云南省番茄莴苣褪绿病毒分子检测及遗传进化分析

doi:10.16420/j.issn.0513-353x.2020-1087

园艺学报 ›› 2022, Vol. 49 ›› Issue (1): 141-147.doi: 10.16420/j.issn.0513-353x.2020-1087

云南省番茄莴苣褪绿病毒分子检测及遗传进化分析

冯丽肖¹, 胡荣¹, 卜姗¹, 张德咏¹^,², 罗香文², 李凡³, 丁铭⁴, 张卓², 张松柏¹^,²^,^*(), 刘勇¹^,²^,^*()

¹湖南大学研究生院隆平分院,长沙410125
²湖南省农业科学院植物保护研究所,长沙 410125
³云南农业大学植物保护学院,昆明 650201
⁴云南省农业科学院,昆明 650231

收稿日期:2021-03-09 修回日期:2021-08-20 出版日期:2022-01-25 发布日期:2022-01-24
通讯作者: 张松柏,刘勇 E-mail:zsongb@hunaas.cn;haoasliu@hunaas.cn
基金资助:
国家自然科学基金项目(31972242);国家现代农业产业技术体系专项资金项目(CARS-23-D-02)

Molecular Detection and Genetic Evolution Analysis of Yunnan Isolates of Lettuce Chlorosis Virus in Tomato

FENG Lixiao¹, HU Rong¹, BU Shan¹, ZHANG Deyong¹^,², LUO Xiangwen², LI Fan³, DING Ming⁴, ZHANG Zhuo², ZHANG Songbai¹^,²^,^*(), LIU Yong¹^,²^,^*()

¹College of Longping,Graduate School of Hunan University,Changsha 410125,China
²Plant Protection Institute,Hunan Academy of Agricultural Sciences,Changsha 410125,China
³Plant Protection College Yunnan Agricultural University,Kunming 650201,China
⁴Yunnan Academy of Agricultural Sciences,Kunming 650231,China

Received:2021-03-09 Revised:2021-08-20 Online:2022-01-25 Published:2022-01-24
Contact: ZHANG Songbai,LIU Yong E-mail:zsongb@hunaas.cn;haoasliu@hunaas.cn

摘要/Abstract

摘要：

采集了77份云南省疑似感染莴苣褪绿病毒（lettuce chlorosis virus,LCV）的番茄样本,采用特异性反转录PCR扩增LCV 次要外壳蛋白基因（minor coat protein,CPm）近全长序列,常规Sanger测序法测定相应片段序列,并对其进行遗传进化分析。结果表明,LCV特异性反转录PCR扩增得到与预期大小（1.38 kb）一致的特异性条带;采集的77份番茄样本中LCV检出率为12.99%。测定的LCV CPm;发育分析表明,所有LCV分离物可分为3个亚簇,具有明显的地域特性,其中LCV云南分离物与中国其他地区LCV分离物聚为一亚簇,来自美洲和欧洲分离物分别聚为一个簇。遗传进化分析表明,LCV云南分离物YN58有1个重组事件,序列中嘧啶（C/T）替换率最高（25.25）,呈现独立进化、加速扩展趋势。这是在云南省首次报道发现LCV。

关键词: 番茄, 莴苣褪绿病毒, 分子检测, 系统发育分析, 进化

Abstract:

The 77 tomato leaves suspected to be infected by lettuce chlorosis virus（LCV）were sampled in Yunnan Province,China. The reverse transcriptional（RT）-PCR amplified near full length of minor coat protein gene（CPm）of LCV,which was sequenced by Sanger sequencing method. The genetic evolution was analyzed based on the sequences of near full length CPm genes. The results showed that the specific 1.38 kb fragment was amplified by RT-PCR,and 12.99% LCV-positive samples were detected in the 77 tomato leaf samples. The sequences of near full length CPm genes of Yunnan Province,China,shared the highest nucleotide sequence identity of 99.27%-99.78% with LCV Shandong isolate（MK370741）. Phylogenetic analysis indicated that all of LCV isolates were divided into three subclusters with region-dependent,LCV Yunnan isolates grouped into same subcluster with other LCV isolates of China,and America isolates and European isolates grouped into other two subclusters separately. Genetic evolution analysis revealed one recombinant event exiting in LCV Yunnan isolate YN58,and the highest nucleotide substitution of 25.25 occurring in pyrimidine C/T,which shown LCV had the tendency of independent evolution and expedited diffusion in China. This is the first report of LCV in Yunnan Province.

Key words: tomaoto, lettuce chlorosis virus, molecular detection, phylogenetic analysis, evolution

中图分类号:

S641.2

冯丽肖, 胡荣, 卜姗, 张德咏, 罗香文, 李凡, 丁铭, 张卓, 张松柏, 刘勇. 云南省番茄莴苣褪绿病毒分子检测及遗传进化分析[J]. 园艺学报, 2022, 49(1): 141-147.

FENG Lixiao, HU Rong, BU Shan, ZHANG Deyong, LUO Xiangwen, LI Fan, DING Ming, ZHANG Zhuo, ZHANG Songbai, LIU Yong. Molecular Detection and Genetic Evolution Analysis of Yunnan Isolates of Lettuce Chlorosis Virus in Tomato[J]. Acta Horticulturae Sinica, 2022, 49(1): 141-147.

图/表 6

图1 疑似被莴苣褪绿病毒侵染的番茄植株田间发病症状 A：植株症状为叶片褪绿;B：下部叶片症状为褪绿、局部褐色类坏死。

Fig. 1 The symptom of a tomato plant in field suspected to be infected by lettuce chlorosis virus A：Chlorosis symptom in a tomato plant;B：Chlorosis and browning necrosis on a tomato leaf.

图2 部分样本LCV RT-PCR琼脂糖凝胶电泳图 1：阴性对照（ddH2O为模板）;2-13：番茄样本;14：阳性对照。

Fig. 2 Electrophoresis of products of LCV-specific RT-PCR 1：Negative control（ddH2O as template）;2-13：Tomato samples;14：Positive control.

图3 LCV分离物次要外壳蛋白基因（minor coat protein,CPm）近全长序列的系统进化树

Fig. 3 Phylogenetic tree based on the near full length sequences of minor coat protein（CPm）gene of LCV isolates

表1 LCV 次要外壳蛋白基因（minor coat protein,CPm）近全长序列重组事件分析

Table 1 Recombinant events analysis of near full length of minor coat of protein gene（CPm）of LCV isolates

重组事件 Recombinant event	重组子 Recombinant	主要来源 Major parent	次要来源 Minor parent	算法Algorithm
重组事件 Recombinant event	重组子 Recombinant	主要来源 Major parent	次要来源 Minor parent	RDP	Geneconv	Bootscan	MaxChi	Chimaera	SiScan	Topal
1	BR （巴西Brazil）	Can1 （以色列Israel）	未知 Unknown	+	-	-	+	+	+	-
2	YN58 （云南Yunnan）	SD （山东Shandong）	未知 Unknown	-	+	+	-	+	+	+

表2 LCV次要外壳蛋白基因（minor coat protein gene,CPm）近全长序列碱基替换

Table 2 Maximum composite likelihood estimate of the pattern of nucleotide substitution of near full length of minor coat protein gene（CPm）of LCV isolates

核苷酸 Nucleotide	A	T	C	G
A	-	3.11	2.02	13.20
T	3.96	-	16.38	2.34
C	3.96	25.25	-	2.34
G	22.31	3.11	2.02	-

表3 LCV分离物次要外壳蛋白基因（minor coat protein,CPm）近全长序列基因流和基因变异

Table 3 Gene flow and genetic differentiation between near full length of minor coat protein（CPm）genes of LCV isolates

亚群Lineage	序列数量Number of sequences	Ss	h	Hd	k	Pi
Ⅰ	13	36	13	1	13.54	0.0099
Ⅱ	3	8	3	1	5.33	0.0039
Ⅲ	2	3	2	1	18.00	0.0670

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云南省番茄莴苣褪绿病毒分子检测及遗传进化分析

Molecular Detection and Genetic Evolution Analysis of Yunnan Isolates of Lettuce Chlorosis Virus in Tomato

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云南省番茄莴苣褪绿病毒分子检测及遗传进化分析

Molecular Detection and Genetic Evolution Analysis of Yunnan Isolates of Lettuce Chlorosis Virus in Tomato

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