作　　者： ; ; ; ; ; ;

机构地区： 西北农林科技大学林学院

出　　处： 《北方园艺》 2013年第22期114-119,共6页

摘　　要： 以毛梾叶片为试材，通过单因素2次循环试验对毛株IYxSR反应体系进行优化，建立了毛株ISSR-PCR的最佳反应体系和扩增程序。结果表明：PCR反应体系总体积为25uL，其中10XBuffer2．5弘L，模板DNA用量50ng，7aqDNA聚合酶用量1．5U，Ag浓度2．0mmol／L，引物浓度0．6μmol／L，dNTPs浓度0．15mmol／L；扩增程序：94℃预变性5min；94℃变性50S，48．8～59．1℃退火60S，72℃延伸1．5min，38个循环；最后72℃延伸10min。在此基础上，从100条ISSR通用引物中筛选出17条多态性好、稳定性高的引物，并优化了它们的退火温度。这一优化体系的建立，为开展毛梾种质资源鉴定、遗传多样性及分子育种方面的研究提供了技术依据和券考。 Using leaves of Comus walteri as material, ISSR reaction conditions of Comus zoalteri were optimized systematically via single factor with two circular tests, and the optimal ISSR reaction system and thermal cycling conditions of Comus walteri were established. The results showed that the optimal ISSR reaction system included a total volume 25 μL containing 2.5 μL 10 X Buffer, 50 ng template DNA, 1.5 U Taq DNA polymerase, 2. 0 rmnol/L MgC12, 0. 6 μmol/L primer, 0. 15 mmol/L dNTPs. Thermal cycling conditions were as follows- denaturation 5 rain at 94℃ 38 cycles of 50 s at 94℃ ,60 s at 48. 8-59. 1℃ ,1.5 min at 72℃;and a final extension of 10 rain at 72℃ at the end of the amplification. Based on the optimized reaction system, 17 primers with superior stability and polymorphism were screened out from 100 ISSR primer candidates. Subsequently,annealing temperatures of selected primers were subjected to further optimization. As a consequence, the optimized system in this study may provide technological base and reference for identification of germplasm resources, genetic diversity and molecular breeding of Comus walter.

关 键 词： 毛梾 单因素 次循环试验 体系优化 引物筛选

领　　域： [农业科学] [农业科学]