Screening of indigenous goats for prolificacy associated DNA markers of sheep

The present study was undertaken to explore the genetic basis of caprine prolificacy and to screen indigenous goats for prolificacy associated markers of sheep in BMPR1B, GDF9 and BMP15 genes. To detect the associated mutations and identify novel allelic variants in the candidate genes, representative samples were collected from the breeding tract of indigenous goat breeds varying in prolificacy and geographic distribution. DNA was extracted and PCR amplification was done using primers designed or available in literature for the coding DNA sequence of candidate genes. Direct sequencing was done to identify the genetic variations. Mutations in the candidate genes associated with fecundity in sheep were not detected in Indian goats. Three non-synonymous SNPs (C818T, A959C and G1189A) were identified in exon 2 of GDF9 gene out of which mutation A959C has been associated with prolificacy in exotic goats. Two novel SNPs (G735A and C808G) were observed in exon 2 of BMP15 gene.     

Genetic diversity of Chinese indigenous sheep breeds inferred from microsatellite markers

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

Mitochondrial DNA Sequence and Phylogenetic Evaluation of Geographically Disparate Sus scrofa Breeds

Next generation sequencing of mitochondrial DNA (mtDNA) facilitates studies into the metabolic characteristics of production animals and their relation to production traits. Sequence analysis of mtDNA from pure-bred swine with highly disparate production characteristics (Mangalica Blonde, Mangalica Swallow-bellied, Meishan, Turopolje, and Yorkshire) was initiated to evaluate the influence of mtDNA polymorphisms on mitochondrial function. Herein, we report the complete mtDNA sequences of five Sus scrofa breeds and evaluate their position within the phylogeny of domestic swine. Phenotypic traits of Yorkshire, Mangalica Blonde, and Swallow-belly swine are presented to demonstrate their metabolic characteristics. Our data support the division of European and Asian breeds noted previously and confirm European ancestry of Mangalica and Turopolje breeds. Furthermore, mtDNA differences between breeds suggest function-altering changes in proteins involved in oxidative phosphorylation such as ATP synthase 6 (MT-ATP6), cytochrome oxidase I (MT-CO1), cytochrome oxidase III (MT-CO3), and cytochrome b (MT-CYB), supporting the hypothesis that mtDNA polymorphisms contribute to differences in metabolic traits between swine breeds. Our sequence data form the basis for future research into the roles of mtDNA in determining production traits in domestic animals. Additionally, such studies should provide insight into how mtDNA haplotype influences the extreme adiposity observed in Mangalica breeds.     

Decrease of MtDNA copy number affects mitochondrial function and involves in the pathological consequences of ischaemic stroke

The mtDNA copy number can affect the function of mitochondria and play an important role in the development of diseases. However, there are few studies on the mechanism of mtDNA copy number variation and its effects in IS. The specific mechanism of mtDNA copy number variation is still unclear. In this study, mtDNA copy number of 101 IS patients and 101 normal controls were detected by qRT‐PCR, the effect of D‐loop variation on mtDNA copy number of IS patients was explored. Then, a TFAM gene KD‐OE PC12 cell model was constructed to explore the effect of mtDNA copy number variation on mitochondrial function. The results showed that the mtDNA copy number level of the IS group was significantly lower than that of the normal control group (p < 0.05). The relative expression of TFAM gene mRNA in the cells of the OGD/R treatment group was significantly lower than that of the control group (p < 0.05). In addition, after TFAM gene knockdown and over‐expression plasmids were transfected into HEK 293T cells, mtDNA copy number and ATP production level of Sh‐TFAM transfection group was significantly decreased (p < 0.05), while mtDNA copy number and ATP production level of OE‐TFAM transfected group were significantly higher than that of blank control group and OE‐ctrl negative control group (p < 0.01). Our study demonstrated that mitochondrial D‐loop mutation and TFAM gene dysfunction can cause the decrease of mtDNA copy number, thus affecting the mitochondrial metabolism and function of nerve cells, participating in the pathological damage mechanism of IS.     

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.     

我国主要地方绵羊品种mtDNA D-loop区PCR-RFLP研究

利用5种限制性内切酶（Hinf I,Msp I,Sau3A I,Xsp I,Taq I）,采用PCR-RFLP技术研究了我国9个地方绵羊品种以及2个引入品种共计83只绵羊个体线粒体DNA D-loop区的多态性。结果表明,我国主要地方绵羊品种线粒体DNA D-loop区存在两种基本单体型,提示我国主要地方绵羊品种起源于两个母系祖先。线粒体DNA D-loop区多态度为0.042 1%,说明我国地方绵羊品种线粒体DNA多态度较为贫乏。
     

Caenorhabditis elegans,a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown.Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C. elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans.This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.     

Maintenance of mitochondrial DNA by the Caenorhabditis elegans ATR checkpoint protein ATL-1

Here we show that inactivation of the ATR-related kinase ATL-1 results in a significant reduction in mitochondrial DNA (mtDNA) copy numbers in Caenorhabditis elegans. Although ribonucleotide reductase (RNR) expression and the ATP/dATP ratio remained unaltered in atl-1 deletion mutants, inhibition of RNR by RNAi or hydroxyurea treatment caused further reductions in mtDNA copy number. These results suggest that ATL-1 functions to maintain mtDNA independently of RNR.     

The Role of Mitochondrial DNA ORIB Polymorphism in Metabolic Syndrome

The development of the metabolic syndrome (MS) involves many genes. Certain evidence exists in the literature on the association of polymorphism in the mitochondrial DNA (mtDNA) oriB site, also known as the polycytosine pathway, with the development of insulin resistance, type 2 diabetes mellitus (T2DM) and other metabolic disorders in various ethnic populations. It is suggested that certain polymorphisms at this site are associated with mtDNA copy number in the cell. In this study, using capillary sequencing, we have identified various allelic variants of the mtDNA oriB site in patients with MS (n = 106) and conditionally healthy donors (n = 71). The mtDNA copy number in blood leukocytes was determined by the droplet digital polymerase chain reaction (ddPCR). It has been shown that the variant of the continuous polycytosine tract is significantly more frequent in MS patients with T2DM (p < 0.01). In general, the mtDNA copy number of blood leukocytes was lower in MS patients than in controls. We did not find any correlations between the oriB site variability and the mtDNA copy number.     

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.     

Runs of homozygosity and signatures of selection: a comparison among eight local Swiss sheep breeds

A dataset consisting of 787 animals with high‐density SNP chip genotypes (346 774 SNPs) and 939 animals with medium‐density SNP chip genotypes (33 828 SNPs) from eight indigenous Swiss sheep breeds was analyzed to characterize population structure, quantify genomic inbreeding based on runs of homozygosity and identify selection signatures. In concordance with the recent known history of these breeds, the highest genetic diversity was observed in Engadine Red sheep and the lowest in Valais Blacknose sheep. Correlation between F_PED and F_ROH was around 0.50 and thereby lower than that found in similar studies in cattle. Mean F_ROH estimates from medium‐density data and HD data were highly correlated (0.95). Signatures of selection and candidate gene analysis revealed that the most prominent signatures of selection were found in the proximity of genes associated with body size (NCAPG, LCORL, LAP3, SPP1, PLAG1, ALOX12, TP53), litter size (SPP1), milk production (ABCG2, SPP1), coat color (KIT, ASIP, TBX3) and horn status (RXFP2). For the Valais Blacknose sheep, the private signatures in proximity of genes/QTL influencing body size, coat color and fatty acid composition were confirmed based on runs of homozygosity analysis. These private signatures underline the genetic uniqueness of the Valais Blacknose sheep breed. In conclusion, we identified differences in the genetic make‐up of Swiss sheep breeds, and we present relevant candidate genes responsible for breed differentiation in locally adapted breeds.     

The Multifaceted Origin of Taurine Cattle Reflected by the Mitochondrial Genome

A Neolithic domestication of taurine cattle in the Fertile Crescent from local aurochsen (Bos primigenius) is generally accepted, but a genetic contribution from European aurochsen has been proposed. Here we performed a survey of a large number of taurine cattle mitochondrial DNA (mtDNA) control regions from numerous European breeds confirming the overall clustering within haplogroups (T1, T2 and T3) of Near Eastern ancestry, but also identifying eight mtDNAs (1.3%) that did not fit in haplogroup T. Sequencing of the entire mitochondrial genome showed that four mtDNAs formed a novel branch (haplogroup R) which, after the deep bifurcation that gave rise to the taurine and zebuine lineages, constitutes the earliest known split in the mtDNA phylogeny of B. primigenius. The remaining four mtDNAs were members of the recently discovered haplogroup Q. Phylogeographic data indicate that R mtDNAs were derived from female European aurochsen, possibly in the Italian Peninsula, and sporadically included in domestic herds. In contrast, the available data suggest that Q mtDNAs and T subclades were involved in the same Neolithic event of domestication in the Near East. Thus, the existence of novel (and rare) taurine haplogroups highlights a multifaceted genetic legacy from distinct B. primigenius populations. Taking into account that the maternally transmitted mtDNA tends to underestimate the extent of gene flow from European aurochsen, the detection of the R mtDNAs in autochthonous breeds, some of which are endangered, identifies an unexpected reservoir of genetic variation that should be carefully preserved.     

Genome-wide analyses reveal population structure and identify candidate genes associated with tail fatness in local sheep from a semi-arid area

Under a climate change perspective, the genetic make-up of local livestock breeds showing adaptive traits should be explored and preserved as a priority. We used genotype data from the ovine 50 k Illumina BeadChip for assessing breed autozygosity based on runs of homozygosity (ROH) and fine-scale genetic structure and for detecting genomic regions under selection in 63 Tunisian sheep samples. The average genomic inbreeding coefficients based on ROH were estimated at 0.017, 0.021, and 0.024 for Barbarine (BAR, n = 26), Noire de Thibar (NDT, n = 23), and Queue fine de l'Ouest (QFO, n = 14) breeds, respectively. The genomic relationships among individuals based on identity by state (IBS) distance matrix highlighted a recent introgression of QFO into the BAR and a genetic differentiation of NDT samples, possibly explained by past introgression of European gene pools. Genome-wide scan for ROH across breeds and within the BAR sample set identified an outstanding signal on chromosome 13 (46.58–49.61 Mbp). These results were confirmed using F_ST index, differentiating fat vs. thin-tailed individuals. Candidate genes under selection pressure (CDS2, PROKR1, and BMP2) were associated to lipid storage and probably preferentially selected in fat-tailed BAR animals. Our findings suggest paying more attention to preserve the genetic integrity and adaptive alleles of local sheep breeds.     

Genome-Wide Specific Selection in Three Domestic Sheep Breeds

Background

Commercial sheep raised for mutton grow faster than traditional Chinese sheep breeds. Here, we aimed to evaluate genetic selection among three different types of sheep breed: two well-known commercial mutton breeds and one indigenous Chinese breed.

Results

We first combined locus-specific branch lengths and d_i statistical methods to detect candidate regions targeted by selection in the three different populations. The results showed that the genetic distances reached at least medium divergence for each pairwise combination. We found these two methods were highly correlated, and identified many growth-related candidate genes undergoing artificial selection. For production traits, APOBR and FTO are associated with body mass index. For meat traits, ALDOA, STK32B and FAM190A are related to marbling. For reproduction traits, CCNB2 and SLC8A3 affect oocyte development. We also found two well-known genes, GHR (which affects meat production and quality) and EDAR (associated with hair thickness) were associated with German mutton merino sheep. Furthermore, four genes (POL, RPL7, MSL1 and SHISA9) were associated with pre-weaning gain in our previous genome-wide association study.

Conclusions

Our results indicated that combine locus-specific branch lengths and d_i statistical approaches can reduce the searching ranges for specific selection. And we got many credible candidate genes which not only confirm the results of previous reports, but also provide a suite of novel candidate genes in defined breeds to guide hybridization breeding.     

Origin, genetic diversity, and population structure of Chinese domestic sheep

To characterize the origin, genetic diversity, and phylogeographic structure of Chinese domestic sheep, we here analyzed a 531-bp fragment of mtDNA control region of 449 Chinese autochthonous sheep from 19 breeds/populations from 13 geographic regions, together with previously reported 44 sequences from Chinese indigenous sheep. Phylogenetic analysis showed that all three previously defined lineages A, B, and C were found in all sampled Chinese sheep populations, except for the absence of lineage C in four populations. Network profiles revealed that the lineages B and C displayed a star-like phylogeny with the founder haplotype in the centre, and that two star-like subclades with two founder haplotypes were identified in lineage A. The pattern of genetic variation in lineage A, together with the divergence time between the two central founder haplotypes suggested that two independent domestication events have occurred in sheep lineage A. Considerable mitochondrial diversity was observed in Chinese sheep. Weak structuring was observed either among Chinese indigenous sheep populations or between Asian and European sheep and this can be attributable to long-term strong gene flow induced by historical human movements. The high levels of intra-population diversity in Chinese sheep and the weak phylogeographic structuring indicated three geographically independent domestication events have occurred and the domestication place was not only confined to the Near East, but also occurred in other regions.     

The regulation of mitochondrial DNA copy number in glioblastoma cells

As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.     

A Candidate Complex Approach to Study Functional Mitochondrial DNA Changes: Sequence Variation and Quaternary Structure Modeling of Drosophila simulans Cytochrome c Oxidase

A problem with studying evolutionary dynamics of mitochondrial (mt) DNA is that classical population genetic techniques cannot identify selected substitutions because of genetic hitchhiking. We circumvented this problem by employing a candidate complex approach to study sequence variation in cytochrome c oxidase (COX) genes within and among three distinct Drosophila simulans mtDNA haplogroups. First, we determined sequence variation in complete coding regions for all COX mtDNA and nuclear loci and their isoforms. Second, we constructed a quaternary structure model of D. simulans COX. Third, we predicted that six of nine amino acid changes in D. simulans mtDNA are likely to be functionally important. Of these seven, genetic crosses can experimentally determine the functional significance of three. Fourth, we identified two single amino acid changes and a deletion of two consecutive amino acids in nuclear encoded COX loci that are likely to influence cytochrome c oxidase activity. These data show that linking population genetics and quaternary structure modeling can lead to functional predictions of specific mtDNA amino acid mutations and validate the candidate complex approach. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.