大熊猫线粒体DNA酶切片段长度多态性研究

用１２种限制性内切酶（ＢａｍＨⅠ，ＥｃｏＲⅠ，ＥｃｏＲⅤ，ＸｈｏⅠ，ＰｓｔⅠ，ＭｓｐⅠ，ＸｂａⅠ，ＰｖｕⅡ，ＨｉｎｄⅢ，ＨａｅⅢ，ＳａｃⅠ，ＨｐａⅡ）对秦岭大熊猫线粒体ＤＮＡ进行了酶切。测定和分析了每种酶所产生片段的数量和大小，并与四川大熊猫的限制性片段进行了比较，发现在相同的６种酶的１６个酶切位点中有一个不同，表明这两地群体间存在着线粒体ＤＮＡ的多态性。其研究结果为进一步研究和保护大熊猫提供了重要依据。     

MGB probe assay for rapid detection of mtDNAl1778 mutation in the Chinese LHON patients by real-time PCR

Objective： Leber＇s hereditary optic neuropathy （LHON） is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA （mtDNA）. Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder （MGB） probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction （PCR）. Methods： Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA 1 1778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones. Results： All 48 LHON patients and their maternal relatives were positive for rntDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min. Conclusion： This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.     

中国部分地方牛种mtDNA D-loop区全序列的遗传多样性与系统进化分析

利用PCR测序及生物信息学分析技术,对我国5个地方黄牛品种、5个地方水牛品种及2个地方牦牛品种的mtDNA D-loop区全序列进行PCR扩增以及核苷酸多样度、单倍型多样度分析,发现中国地方黄牛、水牛与牦牛具有丰富的遗传多样性.对试验牛mtDNA D-loop区全序列与牛亚科代表性物种黄牛、水牛、家牦牛、野牦牛、欧洲普通牛、印度瘤牛以及摩拉水牛相应序列进行系统发育分析.结果显示:黄牛与牦牛的亲缘关系较近,它们与水牛的亲缘关系较远;中国水牛属于沼泽型水牛,也有少量江河型水牛渐渗入中国水牛群体;中国黄牛为普通牛和瘤牛的混合母系起源;进化树显示高原牦牛与野牦牛的亲缘关系较近,环湖牦牛与家牦牛的亲缘关系较近.     

4个群体西太公鱼mtDNAD—loop区段的限制性片段长度多态性分析

应用PCR技术对天津市于桥水库、北京市密云水库、日本北海道和日本青森县4个群体共151尾西太公鱼（Hypomesusnipponensis）的mtDNAD-loop区段进行扩增,得到长度约为1．8kbp的扩增片段．扩增出的DNA片段使用13种核酸内切限制酶进行酶切,其中8种核酸内切限制酶（AluI、DraI、HaeⅢ、Hindm、HinfI、MboI、TaqI和XspI）有酶切位点,5种核酸内切限制酶（AluI、HinfI、MboI、TaqI和XspI）个体间存在变异．不同酶切结果组合后,共得到28种单倍型,于桥水库、密云水库、北海道和青森西太公鱼均以单倍型6为主,分别为36．36％、38．30％、43．33％和26．67％;于桥水库、密云水库、北海道及青森西太公鱼单倍型多样性指数（危）分别为0．8171、0．8141、0．7793和0．8828;核苷酸多样性指数Or）分别为0．007918、0．007326、0．007255和0．009710．于桥水库西太公鱼与密云水库西太公鱼之间的Rogers遗传距离最小,为0．1499;于桥水库西太公鱼与日本青森西太公鱼之间的Rogers遗传距离最大,为0．2215;日本北海道西太公鱼与密云水库西太公鱼之间的遗传距离为0．1511,大于于桥水库西太公鱼与密云西太公鱼之间的遗传距离,但小于日本境内北海道西太公鱼与青森西太公鱼之间的遗传距离．     

Comparative genetic differentiation study of three coexisting mangrove crabs in western Atlantic

ABSTRACT

A multispecies approach may reveal the factors influencing the genetic differentiation of coexisting species along a wide geographic area. Our aim was to compare the genetic differentiation of mangrove crabs coexisting along the western Atlantic in order to verify if there are common barriers, using two mitochondrial DNA markers to quantify genetic differentiation, variance and diversity. In addition, we included a mismatch distribution to check demographic history. Our data revealed that species with similar pelagic larval duration had either high genetic differentiation or the absence of genetic structure. These results can be explained by factors that contribute to genetic differentiation, such as the presence of large estuarine areas (acting as barriers), ocean currents, and larval behaviour. Also, historical events that promoted the isolation of some areas in the past, such as glacial cycles during the Pleistocene, could have caused the observed high levels of genetic divergence in some species.     

上海四膜虫(Tetrahymena shanghaiensis)线粒体DNA的酶切分析

该文以六种限制性内切酶对上海四膜虫ｍｔＤＮＡ进行单酶完全酶切,ＥｃｏＲⅠ、ＨｉｎｄⅢ、ＨｈａⅠ酶切均产生４个片段,ＰｓｔⅠ、ＨｐａⅡ、ＨａｅⅢ酶切分别产生５、６、７个片段。不同的酶切征段大小及总和有一定的差异,酶切结果比较,有较大的差异。同其它方法如四膜虫大核ｒＤＮＡ限制性内切酶分析、同工酶分析及皮层细胞骨架组分分析结果一致,进上步证明上海四膜虫是梨形四膜虫复合种的一种。     

太行山猕猴种群mtDNA遗传多样性研究

2007年11月～2008年2月,利用非损伤性取样法,在太行山猕猴国家级自然保护区采集到3个地理单元、5个野生种群猕猴个体的粪便样品,并从22份样品中提取到DNA,在此基础上,分析和探讨了野生太行山猕猴种群的遗传多样性状况及种群遗传结构.结果表明:①在341 bp的mtDNA控制区序列中发现11个变异位点,其中转换和颠换的位点分别为9个和2个;②在3个地理单元中,共定义了9种单倍型;③AMOVA分析结果表明,88.02％和11.03％的遗传变异分别发生在各地理单元间和各种群间,而地理单元内的各种群间的遗传变异仅占0.95％,且济源、焦作和新乡3个地理单元间存在着显著的遗传分化(P＜0.01),无共享单倍型;④济源黄楝树种群具有最多的单倍型数(3),最高的单倍型多样性(0.833),最高的核苷酸多样性(0.002 93)和最高的平均核苷酸差异数(1.000).因此,从保护物种基因多样性的角度考虑,建议自然保护区管理部门将该种群列为优先保护单元.     

Phylogenetic relationships and divergence times of the family Araucariaceae based on the DNA sequences of eight genes

Araucariaceae is one of the most primitive families of the living conifers, and its phylogenetic relationships and divergence times are critically important issues. The DNA sequences of 8 genes, i.e., nuclear ribosomal 18S and 26S rRNA, chloroplast 16S rRNA, rbcL, matK and rps4, and mitochondrial coxl and atpl, obtained from this study and GenBank were used for constructing the molecular phylogenetic trees of Araucariaceae, indicating that the phyiogenetic relationships among the three genera of this family should be （（Wollemia, Agathis）, Araucaria）. On the basis of the fossil calibrations of Wollemia and the two tribes Araucaria and Eutacta of the genus Araucaria, the divergence time of Araucariaceae was estimated to be （308± 53） million years ago, that is, the origin of the family was in the Late Carboniferous rather than Triassic as a traditional view. With the same gene combination, the diver- gence times of the genera Araucaria and Agathis were （246 ± 47） and （61 ±15） Ma, respectively. Statis- tical analyses on the phylogenetic trees generated by using different genes and comparisons of the divergence times estimated by using those genes suggested that the chloroplast matK and rps4 genes are most suitable for investigating the phylogenetic relationships and divergence times of the family Araucariaceae.     

Genetic typing of mitochondrial DNA in sheep (Ovis aries )

Using PCR-RFLP and DNA sequencing, this study confirmed that HinfI polymorphism in the ovine mitochondrial COI gene resulted from the T-C substitution at the nucleotide 234 but this mutation did not encode another amino acid, which was actually a synonymous mutation. This single nucleotide polymorphism can be used as gene typing marker for mitochondrial genome in the research of the interactions between mitochondria and nucleus, extranuclear gene effects, and as molecular discriminating marker for embryo or individual in the research area of gene transfer and animal cloning.     

The use of museum specimens to reconstruct the genetic variability and relationships of extinct populations

In this review, we discuss the use of DNA from museum specimens to address conservation genetic questions. We provide four examples from our previous studies of the northern hairy-nosed wombat, African wild dog, Ethiopian wolf and red wolf. These species were genetically surveyed using two molecular approaches: first, analysis of short sequences in the mitochondrial genome using species-specific primers, and second, analysis of hypervariable microsatellite loci. The studies demonstrate that museum-derived DNA adds an important dimension to the genetic study of extant populations. Inaccessible populations can be studied, and both the loss of genetic variation and its distribution over space and time can be better understood. Finally, analysis of additional museum material provides definitive evidence for a hybrid origin of the red wolf.