Complete sequence of the Tibetan Mastiff mitochondrial genome and its phylogenetic relationship with other Canids ( Canis, Canidae)

In this study, the complete sequence of the Tibetan Mastiff mitochondrial genome (mtDNA) was determined, and the phylogenetic relationships between the Tibetan Mastiff and other species of Canidae were analyzed using the coyote (Canis latrans) as an outgroup. The complete nucleotide sequence of the Tibetan Mastiff mtDNA was 16 710 bp, and included 22 tRNA genes, 2S rRNA gene, 13 protein-coding genes and one non-coding region (D-loop region), which is similar to other mammalian mitochondrial genomes. The characteristics of the protein-coding genes, non-coding region, tRNA and rRNA genes among Canidae were analyzed in detail. Neighbor-joining and maximum-parsimony trees of Canids constructed using 12 mitochondrial protein-coding genes showed that as the coyotes and Tibetan wolves clustered together, so too did the gray wolves and domestic dogs, suggesting that the Tibetan Mastiff originated from the gray wolf as did other domestic dogs. Domestic dogs clustered into four clades, implying at least four maternal origins (A to D). The Tibetan Mastiff, which belongs to clade A, appears to be closely related to the Saint Bernard and the Old English Sheepdog.     

Strong heterozygote deficit in Tibetan Mastiff of China based on microsatellite loci

Tibetan Mastiff is one of the most archaic, ferocious and the largest dogs in the world. A total of 140 individuals from four geographically separated populations in China (Tibet, Gansu, Qinghai and Beijing) were sampled and genetic diversity was assessed using 10 microsatellite loci on eight different chromosomes. The mean number of alleles per locus ranged from 6 to 13. The mean observed and expected heterozygosities, polymorphism information content and allelic richness were 0.69, 0.79, 0.76 and 7.59, respectively, indicating relatively high genetic diversity in Tibetan Mastiff. However, a highly significant deficiency in heterozygote was observed within populations (mean FIS = 0.11, bootstrap 95% confidence interval (0.06, 0.17)) and total inbreeding (mean FIT = 0.12, bootstrap 95% confidence interval (0.06, 0.18)), along with strong inbreeding coefficients within populations (Fis > 0.09), all of which suggested that intense inbreeding practices occurred in Tibetan Mastiff. Therefore, effective and appropriate breeding management projects in present Tibetan Mastiff will be desirable and urgent. Low genetic differentiation was obtained with a mean FST of 0.01 (bootstrap 95% confidence interval (0.007, 0.019)). Additionally, the four Tibetan Mastiff populations showed close relationships in the neighbor-joining polygenetic tree based on the coancestral genetic distances. Tibetan Mastiff was investigated by using microsatellite loci at the first time, which could facilitate the better understanding of present situation at the molecular level, breed conservation and utilization in Tibetan Mastiff.     

The origin of the Tibetan Mastiff and species identification of Canis based on mitochondrial cytochrome c oxidase subunit I (COI) gene and COI barcoding

DNA barcoding is an effective technique to identify species and analyze phylogenesis and evolution. However, research on and application of DNA barcoding in Canis have not been carried out. In this study, we analyzed two species of Canis, Canis lupus (n = 115) and Canis latrans (n = 4), using the cytochrome c oxidase subunit I (COI) gene (1545 bp) and COI barcoding (648 bp DNA sequence of the COI gene). The results showed that the COI gene, as the moderate variant sequence, applied to the analysis of the phylogenesis of Canis members, and COI barcoding applied to species identification of Canis members. Phylogenetic trees and networks showed that domestic dogs had four maternal origins (A to D) and that the Tibetan Mastiff originated from Clade A; this result supports the theory of an East Asian origin of domestic dogs. Clustering analysis and networking revealed the presence of a closer relative between the Tibetan Mastiff and the Old English sheepdog, Newfoundland, Rottweiler and Saint Bernard, which confirms that many well-known large breed dogs in the world, such as the Old English sheepdog, may have the same blood lineage as that of the Tibetan Mastiff.     

小麦印度腥黑粉菌线粒体DNA提取及其ATP6基因在真菌遗传发育分析中的应用

小麦印度腥黑粉菌(Tilletia indica Mitra)是一种世界范围的重要检疫性有害真菌,该病原菌和近似种之间冬孢子的形态特征极为相似,遗传关系非常相近。为了从分子水平上探讨小麦印度腥黑粉菌和近似种之间线粒体基因序列的差异,从新鲜菌丝中提取总DNA,经两次氯化铯密度梯度超速离心分离线粒体DNA(mtDNA),提取的mtDNA纯度较高,可用于克隆、酶切分析和PCR扩增等分析。选取基因ATP(adenosine triphosphate)6的序列,并结合GenBank中相关种类的ATP6基因DNA序列进行了系统发育分析。结果表明,线粒体基因ATP6可用于科属水平的分类鉴定。     

基于线粒体DNA控制区的斑翅草螽不同地理种群遗传分化研究

采用PCR扩增结合DNA克隆测序技术,分析了斑翅草螽Conocephalus maculates 9个地理种群mtDNA控制区序列的变异及遗传多样性。切除侧翼RNA基因序列后,最终获得的斑翅草螽mtDNA控制区比对后全长为676 bp,平均碱基组成T(37.8%),C(11.7%),A(41.3%)和G(9.1%)。共检测到98个可变位点,占总位点数的14.5%,其中,9处碱基插入/缺失,74处转换(40个T/C,34个A/G),50处颠换(18个A/T,11个T/G,15个A/C,6个C/G)。共定义46个单倍型,其中,4个为种群间共享单倍型(H02、H05、H08和H10),其余42个为各种群独有单倍型,包括6个种群内共享单倍型(H09、H11、H15、H18、H26和H38)。单倍型总数占实验个体总数的69.7%,除四川峨眉山外,其余种群单倍型百分比均﹥50%。通过两两地理种群间的FST值差异显著性检验,将这些群体分为4组,分别为SC+CQ,GX+FLB+HN+YN,XZ和HB。以长瓣草螽C.gladiatus、峨眉草螽C.emeiensis、悦鸣草螽C.melaenus、竹草螽C.bambusanus为外群,构建的斑翅草螽mtDNA控制区单倍型NJ法系统树形成3个自举支持度较高的分支,其中,分支A由28种单倍体组成,包括本研究中除四川峨眉山(SC)和重庆万州(CQ)以外的7个种群;分支B由12种单倍体组成,包含除菲律宾拉乌尼翁(FLB)和江西南昌(JX)以外的7个种群;分支C由6种单倍型组成,全部来自西藏林芝(XZ)的单倍型。聚类结果表明,斑翅草螽不同地理种群间的遗传分化并不明显,即使是两两群体间FST值差异显著的群体,也未能形成完全独立的分支。     

从弹尾纲和原尾纲的亲缘关系质疑缺尾纲（=近昆虫纲）的有效性

鉴于弹尾目（跳虫）和原尾目的尾部都没有尾须（cerci）,Brner于1910年就把这两类归并为一个类群缺尾纲（Ellipura）,这一分类阶元长期被许多昆虫学家沿用至今。Kukalová-Peck(1987 )在讨论化石双尾虫（?）附肢的总体结构（ground plan）时,认为跳虫和原尾虫的腹部侧板更原始,附肢无转节,将二者归纳成近昆虫纲（Parainsecta）。但是从形态特征、内部结构、比较精子学、变态类型和胚后发育等的特点以及线粒体DNA和核糖体DNA的测序分析结果,显示弹尾纲与原尾纲之间存在诸多重要差异,不具备较为密切的亲缘关系,我们不支持（弹尾纲+原尾纲）组成缺尾纲或近昆虫纲。据此建议取消缺尾纲（=近昆虫纲）这一分类阶元。     

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.     

蒙古狼线粒体基因组序列分析及其系统发育地位

应用Long-PCR和克隆测序法得到蒙古狼(Canis lupus chanco)线粒体基因组全序列,结合GenBank中现有犬科动物线粒体基因组数据,应用最大简约法(MP)、最大似然法(ML)和Bayesian分析法对蒙古狼的系统发育地位进行了探讨。结果如下:蒙古狼线粒体基因组全长16709bp,包含13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和1个非编码区。序列碱基的组成存在明显的A-T偏好性。tRNA基因中除tRNA-Ser(AGY)缺少双氢尿嘧啶(DHU)臂以外,其余均能折叠成典型的三叶草二级结构。大多数蛋白质编码基因的起始和终止密码子与犬科动物有报道相同,COXⅡ基因的起始密码子为ATA,与其他犬科动物不同。基于12S rRNA+16S rRNA+H链上的12个蛋白质编码基因的联合数据的系统发育分析发现,在已报道的狼亚种数据中,西藏狼(Canis lupus laniger)的分化时间最早,其次为阿拉伯狼(Canis lupus arabs),蒙古狼与欧亚狼(Canis lupuslupus)的系统发育地位最为接近。     

西藏牦牛线粒体DNA 的遗传多样性及系统进化分析

为从分子水平上探究西藏牦牛类群的遗传多样性、亲缘关系,本研究测定了日多牦牛、类乌齐牦牛、丁青牦牛、错那牦牛、隆子牦牛、仲巴牦牛、聂荣牦牛、申札牦牛等8 个西藏牦牛类群共328 头牦牛mtDNAD-loop区序列,分析其多态性,构建系统进化树。结果表明:本次测定的西藏牦牛mtDNA D-loop 区序列长度为 887 - 895 bp,共检测到135 个变异位点,其中单态突变位点52 个,简约信息位点83 个。在328 个个体中共检测出91 种单倍型,平均单倍型多样性(Hd)、平均核苷酸多样性(π)分别为0. 884、0.010 27,显示西藏牦牛具有丰富的遗传多样性。8 个类群间平均遗传距离为0.011;日多牦牛与错那牦牛间遗传距离最小(0. 006);类乌 齐牦牛与隆子牦牛间遗传距离最大(0.015)。系统进化分析显示西藏牦牛可分为两大类,错那牦牛是较纯的牦牛类群,其它牦牛类群在进化过程中出现相互交流的情况。     

利用12个血液蛋白和非蛋白标记分析同羊起源及系统地位

采用典型群随机抽样在陕西省白水县抽取同羊样本。采用淀粉凝胶和醋酸纤维薄膜检测12个的结构基因座位的遗传多型性,结果在同羊中发现10个多型座位：运铁蛋白（Tf）、碱性磷酸酶（Alp）、亮氨酸氨肽酶（Lap）、芳基酯酶（Ary-Es）、血红蛋白β（Hb-β）、X-蛋白（X-p）、碳酸酐酶（CA）、过氧化氢酶（Cat）、苹果酸脱氢酶（MDH）和赖氨酸（Ly）;而白蛋白（Al）和后白蛋白（Po）为单态。采用遗传贴近度和系统关系聚类分析两种方法分析同羊起源及系统地位。结果表明两种方法均支持同羊属于蒙古羊系统,同羊起源于蒙古羊,这与同羊的育成史实相符。和聚类分析方法相比,遗传贴近度分析方法可以更为有效地用于中亚以东南绵羊群体的血统判别,可以更有效地反映同羊的育成过程。     

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA(RAPD) analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China.A captive population has been established in the Beijing Zoo since 1999.In order to determine the kinship of the offsprings in 2001,randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo.To amplify the genomic DNA of each individual,53 arbitrary primers were selected.The results of amplifica tions showed that 14 primers had clear and distinct RAPD patterns.Totally,226 amplified fragments were generated by RAPD in this study.Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male).This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds.     

Genetic differentiation in pointing dog breeds inferred from microsatellites and mitochondrial DNA sequence

Recent studies presenting genetic analysis of dog breeds do not focus specifically on genetic relationships among pointing dog breeds, although hunting was among the first traits of interest when dogs were domesticated. This report compares histories with genetic relationships among five modern breeds of pointing dogs (English Setter, English Pointer, Epagneul Breton, Deutsch Drahthaar and German Shorthaired Pointer) collected in Spain using mitochondrial, autosomal and Y-chromosome information. We identified 236 alleles in autosomal microsatellites, four Y-chromosome haplotypes and 18 mitochondrial haplotypes. Average F _ST values were 11.2, 14.4 and 13.1 for autosomal, Y-chromosome microsatellite markers and mtDNA sequence respectively, reflecting relatively high genetic differentiation among breeds. The high gene diversity observed in the pointing breeds (61.7–68.2) suggests contributions from genetically different individuals, but that these individuals originated from the same ancestors. The modern English Setter, thought to have arisen from the Old Spanish Pointer, was the first breed to cluster independently when using autosomal markers and seems to share a common maternal origin with the English Pointer and German Shorthaired Pointer, either via common domestic breed females in the British Isles or through the Old Spanish Pointer females taken to the British Isles in the 14th and 16th centuries. Analysis of mitochondrial DNA sequence indicates the isolation of the Epagneul Breton, which has been formally documented, and shows Deutsch Drahthaar as the result of crossing the German Shorthaired Pointer with other breeds. Our molecular data are consistent with historical documents.     

General properties and phylogenetic utilities of nuclear ribosomal DNA and mitochondrial DNA commonly used in molecular systematics

To choose one or more appropriate molecular markers or gene regions for resolving a particular systematic question among the organisms at a certain categorical level is still a very difficult process. The primary goal of this review, therefore, is to provide a theoretical information in choosing one or more molecular markers or gene regions by illustrating general properties and phylogenetic utilities of nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) that have been most commonly used for phylogenetic researches. The highly conserved molecular markers and/or gene regions are useful for investigating phylogenetic relationships at higher categorical levels (deep branches of evolutionary history). On the other hand, the hypervariable molecular markers and/or gene regions are useful for elucidating phylogenetic relationships at lower categorical levels (recently diverged branches). In summary, different selective forces have led to the evolution of various molecular markers or gene regions with varying degrees of sequence conservation. Thus, appropriate molecular markers or gene regions should be chosen with even greater caution to deduce true phylogenetic relationships over a broad taxonomic spectrum.     

Origin and radiation of the house mouse: mitochondrial DNA phylogeny

On the basis of patterns of allele frequency variation in nuclear genes (Din et al., in press) it has been proposed that the house mouse M. musculus originated in the northern Indian subcontinent, from where it radiated in several directions to form the well-described peripheral subspecies (M. m. domesticus, M. m. musculus and M. m. castaneus). Here we use a mitochondrial DNA (mtDNA) phylogeny to test this hypothesis and to analyse the historical and demographic events that have accompanied this differentiation. This marker also provides a powerful means to check for genetic continuity between the central and peripheral populations. We studied restriction site polymorphism of samples from India and the Middle East as well as samples from the rest of Eurasia and northern Africa. M. m. domesticus and M. m. musculus are both monophyletic for mtDNA and belong to the subspecies-specific mtDNA lineages that have been described previously. Average nucleotide diversity is low in M. m. musculus (0.2–5%). It is not only higher in M. m. domesticus (0.7–0.9%) but the distribution of pairwise divergence is wider, and the rate of evolution in this branch appears to be higher than in M. m. musculus. The nucleotide diversity found in M. m. castaneus (0.4%) is due to the existence of two rather divergent linages with little intralineage variation. These two lineages are part of a diversified bush of the phylogenetic tree that also comprises several previously undescribed branches and includes all samples from the northern Indian subcontinent and Iran. The degree of diversity found in each of the samples from this region is high (1.2–2.4%) although they come from small geographic areas. This agrees well with the idea that the origin of the radiation was in the northern Indian subcontinent. However, as neither haplotypes on the M. m. domesticus nor on the M. m. musculus branches were found in this region, there appear to be important phylogeographic discontinuities between this central region and these peripherial subspecies. On the basis of the present result and the nuclear data (Din et al., in press), we propose that M. musculus originated in the north of the indian subcontinent. Our calibration of the evolutionary rate of mtDNA in mice suggests that the mouse settlement in this region could be as old as 900 000 years. Possibly from there, a first radiation could have reach the Middle East and the Caspian Sea, where the M. m. domesticus and M. m. musculus lineages, respectively, would have started to differentiate a few hundred thousand years ago, and from where they could have colonised the peripheral part of their ranges only recently.M. m. castaneus appears from its mtDNA to be recent offshoot of the northern Indian population. This multiple and gradual radiation ultimately led to recent peripheral secondary contacts, such as the well-known European hybrid zone.     

Origin and domestication history of Peking ducks deltermined through microsatellite and mitochondrial marker analysis

In order to elucidate the domestication history of Peking ducks, 190 blood samples from six Chinese indigenous duck breeds were collected with186 individualsgenotyped by 15 microsatellite markers. Both the F_ST and Nei’s standard genetic distances (D_s) from the microsatellite data indicated high genetic differentiation between Peking duck and other Chinese indigenous breeds. The haplotype network with mtDNA data showed that most of the Peking duck haplotypes were distinctly different from those of other domestic breeds. Although the H01 haplotype was shared by all domesticated duck breeds, Peking ducks displayed 12 specific domestic duck haplotypes, including four similar haplotypes H02, H04, H08 and H22, that formed a single haplogroup (A). Both H02 and H22 haplotypes were also shared by mallard and Peking ducks, indicating that Peking ducks originated from wild mallard ducks.     

Geographic population structure and sequence divergence in the mitochondrial DNA control region of the Japanese wild boar (Sus scrofa leucomystax), with reference to those of domestic pigs

Mitochondrial DNA (mtDNA) control regions from 40 Japanese wild boars were examined by direct sequencing after amplification by PCR. From the DNA sequences obtained, we found eight haplotypes, whose differences arose via transitions. The geographical distribution of these different haplotypes indicated that wild boar populations inhabited limited areas and that there was some restricted gene flow between local populations. Eight mtDNA haplotypes from Eastern and Western domestic pigs and the Ryukyu wild boar were also analyzed as references to those from Japanese wild boars. The cluster analyses of the control-region sequences showed that those from Japanese wild boards belong to the Asian type as do those from Eastern domestic pigs and the Ryukyu wild boar, which differed from the European type (Western domestic pigs).     

Analysis of sequence homology between human and mouse mitochondrial DNA

The base sequence homology between human and mouse mitochondrial DNA has been investigated by hybridization of highly labelled mitochondrial DNA probes with restriction fragments of mitochondrial DNA blotted according to the Southern technique. By this analysis, the homologous regions have been found to be widely distributed along the mitochondrial genome. Competition hybridization experiments with unlabelled HeLa mitochondrial RNAs have shown that most of the cross-hybridization involves the ribosomal and 4 S RNA genes.     

Phylogeography of the canyon treefrog, Hyla arenicolor (Cope) based on mitochondrial DNA sequence data

Patterns of phylogeography and gene flow were examined in the canyon treefrog, Hyla arenicolor. A total of 973 bp of mitochondrial cytochrome b sequence data were obtained for 65 individuals from 53 populations, yielding 50 unique haplotypes. Interpopulation sequence variation ranged from 0 to 13.7%. Phylogenetic analysis revealed three deeply divergent mtDNA lineages. These three Clades were mapped onto geography and found to represent completely concordant, nonoverlapping, geographical regions. Levels of sequence divergence between the three Clades were equal to or greater than published levels of divergence found in other vertebrate species and genera. Furthermore, one Clade of H. arenicolor was found to be more closely related to the outgroup H. eximia than to other H. arenicolor, suggesting that the taxonomy of this species may require revision.     

Intra- and interbreed genetic variations of mitochondrial DNA major non-coding regions in Japanese native dog breeds [Canis familiaris)

Mitochondrial DNA (mtDNA) major non-coding regions were amplified from 73 dogs of eight Japanese native dog breeds and from 21 dogs of 16 non-Japanese dog breeds by the polymerase chain reaction and their DNA sequences were determined. A total of 51 nucleotide positions within the non-coding region (969–972 base pairs) showed nucleotide variations of which 48 were caused by transition. These nucleotide substitutions were abundant in the region proximate to tRNA^Pro. In addition to the nucleotide substitutions, the dog mtDNA D-loop sequences had a heteroplasmic repetitive sequence (TACACGTÀCG) involving size variation. The DNA sequences of the non-coding region were classified into four different groups by phylogenetic analysis and the deepest branchpoints of this dog phylogeny was calculated to about 100 000 years before the present. Phylogenetic analysis showed that Japanese native dog breeds could not be clearly delimited as distinct breeds. Many haplotypes found in members of some clustering groups were seen in each dog breed, and interbreed nucleotide differences between Japanese dog breeds were almost the same as the intrabreed nucleotide diversities.