苦楝SSR-PCR反应体系优化及引物筛选

Optimization of SSR-PCR Reaction System and Primer Screening of Melia azedarach

[目的]借鉴以往楝属文献报道的SSR引物,筛选高度多态性、稳定性高、重复性好的苦楝SSR引物,为苦楝遗传图谱构建、QTL定位和分子标记辅助选择育种等研究领域应用奠定基础。[方法]本研究采用单因素法和正交试验设计进行SSR-PCR反应体系优化,并利用该体系以8个不同种源的苦楝基因组DNA为模板,从135对候选SSR引物中进行引物筛选。[结果]苦楝SSR-PCR最优反应体系为:1.0μL 50 ng·μL^(-1)模板DNA,1.2μL 100μmol·L^(-1)引物,1.0μL 10 mmol·L^(-1)d NTPs,0.8μL 25 mmol·L^(-1)Mg^(2+),0.15μL 5 U·μL^(-1)Taq酶,1.5μL 10×Buffer,补dd H2O至15μL;最终筛选出15对具有高度多态性、稳定性高、重复性好的SSR引物。[结论]本研究成功优化了苦楝SSR-PCR反应体系,并成功筛选出15对适用于苦楝的SSR引物。

[Objective]Based on previous achievement on Melia SSR primer to select highly polymorphic,high stability and repeatability Melia azedarach primers,and to lay a foundation for constructing M. azedarach genetic map,QTL mapping and molecular marker- assisted selection and breeding. [Method]The SSR- PCR system was optimized through the single factor experiment and orthogonal test,and by using the genomic DNAs of M. azedarach from 8 provenances as templates,the SSR primers suitable for M. azedarach were screened from 135 pairs of primers. [Result]The optimal SSR- PCR system is as follows： 1. 0 μL 50 ngoμL^（-1）genomic DNA,1. 2 μL 100 μmol·L^（-1）of each primer,1. 0 μL 10 mmol·L^（-1）d NTPs,0. 8 μL 25 mmol·L^（-1）Mg^（2＋）,0. 15 μL 5 U·μL^（-1）Taq polymerase,1. 5 μL 10 × Buffer（ Mg^（2＋）free）,and replenishing dd H_2O to 15 μL. And,15 pairs of SSR primers with high polymorphism,high stability,and good repeatability,were finally screened out. [Conclusion]The SSR-PCR system of M. azedarach was successfully optimized,and 15 pairs of SSR primers applicable to neem were selected.