Screening for Booroola (FecB) and Galway (FecXG) mutations in Indian sheep

This study reports the status of the Booroola (FecB) and Galway (FecX^G) mutations in Indian sheep breeds. The Kendrapada sheep (n = 46) was genotyped for the presence of FecB and FecX^G mutations, while the Garole (n = 34), Malpura (n = 30), and Decanni sheep (n = 15) for the FecX^G mutation. The FecB and FecX^G genotyping was carried out by forced restriction fragment length polymorphism PCR technique. In the present study, FecB mutation was discovered in the Kendrapada sheep of Orissa, which is now the second prolific sheep of India after the Garole. Out of 46 individuals of Kendrapada sheep, 26 were homozygous (BB), 15 heterozygous (B+) and 5 non-carriers (++) for the FecB mutation. The frequency of the FecB allele in this sample was about 0.73. Results indicated that the frequency of the FecB mutation is high, but the gene is not fixed in the population as reported in Garole sheep. None of sheep breeds carried the FecX^G mutation. The discovery of the FecB mutation in Kendrapada sheep will facilitate the use of FecB allele in improving the prolificacy of non-prolific sheep breeds of India.     

None of polymorphism of ovine fecundity major genes FecB and FecX was tested in goat

The polymorphism of mutation Q249R in BMPR-IB gene (FecB) and loci FecX^I, FecX^H, FecX^G, FecX^B in BMP15 gene was analyzed by forced PCR-RFLP method in 550 individuals from 6 flocks or breeds of goats with litter size varied from 1.4 to 2.7 including Boer (209), Haimen (128), second generation of Boer goat crossed with Huanghuai goat (82), Huanghuai (71), Nubi (37) and Matou (23) goat. None of mutations was detected in these goat breeds and their crossbreed. These results suggest that fecundity of goat is not linked to the same loci in BMPR-IB and BMP15 as sheep. Therefore, it is necessary to seek for other genes or loci in order to develop marker assistance selection techniques and study the prolific mechanism of the goat.     

Prolificacy genotypes at BMPR 1B,BMP15 and GDF9 genes in North African sheep breeds

The aim of this research was to investigate the genetic structure at BMPR 1B, BMP15 and GDF9 prolificacy genes in five sheep breeds reared in Tunisia: Barbarine, Queue Fine de L’Ouest, Noire de Thibar, Sicilo-Sarde and D’man. Genomic DNA of 204 sheep was investigated for the FecB^B (BMPR 1B), FecX^R, FecX^H, FecX^I, FecX^L, FecX^G, FecX^B (BMP15) and FecG^H (GDF9) mutations. The sequence variability of the different DNA fragments utilised for genotyping was further investigated by Single Stranded Conformation Polymorphism (SSCP) and sequencing. All the above-mentioned mutations were absent in the five sheep breeds examined. SSCP analysis and sequencing allowed the detection of two nucleotide variations. A non-functional mutation (T/C transition at nt 747 of BMP15 cDNA known as B3) was found at the BMP15 gene, in the Noire de Thibar breed; this mutation was first detected in the Belclare sheep. A new nucleotide change G/A at nt 1159 of BMP15 cDNA, causing the amino acid change A119T in the mature peptide, was detected in the Barbarine breed for the first time. The highly prolific D’man ewes were monomorphic for the absence of all the known prolificacy alleles.     

Polymorphism of fecundity genes (BMPR1B, BMP15 and GDF9) in the Indian prolific Black Bengal goat

The Black Bengal is a prolific goat breed in India. Natural mutations in prolific sheep breeds have shown that the transforming growth factor beta (TGF-β) super family ligands such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor, BMPR1B) are crucial for ovulation and as well as for increasing litter size. Mutations in any of these genes increased prolificacy in sheep. Based on the known mutation information in sheep PCR primers were designed to screen known polymorphism in 88 random Black Bengal goats. Only the BMPR1B gene was polymorphic. Three genotypes of animals were detected in tested animals with mutant (FecB^B) and wild type (FecB⁺) alleles were 0.57 and 0.43, respectively. Non-carrier, heterozygous carrier and homozygous carrier Black Bengal does had 2.7, 3.04 and 3.11 kids, respectively. All known point mutations of BMP15 and GDF9 genes were monomorphic in the animals tested. These results preliminarily showed that the BMPR1B gene might be a major gene that influences prolificacy of Black Bengal goats.     

Investigation of Prolific Sheep from UK and Ireland for Evidence on Origin of the Mutations in BMP15 (FecXG,FecXB) and GDF9 (FecGH) in Belclare and Cambridge Sheep

This paper concerns the likely origin of three mutations with large effects on ovulation rate identified in the Belclare and Cambridge sheep breeds; two in the BMP15 gene (FecX^G and FecX^B) and the third (FecG^H) in GDF9. All three mutations segregate in Belclare sheep while one, FecX^B, has not been found in the Cambridge. Both Belclare and Cambridge breeds are relatively recently developed composites that have common ancestry through the use of genetic material from the Finnish Landrace and Lleyn breeds. The development of both composites also involved major contributions from exceptionally prolific ewes screened from flocks in Ireland (Belclare) and Britain (Cambridge) during the 1960s. The objective of the current study was to establish the likely origin of the mutations (FecX^G, FecX^B and FecG^H) through analysis of DNA from Finnish Landrace and Lleyn sheep, and Galway and Texel breeds which contributed to the development of the Belclare breed. Ewes with exceptionally high prolificacy (hyper-prolific ewes) in current flocks on Irish farms were identified to simulate the screening of ewes from Irish flocks in the 1960s. DNA was obtained from: prolific ewes in extant flocks of Lleyn sheep (n = 44) on the Lleyn peninsula in Wales; hyper-prolific ewes (n = 41); prolific Galway (n = 41) ewes; Finnish Landrace (n = 124) and Texel (n = 19) ewes. The FecX^G mutation was identified in Lleyn but not in Finnish Landrace, Galway or Texel sheep; FecX^B was only found among the hyper-prolific ewes. The FecG^H mutation was identified in the sample of Lleyn sheep. It was concluded from these findings that the Lleyn breed was the most likely source of the FecX^G and FecG^H mutations in Belclare and Cambridge sheep and that the FecX^B mutation came from the High Fertility line that was developed using prolific ewes selected from commercial flocks in Ireland in the 1960′s and subsequently used in the genesis of the Belclare.     

Genetic diversity in Iranian indigenous sheep vis-à-vis selected exogenous sheep breeds and wild mouflon

The heterogeneity of climate and different agro-ecological conditions in Iran have resulted in development of 27 indigenous sheep breeds. Wild Asiatic mouflon (Ovis orientalis) is believed to be the ancestor of Iranian sheep. Evaluation of genetic diversity and population structure within and among domestic breeds has important implications for animal breeding programs and genetic resources management. Based on 50K SNP genotype data, we studied the genetic diversity of five indigenous Iranian sheep breeds: Afshari (n = 37), Moghani (n = 34), Qezel (n = 35), Zel (n = 46) and Lori-Bakhtiari (n = 46), and Asiatic mouflon (n = 8) sampled from Iran. Furthermore, genetic diversity and the breed admixture of Iranian sheep were assessed on a larger geographic scale using a reference panel comprising: three indigenous Afghan breeds – Arabi (n = 15), Balouchi (n = 15) and Gadik (n = 15); three indigenous breeds from Turkey and Cyprus – Cyprus Fat Tail (n = 30), Karakas (n = 18) and Norduz (n = 20); and three commercial European breeds – Suffolk (n = 19), Comisana (n = 24) and Engadine Red Sheep (n = 24). The results revealed that the investigated breeds are divided into five genetically distinct clusters according to their geographic origin. Afshari was closest to the local mouflon population and showed signs of mouflon admixture. Qezel was identified as a hybrid sheep breed. Much evidence supported the Afghan breeds being identical. Inbreeding values, which were estimated based on ROHs, were highest for Suffolk (F_ROH = 0.0544) and lowest for Balouchi (F_ROH = 0.0078). In conclusion, analysis of selected breeds from neighboring countries along with Asiatic mouflon gave a deeper insight into the evolutionary history and origin of Iranian sheep with important implications for future breed management.     

单碱基编辑系统介导欧拉藏绵羊GDF9、FecB多胎基因的定点突变

本研究旨在利用单碱基编辑系统(single base editing system)实现欧拉藏绵羊成纤维细胞FecB和GDF9基因靶位点A到G和C到T的碱基替换并检测其编辑效率。首先设计合成靶向欧拉藏绵羊FecB和GDF9基因的sgRNA序列,再分别连接至epi-ABEmax、epi-BE4max质粒,构建载体并电转至欧拉藏绵羊成纤维细胞,最后对阳性细胞FecB和GDF9基因进行Sanger测序鉴定靶位点突变结果,并通过T-A克隆估算单碱基编辑系统的编辑效率。结果显示获得了靶向欧拉藏绵羊FecB和GDF9基因的sgRNA,并构建使欧拉藏绵羊FecB和GDF9基因单碱基突变的载体,FecB基因靶位点编辑效率为39.13%,GDF9基因靶位点(G260、G721、G1184)编辑效率分别为10.52%、26.67%和8.00%。本研究运用单碱基编辑系统在欧拉藏绵羊成纤维细胞上实现了FecB和GDF9基因靶位点突变,为改良欧拉藏绵羊一胎多羔的繁殖性状奠定理论基础。     

Freemartinism and FecX allele determination in replacement ewes of the Rasa Aragonesa sheep breed by duplex PCR

A new naturally occurring mutation in the fecundity gene BMP15 in the Rasa Aragonesa sheep breed (Ovis aries) has been found to affect prolificacy. This mutation (FecX^R allele) is a deletion of 17 base pairs that leads to an altered amino acid sequence, and this alteration increases prolificacy in heterozygous ewes but causes sterility in homozygous ewes. Selection of repository lambs with the FecX^R allele increases rates of twins and multiple lambing and thereby also increases the probability of lambing freemartins that will become sterile. In this sense, an accurate, reliable, and quick method was developed by duplex polymerase chain reaction (PCR) for sex, amplifying an ovine-specific Y chromosome repetitive fragment, and BMP15 genotype determination in replacement ewe lambs. The BMP15 fragment served as an internal control of the amplification and detected the FecX^R allele, avoiding a false negative and then a mistake in freemartin detection. This assay uncovered 6 freemartin females among 195 replacement ewes from 7 different commercial flocks and 1 experimental flock. Furthermore, 1554 rams from 64 commercial flocks were also analyzed to identify FecX^R rams. This analysis identified 103 rams hemizygous for the FecX^R allele and 1 heterozygous ram. Because this gene is located on the X chromosome, this heterozygous animal is a freemartin ram that is co-amplifying the DNA from XX and XY lymphocytes. These results confirm the usefulness of this multiplex PCR assay for detecting phenotypically sexed females, freemartins, and the BMP15 genotype to detect highly prolific ewes in commercial flocks and to assist breeders in selection of repository lambs.     

The polymorphism of a novel 30 bp-deletion mutation at KAP9.2 locus in the cashmere goat

The keratins and keratin-associated proteins (KAPs) are a large heterogeneous group of proteins that make up about 90% of the cashmere fiber. Keratin-associated proteins 9.2 gene (KAP9.2) is one of the ultra high sulfur KAPs, which might play an important role in the bundling of intermediate filaments. In this study, the deletion/insertion mutation of KAP9.2 gene in 997 cashmere goat samples was firstly detected, at the same time, parts of these samples were sequenced. The results showed that two alleles were detected at this KAP9.2P1 locus, named allele W and D. The frequencies of the KAP9.2-W allele in Inner Mongolia White cashmere (n = 785) and Shaanbei White cashmere goat breeds (n = 212) were 0.878 and 0.790, respectively. The χ²-test showed that the genotype distributions in these two cashmere goat breeds were not in agreement with Hardy–Weinberg equilibrium. According to the classification of polymorphism information content (PIC), Shaanbei White cashmere goat was more polymorphic at this locus. Moreover a 30 bp-deletion mutation was described at KAP9.2P2 locus for the first time and no deletion/insertion was described at KAP9.2P1 locus. The results possibly revealed that the size polymorphism existed in the two Chinese cashmere goat and the 30 bp-deletion mutation was possibly caused by variations in the number of the decapeptide repeat structures.     

Genome-Wide Association Studies Identify Two Novel BMP15 Mutations Responsible for an Atypical Hyperprolificacy Phenotype in Sheep

Some sheep breeds are naturally prolific, and they are very informative for the studies of reproductive genetics and physiology. Major genes increasing litter size (LS) and ovulation rate (OR) were suspected in the French Grivette and the Polish Olkuska sheep populations, respectively. To identify genetic variants responsible for the highly prolific phenotype in these two breeds, genome-wide association studies (GWAS) followed by complementary genetic and functional analyses were performed. Highly prolific ewes (cases) and normal prolific ewes (controls) from each breed were genotyped using the Illumina OvineSNP50 Genotyping Beadchip. In both populations, an X chromosome region, close to the BMP15 gene, harbored clusters of markers with suggestive evidence of association at significance levels between 1E⁻⁰⁵ and 1E⁻⁰⁷. The BMP15 candidate gene was then sequenced, and two novel non-conservative mutations called FecX^Gr and FecX^O were identified in the Grivette and Olkuska breeds, respectively. The two mutations were associated with the highly prolific phenotype (p_FecX^Gr = 5.98E⁻⁰⁶ and p_FecX^O = 2.55E⁻⁰⁸). Homozygous ewes for the mutated allele showed a significantly increased prolificacy (FecX^Gr/FecX^Gr, LS = 2.50±0.65 versus FecX⁺/FecX^Gr, LS = 1.93±0.42, p<1E⁻⁰³ and FecX^O/FecX^O, OR = 3.28±0.85 versus FecX⁺/FecX^O, OR = 2.02±0.47, p<1E⁻⁰³). Both mutations are located in very well conserved motifs of the protein and altered the BMP15 signaling activity in vitro using a BMP-responsive luciferase test in COV434 granulosa cells. Thus, we have identified two novel mutations in the BMP15 gene associated with increased LS and OR. Notably, homozygous FecX^Gr/FecX^Gr Grivette and homozygous FecX^O/FecX^O Olkuska ewes are hyperprolific in striking contrast with the sterility exhibited by all other known homozygous BMP15 mutations. Our results bring new insights into the key role played by the BMP15 protein in ovarian function and could contribute to a better understanding of the pathogenesis of women′s fertility disorders.     

Genotyping of the prion protein (PrP) gene in Red Maasai and Black Head Persian sheep in Tanzania

Susceptibility to scrapie in sheep is largely influenced by four polymorphic amino acid positions of the ovine PrP gene at codons 136, 141, 154 and 171. Genotyping of corresponding DNA polymorphism can be used as a basis for selection decisions. A total of 100 Red Maasai and 79 Black Herd Persian sheep, representative of the widely distributed breeds in Tanzania, were genotyped by real-time PCR. Genomic DNA was extracted from buccal epithelial cells. We report six genotypes derived from four different alleles, with the ARQ/ARQ genotype being more frequent (p = 0.0081) in the Red Maasai than in the Black Head Persian sheep. Our study also demonstrated higher allelic frequency (p = 0.00055) of the ARQ in the Red Maasai than in Black Head Persian sheep, while the AHQ allelic frequency was higher (p = 0.00086) in the Black Head Persian. All the animals were homozygous LL₁₄₁. The highly susceptible VRQ allele was not found in any of the sheep breeds examined in this study. Both breeds were genetically low-level resistant to scrapie (NSP3). Due to absence or very low frequency of the ARR allele in the two breeds, selection for genetic resistance to scrapie through an increase of the ARR allele does not seem very relevant. If new breeds of sheep are to be introduced for crossbreeding in Tanzania, care should be taken to avoid import of the VRQ allele. To our knowledge, this is the first study exploring the PrP genotypes in sub-Saharan African sheep and in the Red Maasai and Black Head Persian sheep breeds in particular. The data provide base-line information on genetic susceptibility of the two sheep breeds to scrapie, which may be useful in policy formulation for prevention and future research on prion diseases.     

Resequencing and signatures of selection scan in two Siberian native sheep breeds point to candidate genetic variants for adaptation and economically important traits

Russian sheep breeds represent an important economic asset by providing meat and wool, whilst being adapted to extreme climates. By resequencing two Russian breeds from Siberia: Tuva (n = 20) and Baikal (n = 20); and comparing them with a European (UK) sheep outgroup (n = 14), 41 million variants were called, and signatures of selection were identified. High‐frequency missense mutations on top of selection peaks were found in genes related to immunity (LOC101109746) in the Baikal breed and wool traits (IDUA), cell differentiation (GLIS1) and fat deposition (AADACL3) in the Tuva breed. In addition, genes found under selection owing to haplotype frequency changes were related to wool traits (DSC2), parasite resistance (CLCA1), insulin receptor pathway (SOCS6) and DNA repair (DDB2) in the Baikal breed, and vision (GPR179) in the Tuva breed. Our results present candidate genes and SNPs for future selection programmes, which are necessary to maintain and increase socioeconomic gain from Siberian breeds.     

Genetic structure and admixture in sheep from terminal breeds in the United States

Selection for performance in diverse production settings has resulted in variation across sheep breeds worldwide. Although sheep are an important species to the United States, the current genetic relationship among many terminal sire breeds is not well characterized. Suffolk, Hampshire, Shropshire and Oxford (terminal) and Rambouillet (dual purpose) sheep (n = 248) sampled from different flocks were genotyped using the Applied Biosystems Axiom Ovine Genotyping Array (50K), and additional Shropshire sheep (n = 26) using the Illumina Ovine SNP50 BeadChip. Relationships were investigated by calculating observed heterozygosity, inbreeding coefficients, eigenvalues, pairwise Wright’s F_ST estimates and an identity by state matrix. The mean observed heterozygosity for each breed ranged from 0.30 to 0.35 and was consistent with data reported in other US and Australian sheep. Suffolk from two different regions of the United States (Midwest and West) clustered separately in eigenvalue plots and the rectangular cladogram. Further, divergence was detected between Suffolk from different regions with Wright’s F_ST estimate. Shropshire animals showed the greatest divergence from other terminal breeds in this study. Admixture between breeds was examined using admixture , and based on cross-validation estimates, the best fit number of populations (clusters) was K = 6. The greatest admixture was observed within Hampshire, Suffolk, and Shropshire breeds. When plotting eigenvalues, US terminal breeds clustered separately in comparison with sheep from other locations of the world. Understanding the genetic relationships between terminal sire breeds in sheep will inform us about the potential applicability of markers derived in one breed to other breeds based on relatedness.     

Genome constitutions of Pseudoroegneriageniculata, P. geniculata ssp. scythica and P. geniculata ssp. pruinifera (Poaceae: Triticeae) revealed by genomic in situ hybridization

Genomic constitutions of three taxa of Pseudoroegneria á. L?ve, P. geniculata, P. geniculata ssp. scythica, and P. geniculata ssp. pruinifera, were characterized using genomic in situ hybridization (GISH). The results indicated that ploidy level and genomic constitution in the three Pseudoroegneria taxa studied were as follows: P. geniculata (2n = 4x = 28; genomic formula StSt ^g ), P. geniculata ssp. scythica (2n = 4x = 28; genomic formula ESt), and P. geniculata ssp. pruinifera (2n = 6x = 42; genomic formula EESt). P. geniculata ssp. scythica and P. geniculata ssp. pruinifera should be transferred to Trichopyrum á. L?ve following L?ve’s principles and treated as T. scythicum and T. pruiniferum, respectively.     

DNA tests in prolific sheep from eight countries provide new evidence on origin of the Booroola (FecB) mutation

Recent discoveries that high prolificacy in sheep carrying the Booroola gene (FecB) is the result of a mutation in the BMPIB receptor and high prolificacy in Inverdale sheep (FecX(I)) is the result of a mutation in the BMP15 oocyte-derived growth factor gene have allowed direct marker tests to be developed for FecB and FecX(I). These tests were carried out in seven strains of sheep (Javanese, Thoka, Woodlands, Olkuska, Lacaune, Belclare, and Cambridge) in which inheritance patterns have suggested the presence of major genes affecting prolificacy and in the prolific Garole sheep of India, which have been proposed as the ancestor of Australian Booroola Merinos. The FecB mutation was found in the Garole and Javanese sheep but not in Thoka, Woodlands, Olkuska, Lacaune, Belclare, and Cambridge sheep. None of the sheep tested had the FecX(I) mutation. These findings present strong evidence to support historical records that the Booroola gene was introduced into Australian flocks from Garole (Bengal) sheep in the late 18th century. It is unknown whether Javanese Thin-tailed sheep acquired the Booroola gene directly from Garole sheep from India or via Merinos from Australia. The DNA mutation test for FecB will enable breeding plans to be developed that allow the most effective use of this gene in Garole and Javanese Thin-tailed sheep and their crosses.     

Prevalence and multilocus genotyping of Giardia duodenalis in Tan sheep (Ovis aries) in northwestern China

Giardia duodenalis is a common intestinal protozoa, which can cause the occurrence of diarrhea, weight loss, and even death in animals or human, this threatens the husbandry industry and public health. It can infect virtually humans and all domestic animals including sheep. Tan sheep is one of the most important sheep breeds, which is short-tailed indigenous sheep breed used for production of high quality meat and pelts in China. However, there are no report regarding the occurrence and multilocus genotyping of G. duodenalis in Tan sheep in northwestern China. Thus, the objective of the present study was to investigate the prevalence and multilocus genotypes of G. duodenalis in Tan sheep. 1014 fecal samples were collected from Tan sheep from Ningxia Hui Autonomous Region, and three loci (β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes) were amplified by nested PCR. The prevalence of G. duodenalis in Tan sheep was 14.5% (147/1014), two assemblages (assemblage A, n = 43; and E, n = 90) were detected, including one novel assemblage A at bg locus, one novel assemblage A at tpi locus, and 10 and 11 novel subtypes of assemblage E were detected at the bg and gdh loci, respectively. One MLGs was formed based on sequence variation among the three loci. Moreover, 9 Tan sheep were infected with two assemblages (A and E) based on the three loci. These findings expand the host range of G. duodenalis and revealed genetic diversity of G. duodenalis assemblages in Tan sheep.     

Relationships between stream habitat and breeding territory length of the Brown Dipper (Cinclus pallasii) in Taiwan

Dippers from the genus Cinclus are highly specialised predators on aquatic invertebrates, and occupy linear territories along rivers where measurements of variations in quality are relatively straightforward. For these reasons, they are ideal model species in which to examine factors affecting territory size. In this paper, we investigated the influence of stream habitats on the territory length of the Brown Dipper (Cinclus pallasii) in Taiwan. The biomass of aquatic insects and other stream habitat variables were analyzed to determine their relationships with the territory length of Brown Dippers from November 1988 to May 1989. Compared with slow-moving waters, riffle areas contained significantly greater insect biomass (paired t test, t ₁₁ = 3.49, P < 0.01), of which trichopteran larvae contributed about 70%. Dippers spent more time foraging in riffles than in slow-moving waters. In addition, dippers preferred foraging in shallow riffles, but avoided deep, slow-moving waters (G = 62.53, df = 3, P < 0.001). Territory length (1,045 ± 165 m [SE], n = 14) was negatively related to proportion of riffles (r ² = 0.5715, P < 0.01), total aquatic insect biomass (r ² = 0.5840, P < 0.01), and altitude (r ² = 0.7176, P < 0.001). In factor analysis, four factors were extracted from the 14 stream variables. However, only factor 1 was significantly related to territory length (r ² = 0.5207, P < 0.01). Factor 1 explained 42.8% of the total variance and collectively revealed the importance of high food abundance. In other words, Brown Dipper territories were the shortest along high-altitude streams with abundant riffles, fewer pools and abundant aquatic insects. These results indicate that abundant supply of accessible invertebrate prey is the most important factor affecting the territory length of Brown Dippers. This is consistent with Cinclus species elsewhere, and reveals the importance of clean, productive river ecosystems.