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韧皮部杆菌亚洲种(‘Candidatus Libriacter 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(short tandem repeat,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0.06~0.81,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。
Candidatus Libriacter asiaticus’, ’Ca.L.asiaticus’ is the most widely endemic and endangered citrus Huanglongbing pathogens. In this study, a systematic method was developed for the genetic diversity of ’Ca.L.asiaticus’ and the molecular epidemiology of the Huanglonglong disease using a complete set of tandem repeat (STR) sequences in the ’Ca.L.asiaticus’ genome. Of the 36 STR loci screened by PCR and PAGE, 33 amplified efficiently. Among them, 20 pairs of primers had better polymorphism in the amplification products of 32 samples from different sources, and up to 9 types of bands could be amplified. The Shannon’s information index of 32 Ca.L.asiaticus positive samples collected from 6 provinces in China was 0.14-1.90 with an average of 0.68. The Nei’s gene diversity index was 0.06-0.81 with an average of 0.38. A higher genetic diversity, especially from Fujian, Guangxi and Yunnan provinces. Clustering analysis revealed that it may have some genetic specificity for the Huanglongbing strain in Guangdong Province, the birthplace of the Chinese Huanglongshan disease. The possibility of exchanging the strains caused by the propagation of the Pipi in other neighboring provinces may explain the inter-provincial spread. Studies have shown that the developed STR-PAGE combined with PAGE method can be used as an efficient method for molecular genetic analysis of disease strains and future genetic diversity.