摘要： <正>AFLP(扩增片段长度多态性)标记技术是高效而可靠的标记技术之一,特别对于那些基因组序列信息很少的物种更为有效。笔者对基因组较大的松属树种的AFLP实验程序进行了优化,提供了松树树种AFLP分析中DNA酶切、引物连接、预扩增及选择性扩增的优化实验程序。结果显示,预扩增反应中利用选择碱基数不同的预扩增引物(E/M+1,E/M+2)制备的模板对后续的选择性扩增结果没有显著影响;在选择性扩增反应中,对碱基数目选择进行了细致的优化,分别测试了15个E+3/M+3引物组合,6个E+4/M+3引物组合及15个E+3/M+4引物组合。对于有相同选择碱基数的引物,扩增结果随选择碱基的组合不同而有较大差异。据总的扩增结果发现,选择碱基增加时,扩增谱带数明显减少,谱带清晰度增加,即当选择碱基增加到E引物末端时(M+4/E+3),大多数引物组合的扩增谱带数要比选择碱基增加到M引物末端(E+3/M+4)的引物组合扩出的谱带数少且带型清晰。总体来看,E+4/M+3的引物组合比较适合于松属树种的AFLP分析。笔者还利用单株树种子的胚乳组织对AFLP标记的遗传方式进行了研究,在所用的两个引物组合获得的标记中,约有30%左右的分离标记,分离标记中偏分离比例约为6%。另外,选用3个松属树种对选出的14个AFLP引物组合进行的验证显示,这些组合在不同种间均获得了较好的扩增结果,说明在不同松属树种中筛选出的引物组合可以为其它松属树种AFLP分析引物组合的选择提供借鉴。

Abstract: The reliable technique for seed and seedling authentic identification is highly desired in forest tree seed and seedling inspection.AFLP(Amplified Fragment Length Polymorphism) is one of the most powerful techniques for this purpose.This paper specially optimized an economic and reliable AFLP analysis protocol for Pinus spp.due to their enormous genome size.The number of selective nucleotides added to the core primers and the primer combinations suitable for AFLP analysis in such species were tested.Some AFLP primer combinations and were screened their utility across different species were tested.The results could also be used as a reference for allowing better amplification in complex templates isolated from plants species with large genomes.

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