymorphic microsatellite (ms) markers to identify quantitative trait loci (QTL) for 11 growth traits in the University of Illinois Meishan × Yorkshire Swine Resource Family. A significant QTL effect was found for post-weaning average daily gain (ADG) between 5.5 and 56 kg of body weight that mapped between markers SW373 and SW1301 near the telomere of Chromosome (Chr) 1 q (SSC1). This QTL effect had a nominal (pointwise) p-value of 0.000007, a genome wide p-value of 0.012, and accounted for 26% of the F2 phenotypic variance. The same chromosome region also had significant effects on ADG between birth and 56 kg body weight (p-value = .000227), and on ADG between 35 and 56 kg (p-value = .00077). These observations suggest that a significant QTL for post-weaning growth resides on SSC1.

A high utility integrated map of the pig genome

BackgroundThe domestic pig is being increasingly exploited as a system for modeling human disease. It also has substantial economic importance for meat-based protein production. Physical clone maps have underpinned large-scale genomic sequencing and enabled focused cloning efforts for many genomes. Comparative genetic maps indicate that there is more structural similarity between pig and human than, for example, mouse and human, and we have used this close relationship between human and pig as a way of facilitating map construction.ResultsHere we report the construction of the most highly continuous bacterial artificial chromosome (BAC) map of any mammalian genome, for the pig (Sus scrofa domestica) genome. The map provides a template for the generation and assembly of high-quality anchored sequence across the genome. The physical map integrates previous landmark maps with restriction fingerprints and BAC end sequences from over 260,000 BACs derived from 4 BAC libraries and takes advantage of alignments to the human genome to improve the continuity and local ordering of the clone contigs. We estimate that over 98% of the euchromatin of the 18 pig autosomes and the X chromosome along with localized coverage on Y is represented in 172 contigs, with chromosome 13 (218 Mb) represented by a single contig. The map is accessible through pre-Ensembl, where links to marker and sequence data can be found.ConclusionThe map will enable immediate electronic positional cloning of genes, benefiting the pig research community and further facilitating use of the pig as an alternative animal model for human disease. The clone map and BAC end sequence data can also help to support the assembly of maps and genome sequences of other artiodactyls.

A high density recombination map of the pig reveals a correlation between sex-specific recombination and GC content

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.

Porcine gene discovery by normalized cDNA-library sequencing and EST cluster assembly.

Genetic and environmental factors affect the efficiency of pork production by influencing gene expression during porcine reproduction, tissue development, and growth. The identification and functional analysis of gene products important to these processes would be greatly enhanced by the development of a database of expressed porcine gene sequence. Two normalized porcine cDNA libraries (MARC 1PIG and MARC 2PIG), derived respectively from embryonic and reproductive tissues, were constructed, sequenced, and analyzed. A total of 66,245 clones from these two libraries were 5?-end sequenced and deposited in GenBank. Cluster analysis revealed that within-library redundancy is low, and comparison of all porcine ESTs with the human database suggests that the sequences from these two libraries represent portions of a significant number of independent pig genes. A Porcine Gene Index (PGI), comprising 15,616 tentative consensus sequences and 31,466 singletons, includes all sequences in public repositories and has been developed to facilitate further comparative map development and characterization of porcine genes (http://www.tigr.org/tdb/ssgi/). The clones and sequences from these libraries provide a catalog of expressed porcine genes and a resource for development of high-density hybridization arrays for transcriptional profiling of porcine tissues. In addition, comparison of porcine ESTs with sequences from other species serves as a valuable resource for comparative map development. Both arrayed cDNA libraries are available for unrestricted public use.

Physical assignments of 68 porcine cosmid and lambda clones containing polymorphic microsatellites

Two lambda phage and 66 cosmids containing informative porcine microsatellites were assigned to 17 of 18 porcine autosomes and the X Chromosome (Chr) by fluorescence in situ hybridization (FISH). These assignments provide additional physically anchored markers to integrate the porcine physical and genetic maps.

A porcine BAC library with tenfold genome coverage: a resource for physical and genetic map integration

Recent advances in porcine genomics have identified quantitative trait loci (QTL) that influence pork production traits such as carcass traits, meat quality, and reproductive efficiency (Rohrer 2000; Cassady 1999). The low resolution to which most of these loci have been defined precludes the accurate application of markerassisted selection (MAS) strategies for increasing production efficiency. Large-insert genomic libraries are an excellent resource for marker development aimed at increasing the resolution of QTL and for the development of contiguous physical maps (contigs) of the chromosomal regions containing them. To facilitate applications requiring genomic clones, several porcine yeast artificial chromosome (YAC) libraries have been developed representing onefold (Leeb et al. 1995), threefold (Rogel-Gaillard et al. 1997), and 5.5-fold (Alexander et al. 1997) coverage of the pig genome. These YAC libraries have been valuable resource owing to their large insert size. However, marker isolation from YAC libraries is confounded by the equimolar representation of the complex yeast genome and YAC DNA, susceptibility to insert rearrangement, and a relatively high degree of chimerism. The development of bacterial artificial chromosome (BAC) libraries (Shizuya et al. 1992; Ioannou et al. 1994) represents a compromise between insert size, stability, and ease of clone DNA isolation. BACs are capable of stably maintaining insert sizes exceeding 200 kb and can be easily isolated by standard alkaline lysis from bacterial genomic DNA by virtue of their closed-circular conformation (Shizuya et al. 1992; Ioannou et al. 1994). The ease of BAC DNA isolation allows for efficient restriction analysis, subcloning, and direct BAC DNA sequencing, procedures invaluable for marker isolation and the development of contiguous physical maps. Recently BAC libraries representing livestock species, including cattle, sheep, and pigs (Cai et al. 1995; Warren et al. 2000; Vaiman et al. 1999; Suzuki et al. 2000; Rogel-Gaillard et al. 1999) have been developed. Among these are two porcine BAC libraries representing fourfold (Suzuki et al. 2000) and fivefold (RogelGaillard et al. 1999) coverage of the porcine genome. We undertook the development of a publicly available pig BAC library, RPCI-44, representing at least a tenfold coverage of the pig genome as a resource for physical map development and pig genome sequencing. High-molecular-weight genomic DNA from four crossbred male pigs (breed composition: 37.5% Yorkshire, 37.5% Landrace, and 25% Meishan) was isolated as previously described (Osoegawa et al. 1998), partially digested with EcoRI (New England Biolabs, Beverly, MA) and EcoRI methylase (New England Biolabs), and fractionated by clamped homogeneous electrical field (CHEF; Chu et al. 1986) electrophoresis (BioRad, Hercules, CA). Digested genomic DNA in the range of 150–200 kpb was gelisolated by electroelution and ligated to EcoRI-cut pTARBAC2 (Wang et al., unpublished; http://www.chori.org/bacpac/ vectorframe.htm). Ligated DNA was drop-dialyzed on floating membranes, first against sterile water, followed by 0.5 × TE containing 30% PEG8000, and introduced by electroporation into DH1OB electrocompetent cells (Life Technologies, Rockville, MD). Transformed bacterial cells were allowed to recover in SOC medium for 1 h at 37°C, and were spread on LB agar plates containing 20 mg/ml chloramphenicol and 5% sucrose. Individual colonies were picked with an automatic colony-picking robot (Qbot; Genetix, Stony Brook, NY) and arrayed into 528 individual 384-well microtiter dishes (Genetix) with LB medium containing 7.5% glycerol and 20 mg/ml chloramphenicol. The library was divided into two segments and gridded onto 11 high-density replica filters, each containing up to 18,432 independent clones that have been spotted in duplicate. To serve as anchor spots, an endsequenced BAC clone (1A1) from a Caenorhabditis brigsea BAC library (RPCI-94), containing DNA apparently unique to nematode genomes, was gridded on the corner of each of the six fields that comprise the high-density filters. The inclusion of a probe directed against the anchor spots can facilitate orientation of hybridized filters. The predicted coverage of RPCI-44 is a function of the total number of insert-containing clones and the size of the genomic DNA fragments they contain. From a total of 202,752 clones picked, 1,764 (segment 1, 1.9%) and 1,519 (segment 2, 1.4%) clones failed to grow, leaving a total of 199,469 clones. Non-recombinant clones containing the original vector were identified by high-density colony filter hybridization with the vector as a probe. The probing identifies two classes of clones with strong versus weak hybridization signals corresponding to the presence or absence of the original vector stuffer fragment (“pUClink”), which contains a high-copy-number replicon. Recombinant clones lack the stuffer fragment, resulting in much weaker hybridization signals. In total, 119 clones (0.06%) were identified as containing intact vector by this approach. The plate and well addresses of these are available at http://www.chori.org/framenonrec.htm. To evaluate the average insert size and insert size distribution in the library, randomly selected clones from each segment were subjected to restriction analysis with Not1, insert size determination by CHEF electrophoresis, and comparison with molecular weight standards (Osoegawa et al. 2000). Segment 1 was composed of 90,396 clones containing inserts averaging 157 kpb * Present address: University of Minnesota, Department of Animal Science, 495 An Sci/Vet Med, 1988 Fitch Ave., St. Paul, MN 55108, USA.

Interval mapping of carcass and meat quality traits in a divergent swine cross.

An autosomal scan of the swine genome with 119 polymorphic microsatellite (ms) markers and data from 116 F2 barrows of the University of Illinois Meishan x Yorkshire Swine Resource Families identified genomic regions with effects on variance in carcass composition and meat quality at nominal significance (p-value <0.05). Marker intervals on chromosomes 1, 6, 7, 8 and 12 (SSC1, SSC6, SSC7, SSC8, SSC12) with phenotypic effects on carcass length, 10th rib backfat thickness, average backfat thickness, leaf fat, loin eye area and intramuscular fat content confirm QTL effects identified previously based on genome wide significance (p-value<0.05). Several marker intervals included nominally significant (p-value<0.05) dominance effects on leaf fat, 10th rib backfat thickness, loin eye area, muscle pH and intramuscular fat content.

Evaluating Evolutionary Divergence with Microsatellites

Abstract. We report the use of microsatellites (MS) to track the recent evolution of swine. Allelic frequencies for nine MS loci linked on swine chromosome 6 (SSC6) representing four western and one Chinese swine breeds were used to estimate genetic distances and times of breed divergence. A phylogenetic tree was constructed which partitioned into western and Meishan breed branches. Yorkshire and Hampshire breeds exhibited the most recent divergence with a calculated distance of 391 years. The oldest divergence, of 2,227 years, was between Meishan and Hampshire swine. Estimates of breed divergence are consistent with historical records. Additional analysis suggests that polymorphic MS linked on a single chromosome are sufficient to determine evolutionary relationships within a single species.

Interrelationships of Porcine X and Y Chromosomes with Pituitary Gonadotropins and Testicular Size

Abstract Endocrine and testicular responses to unilateral castration on 1, 10, 56, or 112 days of age were characterized in 132 Chinese Meishan (MS) × White composite (WC) crossbred boars in which testicular size associates with a quantitative trait locus (QTL) on X chromosome. At 220 days of age, testicles of boars unilaterally castrated on Day 1 or 10 weighed more and had greater total daily sperm production (DSP) than one testicle of bilaterally intact boars (P < 0.05); compensation did not double these two responses. Boars with MS alleles at the X chromosome QTL had smaller testicles, darker colored parenchyma, and lower total DSP than boars with WC alleles (P < 0.05). The MS alleles engendered greater (P < 0.05) plasma FSH and LH during puberty than WC alleles. Plasma FSH increased (P < 0.05) within 48 h of unilateral castration on Days 1, 10, and 56. Subsequent increases occurred earlier during puberty (P < 0.05) after unilateral castration at younger ages than after unilateral castration at older ages. Pubertal increases in plasma FSH and LH were greater (P < 0.05) in boars with MS alleles than in those with WC alleles for the X chromosome QTL. Breed of Y chromosome had no effect on testicular traits, FSH, testosterone, or estrone. For LH, boars with an MS Y chromosome had greater (P < 0.01) plasma LH across all ages than boars with a WC Y chromosome. We conclude that a gene or groups of genes that reside on the porcine X chromosome regulate testicular development and pubertal gonadotropin concentrations.