基于短串联重复和PAGE的柑橘黄龙病菌‘Candidatus Liberibacter asiaticus’种间遗传多样性分析(英文)

Intraspecific Genetic Diversity Analysis of ‘Candidatus Liberibacter asiaticus' by Short Tandem Repeats and PAGE

韧皮部杆菌亚洲种（‘Candidatus Liberibacter asiaticus’，‘Ca．L．asiaticus’）是目前流行范围最广、危害最严重的柑橘黄龙病致病菌。本研究利用‘Ca．L．asiaticus’基因组中全套串联重复基因（shorttandemrepeat，STR）开发了一套系统的方法用于‘Ca．L．asiaticus’遗传多样性和黄龙病分子流行学研究。PCR和PAGE分析筛选到的36个STR位点中，有33个获得了有效扩增。其中20对引物对不同来源的32个样品的扩增产物多态性较好，最多的可扩增出9种条带类型。采自我国6省的32个‘Ca．L．asiaticus’阳性样品之间的Shannon’S信息指数为0．14～1．90，平均为0．68；Nei’S基因多样性指数为0．06。0．8l，平均为0．38，各省的菌株表现出较高的遗传多样性，特别是来自福建、广西和云南三省的。聚类分析发现可能为中国黄龙病发源地的广东省的黄龙病菌株具有一定的遗传特异性；其余邻省之间存在由木虱传播引起的菌株交流的可能性可以解释该病害在省际间传播。研究表明，开发的STR标记结合PAGE的方法可作为今后菌株遗传多样性和病害分子流行学分析的高效方法。

The most prevalent uncultured phloem-restricted bacterial species associated with citrus huanglong- bing （HLB） disease in the world is ‘ Candidatus Libriacter asiaticus＇. To develop a set of short tandem repeat （STR） markers for‘Ca. L. asiaticus＇ and facilitate studies on its genetic diversity and epidemiology of HLB, Tandem Repeats Finder software was applied to identify and analyze the STR loci. STR primers were designed by Primer Premier 5.0. PopGene and PowerMark were used to analyze polyacrylamide gel electrophoresis （PAGE） bands of isolates collected from 6 provinces in China. Thirty-three of the 36 STR loci could be effectively amplified. Twenty polymorphic STR primers were selected and used to differentiate 32 Chinese ‘ Ca. L. asiaticus＇ isolates. The Nei＇ s measure of genetic diversity values for these 20 STRs ranged from 0.06 to 0.81, with a mean of 0.38. The shannon＇ s diversity indexes were between 0.14 and 1.90, with a mean of 0.68. In conclusion, the 20 polymorphic STR loci were used for genetic diversity analysis of‘Ca. L. asiaticus＇ isolates from different provinces in China. Diversity of ‘ Ca. L. asiaticus＇ was related to geographic distribution. The isolates from Guangdong that was considered to be HLB origin were specific. Isolates from Fujian, Guangxi and Yunnan were suggested to be higher in diversity. Psyllid transmission may play roles in the spread of ‘ Ca. L. asiaticus＇ between neighbor provinces.