喜马拉雅旱獭不同地理种群的遗传多态性分析
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摘要
用限制性内切酶Sau3A I酶切喜马拉雅旱獭基因组DNA,从低熔点琼脂糖凝胶中回收长度200-1000bp的片段,与用碱裂解法提取的经BamH I限制性内切酶酶切的质粒pUC19连接,转化用CaC1_2法制备的大肠杆菌DH5α感受态细胞。用蓝白斑方法结合以(CA)_8寡聚核苷酸引物和pUC19质粒多克隆位点两侧的M13primers M3和RV分别配对筛选阳性克隆,通过对经过鉴定的4000多个克隆的筛选,获得61个可能含有微卫星的重组阳性克隆。测定这61个阳性克隆的序列,其中9个克隆共含有13个明显的微卫星序列。9个克隆序列已在GenBank注册,序列号分别为EF555518～EF555519,EF676084～EF676090。利用BLAST序列分析软件对GenBank数据库进行检索,没有发现高度同源性的微卫星序列,表明本文得到的喜马拉雅旱獭的微卫星序列都是首次发现。
根据所测微卫星序列,利用Primer 5.0和Oligo 6.0软件设计微卫星序列引物,并事先设计好相应的参数,共设计出13对不含茎环和二级结构的引物,长度在20bp左右。用PCR法、琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳分析青藏铁路沿线4个不同地理种群喜马拉雅旱獭的多态性。统计分析结果表明,13个微卫星位点在4个种群中观察到的等位基因和有效等位基因数目分别达到2～6个、0.166～3.073个。多态信息含量显示6个微卫星位点(SSR2、SSR3、SSR4、SSR7、SSR10、SSR12)为高度多态(PIC＞0.5),6个位点(SSR1、SSR5、SSR8、SSR9、SSR11、SSR13)为中度多态(0.5＞PIC＞0.25),1个位点(SSR6)为低度多态(PIC＜0.25)。除了SSR1和SSR6外,其它位点Shalmon指数都较高,最高为1.188。各位点平均杂合度大小波动范围为0.164～0.672。4个种群在所有位点的平均Shannon指数均大于0.8,平均PIC均大于0.45,平均观察杂合度较高,显示4个种群都处于高度多态。UPGMA聚类分析显示四个地理种群遗传进化关系明确。德令哈种群与乌兰种群遗传距离最近(0.1504),可归为一个类群。其次,沱沱河种群与这两个种群遗传距离较近。四个种群中,沱沱河种群与安多种群遗传距离最远(0.5104)。因此,这12个微卫星标记(除SSR6外)可用于喜马拉雅旱獭的遗传多态性分析,为进一步开展喜马拉雅旱獭的种群分子生态学研究及其近缘种群的分子生物学研究奠定了基础。
Marmota himalayana belongs to Rodentia,Sciuridae,Marmota.Plague plots virus usually are carried and diffused by Marmota himalayana.However,no researches of Marmota himalayana in the field of molecular ecoloy have been found so far.Meanwhile,the construction of a genomic library is essential to researches on genome structure and function.A comprehensive genomie library makes it possible to screen and isolate any DNA fragments from it.
In this paper,part of the genomie library of Marmota himalayana in four different geographical populations was constructed.The sampling localities include Delingha,Wulan, Tuotuohe,and Anduo,all were alongside the Qinghai-Tibet railway.Genomic DNA extracted from Marmota himalayana by Improved Method of Phenol-Chloroform.Genomic DNA digested by restriction endonuclease Sau3AI.Recovery 200-1000bp fragments from low melting point agarose gel.Plasmid pUC19 was extracted by alkaline lysis method and digested by restriction endonuclease BamH I to come into being the carriers.The fragments and the carriers were linked and transformed into the E.coli DH5αfeel competent cells which were prepared by CaCl_2 method.The positive clones were screened by two methods.One is the blue and white plots screening.The other is screening the positive clones by PCR with oligonucleotide primer(CA) _8 and pUC 19 multiple cloning sites bilateral M13 primers(M3 and RV).4000 clones were screened,61 recombinants were obtained.The sequences of the recombinants were identified. Then nine colones including thirteen high various microsatellite loci were submitted to the GenBank and admitted,recorded.Genbank accession numbers were EF555518～EF555519, EF676084～EF676090.High homologous microsatellite sequences have not been found in Genbank.Indicated the thirteen microsatellite loci were first found.
The corresponding primers were designed and synthesized as molecular markers to detect genetic diversity of the Marmota himalayana in the four populations.The analysis shows that the numbers of the alleles and the effective alleles of all loci are 2～6 and 1.166～3.073.The polymorphism information content(PIC)of all loci show that six loci are high polymorphism (PIC＞0.5)and six loci are middle polymorphism(0.5＞PIC＞0.25)and only one locus is low polymorphism(PIC＜0.25).Average heterozygosity(Het)and Sharmon index(Ⅰ)reveal the same results.PIC and I and Her also show that all of the four populations are high polymorphic populations.The Cluster Analysis based unweighted pair-group method using arithmetic averages show the theorical phylogenetic tree is consistent to the actuátl geographical situation. The genetic distance between Delingha populations and Wulan populations is the smallest value (0.1504).Then the genetic distance between Tuotuohe population and the two is nearer than that of the Anduo and the two.The genetic distance between Anduo population and Tuotuohe population is the biggest value(0.5104).Therefore twelve micro satallite lo ci(except SSR6)and the four populations may be used to the researches of genetic diversity ofMarmota himalayana.
根据所测微卫星序列,利用Primer 5.0和Oligo 6.0软件设计微卫星序列引物,并事先设计好相应的参数,共设计出13对不含茎环和二级结构的引物,长度在20bp左右。用PCR法、琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳分析青藏铁路沿线4个不同地理种群喜马拉雅旱獭的多态性。统计分析结果表明,13个微卫星位点在4个种群中观察到的等位基因和有效等位基因数目分别达到2～6个、0.166～3.073个。多态信息含量显示6个微卫星位点(SSR2、SSR3、SSR4、SSR7、SSR10、SSR12)为高度多态(PIC＞0.5),6个位点(SSR1、SSR5、SSR8、SSR9、SSR11、SSR13)为中度多态(0.5＞PIC＞0.25),1个位点(SSR6)为低度多态(PIC＜0.25)。除了SSR1和SSR6外,其它位点Shalmon指数都较高,最高为1.188。各位点平均杂合度大小波动范围为0.164～0.672。4个种群在所有位点的平均Shannon指数均大于0.8,平均PIC均大于0.45,平均观察杂合度较高,显示4个种群都处于高度多态。UPGMA聚类分析显示四个地理种群遗传进化关系明确。德令哈种群与乌兰种群遗传距离最近(0.1504),可归为一个类群。其次,沱沱河种群与这两个种群遗传距离较近。四个种群中,沱沱河种群与安多种群遗传距离最远(0.5104)。因此,这12个微卫星标记(除SSR6外)可用于喜马拉雅旱獭的遗传多态性分析,为进一步开展喜马拉雅旱獭的种群分子生态学研究及其近缘种群的分子生物学研究奠定了基础。
Marmota himalayana belongs to Rodentia,Sciuridae,Marmota.Plague plots virus usually are carried and diffused by Marmota himalayana.However,no researches of Marmota himalayana in the field of molecular ecoloy have been found so far.Meanwhile,the construction of a genomic library is essential to researches on genome structure and function.A comprehensive genomie library makes it possible to screen and isolate any DNA fragments from it.
In this paper,part of the genomie library of Marmota himalayana in four different geographical populations was constructed.The sampling localities include Delingha,Wulan, Tuotuohe,and Anduo,all were alongside the Qinghai-Tibet railway.Genomic DNA extracted from Marmota himalayana by Improved Method of Phenol-Chloroform.Genomic DNA digested by restriction endonuclease Sau3AI.Recovery 200-1000bp fragments from low melting point agarose gel.Plasmid pUC19 was extracted by alkaline lysis method and digested by restriction endonuclease BamH I to come into being the carriers.The fragments and the carriers were linked and transformed into the E.coli DH5αfeel competent cells which were prepared by CaCl_2 method.The positive clones were screened by two methods.One is the blue and white plots screening.The other is screening the positive clones by PCR with oligonucleotide primer(CA) _8 and pUC 19 multiple cloning sites bilateral M13 primers(M3 and RV).4000 clones were screened,61 recombinants were obtained.The sequences of the recombinants were identified. Then nine colones including thirteen high various microsatellite loci were submitted to the GenBank and admitted,recorded.Genbank accession numbers were EF555518～EF555519, EF676084～EF676090.High homologous microsatellite sequences have not been found in Genbank.Indicated the thirteen microsatellite loci were first found.
The corresponding primers were designed and synthesized as molecular markers to detect genetic diversity of the Marmota himalayana in the four populations.The analysis shows that the numbers of the alleles and the effective alleles of all loci are 2～6 and 1.166～3.073.The polymorphism information content(PIC)of all loci show that six loci are high polymorphism (PIC＞0.5)and six loci are middle polymorphism(0.5＞PIC＞0.25)and only one locus is low polymorphism(PIC＜0.25).Average heterozygosity(Het)and Sharmon index(Ⅰ)reveal the same results.PIC and I and Her also show that all of the four populations are high polymorphic populations.The Cluster Analysis based unweighted pair-group method using arithmetic averages show the theorical phylogenetic tree is consistent to the actuátl geographical situation. The genetic distance between Delingha populations and Wulan populations is the smallest value (0.1504).Then the genetic distance between Tuotuohe population and the two is nearer than that of the Anduo and the two.The genetic distance between Anduo population and Tuotuohe population is the biggest value(0.5104).Therefore twelve micro satallite lo ci(except SSR6)and the four populations may be used to the researches of genetic diversity ofMarmota himalayana.
引文
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