扬子鳄微卫星位点的筛选及其多态性分析
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摘要
扬子鳄(Alligator sinensis)是我国特有的珍稀濒危保护动物。虽然扬子鳄饲养种群近年来有了较大发展,但由于奠基者效应,已发现子代繁殖衰退的迹象。随着重引入工程的启动,正确评价扬子鳄种群的遗传多样性现状,从而制定更加有效的保护策略,已成为当务之急。
本研究利用磁珠富集法,筛选扬子鳄自身的多态性微卫星位点,同时利用这些位点对两个扬子鳄种群的遗传多样性和种群结构进行了比较分析,主要研究结果如下:
1.共挑取4000个克隆,检测后得到260个阳性克隆。随机抽取其中的150个克隆进行测序,其中92个序列可供设计微卫星引物,56对引物能成功扩增出预期大小的产物。对这56对引物进行多态性检测后,最终获得扬子鳄的11个多态性微卫星位点。
2.57只扬子鳄经以上11个多态性微卫星位点检测后,共获得31个等位基因,平均每个位点的等位基因数为2.82个。其中,有4个等位基因(Alsi05-1;Alsi06-1;Alsi07-1;Alsil0-1)为长兴本土种群所特有,1个等位基因(Alsi05-2)为美国重引入种群所特有。11个位点的平均多态信息含量(PIC)为0.384。
3.对所筛选的11个多态性微卫星位点进行了个体识别机率的有效性评估。结果显示,累计个体识别机率达到了0.9998,表明这些微卫星标记可被用于扬子鳄圈养种群的遗传谱系构建和重引入奠基种群的个体筛选。
4.利用8对跨物种的多态性微卫星引物进行了群体检测分析,其中6对引物得到了有效性扩增,从而加上之先前筛选的11个多态性微卫星位点,本文得到的扬子鳄父权鉴定概率达到了0.991,达到了预期的研究效果。
5.利用BAPS(Bayesian Analysis of Population Structure)软件在未知种群信息的前提下对57只扬子鳄个体进行类群分析,发现这些个体可以被划分为2个种群(长兴本土种群和美国重引入种群)。同时遗传混合分析显示,长兴种群和美国重引入种群虽然存在着一定程度的遗传差异,但是两者间出现了一定的混合。
6.微卫星分析结果显示,扬子鳄种群的遗传多样性较低,但长兴本土种群和美国重引入种群均具有自身特有的稀有等位基因。因此,基于保护扬子鳄这一极度濒危物种的遗传多样性之目的,作者建议在野外放归或重引入工程中,管理者以选取不同地域(种群)的个体组成扬子鳄的奠基种群为佳。
Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. Although the captive Chinese alligator populations have been greatly developed in recent years, there has been genetic depression in the filial generation because of the founder effect. As the startup of the reintroduction project for the captive Chinese alligator population, it is urgent to investigate the genetic status of this species and provide feasible strategies for its conservation.
In this study, we isolated polymorphic microsatellite loci from the Chinese alligator based on magnetic beads enrichment protocol, and did comprehensively research on genetic diversity and population structure of this species. The major results are:
1. Approximately 4,000 clones were screened and a total of 260 recombinants that potentially contained microsatellite sequence were obtained. 150 positive clones were chosen randomly for DNA sequencing, out of which 92 clones had sufficient flanking sequence for primer design. The result of PCR amplification revealed that 56 primer pairs could yield consistent specific products. Further experiments showed that 11 pairs of them were polymorphic and well amplificated for each sample.
2. The 11 polymorphic microsatellites presented a total of 31 alleles among 57 individuals scored, yielding an average of 2.82 alleles per locus. One allele was unique to the American reintroduced population(Alsi05-2) but four private alleles(Alsi05-1; Alsi06-1; Alsi07-1; Alsi10-1) were detected in the Changxing indigenous population. The average PIC (polymorphic information content) of the 11 polymorphic loci obtained in this study is 0.384.
3. The individual identity probability was evaluated for the 11 polymophic markers. Results suggested that when the 11 polymorphic loci were combined, the individual identity probability was 0.9998. Therefore, the microsatellite markers could be utilized to genetic pedigree construction for the captive Chinese alligator populations. In addition, it could be helpful in the selecting of proper individuals for the reintroduction program.
4. In this study, we also used 8 polymorphic loci isolated by cross-species amplification to assess the genetic status of this species, of which 6 loci was amplificated effectively. Finally, the exclusion probabilities of paternity testing went up to 0.991 if 11 species-specific polymorphic loci calculated in combination with 6 previous cross-species markers, which achieved the desired results.
5. A complete Bayesian method in BAPS (Bayesian Analysis of Population Structure) was chosen to perform individual clustering without prior population information. The results showed that the 57 individuals were grouped into 2 clusters (named as the Changxing indigenous population and the American reintroduced population). The further genetic admixture analysis revealed that although genetic difference was present between the two populations, there was certain admixture among them.
6. Based on the microsatellite analysis results, we conclude that the Chinese alligator has very low genetic diversity, but the Changxing indigenous population and the American reintroduced population still has some rare genes of their own. Hence, we strongly suggest that individuals from different populations should be chosen as the founders in the reintroduction program to largely protect the genetic diversity of this critically endangered species.
本研究利用磁珠富集法,筛选扬子鳄自身的多态性微卫星位点,同时利用这些位点对两个扬子鳄种群的遗传多样性和种群结构进行了比较分析,主要研究结果如下:
1.共挑取4000个克隆,检测后得到260个阳性克隆。随机抽取其中的150个克隆进行测序,其中92个序列可供设计微卫星引物,56对引物能成功扩增出预期大小的产物。对这56对引物进行多态性检测后,最终获得扬子鳄的11个多态性微卫星位点。
2.57只扬子鳄经以上11个多态性微卫星位点检测后,共获得31个等位基因,平均每个位点的等位基因数为2.82个。其中,有4个等位基因(Alsi05-1;Alsi06-1;Alsi07-1;Alsil0-1)为长兴本土种群所特有,1个等位基因(Alsi05-2)为美国重引入种群所特有。11个位点的平均多态信息含量(PIC)为0.384。
3.对所筛选的11个多态性微卫星位点进行了个体识别机率的有效性评估。结果显示,累计个体识别机率达到了0.9998,表明这些微卫星标记可被用于扬子鳄圈养种群的遗传谱系构建和重引入奠基种群的个体筛选。
4.利用8对跨物种的多态性微卫星引物进行了群体检测分析,其中6对引物得到了有效性扩增,从而加上之先前筛选的11个多态性微卫星位点,本文得到的扬子鳄父权鉴定概率达到了0.991,达到了预期的研究效果。
5.利用BAPS(Bayesian Analysis of Population Structure)软件在未知种群信息的前提下对57只扬子鳄个体进行类群分析,发现这些个体可以被划分为2个种群(长兴本土种群和美国重引入种群)。同时遗传混合分析显示,长兴种群和美国重引入种群虽然存在着一定程度的遗传差异,但是两者间出现了一定的混合。
6.微卫星分析结果显示,扬子鳄种群的遗传多样性较低,但长兴本土种群和美国重引入种群均具有自身特有的稀有等位基因。因此,基于保护扬子鳄这一极度濒危物种的遗传多样性之目的,作者建议在野外放归或重引入工程中,管理者以选取不同地域(种群)的个体组成扬子鳄的奠基种群为佳。
Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. Although the captive Chinese alligator populations have been greatly developed in recent years, there has been genetic depression in the filial generation because of the founder effect. As the startup of the reintroduction project for the captive Chinese alligator population, it is urgent to investigate the genetic status of this species and provide feasible strategies for its conservation.
In this study, we isolated polymorphic microsatellite loci from the Chinese alligator based on magnetic beads enrichment protocol, and did comprehensively research on genetic diversity and population structure of this species. The major results are:
1. Approximately 4,000 clones were screened and a total of 260 recombinants that potentially contained microsatellite sequence were obtained. 150 positive clones were chosen randomly for DNA sequencing, out of which 92 clones had sufficient flanking sequence for primer design. The result of PCR amplification revealed that 56 primer pairs could yield consistent specific products. Further experiments showed that 11 pairs of them were polymorphic and well amplificated for each sample.
2. The 11 polymorphic microsatellites presented a total of 31 alleles among 57 individuals scored, yielding an average of 2.82 alleles per locus. One allele was unique to the American reintroduced population(Alsi05-2) but four private alleles(Alsi05-1; Alsi06-1; Alsi07-1; Alsi10-1) were detected in the Changxing indigenous population. The average PIC (polymorphic information content) of the 11 polymorphic loci obtained in this study is 0.384.
3. The individual identity probability was evaluated for the 11 polymophic markers. Results suggested that when the 11 polymorphic loci were combined, the individual identity probability was 0.9998. Therefore, the microsatellite markers could be utilized to genetic pedigree construction for the captive Chinese alligator populations. In addition, it could be helpful in the selecting of proper individuals for the reintroduction program.
4. In this study, we also used 8 polymorphic loci isolated by cross-species amplification to assess the genetic status of this species, of which 6 loci was amplificated effectively. Finally, the exclusion probabilities of paternity testing went up to 0.991 if 11 species-specific polymorphic loci calculated in combination with 6 previous cross-species markers, which achieved the desired results.
5. A complete Bayesian method in BAPS (Bayesian Analysis of Population Structure) was chosen to perform individual clustering without prior population information. The results showed that the 57 individuals were grouped into 2 clusters (named as the Changxing indigenous population and the American reintroduced population). The further genetic admixture analysis revealed that although genetic difference was present between the two populations, there was certain admixture among them.
6. Based on the microsatellite analysis results, we conclude that the Chinese alligator has very low genetic diversity, but the Changxing indigenous population and the American reintroduced population still has some rare genes of their own. Hence, we strongly suggest that individuals from different populations should be chosen as the founders in the reintroduction program to largely protect the genetic diversity of this critically endangered species.
引文
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