Single nucleotide polymorphisms for pig identification and parentage exclusion

Single nucleotide polymorphisms (SNPs) have become an important type of marker for commercial diagnostic and parentage genotyping applications as automated genotyping systems have been developed that yield accurate genotypes. Unfortunately, allele frequencies for public SNP markers in commercial pig populations have not been available. To fulfil this need, SNP markers previously mapped in the USMARC swine reference population were tested in a panel of 155 boars that were representative of US purebred Duroc, Hampshire, Landrace and Yorkshire populations. Multiplex assay groups of 5-7 SNP assays/group were designed and genotypes were determined using Sequenom's massarray system. Of 80 SNPs that were evaluated, 60 SNPs with minor allele frequencies >0.15 were selected for the final panel of markers. Overall identity power across breeds was 4.6 x 10(-23), but within-breed values ranged from 4.3 x 10(-14) (Hampshire) to 2.6 x 10(-22) (Yorkshire). Parentage exclusion probability with only one sampled parent was 0.9974 (all data) and ranged from 0.9594 (Hampshire) to 0.9963 (Yorkshire) within breeds. Sire exclusion probability when the dam's genotype was known was 0.99998 (all data) and ranged from 0.99868 (Hampshire) to 0.99997 (Yorkshire) within breeds. Power of exclusion was compared between the 60 SNP and 10 microsatellite markers. The parental exclusion probabilities for SNP and microsatellite marker panels were similar, but the SNP panel was much more sensitive for individual identification. This panel of SNP markers is theoretically sufficient for individual identification of any pig in the world and is publicly available.     

Single nucleotide polymorphisms for DNA typing in the domestic horse

Genetic markers are important resources for individual identification and parentage assessment. Although short tandem repeats (STRs) have been the traditional DNA marker, technological advances have led to single nucleotide polymorphisms (SNPs) becoming an attractive alternative. SNPs can be highly multiplexed and automatically scored, which allows for easier standardization and sharing among laboratories. Equine parentage is currently assessed using STRs. We obtained a publicly available SNP dataset of 729 horses representing 32 diverse breeds. A proposed set of 101 SNPs was analyzed for DNA typing suitability. The overall minor allele frequency of the panel was 0.376 (range 0.304–0.419), with per breed probability of identities ranging from 5.6 × 10^?35 to 1.86 × 10^?42. When one parent was available, exclusion probabilities ranged from 0.9998 to 0.999996, although when both parents were available, all breeds had exclusion probabilities greater than 0.9999999. A set of 388 horses from 35 breeds was genotyped to evaluate marker performance on known families. The set included 107 parent–offspring pairs and 101 full trios. No horses shared identical genotypes across all markers, indicating that the selected set was sufficient for individual identification. All pairwise comparisons were classified using ISAG rules, with one or two excluding markers considered an accepted parent–offspring pair, two or three excluding markers considered doubtful and four or more excluding markers rejecting parentage. The panel had an overall accuracy of 99.9% for identifying true parent–offspring pairs. Our developed marker set is both present on current generation SNP chips and can be highly multiplexed in standalone panels and thus is a promising resource for SNP‐based DNA typing.     

Minisatellite mutational processes reduce Fst estimates

We have used a new method for binning minisatellite alleles (semi-automated allele aggregation) and report the extent of population diversity detectable by eleven minisatellite loci in 2,689 individuals from 19 human populations distributed widely throughout the world. Whereas population relationships are consistent with those found in other studies, our estimate of genetic differentiation (F(st)) between populations is less than 8%, which is lower than comparative estimates of between 10%-15% obtained by using other sources of polymorphism data. We infer that mutational processes are involved in reducing F(st) estimates from minisatellite data because, first, the lowest F(st) estimates are found at loci showing autocorrelated frequencies among alleles of similar size and, second, F(st) declines with heterozygosity but by more than predicted assuming simple models of mutation. These conclusions are consistent with the view that minisatellites are subject to selective or mutational constraints in addition to those expected under simple step-wise mutation models.     

Genomic Diversity in Pig (Sus scrofa) and its Comparison with Human and other Livestock

We have reviewed the current pig (Sus scrofa) genomic diversity within and between sites and compared them with human and other livestock. The current Porcine 60K single nucleotide polymorphism (SNP) panel has an average SNP distance in a range of 30 - 40 kb. Most of genetic variation was distributed within populations, and only a small proportion of them existed between populations. The average heterozygosity was lower in pig than in human and other livestock. Genetic inbreeding coefficient (F(IS)), population differentiation (F(ST)), and Nei's genetic distance between populations were much larger in pig than in human and other livestock. Higher average genetic distance existed between European and Asian populations than between European or between Asian populations. Asian breeds harboured much larger variability and higher average heterozygosity than European breeds. The samples of wild boar that have been analyzed displayed more extensive genetic variation than domestic breeds. The average linkage disequilibrium (LD) in improved pig breeds extended to 1 - 3 cM, much larger than that in human (~ 30 kb) and cattle (~ 100 kb), but smaller than that in sheep (~ 10 cM). European breeds showed greater LD that decayed more slowly than Asian breeds. We briefly discuss some processes for maintaining genomic diversity in pig, including migration, introgression, selection, and drift. We conclude that, due to the long time of domestication, the pig possesses lower heterozygosity, higher F(IS), and larger LD compared with human and cattle. This implies that a smaller effective population size and less informative markers are needed in pig for genome wide association studies.     

Using the dog genome to find single nucleotide polymorphisms in red foxes and other distantly related members of the Canidae

Single nucleotide polymorphisms (SNP) are the ideal marker for characterizing genomic variation but can be difficult to find in nonmodel species. We explored the usefulness of the dog genome for finding SNPs in distantly related nonmodel canids and evaluated so-ascertained SNPs. Using 40 primer pairs designed from randomly selected bacterial artificial chromosome clones from the dog genome, we successfully sequenced 80-88% of loci in a coyote (Canis latrans), grey fox (Urocyon cinereoargenteus), and red fox (Vulpes vulpes), which compared favourably to a 60% success rate for each species using 10 primer pairs conserved across mammals. Loci were minimally heterogeneous with respect to SNP density, which was similar, overall, in a discovery panel of nine red foxes to that previously reported for a panel of eight wolves (Canis lupus). Additionally, individual heterozygosity was similar across the three canids in this study. However, the proportion of SNP sites shared with the dog decreased with phylogenetic divergence, with no SNPs shared between red foxes and dogs. Density of interspecific SNPs increased approximately linearly with divergence time between species. Using red foxes from three populations, we estimated F(ST) based on each of 42 SNPs and 14 microsatellites and simulated null distributions conditioned on each marker type. Relative to SNPs, microsatellites systematically underestimated F(ST) and produced biased null distributions, indicating that SNPs are superior markers for these functions. By reconstituting the frequency spectrum of SNPs discovered in nine red foxes, we discovered an estimated 77-89% of all SNPs (within the region screened) present in North American red foxes. In sum, these findings indicate that information from the dog genome enables easy ascertainment of random and gene-linked SNPs throughout the Canidae and illustrate the value of SNPs in ecological and evolutionary genetics.     

Genomic differentiation between swamp and river buffalo using a cattle high-density single nucleotide polymorphisms panel

Buffalo (Bubalus bubalis) is an important livestock species in many tropical and subtropical regions. In recent decades, the interest in buffalo’s milk have expanded and intensive buffalo farms start to emerge. However, breeding programs and population genetics information for this species is scarce or inexistent. The present study aims to test the suitability of the commercial high-density single nucleotide polymorphisms (SNP) genotyping panel, the Illumina BovineHD BeadChip, to estimate population genetics parameters, pedigree control and identification of common variants in major production candidate genes. From a total of 777 962 SNPs included in the panel, 20 479 were polymorphic in water buffalo at a call rate of 86% and an average expected heterozygosity (H_E) of 0.306. From these, 357 were mapped within or around the flanking regions of several major candidate genes. A principal components analysis identified three different clusters, each representing pure swamp buffalo type, pure river buffalo type and admixed river buffalo. The hybrids between swamp and river buffalo were clearly identified as an intermediary cluster. The suitability of these SNPs data set for parentage and identity testing demonstrated that the combination of just 30 to 50 SNPs were enough to attain high probabilities of parentage exclusion (0.9999) in both types and identity (2.3×10⁻⁵ and 2.0×10⁻⁷) for river and swamp buffalo, respectively. Our analysis confirms the suitability of the BovineHD BeadChip to assess population structure, hybridization and identity of the water buffalo populations.     

Analysis of genetic diversity of Russian regional populations based on STR markers used in DNA identification

We conducted the first genetic analysis of a wide a range of rural Russian populations in European Russia with a panel of DNA markers commonly used in Human DNA identification. We examined a total of 647 samples from indigenous ethnic Russian populations in Arkhangelsk, Belgorod, Voronezh, Kursk, Rostov, Ryazan, and Orel regions. We employed a multiplex genotyping kit, COrDIS Plus, to genotype Short Tandem Repeat (STR) loci, which included markers officially recommended for DNA identification in the Russian Federation, the United States, and the European Union. In the course of our study, we created a database of allele frequencies, examined the distribution of alleles and genotypes in seven rural Russian populations, and analyzed the genetic relationships between these populations. We found that, although multidimensional analysis indicated a difference between the Northern Russian gene pool and the rest of the Russian European populations, a pairwise comparison using 19 STR markers among all populations did not reveal significant differences. This is in concordance with previous studies, which examined up to 12 STR markers in urban Russian populations. Therefore, the database of allele frequencies created in this study can be applied for forensic examinations and DNA identification among the ethnic Russian population over European Russia. We also noted a decrease in the levels of heterozygosity in the northern Russian population compared to southern and central Russian populations, which is consistent with trends identified previously using classical gene markers and analysis of mitochondrial DNA.     

Comparison of SSRs and SNPs in assessment of genetic relatedness in maize

Advances in high-throughput SNP genotyping and genome sequencing technologies have enabled genome-wide association mapping in dissecting the genetic basis of complex quantitative traits. In this study, 82 SSRs and 884 SNPs with minor allele frequencies (MAF) over 0.20 were used to compare their ability to assess population structure, principal component analysis (PCA) and relative kinship in a maize association panel consisting of 154 inbred lines. Compared to SNPs, SSRs provided more information on genetic diversity. The expected heterozygosity (He) of SSRs and SNPs averaged 0.65 and 0.44, and the polymorphic information content of these two markers was 0.61 and 0.34 in this panel, respectively. Additionally, SSRs performed better at clustering all lines into groups using STRUCTURE and PCA approaches, and estimating relative kinship. For both marker systems, the same clusters were observed based on PCA and the first two eigenvectors accounted for similar percentage of genetic variations in this panel. The correlation coefficients of each eigenvector from SSRs and SNPs decreased sharply when the eigenvector varied from 1 to 3, but kept around 0 when the eigenvector were over 3. The kinship estimates based on SSRs and SNPs were moderately correlated (r (2)?=?0.69). All these results suggest that SSR markers with moderate density are more informative than SNPs for assessing genetic relatedness in maize association mapping panels.     

Conserved worldwide linkage disequilibrium in the human factor XI gene

We have identified, in four diverse human populations, five common single-nucleotide polymorphisms (SNPs) in the coding region of the gene for the blood coagulation protease factor XI. Each SNP has an allele frequency >5% in at least one population. Three of the SNPs (C472T, A844G, and T1234C), spread out over approximately 10 kb of genomic DNA, are in marked linkage disequilibrium (LD) with one another (P < 10(-4)). Interestingly, haplotypes associated with the linked SNPs are conserved across all populations studied, despite significantly different allele frequencies between populations. The presence of such common, widely dispersed haplotypes could complicate the interpretation of LD studies and emphasizes the need for a better understanding of general patterns of LD to facilitate identification of genes for common disorders.     

De Novo SNP Discovery in the Scandinavian Brown Bear (Ursus arctos)

Information about relatedness between individuals in wild populations is advantageous when studying evolutionary, behavioural and ecological processes. Genomic data can be used to determine relatedness between individuals either when no prior knowledge exists or to confirm suspected relatedness. Here we present a set of 96 SNPs suitable for inferring relatedness for brown bears (Ursus arctos) within Scandinavia. We sequenced reduced representation libraries from nine individuals throughout the geographic range. With consensus reads containing putative SNPs, we applied strict filtering criteria with the aim of finding only high-quality, highly-informative SNPs. We tested 150 putative SNPs of which 96% were validated on a panel of 68 individuals. Ninety-six of the validated SNPs with the highest minor allele frequency were selected. The final SNP panel includes four mitochondrial markers, two monomorphic Y-chromosome sex-determination markers, three X-chromosome SNPs and 87 autosomal SNPs. From our validation sample panel, we identified two previously known parent-offspring dyads with reasonable accuracy. This panel of SNPs is a promising tool for inferring relatedness in the brown bear population in Scandinavia.     

Population differentiation determined from putative neutral and divergent adaptive genetic markers in Eulachon (Thaleichthys pacificus,Osmeridae), an anadromous Pacific smelt

Twelve eulachon (Thaleichthys pacificus, Osmeridae) populations ranging from Cook Inlet, Alaska and along the west coast of North America to the Columbia River were examined by restriction‐site‐associated DNA (RAD) sequencing to elucidate patterns of neutral and adaptive variation in this high geneflow species. A total of 4104 single‐nucleotide polymorphisms (SNPs) were discovered across the genome, with 193 putatively adaptive SNPs as determined by F_ST outlier tests. Estimates of population structure in eulachon with the putatively adaptive SNPs were similar, but provided greater resolution of stocks compared with a putatively neutral panel of 3911 SNPs or previous estimates with 14 microsatellites. A cline of increasing measures of genetic diversity from south to north was found in the adaptive panel, but not in the neutral markers (SNPs or microsatellites). This may indicate divergent selective pressures in differing freshwater and marine environments between regional eulachon populations and that these adaptive diversity patterns not seen with neutral markers could be a consideration when determining genetic boundaries for conservation purposes. Estimates of effective population size (N_e) were similar with the neutral SNP panel and microsatellites and may be utilized to monitor population status for eulachon where census sizes are difficult to obtain. Greater differentiation with the panel of putatively adaptive SNPs provided higher individual assignment accuracy compared to the neutral panel or microsatellites for stock identification purposes. This study presents the first SNPs that have been developed for eulachon, and analyses with these markers highlighted the importance of integrating genome‐wide neutral and adaptive genetic variation for the applications of conservation and management.     

Primers for sequence characterization and polymorphism detection in the Atlantic salmon (Salmo salar) growth hormone 1 (GH1) gene

We report the identification of intraspecific sequence variation in the Atlantic salmon (Salmo salar) growth hormone 1 gene. Rapid and inexpensive assays for polymorphism detection were developed for 10 sites. Five of the assays detected single nucleotide polymorphisms (SNPs) using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses, and five were indel polymorphisms, detected using fragment length analyses. The average within population frequency of the most common allele varied from 0.52 to 0.90, and the average within population heterozygosity varied from 0.02 to 0.37 in seven European salmon populations.     

Discovery and characterization of single nucleotide polymorphisms in Chinook salmon, Oncorhynchus tshawytscha

Molecular population genetics of non-model organisms has been dominated by the use of microsatellite loci over the last two decades. The availability of extensive genomic resources for many species is contributing to a transition to the use of single nucleotide polymorphisms (SNPs) for the study of many natural populations. Here we describe the discovery of a large number of SNPs in Chinook salmon, one of the world's most important fishery species, through large-scale Sanger sequencing of expressed sequence tag (EST) regions. More than 3 Mb of sequence was collected in a survey of variation in almost 132 kb of unique genic regions, from 225 separate ESTs, in a diverse ascertainment panel of 24 salmon. This survey yielded 117 TaqMan (5' nuclease) assays, almost all from separate ESTs, which were validated in population samples from five major stocks of salmon from the three largest basins on the Pacific coast of the contiguous United States: the Sacramento, Klamath and Columbia Rivers. The proportion of these loci that was variable in each of these stocks ranged from 86.3% to 90.6% and the mean minor allele frequency ranged from 0.194 to 0.236. There was substantial differentiation between populations with these markers, with a mean F(ST) estimate of 0.107, and values for individual loci ranging from 0 to 0.592. This substantial polymorphism and population-specific differentiation indicates that these markers will be broadly useful, including for both pedigree reconstruction and genetic stock identification applications.     

Estimating genetic diversity across the neutral genome with the use of dense marker maps

Background

With the advent of high throughput DNA typing, dense marker maps have become available to investigate genetic diversity on specific regions of the genome. The aim of this paper was to compare two marker based estimates of the genetic diversity in specific genomic regions lying in between markers: IBD-based genetic diversity and heterozygosity.

Methods

A computer simulated population was set up with individuals containing a single 1-Morgan chromosome and 1665 SNP markers and from this one, an additional population was produced with a lower marker density i.e. 166 SNP markers. For each marker interval based on adjacent markers, the genetic diversity was estimated either by IBD probabilities or heterozygosity. Estimates were compared to each other and to the true genetic diversity. The latter was calculated for a marker in the middle of each marker interval that was not used to estimate genetic diversity.

Results

The simulated population had an average minor allele frequency of 0.28 and an LD (r²) of 0.26, comparable to those of real livestock populations. Genetic diversities estimated by IBD probabilities and by heterozygosity were positively correlated, and correlations with the true genetic diversity were quite similar for the simulated population with a high marker density, both for specific regions (r = 0.19-0.20) and large regions (r = 0.61-0.64) over the genome. For the population with a lower marker density, the correlation with the true genetic diversity turned out to be higher for the IBD-based genetic diversity.

Conclusions

Genetic diversities of ungenotyped regions of the genome (i.e. between markers) estimated by IBD-based methods and heterozygosity give similar results for the simulated population with a high marker density. However, for a population with a lower marker density, the IBD-based method gives a better prediction, since variation and recombination between markers are missed with heterozygosity.     

Customized multiplexing SNP panel for Korean-specific DNA phenotyping in forensic applications

Due to the recent growth of the alien population in Korea, tracking the ethnic origin of human specimens has gained importance in genetic forensics. To address this issue, we developed a method to analyze single nucleotide polymorphisms (SNPs) based on next-generation sequencing (NGS) technology, which is now being used for personal identification in forensics. We designed a panel of 153 Korean-specific high-performance multiplexed SNPs and performed NGS using 233 DNA samples collected from eight different ethnic groups around the world. Eight Korean-specific genetic markers (rs28777, rs1010872, rs6043841, rs6034433, rs885479, rs2503107, rs4530059, and rs214955) that were screened showed significant variability among the ethnic groups. Three markers were novel SNPs that were absent from our multiplexed SNP panel, and two markers were associated with specific phenotypes. Our high-performance multiplexed SNP panel allows efficient screening of Korean-specific SNP alleles in populations that are genetically similar to the Korean population (e.g., Japanese and northeast Asians including Chinese and Eurasians), which will be useful for personal identification, paternity testing, and forensic investigations.     

Prospects for inferring pairwise relationships with single nucleotide polymorphisms

An extraordinarily large number of single nucleotide polymorphisms (SNPs) are now available in humans as well as in other model organisms. Technological advancements may soon make it feasible to assay hundreds of SNPs in virtually any organism of interest. One potential application of SNPs is the determination of pairwise genetic relationships in populations without known pedigrees. Although microsatellites are currently the marker of choice for this purpose, the number of independently segregating microsatellite markers that can be feasibly assayed is limited. Thus, it can be difficult to distinguish reliably some classes of relationship (e.g. full-sibs from half-sibs) with microsatellite data alone. We assess, via Monte Carlo computer simulation, the potential for using a large panel of independently segregating SNPs to infer genetic relationships, following the analytical approach of Blouin et al. (1996). We have explored a 'best case scenario' in which 100 independently segregating SNPs are available. For discrimination among single-generation relationships or for the identification of parent-offspring pairs, it appears that such a panel of moderately polymorphic SNPs (minor allele frequency of 0.20) will provide discrimination power equivalent to only 16-20 independently segregating microsatellites. Although newly available analytical methods that can account for tight genetic linkage between markers will, in theory, allow improved estimation of relationships using thousands of SNPs in highly dense genomic scans, in practice such studies will only be feasible in a handful of model organisms. Given the comparable amount of effort required for the development of both types of markers, it seems that microsatellites will remain the marker of choice for relationship estimation in nonmodel organisms, at least for the foreseeable future.     

Ethnic and Geographical Aspects of the Prevalence of the Polymorphic Variants of Genes Associated with Tuberculosis

Specificity of the structure of gene pools of different ethno-territorial groups of the human population can underlie the epidemiological features of the spread of tuberculosis (TB) and the structure of the genetic component of the susceptibility to the disease. The variability of 62 genetic variants potentially associated with the risk of the development of TB in the Russian population of the city of Tomsk has been studied and the differentiation of various ethno-territorial groups of the world by these markers has been assessed. The studied sample comprised 445 Russian residents of the city of Tomsk without bronchopulmonary pathology. For comparison, the data on the variability of the genetic markers of interest in 26 populations from the 1000 Genomes Project was involved. In the Tomsk population, only the ancestral allele was found for seven of the 58 SNPs studied; the allele frequencies for 36 markers were within the limits of the values seen in other European populations; for 12 SNPs, the observed frequencies were closer to populations with a significant Mongoloid component. By the total of the SNPs, the Tomsk population, despite the geographical distance from the rest of the European populations, did not differ from them (in genetic distances and G_st statistics), although it had some features of the gene pool. Intergroup differentiation of the world populations by these SNPs reflects mainly interracial differences. The greatest differences in the genetic structure between the studied populations were seen for the markers localized in intergenic regions. Statistically significant differences were found when comparing the levels of the average expected heterozygosity between groups of “L4 carrier populations” of mycobacteria and “non-L4” populations, which indicates the impact of the prevalence of different pathogenic lineages of M. tuberculosis on the formation of population specificity of the allelic frequencies.     

Detection of novel SNPs and mapping of the fatness QTL on pig chromosome 7q1.1-1.4 region

Many QTLs for fatness traits have been mapped on pig chromosome 7q1.1-1.4 in various pig resource populations. Eight novel markers, including seven SNPs and one insertion or deletion within BTNL1, COL21A1, PPARD, GLP1R, MDFI, GNMT, ABCC10, and PLA2G7 genes, as well as two previously reported SNPs in SLC39A7 and HMGA1 genes, were genotyped in Large White and Meishan pig breeds. Except for two SNPs in HMGA1 and ABCC10 genes, allele frequencies of the other eight markers are highly significant different between Chinese indigenous Meishan breeds and Large White pig breeds. Eight polymorphic sites were then used for linkage and QTL mapping to refine the fatness QTL in a Large White × Meishan F(2) resource population. Five chromosome-wise significant QTLs were detected, of which the QTLs for leaf fat weight, backfat thickness at 6-7th rib and rump, and mean backfat thickness were narrowed to the interval between PPARD and GLP1R genes and the QTL for backfat thickness at thorax-waist between GNMT and PLA2G7 genes on SSC7p1.1-q1.4.     

High-throughput SNP genotyping with the GoldenGate assay in maize

Single nucleotide polymorphisms (SNPs) are abundant and evenly distributed throughout the genomes of most plant species. They have become an ideal marker system for genetic research in many crops. Several high throughput platforms have been developed that allow rapid and simultaneous genotyping of up to a million SNP markers. In this study, a custom GoldenGate assay containing 1,536 SNPs was developed based on public SNP information for maize and used to genotype two recombinant inbred line (RIL) populations (Zong3 x 87-1, and B73 x By804) and a panel of 154 diverse inbred lines. Over 90% of the SNPs were successfully scored in the diversity panel and the two RIL populations, with a genotyping error rate of less than 2%. A total of 975 SNP markers detected polymorphism in at least one of the two mapping populations, with a polymorphic rate of 38.5% in Zong3 x 87-1 and 52.6% in B73 x By804. The polymorphic SNPs in B73 x By804 have been integrated with previously mapped simple sequence repeat markers to construct a high-density linkage map containing 662 markers with a total length of 1,673.7 cM and an average of 2.53 cM between two markers. The minor allelic frequency (MAF) was distributed evenly across 10 continued classes from 0.05 to 0.5, and about 16% of the SNP markers had a MAF below 10% in the diversity panel. Polymorphism rates for individual SNP markers in pair-wise comparisons of genotypes tested ranged from 0.3 to 63.8% with an average of 36.3%. Most SNPs used in this GoldenGate assay appear to be equally useful for diversity analysis, marker-trait association studies, and marker-aided breeding.