A High Throughput Single Nucleotide Polymorphism Multiplex Assay for Parentage Assignment in New Zealand Sheep Shannon M. Clarke*, Hannah M. Henry, Ken G. Dodds, Timothy W. D. Jowett, Tim R. Manley, Rayna M. Anderson, John C. McEwan Animal Genomics, AgResearch, Invermay Agricultural Centre, Mosgiel, New Zealand Abstract 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. Citation: Clarke SM, Henry HM, Dodds KG, Jowett TWD, Manley TR, et al. (2014) A High Throughput Single Nucleotide Polymorphism Multiplex Assay for Parentage Assignment in New Zealand Sheep. PLoS ONE 9(4): e93392. doi:10.1371/journal.pone.0093392 Editor: Claire Wade, University of Sydney, Australia Received November 12, 2013; Accepted March 4, 2014; Published April 16, 2014 Copyright: ß 2014 Clarke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this research was provided by Ovita Limited and Beef and Lamb New Zealand. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following interests: Funding for this research was provided by Ovita Limited and Beef and Lamb New Zealand. None of the authors are employees of Ovita nor do they have shares in or consultancy with Ovita. There are no patents or products in development relating to this Parentage SNP assay and all of the parentage SNPs are in the public domain and are freely available. AgResearch is not involved in marketing this Parentage SNP assay. This does not alter the author’s adherence to PLOS ONE polices on sharing data and materials. * E-mail: Shannon.Clarke@agresearch.co.nz Introduction To make genetic gain in animal breeding programs, pedigree information is required to estimate breeding values accurately. The use of incorrect pedigree information has the potential to reduce the rate of genetic gain [1,2]. Furthermore pedigree information is also required for inbreeding management, a crucial element for a successful breeding system resulting in genetic gain [3–6]. Traditionally pedigree information has been achieved by breeder records and more recently via DNA marker tests, for example, using microsatellites (MS), also known as either simple sequence repeats or short tandem repeats [7]. However, with the availability of a wealth of genomic information together with development of high throughput genotyping platforms, single nucleotide polymorphisms (SNPs) are now the DNA marker of choice in genomic selection studies. A SNP is a position in the genome that has at least two different bases at that location. These DNA markers are abundant throughout genomes; in sheep there is on average 4.9 SNPs in every 1 kb [8] and 5.1–5.8 SNPs per kb in domestic chickens [9]. These polymorphisms in dog and human are however found at a lower abundance at approximately 1 SNP per kb [10,11]. The New Zealand (NZ) sheep industry has had the potential to utilize a MS marker test for parentage analysis. Although SNP markers have less polymorphic information (biallelic) compared to MS markers which can have many alleles, this can easily be overcome and superseded by utilizing multiple SNP markers simultaneously. In addition SNPs are superior to MS markers in that, due to utilizing the biallelic SNPs, they are more robust with respect to use in the lab, subsequent interpretation of data and have a lower mutation rate. In addition, short amplicons (, 100 bp) can be achieved and high throughput genotyping technologies are applicable to SNP markers. Due to these qualities, we selected a set of SNP markers that were multiplexed with the aim of producing a reproducible, low cost, high throughput PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e93392