Shannon M. Clarke

SNPs for Parentage Testing and Traceability in Globally Diverse Breeds of Sheep

DNA-based parentage determination accelerates genetic improvement in sheep by increasing pedigree accuracy. Single nucleotide polymorphism (SNP) markers can be used for determining parentage and to provide unique molecular identifiers for tracing sheep products to their source. However, the utility of a particular “parentage SNP” varies by breed depending on its minor allele frequency (MAF) and its sequence context. Our aims were to identify parentage SNPs with exceptional qualities for use in globally diverse breeds and to develop a subset for use in North American sheep. Starting with genotypes from 2,915 sheep and 74 breed groups provided by the International Sheep Genomics Consortium (ISGC), we analyzed 47,693 autosomal SNPs by multiple criteria and selected 163 with desirable properties for parentage testing. On average, each of the 163 SNPs was highly informative (MAF≥0.3) in 48±5 breed groups. Nearby polymorphisms that could otherwise confound genetic testing were identified by whole genome and Sanger sequencing of 166 sheep from 54 breed groups. A genetic test with 109 of the 163 parentage SNPs was developed for matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. The scoring rates and accuracies for these 109 SNPs were greater than 99% in a panel of North American sheep. In a blinded set of 96 families (sire, dam, and non-identical twin lambs), each parent of every lamb was identified without using the other parent’s genotype. In 74 ISGC breed groups, the median estimates for probability of a coincidental match between two animals (PI), and the fraction of potential adults excluded from parentage (PE) were 1.1×10(−39) and 0.999987, respectively, for the 109 SNPs combined. The availability of a well-characterized set of 163 parentage SNPs facilitates the development of high-throughput genetic technologies for implementing accurate and economical parentage testing and traceability in many of the world’s sheep breeds.

A High Throughput Single Nucleotide Polymorphism Multiplex Assay for Parentage Assignment in New Zealand Sheep

Accurate pedigree information is critical to animal breeding systems to ensure the highest rate of genetic gain and management of inbreeding. The abundance of available genomic data, together with development of high throughput genotyping platforms, means that single nucleotide polymorphisms (SNPs) are now the DNA marker of choice for genomic selection studies. Furthermore the superior qualities of SNPs compared to microsatellite markers allows for standardization between laboratories; a property that is crucial for developing an international set of markers for traceability studies. The objective of this study was to develop a high throughput SNP assay for use in the New Zealand sheep industry that gives accurate pedigree assignment and will allow a reduction in breeder input over lambing. This required two phases of development- firstly, a method of extracting quality DNA from ear-punch tissue performed in a high throughput cost efficient manner and secondly a SNP assay that has the ability to assign paternity to progeny resulting from mob mating. A likelihood based approach to infer paternity was used where sires with the highest LOD score (log of the ratio of the likelihood given parentage to likelihood given non-parentage) are assigned. An 84 “parentage SNP panel” was developed that assigned, on average, 99% of progeny to a sire in a problem where there were 3,000 progeny from 120 mob mated sires that included numerous half sib sires. In only 6% of those cases was there another sire with at least a 0.02 probability of paternity. Furthermore dam information (either recorded, or by genotyping possible dams) was absent, highlighting the SNP test’s suitability for paternity testing. Utilization of this parentage SNP assay will allow implementation of progeny testing into large commercial farms where the improved accuracy of sire assignment and genetic evaluations will increase genetic gain in the sheep industry.

Construction of relatedness matrices using genotyping-by-sequencing data

BackgroundGenotyping-by-sequencing (GBS) is becoming an attractive alternative to array-based methods for genotyping individuals for a large number of single nucleotide polymorphisms (SNPs). Costs can be lowered by reducing the mean sequencing depth, but this results in genotype calls of lower quality. A common analysis strategy is to filter SNPs to just those with sufficient depth, thereby greatly reducing the number of SNPs available. We investigate methods for estimating relatedness using GBS data, including results of low depth, using theoretical calculation, simulation and application to a real data set.ResultsWe show that unbiased estimates of relatedness can be obtained by using only those SNPs with genotype calls in both individuals. The expected value of this estimator is independent of the SNP depth in each individual, under a model of genotype calling that includes the special case of the two alleles being read at random. In contrast, the estimator of self-relatedness does depend on the SNP depth, and we provide a modification to provide unbiased estimates of self-relatedness. We refer to these methods of estimation as kinship using GBS with depth adjustment (KGD). The estimators can be calculated using matrix methods, which allow efficient computation. Simulation results were consistent with the methods being unbiased, and suggest that the optimal sequencing depth is around 2–4 for relatedness between individuals and 5–10 for self-relatedness. Application to a real data set revealed that some SNP filtering may still be necessary, for the exclusion of SNPs which did not behave in a Mendelian fashion. A simple graphical method (a ‘fin plot’) is given to illustrate this issue and to guide filtering parameters.ConclusionWe provide a method which gives unbiased estimates of relatedness, based on SNPs assayed by GBS, which accounts for the depth (including zero depth) of the genotype calls. This allows GBS to be applied at read depths which can be chosen to optimise the information obtained. SNPs with excess heterozygosity, often due to (partial) polyploidy or other duplications can be filtered based on a simple graphical method.

Sheep genome functional annotation reveals proximal regulatory elements contributed to the evolution of modern breeds

Domestication fundamentally reshaped animal morphology, physiology and behaviour, offering the opportunity to investigate the molecular processes driving evolutionary change. Here we assess sheep domestication and artificial selection by comparing genome sequence from 43 modern breeds (Ovis aries) and their Asian mouflon ancestor (O. orientalis) to identify selection sweeps. Next, we provide a comparative functional annotation of the sheep genome, validated using experimental ChIP-Seq of sheep tissue. Using these annotations, we evaluate the impact of selection and domestication on regulatory sequences and find that sweeps are significantly enriched for protein coding genes, proximal regulatory elements of genes and genome features associated with active transcription. Finally, we find individual sites displaying strong allele frequency divergence are enriched for the same regulatory features. Our data demonstrate that remodelling of gene expression is likely to have been one of the evolutionary forces that drove phenotypic diversification of this common livestock species.The domestication of plants and animals causes genomic changes underlying various morphologic, physiologic and behavioral changes. Here, Naval-Sanchez et al. provide a ChIP-Seq validated comparative functional annotation of the sheep genome, and show widespread evolution of proximal regulatory elements.

Male–female relatedness at specific SNP-linkage groups influences cryptic female choice in Chinook salmon (Oncorhynchus tshawytscha)

In a range of taxa, the relatedness between mates influences both pre- and post-mating processes of sexual selection. However, relatively little is known about the genetic loci facilitating such a bias, with the exception of the major histocompatibility complex. Here, we performed tightly controlled replicated in vitro fertilization trials to explore the impact of relatedness on two possible mechanisms of cryptic female choice (CFC) in Chinook salmon (Oncorhynchus tshawytscha). We tested (i) whether relatedness of mates, assessed using 682 single nucleotide polymorphisms (SNPs) on 29 SNP-linkage groups (LGs), biases a males sperm velocity in ovarian fluid (a parameter previously shown to predict male fertilization success), and (ii) whether relatedness of mates governs fertilization success via other mechanisms, probably via sperm–egg interactions. We found that relatedness on three LGs explained the variation in sperm velocity, and relatedness on two LGs explained fertilization success, which might indicate the presence of genes important in sperm–ovarian fluid and sperm–egg interactions in these genomic regions. Mapping of the SNPs on these LGs to the rainbow trout genome revealed two genes that affect fertility in humans and represent candidate genes for further studies. Our results thereby provide a novel contribution to the understanding of the mechanism of CFC.

Estimation of linkage disequilibrium and effective population size in New Zealand sheep using three different methods to create genetic maps

BackgroundInvestments in genetic selection have played a major role in the New Zealand sheep industry competitiveness. Selection may erode genetic diversity, which is a crucial factor for the success of breeding programs. Better understanding of linkage disequilibrium (LD) and ancestral effective population size (Ne) through quantifying this diversity and comparison between populations allows for more informed decisions with regards to selective breeding taking population genetic diversity into account. The estimation of Ne can be determined via genetic markers and requires knowledge of genetic distances between these markers. Single nucleotide polymorphisms (SNP) data from a sample of 12,597 New Zealand crossbred and purebred sheep genotyped with the Illumina Ovine SNP50 BeadChip was used to perform a genome-wide scan of LD and Ne. Three methods to estimate genetic distances were investigated: 1) M1: a ratio fixed across the whole genome of one Megabase per centiMorgan; 2) M2: the ratios of genetic distance (using M3, below) over physical distance fixed for each chromosome; and, 3) M3: a genetic map of inter-SNP distances estimated using CRIMAP software (v2.503).ResultsThe estimates obtained with M2 and M3 showed much less variability between autosomes than those with M1, which tended to give lower Ne results and higher LD decay. The results suggest that Ne has decreased since the development of sheep breeds in Europe and this reduction in Ne has been accelerated in the last three decades. The Ne estimated for five generations in the past ranged from 71 to 237 for Texel and Romney breeds, respectively. A low level of genetic kinship and inbreeding was estimated in those breeds suggesting avoidance of mating close relatives.ConclusionsM3 was considered the most accurate method to create genetic maps for the estimation of LD and Ne. The findings of this study highlight the history of genetic selection in New Zealand crossbred and purebred sheep and these results will be very useful to understand genetic diversity of the population with respect to genetic selection. In addition, it will help geneticists to identify genomic regions which have been preferentially selected within a variety of breeds and populations.

Linkage Disequilibrium Estimation in Low Coverage High-Throughput Sequencing Data

High-throughput sequencing methods provide a cost-effective approach for genotyping and are commonly used in population genetics studies. A drawback of these methods, however, is that sequencing and genotyping errors can arise... High-throughput sequencing methods that multiplex a large number of individuals have provided a cost-effective approach for discovering genome-wide genetic variation in large populations. These sequencing methods are increasingly being utilized in population genetic studies across a diverse range of species. Two side-effects of these methods, however, are (1) sequencing errors and (2) heterozygous genotypes called as homozygous due to only one allele at a particular locus being sequenced, which occurs when the sequencing depth is insufficient. Both of these errors have a profound effect on the estimation of linkage disequilibrium (LD) and, if not taken into account, lead to inaccurate estimates. We developed a new likelihood method, GUS-LD, to estimate pairwise linkage disequilibrium using low coverage sequencing data that accounts for undercalled heterozygous genotypes and sequencing errors. Our findings show that accurate estimates were obtained using GUS-LD, whereas underestimation of LD results if no adjustment is made for the errors.

Genome-wide association study of lung lesions and pleurisy in New Zealand lambs1

Abstract Pneumonia is an important issue for sheep production, leading to reduced growth rate and a predisposition to pleurisy. The objective of this study was to identify loci associated with pneumonic lesions and pleurisy in New Zealand progeny test lambs. The lungs from 3,572 progeny-test lambs were scored for presence and severity of pneumonic lesions and pleurisy at slaughter. Animals were genotyped using the Illumina Ovine Infinium HD SNP BeadChip (606,006 markers). The heritability of lung lesion score and pleurisy were calculated using the genomic relationship matrix, and genome-wide association analyses were conducted using EMMAX and haplotype trend regression. At slaughter, 35% of lambs had pneumonic lesions, with 9% showing lesions on more than half of any individual lobe. The number of lambs recorded as having pleurisy by the processing plants was 9%. Heritability estimates for pneumonic lesions and pleurisy scores adjusted for heteroscedasticity (CPSa and PLEURa) were 0.16 (± 0.03) and 0.05 (± 0.02), respectively. Five single-nucleotide polymorphisms (SNPs) were significantly associated with pneumonic lesions at the genome-wide level, and additional 37 SNPs were suggestively significant. Four SNPs were significantly associated with pleurisy, with an additional 11 SNPs reaching the suggestive level of significance. There were no regions that overlapped between the 2 traits. Multiple SNPs were in regions that contained genes involved in either the DNA damage response or the innate immune response, including several that had previously been reported to have associations with respiratory disease. Both EMMAX and HTR analyses of pleurisy data showed a significant peak on chromosome 2, located downstream from the transcription factor SP3. SP3 activates or suppresses the expression of numerous genes, including several genes with known functions in the immune system. This study identified several SNPs associated with genes involved in both the innate immune response and the response to DNA damage that are associated with pneumonic lesions and pleurisy in lambs at slaughter. Additionally, the identification in sheep of several SNPs within genes that have previously been associated with the respiratory system in cattle, pigs, rats, and mice indicates that there may be common pathways that underlie the response to invasion by respiratory pathogens in multiple species.

Reduced representation sequencing detects only subtle regional structure in a heavily exploited and rapidly recolonizing marine mammal species

Abstract Next‐generation reduced representation sequencing (RRS) approaches show great potential for resolving the structure of wild populations. However, the population structure of species that have shown rapid demographic recovery following severe population bottlenecks may still prove difficult to resolve due to high gene flow between subpopulations. Here, we tested the effectiveness of the RRS method Genotyping‐By‐Sequencing (GBS) for describing the population structure of the New Zealand fur seal (NZFS, Arctocephalus forsteri), a species that was heavily exploited by the 19th century commercial sealing industry and has since rapidly recolonized most of its former range from a few isolated colonies. Using 26,026 neutral single nucleotide polymorphisms (SNPs), we assessed genetic variation within and between NZFS colonies. We identified low levels of population differentiation across the species range (<1% of variation explained by regional differences) suggesting a state of near panmixia. Nonetheless, we observed subtle population substructure between West Coast and Southern East Coast colonies and a weak, but significant (p = 0.01), isolation‐by‐distance pattern among the eight colonies studied. Furthermore, our demographic reconstructions supported severe bottlenecks with potential 10‐fold and 250‐fold declines in response to Polynesian and European hunting, respectively. Finally, we were able to assign individuals treated as unknowns to their regions of origin with high confidence (96%) using our SNP data. Our results indicate that while it may be difficult to detect population structure in species that have experienced rapid recovery, next‐generation markers and methods are powerful tools for resolving fine‐scale structure and informing conservation and management efforts.