Ovine reference materials and assays for prion genetic testing

<p>Abstract</p> <p>Background</p> <p>Genetic predisposition to scrapie in sheep is associated with several variations in the peptide sequence of the prion protein gene (<it>PRNP</it>). DNA-based tests for scoring <it>PRNP </it>codons are essential tools for eradicating scrapie and for evaluating rare alleles for increased resistance to disease. In addition to those associated with scrapie, there are dozens more <it>PRNP </it>polymorphisms that may occur in various flocks. If not accounted for, these sites may cause base-pair mismatching with oligonucleotides used in DNA testing. Thus, the fidelity of scrapie genetic testing is enhanced by knowing the position and frequency of <it>PRNP </it>polymorphisms in targeted flocks.</p> <p>Results</p> <p>An adaptive DNA sequencing strategy was developed to determine the 771 bp <it>PRNP </it>coding sequence for any sheep and thereby produce a consensus sequence for targeted flocks. The strategy initially accounted for 43 known polymorphisms and facilitates the detection of unknown polymorphisms through an overlapping amplicon design. The strategy was applied to 953 sheep DNAs from multiple breeds in U.S. populations. The samples included two sets of reference sheep: one set for standardizing <it>PRNP </it>genetic testing and another set for discovering polymorphisms, estimating allele frequencies, and determining haplotype phase. DNA sequencing revealed 16 previously unreported polymorphisms, including a L237P variant on the F₁₄₁haplotype. Two mass spectrometry multiplex assays were developed to score five codons of interest in U.S. sheep: 112, 136, 141, 154, and 171. Reference tissues, DNA, trace files, and genotypes from this project are publicly available for use without restriction.</p> <p>Conclusion</p> <p>Identifying ovine <it>PRNP </it>polymorphisms in targeted flocks is critical for designing efficient scrapie genetic testing systems. Together with reference DNA panels, this information facilitates training, certification, and development of new tests and knowledge that may expedite the eradication of sheep scrapie.</p

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

Reduced Lentivirus Susceptibility in Sheep with TMEM154 Mutations

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10−9). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5–1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36–3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection

Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep

Stefan Hiendleder is a member of the International Sheep Genomics ConsortiumIn 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 (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization–time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.Michael P. Heaton, Theodore S. Kalbfleisch, Dustin T. Petrik, Barry Simpson, James W. Kijas, Michael L. Clawson, Carol G. Chitko-McKown, Gregory P. Harhay, Kreg A. Leymaster, the International Sheep Genomics Consortiu

Viral Infection in Sheep Linked to Gene

The discovery of a gene associated with a persistent viral infection that causes an incurable disease—ovine progressive pneumonia (OPP)—in sheep has led to the development of a genetic test that can be used to help reduce the impact of the disease. A slow-acting, wasting disease, OPP affects millions of sheep worldwide and is one of the most costly diseases to producers in North America. Previous research revealed that 36 percent of sheep operations and 24 percent of sheep tested in the United States were infected with ovine progressive pneumonia virus (OPPV)—lentivirus strains that target the immune system and cause OPP. Once infected, sheep are infected for life. Infected ewes are about 20 percent less productive, having fewer lambs and that also weigh less than lambs born to uninfected ewes. In addition to pneumonia, they show signs of wasting, lameness, and “hard bag” syndrome, in which udders become hard and contain barely any milk. Infected ewes are often culled—removed from the flock. Scientists at the Agricultural Research Service Roman L. Hruska U.S. Meat Animal Research Center (USMARC) in Clay Center, Nebraska, along with their colleagues at the Animal Disease Research Unit in Pullman, Washington, and the U.S. Sheep Experiment Station near Dubois, Idaho, have found and verified that the gene— TMEM154— affects susceptibility to OPPV infection in sheep

Genetic correlations for daily gain between ram and ewe lambs fed in feedlot conditions and ram lambs fed in Pinpointer units

When performance is recorded in automated facilities that measure feed intake of individual lambs that are penned in a group, such as Pinpointer units, a legitimate question is the degree to which daily gain is genetically correlated with daily gain achieved under feedlot conditions. Lambs were from a composite population (½Columbia, ¼ Suffolk, and ¼ Hampshire germplasm) and of the F2 or more advanced generations. Data were daily gains of 1,101 rams (PR) fed in Pinpointer units (11 to 17 wk of age) and 2,021 rams (FR) and 3,513 ewes (FE) fed under feedlot conditions (9- or 10-wk period starting at 9 wk of age). The FR and FE lambs were born from 1983 through 1995, whereas the PR lambs were born from 1986 through 1995. Measurements of daily gain in PR, FR, and FE lambs were considered to represent three correlated traits. Unadjusted means were .411, .406, and .326 kg/ d for PR, FR, and FE, respectively. Random effects in the model were animal direct genetic, maternal genetic, and maternal permanent environmental. Fixed effects were associated with age of dam (1 to 6 yr), type of rearing (1 to 4), and contemporary group (test date). Variances due to maternal genetic effects with single-trait analyses were near zero, so those effects were eliminated from the three-trait analysis although a random uncorrelated effect due to dam was included in the model. Estimates of heritability were .22, .14, and .23 for PR, FR, and FE, respectively, with fractions of variance due to dam effects ranging from .02 to .05. Estimates of genetic correlations were .86 for PR with FR, .83 for PR with FE, and 1.00 for FR with FE. Estimated phenotypic variances were similar for PR and FR, but one-third less for FE. The similarity of heritability estimates and estimates of genetic correlations all exceeding .83 suggest that daily gain of rams fed in Pinpointer units will reflect genetic expression for daily gain in both ram and ewe lambs fed under feedlot conditions

Evaluation of the Ovine \u3ci\u3eCallipyge\u3c/i\u3e Locus: II. Genotypic Effects on Growth, Slaughter, and Carcass Traits

A resource flock of 362 F2 lambs provided phenotypic and genotypic data to estimate effects of callipyge ( CLPG) genotypes on growth, slaughter, and carcass traits. Lambs were serially slaughtered in six groups at 3-wk intervals starting at 23 wk of age to allow comparisons at different end points. Probabilities of CLPG genotypes were calculated at a position 86 cM from the most centromeric marker of chromosome 18. A contrast of CLPG genotypic effects, based on the paternal polar over-dominance model, was used to evaluate callipyge and normal phenotypes. Relationships of traits with slaughter age, carcass weight, or 12th-rib fat depth for callipyge and normal phenotypic groups were estimated by regression. callipyge and normal lambs did not differ for growth traits measured from birth to slaughter. Callipyge lambs produced 55.9% of live weight as chilled carcass weight compared with 51.7% for normal lambs at the same mean live weight of 48.32 kg. Lighter pelt, kidney-pelvic fat, and liver weights contributed to this advantage of callipyge lambs for dressing percentage ( P \u3c .001). Estimated accretion rates of carcass protein at the mean slaughter age were 12.5 and 10.2 g/d for callipyge and normal carcasses, respectively. Corresponding values for carcass fat were 35.2 and 42.1 g/d. Compositional differences in favor of callipyge carcasses were detected at constant values of slaughter age, carcass weight, and 12th-rib fat depth. Callipyge carcasses had 2.56 kg greater fat-free lean and 1.39 kg less fat than normal carcasses at the same mean age of 214.9 d (P \u3c .001). The majority of these differences were established before the initial group was slaughtered and were maintained as age increased. Callipyge carcasses consisted of 24.3% fat and 71.3% fat-free lean, compared with 31.5 and 64.0% for normal carcasses at 25.6 kg of carcass weight. When evaluated at .49 cm of 12th-rib fat depth, callipyge lambs were 15.4 d older and produced 4.1 kg heavier carcasses with 4.3% less fat ( P \u3c .001). Effects of CLPG genotypic groups on carcass composition were greater than virtually all reported breed substitution effects. Use of the CLPG mutant allele in structured mating systems can dramatically increase production of lean lamb

A Simulation Model Including Ovulation Rate, Potential Embryonic Viability, and Uterine Capacity to Explain Litter Size in Mice: II. Responses to Alternative Criteria of Selection

Direct selection for litter size was compared with selection for ovulation rate, ova success, or uterine capacity and for indexes of ovulation rate with ova success or uterine capacity. Selection was simulated for 10 generations in a mouse population based on a model integrating ovulation rate, potential embryonic viability, and uterine capacity. Two indexes including ovulation rate (OR) and ova success (OS) were I = .291 x OR + 2.19 x OS and I = .165 x OR + .736 x OS. Heritabilities for ovulation rate and ova success, assumed in the simulation and to derive the indexes, were .25 and .06, respectively. Both indexes resulted in the same response in litter size, 12.9% greater than response to direct selection for litter size. Two indexes including OR and uterine capacity (TUC = true total uterine capacity; UC = uterine capacity measured as number born for a female with right ovary excised) were I = .881 x OR + .223 x TUC and I = .876 x OR + .568 x UC. Heritabilities assumed for uterine capacity were .09 (TUC) and .065 (UC). The first index assumed true parameters for uterine capacity (TUC) and resulted in a response in litter size that was 23.9% greater than direct selection. The second index was calculated using parameters estimated under a unilateralovariectomy model and resulted in response that was 14.7% greater than direct selection. Selection for OR, TUC, UC, or OS resulted in responses that were 4.5, 48.5, 38.7, or 74.8%, respectively, less than that from direct selection for litter size

A Simulation Model Including Ovulation Rate, Potential Embryonic Viability, and Uterine Capacity to Explain Litter Size in Mice: I. Model Development and Implementation

Litter size in mice was studied using a model including ovulation rate, potential embryonic viability, and uterine capacity. Simulated results were compared with experimental results from a selection experiment with mice. The four criteria of selection were selection on number born (LS), selection on an index of ovulation rate and ova success (IX), selection on number born to unilaterally ovariectomized females (UT), and unselected control (LC). Comparisons were made to statistics of the base generation and to responses after 13 generations of selection. Phenotypic and genetic statistics for uterine capacity were generated so that simulations produced the experimental means, standard deviations, and correlations between left and right litter size, as well as responses in number born using the LS, IX, and UT criteria. Statistics for the simulated data generally agreed with observed values. Simulated heritability in the base generation for uterine capacity was .065. Experimental and simulated responses per generation in litter size through 13 generations of selection were .15 and .16, .17 and .18, and .10 and .11 for LS, IX, and UT, respectively. Simulated responses in uterine capacity after 13 generations were 2.19, 1.60, and 3.40 for LS, IX, and UT, respectively. Simulated means for the base generation were 13.22 and 16.30 for ovulation rate and uterine capacity, respectively. Uterine capacity was an important component of the variability in litter size; however, ovulation rate was the more limiting component

Estimates of Genetic Parameters for Feed Intake, Feeding Behavior, and Daily Gain in Composite Ram Lambs

Our objective was to estimate genetic parameters for feed intake, feeding behavior, and ADG in composite ram lambs (¹⁄₂ Columbia, ¹⁄₄ Hampshire, ¹⁄₄ Suffolk). Data were collected from 1986 to 1997 on 1,239 ram lambs from approximately 11 to 17 wk of age at the U.S. Meat Animal Research Center near Clay Center, NE. Feeding equipment consisted of an elevated pen with an entrance chute that permitted access to the feeder by only one ram lamb at a time, with disappearance of feed measured by an electronic weighing system. Ram lambs were grouped 11 per pen from 1986 to 1989, and nine per pen from 1990 to 1997. Data were edited to exclude invalid feeding events, and approximately 80% of the data remained after edits were applied. Traits analyzed were daily feed intake (DFI), event feed intake (EFI), residual feed intake (RFI), daily feeding time (DFT), event feeding time (EFT), number of daily feeding events (DFE), and ADG. Feed intake traits of DFI and EFI had estimated heritabilities of 0.25 and 0.33, respectively, whereas estimated heritability of RFI was 0.11. Heritability estimates for feeding behavior traits, including DFT, EFT, and DFE, ranged from 0.29 to 0.36. Average daily gain had an estimated heritability of 0.26. Genetic correlations were positive between all pairs of traits, except for RFI and ADG, and that estimate was essentially zero. Phenotypic correlations were generally similar to genetic correlations. Genetic correlations were large (0.80) between DFI and ADG, intermediate between DFI and RFI (0.61) and between DFT and DFE (0.55), and low (0.17 to 0.31) for the other pairs of traits, with the exception of RFI and ADG (−0.03). Genetic correlations between behavioral traits were greater than correlations between behavioral traits and measures of feed intake or ADG; however, selection for ADG and/or feed intake would be expected to cause some changes in feeding behavior