SNPs for parentage testing and traceability in globally diverse breeds of sheep

Abstract

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.

Figure 1. Venn diagram of SNP sets in this study and genome distribution of 163 parentage SNPs. Venn diagram:

Set A, autosomal SNPs from the Ovine SNP50k Bead Array; Set B, SNPs with a MAF greater than or equal to 0.3 in at least 36 of the 74 ISGC breed groups; Set C, SNPs from four preexisting ovine parentage SNP panels (425 inside set B); Set D, SNPs with MAF greater than or equal to 0.3 in a U.S. sheep panel; Set E, 163 USDA parentage SNPs from the present report with 42 outside Set C; Sets F and G, 57 and 52 USDA parentage SNPs used in two respective multiplex assays developed for use in North American sheep (12 and 17 SNPs outside Set C, respectively). Graph: distribution of 163 parentage SNPs across 26 autosomal chromosomes. A SNP was classified as highly-informative in a breed if the MAF was greater or equal to 0.3.

Figure 2. Average MAFs for the set of 163 parentage SNPs by ISGC breed group.

Panel A: average MAFs for 163 parentage SNPs by breed group. MSDPv2.4 is the USMARC Sheep Diversity Panel version 2.4 (Materials and Methods). The number in parentheses for each breed group is the number of animals used. Panel B: breeds with five or more parentage SNPs having with fixed alleles (i.e., MAF = 0).

Figure 3. Physical maps of five representative amplicons with parentage SNPs.

High resolution map of five regions on ovine chromosome 1 that were targeted for in silico NGS analysis and PCR-amplification for Sanger sequencing and analysis. The parentage SNP is boxed in yellow. SNP positions are indicated by blue and red vertical bars and denote frequency of SNPs in an international panel of 70 sheep and a panel of 96 U.S. sheep, respectively and IUPAC/IUBMB ambiguity codes for nucleotides (r = a/g, y = c/t, m = a/c, k = g/t, s = c/g, w = a/t) . Other symbols: red triangles, indel polymorphisms; black rectangles, repetitive elements grey rectangles, intergenic regions; orange arrows, exons.

Figure 4. Parentage exclusion in 95 tetrad families with 109 parentage SNPs.

Panel A: Structure of the USMARC Sheep Diversity Family Panel version 2.46. Panel B: Distribution of the opposing homozygous SNPs genotypes in a pair-wise comparison of all possible combinations of parents and offspring (36,864 comparisons between an adults and an offspring). Panel C: Distribution of opposing homozygotes between the true parents and offspring (380 comparisons between lambs and sires/dams). Panel D: Distribution of the opposing homozygotes in a pair-wise SNP comparison of the 190 lambs and 95 each of the closest matching ram and ewe that were not parents of the lambs (380 comparisons between lambs and rams/ewes).