Sheep Chromosome Research Articles

Imprinted Dlk1-Gtl2 cluster miRNAs are potential epigenetic regulators of lamb fur quality

BackgroundTan and Hu sheep are well-known local breeds in China, producing lamb fur with unique ornamental and practical values highly appreciated by consumers worldwide. Fur quality is optimal at one month of age and gradually declines with time. Despite active research on its genetic mechanism using transcriptomic and whole genome bisulfite sequencing analysis, the main effective gene locus has not been found, and its regulatory mechanism is still unclear, which limits the breeding and improvement of fur traits.ResultsScapular skin samples from newborn (1-month old) and adult (24-month old) Tan sheep were utilized for small ribonucleic acid (RNA) sequencing Principal Component Analysis (PCA) showed that the newborn and adult groups were completely separated. Differential expression analysis of micro-RNAs (miRNAs) identified 32 up-regulated miRNAs and 48 down-regulated miRNAs in the newborn groups. All up-regulated miRNAs were located in the imprinted.Dlk1-Gtl2 locus on chromosome 18, whereas all down-regulated miRNAs were distributed across the sheep chromosomes, without a clear pattern of positional consistency. Further, by systematically analyzing the target genes and signaling pathways of all 32 up-regulated miRNAs, we found that the PI3K-AKT signaling pathway has the potential to be targeted and regulated by most of the miRNAs in the Dlk1-Gtl2 region. In addition, we also re-analyzed miRNA sequencing data from public databases on Hu lambs (full sibling Hu lambs with high- and low-quality fur characteristics). Again, it was found that most of the up-regulated miRNAs in lambs with high-quality fur were also located in the Dlk1-Gtl2 region, whereas this patter was not present for down-regulated miRNAs.ConclusionSequencing of miRNAs in conjunction with public databases was employed to identify miRNAs within the imprinted Dlk1-Gtl2 region on chromosome 18, suggesting their potential roles as epigenetic regulators of fur traits. Small RNAs located at the Dlk1-Gtl2 locus were identified as having the potential to systematically regulate the PI3K-AKT signaling pathway, thereby indicating the relevance of the Dlk1-Gtl2/PI3K-AKT axis in the context of fur traits. Selection of parental specific expressed imprinted genes in the process of conserving and exploiting lamb fur traits should be emphasized.

Epigenetic mechanism of Gtl2-miRNAs causes the primitive sheep characteristics found in purebred Merino sheep

BackgroundIt is not uncommon for some individuals to retain certain primitive characteristics even after domestication or long-term intensive selection. Wild ancestors or original varieties of animals typically possess strong adaptability to environmental preservation, a trait that is often lacking in highly artificially selected populations. In the case of the Merino population, a world-renowned fine wool sheep breed, a phenotype with primitive coarse wool characteristic has re-emerged. It is currently unclear whether this characteristic is detrimental to the production of fine wool or whether it is linked to the adaptability of sheep. The underlying genetic/epigenetic mechanisms behind this trait are also poorly understood.ResultsThis study identified lambs with an ancestral-like coarse (ALC) wool type that emerged during the purebred breeding of Merino fine wool sheep. The presence of this primitive sheep characteristic resulted in better environmental adaptability in lambs, as well as improved fine wool yield in adulthood. Reciprocal cross experiments revealed that the ALC phenotype exhibited maternal genetic characteristics. Transcriptomic SNP analysis indicated that the ALC phenotype was localized to the imprinted Gtl2-miRNAs locus, and a significant correlation was found between the ALC wool type and a newly identified short Interstitial Telomeric Sequences (s-ITSs) at this locus. We further confirmed that a novel 38-nt small RNA transcribed from these s-ITSs, in combination with the previously reported 22-nt small RNAs cluster from the Gtl2-miRNAs locus, synergistically inhibited PI3K/AKT/Metabolic/Oxidative stress and subsequent apoptotic pathways in wool follicle stem cells, resulting in the ALC wool type. The necessity of Gtl2-miRNAs in controlling primary hair follicle morphogenesis, as well as the wool follicle type for ALC wool lambs, was verified using intergenic differentially methylated region-knockout mice.ConclusionThe ALC wool type of Merino sheep, which does not reduce wool quality but increases yield and adaptability, is regulated by epigenetic mechanisms in the imprinted Gtl2-miRNAs region on sheep chromosome 18, with the maternally expressed imprinted gene responsible for the ALC phenotype. This study highlights the significance of epigenetic regulation during embryonic and juvenile stages and emphasizes the advantages of early adaptation breeding for maternal parents in enhancing the overall performance of their offspring.

Genetic resistance to gastrointestinal parasites in sheep

Abstract Gastrointestinal nematode (GIN) infection is the most common disease affecting sheep production systems throughout the world, causing significant productive and economic losses. The control of these parasites was traditionally based on the use of anthelmintic drugs. However, continuous, extensive, and indiscriminate use of these drugs has led to the emergence and spread of strains of parasites resistant to the major chemical compounds used. This situation has led to considering alternative strategies for worm control. One of them is the selection of individuals for greater resistance to GINs. A number of phenotypic traits have been reported in the bibliography, including parasitological, biochemical, hematological, and immunological traits, but fecal egg count (FEC) is considered the primary and most practical measure of resistance. Genomic studies have reported polymorphisms associated with GIN resistance traits on almost all sheep chromosomes. Among other loci, FEC has been associated with the major histocompatibility complex (MHC) through quantitative trait loci (QTL) analysis and genome-wide association studies (GWAS). This review covers the principal aspects reported in the literature on several parameters considered to evaluate the resistance status of sheep to nematodes, mainly Haemonchus contortus , the correlation between resistance markers with economically important production traits, and the main genomic regions identified as relevant in determining the phenotype for resistance or susceptibility to GINs.

Genetic Parameters and Genomic Regions Underlying Growth and Linear Type Traits in Akkaraman Sheep.

In the current study, the genetic architecture of growth and linear type traits were investigated in Akkaraman sheep. Estimations of genomic heritability, genetic correlations, and phenotypic correlations were implemented for 17 growth and linear type traits of 473 Akkaraman lambs by the univariate and multivariate analysis of animal mixed models. Correspondingly, moderate heritability estimates, as well as high and positive genetic/phenotypic correlations were found between growth and type traits. On the other hand, 2 genome-wide and 19 chromosome-wide significant single nucleotide polymorphisms were found to be associated with the traits as a result of animal mixed model-based genome-wide association analyses. Accordingly, we propose several genes located on different chromosomes (e.g., PRDM2, PTGDR, PTPRG, KCND2, ZNF260, CPE, GRID2, SCD5, SPIDR, ZNF407, HCN3, TMEM50A, FKBP1A, TLE4, SP1, SLC44A1, and MYOM3) as putative quantitative trait loci for the 22 growth and linear type traits studied. In our study, specific genes (e.g., TLE4, PTGDR, and SCD5) were found common between the traits studied, suggesting an interplay between the genetic backgrounds of these traits. The fact that four of the proposed genes (TLE4, MYOM3, SLC44A1, and TMEM50A) are located on sheep chromosome 2 confirms the importance of these genomic regions for growth and morphological structure in sheep. The results of our study are therefore of great importance for the development of efficient selection indices and marker-assisted selection programs, as well as for the understanding of the genetic architecture of growth and linear traits in sheep.

NEW CANDIDATE GENES ASSOCIATED WITH SHOULDER HEIGHT OF SHEEP OF RUSSIAN MEAT MERINO BREED

The search for candidate genes and genetic markers associated with economically valuable traits of productive animals is of great scientific and practical importance for the agro-industrial complex. Of particular interest are studies aimed at finding associations for parametres which characterize features of build and growth of animals. The aim of this work was to search for single nucleotide polymorphisms and candidate genes associated with height at the shoulder, one of the main measurements that allows to judge the conformation characteristics of sheep. The object of the study was 12-month-old Russian Merino meat rams (n = 50), bred on the Stavropol Territory. Animal genotyping was performed using DNA biochips of Ovine Infinium HD BeadChip 600K; genome-wide association analysis was performed using PLINK V.1.07 software. In the course of the work done, significant associations were found between the height at the shoulder and single nucleotide substitutions of 3, 10 and 18 chromosomes of sheep of Russian meat merino breed. Polymorphisms are located in the region of introns of protein-coding genes (rs422409900, rs413412092) and intergenic regions (rs414158152, rs430384069, rs415673748). A negative regression coefficient is observed in five analyzed substitutions. Location of analyzed substitutions in relation to the latest Oar_rambouillet_v1.0 genome annotation allowed to reveal 4 new candidate genes associated with height at the shoulder of sheep: RALGAPA1, MEIS1, AFTPH, SLC25A21. Further studies should be aimed at confirming the effect of the proposed candidate genes on the phenotype of animals, as well as confirming the relationship of rs414158152, rs430384069, rs42240990, rs415673748, and rs413412092 polymorphisms with conformation parametres of sheep.

State of the art on the physical mapping of the Y-chromosome in the Bovidae and comparison with other species - A review.

The next generation sequencing has significantly contributed to clarify the genome structure of many species of zootechnical interest. However, to date, some portions of the genome, especially those linked to a heterogametic nature such as the Y chromosome, are difficult to assemble and many gaps are still present. It is well known that the fluorescence in situ hybridization (FISH) is an excellent tool for identifying genes unequivocably mapped on chromosomes. Therefore, FISH can contribute to the localization of unplaced genome sequences, as well as to correct assembly errors generated by comparative bioinformatics. To this end, it is necessary to have starting points; therefore, in this study, we reviewed the physically mapped genes on the Y chromosome of cattle, buffalo, sheep, goats, pigs, horses and alpacas. A total of 208 loci were currently mapped by FISH. 89 were located in the male-specific region of the Y chromosome (MSY) and 119 were identified in the pseudoautosomal region (PAR). The loci reported in MSY and PAR were respectively: 18 and 25 in Bos taurus, 5 and 7 in Bubalus bubalis, 5 and 24 in Ovis aries, 5 and 19 in Capra hircus, 10 and 16 in Sus scrofa, 46 and 18 in Equus caballus. While in Vicugna pacos only 10 loci are reported in the PAR region. The correct knowledge and assembly of all genome sequences, including those of genes mapped on the Y chromosome, will help to elucidate their biological processes, as well as to discover and exploit potentially epistasis effects useful for selection breeding programs.

A comprehensive genome-wide scan detects genomic regions related to local adaptation and climate resilience in Mediterranean domestic sheep

BackgroundThe management of farm animal genetic resources and the adaptation of animals to climate change will probably have major effects on the long-term sustainability of the livestock sector. Genomic data harbour useful relevant information that needs to be harnessed for effectively managing genetic resources. In this paper, we report the genome characterization of the highly productive Mediterranean Chios dairy sheep and focus on genetic diversity measures related with local adaptation and selection and the genetic architecture of animal resilience to weather fluctuations as a novel adaptative trait linked to climate change.ResultsWe detected runs of homozygosity (ROH) and heterozygosity (ROHet) that revealed multiple highly homozygous and heterozygous hotspots across the Chios sheep genome. A particularly highly homozygous region was identified on chromosome 13 as a candidate of directional genetic selection associated with milk traits, which includes annotated genes that were previously shown to be linked to local adaptation to harsh environmental conditions. Favourable heterozygosity related with a potentially protective role against livestock diseases and enhanced overall fitness was revealed in heterozygous-rich regions on sheep chromosomes 3, 10, 13 and 19. Furthermore, genomic analyses were conducted on sheep resilience phenotypes that display changes in milk production in response to weather variation. Sheep resilience to heat stress was a significantly heritable trait (h2 = 0.26) and genetically antagonistic to milk production. Genome-wide association and regional heritability mapping analyses revealed novel genomic markers and regions on chromosome 5 that were significantly associated with sheep resilience to climate change. Subsequently, an annotation analysis detected a set of genes on chromosome 5 that were associated with olfactory receptor complexes that could participate in heat stress mitigation through changes in respiration rate and respiratory evaporation. Other genes were grouped in previously reported biological processes relevant to livestock heat dissipation, including stress and immune response.ConclusionsOur results may contribute to the optimal management of sheep genetic resources and inform modern selective breeding programmes that aim at mitigating future environmental challenges towards sustainable farming, while better balancing animal adaptation and productivity. Our results are directly relevant to the studied breed and the respective environmental conditions; however, the methodology may be extended to other livestock species of interest.

Association Analysis between SPP1, POFUT1 and PRLR Gene Variation and Milk Yield, Composition and Coagulation Traits in Sarda Sheep.

Simple SummaryThe purpose of our research was to analyze the association between the three candidate genes secreted phosphoprotein 1 (SPP1), protein O-fucosyltransferase 1 (POFUT1) and prolactin receptor (PRLR) with milk production, quality and coagulation properties in 380 Sarda dairy sheep. Results revealed an association between SPP1 and somatic cells count, in line with the function of this gene and with its genomic position. We revealed an association of POFUT1 variation with milk coagulation properties, and PRLR with quality. This information can be useful for future breeding schemes in sheep.Many studies focus on the identification of genomic regions that undergo selective processes, where evidence of selection is revealed and positional candidate genes are identified. The aim of the research was to evaluate the association between positional candidate genes, namely secreted phosphoprotein 1 (SPP1, sheep chromosome Ovis aries OAR6, 36.651–36.658 Mb), protein O-fucosyltransferase 1 (POFUT1, OAR13, 61.006–61.027 Mb) and prolactin receptor (PRLR, OAR16, 38.969–39.028 Mb) with milk yield, composition and coagulation traits. Eight single nucleotide polymorphisms (SNPs) mapping to the three genes were genotyped in 380 Sarda dairy sheep. Statistical analysis revealed an association between SNP rs161844011 at SPP1 (chromosome position Oar_v3 OAR6:36651870, gene region exon 7) and somatic cell score, while POFUT1 SNP rs424501869 (OAR13:61007495, intron 1) was associated with curd firmness both 45 and 60 min after rennet addition (p = 0.015 and p = 0.007, respectively). SNP rs400874750 at PRLR gene (OAR16:39004070, intron 2) had a significant association with lactose content (p = 0.020), somatic cell score (p = 0.038), rennet coagulation time (p = 0.018) and curd firming time (p = 0.047). The outcome of this research confirmed predictions based on genomic studies, producing new information regarding the SPP1, POFUT1 and PRLR genes, which may be useful for future breeding schemes.

Highly efficient synchronization of sheep skin fibroblasts at G2/M phase and isolation of sheep Y chromosomes by flow cytometric sorting

At present, based on whole genome sequencing, sequences and genes annotation of the sheep (Ovis aries) Y chromosome are still absent. The isolation of Y chromosomes followed by sequencing has been approved as an effective approach to analyze this complex chromosome in other species. In this study, we established a highly efficient synchronization method for G2/M phase of sheep fibroblasts, which was successfully applied to flow-sorting chromosomes of sheep, with a focus on isolation and sequencing of the ovine Y chromosome. The isolated (~80,000) Y chromosomes were verified by fluorescence quantitative real-time polymerase chain reaction, further confirmed by fluorescence in situ hybridization, and amplified by the MALBAC method before next-generation sequencing. The sequence results indicated that 68.90% of reads were Y chromosome-related sequences as they are homologous to the bovine Y chromosome. The remaining 31.1% of reads were aligned to the sheep reference genome, including 13.57% reads to chromosome X and 6.68% to chromosome 17. Importantly, the paired-end reads that are properly aligned to the bovine Y sequence assembly accounted for 46.49%, indicating the success in the ovine Y chromosome isolation and the high quality of the Y chromosome sequences. This study not only set up a foundation for future sequencing, assembly and annotation of the ovine Y chromosome, but also provide a validated approach to overcoming difficulties in sequencing Y chromosome in other mammalian species.

Cytogenetic Characterization of a Small Evolutionary Rearrangement Involving Chromosomes BTA21 and OAR18

Both cattle (Bos taurus) and sheep (Ovis aries) belong to the Bovidae family but to different subfamilies, Bovinae and Caprinae, respectively. From a chromosomal point of view, apart from the already known centric fusions (that occurred during the evolutionary process in the Bovidae family) and the small differences in the chromosome classification, the 2 karyotypes are very similar in banding patterns. In this study, the combination of bioinformatics techniques and physical mapping of DNA markers enabled the identification of a micro-rearrangement, a small inversion involving bovine chromosome 21 (BTA21) and the corresponding sheep chromosome 18 (OAR18). The aim of this study was the cytogenetic characterization of this difference in genomic assemblies between cattle and sheep in this single chromosome region. To verify the inversion in FISH experiments, we used the BACs 442H08 and 222H03 from the INRA library and BACs 134H22 and 436P08 from the sheep-specific CHORI library. The results confirmed the presence of the inverted fragment in sheep compared to the cattle genome. Genomic rearrangements may have consequences depending on their influence on gene activity, but in this case no gene or transcribed DNA portion seemed to be involved. In conclusion, we showed for the first time, concerning autosomes, that besides the already known centric fusions also other differences exist between the bovine and sheep karyotypes. Furthermore, we demonstrated that the combination of a bioinformatics approach and physical mapping is a valid tool for the identification of currently unknown rearrangements between related species.

Karyotype variability of the Ukrainian Mountain-Carpathian sheep breed

Abstract. The main purpose of this work was to describe the karyotype variability of the Ukrainian Mountain-Carpathian sheep breed. Cytogenetic studies were carried out on a group of 25 sheep from that breed. The cultivation of lymphocytes, preparing the cytogenetic samples, classification and registration of chromosome aberrations were held using conventional methods. It was established that the number and structure of the chromosomes of sheep of the Ukrainian Mountain-Carpathian breed correspond to the norm typical for this species of animals. Chromosomal variability of the studied population includes 19.22% of numerical and structural aberrations. By the number and structure of the chromosome set the Ukrainian Mountain-Carpathian sheep breed does not differ from other breeds of sheep. Accurate identification of individual chromosomes with routine coloring allows using cytogenetic studies in breeding practice in sheep breeding.

New insights into mammalian sex chromosome structure and evolution using high-quality sequences from bovine X and Y chromosomes

BackgroundMammalian X chromosomes are mainly euchromatic with a similar size and structure among species whereas Y chromosomes are smaller, have undergone substantial evolutionary changes and accumulated male specific genes and genes involved in sex determination. The pseudoautosomal region (PAR) is conserved on the X and Y and pair during meiosis. The structure, evolution and function of mammalian sex chromosomes, particularly the Y chromsome, is still poorly understood because few species have high quality sex chromosome assemblies.ResultsHere we report the first bovine sex chromosome assemblies that include the complete PAR spanning 6.84 Mb and three Y chromosome X-degenerate (X-d) regions. The PAR comprises 31 genes, including genes that are missing from the X chromosome in current cattle, sheep and goat reference genomes. Twenty-nine PAR genes are single-copy genes and two are multi-copy gene families, OBP, which has 3 copies and BDA20, which has 4 copies. The Y chromosome X-d1, 2a and 2b regions contain 11, 2 and 2 gametologs, respectively.ConclusionsThe ruminant PAR comprises 31 genes and is similar to the PAR of pig and dog but extends further than those of human and horse. Differences in the pseudoautosomal boundaries are consistent with evolutionary divergence times. A bovidae-specific expansion of members of the lipocalin gene family in the PAR reported here, may affect immune-modulation and anti-inflammatory responses in ruminants. Comparison of the X-d regions of Y chromosomes across species revealed that five of the X-Y gametologs, which are known to be global regulators of gene activity and candidate sexual dimorphism genes, are conserved.

Genome-wide association study of first lambing age and lambing interval in sheep

First lambing age and lambing interval are among the most important reproductive traits affecting profitability of sheep breeding systems. Genome-wide association studies (GWAS) were performed on estimated breeding values (EBVs) for age at first lambing and the lambing interval between first and second lambing in Lori-Bakhtiari ewes. Animals were genotyped using the Illumina Ovine SNP50 K genotyping array. Two single nucleotide polymorphisms (SNPs) associated with lambing interval were identified on chromosomes 1 and 2 (Bonferroni adjusted P-value<0.05). No SNP associated with first lambing age was detected. SNP s50067.1 on sheep chromosome 2: 86798921 is within a candidate gene; ADAMTS like 1 (ADAMTSL1), known to be involved in proteolysis, peptidase activity, biological process and reproductive pathways in mammalian species. No candidate gene was detected for SNP OAR1_57568179.1 on chromosome 1. The results of the present study may provide new insights into the genomic regions involved in reproductive traits of sheep.

Detection of copy number variation and selection signatures on the X chromosome in Chinese indigenous sheep with different types of tail.

ObjectiveChinese indigenous sheep breeds can be classified into the following three categories by their tail morphology: fat-tailed, fat-rumped and thin-tailed sheep. The typical sheep breeds corresponding to fat-tailed, fat-rumped, and thin-tailed sheep are large-tailed Han, Altay, and Tibetan sheep, respectively. Detection of copy number variation (CNV) and selection signatures provides information on the genetic mechanisms underlying the phenotypic differences of the different sheep types.MethodsIn this study, PennCNV software and F-statistics (FST) were implemented to detect CNV and selection signatures, respectively, on the X chromosome in three Chinese indigenous sheep breeds using ovine high-density 600K single nucleotide polymorphism arrays.ResultsIn large-tailed Han, Altay, and Tibetan sheep, respectively, a total of six, four and 22 CNV regions (CNVRs) with lengths of 1.23, 0.93, and 7.02 Mb were identified on the X chromosome. In addition, 49, 34, and 55 candidate selection regions with respective lengths of 27.49, 16.47, and 25.42 Mb were identified in large-tailed Han, Altay, and Tibetan sheep, respectively. The bioinformatics analysis results indicated several genes in these regions were associated with fat, including dehydrogenase/reductase X-linked, calcium voltage-gated channel subunit alpha1 F, and patatin like phospholipase domain containing 4. In addition, three other genes were identified from this analysis: the family with sequence similarity 58 member A gene was associated with energy metabolism, the serine/arginine-rich protein specific kinase 3 gene was associated with skeletal muscle development, and the interleukin 2 receptor subunit gamma gene was associated with the immune system.ConclusionThe results of this study indicated CNVRs and selection regions on the X chromosome of Chinese indigenous sheep contained several genes associated with various heritable traits.

Chromosome synapsis, recombination and epigenetic modification in rams heterozygous for metacentric chromosome 3 of the domestic sheep Ovis aries and acrocentric homologs of the argali Ovis ammon

Hybridization of domestic animal breeds with their wild relatives is a promising method for increasing the genetic diversity of farm animals. Resource populations derived from the hybridization of various breeds of domestic sheep with mouflon and argali are an important source of breeding material. The karyotypes of argali and domestic sheep differ for a Robertsonian translocation, which occurred in the common ancestor of mouflon and domestic sheep (Ovis aries) due to the centric fusion of chromosomes 5 and 11 of the argali (O. ammon) into chromosome 3 of sheep. It is known that heterozygosity for translocation can lead to synapsis, recombination and chromosome segregation abnormalities in meiosis. Meiosis in the heterozygotes for translocation that distinguishes the karyotypes of sheep and argali has not yet been studied. We examined synapsis, recombination, and epigenetic modification of chromosomes involved in this rearrangement in heterozygous rams using immunolocalization of key proteins of meiosis. In the majority of cells, we observed complete synapsis between the sheep metacentric chromosome and two argali acrocentric chromosomes with the formation of a trivalent. In a small proportion of cells at the early pachytene stage we observed delayed synapsis in pericentromeric regions of the trivalent. Unpaired sites were subjected to epigenetic modification, namely histone H2A.X phosphorylation. However, by the end of the pachytene, these abnormalities had been completely eliminated. Asynapsis was replaced by a nonhomologous synapsis between the centromeric regions of the acrocentric chromosomes. By the end of the pachytene, the γH2A.X signal had been preserved only at the XY bivalent and was absent from the trivalent. The translocation trivalent did not differ from the normal bivalents of metacentric chromosomes for the number and distribution of recombination sites as well as for the degree of centromeric and crossover interference. Thus, we found that heterozygosity for the domestic sheep chromosome 3 and argali chromosomes 5 and 11 does not cause significant alterations in key processes of prophase I meiosis and, therefore, should not lead to a decrease in fertility of the offspring from interspecific sheep hybridization.

The search for the Booroola (FecB) mutation

Sheep derived from the Booroola Merino strain carry an autosomal mutation (FecB) that increases ovulation rate and litter size. One approach to characterize the genetic mutation is to locate the gene using positional cloning. The locus has been mapped to a region between genes for secreted phosphoprotein 1 (SPP1) and epidermal growth factor (EGF) on sheep chromosome 6. Analysis of possible candidate genes have excluded a number of genes associated with control of reproduction including genes from chromosome 6. Attempts to define close flanking markers and clone the region of DNA containing the mutation are now in progress. We have cloned additional markers and developed a linkage map showing that the FecB locus maps towards the centromere on chromosome 6. We have developed a yeast artificial chromosome (YAC) library for the sheep and begun screening the library to identify large DNA clones spanning the FecB region. These will be used to locate the mutation and shed light on how the mutation increases ovulation rate in Booroola sheep.

Dosage Compensation and Gene Expression of the X Chromosome in Sheep.

Ohno’s hypothesis predicts that the expression of the single X chromosome in males needs compensatory upregulation to balance its dosage with that of the diploid autosomes. Additionally, X chromosome inactivation ensures that quadruple expression of the two X chromosomes is avoided in females. These mechanisms have been actively studied in mice and humans but lag behind in domestic species. Using RNA sequencing data, we analyzed the X chromosome upregulation in sheep fetal tissues from day 135 of gestation under control, over or restricted maternal diets (100%, 140% and 60% of National Research Council Total Digestible Nutrients), and in conceptuses, juvenile, and adult somatic tissues. By computing the mean expression ratio of all X-linked genes to all autosomal genes (X:A), we found that all samples displayed some levels of X chromosome upregulation. The degrees of X upregulation were not significant (P-value = 0.74) between ovine females and males in the same somatic tissues. Brain, however, displayed complete X upregulation. Interestingly, the male and female reproduction-related tissues exhibited divergent X dosage upregulation. Moreover, expression upregulation of the X chromosome in fetal tissues was not affected by maternal diets. Maternal nutrition, however, did change expression levels of several X-linked genes, such as sex determination genes SOX3 and NR0B1. In summary, our results showed that X chromosome upregulation occurred in nearly all sheep somatic tissues analyzed, thus support Ohno’s hypothesis in a new species. However, the levels of upregulation differed by different subgroups of genes such as those that are house-keeping and “dosage-sensitive”.

Genome-wide association study to identify genomic regions affecting prolificacy in Lori-Bakhtiari sheep.

Several causative mutations in candidate genes affecting prolificacy have been detected in various sheep breeds. A genome-wide association study was performed on estimated breeding values for litter size in Lori-Bakhtiari sheep. Prolific ewes with twinning records and others with only singleton records were genotyped using the medium-density Illumina Ovine SNP50 array. Four single nucleotide polymorphisms (SNPs) associated with litter size were identified on chromosomes 3, 6 and 22. The region on sheep chromosome 3 between 75739167 and 75745152bp included two significant SNPs (s52383.1 and OAR3_80038014_X.1) in high linkage disequilibrium with each other. The region that surrounds these SNPs contains a novel putative candidate gene: luteinizing hormone/choriogonadotropin receptor (LHCGR), known to be involved in ovarian steroidogenesis and organism-specific biosystem pathways in sheep. Known prolificacy genes BMPR1B, BMP15 and GDF9 were not associated with litter size in Lori-Bakhtiari sheep, suggesting that other biological mechanisms could be responsible for the trait's variation in this breed.

Complete mitogenomes from Kurdistani sheep: abundant centromeric nuclear copies representing diverse ancestors

The geographical centre of domestication and species diversity for sheep (Ovis aries) lies around the Kurdistan region of Northern Iraq, within the ‘Fertile Crescent’. From whole genome sequence reads, we assembled the mitochondrial genomes (mtDNA or mitogenome) of five animals of the two main Kurdistani sheep breeds Hamdani and Karadi and found they fitted into known sheep haplogroups (or matrilineages), with some SNPs. Haplotyping 31 animals showed presence of the main Asian (hpgA) and European (hpgB) haplogroups, as well as the rarer Anatolian haplogroup hpgC. From the sequence reads, near-complete genomes of mitochondria from wild sheep species (or subspecies), and even many sequences similar to goat (Capra) mitochondria, could be extracted. Analysis suggested that these polymorphic reads were nuclear mitochondrial DNA segments (numts). In situ hybridization with seven regions of mitochondria chosen from across the whole genome showed strong hybridization to the centromeric regions of all autosomal sheep chromosomes, but not the Y. Centromeres of the three submetacentric pairs and the X chromosomes showed fewer copies of numts, with varying abundance of different mitochondrial regions. Some mitochondrial-nuclear transfer presumably occurred before species divergence within the genus, and there has been further introgression of sheep mitochondrial sequences more recently. This high abundance of nuclear mitochondrial sequences is not reflected in the whole nuclear genome assemblies, and the accumulation near major satellite sequences at centromeres was unexpected. Mitochondrial variants including SNPs, numts and heteroplasmy must be rigorously validated to interpret correctly mitochondrial phylogenies and SNPs.

Mapping the four-horned locus and testing the polled locus in three Chinese sheep breeds.

Four-horned sheep are an ideal animal model for illuminating the genetic basis of horn development. The objective of this study was to locate the genetic region responsible for the four-horned phenotype and to verify a previously reported polled locus in three Chinese breeds. A genome-wide association study (GWAS) was performed using 34 two-horned and 32 four-horned sheep from three Chinese indigenous breeds: Altay, Mongolian and Sishui Fur sheep. The top two significant single nucleotide polymorphisms (SNPs) associated with the four-horned phenotype were both located in a region spanning positions 132.6 to 132.7Mb on sheep chromosome 2. Similar locations for the four-horned trait were previously identified in Jacob, Navajo-Churro, Damara and Sishui Fur sheep, suggesting a common genetic component underlying the four-horned phenotype. The two identified SNPs were both downstream of the metaxin 2 (MTX2) gene and the HOXD gene cluster. For the top SNP-OAR2:g.132619300G>A-the strong associations of the AA and AG genotypes with the four-horned phenotype and the GG genotype with the two-horned phenotype indicated the dominant inheritance of the four-horned trait. No significant SNPs for the polled phenotype were identified in the GWAS analysis, and a PCR analysis for the detection of the 1.8-kb insertion associated with polled sheep in other breeds failed to verify the association with polledness in the three Chinese breeds. This study supports the hypothesis that two different loci are responsible for horn existence and number. This study contributes to the understanding of the molecular regulation of horn development and enriches the knowledge of qualitative traits in domestic animals.