The authors have the following interests: co-authors D.T.P. and B.S. are full-time employees at GeneSeek, a Neogen company that provides agrigenomic and veterinary diagnostic services. T.S.K. is the CEO of Intrepid Bioinformatics, a company that provides web-based systems to privately store, analyze, curate, share, and remotely access genetic data. International Sheep Genomics Consortium member K.G. is employed by Illumina Inc. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Performed the experiments: MPH TSK DTP BS. Conceived and designed the experiments: MPH TSK DTP BS MLC CGC-M GPH KAL. Analyzed the data: MPH TSK DTP BS KAL. Wrote the paper: MPH TSK DTP JWK MLC CGC-M GPH KAL. Contributed reagents/materials/analysis tools: MPH TSK DTP BS JWK KAL ISGC.
¶ Membership of the International Sheep Genomics Consortium is provided in the Acknowledgments and at
In sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal’s health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (
Visna/Maedi virus (VMV) and its closely related North American counterpart, ovine progressive pneumonia virus (OPPV), are small ruminant lentiviruses (SRLV) of the retroviridae family that infect sheep around the world (for review see
The impact of SRLV infection is considerable when the virus is introduced into a naïve flock with susceptible sheep. The mortality in such flocks may reach 30% per year after a few years
Stepwise strategies for SRLV disease control typically begin with the removal of infected sheep and focus on lowering the infection prevalence
The discovery of ovine transmembrane protein gene 154 (
While these findings show promise for improving U.S. sheep populations, it is important to identify which other populations around the world may be impacted by highly-susceptible
The availability of next-generation whole genome sequence data from the International Sheep Genomics Consortium (ISGC) provided the opportunity to directly determine
In a collection of 2,759 sheep DNAs from 74 breeds from around the world, the frequency of the “c” nucleotide allele of the c/t SNP OAR17_5388531 ranged from 0.0 to 1.0 with a mean of 0.51 and median of 0.50 among breeds (
The “c” allele of SNP OAR17_5388531 is in linkage disequilibrium with the “g” nucleotide allele in codon 35 (gaa) of
The fidelity of genetic testing is enhanced by knowing the position and frequency of polymorphisms in the populations to be tested. If not accounted for, nucleotide variation at neighboring sites may cause base-pair mismatching with oligonucleotides used in DNA testing and significantly decrease the genotyping accuracy in some populations. Moreover, characterizing nucleotide variation in many previously untested breeds allows discovery of
Panel A, genomic map of
Four of the previously unreported SNPs were coding mutations located in the predicted signal peptide (A13V) and extracellular domains (E31Q, I74F, and I102T) of
The A13V variant was observed as a heterozygote in a single Changthangi sheep, a local breed in the Changthang area of Leh district of Jammu and Kashmir state (CHA02,
Computer screen images of Integrated Genome Viewer software
The I102T variant was observed as a compound heterozygote in a single Santa Inês sheep, a breed of hair sheep found in Brazil (BSI4,
The most frequent of the new variants, I74F, was identified in wild sheep (
Breed or species group | Region of prominence | Animals diplotyped | Novel missense variants | |
African White Dorper | South Africa | 2 | 1, 2, 3 | – |
Afrikaner, Namaqua | South Africa | 1 | 1, 3 | – |
Afrikaner, Ronderib | South Africa | 2 | 2, 3 | – |
Afshari | NW Iran | 2 | 1, 2, 3, 4 | – |
Awassi | Middle east | 1 | 2, 3 | – |
Awassi, Turkish | Turkey | 2 | 3 | – |
Bangladeshi | Bangladesh | 2 | 2, 3 | – |
Brazilian Creole | Brazil | 2 | 1, 3 | – |
Castellana | Spain | 2 | 1 | – |
Changthangi | Jammu and Kashmir | 2 | 3, 9 | A13V |
Cheviot | England-Scotland | 2 | 1, 2 | – |
Churra | Spain | 2 | 1 | – |
Cine Capari | Turkey | 1 | 3, 9 | – |
Dorset, Poll | USA | 1 | 1 | – |
Ethiopian Menz | Ethiopia | 1 | 3 | – |
Finnsheep | Finland | 2 | 1, 2, 3 | – |
Garole, Banglegdeshi | Bangledesh | 1 | 2 | – |
Garole, Indian | India | 1 | 2, 3 | – |
Garut | Indonesia | 2 | 3 | – |
Gulf Coast native | USA Gulf Coast | 2 | 1, 2 | – |
Karakas | Turkey | 2 | 1, 2, 3 | – |
Karya | Turkey | 1 | 2, 4 | – |
Lacaune, Meat | France | 1 | 1 | – |
Lacaune, Milk | France | 1 | 1, 4 | – |
Merino | Spain | 3 | 1, 3 | – |
Morada Nova | Brazil | 2 | 1, 3 | – |
Norduz | Turkey | 2 | 1, 3, 4 | – |
Ojalada | Spain | 2 | 1, 4 | – |
|
Canada-USA | 3 | 3 | I74F |
|
Canada-USA | 2 | 3 | E31Q, I74F |
Romney | England | 1 | 1, 2 | – |
Sakiz | Turkey | 2 | 1, 2, 9 | – |
Salz | Spain | 3 | 1, 2, 3 | – |
Santa Inês | Brazil | 2 | 1, 2, 3 | I102T |
Scottish Blackface | United Kingdom | 1 | 1, 2 | – |
Sumatra | Sumatra | 2 | 1, 2 | – |
Swiss Mirror | Switzerland | 1 | 1 | – |
Swiss White Alpine | Switzerland | 4 | 1, 2, 3 | – |
Texel | Netherlands | 1 | 2, 3 | – |
Tibetan, Eastern | Tibet | 1 | 2, 9 | – |
Tibetan, Northern | Tibet | 1 | 3 | – |
Valais Blacknose | Switzerland | 1 | 1 | – |
Welsh Hardy Speckled Face | Wales | 1 | 1 | – |
Welsh Mountain, Dolgellau | Wales | 1 | 1 | – |
Welsh Mountain, Tregaron | Wales | 1 | 2 | – |
Not detected.
Haplotype code | Key feature of haplotype | Haplotypes observed |
Groups with haplotype |
1 | K35 | 56 | 26 |
2 | I70 | 34 | 22 |
3 |
E35 | 42 | 25 |
4 | A4(delta53) | 5 | 5 |
6 | Y82(delta82) | 0 | 0 |
9 | N33 | 4 | 4 |
10 | H14 | 0 | 0 |
11 | I25 | 0 | 0 |
12 | F74 | 6 | 2 |
13 | V13 | 1 | 1 |
14 | T102 | 1 | 1 |
15 | Q31 | 1 | 1 |
In 75 sheep.
Of 45 total groups.
Ancestral haplotype.
The development of genotyping assays for routine high-throughput testing required a set of reference DNAs. Homozygous DNAs were useful as PCR and genotyping controls because they provided uncomplicated results. A minimal set of DNAs was available from animals with the four most common homozygous diplotypes (1,1; 2,2; 3,3; and 4,4) and three rare diplotypes (1,6; 1,9; and 1,10). Together, these seven DNAs provided examples of the eight most common polymorphisms: R4A(delta53), L14H, T25I, D33N, E35K, T44M, N70I, and E82Y(delta82). DNA is not readily available from sheep with the four newly-discovered rare alleles (A13V, E31Q, I74F, and I102T) and thus representative DNA sequences were synthesized and used as controls (Materials and Methods). An empirically determined mixture of synthetic control DNA and reference DNA with
Based on the locations of missense mutations in
A three-phase iterative strategy was used to validate the assay development and check concordance of diplotypes derived from MALDI-TOF MS with those derived from Sanger sequencing. In each phase, the samples were blinded, scored, and decoded. Adjustments in assay conditions were made between phases of development. In the first phase, a U.S. panel of 96 rams was genotyped and showed 100% concordance between MALDI-TOF MS and Sanger genotyping. In the second phase, a U.S. panel of 95 tetrad families was used to detect genotyping errors as revealed by non-Mendelian inheritance patterns. One error was detected where the R4 allele was not evident in some animals known to be heterozygous for R4 and A4(delta53) alleles. This apparent allele “dropout” phenomenon was also occasionally observed with R4 alleles scored by Sanger sequencing (data not shown). Although the cause(s) of the R4 allele dropout was unknown, this region of
In the last phase of validation, results were obtained for 499 sheep in a single blinded genotyping trial to measure efficiency and accuracy of the genotype assay. Genotyping statistics were calculated for the eight most common polymorphic sites because the remaining four SNPs were monomorphic in these sheep. The call rate for the eight sites was 99.4% for the 499 sheep tested and 96.0% of the animals received a
Animal group | Yearsampled | Sheep | Missing SNPgenotypes | SNP callrate |
Missingdiplotypes | Diplotypecall rate | Discordant diplotypes | MALDI-TOFMS errors | Sanger errors |
4- to 9-year-old ewes |
2003 | 260 | 7 | 99.7 | 7 | 0.973 | 3 | 0 | 3 |
Composite lambs |
2011 | 239 | 16 | 99.2 | 13 | 0.946 | 4 | 0 | 4 |
Total | na |
499 | 23 | 99.4 | 20 | 0.960 | 7 | 0 | 7 |
Genoptypic data were collected in a single pass for R4A(delta53), H14L, T25I, D33N, E35K, T44M, N70I, and E82Y(delta82).
This animal group was composed of 160 OPP case-control pairs as previously described
Breed composition: 1/2 Romanov, 1/4 Rambouillet, 1/8 White Dorper, and 1/8 Katahdin.
Not applicable.
This report characterizes
The distribution of rare mutant haplotype alleles in populations may provide insight to their history. For example,
The diversity of protein isoforms encoded by the
The genetic information and assay designs described here were developed so they could be adapted to other platforms and technologies around the world to measure the effects of
Prior to their implementation, all animal procedures were reviewed and approved by the Care and Use Committee at the United States Department of Agriculture (USDA), Agricultural Research Service, Meat Animal Research Center (USMARC) in Clay Center, Nebraska.
The ISGC collected and genotyped 2,819 sheep from 74 breeds as part of a large study into genetic diversity and the impact of selection after domestication
The ISGC selected 75 animals for whole genome sequencing to extend its investigation of genetic diversity and selection in the world’s sheep breeds
The USMARC Sheep Diversity Panel, version 2.4 consists of 96 rams from nine breeds, a composite population, and one Navajo-Churro ram with a rare prion haplotype allele (ARK) as previously described
Sheep used for blinded MALDI-TOF MS genotyping trials included 260 USMARC OPP case-control sheep composed of 130 pairs of 4- to 9-year-old ewes
The Wyoming, USA bighorn sheep samples consisted of DNA from 10 wild animals taken from different wildlife management areas across Wyoming prior to 2001.
DNA sequence reads from sheep representing 39
Genotyping was performed at GeneSeek with the Sequenom MassARRAY platform and iPLEX GOLD chemistry according to the manufacturer’s instructions (Sequenom, San Diego, CA USA). Briefly, multiplex assays were designed with commercial software and adjusted manually (see
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We thank J. Carnahan for outstanding technical assistance, J. Watts for secretarial support, the USMARC sheep crew for production and management of sheep, and D.A Hawk of the Wyoming Game and Fish Department for providing bighorn sheep samples. This work was conducted in part using the resources of the University of Louisville’s research computing group and the Cardinal Research Cluster.
The members of the International Sheep Genomics Consortium who contributed samples and expertise towards the design and execution of ovine SNP50k genotyping and next generation sequencing, and/or coauthors of