鲢微卫星标记的荧光多重PCR体系建立及其应用

摘要

为了提高鲢微卫星(SSR)分型效率,本研究从已开发SSR标记中筛选出14对具有高多态性和扩增稳定性的SSR引物,并组成一个四重PCR(A)和两个五重PCR(B和C).以单对引物PCR为对照,对多重PCR的条件进行优化,包括退火温度、Taq DNA聚合酶浓度、dNTPs浓度、Mg2+浓度、PCR Buffer使用量等.优化结果为,各体系最佳退火温度时,25 μL体系中包括2 UTaq DNA聚合酶,0.4 mmol/LdNTPs,2.3 mmol/L Mg2+,以及3μL的10×PCR Buffer.优化后的多重PCR扩增稳定,各对引物扩增良好,SSR分型效率显著提高.利用建立的多重PCR体系对采自石首老河四大家鱼原种场和监利老江河四大家鱼原种场各100尾鲢(Hypophthalmichthys molitrix)样本进行SSR分析.结果显示,石首和监利两个原种场的样本都保持有较高的遗传多样性,其中观测杂合度分别是0.8479和0.9443,期望杂合度分别为0.7967和0.8134.群体间分子方差分析(AMOVA)FST=0.00613,说明两个原种场鲢群体间没有发生遗传分化.本研究最终建立了三个准确、稳定的荧光标记多重PCR体系,具有较大应用价值.运用建立好的3个荧光标记多重PCR对2个鲢群体进行遗传多样性分析,结果为两处原种场的进一步科学管理提供了可靠依据.%Silver carp (Hypophthalmichthys molitrix) is a commercially important fish in China, and its natural resource declined seriously in recent years. Several molecular markers had been used to evaluate population genetics of silver carp, and more markers and samples are still necessary for long term conservation issue. In order to enhance genotypic efficiency of microsatellite DNA (SSR) in silver carp, 14 SSR primer sets with characters of high polymorphism and stable amplification were screened out. Those primers were grouped to three multiplex PCR with four or five primer sets. Parameters of multiplex PCR such as annealing temperature, contents of Taq DNA polymerase,dNTPs.Mg2+ and PCR Buffer were optimized. When the annealing temperature were optimized, the PCR content including 2 units of Taq DNA polymerase, 0.4 mmol/L dNTPs,2.3 mmol/L Mg2+ and 3 μL 10×PCR Buffer in a total volume of 25 |xL could work preferably for different multiplex PCR. The optimized multiplex PCR then were used to evaluate genetic diversity of silver carp from National Original Breeding Farms in Laojianghe of Jianli and Laohe of Shishou. Two hundred specimens were collected from the two farms and used in this study. Number of alleles ranged from 7 to 24 and from 8 to 20 with mean 13.36 and 12.71 per locus for Jianli and Shishou Farms, respectively. The observed heterozygosity ranged from 0.83 to 1 and from 0.66 to 0.99, with means 0.8479 and 0.9443, and the expected heterozygosity from 0.6462 to 0.9327 and from 0.6075 to 0.9063, with means 0.7967 and 0.8134, respectively. There were 1 and 4 loci being deviations from Hardy-Weinberg equilibrium for Jianli and Shishou Farms, respectively. Analysis of molecular variance (AMOVA) among populations revealed FST=0.0864. It suggested that the genetic diversity level of H.molitrix in both Shishou and Jianli farms were high and no genetic divergence have taken place between H.molitrix populations in the two farms. The results . Show that the use of those three multiplex PCR can be more rapid and more economic in genetics analysis of H.molitrix.