中国17个黄牛品种mtDNA变异特征与多态性分析

中国黄牛品种资源丰富,尚有28个地方固有品种.为了进一步深入了解这些宝贵遗传资源,本研究通过mtDNA变异特征与多态性分析揭示这些来自中国不同地域地方黄牛的母系起源与分子系统学特征.在17个品种84个体的mtDNA D-loop全序列中,一共检测到了102个核苷酸替代突变位.由此定义的53个单倍型被类聚为2个明显的单倍群：普通牛和瘤牛.mtDNA D-loop全序列变异的第一个特征是转换发生的频率远高于颠换;第二个特征是缺失与替代突变共存;第三个特征是缺失突变率比较高.所有D-loop全序列的核苷酸多样性和单倍型多样性分别为0.026 78±0.000 50和0.919±0.027.普通牛D-loop单倍型在北方牛种群中占有优势(80%～100%),而瘤牛单倍型在南方牛种群中占有优势(42.9%～100%),2种不同单倍型在中原牛种群中的分布也存在差异.2种不同单倍型在中国不同地域17个黄牛品种中的差异性分布揭示出了瘤牛mtDNA基因在中国黄牛中自南而北、由高到低的流动模式,这种基因流动模式的形成可能是由历史事件、地理隔离以及气候环境差异等造成的.     

贵州黄牛mtDNA D-loop 遗传多样性研究

对贵州4个地方黄牛品种共计82个个体的线粒体DNA D-loop区全序列910 bp进行分析,检测到31种单倍型,其核苷酸多态位点65个,约占所测核苷酸总长的7.14%,其中有62个转换,2个颠换,1个转换/颠换共存。贵州4个黄牛品种mtDNA D-loop区核苷酸多样度（π值）为2.16%～2.61%,单倍型多样度（H）为0.695～0.909,表明贵州黄牛mtDNA遗传多样性比较丰富。根据单倍型构建了贵州4个黄牛品种的NJ分子系统树。聚类表明,贵州黄牛有普通牛和瘤牛2大母系起源,其影响较为均一。并探讨了用核苷酸多样度π值的大小来衡量黄牛群体遗传分化程度的可行性。      

中国黄牛mtDNA D-loop遗传多样性及起源

黄牛自古以来就是我国一个重要的畜种,其经济、文化价值很高。我国是世界上黄牛品种资源最丰富的国家之一。据《中国牛品种志》介绍,把一些地区同种异名的黄牛品种合并以后,尚有28个地方黄牛品种,按照其地理分布区域分为北方黄牛、中原黄牛和南方黄牛三大类型(邱怀,1986)。如果把中国地方黄牛品种分得更细,则有49个固有品种(常洪,1995)。关于中国黄牛的起源,历来有不同的观点。一般认为,中国黄牛是多元起源的,但究竟起源于哪几个牛种,观点不一(陈宏等,1993;于汝梁等,1993;Yu et al.,1999;陈幼春,1990)。主要的观点有:(1)中国黄牛主要起源于…     

Multiple Origins of a Mitochondrial Mutation Conferring Deafness

A point mutation (1555G) in the smaller ribosomal subunit of the mitochondrial DNA (mtDNA) has been associated with maternally inherited traits of hypersensitivity to streptomycin and sensorineural deafness in a number of families from China, Japan, Israel, and Africa. To determine whether this distribution was the result of a single or multiple mutational events, we carried out genetic distance analysis and phylogenetic analysis of 10 independent mtDNA D-loop sequences from Africa and Asia. The mtDNA sequence diversity was high (2.21%). Phylogenetic analysis assigned 1555G-bearing haplotypes at very divergent points in the human mtDNA evolutionary tree, and the 1555G mutations occur in many cases on race-specific mtDNA haplotypes, both facts are inconsistent with a recent introgression of the mutation into these races. The simplest interpretation of the available data is that there have been multiple origins of the 1555G mutation. The genetic distance among mtDNAs bearing the pathogenic 1555G mutation is much larger than among mtDNAs bearing either evolutionarily neutral or weakly deleterious nucleotide substitutions (such as the 4336G mutation). These results are consistent with the view that pathogenic mtDNA haplotypes such as 1555G arise on disparate mtDNA lineages which because of negative natural selection leave relatively few related descendants. The co-existence of the same mutation with deafness in individuals with very different nuclear and mitochondrial genetic backgrounds confirms the pathogenicity of the 1555G mutation.     

尾斑瘰螈种群的D-loop区遗传多样性分析

以线粒体DNA D-loop序列为分子标记,研究了采自雷公山和武陵山的尾斑瘰螈(Paramesotriton caudopunclatus)5个种群的遗传多样性.获得42个个体mtDNA D-loop区序列,长度为690 bp,其中有30个变异位点,由此定义了13个单倍型.5个种群中未出现共享单倍型的现象.尾斑瘰螈种群...     

Variation in mitochondrial DNA and maternal genetic ancestry of Ethiopian cattle populations

This study describes complete control region sequences of mitochondrial DNA (mtDNA) from 117 Ethiopian cattle from 10 representative populations, in conjunction with the available cattle sequences in GenBank. In total, 79 polymorphic sites were detected, and these defined 81 different haplotypes. The haplotype and nucleotide diversity of Ethiopian cattle did not vary among the populations studied. All mtDNA sequences from Ethiopian cattle converged into one main maternal lineage (T1) that corresponds to African Bos taurus cattle. According to the results of this study, no zebu mtDNA haplotypes have been found in Ethiopia, where the most extensive hybridization took place on the African continent.     

西藏小型猪的线粒体DNA控制区分析

目的研究西藏小型猪的遗传标记以及与其他国内地方猪的亲缘关系。方法扩增102头西藏小型猪以及16头巴马小型猪、17头贵州香猪的线粒体DNA控制区,测序并与国内其他猪进行比较。结果西藏小型猪线粒体DNAD-loop区分三个区域。串联重复序列区处于中间位置,包含有15～29个10 bp的重复片段,分为A、B两种类型。D-loop 3′端340 bp,与国内其他猪的序列相同比较保守;5′端704 bp,共有22个变异位点。由22个变异位点中归纳出25个单倍型,其中有两种主要的单倍型,分别占34.4%和36.6%。根据三个转换位点：305、500、691,将西藏小型猪分成了两组,几乎与串联重复序列所分的A、B两组类型相对应。与西藏小型猪相比,巴马小型猪和贵州香猪D-loop 5′端变异位点较少,分别只有4种和2种单倍型,串联重复区也只有一个类型。结论西藏小型猪可能有两个母系祖先并且与我国西南地区的品种猪有较近的亲缘关系;不同的串联重复片段类型和5′端的变异位点可以联合组建西藏小型猪的遗传标记。     

三种小型猪线粒体DNA控制区的比较研究

目的分析五指山小型猪、巴马小型猪和贵州香猪线粒体DNA控制区碱基序列,比较研究不同猪种的遗传标志。方法应用PCR技术分别对这三种小型猪的血液总DNA样品中线粒体DNA D-loop区进行扩增,测序比对。结果猪的线粒体DNA D-loop区分三个区域。I区（靠近5’端区域）704bp,五指山小型猪在此区共有6个变异位点,通过6个变异位点中归纳出3个单倍体,而巴马小型猪在此区有9个变异位点,通过9个变异位点归纳出4个单倍体,贵州香猪在此区共有6个变异位点,通过6个变异位点归纳出3个单倍体。Ⅱ区（串联重复序列区）,五指山小型猪、巴马小型猪和贵州香猪序列相同。Ⅲ区（靠近3’端区域）三种小型猪的序列几乎相同。结论五指山小型猪、巴马小型猪和贵州香猪三种小型猪之间线粒体DNA碱基序列变异位点较少,五指山小型猪和巴马小型猪亲缘关系较近。     

A new insight into cattle's maternal origin in six Asian countries

The domestication of cattle fuelled the development of agricultural society in the history of human being.The evolution and genetic relationship of cattle can be elucidated by investigating the variation of mitochondrial DNA (mtDNA) D-loop sequence.In this study,we built a cattle phylogeny with a pool of 856 individual D-loop sequences,of which 264 Chinese cattle D-loop sequences were obtained in this study (141 ones were first analyzed,and 123 were first submitted) and the rest sequences of cattle from six Asian countries (Japan,Korea,Mongolia,Nepal,India and China) were retrieved from GenBank.Our results indicated that cattle from six Asian countries fell into three clades,Bos taurus (taurine),Bos indicus (zebu) and yak.Four main haplogroups T1A,T2,T3 (including T3A and T3B) and T5 were found in taurine,and two haplogroups I1 and I2 in zebu.Furthermore,we found that I1 and I2 haplogroups were separated by four variable sites rather than five ones and four haplogroups or sub-haplogroups of T1A,T3A,T3B and T5 were found for the first time in these Asian cattle.These data brought us a new insight into cattle's genetic structure in these six Asian countries.The geographical distribution of haplogroups was also outlined to provide systematic information on cattle genetic resources.     

舟山小黄鱼线粒体DNA D-loop区序列变异的遗传多样性分析

小黄鱼(Pseudosciaena polyactis)为我国重要海产经济鱼类之一,过度捕捞和环境污染等因素造成其资源日益衰退。研究小黄鱼种群遗传结构对其资源的保护及其可持续利用有十分重要的意义。该研究采用聚合酶链式反应(PCR)技术对浙江舟山附近海域小黄鱼种群53个个体的mtDNA D-loop区全序列进行扩增,序列长度在795~801bp之间,长度差异不大。采用ClustalX1.83、MEGA3.1、DnaSP4.0等生物信息学软件进行遗传多样性分析,结果显示:53条小黄鱼线粒体DNA D-loop区的T、C、A和G碱基平均含量分别为30.3%、23.1%、32.3%和14.3%,排除13处核苷酸的插入或缺失后,共检测到93处转换和颠换位点,约占分析序列总长度的11.6%,其中包括53个单一多态位点和40个简约信息位点,共确定了52种单倍型,单倍型多样性(hd)为0.9993,单倍型间的平均遗传距离为0.012,转换/颠换平均值为4.305,平均核苷酸差异数(k)为9.73875,核苷酸多样性(π)为0.01233,表明舟山小黄鱼遗传多样性处于中等水平。     

Co-amplification of mitochondrial pseudogenes in Calomys musculinus (Rodentia, Cricetidae): a source of error in phylogeographic studies.

In a previous phylogeographic study of the rodent Calomys musculinus, 24 haplotypes of the mitochondrial DNA D-loop region were detected using the restriction fragment length polymorphism technique (PCR-RFLP). Seven percent of the individuals showed patterns in which the sum of the sizes of the restriction fragments exceeded the size of the original PCR product. In the present paper we analyze possible causes of these atypical haplotypes. PCR products were cloned, and two or three different clones from a single individual were detected by their RFLP patterns. Nine clones with different restriction patterns were selected for sequence analyses. A maximum parsimony phylogenetic analysis revealed two well-supported paraphyletic groups. One group comprised sequences showing low nucleotide divergence compared with the most common haplotypes detected in the phylogeographic study. The other group was basal to the three species of Calomys other than C. musculinus included in the study; the mutations in the short portion of the cytochrome b gene amplified corresponded to 12 amino acid substitutions. The results suggest that two independent insertions of mtDNA sequences into the nucleus occurred; these sequences would co-amplify in the PCR procedure. Identification of pseudogenes is crucial to obtain reliable reconstruction of the intraspecific genealogy in phylogeographic studies.     

Terminal branch haplotype analysis: a novel approach to investigate newly arisen variants of mitochondrial DNA in natural populations.

The discrimination of recent mutational derivatives from ancestral variation is a critical antecedent to any effort which aims to identify the factors modulating the rates of origin and persistence of new mutants. We propose that newly arisen mtDNA variants, which we designate as terminal branch haplotypes (TBHs), can be recognized by joint sequencing and phylogenetic analysis. This study examined mtDNA diversity in natural populations of the brown bullhead (Ameiurus nebulosus) from four heavily contaminated sites and three relatively pristine locations. While sequence analysis of the mtDNA D-loop region revealed that TBHs were prevalent in these populations, contaminant exposure appeared to play a minor role in their generation. Instead, most TBHs likely arose due to spontaneous mutations with variation in their incidence among sites reflecting the impact of demographic factors.     

Origin of Hungarian indigenous chicken breeds inferred from mitochondrial DNA D-loop sequences

In this study, we assessed the maternal origin of six Hungarian indigenous chicken breeds using mitochondrial DNA information. Sequences of Hungarian chickens were compared with the D-loop chicken sequences annotated in the GenBank and to nine previously described reference haplotypes representing the main haplogroups of chicken. The first 530 bases of the D-loop region were sequenced in 74 chickens of nine populations. Eleven haplotypes (HIC1-HIC11) were observed from 17 variable sites. Three sequences (HIC3, HIC8 and HIC9) of our chickens were found as unique to Hungary when searched against the NCBI GenBank database. Hungarian domestic chicken mtDNA sequences could be assigned into three clades and probably two maternal lineages. Results indicated that 86% of the Hungarian haplotypes are related to the reference sequence that likely originated from the Indian subcontinent, while the minor part of our sequences presumably derive from South East Asia, China and Japan.     

Microsatellite and Mitochondrial DNA Study of Native Eastern European Cattle Populations: The Case of the Romanian Grey

The Eastern European Grey cattle are regarded as the direct descendants of the aurochs (Bos taurus primigenius). Nowadays in Romania, less than 100 Grey animals are being reared and included in the national gene reserve. We examined the genetic diversity among Romanian Grey, Brown, Spotted and Black and White cattle breeds, with a particular focus on Romanian Grey through the use of (i) 11 bovine specific microsatellite markers on 83 animals and (ii) 638 bp length of mitochondrial DNA (mtDNA) D-loop region sequence data from a total of 81 animals. Both microsatellite and mtDNA analysis revealed a high level of genetic variation in the studied breeds. In Romanian Grey a total of 100 alleles were found, the mean number of observed alleles per locus was 9.091; the average observed heterozygosity was 0.940; the Wright’s fixation index (F_IS) was negative (-0.189) and indicates that there is no inbreeding and no selection pressure. MtDNA analysis revealed 52 haplotypes with 67 variable sites among the Romanian cattle breeds without any insertion or deletion. Haplotype diversity was 0.980 ± 0.007 and ranged from 0.883 ± 0.056 (Brown) to 0.990 ± 0.028 (Spotted and Black and White). The highest genetic variability of the mtDNA was recorded in the Grey breed, where 18 haplotypes were identified. The most frequent mtDNA D-loop region belonged to T3 haplogroup (80.247%), which was found across all studied breeds, while T2 haplotypes (16.049%) was only found in Grey, Spotted and Black and White genotypes. The T1 haplotypes (3.704%) were found in the Grey and Spotted. The current results contribute to the general knowledge on genetic diversity found in Eastern European cattle breeds and could prove a valuable tool for the conservation efforts of animal genetic resources (FAnGR).     

凉地方牛群体母系遗传背景研究

黄牛（Bos taurus）的驯养作为农耕文化重要内容之一,在我国历史悠久,长久以来形成的役用型逐步被肉用型所替代。为了揭示平凉地方牛群体的遗传背景,分析其是否具有生产优质牛肉的遗传基础,本研究测定了88头平凉地方牛群体mtDNA D-loop HVS序列,并对包括平凉地方牛群体在内的我国23个地方牛群体单倍型分布及系统发生关系进行了分析。结果表明,mtDNA D-loop 高变区,在平凉地方牛群体共有52个单倍型,而23个地方牛群体共有95个单倍型,这些单倍型在系统发生树和中介网络关系中分布于两个分枝,即瘤牛型和普通牛进化枝。因此,我们认为平凉地方牛群体和我国其他牛种一样,存在普通牛和瘤牛两个母系起源的遗传背景。     

High frequency of mitochondrial DNA D-loop mutations in Ewing’s sarcoma

Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing’s sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations’ possible involvement in the initiation and/or progression of this rare malignancy.     

中国狼不同地理种群遗传多样性及系统进化分析

为了解中国狼不同地理种群遗传多样性及系统发育情况,从中国境内狼的主要分布区青海、新疆、内蒙古和吉林4个地区采集样品,用分子生物学技术手段成功地获得44个个体线粒体DNA控制区第一高变区(HVRⅠ)序列和40个线粒体Cyt b部分序列。线粒体控制区HVRⅠ共检测到51个变异位点,位点变异率为8.76%;线粒体Cyt b部分序列发现31个变异位点,位点变异率为5.33%,未见插入及缺失现象,变异类型全部为碱基置换。共定义了16个线粒体HVRⅠ单倍型,其中吉林与内蒙种群存在共享单倍型,估计这两地间种群亲缘关系较近。4个地理种群中新疆种群拥有较高的遗传多样性(0.94)。中国狼种群总体平均核苷酸多态性为2.27%,与世界其他国家地区相比,中国狼种群拥有相对较高的遗传多样性。通过线粒体HVRⅠ单倍型构建的系统进化树可以看出,中国狼在进化上分为2大支,其中位于青藏高原的青海种群独立为一支,推测其可能长期作为独立种群进化。基于青海种群与新疆,内蒙种群的线粒体Cyt b遗传距离,推测中国狼2个世系可能在更新世冰川时期青藏高原受地质作用急速隆起后出现分歧,分歧时间大约在1.1 MY前。     

我国部分黄牛品种线粒体D-loop区遗传多样性与起源分化

为了解我国地方黄牛品种线粒体DNA的遗传变异情况, 文章测定了16个地方黄牛品种206个个体线粒体D-loop区的全序列, 共检测到101个变异位点; 99种单倍型, 其中73种是普通牛单倍型, 26种是瘤牛单倍型; 平均核苷酸差异为22.6920, 单倍型多样度为0.9320, 核苷酸多样度为0.0227, 表明我国黄牛品种遗传多样性非常丰富。构建的NJ进化树显示16个品种来源于两大母系: 普通牛和瘤牛; 构建的Network图表明73种普通牛单倍型可以分为3大单倍型群; 26种瘤牛单倍型分为5种单倍型群, 推测我国瘤牛在迁移过程中, 至少经历了4次群体扩张事件。通过分析比较地方黄牛品种与内罗门牛共有的 H3单倍型, 发现其中只有16%的序列与内罗门牛的H3单倍型非常相似, 其余84%的序列均发生了鸟嘌呤变异, 推测这些变异很可能是我国瘤牛固有的变异。     

Somatic mutations in the D-loop and decrease in the copy number of mitochondrial DNA in human hepatocellular carcinoma

Somatic mutations in mitochondrial DNA (mtDNA) have been detected in many human cancers, including hepatocellular carcinoma (HCC). The D-loop region was found to be a "hot spot" for mutation in mtDNA of the tumors. However, effects of the D-loop mutations on the copy number of mtDNA in tumor tissues are poorly understood. Using direct sequencing, we examined mutations in the D-loop region of mtDNA in 61 HCCs and the corresponding non-tumor liver tissues. The results revealed that 39.3% of the HCCs carried somatic mutation(s) in the D-loop of mtDNA, and most of these mutations were homoplasmic. Moreover, 37.0% (10/27) of these mutations were T-to-C and G-to-A transitions and 40.7% (11/27) of them were located in the polycytidine stretch between nucleotide position (np) 303 and 309 of mtDNA. In addition, we found that mtDNA copy number of HCC was significantly decreased in 60.5% of the patients with hepatoma, especially in those with somatic mutation(s) in the D-loop of mtDNA (17/24). This decrease in mtDNA copy number was highly associated with the occurrence of point mutations near the replication origin of the heavy-strand of mtDNA. Interestingly, we found that 42.9% (6/14) of the HCCs without mutation in the D-loop had a reduced copy number of mtDNA, indicating that other unidentified factors involved in mitochondrial biogenesis might be defective in the tumor. The results obtained in this study strongly suggest that somatic mutations in the D-loop together with the decrease in the copy number of mtDNA may be an important event during the early phase of liver carcinogenesis.     

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell

Adult somatic cell cloned calves were produced by somatic cell nuclear transfer prepared by fusion of cultured ear fibroblast from a Holstein cow into enucleated oocytes of Luxi Yellow cow. In order to determinate the source of mitochondrial DNA of cloned calves, we designed the breed-specific PCR primers by aligning the known D-loop sequences of Bos taurus and analyzed the displacement loop sequences of five live cloned calves by breed-specific primers PCR. The results demonstrated that mtDNA originated from Holstein breed and that from Luxi breed co-exist in all five live calves.