Genetic diversity of selected genes that are potentially economically important in feral sheep of New Zealand

Background

Feral sheep are considered to be a source of genetic variation that has been lost from their domestic counterparts through selection.

Methods

This study investigates variation in the genes KRTAP1-1, KRT33, ADRB3 and DQA2 in Merino-like feral sheep populations from New Zealand and its offshore islands. These genes have previously been shown to influence wool, lamb survival and animal health.

Results

All the genes were polymorphic, but no new allele was identified in the feral populations. In some of these populations, allele frequencies differed from those observed in commercial Merino sheep and other breeds found in New Zealand. Heterozygosity levels were comparable to those observed in other studies on feral sheep. Our results suggest that some of the feral populations may have been either inbred or outbred over the duration of their apparent isolation.

Conclusion

The variation described here allows us to draw some conclusions about the likely genetic origin of the populations and selective pressures that may have acted upon them, but they do not appear to be a source of new genetic material, at least for these four genes.     

Polymorphism of the ovine β3-adrenergic receptor gene (ADRB3) and its association with wool mean staple strength and yield

We investigated the possibility that variation in ovine ADRB3 is associated with various wool traits, in particular mean staple strength (MSS). Polymerase chain reaction-single strand conformational polymorphism analysis of part of the ADRB3 intron was used to genotype 695 Merino lambs born on three farms in the South Island of New Zealand and which were shorn as 2-tooths. For each fleece, MSS, mean fibre diameter, mean staple length and yield were measured. The results from mixed-effects models and half-sib analyses suggest that ADRB3 alleles A and D have a negative impact on some wool traits, whereas ADRB3 alleles C and E appear to have a positive impact, with allele C potentially having a greater impact than allele E on MSS. This variation in the ADRB3 may assist in the genetic selection for increased MSS and yield in Merino sheep.     

Polymorphism of the bovine ADRB3 gene

The β₃-adrenergic receptor (ADRB3) is predominantly expressed in white and brown adipose tissue and mediates the lipolytic and thermogenic effects of high catecholamine concentrations. Variation in the ADRB3 gene (ADRB3) has been associated with obesity and the earlier onset of non-insulin-dependent diabetes mellitus in some ethnic groups, as well as some production traits of sheep, but to date variation of bovine ADRB3 has not been reported. In this study, variation in the promoter region of bovine ADRB3 was investigated in 737 cattle by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. Six PCR-SSCP patterns representing six allelic variations and containing four single nucleotide polymorphisms (SNPs) and three nucleotide deletions/insertions were observed. Allele A was the most common allele (93.83%), whereas alleles C, D, E and F were rare (0.07, 1.09, 0.41, and 0.34%, respectively). The variation identified here might have an impact on both the function and level of expression of bovine ADRB3.     

Comparative Susceptibility of Sheep of Different Origins,Breeds and PRNP Genotypes to Challenge with Bovine Spongiform Encephalopathy and Scrapie

Sheep are natural hosts of the prion disease, scrapie. They are also susceptible to experimental challenge with various scrapie strains and with bovine spongiform encephalopathy (BSE), which affects cattle and has been accidentally transmitted to a range of other species, including man. Incidence and incubation period of clinical disease in sheep following inoculation is controlled by the PRNP gene, which has different alleles defined on the basis of polymorphisms, particularly at codons 136, 154 and 171, although other codons are associated with survival time, and the exact responses of the sheep may be influenced by other breed-related differences. Here we report the results of a long term single study of experimental scrapie and BSE susceptibility of sheep of Cheviot, Poll Dorset and Suffolk breeds, originating from New Zealand and of a wide range of susceptible and resistant PRNP genotypes. Responses were compared with those of sheep from a closed Cheviot flock of UK origin (Roslin Cheviot flock). The unusually long observation period (6–8 years for most, but up to 12 years for others) allows us to draw robust conclusions about rates of survival of animals previously regarded as resistant to infection, particularly PRNP heterozygotes, and is the most comprehensive such study reported to date. BSE inoculation by an intracerebral route produced disease in all genotype groups with differing incubation periods, although M112T and L141F polymorphisms seemed to give some protection. Scrapie isolate SSBP/1, which has the shortest incubation period in sheep with at least one VRQ PRNP allele, also produced disease following sub-cutaneous inoculation in ARQ/ARQ animals of New Zealand origin, but ARQ/ARQ sheep from the Roslin flock survived the challenge. Our results demonstrate that the links between PRNP genotype and clinical prion disease in sheep are much less secure than previously thought, and may break down when, for example, a different breed of sheep is moved into a new flock.     

Polymorphism at the beta-adrenergic receptor (ADRB3) locus of Merino sheep and its association with lamb mortality

Beta3-adrenergic receptors are predominantly found on the surface of adipocytes and are major mediators of the lipolytic and thermogenic effects of high catecholamine concentrations. Recently, variation in the ovine beta3-adrenergic receptor (ADRB3) gene has been reported to be associated with lamb survival. In this study, polymerase chain reaction-single strand conformational polymorphism analysis of part of the ADRB3 intron was used to genotype 4488 Merino lambs born at 10 farms throughout the South Island of New Zealand. Univariate and multivariate odds ratios for each allele revealed a significant association of the E allele with cdd survival and of the D allele with mortality. This variation at the ADRB3 locus may assist in the genetic selection for survival in Merino sheep.     

Prion gene (PRNP) haplotype variation in United States goat breeds (Open Access publication)

Scrapie eradication efforts cost 18 million dollars annually in the United States and rely heavily upon PRNP genotyping of sheep. Genetic resistance might reduce goat scrapie and limit the risk of goats serving as a scrapie reservoir, so PRNP coding sequences were examined from 446 goats of 10 breeds, 8 of which had not been previously examined at PRNP. The 10 observed alleles were all related to one of two central haplotypes by a single amino acid substitution. At least five of these alleles (M142, R143, S146, H154, and K222) have been associated with increased incubation time or decreased odds of scrapie. To the best of our knowledge, neither S146 nor K222 has been found in any goats with scrapie, though further evaluation will be required to demonstrate true resistance. S146 was more common, present in several breeds at widely varying frequencies, while K222 was observed only in two dairy breeds at low frequency. Overall, this study provides frequency data on PRNP alleles in US goats, shows the pattern of relationships between haplotypes, and demonstrates segregation of multiple scrapieassociated alleles in several breeds not examined before at PRNP.     

Extended haplotype analysis of ovine ADRB3 using polymerase chain reaction single strand conformational polymorphism on two regions of the gene

The β3 adrenergic receptor (ADRB3) plays a critical role in the regulation of energy metabolism in mammals. In sheep, intronic polymorphism of the ADRB3 gene has been associated with lamb survival and various production traits. This study investigates variation in the ovine ADRB3 3' untranslated region (3'UTR), a region that may impact expression of the gene. Using PCR- single strand conformational polymorphism (SSCP), six unique patterns (named a-f) were observed in an approximately 304-bp amplicon. Sequencing revealed three single-nucleotide polymorphisms (c.*233A>C, c.*271G>C, c.*357A>T) and a single-nucleotide deletion (c.*257delG). Haplotype analyses showed that the previously described allele A defined by variation in the ovine ADRB3 intron can be divided into three haplotypes (Aa, Ab, and Ac). In total, 16 haplotypes through ovine ADRB3 were detected. This study suggests that ovine ADRB3 is highly polymorphic and that the extended haplotype analysis through the promoter, 5'UTR, coding sequence, intron, and 3'UTR needs to be performed to define the full extent of variation in this gene.     

Single-strand conformational polymorphism and sequence polymorphism of Mhc-DRB in Latxa and Karrantzar sheep: implications for Caprinae phylogeny

Single-strand conformational polymorphism analysis and DNA sequencing were used to characterize Mhc-DRB second exon variability in the Latxa and Karrantzar breeds of sheep. The presence of more than two sequences in some animals indicates that alleles of two different loci have been amplified. Six new alleles were identified by sequencing. The allele frequency distribution of the DRB1 gene is striking, with two alleles accounting for half of the gene pool in both breeds under study. The most frequent allele in both breeds was the same (named DRB1*0702), with some specific amino acids: Tyr in position 31 and Thr in 51. A species variability analysis was also performed including the entire set of sheep DRB exon 2 sequences. Based on the patchwork patterns of different alleles, interallelic recombination appears to be playing a significant role in the generation of allelic diversity at this locus in sheep. The phylogenetic tree of all known Caprinae DRB sequences shows that certain alleles from one species are more closely related to those from other species than they are to each other. Allele DRB1*0702 merits special attention due to its high similarity to the Mufflon allele. As this is the most frequent in both breeds analyzed, one can hypothesize that in sheep, both Mufflon and Argali have had different influences depending on the sheep breed under study and that the relationship between domestic sheep and Mufflon is greater than previously thought. The data generated in this study can serve as a basis for developing a typing assay for the sheep DRB genes in the Latxa and Karrantzar populations.     

Genetic diversity of three Chinese native sheep breeds

The sheep (Ovis aries L.) has been an important farm animal species since its domestication. A wide array of indigenous sheep breeds with abundant phenotypic diversity exists for domestication and selection. Therefore, assessing the genetic diversity of a local sheep resource using a multi-molecular system is helpful for maintaining and conserving those breeds. This study aimed to investigate the genetic diversity of three native Chinese sheep breeds (Tibetan sheep, Sishui Fur sheep, and Small-tailed Han sheep) using 15 microsatellite markers and the second exon of the DRA gene. In regards to the microsatellites, on average, 19 alleles per loci were observed among all individuals. Across loci, the HO within the population was 0.652 ± 0.022 in Tibetan sheep, 0.603 ± 0.023 in Small-tailed Han sheep and 0.635 ± 0.022 in SFS, and for most populations, the H _E and H _O were inconsistent. In addition, affluent private alleles within the breed indicated that the breeds have different domestication histories or sites. In regards to the 2 exon of the DRA gene, three haplotypes were constructed by seven single-nucleotide polymorphisms (SNPs), which were identified in the second DRA exon and inferred the potential for phenotypic variety in these Chinese native sheep. In summary, the current study reveals the importance of implementing effective conservation strategies for these three native Chinese sheep.     

Genetic diversity of <Emphasis Type="Italic">Ovis aries</Emphasis> populations near domestication centers and in the New World

Domestic sheep in Kazakhstan may provide an interesting source of genetic variability due to their proximity to the center of domestication and the Silk Route. Additionally, those breeds have never been compared to New World sheep populations. This report compares genetic diversity among five Kazakhstan (KZ) and 13 United States (US) sheep breeds (N = 442) using 25 microsatellite markers from the FAO panel. The KZ breeds had observed and expected measures of heterozygosity greater than 0.60 and an average number of alleles per locus of 7.8. In contrast, US sheep breeds had observed heterozygosity ranged from 0.37 to 0.62 and had an average number of alleles of 5.7. A Bayesian analysis indicated there were two primary populations (K = 2). Surprisingly, the US breeds were near evenly split between the two clusters, while all of the KZ breeds were placed in one of the two clusters. Pooling breeds within country of sample origin showed KZ and US populations to have similar levels of expected heterozygosity and the average number of alleles per locus. The results of breeds pooled within country suggest that there was no difference between countries for these diversity measures using this set of neutral markers. This finding suggests that populations’ geographically isolated from centers of domestication can be more diverse than previously thought, and as a result, conservation strategies can be adjusted accordingly. Furthermore, these results suggest there may be limited need for countries to alter the protocols for trade and exchange of animal genetic resources that are in place today, since no one population has a unique set of private alleles.     

Gene Frequency Distribution of the BoLA-DRB3 Locus in Saavedreño Creole Dairy Cattle

The objective of this study is to describe the gene frequency distribution of the bovine lymphocyte antigen (BoLA)-DRB3 locus in Saavedreño Creole dairy cattle and to compare it with previously reported patterns in other cattle breeds. One hundred and twenty-five Saavedreño Creole dairy cattle were genotyped for the BoLA-DRB3.2 allele by polymerase chain reaction and restriction fragment length polymorphism. Twenty-two out of 53 previously identified BoLA-DRB3.2 alleles were detected, with gene frequencies ranging from 0.4 to 16.8%. Seventy percent of the variation corresponded to the seven most frequent alleles (BoLA-DRB3.2*7, *8, *11, *16, *27, *36, and *37). The studied population exhibits a high degree of expected heterozygosity (h _e = 0.919). The F _IS index did not show significant deviation from Hardy-Weinberg equilibrium. However, the neutrality test showed an even gene frequency distribution. This result could be better explained assuming balancing selection instead of neutral or positive selection for one or a few alleles. In conclusion, the results of this study demonstrated that BoLA-DRB3.2 is a highly polymorphic locus in Saavedreño Creole dairy cattle, with significant variation in allele frequency among cattle breeds.     

Genetic variations in the myostatin gene (MSTN) in New Zealand sheep breeds

Myostatin, which is also known as growth and differentiation factor 8 (GDF8), acts as a negative regulator of skeletal muscle growth. Variation in the myostatin gene (MSTN) has been associated with variation in muscularity in many animals including sheep. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used to investigate MSTN in a diverse range of sheep breeds including the New Zealand (NZ) Romney, Coopworth, Corriedale, Dorper, Perendale, Suffolk, Merino, Dorset Down, Poll Dorset, Texel and other NZ cross-bred sheep. A total of 28 nucleotide substitutions were identified from nucleotide c.-1199 in the promoter region to c.*1813 (based on NCBI GenBank accession number DQ530260) and including the well-described substitution c.*1232G>A (MSTN g+6223G>A). Of these 28 substitutions, 3 were located in the promoter region, 3 in the 5′UTR, 11 in intron 1, 5 in intron 2 and 5 in the 3′UTR. One substitution in exon 1 (c.101G>A) potentially results in an amino acid substitution of glutamic acid (Glu) with glycine (Gly) at codon 34. Ten of these substitutions have not been reported previously. The genetic variation revealed in this study suggests this gene is more variable than hitherto reported and provides a foundation for future research into how this variation affects muscle and growth traits.     

Molecular evolution of the leptin exon 3 in some species of the family Canidae

The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris) – 16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides), the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus)) were studied with the use of single strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical.     

Characterization of bovine MHC DRB3 diversity in Latin American Creole cattle breeds

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for diseases and immunological traits. However, none of the highly adapted Latin American Creole breeds have been characterized for BoLA gene polymorphism by high resolution typing methods. In this work, we sequenced exon 2 of the BoLA class II DRB3 gene from 179 cattle (113 Bolivian Yacumeño cattle and 66 Colombian Hartón del Valle cattle breeds) using a polymerase chain reaction sequence-based typing (PCR-SBT) method. We identified 36 previously reported alleles and three novel alleles. Thirty-five (32 reported and three new) and 24 alleles (22 reported and two new) were detected in Yacumeño and Hartón del Valle breeds, respectively. Interestingly, Latin American Creole cattle showed a high degree of gene diversity despite their small population sizes, and 10 alleles including three new alleles were found only in these two Creole breeds. We next compared the degree of genetic variability at the population and sequence levels and the genetic distance in the two breeds with those previously reported in five other breeds: Holstein, Japanese Shorthorn, Japanese Black, Jersey, and Hanwoo. Both Creole breeds presented gene diversity higher than 0.90, a nucleotide diversity higher than 0.07, and mean number of pairwise differences higher than 19, indicating that Creole cattle had similar genetic diversity at BoLA-DRB3 to the other breeds. A neutrality test showed that the high degree of genetic variability may be maintained by balancing selection. The F_ST index and the exact G test showed significant differences across all cattle populations (F_ST = 0.0478; p < 0.001). Results from the principal components analysis and the phylogenetic tree showed that Yacumeño and Hartón del Valle breeds were closely related to each other. Collectively, our results suggest that the high level of genetic diversity could be explained by the multiple origins of the Creole germplasm (European, African and Indicus), and this diversity might be maintained by balancing selection.     

Identification of Allelic Polymorphism in the Ovine Leptin Gene

Leptin is an adipocyte-derived hormone/cytokine that influences the physiological control of numerous biological functions and links nutritional status with both neuroendocrine and immune functions. In livestock, variation in the leptin (LEP) gene has been characterized in cattle and pig, but it has not been reported in sheep. In this study, variation in the exon 3 coding sequence of the ovine LEP gene was investigated by polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) analysis and DNA sequencing. Five novel SSCP patterns, representing five different sequences, were identified under a combination of two different electrophoresis conditions. Either one or two different sequences were detected in individual sheep and all the sequences identified shared high homology with the LEP sequences from a variety of species, suggesting that these sequences represent alleles of the ovine LEP gene. Four single nucleotide polymorphisms (SNPs) were detected, and three of these resulted in amino acid changes. Variation detected here might have an impact on leptin activity and function.     

Ovine prion protein genotype frequencies in northwestern China

Anti-scrapie breeding programs have been initiated to screen for scrapie-resistant sheep based on ovine prion protein gene (PRNP) genotypes at codons 136, 154 and 171 in many countries, especially European Union member states. However, investigation of sheep PRNP genotypes is limited in China, despite the large number of sheep breeds. We analyzed 432 sheep of five different breeds from farms in northwestern China, using PCR-single-strand conformational polymorphism analysis (PCR-SSCP); the corresponding haplotypes of different PRNP alleles were cloned. PRNP allele genotyping was done by amplification refractory mutation system-PCR (ARMS-PCR), according to the haplotype clones of each PRNP allele. The validity of ARMS-PCR was checked by PCR-SSCP. Another 325 unknown PRNP genotypes of other sheep breeds were analyzed according to the established ARMS-PCR. Genotype frequencies of 757 sheep were analyzed with these two methods to evaluate susceptibility to scrapie in northwestern China. Relevant mutations were also detected at other sites. Both methods were effective for ovine PRNP allele genotyping, and the results of the analysis completely coincided. Scrapie-resistant genotypes were found to be uncommon, indicating a high risk for ovine scrapie in northwest China. In addition to codons 136, 154 and 171, we found numerous new mutations; nearly half of them were previously unreported. These sheep populations have a high degree of polymorphism at the PRNP locus.     

Halplotypes of the ovine ADRB3 gene (ADRB3) and their association with post-weaning growth in New Zealand Suffolk sheep

The β₃-adrenergic receptor (ADRB3) regulates thermogenesis and lipolysis in brown and white adipose tissue. Previously, sixteen ovine ADRB3 haplotypes have been defined. In this study, the relationship between these ADRB3 haplotypes and variation in post-weaning growth was investigated in 797 New Zealand Suffolk lambs from 38 sire lines and eight studs, using PCR-SSCP and General Linear Mixed-effects Models. Seven haplotypes were found in these sheep and they comprised five previously reported intron sequences and four previously reported 3′UT sequences. The frequencies of the various diplotypes ranged from 0.1 to 17.6 % and individual haplotypes from 0.8 to 32.5 %. The presence of haplotype A-b was associated with a decreased weaning-weight (P = 0.001). Sheep with the B-c/F-e diplotype had a higher mean weaning-weight than those with A-b/B-c or A-b/E-e (P < 0.05). The presence of C-a was found to be associated with increased post-weaning growth (P = 0.008), while the presence of B-c was associated with decreased post-weaning growth (P = 0.005). Sheep with A-b/C-a had higher mean post-weaning growth than those with A-b/A-b, A-b/B-c, B-c/B-c, B-c/E-e or B-c/F-e (P < 0.05). Sheep with B-c/B-c had lower mean post-weaning growth than those with A-b/C-a or B-c/C-a (P < 0.05). Additive effects for the different forms of the B-c haplotypes on post-weaning growth were identified. The effects of the ovine ADRB3 haplotypes on weaning-weight and post-weaning growth confirm that they could be used as a candidate gene-marker for improving sheep growth.     

Genetic diversity of Chinese indigenous sheep breeds inferred from microsatellite markers

To determine the genetic diversity and phylogenetic relationships among Chinese sheep, 10 indigenous breeds and one introduced breed were genotyped for 19 microsatellite loci. The mean number of alleles per breed ranged from 5.44 (Guide Black Fur sheep) to 9.13 (Ujumqin sheep and Hulunbeier sheep), the expected heterozygosity varied from 0.623 (Guide Black Fur sheep) to 0.737 (Zhaotong sheep), and the allelic richness ranged from 5.169 (Guide Black Fur sheep) to 7.610 (Zhaotong sheep). The deviation from Hardy–Weinberg equilibrium (HWE) was statistically significant (P < 0.05) at three loci (SRCRSP5, OarAE129 and DYMS1) in most of the breeds. Chinese sheep breeds had maintained a high level of within-population genetic differentiation (95.23%), with the remainder explained by differentiation among populations (4.77%). The genetic differentiation pattern and genetic relationships among Chinese sheep breeds displayed a high consistency with the traditional classification. Both the Bayesian cluster and principal component analyses showed a reliable clustering pattern, which revealed three major clusters in Chinese indigenous sheep (Mongolian sheep, Kazakh sheep and Tibetan sheep), except Zhaotong and Guide Black Fur sheep. There were probably caused by different breeding history, geography isolation and different levels of inbreeding. This study will help to interpret the genetic characters of Chinese indigenous sheep and benefit to the future conservation programs.     

Serum antigens of cattle

We have produced an alloimmune serum that detects a new cattle immunoglobulin light chain allotype. Genetic analysis involving more than 600 offspring showed that this allotype is controlled by an autosomal codominant allele [Ig(L)b ²], of theIg(L)b ¹ gene previously reported from this laboratory. This system also contains a recessive allele for which no product can now be detected. The frequencies of the three alleles in 5 dairy breeds are reported. The Ig(L)B1 and Ig(L)B2 allotypes are expressed as complexes of 2 or 3 precipitin bands in double diffusion gels containing polyethylene glycol-6000. The complexes are transmitted as genetic units.     

Impact of strong selection for the PrP major gene on genetic variability of four French sheep breeds (Open Access publication)

Effective selection on the PrP gene has been implemented since October 2001 in all French sheep breeds. After four years, the ARR "resistant" allele frequency increased by about 35% in young males. The aim of this study was to evaluate the impact of this strong selection on genetic variability. It is focussed on four French sheep breeds and based on the comparison of two groups of 94 animals within each breed: the first group of animals was born before the selection began, and the second, 3–4 years later. Genetic variability was assessed using genealogical and molecular data (29 microsatellite markers). The expected loss of genetic variability on the PrP gene was confirmed. Moreover, among the five markers located in the PrP region, only the three closest ones were affected. The evolution of the number of alleles, heterozygote deficiency within population, expected heterozygosity and the Reynolds distances agreed with the criteria from pedigree and pointed out that neutral genetic variability was not much affected. This trend depended on breed, i.e. on their initial states (population size, PrP frequencies) and on the selection strategies for improving scrapie resistance while carrying out selection for production traits.