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微卫星多重PCR技术在生物的亲子鉴定、群体遗传分析以及家系管理等方面均得到了广泛的应用。长鳍吻鮈(Rhinogobio ventralis)是我国长江上游特有珍稀鱼类,目前已达3级急切保护状,被列为低危鱼类。为了提高长鳍吻鮈微卫星分型效率,本研究从已开发的微卫星中筛选出15个多态性较高的微卫星位点进行组合扩增,并对位点组合的引物浓度、退火温度和反应体系等条件进行优化,建立了5组各含3个微卫星位点多重PCR体系。利用5组微卫星多重PCR,通过ABI 3730测序仪和Cervus v.3.0软件对136尾长鳍吻鮈进行个体基因型检测以及遗传多样性分析,结果显示,群体的平均等位基因数(number of allele,Na)为15.600,平均期望杂合度(expected heterozygosity,He)为0.825,平均观测杂合度(observed heterozygosity,Ho)为0.813,平均多态信息含量(polymorphism information content,PIC)为0.801。对40尾候选亲本和96尾子代进行亲子鉴定分析,结果显示,第一亲本的累积排除概率(combined exclusion probability of the first parent,CE-1P)、第二亲本累积排除概率(combined exclusion probability of the second parent,CE-2P)和双亲累积排除概率(combined exclusion probability of a parent pair,CE-PP)分别为0.999986 07、0.999 999 97和1.000 000 00。使用该5组微卫星多重PCR体系进行亲子鉴定准确率为100%。长鳍吻鮈微卫星多重PCR技术的建立为亲子鉴定、分析群体遗传多样性和辅助家系管理提供了一种高效的技术手段。
Microsatellite multiplex PCR technology has been widely used in biological paternity testing, population genetic analysis and pedigree management. Rhinogobio ventralis is endemic to China’s upper reaches of the Yangtze River. It has reached grade 3 in urgent protection and is classified as low-risk fish. In order to improve the efficiency of SSR microsatellite typing, 15 microsatellite loci with high polymorphism were screened from the developed microsatellites for combined amplification. The primer combinations of the loci were annealed Temperature and reaction conditions were optimized, and five groups of 3 microsatellite loci multiplex PCR systems were established. Five genotypes of microsatellite multiplex PCR were used to analyze the genotypes and genetic diversity of 136 long-finned iguanas by ABI 3730 sequencer and Cervus v.3.0 software. The results showed that the average number of allele, Na) was 15.600. The average expected heterozygosity (He) was 0.825, the observed heterozygosity (Ho) was 0.813, and the polymorphism information content (PIC) was 0.801. Paternity analysis of 40 candidate and 96 progeny showed that the combined exclusion probability of the first parent (CE-1P), the second parent’s total exclusion probability of the second parent, CE-2P) and the combined exclusion probability of a parent pair (CE-PP) were 0.999986 07, 0.999 999 97 and 1.000 000 00, respectively. The 5 sets of microsatellite multiplex PCR system for paternity testing accuracy of 100%. The establishment of microsatellite multiplex PCR of Long-fingered Anopheles stephensi provides an efficient technique for paternity testing, analyzing population genetic diversity and assisting pedigree management.