【摘要】 以初展开的板栗嫩叶为材料,利用改进的CTAB法,提取到高质量的板栗叶片总DNA。通过优化酶切连接、预扩增、选择性扩增等试验条件,建立了板栗AFLP银染反应体系,得到了清晰的板栗AFLP指纹图谱。为板栗品种的分子标记和板栗品种间亲缘关系等研究奠定了基础。研究结果表明:①DNA模板的质量影响酶切以及后续的连接扩增反应。以初展开的嫩叶提取的板栗叶片总DNA纯度最高,所含蛋白质、小分子杂质少,改良的CTAB提取法可用于板栗AFLP分析,形成清晰的AFLP指纹。②先进行酶切后再进行酶连的反应体系比酶切酶连一起进行的体系效果更好。板栗基因组DNA最佳酶切反应体系为:模板DNA总量500ng,反应体积为25μl,10×Y+/TanqoTMBuffer2.5μl,BSA0.8μl,EcoRI5U,MseI5U。连接反应体系中T4DNA连接酶浓度2U即达最佳效果。酶切、酶连反应最佳温度均为37℃。③以板栗为材料进行AFLP标记时,E AAC+M CAA、E AAC+M CAT和E AGT+M CAT三个引物均获得较好的多态性。其中以E AGT+M CAT引物组合的扩增条带信号强度一致性好,条带分布均匀,能够得到稳定、清晰、分辨率较高的指纹谱带,可进行中国板栗的遗传变异分析。更多还原

【Abstract】 The improved method of CTAB was used to extract total genic DNA from tender leaves of Castanea mollissima Bl.in this study.High quality total genic DNA from chestnut leaf was isolated.DNA restriction-ligase reaction, pre-amplification and selective amplification were optimized , the AFLP reaction system was established and clear DNA fingerprints were obtained.This paper provided a method for the further study in the field of molecular marker in genetic diversity, new cultivars breeding and genenic relationship in Castanea mollissima Blume.The results indicated:①The effect of restrction of DNA and lingation of oligonucleotide adapters and amplification was influenced by the quality of total genic DNA.Compared with the conventional method,high quality and ligher yield as well as clear finger map were obtained by the improved CTAB method.②Lingating oligonucleotide adapters DNA after restricting was better than restricting while lingating of DNA.There were 500 ng DNA template,EcoRI 5 U,MseI 5 U,10×Y⁺/Tanqo^TM Buffer 2.5 μl,BSA 0.8 μl in the best 25 μl system of restriction and 2 U T （4）-DNA polymerase in the lingation systerm.The optimum temperature was 37 ℃.③The polymorphism bands could be obtained from three Primer pairs（E-AAC + M-CAA,E-AAC + M-CAT,E-AGT + M-CAT）,and E-AGT + M-CAT was best in studies of chestnut genetic diversification,variety identification,finger map building, identification and classification with AFLP molecular markers.The silver-staining AFLP system suitable for Castanea mollissima Blume was developed, clear and high resolution bands were obtained. The developed techniques could be applied in the analysis of genetic diversity in Chinese chestnut. reePrimerpairs(E A asbestinstudiesof更多还原