中国野生葡萄果实抗白腐病基因RAPD标记的克隆及序列分析

Cloning and sequencing of RAPD maker linked to white rot disease resistant gene in wild grapes native to China

以感病×抗病的葡萄种间杂交组合白玉霓×塘尾的F1代及塘尾自交一代33个单株为试验材料,采用BSA法和RAPD技术,对155个随机引物进行筛选,从中获得了1个与中国野生葡萄抗白腐病基因连锁的RAPD标记OPP09-760。用WizardDNAClean-upSystem试剂盒回收纯化RAPD遗传标记OPP09-760,连接于pGME-T-EasyWector上并克隆测序,得到OPP09-760的全序列,其长度为766bp。根据两端序列设计特异PCR扩增引物作为合成中国野生葡萄抗白腐病检测探针的基础,用于检测葡萄抗白腐病种质种和分子标记辅助育种。

Bulk segregant analysis (BSA),randomly amplified polymorphic DNA (RAPD) methords were used to tag the white rotresistant genes of grape molecular markers,parents and their 33 individuals of F1 and F1 progeny resulting from a cross 915 between Tangwei (vitis davidii),white rotresistant and Ugni Blanc (V.vinifera),white rotsusceptible.Among 155 Operon primers giving distinct band patterns,one RAPD marker OPP09760 was tightly linked to a major gene resistant to Coniothyrium diplodiella.OPP09760 fragment was reclaimed by Wizard DNA Cleanup system from electrophoresis gel,and cloned in Teasy Vector,then sequenced from two sides.The DNA fragment OPP09760 was actually 766 bp.It is suggested that the sequence of RAPD marker OPP09760 might be used as a basis for synthesizing the specific PCR primers and the probe for detecting grape white rotresistant in disease resistant breeding and molecular marker assisted selection (MAS).