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林业科学 ›› 2017, Vol. 53 ›› Issue (3): 105-118.doi: 10.11707/j.1001-7488.20170312

• 论文与研究报告 • 上一篇    下一篇

多位点序列分析揭示我国16SrI组植原体不同株系间遗传变异和系统发育关系

于少帅, 李永, 任争光, 宋传生, 林彩丽, 朴春根, 田国忠   

  1. 中国林业科学研究院森林生态环境与保护研究所 国家林业局森林保护学重点实验室 北京 100091
  • 收稿日期:2016-02-02 修回日期:2016-03-10 出版日期:2017-03-25 发布日期:2017-04-25
  • 通讯作者: Tian Guozhong
  • 基金资助:
    The National High Technology Research and Development Program of China (2012AA101501); National Infrastructure of Microbial Resources of China (NIMR2014-7).

Multilocus Sequence Analysis for Revealing Finer Genetic Variation and Phylogenetic Interrelatedness of Phytoplasma Strains in 16SrI Group in China

Yu Shaoshuai, Li Yong, Ren Zhengguang, Song Chuansheng, Lin Caili, Piao Chungen, Tian Guozhong   

  1. Key Laboratory of Forest Protection of State Forestry Administration Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry Beijing 100091
  • Received:2016-02-02 Revised:2016-03-10 Online:2017-03-25 Published:2017-04-25
  • Supported by:
    The National High Technology Research and Development Program of China (2012AA101501); National Infrastructure of Microbial Resources of China (NIMR2014-7).

摘要: [目的] 16SrI组植原体对我国作物和生态植物危害严重。目前植原体尚不能分离培养,对于我国植原体不同株系间遗传变异和种群结构尚不清晰,通过多位点序列分析(MLSA)揭示我国不同地区16SrI组不同植原体株系的遗传多样性及其地理分布特征,比较不同植原体株系间不同持家基因的变异程度,为我国不同植原体株系的检测、分类鉴定和系统发育研究提供一定的参考方法和依据。[方法] 应用rp,tuf,secA,secY,ipt,dnaK,fusA,gyrB,pyrGrpoB共10个持家基因序列,结合16S rDNA序列,以全基因序列已完成的洋葱黄化植原体(OY-M)、翠菊黄化丛枝植原体(AYWB)、澳大利亚葡萄黄化植原体(CPA)、草莓致死黄化植原体(SLY)和苹果簇生植原体(CPM)共5种植原体株系为参照,分析我国10个省(市)苦楝丛枝、莴苣黄化、桑萎缩、泡桐丛枝和长春花绿变植原体株系(共18株)的遗传变异规律和系统发育关系,通过多重序列比对分析不同基因片段的序列多态性和变异程度。[结果] 我国18株苦楝丛枝、莴苣黄化、桑萎缩、泡桐丛枝和长春花绿变植原体株系的rp,tuf,secA,secY,ipt,dnaK,fusA,gyrB,pyrGrpoB多位点序列共有15种序列类型,从而揭示16SrI组不同植原体株系间丰富的遗传多样性。基于rp等10个基因多位点序列分析可将16SrI-B、D亚组的不同植原体株系清晰地分开。10株苦楝丛枝植原体株系与2株桑萎缩植原体株系系统发育关系最近,多位点序列分析可将这2种基于16S rDNA序列难以区分的植原体株系清晰地区分。10株苦楝丛枝植原体株系分为4个进化枝,其多位点序列存在8种序列类型,这4个分枝与植原体株系的地理分布关系密切。与我国长春花绿变和泡桐丛枝植原体株系相比,福建三明2株莴苣黄化植原体株系与日本洋葱黄化植原体株系OY-M系统发育关系较近。在已检测分析的植原体不同基因序列片段中,dnaK基因序列片段的变异水平最高。[结论] 多位点序列分析可做为一种对植原体鉴定、区分以及株系遗传多样性全面检测的有效、可靠方法。在以后的研究中该方法可广泛应用于深入探讨植原体不同组间或亚组间株系的遗传变异和系统发育关系。

关键词: 植原体, 多位点序列分析(MLSA), 遗传变异, 序列类型(ST), 系统发育

Abstract: [Objective] The phytoplasmas in 16SrI group cause severe diseases of many crops and ecological plants in China. At present, the genetic variation and population structure of the yet-uncultured and very closely related phytoplasma strains in China are still not fully understood. A multilocus sequence analysis (MLSA) scheme was used to elucidate genetic diversity of phytoplasma strains in 16SrI group from different regions in China and their relationships with geographical distribution, and to compare the levels of variation in different housekeeping gene of different phytoplasma strains. The study aimed to provide some reference approach and evidence for finer detection, identification, classification and phylogenetic analysis of different phytoplasma strains in China.[Method] Ten housekeeping gene (rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB) fragments combined with 16S rDNA sequences were employed to analyze the genetic variation and phylogenetic relationships of 18 phytoplasma strains infecting chinaberry, lettuce, mulberry, paulownia and periwinkle from ten provinces in China, using five phytoplasma strains, onion yellows phytoplasma (OY-M), aster yellows witches'-broom phytoplasma (AYWB), Candidatus Phytoplasma australiense (CPA), strawberry lethal yellows phytoplasma (SLY) and Candidatus Phytoplasma mali (CPM), of which whole genomes have been sequenced, as references. Sequence polymorphism and variation levels of different gene fragments were analyzed by multiple sequence alignment.[Result] The nucleotide site polymorphisms of rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB gene fragments were used to resolve all strains into 15 sequence types (STs), demonstrating extensive genetic diversity among the 16SrI group strain population. All the strains, classified into 16SrI-B and -D subgroup by 16S rDNA analysis, were clustered into one clade and clearly differentiated into discrete subclades by phylogenetic analysis of the concatenated gene sequences. Ten chinaberry witches'-broom strains, which were most closely related to two mulberry dwarf strains and hardly distinguished with 16S rDNA, were definitely split into four distinct clusters and 8 STs apparently relative to their geographical origins. Two lettuce yellows strains in Sanming, Fujian province, China, were more closely related to the onion yellows OY-M strain in Japan than the periwinkle virescence and paulownia witches'-broom strains in China. The levels of variation in dnaK locus were higher than those in 16S rDNA and other genes tested.[Conclusion] It is suggested that the MLSA should potentially be a useful and reliable approach for phytoplasma identification and differentiation as well as for depth examination of strain diversity and this method can be wildly used to study the genetic variation and evolutionary relationships of phytoplasma strains among various groups or subgroups in the future.

Key words: phytoplasma, multilocus sequence analysis (MLSA), genetic variation, sequence type (ST), phylogenetic evolution

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