基于微卫星多重PCR技术的兰州鲇亲子鉴定

针对目前兰州鲇(Silurus lanzhouensis)种质资源救护保存和良种选育等研究工作中面临的亲子鉴定及系谱管理等问题,研究应用微卫星荧光标记多重PCR与自动测序分型技术,建立了2组四重PCR和2组三重PCR体系,并成功应用于3个家系亲子鉴定中。利用Cervus v.3.0软件对110尾兰州鲇进行遗传多样性分析,结果显示:研究筛选的14个微卫星标记的平均观测杂合度(H_o)为0.750,平均期望杂合度(H_e)为0.667,平均多态信息含量(PIC)为0.624,具有丰富的遗传多样性。对已知系谱信息的3个兰州鲇家系的90尾子代和20尾候选亲本进行亲子鉴定分析,结果表明,双亲基因型未知累积排除概率(CE-1P)、单亲基因型已知累积排除概率(CE-2P)和双亲基因型已知累积排除概率(CE-PP)分别为0.99753092、0.99983971和0.99999964。4组多重PCR累积模拟鉴定率为100%,累积实际鉴定率为83%。采用50尾个体进行双盲验证,利用MEGA7.0对3个家系50尾个体进行聚类分析,结果表明同一家系94%的个体聚类分析...

PARENTAGE ASSIGNMENT OF SILURUS LANZHOUENSIS USING MULTIPLEX PCR OF MICROSATELLITES

Silurus lanzhouensis, the name card of the aquatic organism of the Yellow River, is an ecological resource of important value. As a key protected commercial fish in the Upper and Middle Yellow River, S. lanzhouensis has a broad breeding prospect. For the ecological civilization construction of the Yellow River, strengthening the rescue practices, breeding strategies, and scientific utilization of the S. lanzhouensis germplasm resources are of great significance. Aiming to figure out problems of paternity identification and genealogy management, to rescue and preserve S. lanzhouensis germplasm resources and thus to improve variety selection, two sets of quadruple PCR and two sets of triple PCR systems have been established using fluorescent primers and automatic sequencing technology, which have been successfully applied to the paternity test in three families. Performing the analysis of the genetic diversity of 110 fish of S. lanzhouensis using Cervus v.3.0 software, the results obtained from the 14 microsatellite markers screened in this study revealed the average of observed heterozygosity (H_o) 0.750, the average of expected heterozygosity (H_e) 0.667, and the average of polymorphic information content (PIC) 0.624, indicating rich igenetic diversity. The parentage assignment was performed on 90 offspring and 20 candidate parents from three S. lanzhouensis family with known pedigree information. The results showed that the combined exclusion probabilities of the first parent (CE-1P), the second parent (CE-2P), and a parent pair (CE-PP) 0.99753092, 0.99983971, and 0.99999964, respectively. The cumulative simulated identification rate of the four sets of multiplex PCR was 100%, and the cumulative actual identification rate was 83%. Double-blind validation was performed on 50 offspring from 3 families, and cluster analysis was performed using MEGA7.0. The result that the cluster analysis results of 94% of the individuals from the same family was consistent with the genealogical relationship. It is concluded that the four sets of technical system for paternity test using multiplex PCR can provide important technical supports for the polyculture of S. lanzhouensis with different germplasm, germplasm selection, pedigree management and molecular marker-assisted selection.