丁坝对鱼类栖地的影响范围评估

利用磁珠富集法和5’锚定PCR法开发背瘤丽蚌的微卫星标记,将获得的多态性引物用于群体的遗传多态性分析,以期在比较两种开发微卫星标记方法的基础上同时获得一批有用的微卫星引物。从磁珠富集法获得的微卫星序列阳性克隆率为69.2%,重复次数超过10的占总数的70.2%,从设计的28对引物中筛选得到多态性引物11对,开发效率为39.3%。这11对引物用于养殖群体的遗传多样性分析,结果显示,等位基因数范围为4～13,观测杂合度、期望杂合度范围分别为0.205～0.738、0.566～0.839。而5’锚定PCR法获得的微卫星序列阳性克隆率为97.8%,重复次数超过10的占总数的24.7%,从设计的56对引物中筛选得到多态性引物19对,开发效率为30.4%。这19对引物用于养殖群体的遗传多样性分析,结果显示,等位基因数范围为3～10,观测杂合度、期望杂合度范围分别为0.208～0.894、0.431～0.896。实验结果表明,磁珠富集法所获微卫星序列质量高,开发微卫星标记效率较高;而5’锚定PCR法实验操作更简便,所获得的引物遗传多样性指数更高。两种方法开发的引物均可用于背瘤丽蚌和近缘种的野生种质资源遗传多样性研究。

Development of microsatellite markers from Lamprotula leai: A comparison of magnetic beads hybridization and 5' anchored PCR methods

The microsatellite DNA markers from Lamprotula leai were developed by using magnetic beads hybridization and 5’ anchored PCR methods,and applied in genetic diversity analysis,on the purpose of obtaining a batch of microsatellite primers based on the comparison of the two microsatellite development methods.The colonies from library constructed by magnetic beads hybridization method were selected and screened,of which,69.2% were positive.The number of the replications over 10 occupied 70.2% of the total,and 28 pairs of the primers were designed,according to the sequences obtained.Eleven of the primers could be applied in DNA polymorphism analysis,rating 39.3%.The analysis of the genetic diversity in cultured population of L.leai by using the 11 primers indicated that the number of the alleles per locus ranged from 4 to 13.The observed heterozygosity(Ho) and expected heterozygosity(He) ranged from 0.205-0.738 and 0.566-0.839,respectively.The colonies from library constructed by 5’ anchored PCR method were selected and screened,of which,97.8% were positive.The number of the replications over 10 occupied 24.7% of the total,and 56 pairs of the primers were designed,according to the sequences obtained.Nineteen of the primers could be applied in DNA polymorphism analysis,rating 30.4%.The analysis of the genetic diversity in cultured population of L.leai by using the 19 primers indicated that the number of the alleles per locus ranged from 3-10.The observed Ho and He ranged from 0.208-0.894 and 0.431-0.896,respectively.Our results indicated that the microsatellite sequences obtained by magnetic beads method had higher quality,and this method could be a higher efficient method,whereas the 5’ anchored PCR method was more easily performed,and the genetic diversity index of the primers obtained was higher.All the microsatellite markers developed by the two methods could be useful in the genetic diversity analysis of L.leai and related species.