Assessment of genetic diversity and population structure of U.S. Polypay sheep from breed origins to future genomic selection.

Knowledge of past and present genetic diversity within a breed is critical for the design and optimization of breeding programs as well as the development of strategies for the conservation of genetic resources. The Polypay sheep breed was developed at the U.S. Sheep Experiment Station (USSES) in 1968 with the goal of improving productivity in Western U.S. range flocks. It has since flourished in the more intensively managed production systems throughout the U.S. The genetic diversity of the breed has yet to be documented. Therefore, the primary objective of this study was to perform a comprehensive evaluation of the genetic diversity and population structure of U.S. Polypay sheep using both pedigree- and genomic-based methods. Pedigree data from 193 Polypay flocks participating in the National Sheep Improvement Program (NSIP) were combined with pedigree records from USSES (n = 162,997), tracing back to the breed's origin. A subset of these pedigreed sheep from 32 flocks born from 2011 to 2023 were genotyped with the GGP Ovine 50K BeadChip containing 51,867 single nucleotide polymorphisms (SNPs). Four subgroups were used for the pedigree-based analyses: 1) the current generation of animals born in 2020-2022 (n = 20,701), 2) the current generation with a minimum of four generations of known ancestors (n = 12,685), 3) only genotyped animals (n = 1,856), and 4) the sires of the current generation (n = 509). Pedigree-based inbreeding for the full population was 2.2%, with a rate of inbreeding of 0.22% per generation. Pedigree-based inbreeding, Wright's inbreeding, and genomic inbreeding based on runs of homozygosity were 2.9%, 1.3%, and 5.1%, respectively, for the genotyped population. The effective population size ranged from 41 to 249 for the pedigree-based methods and 118 for the genomic-based estimate. Expected and observed heterozygosity levels were 0.409 and 0.403, respectively. Population substructure was evident based on the fixation index (FST), principal component analysis, and model-based population structure. These analyses provided evidence of differentiation from the foundation flock (USSES). Overall, the Polypay breed exhibited substantial genetic diversity and the presence of a population substructure that provides a basis for the implementation of genomic selection in the breed.

Comparison of three maternal composite sheep breeds managed under pasture lambing and purebred or terminal mating systems: ewe body weight, reproductive efficiency, and longevity.

Extensive sheep systems have lower inputs (e.g., feed, labor, infrastructure) but, generally, lower outputs per ewe than intensively managed flocks. Average ewe prolificacy is low (< 1.1 lambs per ewe) across the United States and traditional expectation is that costs required to support larger litters will not be recovered due to greater lamb mortality. The Composite-IV (C-IV) is a 50% Romanov, 25% Katahdin, and 25% White Dorper hair sheep developed and selected for enhanced reproductive efficiency at the U.S. Meat Animal Research Center. Objectives of the current study were to compare productivity of C-IV ewes (N = 404) to Katahdin (N = 342) and Polypay (N = 390), two popular maternal composite breeds, in a lower-input system. Experimental ewes were exposed to either rams of their own breed or Texel rams for the first time at 7 mo of age and remained in the same mating system for up to 4 yr. Ewes lambed on pasture from May to June and were expected to rear their lambs in the absence of supplemental feed and with minimal intervention. The ewe age × breed interaction effect impacted all annually recorded traits (P < 0.01) including number and total weight of lamb marketed at 25 wk per ewe exposed to mating (LS25 and LW25, respectively). Within all ages, LS25 of C-IV was greater (1.2 to 1.63 lambs; P < 0.01) than Katahdin (0.71 to 1.17 lambs) and Polypay (0.68 to 1.26 lambs) which were similar. At 1, 2, and 3-yr of age, C-IV ewes had, on average, 12 to 17 kg greater LW25 (P ≤ 0.01) than other breeds. Polypay LW25 was similar to Katahdin at 1 yr of age, but 8 to 13 kg greater (P < 0.01) at older ages. At 4 yr of age, LW25 was similar for C-IV and Polypay (63 and 62 kg, respectively). Overall, Texel mating improved LW25 by 3 kg compared to purebred mating (P < 0.01). Twin litters were most common within mature ewes (> 1 yr) of all breeds (52% to 65%); however, the proportion of triplet litters was numerically greater for C-IV (13%) than Katahdin (1%) or Polypay (4%). The frequency of mature ewes that gave birth to and reared twins (75% to 82%) was high for all breeds. Most triplet-bearing mature C-IV ewes reared twins (43%) though many reared triplets (39%). Ewe reproductive efficiency can be enhanced in lower-input systems by utilizing additive breed effects and heterosis in a complimentary crossbreeding program. More research is needed to identify indicator traits associated with lamb survival and to derive appropriate levels of prolificacy to fit specific management constraints.

Production systems across the U.S. sheep industry are diverse, ranging from intensive management to lower-input and extensive operations. Extensive flocks are more nutritionally reliant on grazing, have lower labor costs, and have limited housing for animals. Most extensively managed sheep operations in the United States are in the western states, where approximately 60% of lambs are born outside and with little intervention. Predation, recurrent drought, and limited nutritional value of forages are common in this region and are antagonistic to rearing multiple lambs. However, large breed differences in ewe maternal ability exist and can be utilized to improve lamb production through strategic crossbreeding. The Composite-IV is a sheep population developed at the U.S. Meat Animal Research Center and has been selected for enhanced reproductive efficiency in a lower-input system. Composite-IV ewes were evaluated alongside Katahdin and Polypay ewes, two other popular maternal sheep breeds, over 4 yr in a lower-input, pasture lambing system. Total number and weight of lamb marketed per ewe was greatest for Composite-IV, but all three breeds had much greater reproductive performance than the current U.S. average. Results highlight the importance of applying existing and novel genetic tools to optimize sheep performance for specific production systems.

Impact of Four Ovine TMEM154 Haplotypes on Ewes during Multiyear Lentivirus Exposure.

Polypeptide variation encoded by the ovine transmembrane protein 154 gene (TMEM154) is associated with susceptibility to ovine lentivirus, the causative agent of Ovine Progressive Pneumonia (OPP) and Visna/Maedi. Our aim was to compare the four most prevalent TMEM154 haplotypes on the incidence of infection and ewe productivity during natural multiyear virus exposure. Prospective cohort studies were designed to test gene action and estimate effects of TMEM154 haplotypes encoding distinctive variant residues: K35 ("1"), I70 ("2"), ancestral ("3"), and A4del/M44 ("4"). Exposure consisted of co-mingling infected ewes at a rate greater than 30% with serological status evaluated every four months. For ewes with one or two copies of the highly susceptible haplotypes "2" and "3", the infection prevalence steadily increased to nearly 100% at 55 months. Haplotypes "2" and "3" were equally susceptible and dominant to haplotype "1". A difference was not detected (p < 0.53) in the magnitude of effect with haplotype combinations of "1" and "4". The ewe infection prevalence with "1,1"; "1,4"; and "4,4" was 10% to 40% at 55 months. The latter suggested that two copies of the K35 amino acid substitution ("1") were as effective as a homozygous TMEM154 "knockout" with the frame-shift deletion mutation ("4") in reducing infection susceptibility. When considering ewe reproductive performance, a difference was not detected when comparing haplotypes "2", and "3" to each other, or "1" and "4" to each other. Our study indicated that ewes with two copies of the severely truncated versions of TMEM154 ("4,4") had normal lamb productivity. Without complete understanding of the natural function of TMEM154 our recommendations to producers interested in using TMEM154 selection to reduce their flock's genetic predisposition to OPP are encouraged to increase the frequency of TMEM154 haplotype K35 ("1") since it encodes a full-length protein with minimal difference to the ancestral polypeptide.

Effect of TMEM154 E35K variant (haplotypes 1 and 3) on the incidence of ovine lentivirus infection and ewe productivity during lifetime exposure.

Ovine progressive pneumonia virus (OPPV) is a small ruminant lentivirus that is widespread throughout U.S. sheep flocks. Infections with OPPV are lifelong and effects are multi-systemic with significant implications for animal well-being and productivity. A protein isoform with lysine at position 35 (K35, haplotype "1") encoded by the ovine transmembrane protein 154 (TMEM154) gene has been associated with reduced susceptibility to infection when two copies are present (i.e., diplotype "1,1"). Conversely, the ancestral protein isoform with glutamate at position 35 (E35, haplotype "3") is associated with high susceptibility to infection when at least one copy is present. The beneficial effect of TMEM154 K35 alleles on ewe productivity has not been previously measured in controlled challenge experiments and was a major objective of this study. Ewes with TMEM154 diplotypes "1,1"; "1,3"; and "3,3" (n = 31, 47, and 30, respectively) were born and reared by OPPV-infected dams and managed under continual natural exposure to OPPV. Ewes were tested for serological status at 4-mo intervals for up to 5.5 yr. The incidence of infection in ewes with diplotype "1,1" was 6.5% to 9.7% and significantly lower (P < 0.001) than ewes with diplotype "1,3" (60.5 to 97.3%) or "3,3" (64.0 to 91.4%). Furthermore, the incidence among ewes with diplotype "1,1" did not increase from 10 to 67 mo of age (P > 0.99), whereas the incidence among diplotype "1,3" and "3,3" ewes increased steadily until reaching an asymptote at approximately 52 mo of age. Total number and weight of lamb weaned per ewe exposed through 5.5 yr from ewes with diplotype "1,1" far exceeded (P ≤ 0.05) those with diplotypes "1,3" and "3,3" by, on average, 2.1 lambs and 40 kg, respectively. The present study confirmed that TMEM154 diplotype "1,1" animals have reduced incidence of OPPV infection and, correspondingly, improved productivity. In flocks with a high frequency of TMEM154 haplotype "3," selection for haplotype "1" appears to be a cost-effective approach to mitigate the impact of this economically important disease.

Comparison of performance of F1 Romanov crossbred ewes with wool and hair breeds during fall lambing and body weight and longevity through six production years.

The objective of this study was to evaluate wool (Dorset and Rambouillet) and hair (Dorper, Katahdin, and White Dorper) breeds for their ability to complement Romanov germplasm in an annual fall lambing system by estimating direct maternal grandsire and sire breed effects on economically important lamb and ewe traits. After 3 yr of evaluation under spring lambing, ewes of the five F1 types were transitioned to spring mating, exposed to composite terminal sires, and evaluated under a barn lambing system at 4, 5, and 6 yr of age. A total of 527 first generation crossbred (F1) ewes produced 1,151 litters and 2,248 lambs from 1,378 May exposures. After accounting for differences in dam age, birth type, and sex, lamb survival to weaning was unaffected by maternal grandsire breed (P = 0.30). However, lambs born to 50% Dorset (16.8 ± 0.21 kg) or 50% White Dorper ewes (16.8 ± 0.28 kg) were heavier at weaning than those born to 50% Katahdin dams (13.8 ± 0.32 kg; P < 0.001). Additionally, lambs born to 50% Dorset ewes were heavier than those born to 50% Rambouillet (16.0 ± 0.22 kg) and 50% Dorper ewes (15.7 ± 0.33; P ≤ 0.03), but no other pairwise maternal grandsire breed differences were observed (P ≥ 0.06). Ewe body weight (n = 3,629) was recorded prior to each of six possible mating seasons and, across ages, was greatest for Dorset- and Rambouillet-sired ewes (56.7 ± 0.44 and 56.5 ± 0.45 kg, respectively), intermediate for Dorper- and White Dorper-sired ewes (54.7 ± 0.78 and 54.1 ± 0.64 kg, respectively), and least for Katahdin-sired ewes (51.5 ± 0.45 kg). Fertility after spring mating (0.80 ± 0.03 to 0.87 ± 0.02), litter size at birth (1.46 ± 0.09 to 1.71 ± 0.07), and litter size at weaning (1.25 ± 0.06 to 1.46 ± 0.06) were not impacted by sire breed (P ≥ 0.16). Ewe longevity, assessed as the probability of being present after 6 production years, was also not affected by sire breed (0.39 ± 0.03 to 0.47 ± 0.03; P = 0.44). Rambouillet-sired ewes weaned more total weight of lamb (21.5 ± 0.94 kg) than Katahdin-sired ewes (17.8 ± 0.94 kg; P = 0.05), but no other sire breed differences were detected (P ≥ 0.07). Results demonstrated that incorporating the Romanov into a crossbreeding system is a practical means of improving out-of-season ewe productivity.

Comparison of performance of F1 Romanov crossbred ewes with wool and hair breeds during spring lambing under intensive and extensive production systems.

The objective was to evaluate wool (Dorset and Rambouillet) and hair (Dorper, Katahdin, and White Dorper) breeds for their ability to complement Romanov germplasm in two distinct production systems by estimating direct sire and grandsire effects on lamb growth, survival, and ewe productivity traits. Rams of the five breeds (n = 75) were mated to Romanov ewes (n = 459) over a 3-yr period to produce five types of crossbred lambs (n = 2,739). Sire breed (P > 0.06) did not impact body weight or survival traits of the first-generation crossbred (F1) lambs. The productivity of retained crossbred ewes (n = 830) mated to Suffolk and Texel terminal sires was evaluated at 1, 2, and 3 yr of age in each production system. In the intensive production system, labor and harvested feed were provided for sheep that lambed in March in barns, and ewes were limited to rearing two lambs with additional lambs reared artificially. Ewes in the extensive production system lambed in May on pasture and were responsible for rearing all lambs born with no labor or supplemental feed provided before weaning. A total of 1,962 litters and 4,171 lambs from 2,229 exposures to two terminal sire breeds (Suffolk and Texel) were produced in the experiment. Crossbred ewes in the intensive production system were mated in October, resulting in larger litter sizes than crossbred ewes mated in December for the extensive production system. However, single- and twin-born lamb mortality was similar between the two systems that differed greatly in labor, feed, and facilities. Lambs produced in the intensive system received concentrate feed from an early age and were heavier at 24 wk of age than lambs produced in the extensive system. These outcomes resulted in greater 24-wk litter weight in the intensive than in the extensive system (P < 0.0001). Unexpectedly, the relative performance of crossbred types did not differ importantly between production systems. White Dorper × Romanov crossbred ewes had numerical advantages in productivity in each system; however, differences between ewe types were not significant. In the extensive system, without labor and shelter at lambing or supplemental feed until weaning, 3-yr-old crossbred ewes of all types averaged 1.78 lambs marketed per ewe lambing, and 40% of the ewes that gave birth to triplets weaned their entire litters. These results document that prolific sheep and extensive systems can be successfully combined if appropriate crossbred types are used.

Genetic and nongenetic factors influencing ewe prolificacy and lamb body weight in a closed Romanov flock.

The U.S. Meat Animal Research Center was the first entity in the United States to import the Romanov breed and it has been maintained as a closed flock for over 30 yr. Incorporating this super-prolific breed into crossbred and composite populations has resulted in large improvements in ewe productivity. However, few have quantified factors contributing to genetic and nongenetic variation in ewe reproduction and lamb growth within purebred Romanov populations, which were the objectives of this study. The pedigree contained a total of 8,683 lambs born to 218 and 1,600 unique sires and dams, respectively. Number of lambs born on a per ewe exposed (NLBE) and lambing (NLBL) basis were analyzed in univariate repeatability animal models. As expected, the proportion of phenotypic variance (σP2) in litter size attributable to additive genetic (0.06 to 0.08) and permanent environmental (0.05 to 0.07) effects of the ewe was low. The service sire permanent environmental effect contributed to a small but significant amount of σP2 in NLBE (0.03) but not NLBL. However, the service sire additive genetic effect did not influence σP2 in NLBE or NLBL. Lamb body weight was recorded at birth (BWB) and upon weaning from either milk replacer (~30 d; BWW-N) or their dam (~60 d; BWW-D) and were analyzed in a three-trait model with random additive direct and maternal effects. Estimated direct heritabilities were low for all body weight (BW) traits (0.07 to 0.10). Maternal heritability was moderate for BWB (0.34) but low for weaning BW (0.11 to 0.18). This was the first to report direct and maternal genetic correlations between BW of nursery- and dam-reared lambs, and both were estimated to be moderate (0.43 to 0.47). Additionally, the direct and maternal effects of BWB were more strongly correlated with BWW-N (0.74 to 0.82) than BWW-D (0.17 to 0.33). Despite inbreeding coefficients having increased at a rate of 0.33% per birth year (1986 to 2019) in this flock, they were not consistently associated with reductions in ewe or lamb performance. Parameter estimates generally agreed with those from less-prolific breeds, and results indicate that selection can be an effective means of improving subcomponents of ewe productivity.

Rambouillet and Romanov reciprocal breed effects on survival and growth traits of F1 lambs and on reproductive traits of F1 ewes.

Objectives were to estimate reciprocal effects of Romanov and Rambouillet breeds on survival, growth, and reproductive traits of F1 progeny and direct breed effects (Suffolk and Composite - ½ Columbia, » Hampshire, and » Suffolk) on survival and growth traits of the subsequent terminally sired lambs. Mature Rambouillet ewes (n = 243) were exposed to 20 Romanov rams over two seasons producing 621 lambs for evaluation of growth and survival traits with 274 F1 ewes being evaluated for reproduction traits through 4 yr of age. Similarly, mature Romanov ewes (n = 116) were exposed to 20 Rambouillet rams producing 601 lambs for evaluation of growth and survival traits with 176 F1 ewes being evaluated for reproduction traits through 4 yr of age. A total of 433 of those F1 ewes produced 3,431 lambs (1,552 litters) from 1,634 exposures to terminal sires over 4 yr. Terminal sires consisted of 38 Suffolk and 44 Composite rams. Reciprocal crossbred ewe lambs were produced from dramatically different uterine and neonatal environments, with litter size at birth from Romanov dams exceeding those from Rambouillet dams by 1.52 lambs (P < 0.001) and birth weight of lambs from Romanov dams averaged 3.41 kg compared with 4.26 kg from Rambouillet dams. Differences in BW were still evident at 140 d (P < 0.001) for dam-reared lambs. However, reciprocal ewe first breeding BW of both types were similar (P = 0.38). Minimal differences were observed in performance of reciprocal cross ewes through 4 yr for productivity, longevity, or progeny growth and survival. One exception was BW at 140 d where an interaction of dam breed with terminal sire breed reached significance for both dam-reared (P = 0.05) and nursery-reared (P = 0.02) lambs. This interaction was due to the lower weight of Composite-sired lambs out of reciprocal cross ewes born from Rambouillet dams. Composite rams increased number born (P < 0.01) and number weaned (P < 0.05) of the reciprocal cross ewes. Suffolk rams increased (P < 0.001) BW and growth rates from birth to 140 d of terminal progeny. Thus, there were little cumulative differences accrued over the 4 yr and no differences were detected for cumulative kilogram of lamb generated at 140 d per ewe exposed. The practical outcome of this evaluation was that performance levels of both types of Romanov crossbred ewes was similar allowing the industry to produce the desired crossbred ewes without needing large purebred ewe flocks of the less numerous Romanov breed.

Effects and interactions of myostatin and callipyge mutations: I. Growth and carcass traits.

Objectives were to document effects of the Texel myostatin mutation (MSTN) on growth and carcass traits and also test whether or not interactions with the callipyge mutation (CLPG) could be detected. Twelve rams heterozygous at both loci on the two different chromosomes were mated to 215 terminal-sire type composite crossbred ewes genotyped as non-carriers for both loci. A total of 365 lambs were born, 362 of those were genotyped and 236 lambs contributed carcass data to estimate effects and interactions among the four genotype combinations produced. The four genotype combinations were defined as follows: ++/++ for wild-type at both loci; ++/C+ for wild-type at MSTN and heterozygous at CLPG; M+/++ for heterozygous at MSTN and wild-type at CLPG; and M+/C+ for heterozygous at both loci. The two independently segregating sire-derived alleles represent different breed-of-origin contrasts at each locus (Texel vs. composite origin for MSTN and Dorset vs. Texel origin for CLPG). Birth weight was recorded on all lambs, and subsequent body weights were adjusted to 56 (weaning), 70, and 140 d of age. Within sire-sex-genotype subgroups, naturally reared lambs were assigned to one of eight slaughter groups accounting for variation in birth date. Lambs were serially slaughtered at weekly intervals, 30 lambs per group, from roughly 26 to 33 wk of age. In addition to standard carcass traits, subjective leg scores were assigned and widths of carcasses were measured at the widest points of the shoulder and rump. Differences in birth weight were detected (P < 0.01) for the combination of the two loci and birth type, with single-born differences among genotypes exceeding differences among twin born progeny. Those interaction differences among genotypes were not as important at weaning (P = 0.36). Impact on growth rate differences among the genotypes during the post-weaning period were variable and dependent on sex of the lamb (P < 0.01). A synergistic interaction between MSTN and CLPG was observed for leg muscling scores (P < 0.05) but no other measures of carcass shape were affected. One copy of MSTN had a more modest impact on fat deposition and muscle conformation than did CLPG and did not interact (all values P > 0.20). Although some non-additive interactions that vary by trait and sex were detected, in general the data are consistent with the two mutations acting on muscle growth through independent pathways.

Association of porcine heparanase and hyaluronidase 1 and 2 with reproductive and production traits in a landrace-duroc-yorkshire population.

The ovary and placenta are dynamic structures requiring constant modification both structurally and through cell-cell communication capabilities. The extracellular matrix and basement membranes are primarily composed of a milieu of glycosaminoglycans, including heparan sulfate and hyaluronan. Heparanase (HPSE) and hyaluronidases (HYAL) are responsible for degrading heparan sulfate and hyaluronan, respectively. Therefore, the objective of this study was to evaluate the relationship of SNPs distinct to HPSE, HYAL1, and HYAL2 with measurements of reproduction and production traits in swine. Single trait associations were performed on a Landrace-Duroc-Yorkshire population using SNPs discovered and identified in HPSE, HYAL1, and HYAL2. Analyses were conducted on an extended pedigree and SNPs were found to be associated with reproductive and production traits. Prior to multiple-testing corrections, SNPs within HPSE were weakly associated (P < 0.03) having additive effects with age at puberty (-2.5 ± 1.08 days), ovulation rate (0.5 ± 0.24 corpora lutea), and number of piglets born alive (0.9 ± 0.44 piglets). A HYAL1 and two HYAL2 SNP were nominally associated (P ≤ 0.0063) with number of piglets born alive after multiple-testing corrections (effects between 1.02 and 1.44 piglets), while one of the same HYAL2 markers maintained a modest association (P = 0.0043) having a dominant effect with number of piglets weaned (1.2 ± 0.41 piglets) after multiple-testing correction. Functionally, HPSE and HYAL1 and 2 have been shown to participate in events related to ovarian and placental activity. SNPs from these studies could potentially assist with understanding genetic components underlying sow lifetime productivity as measured by piglet survivability based on number born alive and number weaned, thereby contributing to a greater number of pigs/sow/year.

Ovine reference materials and assays for prion genetic testing.

BACKGROUND: Genetic predisposition to scrapie in sheep is associated with several variations in the peptide sequence of the prion protein gene (PRNP). DNA-based tests for scoring PRNP 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 PRNP 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 PRNP polymorphisms in targeted flocks. RESULTS: An adaptive DNA sequencing strategy was developed to determine the 771 bp PRNP 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 PRNP 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 F141 haplotype. 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. CONCLUSION: Identifying ovine PRNP 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.

Effect of fetal size on fetal placental hyaluronan and hyaluronoglucosaminidases throughout gestation in the pig.

The trophoblast-endometrial epithelial cell bilayer of porcine placenta undergoes microscopic folding during gestation, and the folded bilayer is embedded in fetal placental stroma. We hypothesized that hyaluronan was a component of fetal placental stroma, and that hyaluronoglucosaminidases played a role in bilayer folding. Gilts were unilaterally hysterectomized-ovariectomized (UHO) at 160 days of age, mated at estrus and killed on days 25, 45, 65, 85 or 105 of gestation. Fetal placental tissues were collected to evaluate hyaluronan and hyaluronoglucosaminidase content. Fetal placental hyaluronan concentration increased (P<0.01) between day 25 and 45 of gestation, remained high throughout gestation, and was greater (P<0.05) in the fetal placenta of the smallest compared to the largest fetuses on day 105 of gestation. Hyaluronan was localized to fetal placental stroma. Three cDNAs for hyaluronoglucosaminidase 1 (two 1379 and one 1552bp) and one cDNA (1421bp) for hyaluronoglucosaminidase 2 were cloned from day-85 fetal placental RNA. Gene expression analysis indicated that the 1379bp form of hyaluronoglucosaminidase 1 mRNA did not differ, the 1552bp form increased, and the 1421bp form of hyaluronoglucosaminidase 2 decreased during pregnancy. Amount of all three mRNAs was greater (P<0.05) in fetal placenta of the smallest compared to the largest fetuses. Zymography indicated 70 and 55kd protein isoforms of hyaluronoglucosaminidase in fetal placental tissue. Both forms increased with advancing gestation and were greater in fetal placenta of the smallest compared to the largest fetuses (P<0.05). These results are consistent with a role for hyaluronan and hyaluronoglucosaminidases in the development of the microscopic folds of the pig placenta during gestation.

Molecular cloning and characterisation of heparanase mRNA in the porcine placenta throughout gestation.

Heparanase (HPSE) is an endoglycosidase that specifically degrades heparan sulfate, which is an abundant glycosaminoglycan of the pig placenta. The aim of the present study was to clone cDNA encoding porcine HPSE and characterise the expression level and localisation of HPSE mRNA in porcine placentas throughout gestation. Placental tissues were collected from litters on Days 25, 45, 65, 85 and 105 of gestation. Three transcript variants similar to HPSE were identified in the pig placenta. In addition, the HPSE gene was mapped to pig chromosome 8 in close proximity to quantitative trait loci for litter size and prenatal survival. Real-time polymerase chain reaction and in situ hybridisation were used to characterise the expression of two HPSE variants, namely HPSE v1 and v2, in the pig placenta throughout gestation. The expression of HPSE v1 and v2 was elevated (P < 0.01) in placentas during very early gestation (Day 25) as well as during late gestation (Days 85 and 105). Finally, HPSE v1 and v2 mRNA were localised to the cuboidal trophoblast cells of the folded bilayer located nearest to the maternal endometrium. These findings illustrate that HPSE likely plays a role in the development and modification of the pig placenta, which has implications for litter size and prenatal survival.

Characterization of conserved and nonconserved imprinted genes in swine.

To increase our understanding of imprinted genes in swine, we carried out a comprehensive analysis of this gene family using two complementary approaches: expression and phenotypic profiling of parthenogenetic fetuses, and analysis of imprinting by pyrosequencing. The parthenote placenta and fetus were smaller than those of controls but had no obvious morphological differences at Day 28 of gestation. By Day 30, however, the parthenote placentas had decreased chorioallantoic folding, decreased chorionic ruggae, and reduction of fetal-maternal interface surface in comparison with stage-matched control fetuses. Using Affymetrix Porcine GeneChip microarrays and/or semiquantitative PCR, brain, fibroblast, liver, and placenta of Day 30 fetuses were profiled, and 25 imprinted genes were identified as differentially expressed in at least one of the four tissue types: AMPD3, CDKN1C, COPG2, DHCR7, DIRAS3, IGF2 (isoform specific), IGF2AS, IGF2R, MEG3, MEST, NAP1L5, NDN, NNAT, OSBPL1A, PEG3, APEG3, PEG10, PLAGL1, PON2, PPP1R9A, SGCE, SLC38A4, SNORD107, SNRPN, and TFPI2. For DIRAS3, PLAGL1, SGCE, and SLC38A4, tissue-specific differences were detected. In addition, we examined the imprinting status of candidate genes by quantitative allelic pyrosequencing. Samples were collected from Day 30 pregnancies generated from reciprocal crosses of Meishan and White Composite breeds, and single-nucleotide polymorphisms were identified in candidate genes. Imprinting was confirmed for DIRAS3, DLK1, H19, IGF2AS, NNAT, MEST, PEG10, PHLDA2, PLAGL1, SGCE, and SNORD107. We also found no evidence of imprinting in ASB4, ASCL2, CD81, COMMD1, DCN, DLX5, and H13. Combined, these results represent the most comprehensive survey of imprinted genes in swine to date.

Identification of SNPs and INDELS in swine transcribed sequences using short oligonucleotide microarrays.

BACKGROUND: Genome-wide detection of single feature polymorphisms (SFP) in swine using transcriptome profiling of day 25 placental RNA by contrasting probe intensities from either Meishan or an occidental composite breed with Affymetrix porcine microarrays is presented. A linear mixed model analysis was used to identify significant breed-by-probe interactions. RESULTS: Gene specific linear mixed models were fit to each of the log2 transformed probe intensities on these arrays, using fixed effects for breed, probe, breed-by-probe interaction, and a random effect for array. After surveying the day 25 placental transcriptome, 857 probes with a q-value < or = 0.05 and |fold change| > or = 2 for the breed-by-probe interaction were identified as candidates containing SFP. To address the quality of the bioinformatics approach, universal pyrosequencing assays were designed from Affymetrix exemplar sequences to independently assess polymorphisms within a subset of probes for validation. Additionally probes were randomly selected for sequencing to determine an unbiased confirmation rate. In most cases, the 25-mer probe sequence printed on the microarray diverged from Meishan, not occidental crosses. This analysis was used to define a set of highly reliable predicted SFPs according to their probability scores. CONCLUSION: By applying a SFP detection method to two mammalian breeds for the first time, we detected transition and transversion single nucleotide polymorphisms, as well as insertions/deletions which can be used to rapidly develop markers for genetic mapping and association analysis in species where high density genotyping platforms are otherwise unavailable.SNPs and INDELS discovered by this approach have been publicly deposited in NCBI's SNP repository dbSNP. This method is an attractive bioinformatics tool for uncovering breed-by-probe interactions, for rapidly identifying expressed SNPs, for investigating potential functional correlations between gene expression and breed polymorphisms, and is robust enough to be used on any Affymetrix gene expression platform.

Callipyge mutation affects gene expression in cis: a potential role for chromatin structure.

Muscular hypertrophy in callipyge sheep results from a single nucleotide substitution located in the genomic interval between the imprinted Delta, Drosophila, Homolog-like 1 (DLK1) and Maternally Expressed Gene 3 (MEG3). The mechanism linking the mutation to muscle hypertrophy is unclear but involves DLK1 overexpression. The mutation is contained within CLPG1 transcripts produced from this region. Herein we show that CLPG1 is expressed prenatally in the hypertrophy-responsive longissimus dorsi muscle by all four possible genotypes, but postnatal expression is restricted to sheep carrying the mutation. Surprisingly, the mutation results in nonimprinted monoallelic transcription of CLPG1 from only the mutated allele in adult sheep, whereas it is expressed biallelically during prenatal development. We further demonstrate that local CpG methylation is altered by the presence of the mutation in longissimus dorsi of postnatal sheep. For 10 CpG sites flanking the mutation, methylation is similar prenatally across genotypes, but doubles postnatally in normal sheep. This normal postnatal increase in methylation is significantly repressed in sheep carrying one copy of the mutation, and repressed even further in sheep with two mutant alleles. The attenuation in methylation status in the callipyge sheep correlates with the onset of the phenotype, continued CLPG1 transcription, and high-level expression of DLK1. In contrast, normal sheep exhibit hypermethylation of this locus after birth and CLPG1 silencing, which coincides with DLK1 transcriptional repression. These data are consistent with the notion that the callipyge mutation inhibits perinatal nucleation of regional chromatin condensation resulting in continued elevated transcription of prenatal DLK1 levels in adult callipyge sheep. We propose a model incorporating these results that can also account for the enigmatic normal phenotype of homozygous mutant sheep.

Abnormal postnatal maintenance of elevated DLK1 transcript levels in callipyge sheep.

The underlying mechanism of the callipyge muscular hypertrophy phenotype in sheep (Ovis aries) is not presently understood. This phenotype, characterized by increased glycolytic type II muscle proportion and cell size accompanied by decreased adiposity, is not visibly detectable until approximately three to eight weeks after birth. The muscular hypertrophy results from a single nucleotide change located at the telomeric end of ovine Chromosome 18, in the region between the imprinted MATERNALLY EXPRESSED GENE 3 (MEG3) and DELTA, DROSOPHILA, HOMOLOG-LIKE 1 (DLK1) genes. The callipyge phenotype is evident only when the mutation is paternally inherited by a heterozygous individual. We have examined the pre- and postnatal expression of MEG3 and DLK1 in sheep of all four possible genotypes in affected and unaffected muscles as well as in liver. Here we show that the callipyge phenotype correlates with abnormally high DLK1 expression during the postnatal period in the affected sheep and that this elevation is specific to the hypertrophy-responsive fast-twitch muscles. These results are the first to show anomalous gene expression that coincides with both the temporal and spatial distribution of the callipyge phenotype. They suggest that the effect of the callipyge mutation is to interfere with the normal postnatal downregulation of DLK1 expression.

Prion gene sequence variation within diverse groups of U.S. sheep, beef cattle, and deer.

Prions are proteins that play a central role in transmissible spongiform encephalopathies in a variety of mammals. Among the most notable prion disorders in ungulates are scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer. Single nucleotide polymorphisms in the sheep prion gene ( PRNP) have been correlated with susceptibility to natural scrapie in some populations. Similar correlations have not been reported in cattle or deer; however, characterization of PRNP nucleotide diversity in those species is incomplete. This report describes nucleotide sequence variation and frequency estimates for the PRNP locus within diverse groups of U.S. sheep, U.S. beef cattle, and free-ranging deer ( Odocoileus virginianus and O. hemionus from Wyoming). DNA segments corresponding to the complete prion coding sequence and a 596-bp portion of the PRNP promoter region were amplified and sequenced from DNA panels with 90 sheep, 96 cattle, and 94 deer. Each panel was designed to contain the most diverse germplasm available from their respective populations to facilitate polymorphism detection. Sequence comparisons identified a total of 86 polymorphisms. Previously unreported polymorphisms were identified in sheep (9), cattle (13), and deer (32). The number of individuals sampled within each population was sufficient to detect more than 95% of all alleles present at a frequency greater than 0.02. The estimation of PRNP allele and genotype frequencies within these diverse groups of sheep, cattle, and deer provides a framework for designing accurate genotype assays for use in genetic epidemiology, allele management, and disease control.

Identification of the single base change causing the callipyge muscle hypertrophy phenotype, the only known example of polar overdominance in mammals.

A small genetic region near the telomere of ovine chromosome 18 was previously shown to carry the mutation causing the callipyge muscle hypertrophy phenotype in sheep. Expression of this phenotype is the only known case in mammals of paternal polar overdominance gene action. A region surrounding two positional candidate genes was sequenced in animals of known genotype. Mutation detection focused on an inbred ram of callipyge phenotype postulated to have inherited chromosome segments identical-by-descent with exception of the mutated position. In support of this hypothesis, this inbred ram was homozygous over 210 Kb of sequence, except for a single heterozygous base position. This single polymorphism was genotyped in multiple families segregating the callipyge locus (CLPG), providing 100% concordance with animals of known CLPG genotype, and was unique to descendants of the founder animal. The mutation lies in a region of high homology among mouse, sheep, cattle, and humans, but not in any previously identified expressed transcript. A substantial open reading frame exists in the sheep sequence surrounding the mutation, although this frame is not conserved among species. Initial functional analysis indicates sequence encompassing the mutation is part of a novel transcript expressed in sheep fetal muscle we have named CLPG1.