中国对虾SRAP分子标记体系正交优化及在遗传多样性分析中的应用

Optimization of SRAP-PCR System Using Orthogonal Design and its Application to Genetic Analysis in Fenneropenaeus chinensis

中文关键词: 中国对虾  正交设计  SRAP  遗传多样性

英文关键词: Fenneropenaeus chinensis  Orthogonal design  SRAP  Genetic diversity

中文摘要:

      利用正交设计L16(45)对影响中国对虾SRAP分子标记分析的5个因素（Taq酶，Mg2+，模板DNA，dNTP和引物浓度）在4个水平上进行了优化，筛选出各反应因素的最佳反应水平为：20 μL反应体系中包含1.0 U Taq酶，2.0 mmol/L的Mg2+，40.0 ng的模板DNA，0.125 mmol/L的dNTP以及0.4 μmol/L的引物，引物的退火温度为53.5 ℃。研究表明各因素的不同水平对扩增结果有显著影响，其中Mg2+影响最大，影响大小顺序依次为Mg2+，Taq酶，引物，dNTP和模板DNA。利用优化的SRAP分子标记体系对中国对虾“黄海1号”第12世代选育群体进行了遗传多样性分析的应用，实验结果表明此标记技术可作为中国对虾遗传分析的良好技术工具。遗传多样性分析中共筛选出8对可以扩增出清晰稳定条带的引物组合，共获得171个位点；其中多态性位点154个，占90.08%；有效等位基因数为1.7741，期望杂合度均值为0.4219，shannon多样性指数为0.6055。上述参数值均高于应用AFLP技术对前几代选育群体的遗传多样性分析结果。

英文摘要:

      The orthogonal design L16(45) was used to optimize SRAP-PCR amplification system in marine shrimp Fenneropenaeus chinensis on four levels of five factors (Taq DNA polymerase, Mg2+, DNA template, dNTP, and primer). The results showed that the optimum concentrations were 1.0 U Taq enzyme, 2.0 mM Mg2+, 40.0 ng DNA template, 0.125 mM dNTP and 0.4 μM primer, respectively in this 20 μL SRAP-PCR system., and the annealing temperature was 53.5 ℃ via setting temperature gradient. Significant effects of each factor in different levels were observed and the concentration of Mg2+ was the most dominant factor affecting the result of PCR, while Taq polymerse, primer, dNTP, and template DNA followed in terms of dominance. Then, the established amplification system was used to analyze the genetic diversity of the 12th generation of selected new variety “Huanghai No.1” of F. chinensis. As a result, 171 bands were generated with 8 primer sets, of which 154 bands were polymorphic bands, accounting for 90.08%. The effective number of alleles, genetic diversity and Shannon's information index were counted as 1.7741, 0.4219 and 0.6055, respectively. The parameters mentioned above were higher than those reported by authors applying AFLP technique.l