摘要： 以西瓜品种中华拳王为试材,探索了西瓜SRAP-PCR反应程序,并对西瓜SRAP-PCR反应体系的各影响因子进行了梯度设置试验,筛选和建立了可扩增多态性高、重复性好、带型清晰的最佳SRAP-PCR反应程序和体系。反应程序为:94℃预变性5 min;94℃变性1 min,35℃退火1 min,72℃延伸1 min,共5个循环;94℃变性1 min,50℃退火1min,72℃延伸1 min,共35个循环;72℃延伸5 min;4℃保存。反应体系(Total为15μL):DNA 100 ng,Mg2+2.0 mmol/L,dNTPs 0.3 mmol/L,Primer 60 ng,Taqpolymerase 0.75 U。结果表明,该程序和体系能很好地满足西瓜基因组SRAP扩增的要求,SRAP标记应用于西瓜遗传研究是可行的。
关键词:
西瓜,
SRAP,
体系优化
Abstract: The SRAP-PCR reaction procedure and system for amplifying the watermelon genomic DNA was optimizedusing gradient experiment,so as to establish the optimum SRAP-PCR reaction conditions with high polytmrphism,good re-peatability and clear band pattern. The optimum SRAP-PCR reaction procedure was:pre-denaturation at 94 for 5 minfollowed by 5 cycles of denaturation at 94 0Cfor 1 min,anneal at 35 ?Cfor 1 min and extension at 72 ?Cfor 1 min;then 35cycles of 94 ?Cfor 1 min,50 for 1 min and 72 ?Cfor 1 min;and a final extension at 72 ?Cfor 5 min;kept at 4 ?C. Theoptimum SRAP-PCR system for a plume of 15 μL was:DNA 100 ng,Mg2+2. 0 mtml/L,dNTPs 0.3 mmol/ L,primer 60ng，Taq polymerase 0. 75 U. The procedure and system could meet the demands for genome SRAP amplification in C.lanatus. It was feable to apply the SRAP marker in genetic research in C.lanatus.
Key words:
Watermelon,
SRAP,
System optimization
中图分类号:
李晓慧, 王从彦, 徐小利, 常高正, 张四普. 西瓜SRAP-PCR反应程序的建立与体系的优化[J]. 华北农学报, 2008, 23(3): 38-41. doi: 10.7668/hbnxb.2008.03.011.
LI Xiao-hui, WANG Cong-yan, XU Xiao-li, CHANG Gao-zheng, ZHANG Si-pu. The Optimization of SRAP-PCR Amplification Program and System for Citrullus lanatus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2008, 23(3): 38-41. doi: 10.7668/hbnxb.2008.03.011.