甜橙和柚中CTV强弱毒株系p20的遗传变异

doi:10.3864/j.issn.0578-1752.2017.07.017

摘要/Abstract

摘要： 【目的】对7个柑橘衰退病毒（CTV）株系进行遗传变异研究,明确寄主甜橙和柚中CTV强弱毒株系p20的变异水平。【方法】运用RT-PCR、克隆及测序等技术建立CTV p20种群，并借助MEGA6构建单倍型系统发育树，运用软件DNAStar对两种寄主中CTV强弱毒种群的遗传结构、变异水平进行分析，运用DnaSP软件对各种群进行单倍型多样性、核苷酸多样性分析和中性检验分析。【结果】构建了7个CTV p20种群，由162条序列构成，包含11个单倍型，单个种群有1个或更多单倍型出现。序列分析发现，来自不同种群的单倍型对应的原始核苷酸序列一致性为88.2%—100.0%，对应的氨基酸序列的一致性为92.3%—100.0%，最低的氨基酸序列一致性发生在CT23-1和CT9-2之间；其中单倍型PeraIAC-4、CT22和CT9-1共有50条序列，对应的原始核苷酸序列一致性为100.0%，属优势单倍型，与标准株系T36亲缘关系较近；单倍型多样性最丰富的是甜橙种群PeraIAC，单倍型多样性为0.800，而单倍型多样性最低的是柚种群CT22，单倍型多样性为0.170；相比柚种群的单倍型多样性（0.170—0.552）和核苷酸多样性（0.00032—0.05919），甜橙种群具有更为丰富的单倍型多样性（0.513—0.800）和核苷酸多样性（0.04208—0.05677）。系统发育树分析表明，来自甜橙的分离株种群结构复杂，甜橙种群中检测到的单倍型与标准株系T30、T36、VT和T3均有相关性；与标准株系T3相距很近的CT31-2与优势单倍型在系统发育树上距离最远，对应的原始核苷酸序列一致性仅为88.3%。中性检验结果表明，CTV甜橙种群趋于平衡或收缩状态，而CTV柚种群除CT23外则趋于扩张状态；其中TR-514Y、CT31和CT23种群的Tajima’s D值、Fu和Li’s D*值以及Fu和Li’s F*值均为正值且达到显著水平，而CT9种群的Tajima’s D值、Fu和Li’s D*值以及Fu和Li’s F*值均为负值且达到显著水平。运用DnaSP软件对各种群进行重组分析表明，在各种群中均未检测到重组事件发生。种群变异分析发现，各种群突变克隆百分比在0—30.8%，碱基突变频率在0—7.706×10^-4，其中CTV甜橙强毒种群有最高的突变克隆百分比（30.8%），最高的碱基突变频率（7.706×10^-4），最多的碱基突变数量（11个）和最多的突变位点（7个）；CTV甜橙弱毒种群的突变克隆百分比和碱基突变频率均明显低于甜橙强毒种群，CTV柚弱毒种群的突变克隆百分比和碱基突变频率略低于柚强毒种群。碱基突变类型分析发现碱基突变以碱基替代为主，其中A→G突变为优势类型，仅在柚强毒种群CT3的156和157位点间检测到一个碱基插入突变类型，为碱基A插入，未检测到碱基缺失突变类型。【结论】在寄主甜橙和柚中CTV强弱毒p20种群结构及变异存在差异，CTV甜橙种群有着更复杂的种群结构和更高的种群变异水平，且CTV强毒种群变异更大。

关键词: 柑橘衰退病毒, p20, 种群, 遗传变异

Abstract: 【Objective】 In order to clarify the genetic variation of p20 of the severe and mild strains of Citrus tristeza virus (CTV) in sweet orange and pummelo, seven CTV strains were studied. 【Method】The CTV p20 populations were established by RT-PCR, cloning and sequencing, and the phylogenetic tree was constructed with MEGA6. The genetic structure and mutation level of the CTV populations were analyzed by DNAStar. Haplotype diversity, nucleotide diversity and neutral test of the CTV populations were analyzed by DnaSP. 【Result】 Seven CTV p20 populations consisted of 162 sequences with 11 haplotypes were established. One or more haplotypes were found in each population. Sequence analysis showed that the original nucleotide sequence identity of the haplotypes from different populations was 88.2%-100.0%, the corresponding amino acid sequence identity was 92.3%-100.0% and the lowest amino acid sequence identity was found between CT23-1 and CT9-2. There were 50 sequences of haplotypes PeraIAC-4, CT22 and CT9-1, the corresponding nucleotide sequence identity was 100.0%, of which were dominant haplotypes and close to the standard strain T36. PeraIAC possessed the most abundant haplotype diversity (0.800±0.051) and CT22 had the lowest haplotype diversity (0.170±0.102). Compared with haplotype diversity (0.170-0.552) and nucleotide diversity (0.00032-0.05919) of the populations from pummelo, the populations from sweet orange had richer haplotype diversity (0.513-0.800) and nucleotide diversity (0.04208-0.05677). Phylogenetic analysis showed that the population structure of the isolates from sweet orange was complex, and the haplotypes detected in the populations from sweet orange were related to the standard strains T30, T36, VT and T3. CT31-2, which is close to the strain T3, was the most distant to the dominant haplotype in the phylogenetic tree, and the identity of the corresponding nucleotide sequences was 88.3%. The results of neutrality test showed that the populations from sweet orange tended to be balanced or contracted, while the populations from pummelo tended to be expanded except for CT23. The Tajima’s D values, Fu and Li’s D* values and Fu and Li's F* values of the TR-514Y, CT31 and CT23 populations were positive and significantly different, while those of the CT9 population were negative and significantly different. Recombination analysis of each population showed that no recombination occurred in any group. The population variation analysis showed that the percentage of mutant clones was 0-30.8% and the base mutation frequency was 0-7.706×10^-4. The highest percentage of mutant clones (30.8%), the highest mutation frequency (7.706×10^-4), the most base mutations (11) and the most mutation sites (7) happened in p20 populations of CTV severe strains from sweet orange. The percentage of mutated clones and mutation frequency of CTV mild populations from sweet orange were significantly lower than those of CTV severe populations from sweet orange. The percentage of mutated clones and mutation frequency of CTV mild populations from pummelo were slightly lower than those of severe populations from pummelo. The analysis of base mutation types showed that the base mutation types were mainly base substitution, and substitution from A to G was dominant. The sole insertion of A was detected between the 156 and 157 sites of CT3 and no deletion was found in p20 populations. 【Conclusion】 The variations of severe and mild populations of CTV p20 from sweet orange and pummelo were different. CTV p20 populations from sweet orange had more complex population structure and higher variation level than those from pummelo, and the severe populations of CTV p20 had higher variation level than those of mild populations.

Key words: Citrus tristeza virus (CTV), p20, population, genetic variation

王亚飞，阮涛，周彦，王雪峰，吴根土，孙现超，周常勇，青玲. 甜橙和柚中CTV强弱毒株系p20的遗传变异[J]. 中国农业科学, 2017, 50(7): 1343-1350.

WANG YaFei, RUAN Tao, ZHOU Yan, WANG XueFeng, WU GenTu, SUN XianChao, ZHOU ChangYong, QING Ling. Genetic Variation of p20 of the Severe and Mild Strains of CTV in the Sweet Orange and Pummelo[J]. Scientia Agricultura Sinica, 2017, 50(7): 1343-1350.

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