Genome-wide analysis reveals population structure and selection in Chinese indigenous sheep breeds

Background

Traditionally, Chinese indigenous sheep were classified geographically and morphologically into three groups: Mongolian, Kazakh and Tibetan. Herein, we aimed to evaluate the population structure and genome selection among 140 individuals from ten representative Chinese indigenous sheep breeds: Ujimqin, Hu, Tong, Large-Tailed Han and Lop breed (Mongolian group); Duolang and Kazakh (Kazakh group); and Diqing, Plateau-type Tibetan, and Valley-type Tibetan breed (Tibetan group).

Results

We analyzed the population using principal component analysis (PCA), STRUCTURE and a Neighbor-Joining (NJ)-tree. In PCA plot, the Tibetan and Mongolian groups were clustered as expected; however, Duolang and Kazakh (Kazakh group) were segregated. STRUCTURE analyses suggested two subpopulations: one from North China (Kazakh and Mongolian groups) and the other from the Southwest (Tibetan group). In the NJ-tree, the Tibetan group formed an independent branch and the Kazakh and Mongolian groups were mixed. We then used the d_i statistic approach to reveal selection in Chinese indigenous sheep breeds. Among the 599 genome sequence windows analyzed, sixteen (2.7%) exhibited signatures of selection in four or more breeds. We detected three strong selection windows involving three functional genes: RXFP2, PPP1CC and PDGFD. PDGFD, one of the four subfamilies of PDGF, which promotes proliferation and inhibits differentiation of preadipocytes, was significantly selected in fat type breeds by the Rsb (across pairs of populations) approach. Two consecutive selection regions in Duolang sheep were obviously different to other breeds. One region was in OAR2 including three genes (NPR2, SPAG8 and HINT2) the influence growth traits. The other region was in OAR 6 including four genes (PKD2, SPP1, MEPE, and IBSP) associated with a milk production quantitative trait locus. We also identified known candidate genes such as BMPR1B, MSRB3, and three genes (KIT, MC1R, and FRY) that influence lambing percentage, ear size and coat phenotypes, respectively.

Conclusions

Based on the results presented here, we propose that Chinese native sheep can be divided into two genetic groups: the thin type (Tibetan group), and the fat type (Mongolian and Kazakh group). We also identified important genes that drive valuable phenotypes in Chinese indigenous sheep, especially PDGFD, which may influence fat deposition in fat type sheep.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1384-9) contains supplementary material, which is available to authorized users.     

IGFBP-3基因多态性及其与中国美利奴羊部分羊毛性状的关联性分析

采用PCR-SSCP方法对中国美利奴羊和哈萨克羊中IGFBP-3基因的多态性进行了检测, 并对不同基因型与中国美利奴羊部分羊毛性状间的关联性进行了分析。结果在位于内含子1区的一段178 bp的扩增产物经SSCP分析后出现了3种基因型, 基因型AA、AB和BB及等位基因A、B在中国美利奴羊中的频率分别为0.70、0.24、0.06和0.82、0.18; 在哈萨克羊中的频率分别为0.87、0.13、0.00和0.93、0.07。序列分析发现: 在该序列的122位碱基表现多态性 (g.122 G>T)。所研究的两个群体在该位点上均处于Hardy-Weinberg不平衡状态(P<0.01)。不同基因型对部分羊毛性状有一定的影响: 不同基因型个体在剪毛后体重和净毛率上没有明显差异。AA、AB及BB基因型个体的羊毛伸直长度逐渐变短, 其中AA与AB基因型之间差异极显著(P<0.01)。AA型个体的剪毛量和羊毛密度要明显低于AB型(P<0.01)和BB型个体(P<0.05); 羊毛纤维直径则明显高于AB型(P<0.01)和BB型(P<0.05)个体。     

Genetic variation of 5 SNPs of MC1R gene in Chinese indigenous sheep breeds

The purpose of this study was to assess genetic diversity, genetic differentiation relationship and population structure among 10 Chinese sheep populations using 5 single nucleotide polymorphisms (SNPs) in MC1R gene. The genetic diversity indices suggested that the intra-population variation levels of Chinese Merino and Large-tailed Han breeds were lowest than Kazakh Fat-Rumped. Chinese sheep breeds have maintained a high intra-population variation levels (95.23%). The genetic differentiation patterns and genetic relationships among Chinese sheep breeds displayed a high consistency with the traditional classification. The cluster trees were constructed by UPMGA method. The results showed that Chinese indigenous sheep populations have distinct genetic differentiation. The inter-population variation levels in Chinese sheep populations indicated three geographically independent domestication events have occurred. The Bayesian cluster analyses also showed a reliable clustering pattern, which revealed three major clusters in Chinese indigenous sheep populations (Mongolian group, Kazakh group and Tibetan group), except for Duolang and Minxian Black-fur. There were probably caused by different breeding history, geography isolation and different levels of inbreeding. The findings supported the related records in literature, ten sheep populations originated on different time stage from the primogenitor population and communicated genetically with each other in the process of natural and artificial selection, and in different ecological environment. It is concluded that Chinese indigenous sheep have higher genetic variation and diversity, genetic differentiation exist between Chinese sheep populations.     

Mitochondrial diversity and the origin of Iberian sheep

Mitochondrial DNA diversity was analysed in 19 Iberian and six foreign sheep breeds. Three mtDNA lineages (B, A and C) were found in the Iberian sheep, with type B clearly predominating over the others. The results were analysed for each of the morphologically determined breed groups in Iberian sheep: Merino, Entrefino, Churro and Iberian trunks. MtDNA lineage C was found only in the Iberian trunk composed of Montesina and Ojalada. These two populations had high mtDNA variability, and in the Iberian sheep only Merino Branco had more variation. The other three Merino types studied showed moderate variability, including the most authentic Merino, the Spanish Merino. These three Merinos clustered closely in a multidimensional scaling representation of distances, while the fourth breed (Merino Branco) showed a clear separation. As for the other two trunks, breeds from the Churro group showed greater maternal uniformity while results for populations included in the so-called Entrefino trunk seemed to have a more heterogeneous maternal origin. The results obtained are discussed with available data from nuclear markers and with morphological classifications, and all this information is analysed in relation to the origin of the different Iberian sheep breeds.     

On-station comparative analysis of reproductive and survival performance between Red Maasai,Dorper, and Merino sheep breeds

The reproductive performance of ewes and the survivability of lambs to weaning have a critical economic impact on sheep farming worldwide. Further, knowledge of major mortality causes allows an opportunity for improved flock management to evade financial losses. The maximum likelihood estimates for generalised linear mixed models and chi-square test methods were used to examine 971 mating records, 839 and 763 lambs born and weaned (singles or twins) from the Naivasha Sheep and Goats station in Kenya for the years 2011 to 2020 consisting of Dorper, Red Maasai (RedM), and Merino breeds. The RedM (P < 0.05) outperformed Dorper and Merino in weaning rate, whereas reproductive performance between the three breeds was not significantly different (P > 0.05) in litter size and multiple lambings per ewe lambing. On the one hand, Dorper significantly (P < 0.05) outperformed the other two breeds only in weaning weight per lamb born. In addition, among all the major causes of death, pneumonia appeared to be the one to which Dorper breeds were most susceptible (chi-square test, P < 0.05). According to the findings of this study, neither the Dorper nor the Merino sheep breeds were reproductively superior to the RedM in an extensive semi-arid production environment. In addition, Dorper's susceptibility to the leading causes of mortality, particularly pneumonia and sheep pox, were relatively high compared to other breeds and could be a precursor to massive economic losses for Dorper sheep producers. In contrast to the indigenous RedM breed, imported sheep breeds appeared to be more susceptible to major mortality-related under an extensive production system. Therefore, regardless of weaning weight, RedM breed production appears to be a more viable investment for small-scale farmers, particularly in semi-arid regions.     

绵羊ILK基因的克隆及其在毛囊生长期的表达

整合素连接激酶(ILK)是一种支架蛋白,在毛囊发育过程中发挥重要作用。文章利用PCR技术,首次获得绵羊ILK基因的编码区全长序列,并进行了生物信息学分析;同时对该基因的组织表达谱及其在不同绵羊品种毛囊生长期皮肤组织中的表达变化进行了研究。结果表明,绵羊ILK基因ORF全长1 359 bp,编码452个氨基酸。ILK蛋白结构经预测含有3个锚定重复序列和1个激酶结构域,并存在多个磷酸化位点和蛋白激酶C的磷酸化位点。半定量RT-PCR结果显示该基因在绵羊心脏、肝脏、脾脏、肺脏、肾脏、骨骼肌、皮肤和小肠组织中均有表达,皮肤、脾脏和肝脏中表达量较高;实时荧光定量PCR结果表明在3~5月份(毛囊生长起始期),ILK基因在中国美利奴超细型和哈萨克羊皮肤组织中的表达水平较高并均呈逐月上升趋势;在6~10月份(毛囊生长期),中国美利奴超细型皮肤ILK基因表达水平高于同期的哈萨克羊。分析认为ILK可能在调控绵羊次级毛囊生长发育过程中起一定作用。     

11个绵羊品种MSTN基因非翻译区的变异

利用PCR-RFLP技术对特克塞尔羊、夏洛莱羊、小尾寒羊、蒙古羊、乌珠穆沁羊、阿勒泰羊、呼伦贝尔羊、塔什库尔干羊、多浪羊、湖羊和岗巴羊11个品种的345个个体的肌肉生长抑制素(Myostatin, MSTN)基因非翻译区(UTR)的变异进行了多态性分析。结果表明大小为271 bp和1 003 bp的扩增片段经限制性内切酶MboⅡ和BsaⅠ酶切后表现多态, 经卡方检验所有品种在该基因座位均处于平衡状态(P>0.05), 3种基因型在11个绵羊品种中的分布差异极显著(P<0.01)。通过限制性内切酶HpyCH4Ⅳ 酶切实验, 证明我国9个地方绵羊品种不存在特克塞尔绵羊中发现的导致肌肉发达的SNP位点, 并在3′UTR区发现了个别碱基突变位点能够形成miRNA作用的靶基序, 测序表明3′UTR区的突变频率较高。     

绵羊皮肤中GHR、IGF-1和IGF-1R基因表达的发育性变化及品种特点

采用相对定量反转录多聚酶链式反应 (RT-PCR)方法, 以18S rRNA作内标, 研究了罗米丽(Romilly Hillys)×中国美利奴(新疆军垦型)杂交一代优质细毛羊和哈萨克粗毛羊皮肤中生长激素受体(GHR)、胰岛素样生长因子1(IGF-1)和胰岛素样生长因子1受体(IGF-1R) mRNA发育性变化并进行了品种间比较。分别于30、60、90、135、180和255日龄称重、采毛样, 并于30、90、135和255日龄采皮样。结果表明: 粗毛羊和细毛羊体重、羊毛生长的发育模式没有明显的差异, 30~135日龄体重迅速增加, 135~255日龄增重十分缓慢; 30~135日龄羊毛日增长逐渐增加, 135~180日龄羊毛生长十分缓慢, 而180~255日龄又上升到较高水平。粗毛羊皮肤中GHR mRNA在30~90日龄显著增加 (P<0.05), 90日龄达到高峰, 此后显著下降(P<0.05); 细毛羊在135日龄时GHR mRNA极显著地升高(P<0.01), 此后又极显著地下降。粗毛羊皮肤中IGF-1、IGF-1R mRNA 30~90日龄上升, 90日龄之后极显著下降(P<0.01); 细毛羊皮肤中IGF-1、IGF-1R mRNA出生时较高, 然后逐渐下降。品种之间比较, 细毛羊GHR mRNA出现高峰晚于粗毛羊, 135日龄高峰时显著地高于粗毛羊; 粗毛羊IGF-1、IGF-1R mRNA在90日龄出现高峰, 并极显著或显著地高于细毛羊; 粗毛羊90日龄前GHR、IGF-1和IGF-1R mRNA高于细毛羊, 之后低于细毛羊。结果提示: 绵羊皮肤中GHR、IGF-1和IGF-1R基因表达有特定的发育模式, 并存在品种差异。     

优良的BMPR-IB基因型可提高绵羊冷冻胚胎的受胎效果

以控制BooroolaMerino羊高繁殖力的BMPR-IB基因为候选基因,以小尾寒羊及其杂交羊、东北半细毛羊、澳洲美利奴羊、德国肉用美利奴羊、萨福克羊、特克塞尔羊、夏洛莱羊为试验对象,采用PCR-限制性片段长度多态性(PCR-RFLP)方法进行基因单核苷酸多态性(SNP)检测和基因型分析,同时研究基因对高繁殖力的影响.研究结果表明:小尾寒羊及其杂交羊、东北半细毛羊和夏洛莱羊群体中发现了与BooroolaMerino羊相同的A746G碱基突变,而小尾寒羊及其杂交羊群体的B等位基因频率明显高于其他2个品种.另外4个品种中未发现此突变.携带B等位基因的群体较非携带B等位基因群体排出更多的卵子,排卵后黄体直径较小.移植入冷冻胚胎后, 、B 和BB3种基因型群体的妊娠率分别为38.78%、45.71%和66.67%.由此推断,BMPR-IB基因突变很有可能从增加卵巢排卵数和提高胚胎着床及妊娠建立效率两个方面同时影响绵羊高繁殖力性状.所得BB型群体冻胚移植妊娠率明显高于 和B 型群体,已接近鲜胚移植水平,通过PCR-RFLP方法进行基因型分析,选用合适基因型群体作为胚胎移植受体,有可能为提高绵羊胚胎移植受胎率提供新的方向.     

DNA fingerprints of sheep using an M13 probe

The bacteriophage M13 DNA was used to detect hypervariable minisatellites in several families of Booroola sheep as well as Merino and Suffolk sheep. Digestion of sheep DNA gave rise to three to eight fragments with different restriction enzymes demonstrating considerable polymorphism between the different breeds. The length of informative DNA fragments varied in size from 6 to 20kb. The DNA fingerprints generated were individual specific and allowed for differentiation between closely related animals. The pattern obtained with sheep DNA was different from that observed with humans and other vertebrates in the proportion of high molecular weight DNA fragments present. Pedigree analysis of DNA patterns of dams and their offspring for several sets of twins and triplets showed a clear distinction between individuals and failed to reveal the presence of monozygosity.     

Genetic relationships among Spanish sheep using microsatellites

Five indigenous Spanish breeds of sheep, Churra, Latxa, Manchega, Rasa–Aragonesa and Merino, with Awassi sheep as a reference breed were genotyped for 19 DNA microsatellites. Allele frequencies and mean heterozygosities revealed the greatest genetic variation in Merino sheep and the lowest in Awassis. Differences in variability were not great in the other breeds studied. The dendrograms obtained based on genetic distances showed a large differentiation between Awassi sheep and the Spanish breeds, as was to be expected from their distinct genetic origin. Merinos appeared separated from the other four breeds, of which, according to a classification based on the fleece characteristics, Churra and Latxa belong to the churro type and Manchega and Rasa–Aragonesa to the so called entrefino type, though no clear separation was evident between the two types. These results suggest that morphological data alone are insufficient for determining relationships between breeds and that studies involving genetic markers may be of great assistance.     

The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep

Male Kazak sheep and Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90 and 120), were used in the present study to investigate the tissue distribution and developmental changes of ghrelin mRNA expression in abomasum; however, there was no 120-day-old Kazak sheep. After measurement of body weight, the tissues such as hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle were sampled. And the total RNA of different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR and real-time PCR. The results showed that (1) for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight of Kazak was significantly higher than that of Xinjiang (P<0.01); (2) Ghrelin mRNA existed in all the above tissues and was significantly higher in the abomasum than in other tissues (P<0.05); (3) the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results also demonstrated that from birth to day 90, ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01).     

The growth of sheep scab lesions in relation to sheep breed and time of the year

Numerically equal groups of between five and ten young Merino and Dorper sheep were artificially infested with sheep scab mites, Psoroptes ovis. Separate groups of sheep were infested during the early winter of two successive years as well as during the early summer of the second year. Lesion growth was measured at two-weekly intervals for a period of six weeks during the first winter and for eight weeks during the second winter and in the summer. The rate of lesion growth and mean lesion size on Merino sheep was significantly greater than that on Dorper sheep at each two-weekly recording during the winter of both years. The rate of growth of the lesions did not differ significantly between the two sheep breeds during summer, although, with the exception of eight weeks post-infestation, the mean lesion size on Merino sheep was significantly larger than that on Dorper sheep at each assessment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Merino and Merino-derived sheep breeds: a genome-wide intercontinental study

Background

Merino and Merino-derived sheep breeds have been widely distributed across the world, both as purebred and admixed populations. They represent an economically and historically important genetic resource which over time has been used as the basis for the development of new breeds. In order to examine the genetic influence of Merino in the context of a global collection of domestic sheep breeds, we analyzed genotype data that were obtained with the OvineSNP50 BeadChip (Illumina) for 671 individuals from 37 populations, including a subset of breeds from the Sheep HapMap dataset.

Results

Based on a multi-dimensional scaling analysis, we highlighted four main clusters in this dataset, which corresponded to wild sheep, mouflon, primitive North European breeds and modern sheep (including Merino), respectively. The neighbor-network analysis further differentiated North-European and Mediterranean domestic breeds, with subclusters of Merino and Merino-derived breeds, other Spanish breeds and other Italian breeds. Model-based clustering, migration analysis and haplotype sharing indicated that genetic exchange occurred between archaic populations and also that a more recent Merino-mediated gene flow to several Merino-derived populations around the world took place. The close relationship between Spanish Merino and other Spanish breeds was consistent with an Iberian origin for the Merino breed, with possible earlier contributions from other Mediterranean stocks. The Merino populations from Australia, New Zealand and China were clearly separated from their European ancestors. We observed a genetic substructuring in the Spanish Merino population, which reflects recent herd management practices.

Conclusions

Our data suggest that intensive gene flow, founder effects and geographic isolation are the main factors that determined the genetic makeup of current Merino and Merino-derived breeds. To explain how the current Merino and Merino-derived breeds were obtained, we propose a scenario that includes several consecutive migrations of sheep populations that may serve as working hypotheses for subsequent studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0139-z) contains supplementary material, which is available to authorized users.     

Polymorphism of FecB gene in nine sheep breeds or strains and its effects on litter size, lamb growth and development

Nine sheep breeds or strains, including 615 individuals were screened with forced PCR RFLP method for the FecB gene to study the polymorphism and its effects on litter sizes, body weights and body sizes. Results show that the polymorphism frequencies of FecB gene are significantly imbalanced in these breeds or strains. The Hu sheep were all homozygous carriers (BB). In the Chinese Merino prolific meat strain, the genotype frequencies of BB, B+ and ++ are 51%, 30% and 19%, respectively, whereas all the other flocks had only the wild-type (++) genotype. Results within Chinese Merino prolific meat strain showed that mean litter sizes of ewes with genotype BB and B+ are 2.8 (+/-0.74) and 2.3 (+/-0.63) (P > 0.05), whereas ++ ewes had a litter size of only 1.2 (+/-0.68) (P < 0.01). At 90 days after birth, the body weights of BB/B+ lambs were higher than that of ++ lambs (18.6 +/- 3.70 kg, 18.0 +/- 3.71 kg versus 15.6 +/- 2.22 kg, P < 0.05). In addition, the heart girth and chest width of BB/B+ lambs were significantly longer than ++ lambs (P < 0.05). No significant differences were observed in either body weight or body size at day 120. Litter size at first lambing from Hu at Natural Source Conservative Region was found to be significantly higher than that from the other two regions sampled (P < 0.05). In addition to the additive effect on litter size, these findings show for the first time that the FecB gene had a positive effect on early postnatal body growth.     

多浪羊mtDNA D-loop区遗传多样性及系统发育分析

以多浪羊为研究对象,分析绵羊线粒体D-loop区的遗传多样性,为研究多浪羊的起源和进化历史奠定基础。结果显示,多浪羊线粒体DNA D-loop序列长度为945~1 039 bp,A、T、G和C含量分别为29.4%、27.7%、17.7%和25.1%,其中A+T为57.1%,G+C为42.8%。研究获得了26种单倍型,56个多态位点,其中单一多态位点42个,简约信息位点14个。平均核苷酸差异数k为5.289,核苷酸多样度Pi为0.02 415,核苷酸多样度较低,说明多浪羊的遗传多样性贫乏,应采取重点措施予以保护。另外研究发现多浪羊经历过群体扩张,其母系起源除A、B和C世系外,可能存在D或E世系。     

Polymorphism of FecB Gene in Nine Sheep Breeds or Strains and Its Effects on Litter Size, Lamb Growth and Development

Nine sheep breeds or strains, including 615 individuals were screened with forced PCR RFLP method for the FecB gene to study the polymorphism and its effects on litter size, body weight and body size. Results showed that the polymorphism frequencies of FecB gene were significantly imbalanced in these breeds or strains. The Hu sheep were all homozygous carriers of FecB gene(BB). In the Chinese Merino prolific meat strain, the genotype frequencies of BB, B+ and ++ were 51%, 30% and 19%, respectively, whereas all the other flocks had only the wild-type (++) genotype. Results within the Chinese Merino prolific meat strain showed that the mean litter size of ewes with genotype BB and B+ were 2.8 (±0.74) and 2.3 (±0.63) (P<0.05). whereas ++ genotype ewes had a litter size of only 1.2 (±0.68) (P<0.01). At day 90 after birth, the body weights of BB/B+ genotype lambs were higher than that of ++ genotype lambs (18.6±3.70 kg. 18.0±3.71 kg vs 15.6±2.22 kg, P<0.05). In addition, the heart girth and chest width of BB/B+ genotype lambs were significantly longer than those of the ++ lambs (P<0.05). No significant differences were observed in either body weight or body size at day 120. Litter size at first lambing from Hu at Natural Source Conservative Region was found to be significantly higher than that from the other two regions sampled (P<0.05). In addition to the additive effect on litter size, these findings showed for the first time that the FecB gene had a positive effect on early postnatal body growth.     

Prion‐like Doppel gene polymorphisms and scrapie susceptibility in portuguese sheep breeds

The establishment of an association between prion protein gene (PRNP) polymorphisms and scrapie susceptibility in sheep has enabled the development of breeding programmes to increase scrapie resistance in the European Union. Intense selection for PRNP genotype may lead to correlated selection for genes linked to PRNP. We intended to investigate if any association exists between genetic variation in prion‐like protein Doppel gene (PRND) and scrapie susceptibility, determined through PRNP genotyping. Sampling included 460 sheep from eight Portuguese breeds and the PRND gene coding region was analysed by multiple restriction fragment‐single strand conformation polymorphism (MRF‐SSCP), whereas PRNP genotyping was carried out by primer extension. A synonymous substitution (c.78G>A) was detected in codon 26 of the PRND gene, in all breeds except Churra Mondegueira. Linkage disequilibrium was found between the PRND and PRNP loci (P = 0.000). Specifically, PRND was monomorphic in the 45 animals with the more resistant ARR/ARR PRNP genotype (P = 0.003), whereas a higher frequency of PRND heterozygotes (GA) was associated with ARQ/AHQ (P = 0.029). These results constitute preliminary evidence of an association between a polymorphism in the PRND gene and scrapie susceptibility, and indicate that the possibility of undesirable consequences from widespread selection for PRNP genotype on genetic diversity and reproduction traits needs to be further investigated.