基于SSR标记的中国亚洲韧皮杆菌种群结构研究

doi:10.3864/j.issn.0578-1752.2014.22.014

摘要/Abstract

摘要： 【目的】亚洲韧皮部杆菌是柑橘黄龙病病原。通过筛选SSR（Simple sequence repeat）位点，分析中国境内亚洲韧皮部杆菌（‘Candidatus Liberibacter asiaticus’）的群体结构。【方法】根据已报道亚洲韧皮部杆菌基因组上的25个SSR位点，使用中国不同地理来源的样品，对报道的SSR位点进行筛选，找出其中适宜进行中国样品群体结构分析的位点。应用PCR的方法，对收集的中国境内的285个亚洲韧皮部杆菌相应SSR位点进行扩增，聚丙烯酰胺凝胶电泳。所得电泳图导入软件Quantity One 4.5.0读取产物的碱基数。通过PopGen version 1.31软件评估选出的位点和不同地理来源的样品的多态性，分别使用软件Powermarker 3.25和structure 2.3.4进行聚类分析。【结果】共选出5个SSR位点可用于中国种群结构研究，Nei's多样性指数0.1542—0.9556。其中LasA位点多态性最高，有效等位基因数NE=22.5，Nei's多样性指数H=0.9556。通过这5个位点分析中国境内8个不同地理来源的样品，发现云南省样品存在较高多态性，NE=5.7，H=0.6580。8个地理种群间的遗传距离0.0236—0.5786，遗传相似度0.5607—0.9767，广西和四川遗传距离最大，遗传相似度最小。Powermarker软件以UPGMA方法进行聚类分析的结果显示，所有样品分为两个组群，四川和云南为一个组群；福建、浙江、贵州、广东、广西和江西为一个组群。Structure软件分析也得出了相似的结果，云南和四川样品的种群构成较为单一，90%以上个体为同一组群，而其他6个地理来源不同程度的为两个组群的混合。【结论】基于5个SSR位点分析中国境内的Ca. L. asiaticus表明其可能存在两种不同的群体结构，为进一步研究中国柑橘黄龙病的发生和流行规律提供了有价值的线索。

关键词: 柑橘黄龙病, SSR, 种群结构

Abstract: 【Objective】 ‘Candidatus Liberibacter asiaticus’ is the pathogeny of Citrus Huanglongbing. The purpose of this research was to investigate the population structure of ‘Candidatus Liberibacter asiaticus’ in China by multi SSR (simple sequence repeat) loci. 【Method】 To identify the polymorphism of SSR loci in the genome of Ca. L. asiaticus, 25 reported SSR primer sets were tested using DNA samples obtained from HLB-affected plants from 8 provinces of China. The selected SSR loci were used to analyze 285 samples by PCR and PAGE electrophoresis. The software Quantity One 4.5.0 was applied to estimate the base pairs of the amplicons. The polymorphism of 285 samples in the selected loci were evaluated via the software PopGen version 1.31, and the clustering analysis was performed by software Powermarker 3.25 and structure 2.3.4. 【Result】 A total of 5 SSR loci were identified, with Nei’s gene diversity index ranging from 0.1542 to 0.9556. The locus LasA showed high diversity. Its effective number of allele (NE) and Nei’s gene diversity (H) were 22.5 and 0.9556, respectively. Analysis on population structure of Ca. L. asiaticus of China from different geographical samples revealed that the isolates from Yunnan province had high diversity, (NE=5.7, H=0.6580). The genetic distance of different geographical populations ranged from 0.0236 to 0.5786 and genetic identity ranged from 0.5607 to 0.9767. The longest genetic distance and lowest genetic identity were identified from the geographical population between Guangxi and Sichuan. The clustering analysis indicated all isolates from China were clustered into two groups. One is from Sichuan and Yunnan, the other is from Fujian, Zhejiang, Guizhou, Guangxi, Guangdong and Jiangxi. Structure analysis also showed the populations of Yunnan and Sichuan were constituted nearly by a single group and the rest of them were made up by mixed groups. 【Conclusion】There are possible two kinds of different population structures of Ca. L. asiaticus in China by using the selected SSR markers and the results of this study will provide better insight on the origin and epidemiology of HLB in China.

Key words: Citrus Huanglongbing, SSR, population structure

黄爱军，苏华楠，王雪峰，唐科志，李中安，周常勇. 基于SSR标记的中国亚洲韧皮杆菌种群结构研究[J]. 中国农业科学, 2014, 47(22): 4488-4494.

HUANG Ai-jun, SU Hua-nan, WANG Xue-feng, TANG Ke-zhi, LI Zhong-an, ZHOU Chang-yong. Population Structure of ‘Candidatus Liberibacter asiaticus’ in China Revealed by SSR Markers[J]. Scientia Agricultura Sinica, 2014, 47(22): 4488-4494.

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