云南松SSR-PCR反应体系的建立与优化

为了建立适宜云南松SSR-PCR的反应体系和扩增程序,利用近缘种火炬松的引物,采用正交设计L16(45)对云南松SSR-PCR反应体系的5因素(Taq酶、Mg2+、模板DNA、dNTP、引物)在4个水平上进行优化,筛选出各反应因素的最佳水平,建立了适于云南松的SSR反应体系.在10μL的反应体系中,模板DNA的用量为30.0 ng,Taq DNA聚合酶的用量为1.0 U,Mg2+的浓度为2.0 mmol/L,dNTPs浓度为0.4 mmol/L,引物的浓度为0.2 μmol/L.扩增程序为:94℃预变性4 min；94℃变性45 s,48℃退火30 s,72℃延伸30 s,30个循环；72℃延长10 min,4℃保存.最后利用1个居群对该体系进行稳定性验证,结果可用于云南松SSR标记的研究.

Establishment and Optimization of the SSR Reaction System for Pinus yunnanensis Using Orthogonal Design

In order to establish and optimize the SSR-PCR reaction amplification system and procedures of Pinus yunnanensis,the known primers from related species-Pinus taeda were employed,orthogonal design L16(45)on Yunnan pine SSR-PCR reaction system of 5 elements(Taq enzyme,Mg2+,template DNA,dNTP,primers)in the four levels of optimization was established,filtering out the best level of response factors established for the SSR Pinus reaction.An optimal 10 μL volumes SSR-PCR system consists of template DNA was 30.0 ng,Taq DNA polymerase was 1.0 U,primer was 0.2 μmol/L,Mg2+ was 2.0 mmol/L,and dNTPs was 0.4 mmol/L.PCR reaction amplification procedures was: pre-denaturation for 4 min at 94℃,followed by 30 cycles of denaturation for 45 s at 94℃,anneal for 30 s at 48℃,extension for 30 s at 72℃,the amplification was completed after extension for 10 min at 72℃,then stored at 4℃.Finally,24 DNA was used to verify the stability of the system,which can be as reference for studyPinus yunnanensisSSR markers.