广西莪术SSR-PCR反应体系优化和引物筛选研究

摘要

以广西莪术(Curcuma kwangsiensis S.G.Lee et C.F.Liang)嫩叶为材料，采用单因素和正交试验设计建立和优化SSR-PCR反应体系和反应程序，对最适宜退火温度(Tm)进行筛选优化，对17对已公布的姜黄属通用性引物进行扩增试验，筛选出多态性好、条带清晰的引物。结果表明，建立了适合广西莪术SSR分析的反应体系和扩增程序，即在15μL体系中，DNA模板为30 ng/μL，3μL，Mix(包含DNTP、Mg2+、1× buffer、Taq酶)为5μL，ddH2O为6μL，引物为各0.5μL时，分析效果较好，扩增程序为94℃预变性5 min；94℃变性30 s，根据不同引物的退火温度复性30 s，72℃延伸1.5 min，共35个循环；最后72℃延伸5 min；反应结束后，4℃保存。同时筛选出在试验材料中能产生较清晰主带的引物共12对，初步验证了应用SSR分子标记分析在广西莪术遗传多样性的可行性。%The single factor analysis and orthogonal experimental design were used to establish and optimize SSR-PCR reaction system andprocedures of Curcuma kwangsiensis S.G.Lee et C.F.Liang,and optimum annealing temperature ﹙Tm﹚ optimization. The 17 published universal primers of Curcuma longa L. were used for amplification test,screened polymorphic and clear bands of primers. The results showed that the establishment of a suitable Curcuma kwangsiensis S.G.Lee et C.F.Liang SSR analysis of the reaction system and amplified procedure,namely in 15μL system,DNA template 30 ng/μL,3μL,Mix﹙containing DNTP,Mg2+,1×buffer,Taq enzyme﹚ of 6μL,ddH2O to 5μL,primers for the 0.5μL,the analysis is better,amplification program was 94℃ for 5 min,94℃ denaturation 30 s,depending on the primer annealing temperature annealing 30 s,72℃ extension 1.5 min,35 cycles,and finally 72℃ for 5 min,after completion of the reaction,4℃ preservation. While filtering out the test mate-rial can produce clearer master with a total of 12 primer pairs validated the application of SSR markers in Curcuma kwangsiensis S.G.Lee et C.F.Liang feasibility analysis of genetic diversity.