摘要
[目的]为利用ISSR标记研究萱草的遗传多样性和和辅助育种奠定基础。[方法]从萱草中提取基因组DNA,建立萱草的ISSR-PCR反应体系,并通过正交试验对其进行优化。[结果]萱草的最佳ISSR-PCR反应体系为:ddH2O16.8μl、2.5 mmol/LdNTP2.0μl、10×buffer 2.5μl、10μmol/LPrimer0.3μl、50 ng/LDNA3μl、Taq酶1 U,总体积25μl。萱草的最佳ISSR扩增反应程序为:94℃变性5 min,94℃变性45 s、48.3℃退火1 min、72℃延伸2 min,共38个循环,最后72℃延伸7 min。该反应程序下进行的ISSR扩增,扩增产物最多,银染的效果最好。[结论]该研究建立了萱草ISSR-PCR的最佳反应体系和反应程序,通过ISSR扩增可获得清晰、稳定的条带。
[Objective] The research aimed to lay the foundation for studying the genetic diversity and the assistant breeding of Hemerocallis fulva L.by using ISSR markers.[Method] The genomic DNA was extracted from H.fulva.ISSR-PCR reaction system of H.fulva was established and optimized in the orthogonal test.[Result] The optimum reaction system for ISSR-PCR of H.fulva was as follows: ddH2O 16.8 μl,2.5 mmol/L dNTP 2.0 μl,10×buffer 2.5 μl,10 μmol/L Primer 0.3 μl,50 ng/L DNA 3 μl,Taq enzyme 1U,with the total volume of 25 ul.The optimum reaction procedure for ISSR amplification of H.fulva was as follows: denaturation for 5 min at 94 ℃,30 cycles of denaturation for 45 s at 94 ℃,anneal for 1 min at 48.3 ℃、extension for 2 min at 72 ℃,finally extension for 7 min at 72 ℃.Through ISSR amplification under this reaction procedure,the amplified products were most and the sliver-staining effect was best.[Conclusion] The optimum reaction system and reaction procedure for ISSR-PCR of H.fulva was established in this r
esearch and clear and stable bands could be obtained though ISSR amplification.
出处
《安徽农业科学》
CAS
北大核心
2008年第12期4884-4885,共2页
Journal of Anhui Agricultural Sciences
基金
国家林业局948项目[2002-08(2)]
关键词
萱草
ISSR
最佳反应体系
Hemerocallis fulva L.
ISSR
Optimum reaction system