正交设计优化番石榴属(Psidium)植物SSR-PCR反应体系和引物筛选

Optimization for SSR-PCR Reaction System of Psidium Trees by Orthogonal Design Method and Primers Screening

以4份番石榴属植物嫩叶为材料,利用L(16)(4~3)正交试验设计方法,对2×Taq Mix、模板DNA、引物浓度3个因素进行4个水平筛选。结果表明,反应各因素对SSR-PCR体系的影响程度为2×Taq Mix用量>DNA模板量>引物浓度,适合番石榴属植物SSR-PCR反应的最佳体系为20μL。反应体系包含了DNA模板40 ng、引物各0.3μmol/L、2×Taq Mix 10μL,ddH2O 4.8μL;运用该优化体系从23对SSR引物中筛选出21对扩增条带清晰、多态性良好的SSR引物,多态引物占比91.30%;利用9份番石榴种质验证了SSR-PCR体系具有较高的稳定性和重复性。

In or der to further study on the genetic diversity and variety identification of Psidium by molecular marker technology,the young leaves of 4 Psidium germplasm as extraction materials applied for DNA template,an L16(43)orthogonal design was applied to optimized PCR system of SSR marker for Psidium trees in 4 levels of 3 factors(2×Taq Mix,primer dosage and template DNA concentration)and the pairs of primers of screening were explored.The results showed that the most remarkable factor that made influence on the SSR-PCR reaction system of Psidium trees was 2×Taq Mix followed by template DNA and primers.The best reaction system for SSRPCR of Psidium was established,a total of 20μL systems containing template DNA 40 ng,each primer 0.3μmol/L,2×Taq Mix 10μL and ddH2O 4.8μL.21 pairs of SSR primers with high polymorphism and good repeatability were successfully screened out by using this optimal system,and the polymorphic ratio was 91.30%.And then 9 different guava materials were tested which proved that the optimized PCR reaction system had high repeatability and stability.