辣椒SRAP-PCR反应体系优化及杂交群体多态性引物筛选

Optimization of SRAP-PCR and Selection for Polymorphic Primers in Hybrid Populations

[目的]对辣椒SRAP反应体系进行研究和优化,并利用优化体系对164对多态性引物进行筛选。[方法]采用单因素随机试验设计和L25(65)正交试验设计对辣椒SRAP反应体系中6种关键因素(退火温度、Taq DNA聚合酶、Mg2+、dNTPs、引物、模板DNA)进行体系优化,并以"泡椒"×"青皮大椒"的亲本和F1代为材料,利用聚丙烯胺酰胺凝胶进行筛选。[结果]辣椒SRAP-PCR的最佳反应体系为:退火温度35.5℃,Taq DNA聚合酶1.0 U,模板DNA 2.25 ng/μl,dNTPs 0.2 mmol/L,引物0.4μmoL/L,Mg2+2 mmol/L,总体积为20μl。利用该体系,从164对SRAP引物组合中筛选出扩增条带清晰、多态性丰富、条带稳定的31对引物组合。[结论]该体系的建立与多态性引物组合的筛选为SRAP标记技术在辣椒育种中的应用奠定了基础。

[ Objective ] To study and optimize pepper SRAP reaction system, 164 ~polymorphie primers were selected by using optimization system. [ Method] Single-factor random experiment design and L25 （6^5 ） orthogonal test were used optimize 6 key factors in pepper SRAP reac- tion system（ annealing temperature, amounts of Taq DNA polymerase, concentrations of Mg2＋ , concentrations of dNTPs, concentrations of primer, concentrations of template DNA）, with Pickled pepper x Green pepper parents and F1 generation as material, screening was conduc- ted by polypropylene amine amide gel. [ Result] The results showed that, the optimized SRAP-PCR system for pepper was： 35.5℃ annealing temperature, 1.0 U Taq DNA polymerase, 2 mmol/L Mg2＋ , 0.2 mmol/L dNTPs, 0.4 μmol/L primer, 2.25 ng/μl template DNA in a total of 20 p,L reaction solution. The optimized SRAP- PCR system was used to select polymorphic primer combinations, 31 primer combinations were selected with distinct, abundant and steady polymorphism from 164 primer combinations. [ Conclusion] The optimized SRAP-PCR system and selected polymorphism primer combinations could be applied to facilitated breeding work of pepper.