山葡萄SRAP技术体系的建立及其在品种鉴定中的应用

Establishment of SRAP-PCR System and Variety Identification on Vitis amurensis

以6个不同的山葡萄品种为材料,对山葡萄SRAP分子标记技术体系进行优化,探讨SRAP标记在山葡萄品种鉴定中的可行性。结果表明:SRAP扩增程序为94℃预变性5 min;前5个循环,94℃变性45 s,35℃退火45 s,72℃延伸90 s;后35个循环,94℃变性45 s,50℃退火45 s,72℃延伸90 s;最后72℃延伸7min。反应体系:25μL总反应体系中含模板DNA 20 ng,dNTP 1.0μL,上下游引物各1μL,rTaq酶0.2μL,10倍Buffer1.0μL,其余用灭菌的ddH2O补充。应用筛选出的16对引物对6个山葡萄品种进行扩增,共扩增出96条条带,其中多态性带占67.71%。每个品种都能找出其特有条带,最少用2对引物就可鉴别6个山葡萄品种。

With ＂zuoyouhong＂ and five different cultivars of grape as materials, the optimization of SRAP molecular marker system of Vitis amurensis was carried out, and the feasibility of SRAP marker in identi- fying grape cultivars was discussed. The main results are as follows： SRAP amplification program： 94℃ denaturation for 5 min; the first 5 cycles, 94 ℃ denaturation 45 s, 35 ℃ annealing 45 s, 72℃ exten- sion 90 s; after 35 cycles, 94 ℃ denaturation 45 s, 50 ℃ annealing 45 s, 72 ℃ for 90 s; last 72 ℃ for 7 min. Reaction system： 25 ~tL total reaction system containing template DNA 20 ng, dNTPs 1.0 μL, upstream and downstream primers each 1 μL, Taq DNA polymerase 0.2 μL, 10 x Buffer 1.0 μL, the other with ddH20 added. Ninety-six bands were produced from six different cultivars of Vitis amurensis by 16 pairs of selected primers, and the percentage of polymorphie bands （PPB） was 67.71%. Each species has its unique band, at least with 2 pairs of primers Vitis amurensis cultivars can be identified.