基于STR位点对广东省柑橘溃疡病菌种群遗传结构的分析

doi:10.16420/j.issn.0513-353x.2021-0297

摘要/Abstract

摘要：

柑橘溃疡病是柑橘生产上最严重的病害之一,由柑橘黄单胞杆菌柑橘亚种（Xanthomonas citri pv. citri,Xcc）引起。为了全面系统地研究广东省柑橘溃疡病菌的种群遗传结构,通过比对分析已报道的中国8个柑橘溃疡病菌全基因组序列的串联重复位点（Short Tandem Repeat,STR）,筛选出6个高度变异位点,对广东省的15个市（县）14个品种共352个柑橘溃疡病菌菌株进行遗传多样性分析。结果表明,基于遗传多样性和遗传距离对广东省不同地区的柑橘溃疡病菌株鉴定出6组类群：潮州和汕尾为Ⅰ组,梅州和河源为Ⅱ组,肇庆、广州、江门、佛山和惠州为Ⅲ组,阳江、茂名和湛江为Ⅳ组,云浮为Ⅴ组,韶关和清远为Ⅵ组。基于遗传多样性和遗传距离对广东省不同品种的柑橘溃疡病菌株鉴定出5组类群：沃柑、贡柑、朱砂橘、砂糖橘、茶枝柑、红江橙、马水橘为Ⅰ组,脐橙和柠檬为Ⅱ组,蕉柑、茂谷柑和春甜橘为Ⅲ组,沙田柚为Ⅳ组。

关键词: 柑橘, 柑橘溃疡病菌, 短串联重复位点, 遗传多样性

Abstract:

Citrus canker is one of the most serious diseases in citrus production,which is caused by Xanthomonas citri pv. citri（Xcc）. In order to systematically and comprehensively learn the genetic structure of citrus canker strains in Guangdong Province,six highly variable locus were selected by comparing and analyzing the short tandem repeat loci of the whole genome sequence of eight Chinese strains,and the genetic diversity of 352 strains of 14 varieties from 15 cities（counties）in Guangdong Province was analyzed. The results showed that based on the genetic diversity and genetic distance of Guangdong Province in different parts of the citrus canker strains identified six groups：Ⅰgroup（Chaozhou and Shanwei）,Ⅱ group（Meizhou and Heyuan）,Ⅲ group（Zhaoqing,Guangzhou,Jiangmen,Foshan and Huizhou）,Ⅳ group（Yangjiang,Maoming and Zhanjiang）Ⅴ group（Yunfu）,Ⅵ group（Shaoguan and Qingyuan）. Based on the genetic diversity and genetic distance on the different varieties of citrus canker in Guangdong strains identified five groups：Ⅰgroup（Wogan,Gonggan,Zhushaju,Shatamhju,Chazhigan,Hongjiangcheng and Mashuiju）,Ⅱ group（Qicheng and Ningmeng）,Ⅲ group（Jiaogan,Maogugan and Chuntianju）,Ⅳ group（Shatianyou）. The results can provide a theoretical basis for the epidemic regularity of citrus canker disease in Guangdong province.

Key words: citrus, Xanthomonas citri pv. citri, short tandem repeat, genetic diversity

中图分类号:

S666

李文婷, 李翠晓, 林小清, 郑永钦, 郑正, 邓晓玲. 基于STR位点对广东省柑橘溃疡病菌种群遗传结构的分析[J]. 园艺学报, 2022, 49(6): 1233-1246.

LI Wenting, LI Cuixiao, LIN Xiaoqing, ZHENG Yongqin, ZHENG Zheng, DENG Xiaoling. Population Genetic Structure of Xanthomonas citri pv. citri in Guangdong Province Based on the STR Locus[J]. Acta Horticulturae Sinica, 2022, 49(6): 1233-1246.

图/表 12

参考文献 30

[1]	Behlau F, Belasque J, Bergamin F A, Graham J H, Leite R P, Gottwald T R. 2008. Copper sprays and windbreaks for control of citrus canker on young orange trees in southern Brazil. Crop Protection, 27 (3-5):807-813. doi: 10.1016/j.cropro.2007.11.008 URL
[2]	Botstein D R, White R L, Skolnick M H, Davis R W. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics, 32 (3):314-331. pmid: 6247908
[3]	Brunings A M, Gabriel D W. 2010. Xanthomonas citri:breaking the surface. Molecular Plant Pathology, 4 (3):141-157. doi: 10.1046/j.1364-3703.2003.00163.x URL
[4]	Flávia M S C, Lázara P C C, Rui P L J. 2005. Genetic diversity of Xanthomonas axnopodis pv. citri based on plasmid profile and pulsed field gel electrophoresis. Genetics and Molecular Biology, 28 (3):446-451. doi: 10.1590/S1415-47572005000300020
[5]	Gary B. 1999. Tandem repeats finder:a program to analyze DNA sequences. Nucleic Acids Research, 27 (2):573-580. doi: 10.1093/nar/27.2.573 URL
[6]	Gottwald T R, Graham J H, Civerolo E L, Barrett H C, Hearn C J. 1993. Differential host range reaction of citrus and citrus relatives to citrus canker and citrus bacterial spot determined by leaf mesophyll susceptibility. Plant Disease, 77 (10):1004-1009. doi: 10.1094/PD-77-1004 URL
[7]	Gottwald T R, Graham J H, Schubert T S. 2002. Citrus canker:the pathogen and its impact. Plant Health Progress, 10 (1):15-49. doi: 10.1094/PHP-2009-0615-01-RS URL
[8]	Guo Qingyun, Li Fangzhen, Fang Wei, Chen Ling, Zhang Huiqin. 2011. Preliminary study on differentiation of navel orange ulcerative bacteria in southern Jiangxi. Journal of Jiangxi Agricultural University, 33 (2):278-282. (in Chinese)
	郭青云, 李坊贞, 方玮, 陈玲, 张慧琴. 2011. 赣南脐橙溃疡病菌菌系分化初步研究. 江西农业大学学报, 33 (2):278-282.
[9]	Jiang Xinxiang, Zhou Canfang, Wan Zhong, Liu Yang, Ma Peiqia. 2011. Analysis on the current situation of citrus industry development in Guangdong in 2010. Guangdong Agricultural Sciences, 38 (5):15-17. (in Chinese)
	蒋新祥, 周灿芳, 万忠, 刘洋, 马培恰. 2011. 2010年广东柑桔产业发展现状分析. 广东农业科学, 38 (5):15-17.
[10]	Khodakaramian G, Swings J. 2011. Genetic diversity and pathogenicity of Xanthomonas axonopodis strains inducing citrus canker disease in Iran and South Korea. Indian Journal of Microbiology, 51 (2):194-199. doi: 10.1007/s12088-011-0103-8 pmid: 22654164
[11]	Kumar S, Stecher G, Tamura K. 2016. MEGA7:Molecular Evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology & Evolution, 33 (7):1870-1874.
[12]	Lee Y H, Lee D H. 2008. Differentiation of citrus bacterial canker strains in Korea by host range,rep-PCR fingerprinting and 16S rDNA analysis. European Journal of Plant Pathology, 121 (1):97-102. doi: 10.1007/s10658-007-9242-5 URL
[13]	Long Qin, Xie Yu, Lei Tiangang, He Yongrui, Xu Lanzhen, Zou Xiuping, Chen Shanchun. 2020a. Bioinformatics analysis of MAPK genes in citrus and their expression in response to canker disease. Acta Horticulturae Sinica, 47 (11):2095-2106. (in Chinese)
	龙琴, 谢宇, 雷天刚, 何永睿, 许兰珍, 邹修平, 陈善春. 2020a. 柑橘MAPK基因的生物信息及其响应溃疡病菌侵染的表达分析. 园艺学报, 47 (11):2095-2106.
[14]	Long Qin, Xie Yu, Xu Lanzhen, He Yongrui, Zou Xiuping, Chen Shanchun. 2020b. Characteristics and mechanism of programmed cell death in response to citrus canker pathogen in the early stage of infection. Acta Horticulturae Sinica, 47 (6):1047-1058. (in Chinese)
	龙琴, 谢宇, 许兰珍, 何永睿, 邹修平, 陈善春. 2020b. 溃疡病菌侵染早期柑橘细胞程序性死亡的响应特征及机制. 园艺学报, 47 (6):1047-1058.
[15]	Lu Xiaolin. 2013. Study on genetic diversity of Chinese citrus ulcer pathogen based on SSR[M. D. Dissertation]. Chongqing: Chongqing University. (in Chinese)
	卢小林. 2013. 基于SSR的中国柑桔溃疡病菌遗传多样性研究[硕士论文]. 重庆: 重庆大学.
[16]	Ngoc L B T, Verniere C, Vital K, Guerin F, Pruvost O. 2010. Development of 14 minisatellite markers for the citrus canker bacterium,Xanthomonas citri pv. citri. Molecular Ecology Resources, 9 (1):125-127. doi: 10.1111/j.1755-0998.2008.02242.x URL
[17]	Peng Yaowu. 2014. Polymorphism of pathogenic bacteria of citrus ulcer in China[M. D. Dissertation]. Chongqing: Southwest University. (in Chinese)
	彭耀武. 2014. 中国柑橘溃疡病病原菌多态性研究[硕士论文]. 重庆: 西南大学.
[18]	Rademaker J L W, Louws F J, Schultz M H, Rossbach U, Bruijn F J D. 2005. A comprehensive species to strain taxonomic framework for Xanthomonas. Phytopathology, 95 (9):1098-1111. doi: 10.1094/PHYTO-95-1098 pmid: 18943308
[19]	Ren Jianguo, Huang Siliang, Li Yangrui, Nong Shifeng, Chen Yongning. 2007. Study on the bacterial strain of citrus ulcer in Guangxi. Microbiology Bulletin, 34 (2):216-220. (in Chinese)
	任建国, 黄思良, 李杨瑞, 农时锋, 陈永宁. 2007. 广西柑橘溃疡病菌菌系分化研究. 微生物学通报, 34 (2):216-220.
[20]	Rychlik W. 2007. OLIGO 7 Primer analysis software. Methods in Molecular Biology, 402:36-60.
[21]	Schubert T S, Rizvi S A, Sun X, Gottwald T R, Graham J H, Dixon W N. 2001. Meeting the challenge of eradicating citrus canker in Florida again. Plant Disease, 85 (4):340-356.
[22]	Tang Xiaolang, Ma Peiqia, Wu Wen, Gan liansheng, Peng Chengji, Huang Yongjing. 2005. Development status and countermeasures of citrus industry in Guangdong. China Fruit Industry Information, 22 (11):7-9. (in Chinese)
	唐小浪, 马培恰, 吴文, 甘廉生, 彭成绩, 黄永敬. 2005. 广东柑桔业发展现状与对策探讨. 中国果业信息, 22 (11):7-9.
[23]	Wang A, Yu Z, Ding Y. 2009. Genetic diversity analysis of wild close relatives of barley from Tibet and the Middle East by ISSR and SSR markers. Comptes Rendus Biologies, 332 (4):393-403. doi: 10.1016/j.crvi.2008.11.007 URL
[24]	Wang L, Guo J, Zhao G F. 2006. Genetic diversity of the endangered and endemic species Psathyrostachys huashanica natural populations using simple sequence repeats(SSRs)markers. Biochemical Systematics & Ecology, 34 (4):310-318.
[25]	Wang Z K, Sun X Y, Xia Y X, Zhou C Y, Yin Y P. 2004. Rapid detection of citrus bacterial canker disease(Xanthomonas axonopodis pv. citri)with polymerase chain reaction(PCR). Acta Phytopathologica Sinica, 34 (1):14-20.
[26]	Wei Chudan, Ding Tian, Ye Gan, Liu Qiongguang. 2014. Analysis of genetic diversity of citrus ulcer bacteria in Guangdong and Jiangxi Province. Journal of South China Agricultural University, 35 (4):71-76. (in Chinese)
	魏楚丹, 丁钿, 叶淦, 刘琼光. 2014. 广东和江西省柑橘溃疡病菌的遗传多样性分析. 华南农业大学学报, 35 (4):71-76.
[27]	Ding Liang, Cong Yu, Xu Xuling, Qiao Huan, He Silong, Xu Wenjian, Xu Tianshun. 2021. Isolation and identification of a lytic bacteriophage infecting Xanthomonas axonopodis pv. citri. Acta Horticulturae Sinica, 48 (12):2349-2359. (in Chinese)
	肖逍, 丁良, 丛郁, 徐旭凌, 乔欢, 何四龙, 许文建, 徐天舜. 2021. 柑橘溃疡病菌噬菌体的分离鉴定. 园艺学报, 48 (12):2349-2359.
[28]	Yao Tingshan, Hu Junhua, Tang Zhike, Ran Chun, Li Zhongan, Zhou Changyong. 2010. Genetic diversity of citrus ulcer bacteria in China using REP-PCR. Journal of Fruit Trees, 27 (5):819-822. (in Chinese)
	姚廷山, 胡军华, 唐科志, 冉春, 李中安, 周常勇. 2010. 利用REP-PCR技术研究我国9省柑橘溃疡病菌遗传多样性. 果树学报, 27 (5):819-822.
[29]	Zhang Gebi, Yang Tingmi, Li Xiqing, Chen Zhusheng. 2004. The resistance of citrus varieties to citrus canker caused by Xanthomonas citri. Acta Phytophylacica Sinica, 31 (2):221-222. (in Chinese)
	张戈壁, 阳廷密, 李喜庆, 陈竹生. 2004. 不同柑桔品种对柑橘溃疡病抗病能力的测定. 植物保护学报, 31 (2):221-222.
[30]	Zhuo Tongxun, Gu Dingqiu, Wu Qionghui. 2006. Discussion on the sustainable development of citrus industry in Guangdong. Economic Forest Research, 24 (3):96-98. (in Chinese)
	卓铜勋, 古定球, 吴琼辉. 2006. 广东柑橘产业可持续发展的探讨. 经济林研究, 24 (3):96-98.

地理来源 Geographical region	数量 Number	柑橘品种 Citrus varieties
茂名 Maioming	29	橘红,沃柑,贡柑 Juhong,Orah,Gonggan
梅州 Meizhou	28	沙田柚,柠檬,脐橙 Shatian pomelo,Lemon,Sweet organge
江门 Jiangmen	26	茶枝柑,沃柑 Chazhigan,Orah
阳江 Yangjiang	26	马水橘,贡柑 Mashuiju,Gonggan
云浮 Yunfu	26	沃柑,砂糖橘 Orah,Shatangju
湛江 Zhanjiang	25	红江橙,沃柑,茂谷柑 Hongjiangcheng,Orah,Maogugan
肇庆 Zhaoqing	24	砂糖橘,贡柑 Shatangju,Gonggan
惠州 Huizhou	24	砂糖橘,春甜橘,沃柑,沙田柚 Shatangju,Chuntianju,Orah,Shatian pomelo
汕尾 Shanwei	24	贡柑,脐橙 Gonggan,Qicheng
佛山 Foshan	23	脐橙,朱砂橘 Qicheng,Zhushaju
韶关 Shaoguan	21	贡柑,沃柑 Gonggan,Orah
广州 Guangzhou	21	沃柑,贡柑,茂谷柑,砂糖橘 Orah,Gonggan,Maogugan,Shatangju
清远 Qingyuan	20	砂糖橘,沃柑 Shatang,Orah
潮州 Chaozhou	18	沃柑,蕉柑 Orah,Jiaogan
河源 Heyuan	17	贡柑,马水橘,砂糖橘 Gonggan,Mashuiju,Shatangju

地理来源 Geographical region	数量 Number	柑橘品种 Citrus varieties
茂名 Maioming	29	橘红,沃柑,贡柑 Juhong,Orah,Gonggan
梅州 Meizhou	28	沙田柚,柠檬,脐橙 Shatian pomelo,Lemon,Sweet organge
江门 Jiangmen	26	茶枝柑,沃柑 Chazhigan,Orah
阳江 Yangjiang	26	马水橘,贡柑 Mashuiju,Gonggan
云浮 Yunfu	26	沃柑,砂糖橘 Orah,Shatangju
湛江 Zhanjiang	25	红江橙,沃柑,茂谷柑 Hongjiangcheng,Orah,Maogugan
肇庆 Zhaoqing	24	砂糖橘,贡柑 Shatangju,Gonggan
惠州 Huizhou	24	砂糖橘,春甜橘,沃柑,沙田柚 Shatangju,Chuntianju,Orah,Shatian pomelo
汕尾 Shanwei	24	贡柑,脐橙 Gonggan,Qicheng
佛山 Foshan	23	脐橙,朱砂橘 Qicheng,Zhushaju
韶关 Shaoguan	21	贡柑,沃柑 Gonggan,Orah
广州 Guangzhou	21	沃柑,贡柑,茂谷柑,砂糖橘 Orah,Gonggan,Maogugan,Shatangju
清远 Qingyuan	20	砂糖橘,沃柑 Shatang,Orah
潮州 Chaozhou	18	沃柑,蕉柑 Orah,Jiaogan
河源 Heyuan	17	贡柑,马水橘,砂糖橘 Gonggan,Mashuiju,Shatangju

引物名称 Primer name	引物（5′-3′） Sequence	退火温度/℃ Annealing temperature
GT1-F	TTGCCACGGGCCAGGGTCGC	64
GT1-R	GCCCAGATGCTCGATCAAGG
GT2-F	CTGTGGTGTTGCGAAGTCAT	55
GT2-R	GTTCGCTTGCTTGAGACTGA
GT3-F	TCGGCGAGTTCGATCGGGTGCT	60
GT3-R	CTGGCGTGCTGCAGGATGTT
GT4-F	ATCGAAACTGTGCAGGCAAT	55
GT4-R	TGGAACAGCATGAGCAGAAC
GT5-F	GGACATCGTCTCCCAGCAACCG	58
GT5-R	GCCTGCCACGCTTTTCCGATT
GT6-F	GCTCGGCGACTTCTTGATAG	55
GT6-R	AAGGTAAAATCGGGCGACTC

引物名称 Primer name	引物（5′-3′） Sequence	退火温度/℃ Annealing temperature
GT1-F	TTGCCACGGGCCAGGGTCGC	64
GT1-R	GCCCAGATGCTCGATCAAGG
GT2-F	CTGTGGTGTTGCGAAGTCAT	55
GT2-R	GTTCGCTTGCTTGAGACTGA
GT3-F	TCGGCGAGTTCGATCGGGTGCT	60
GT3-R	CTGGCGTGCTGCAGGATGTT
GT4-F	ATCGAAACTGTGCAGGCAAT	55
GT4-R	TGGAACAGCATGAGCAGAAC
GT5-F	GGACATCGTCTCCCAGCAACCG	58
GT5-R	GCCTGCCACGCTTTTCCGATT
GT6-F	GCTCGGCGACTTCTTGATAG	55
GT6-R	AAGGTAAAATCGGGCGACTC

位点名称 Loci Name	位置 Position	长度 Period size	重复数量 Copy number
GT1	241859 ~ 241926	7	9.7
GT2	1065817 ~ 1065895	7	11.3
GT3	1441963 ~ 1442079	7	16.7
GT4	1884560 ~ 1884621	7	8.9
GT5	565742 ~ 565790	7	7
GT6	4367068 ~ 4367102	7	5