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紫貂遗传多样性及貂类系统发育研究
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摘要
紫貂是珍贵的毛皮兽类,被我国列为Ⅰ级保护动物,但由于具有高昂的经济价值而驱使人们对其进行大量捕杀,加之人类经济活动强度的大幅增加导致栖息地环境破坏和生境呈斑块状分布,目前野生种群已经处于濒危状态。紫貂主要分布于欧亚大陆,我国东北和新疆阿尔泰山区为其分布的南界,北欧地处紫貂分布区的西界,也有一个紫貂种群。由于我国和北欧分布的紫貂种群均属于边缘种群,对于它们的种群遗传结构及遗传多样性信息的研究将有助于探讨紫貂物种起源及演化历程。
     现生种类黄喉貂仅分布于南亚次大陆,与其它貂属动物的分布区截然不同,不仅如此,黄喉貂在形态特征和化石记录等方面均表现出与其它貂类不同。正因为这种显著的差异导致黄喉貂的分类地位及起源问题一直存在争议。
     鉴于此,本研究通过测定国内唯一一个具有18只个体的紫貂种群的线粒体DNA控制区全序列及紫貂和黄喉貂的线粒体基因组全序列,结合GenBank公布的近缘种序列数据,通过分析各序列的结构特征研究了该紫貂种群的遗传多样性和种群分化问题;鼬科动物线粒体DNA控制区序列的结构特点;紫貂和黄喉貂线粒体基因组结构特点以及它们的起源和进化史。得出如下结论:
     1、18只紫貂包含7个单倍型。利用分子生物学手段验证了基于形态学特征划分的亚种分类系统的正确性,即单倍型M3、M5是长白山亚种,单倍型M1、2、4和7是大兴安岭亚种,单倍型M6是阿尔泰亚种;当前,该紫貂种群的遗传多样性水平较高(Pi=3.94%),但自然条件下的各亚种群的遗传多样性水平均较低(Pi<1%),据此提出了相应的紫貂物种保护措施。分析紫貂大兴安岭亚种、长白山亚种、阿尔泰亚种和北欧亚种间的基因流及进化历史得知:大兴安岭种群与新疆种群和长白山种群间的基因流水平最高(Nm=0.1260和0.1427),新疆与长白山种群间最低(Nm=0.0053);紫貂种群在进化过程中可能发生过种群膨胀,经历过种群增长过程。
     2、对紫貂和黄喉貂的线粒体全基因组结构进行分析发现:全长分别为16 523bp和16549bp,均包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码序列区(D-Loop区),紫貂全序列中碱基组成为A-32.0%,C-27.6%,G-14.7%,T-25.8%,黄喉貂为A-32.5%,C-26.9%,G-14.1%,T-26.5%;基因排列顺序与日本貂和貂熊的一致,但碱基组成、起始密码子和终止密码子的使用及控制区中串联重复序列模式等均存在一定差异。貂属物种的整个基因组密码子使用具有偏向性,编码蛋白基因第三位点利用A和C频率较高,推测可能与控制区A和C的含量具有一定的相关性,即与重复序列模式相关。将紫貂和黄喉貂线粒体基因组全序列提交到GenBank,并获得检索号分别为FJ429093和FJ719367。
     3、结合GenBank中已公布的鼬科其它6种动物的线粒体基因组全序列及貂属6种D-loop区部分序列,分别以虎和狗獾为外类群,应用最大简约法构建鼬科和貂属物种的系统进化树,结果表明:鼬科7个物种分成三大支系,分别为水獭亚科和臭鼬亚科群,日本貂和貂熊群以及紫貂和狗獾群,说明鼬科并非是一个单系群,紫貂与鼬科中的獾亚科亲缘关系较近,而与貂属中的美洲貂和石貂亲缘关系最近。而基于鼬亚科动物线粒体DNA12种重链编码蛋白基因序列及石貂和渔貂线粒体ND2和cyt b全序列,以狼、狗獾和水獭为外类群,应用NJ、MP和ML法构分别构建貂属和貂熊的系统进化树,结果表明:紫貂与日本貂亲缘关系较美洲貂近,石貂与真貂亚属亲缘关系较近,黄喉貂介于石貂和渔貂之间;渔貂是最早从貂属分化出的种类,其次是黄喉貂和石貂;结合化石记录分析,貂属起源于中欧地区,渔貂、黄喉貂和石貂属于一个向温带进化的分支,而真貂亚属为另外一个向寒冷环境进化的分支,建议将黄喉貂和渔貂分别定为单独的属。
     4、比对并分析11种鼬科动物线粒体DNA控制区序列,识别出延长终止序列区、中央区和保守序列区3个区域,指出了一个终止相关序列ETAS1及8个保守序列(CSB-F、E、D、C、B、1、2和3),并给出了序列通式,在CSB1和CSB2之间发现具有不同形式的短串联重复序列。以狼为外类群,应用邻接法构建鼬科线粒体控制区全序列的系统进化树,结果表明:臭鼬亚科最先从鼬科中分化出来,随后剩余类群分为两大支系,即貂属种类与貂熊聚为一支,并与獾亚科的狗獾形成姐妹群;另一支为水獭亚科的物种与鼬属的林鼬形成姐妹群,再与虎鼬聚在一起,狗獾与貂属的紫貂亲缘关系最近,水獭亚科与鼬属亲缘关系最近。
Sable is the rare animal with fur,and was under I rank protection in China.People prey upon them largely for the high economic value.Because of people's economic activity increased largely,the environment of sable's habitats was destroyed and presented speckle appearance,so the wild sable has been in severe danger.Sable mainly distributed in Eurasia, The south finitude of its distributing is northeast and Altai of Sinkiang in our country.Northern Europe which located in the west finitude of sable's distributing,also has a sable population. China and northern Europe are borders of the distribution of Sable.So the study for the genetic frame and diversity information of sable will redound to discuss the sable's species origin and evolvement.
     Yellow-throated Marten which is an exist species and different from other species martes in distributing.It only distributed India.Ney,there are other differences in shape and fossil.Because of these remarkable differences,there are always dissension in classifying status and origin of Yellow-throated Marten.
     Whereas the actuality,based on the determining for the mtDNA control region sequences of the only sable population in captivity and the mitochondrial genome sequences of Sable and Yellow-throated Marten,and the sequence data of sibling species from GenBank,the author studied the genetic diversity and population differentiation of the sable in captivity,the structure of mtDNA control region sequences of weasel,the mitochondrial genetic structure and the origin and evolvement of sable and Yellow-throated Marten.So,we can got these conclusion:
     1、there are 18 sable population which were in captivity in JinZhou of Dalian contain 7 haplotype.The legitimacy of assoeted system for subspecies which was plotted out by morphologic was validated with molecular biology method,namely,haplotypeM3、M5 are M.Z.hamgyenensis,haplotypeM1、M2、M4 and M7 are M.Z.princeps,haplotype M6 is M.Z.averini;Presently,there was higher level in the genetic diversity of the captived sable in JinZhou(Pi=3.94%),but the mean level of genetic diversity for every subspecies is lower in nature.Relevant protection measures were put forward for sable in the article.Based on the analysis for the gene flow between the four subspecies and the evolvement history,we know that the gene flow among the M.Z.princeps and M.Z.hamgyenensis has the highest level(Nm= 0.1427),the one between M.Z.averini and M.Z.hamgyenensis has the lowest level(Nm=0.0053).The sable population expanded in the evolution progress and the number increased.
     2、Based on analyzing the mitochondrial genome structure of sable and Yellow-throated Marten,we can find that the span of two animals were 16 523bp and 16549bp respectively. They all contain 13 protein coding gene,2 rRNA gene,22 tRNA gene and 1 D-Loop.The base composition for the four nucleotides is A-32.0%,C-27.6%,G-14.7%,T-25.8%,And it is A-32.5%,C-26.9%,G-14.1%,T-26.5%for Yellow-throated Marten.But there are some definite differences in base composition,the using of Initiation codon and Stop codon,and the mode of repeat sequences in control region.
     The codon usages of Manes have bias,and the ttiird locations of codon of protein-coding genes have the higher frequency in using A and C.There may be some relativity with the content of A and C in D-loop,namely,it has relation with the mode of repeat sequences.The complete mitochondrial genome of the Sable and Yellow-throated Marten were submitted to GenBank,and the accession number are FJ429093 and FJ719367 respectively.
     3、The complete mitochondrial genome of 6 other species of Mustelidae from GenBank and some sequences of D-loop from the 6 species were aligned.With tiger and Eurasian badger as the outgroup,the phylogenetic relationship of Mustelidae and Martes was analyzed using the maximum parsimony method.The results showed that 7 species of Mustelidae were divided into 3 branches,they were Lutrinae and Mephitinae,Japanese marten and Wolverine,sable and Eurasian badger.This situation also indicated that Mustelidae was not a monophly,Sable which has nearer relationship with Melinae of Mustelidae also has the closest relationship with American Marten and Stone Marten of Martes.Based on the 12 H-strand protein genes sequences of mtDNA of Mustelinae and the mitochondrial ND2 and cytb sequences of Stone marten and Fisher marten,with the wolf、Eurasian badger and European river otter as the outgroup,the phylogenetic relationship of Martes and Wolverine was analyzed respectively using NJ、MP and ML methods.The results showed that sable has the nearer relationship with Japanese marten than American marten.Stone marten and the boreal forest martens have near relationship.Yellow-throated marten's relationship is between Stone marten and Fisher marten.Fisher marten is the first species who was divided from Martes.The second one were Yellow-throated marten and Stone marten.Based on the analysis of the fossil note,Martes came from Central Europe,Fisher marten,Yellow-throated marten and Stone marten belonged to a branch which evolved to temperate zone.Boreal forest martens was another branch which evolved to cold environment.It is suggested that Yellow-throated marten and Fisher martenwere ascertained genus separately.
     4、By contrasting and analyzing 11 complete mitochondrial control region gene sequences of Mustelidae,the extended termination associated sequence domain(ETAS)、the central domain(CD) and the conserved sequence block domain(CSB) were identified.A extended termination associated sequence(ETAS1) and eight conserved sequence(CSB-F、E、D、C、B、1、2、3) was indicated,and sequence mode was presented.Different kind of short repeat sequences were found between CSB1 and CSB2.With wolf as the outgroup,the phylogenetic evolution relationship of Mustelidae was analyzed using the neighbor-joining(NJ) method.The results showed that Mephitinae was divided from Mustelidae firstly,and then the remain were divided into two branches.Martes species and Wolverine were contained in one branch which was formed a sister group with Eurasian badger of Melinae;Lutrinae species and Polecat of Mustelidae formed a sister group,and Marbled polecat was sister to the clade of Lutrinae species and Polecat.They formed another branch.Eurasian badger has the closest relationship with the Sable of Martes,and Lutrinae has the closest relationship with Mustelidae.
引文
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