云南栘[木衣]SRAP-PCR反应体系的建立及引物筛选  

Establishment of SRAP-PCR Reaction System on Docynia delavayi(Franch.)Schneid.and Selection of Primers

作　　者：陈璨 石辰 王大玮[1,2] 彭劲谕 段安安[1,2] CHEN Can;SHI Chen;WANG Dawei;PENG Jingyu;DUAN An'an(Southwest Forestry University,Key Laboratory for Forest Resources Conservation and Use in the Southwest Mountains of China,Ministry of Education,Kunming 650224,China;Southwest Forestry University,Key Laboratory for Forest Genetic and Tree Improvement&Propagation in Universities of Yunnan Province,Kunming 650224,China)

机构地区：[1]西南林业大学,西南山地森林资源保育与利用教育部重点实验室,云南昆明650224 [2]西南林业大学,云南省高校林木遗传改良与繁育重点实验室,云南昆明650224

出　　处：《云南农业大学学报（自然科学版）》2021年第6期1037-1043,共7页Journal of Yunnan Agricultural University:Natural Science

基　　金：国家自然科学基金项目(32060350);国家林业和草原局林业科技发展项目(KJZXSA2019036)。

摘　　要：【目的】建立云南栘[木衣]SRAP-PCR最佳反应体系并筛选SRAP-PCR多态性引物组合。【方法】以木里、澜沧和盈江3个天然群体的云南栘[木衣]嫩叶为试验材料,采用改进后的CTAB法提取云南栘[木衣]基因组DNA。利用L16(45)的正交设计对Taq DNA聚合酶、Mg^(2+)、dNTPs、模板DNA及引物5种因素进行优化,建立云南栘[木衣]SRAP-PCR最佳反应体系,并从100对引物组合中筛选SRAP-PCR多态性较高的引物组合。【结果】云南栘[木衣]SRAP-PCR最佳反应体系(25μL):Taq DNA聚合酶0.5 U,Mg^(2+)2.5 mmol/L,dNTPs 0.25 mmol/L,模板DNA 80 ng,引物0.8μmol/L。各因素对云南栘[木衣]SRAP-PCR反应体系的影响由小到大排列为:dNTPs<模板DNA<Taq DNA聚合酶<Mg^(2+)<引物。利用木里、澜沧和盈江3个天然群体的云南栘[木衣]基因组DNA对建立的体系进行验证,筛选出的20对引物组合共扩增出212个位点,其中多态性位点187个,占比为88.21%。【结论】建立了稳定可靠的云南栘[木衣]SRAP-PCR体系,为后续云南栘[木衣]遗传多样性的研究提供支持。[Purpose]To establish the optimal SRAP-PCR reaction system in Docynia delavayi and selection of SRAP-PCR polymorphism primer combinations.[Method]The improved CTAB method was used to extract DNA from young leaves of D.delavayi from three natural populations of Muli,Lancang and Yingjiang.In order to establish the SRAP-PCR reaction system applied to D.delavayi,the orthogonal design L16(45)was used to optimize five important influencing factors such as Taq DNA polymerase,Mg^(2+),dNTPs,template DNA and primers.And the primer combinations with high SRAP-PCR polymorphism were screened from 100 primer combinations.[Result]The optimal reaction system for the SRAP-PCR of D.delavayi as(25μL):Taq DNA polymerase 0.5 U,Mg^(2+)2.5 mmol/L,dNTPs 0.25 mmol/L,template DNA 80 ng,primers 0.8μmol/L.In addition,the effect degrees of the factors on the SRAP-PCR reaction were in the order of dNTPs<template DNA<Taq DNA polymerase<Mg^(2+)<primers.Using the established and optimized SRAP-PCR system,20 pairs of primers were used to amplify DNA of D.delavayi,and a total of 212 amplified bands were obtained,the polymorphism sites was 187,the percentage was 88.21%.[Conclusion]A stable and reliable SRAP-PCR system was established to support the subsequent study of genetic diversity in D.delavayi.

关 键 词：云南栘[木衣] SRAP标记 引物筛选 

分 类 号：Q949.751.8[生物学—植物学]