Optimization of ISSRPCR system and its application in the identification of sainfoingermplasm of space irradiation

Based on the orthogonal design, the optimized ISSRPCR system of sainfoin (Onobrychis viciaefolia) germplasm was established by comparing the 4 factors (dNTP, Taq polymerase, primer and Mg2+) and the 3 levels. This established system was used to select the primer combination and the optimum Tm and the ISSR Marker System was applied to identify the variation of hereditary substance of sainfoin offspring from space irradiation seeds. The results of this study showed that the optimized ISSRPCR system for sainfoin was 2.5 L 10PCR buffer, 50 ng template DNA, dNTP 0.2 mmol/L, Taq DNA polymerase 1.5 U, primer 0.4 mol/L, Mg2+ 2.0 mmol/L in a total of 25 L reaction solution. 12 primer combinations were selected with abundant polymorphism from 53 primers. 201 DNA bands were amplified by 12 ISSR primers, in which 142 bands were polymorphic, and polymorphic ratio (PPB) was 73.6%. This study also indicated that the percentage of polymorphic loci(P)，Nei gene diversity(h) and Shannon index(I) in Sainfoin seeds of the control was lower than that of the seeds carried by spaceflight, implying that space environment caused the variation of hereditary substance DNA of Sainfoin. This study suggested that the space radiation would be an effective approach to developing new sainfoin germplasm.