茶树多酚氧化酶基因的PCR-RFLP多态性分析

Detection the Polymorphism of Polyphenols Oxidase Gene in Tea Plant (Camellia Sinensis) by PCR-RFLP

采用PCR-RFLP技术,检测茶树多酚氧化酶(PPO)基因中4个限制性核酸内切酶酶切位点在不同品种中的多态性,以研究其与品种适制性及遗传背景的关系。遗传参数分析表明:4个位点均处于Hardy-Weinberg平衡状态(P>0.05),HpaⅡ酶切位点(引物L7/L8扩增区段)呈现高度多态,BsuRⅠ酶切位点呈现中度多态,这2个酶切位点在不同品种中的基因型分布与品种的遗传背景有直接的关联,可作为遗传标记应用于茶树品种遗传亲源关系分析与亲子鉴定。HpaⅡ酶切位点在引物L7/L8扩增区段存在丰富的多态性,且与品种适制性具有明显关联,适制红茶的品种多为AA基因型,此位点能被HpaⅡ内切酶完全酶切;适制绿茶与乌龙茶的品种多为BB型或AB型,此位点不能被酶切或不能完全被酶切,该HpaⅡ酶切位点可作为茶树品种适制性早期鉴定的遗传标记。对引物L7/L8扩增区域的HpaⅡ酶切位点在祁门4号×潮安大乌叶F1代群体进行分型检测的结果表明,该位点在F1代的分离符合孟德尔遗传规律(x2=0.23)。

Restriction fragment length polymorphism （RFLP） analysis of the PCR amplified fragments of Polyphenol Oxidase （PPO） genes was applied for the characterization of genetic relationship and processing property of four tea accessions. Four restriction sites were investigated. The results of genetic parameters indicated that 4 restriction sites fitted Hardy-Weinberg equilibrium （P 〉 0.05）. The Hpa Ⅱ restriction site showed high polymorphism and BsuR Ⅰ restriction site showed moderate polymorphism. There was a high relationship between genotype distribution of BsuR Ⅰ, Hpa Ⅱ restriction sites and genetic background of accessions. The Hpa Ⅱ restriction site in primer L7/L8 amplified region was of abundant polymorphism and a high relationship between genotype and processing property of different accessions. If the genotype was AA, existing of Hpa Ⅱ restriction site, the cultivar was good for processing black tea. If the genotype was BB or AB, this restriction site could not be digested or could partly be digested by Hpa Ⅱ restriction endonucleases, and the cultivar was good for processing green tea or oolong tea. The restriction patterns of F1 progenies of Qi Men No.4 x Chao An Da Wu Ye at Hpa Ⅱ restriction site in primer L7/L8 amplified region showed that the segregation of Hpa Ⅱ restriction site in F1 population was in accord with Mendelian heredity law （x^2=0,23）. From this research, BsuR Ⅰ and Hpa Ⅱ （in primer L7/L8 amplified region） restriction sites in PPO gene might be considered as two useful genetic markers for characterization of genetic relationship and identification of parents. Hpa Ⅱ restriction site （in primer L7/L8 amplified region） could be recommended as a genetic marker to identify processing property of tea cultivars at forepart of tea plant breeding.