Characterization of microRNAs from sheep (<Emphasis Type="Italic">Ovis aries</Emphasis>) using computational and experimental analyses

Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA* and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA* had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3′UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA* in sheep.     

All sheeps and sizes: a genetic investigation of mature body size across sheep breeds reveals a polygenic nature

Mature body size is genetically correlated with growth rate, an important economic trait in the sheep industry. Mature body size has been studied extensively in humans as well as cattle and other domestic animal populations but not in sheep. Six‐hundred and sixteen ewes, across 22 breeds, were measured for 28 linear measurements representing various skeletal parts. PCA from these measures generated principal components 1 and 2 which represented 66 and 7% of the phenotypic variation respectively. Two‐hundred and twenty sheep were genotyped on the Illumina Ovine HD beadchip for a GWAS investigating mature body size and linear body measurements. Forty‐six (Bonferroni P < 0.05) SNP associations across 14 chromosomes were identified utilizing principal component 1, representing overall body size, revealing mature body size to have fewer loci of large effect than other domestic species such as dogs and horses. Genome‐wide associations for individual linear measures identified major quantitative trait loci for withers height and ear length. Withers height was associated (Bonferroni P < 0.05) with 12 SNPs across six chromosomes whereas ear length was associated with a single locus on chromosome 3, containing MSRB3. This analysis identified several loci known to be associated with mature body size in other species such as NCAPG, LCORL, and HMGA2. Mature body size is more polygenic in sheep than other domesticated species, making the development of genomic selection for the trait the most efficient option for maintaining or reducing mature body size in sheep.     

QTL and association analysis for skin and fibre pigmentation in sheep provides evidence of a major causative mutation and epistatic effects

The pursuits of white features and white fleeces free of pigmented fibre have been important selection objectives for many sheep breeds. The cause and inheritance of non‐white colour patterns in sheep has been studied since the early 19th century. Discovery of genetic causes, especially those which predispose pigmentation in white sheep, may lead to more accurate selection tools for improved apparel wool. This article describes an extended QTL study for 13 skin and fibre pigmentation traits in sheep. A total of 19 highly significant, 10 significant and seven suggestive QTL were identified in a QTL mapping experiment using an Awassi × Merino × Merino backcross sheep population. All QTL on chromosome 2 exceeded a LOD score of greater than 4 (range 4.4–30.1), giving very strong support for a major gene for pigmentation on this chromosome. Evidence of epistatic interactions was found for QTL for four traits on chromosomes 2 and 19. The ovine TYRP1 gene on OAR 2 was sequenced as a strong positional candidate gene. A highly significant association (P < 0.01) of grandparental haplotypes across nine segregating SNP/microsatellite markers including one non‐synonymous SNP with pigmentation traits could be shown. Up to 47% of the observed variation in pigmentation was accounted for by models using TYRP1 haplotypes and 83% for models with interactions between two QTL probabilities, offering scope for marker‐assisted selection for these traits.     

Ear type in sheep is associated with the MSRB3 locus

Ear morphology is an important determinant of sheep breeds. It includes different variable traits such as ear size and erectness, suggesting a polygenic architecture. Here, we performed a comprehensive genome-wide analysis to identify regions under selection for ear morphology in 515 sheep from 17 breeds fixed for diverse ear phenotypes using 34k SNP genotyping data. GWASs for two ear type traits, size and erectness, revealed a single genome-wide significant association on ovine chromosome 3. The derived marker alleles were enriched in sheep with large and/or floppy ears. The GWAS signal harboured the MSRB3 gene encoding methionine sulphoxide reductase B3, which has already been found to be associated with different ear types in other species. We attempted whole-genome resequencing to identify causal variant(s) within a 1 Mb interval around MSRB3. This experiment excluded major copy number variants in the interval, but failed to identify a compelling candidate causal variant. Fine-mapping suggested that the causal variant for large floppy ears most likely resides in a 175 kb interval downstream of the MSRB3 coding region.     

Cloning and ontogenetic expression of the uncoupling protein 1 gene <Emphasis Type="Italic">UCP1</Emphasis> in sheep

The uncoupling protein 1 (UCP1) is an indicator of brown adipocytes and is involved in the control of body temperature and regulation of energy balance. It abundantly expresses in newborns and has important functions in adults. However, little information was known on UCP1 gene expression in young and adolescent sheep. In this study, we cloned and identified the full-length DNA and cDNA sequences of the ovine UCP1 gene, which were 6659 bp and 1621 bp, respectively, and predicted the location of the gene on chromosome 17. Forty-eight animals with an equal number of males and females each for both Guangling Large Tail sheep (GLT) and Small Tail sheep Han (STH) sheep were used to study the ontogenetic expression of UCP1 mRNA in eight adipose tissues by quantitative real-time polymerase chain reaction (PCR). The results showed that the mRNA was expressed in all tissues studied and at all stages from 2 to 12 months of age. Nevertheless, the mRNA in perirenal fat was expressed significantly higher than that in other tissues and lower in superficial fat than in deep deposits. The highest expression was observed in animals at 2 months of age and then decreased gradually with age. Global expression in GLT was significantly higher than that in STH. Interactions between tissue and breed and age also influenced the mRNA expression significantly. In addition, the mRNA expression was associated with the single nucleotide polymorphism (SNP) haplotypes detected in the cDNA of the gene.     

Maternal and paternal genetic diversity of ancient sheep in Estonia from the Late Bronze Age to the post‐medieval period and comparison with other regions in Eurasia

Sheep were among the first domesticated animals to appear in Estonia in the late Neolithic and became one of the most widespread livestock species in the region from the Late Bronze Age onwards. However, the origin and historical expansion of local sheep populations in Estonia remain poorly understood. Here, we analysed fragments of the hypervariable D‐loop of mitochondrial DNA (mtDNA; 213 bp) and the Y‐chromosome SRY gene (130 bp) extracted from 31 archaeological sheep bones dated from approximately 800 BC to 1700 AD. The ancient DNA data of sheep from Estonia were compared with ancient sheep from Finland as well as a set of contemporary sheep breeds from across Eurasia in order to place them in a wider phylogeographical context. The analysis shows that: (i) 24 successfully amplified and analysed mtDNA sequences of ancient sheep cluster into two haplogroups, A and B, of which B is predominant; (ii) four of the ancient mtDNA haplotypes are novel; (iii) higher mtDNA haplotype diversity occurred during the Middle Ages as compared to other periods, a fact concordant with the historical context of expanding international trade during the Middle Ages; (iv) the proportion of rarer haplotypes declined during the expansion of sheep from the Near Eastern domestication centre to the northern European region; (v) three male samples showed the presence of the characteristic northern European haplotype, SNP G‐oY1 of the Y‐chromosome, and represent the earliest occurrence of this haplotype. Our results provide the first insight into the genetic diversity and phylogeographical background of ancient sheep in Estonia and provide basis for further studies on the temporal fluctuations of ancient sheep populations.     

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.     

Conserved and novel miRNAs in the legume <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> in response to stress

MicroRNAs (miRNAs) are small RNA molecules recognized as important regulators of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data. We are interested in the identification of miRNAs in Phaseolus vulgaris (common bean) to uncover different plant strategies to cope with adverse conditions and because of its relevance as a crop in developing countries. Here we present the identification of conserved and candidate novel miRNAs in P. vulgaris present in different organs and growth conditions, including drought, abscisic acid treatment, and Rhizobium infection. We also identified cDNA sequences in public databases that represent the corresponding miRNA precursors. In addition, we predicted and validated target mRNAs amongst reported EST and cDNAs for P. vulgaris. We propose that the novel miRNAs present in common bean and other legumes, are involved in regulation of legume-specific processes including adaptation to diverse external cues. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A genome-wide significant association on chromosome 15 for congenital entropion in Swiss White Alpine sheep

Entropion is a known congenital disorder in sheep presumed to be heritable but no causative genetic variant has been reported. Affected lambs show a variable inward rolling of the lower eyelids leading to blindness in severe cases. In Switzerland, the Swiss White Alpine (SWA) breed showed a significantly higher prevalence for entropion than other breeds. A GWAS using 150 SWA sheep (90 affected lambs and 60 controls), based on 600k SNP data, revealed a genome-wide significant signal on chromosome 15. The 0.2 Mb associated region contains functional candidate genes, SMTNL1 and CTNND1. Pathogenic variants in human CTNND1 cause blepharocheilodontic syndrome 2, a rare disorder including eyelid anomalies, and SMTNL1 regulates contraction and relaxation of skeletal and smooth muscle. WGS of a single entropion-affected lamb revealed two private missense variants in SMTNL1 and CTNND1. Subsequent genotyping of both variants in 231 phenotyped SWA sheep was performed. The SMTNL1 variant p.(Asp452Asn) affects an evolutionary conserved residue within an important domain and represents a rare allele, which occurred also in controls. The p.(Glu943Lys) variant in CTNND1 represents a common variant unlikely to cause entropion as the mutant allele occurred more frequently in non-affected sheep. Therefore, we propose that these protein-changing variants are unlikely to explain the phenotype. Additionally, WGS of three further disconcordant pairs of full siblings was carried out but revealed no obvious causative variant. Finally, we conclude that entropion represents a more complex disease caused by different non-coding regulatory variants.     

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 75 739 167 and 75 745 152 bp 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.     

Genome‐wide association analysis identifies the genetic basis of fat deposition in the tails of sheep (Ovis aries)

Fat‐tailed sheep (Ovis aries) can survive in harsh environments and satisfy human's intake of dietary fat. However, the animals require more feed, which increases the cost of farming. Thus, most farmers currently prefer thin‐tailed, short‐tailed or docked sheep. To date, the molecular mechanism of the formation of fat tails in sheep has not been completely elucidated. Here, we conducted a genome‐wide association study using phenotypes and genotypes (the Ovine Infinium HD SNP BeadChip genotype data) of two breeds of contrasting tail types (78 Small‐tailed and 78 Large‐tailed Han sheep breeds) to identify functional genes and variants associated with fat deposition. We identified four significantly (rs416433540, rs409848439, rs408118325 and rs402128848) and three approximately associated autosomal SNPs (rs401248376, rs402445895 and rs416201901). Gene annotation indicated that the surrounding genes (CREB1, STEAP4, CTBP1 and RIP140, also known as NRIP1) function in lipid storage or fat cell regulation. Furthermore, through an X‐chromosome‐wide association analysis, we detected significantly associated SNPs in the OARX: 88–89 Mb region, which could be a strong candidate genomic region for fat deposition in tails of sheep. Our results represent a new genomic resource for sheep genetics and breeding. In addition, the findings provide novel insights into genetic mechanisms of fat deposition in the tail of sheep and other mammals.     

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.     

A genome‐wide association study reveals candidate genes for the supernumerary nipple phenotype in sheep (Ovis aries)

Genome‐wide association studies (GWASs) have been widely applied in livestock to identify genes associated with traits of economic interest. Here, we conducted the first GWAS of the supernumerary nipple phenotype in Wadi sheep, a native Chinese sheep breed, based on Ovine Infinium HD SNP BeadChip genotypes in a total of 144 ewes (75 cases with four teats, including two normal and two supernumerary teats, and 69 control cases with two teats). We detected 63 significant SNPs at the chromosome‐wise threshold. Additionally, one candidate region (chr1: 170.723–170.734 Mb) was identified by haplotype‐based association tests, with one SNP (rs413490006) surrounding functional genes BBX and CD47 on chromosome 1 being commonly identified as significant by the two mentioned analyses. Moreover, Gene Ontology enrichment for the significant SNPs identified by the GWAS analysis was functionally clustered into the categories of receptor activity and synaptic membrane. In addition, pathway mapping revealed four promising pathways (Wnt, oxytocin, MAPK and axon guidance) involved in the development of the supernumerary nipple phenotype. Our results provide novel and important insights into the genetic mechanisms underlying the phenotype of supernumerary nipples in mammals, including humans. These findings may be useful for future breeding and genetics in sheep and other livestock.