不同赤眼蜂品系ISSR-PCR条件优化及分子鉴定

赤眼蜂Trichogramma不同品系在寄生能力等生物学特性上存在差异,而这些差异可能被用于优良品系的筛选。通过优化影响赤眼蜂不同品系ISSR-PCR的主要参数,建立适于鉴别赤眼蜂不同品系的ISSR-PCR反应体系和扩增程序。在20μL反应体系中,各反应物的最适含量为10×PCR Buffer 2.0μL,MgCl22.0μL（25mmol/L）,dNTPs1.6μL（2.5mmol/Leach）,正反向引物各1μL（25μmol/L）,DNA模板1.0μL,Taq酶0.4μL（2.5U/μL）,和ddH2O。ISSR-PCR的扩增程序为：95℃预变性5min,94℃变性50s,52℃退火1min,72℃延伸1min20s,35个循环,72℃延伸10min。结果表明,在上述优化条件下,采用ISSR-PCR可实现对7个赤眼蜂优良品系的分子鉴定。该研究对于赤眼蜂优良品系的筛选具有潜在的应用价值。

Optimization of ISSR-PCR analyses and molecular identification of different strains of Trichogramma spp

Different Trichogramma strains are differentiated from each other in biological characteristics, such as parasitic capability, which might be used for screening of superior Trichogramma strains. Here Inter-Simple Sequence Repeats （ISSR）-PCR was used to investigate the genetic variation of 7 candidate strains, and the molecular markers specific to different strains were subsequently identified. By optimizing the main parameters, the optimal ISSR-PCR reaction conditions for Trichogramma were firstly established. The results showed that the optimum concentrations of different reagents in a total of 20 μL volume were： 2.0 μL MgCl2 （20 mmol/L）, 1.6 μL dNTPs （2.5 mmol/L each）, 1 μL ISSR primer （10 μmmol/L）, 1.5 μL genomic DNA and 0.4 μL Taq DNA polymerase （2.5 U/μL）. The suitable PCR procedures were determined to be： pre-denaturing at 95℃ for 5 min, followed by 35 cycles of denaturing at 94℃ for 50 s, annealing at 52℃ for 1min and extension at 72℃ for 1 min 20 s, with a final extension at 72℃ for 10 min. Under the optimal conditions, the 7 Trichogramma strains tested eould be discriminated by using ISSR-PCR. It is therefore suggested that this technique is helpful in identification and screening of Trichogramma strains.