SSR在普氏原羚个体识别和分子保护遗传学中的应用
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摘要
普氏原羚仅分布于我国青海湖地区，数量仅为300只左右。1996年，国际自然保护联盟红皮书将其列为极濒危级动物。因此，应用分子保护遗传学对我国普氏原羚进行个体识别、种群数量估计、亲缘关系鉴定和种群生存力评估，都具有非常重要意义。
本研究发展了一套完整的普氏原羚粪便DNA的提取方法，将野外普氏原羚的粪便样品中提取的DNA与血液、皮张样品中提取的DNA，进行PCR扩增比较，结果完全一致。与其它从粪便中提取DNA的方法相比较，该方法具有操作简单、易于掌握、省时省力、经济实用、高度灵敏的特点，且操作过程中减少了对有毒化学物质的接触。另外，粪便DNA提取方法对动物不造成任何伤害，是对珍稀濒危物种进行分子保护遗传学研究中获取DNA的首选方法。
本研究从10对非州糜羚的微卫星引物中，筛选出在普氏原羚的基因组DNA中能扩增出明显多态性位点的5对微卫星引物，即TGLA54、BM1392、OarFCB304、SMHCC和BM1862；同时从10对绵羊的微卫星引物中筛选出在普氏原羚基因组DNA中能扩增出多态性位点的3对微卫星引物，即MNS61、MNS64和Oare133。在这20对引物中，共得到8个含多态性位点的引物，占所用引物数的40％，表明微卫星序列两侧是高度保守的，而中间的重复片段具有高度多态性。同时，也表明微卫星位点是一种适合于种内个体遗传分析的分子标记。
利用筛选出的8个微卫星位点，结合粪便DNA提取技术，成功地对采自青海省共和县小北湖地区的野生普氏原羚的39个粪便样品进行了个体识别，分析结果表明：这些粪便至少来自35个不同的个体。同时根据各位点的等位基因频率计算出各位点的基因杂合度、有效等位基因数及其平均值，以及各位点的个体识别能力和累计个体识别能力和多态信息含量。结果表明：各位点基因杂合度介于0.704-0.848，平均杂合度为0.798，有效等位基因数介于3.375-6.592，平均有效等位基因为4.846，各位点个体识别能力0.704-0.848，累计个体识别能力0.999，多态信息含量介于0.657-0.789，平均为0.731。其中OarFCB304的个体识别能力最强，为0.848；而MNS61个体识别能力仅为0.704。表明遗传标记的多态性越高，个体识别能力就越强。
本研究还利用线粒体DNA中的12SrRNA、16SrRNA和D-Loop分子标记，对青海湖国家级自然保护区救护的一只的小羚羊灵灵和青海省动物园饲养的一只公羊，进行了物种鉴定。12SrRNA的测序结果表明：灵灵为普氏原羚，公羊为鹅喉羚。
Procapra przewalskii is endemic to China, and just live around Qinghai Lake region, Qinghai Province, with a population about 300, so it was appointed as CR (Critically Endangered) in the IUCN (The World Conservation Union) Red List in 1996. The conservation genetics research is a key step to conserve the endangered species in individual identification, population size estimation, pedigree analysis and population viability analysis.
First of all, we explored a set of techniques for extracting fecal DNA, and established a new extracted DNA method successfully. Compared the extracted fecal DNA with blood DNA and skin DNA by agar electrophoresis, we found this new method is easy operate high yield, practicable and safety to operator because it reduced chance to touch poisonous chemical drugs, and the method was a high veracity and non-invasive DNA technology. Therefore, this new extracted DNA method imply the non-invasive DNA technology can use to study of genetic diversity of endangered species.
Microsatellite locus is a highly variable nuclear marker. The two sides of the sequence of microsatellite markers were high conservative, and sequences in the middle part were polymorphic. Thus microsatellite markers are one of the better molecular markers for inter-species genetic diversity study and individual analysis. In our study, 10 polymorphic microsatellite DNA primers which identified in Grant gazelle were amplified in Procapra przewalskii genomes, and we found that five of the loci (TGLA54, BM1392, Oarfcb304, SMHCC, BM1862) were polymorphic. At the same time, we used 10 polymorphic microsatellite DNA primers identified in sheep to amplify in Procapra przewalskii genomes, the amplified results showed that 3 loci were polymorphic (MNS61, MNS64, Oarfel33). We identified individuals and tested the genetic diversity in 39 Procapra przewalskii fecal samples using these eight polymorphic microsatellite loci. The PCR amplified products of microsatellite loci were detected by non-denatured polyacry lamide gel electrophoresis. The results showed these fecal samples belonged to 35 different individuals; we calculated the gene heterozygosity, individual identification potential, polymorphism information content (PIC), and effective number of alleles of each locus. The values of gene heterozygosity was 0.7089-0.8488, the values of effective number of alleles is 3.375-6.592, PIC is 0.657-0.789, TDP =0.999; which showed these loci were high polymorphism and fitted to individual identification. These results implied the microsatellite DNA markers could help study the genetic diversity of endangered species.
Finally, We used MtDNA to identify successfully a small captive gazelle (Ling Ling) in the Bird Island in Qinghai National Nature Reserve and a ram that was fed in Qinghai Zoo. The result showed that Ling Ling belonged to Procapra przewalskii i, and ram belonged to Gazella gutturosa.
本研究发展了一套完整的普氏原羚粪便DNA的提取方法，将野外普氏原羚的粪便样品中提取的DNA与血液、皮张样品中提取的DNA，进行PCR扩增比较，结果完全一致。与其它从粪便中提取DNA的方法相比较，该方法具有操作简单、易于掌握、省时省力、经济实用、高度灵敏的特点，且操作过程中减少了对有毒化学物质的接触。另外，粪便DNA提取方法对动物不造成任何伤害，是对珍稀濒危物种进行分子保护遗传学研究中获取DNA的首选方法。
本研究从10对非州糜羚的微卫星引物中，筛选出在普氏原羚的基因组DNA中能扩增出明显多态性位点的5对微卫星引物，即TGLA54、BM1392、OarFCB304、SMHCC和BM1862；同时从10对绵羊的微卫星引物中筛选出在普氏原羚基因组DNA中能扩增出多态性位点的3对微卫星引物，即MNS61、MNS64和Oare133。在这20对引物中，共得到8个含多态性位点的引物，占所用引物数的40％，表明微卫星序列两侧是高度保守的，而中间的重复片段具有高度多态性。同时，也表明微卫星位点是一种适合于种内个体遗传分析的分子标记。
利用筛选出的8个微卫星位点，结合粪便DNA提取技术，成功地对采自青海省共和县小北湖地区的野生普氏原羚的39个粪便样品进行了个体识别，分析结果表明：这些粪便至少来自35个不同的个体。同时根据各位点的等位基因频率计算出各位点的基因杂合度、有效等位基因数及其平均值，以及各位点的个体识别能力和累计个体识别能力和多态信息含量。结果表明：各位点基因杂合度介于0.704-0.848，平均杂合度为0.798，有效等位基因数介于3.375-6.592，平均有效等位基因为4.846，各位点个体识别能力0.704-0.848，累计个体识别能力0.999，多态信息含量介于0.657-0.789，平均为0.731。其中OarFCB304的个体识别能力最强，为0.848；而MNS61个体识别能力仅为0.704。表明遗传标记的多态性越高，个体识别能力就越强。
本研究还利用线粒体DNA中的12SrRNA、16SrRNA和D-Loop分子标记，对青海湖国家级自然保护区救护的一只的小羚羊灵灵和青海省动物园饲养的一只公羊，进行了物种鉴定。12SrRNA的测序结果表明：灵灵为普氏原羚，公羊为鹅喉羚。
Procapra przewalskii is endemic to China, and just live around Qinghai Lake region, Qinghai Province, with a population about 300, so it was appointed as CR (Critically Endangered) in the IUCN (The World Conservation Union) Red List in 1996. The conservation genetics research is a key step to conserve the endangered species in individual identification, population size estimation, pedigree analysis and population viability analysis.
First of all, we explored a set of techniques for extracting fecal DNA, and established a new extracted DNA method successfully. Compared the extracted fecal DNA with blood DNA and skin DNA by agar electrophoresis, we found this new method is easy operate high yield, practicable and safety to operator because it reduced chance to touch poisonous chemical drugs, and the method was a high veracity and non-invasive DNA technology. Therefore, this new extracted DNA method imply the non-invasive DNA technology can use to study of genetic diversity of endangered species.
Microsatellite locus is a highly variable nuclear marker. The two sides of the sequence of microsatellite markers were high conservative, and sequences in the middle part were polymorphic. Thus microsatellite markers are one of the better molecular markers for inter-species genetic diversity study and individual analysis. In our study, 10 polymorphic microsatellite DNA primers which identified in Grant gazelle were amplified in Procapra przewalskii genomes, and we found that five of the loci (TGLA54, BM1392, Oarfcb304, SMHCC, BM1862) were polymorphic. At the same time, we used 10 polymorphic microsatellite DNA primers identified in sheep to amplify in Procapra przewalskii genomes, the amplified results showed that 3 loci were polymorphic (MNS61, MNS64, Oarfel33). We identified individuals and tested the genetic diversity in 39 Procapra przewalskii fecal samples using these eight polymorphic microsatellite loci. The PCR amplified products of microsatellite loci were detected by non-denatured polyacry lamide gel electrophoresis. The results showed these fecal samples belonged to 35 different individuals; we calculated the gene heterozygosity, individual identification potential, polymorphism information content (PIC), and effective number of alleles of each locus. The values of gene heterozygosity was 0.7089-0.8488, the values of effective number of alleles is 3.375-6.592, PIC is 0.657-0.789, TDP =0.999; which showed these loci were high polymorphism and fitted to individual identification. These results implied the microsatellite DNA markers could help study the genetic diversity of endangered species.
Finally, We used MtDNA to identify successfully a small captive gazelle (Ling Ling) in the Bird Island in Qinghai National Nature Reserve and a ram that was fed in Qinghai Zoo. The result showed that Ling Ling belonged to Procapra przewalskii i, and ram belonged to Gazella gutturosa.
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