芒果AFLP分子标记体系的建立

摘要

[目的]构建我国芒果主要栽培品种的AFLP分子标记体系.[方法]供试31个芒果品种为:Banganapalli、台农1号、桂热10号、Carabao、Nang Klang Wun、泰国506、紫花、Keitt、粤西1号、斯里兰卡811、龙井大芒、红象牙、小菲、泰国504、Spooner、R2E2、串芒、鹦鹉芒、Irwin、金煌、Kent、海豹、Haden、Dashehari、Neelum、桂香、Ono、白象牙、Bambaroo、Macheso、Zill.以粤西1号芒果幼嫩叶片为材料探索提取高质量DNA的方法,为了获得更高质量和数量的基因组DNA,对曾杰的方法进行了下列改良:A、用2%的CTAB提取液,其他不变;B、用3%的CTAB提取液,但在提取后只用氯仿异戊醇提取2次;C、用3%的CTAB提取液,但在提取后用氯仿异戊醇提取1次;D、用3%的CTAB提取液,但在提取后用酚氯仿异戊醇提取1次.用供试品种中的台农1号、Carabao、Keitt、Kent对64对引物组合进行筛选,其中8个EcoR I引物分别是E-AAC、E-AAG、E-ACA、E-ACT、E-ACC、E-ACG、E-AGC、E-AGG,8个Mse I引物分别是M-CAA、M-CAC、M-CAG、M-CAT、M-CTA、M-CTC、M-CTG、M-CTT.利用AFLP分子标记在DNA水平上对芒果进行遗传多样性研究.[结果]4种方法提取的DNA较完整,均可得到明亮清晰的主带.且综合比较,用B方法(即采用较高浓度的提取液,提取后用氯仿异戊醇提取2次)可获得高质量、高纯度DNA,可用于芒果AFLP标记分析.供试64对引物组合中适用于芒果种质资源AFLP分析的引物组合共14对,分别是E-ACC/M-CAC、E-AAC/M-CAT、E-AAG/M-CAC、E-AAG/M-CAT、E-ACA/M-CAC、E-ACA/M-CAG、E-ACA/M-CAT、E-ACA/M-CTA、E-ACT/M-CAC、E-ACC/M-CTC、E-ACC/M-CTG、E-AGC/M-CAG、E-AGC/M-CTA、E-AGC/M-CTC.这14对引物组合在31份芒果种质中共扩增出1 761 条带, 其中多样性带比例为97%.平均每对引物产生125.8 条带和121.6 条多样性带.14对引物均可将上述31个品种完全区别开来,其中E-ACA/M-CAT和E-AGC/M-CTC引物组合多态性最大,达100%,可用于芒果品种指纹图谱构建.[结论]利用AFLP可以高效检测芒果种质资源分子标记多样性.%[Objective]The study aimed to construct the AFLP molecular marking system in Mangifera indica. [Method] Four varieties of Mangifera indica were used to explore new ways for high-quality DNA, and AFLP analysis of 31 varieties of Mangifera indica was carried out to detect the varietal genetic diversity. [Result] 14 pairs of primers with stronger polymorphism, better banding patterns and higher resolution were screened out from 64 pairs of selective amplification primers. Then they were used to analyse the fingerprint of 31 varieties of Mangifera indica, the results showed that the ratio of polymorphic bands amplificated by the 14 pairs of primers reached 97% in 31 varieties of Mangifera. [Conclusion] It was suggested that AFLP was suitable for detecting the polymorphism of Mangifera indica resources.