利用SRAP分析核盘菌遗传多样性

doi:10.3724/SP.J.1003.2010.509

生物多样性 ›› 2010, Vol. 18 ›› Issue (5): 509-515. DOI: 10.3724/SP.J.1003.2010.509

利用SRAP分析核盘菌遗传多样性

陈碧云¹, 胡琼¹, Christina Dixelius², 李国庆³, 伍晓明¹^,^*()

1 中国农业科学院油料作物研究所, 农业部油料作物生物学重点开放实验室, 武汉 430062
3 华中农业大学, 武汉 430070

收稿日期:2010-02-25 接受日期:2010-07-27 出版日期:2010-09-20 发布日期:2010-09-20
通讯作者: 伍晓明
作者简介:* E-mail: wuxm@oilcrops.cn
基金资助:
863项目(2006AA10Z1E4);SIDA项目“抗病和抗虫油菜的遗传改良”和农业部油料作物生物学重点开放实验室开放课题(200503)

Genetic diversity in Sclerotinia sclerotiorum assessed with SRAP markers

Biyun Chen¹, Qiong Hu¹, Christina Dixelius², Guoqing Li³, Xiaoming Wu¹^,^*()

1 Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Wuhan 430062
2 Swedish University of Agricultural Sciences, Box 7080, 75007 Uppsala, Sweden

Received:2010-02-25 Accepted:2010-07-27 Online:2010-09-20 Published:2010-09-20
Contact: Xiaoming Wu

1. 附录Ⅰ 76个核盘菌菌株、寄主及来源 .pdf(190KB)

摘要/Abstract

摘要：

核盘菌(Sclerotinia sclerotiorum)是危害油菜等多种经济作物的重要病原真菌。研究不同地区、相同或不同寄主核盘菌的遗传多样性对了解核盘菌的遗传演化过程和指导病害防控具有重要意义。我们采用序列相关扩增多态性(sequence-related amplified polymorphism, SRAP)标记对不同地理来源、不同寄主来源的76个核盘菌菌株的遗传多样性进行了分析。7对SRAP引物共获得260个位点, 其中114个为多态位点, 占43.85%。UPGMA聚类结果显示, 在相似性系数为0.64时, 76个核盘菌菌株分为4个组, 每组包含的菌株数分别为54、18、2和2。聚类及主成分分析结果显示, 来源于春油菜生态区和冬油菜生态区油菜上的核盘菌菌株可以明显分为两簇, 而油菜、大豆、莴苣等不同寄主植物上的核盘菌菌株没有明显的遗传分化。分子变异分析(AMOVA)结果显示, 不同地理来源、不同油菜生态区和不同寄主来源的核盘菌群体内的变异率分别为75.2%、81.2%和97.6%, 均达到极显著水平(P<0.001); 不同地理来源和不同油菜生态区的核盘菌群体间的变异率分别为24.8%和18.8%, 也达到极显著水平(P<0.001); 不同寄主来源的核盘菌群体间的变异率仅为2.4%, 变异不显著(P = 0.8673)。研究结果表明, 来源于春油菜生态区的核盘菌的遗传多样性高于冬油菜生态区。

关键词: Sclerotinia sclerotiorum, 油菜, SRAP标记, 遗传变异, 遗传分化

Abstract

Assessing the genetic diversity of Sclerotinia sclerotiorum isolates from different eco-geographi- cal regions and host plants is crucially important for understanding the evolution of this fungal pathogen and its control. We detected DNA polymorphism of 76 S. sclerotiorum isolates from different regions and host plants using sequence-related amplified polymorphism (SRAP) markers. A total of 260 scorable fragments were identified with seven SRAP primer combinations; among them 114 were polymorphic loci (43.85%). UPMGA (Unweighted Pair Group Method with Arithmetic Mean) indicated that the dendrogram consisted of four groups, which included 54, 18, 2, and 2 isolates, respectively, when the genetic similarity coefficient was 0.64. Little genetic difference was identified among the isolates from different host species by cluster and principal component analyses. AMOVA (Analysis of Molecular Variance) revealed that the percentage of variation among populations differed by geographical region (24.8%, P<0.001) and by ecological region (18.8%, P<0.001), but variation among populations of different host plants was not different than expected at random (2.4%, P = 0.8673). One of our most significant results was that S. sclerotiorum isolates on rapeseed plants from winter and spring ecological regions can be divided into two clusters, and that genetic diversity in isolates of spring rapeseed ecological regions was higher than that of winter rapeseed ecological regions.

Key words: Sclerotinia sclerotiorum, rape, SRAP markers, genetic variation, genetic differentiation

陈碧云, 胡琼, Christina Dixelius, 李国庆, 伍晓明 (2010) 利用SRAP分析核盘菌遗传多样性. 生物多样性, 18, 509-515. DOI: 10.3724/SP.J.1003.2010.509.

Biyun Chen, Qiong Hu, Christina Dixelius, Guoqing Li, Xiaoming Wu (2010) Genetic diversity in Sclerotinia sclerotiorum assessed with SRAP markers. Biodiversity Science, 18, 509-515. DOI: 10.3724/SP.J.1003.2010.509.

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链接本文: https://www.biodiversity-science.net/CN/10.3724/SP.J.1003.2010.509

https://www.biodiversity-science.net/CN/Y2010/V18/I5/509

图/表 7

参考文献 19

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编号 No.	菌株名称 Isolate	寄主 Host		来源 Origin	编号 No.		菌株名称 Isolate		寄主 Host	来源 Origin
1	RSC-1	油菜 Rape		陕西勉县 Mianxian, Shaanxi	31		F5-219-S2 clone 39		大豆 Soybean	加拿大 Canada
2	RSC-2	油菜 Rape		陕西咸阳 Xianyang, Shaanxi	32		F5-138-L4 clone 321		大豆 Soybean	加拿大 Canada
3	RSC-3	油菜 Rape		陕西大荔 Dali, Shaanxi	33		LMK211 clone 2		油菜 Rape	加拿大 Canada
4	RSC-4	油菜 Rape		湖北武汉 Wuhan, Hubei	34		CW312 clone 1		大豆 Soybean	加拿大 Canada
5	RSC-5	油菜 Rape		湖北武汉 Wuhan, Hubei	35		CW317 clone 2		大豆 Soybean	加拿大 Canada
6	RSC-6	油菜 Rape		湖北武汉 Wuhan, Hubei	36		SC-13 Sweden		油菜 Rape	瑞典 Sweden
7	RSC-7	油菜 Rape		湖北襄樊 Xiangfan, Hubei	37		IC16		油菜 Rape	挪威 Norway
8	RSC-8	油菜 Rape		河南确山 Queshan, Henan	38		IC6		油菜 Rape	挪威 Norway
9	RSC-9	油菜 Rape		湖南汉寿 Hanshou, Hunan	39		824 clone 1		油菜 Rape	加拿大 Canada
10	RSC-10	油菜 Rape		河南遂平 Suiping, Henan	40		Xbz-4		小白菜 Pakchoi	湖北罗田 Luotian, Hubei
11	RSC-11	油菜 Rape		河南信阳 Xinyang, Henan	41		Xbz-5		小白菜 Pakchoi	湖北十堰 Shiyan, Hubei
12	RSC-12	油菜 Rape		河南郑州 Zhengzhou, Henan	42		Xbz-9		小白菜 Pakchoi	湖北洪湖 Honghu, Hubei
13	RSC-13	油菜 Rape		河南郑州 Zhengzhou, Henan	43		Sb-2		大豆 Soybean	黑龙江 Heilongjiang
14	RSC-14	油菜 Rape		江西宜春 Yichun, Jiangxi	44		Sb-4		大豆 Soybean	美国 USA
15	RSC-15	油菜 Rape		浙江杭州 Hangzhou, Zhejiang	45		Sb-6		大豆 Soybean	加拿大 Canada
16	RSC-16	油菜 Rape		浙江杭州 Hangzhou, Zhejiang	46		Cor-3		芫荽 Coriander	湖北神农架 Shennongjia, Hubei
17	RSC-17	油菜 Rape		贵州思南 Sinan, Guizhou	47		Cor-6		芫荽 Coriander	湖北通城 Tongcheng, Hubei
18	RSC-18	油菜 Rape		湖北罗田 Luotian, Hubei	48		Brb-1		蚕豆 Horsebean	湖北武汉 Wuhan, Hubei
19	RSC-19	油菜 Rape		湖北英山 Yingshan, Hubei	49		Brb-9		蚕豆 Horsebean	湖北通城 Tongcheng, Hubei
20	RSC-20	油菜 Rape		江苏南京 Nanjing, Jiangsu	50		Rap-1		油菜 Rape	湖北云梦 Yunmeng, Hubei
21	RSC-21	油菜 Rape		江苏南京 Nanjing, Jiangsu	51		Rap-2		油菜 Rape	湖北沙市 Shashi, Hubei
22	RSC-22	油菜 Rape		江西南昌 Nanchang, Jiangxi	52		Rap-6		油菜 Rape	四川雅安 Yaan, Sichuan
23	RSC-23	油菜 Rape		四川绵阳 Mianyang, Sichuan	53		Rap-20		油菜 Rape	湖北罗田 Luotian, Hubei
24	RSC-24	油菜 Rape		陕西杨凌 Yangling, Shaanxi	54		Rap-21		油菜 Rape	湖北宣恩 Xuanen, Hubei
25	RSC-25	油菜 Rape		湖北武汉 Wuhan, Hubei	55		Rap-31		油菜 Rape	湖北宜昌 Yichang, Hubei
26	RSC-26	油菜 Rape		黑龙江佳木斯 Jiamusi, Heilongjiang	56		Rap-35		油菜 Rape	湖北五峰 Wufeng, Hubei
27	RSC-27	油菜 Rape		青海西宁 Xining, Qinghai	57		Rap-36		油菜 Rape	英国 Britain
28	RSC-28	油菜 Rape		青海西宁 Xining, Qinghai	58		Rap-37		油菜 Rape	加拿大 Canada
29	RSC-29	油菜 Rape		青海西宁 Xining, Qinghai	59		Hzd-3		小白菜 Pakchoi	湖北仙桃 Xiantao, Hubei
30	RSC-30	油菜 Rape		青海西宁 Xining, Qinghai	60		Hzd-4		小白菜 Pakchoi	湖北黄梅 Huangmei, Hubei
编号 No.	菌株名称 Isolate	寄主 Host	来源 Origin			编号 No.		菌株名称 Isolate	寄主 Host	来源 Origin
61	Nic-1	烟草 Tobacco	湖北咸丰 Xianfeng, Hubei			69		Let-46	莴苣 Lettuce	湖北神农架 Shennongjia, Hubei
62	Zxd-1	红豆 Red bean	内蒙古扎兰屯 Zalanten, Inner Monglia			70		Brb-8	蚕豆 Horsebean	湖北通山 Tongshan, Hubei
63	Rad-1	萝卜 Radish	湖北竹山 Zhushan, Hubei			71		Let-9	莴苣 Lettuce	湖北五峰 Wufeng, Hubei
64	clone-22	油菜 Rape	加拿大 Canada			72		Let-3	莴苣 Lettuce	湖北武汉 Wuhan, Hubei
65	UK-2	不清 Unknown	湖北武汉 Wuhan, Hubei			73		Let-27	莴苣 Lettuce	台湾 Taiwan
66	Can02-6	油菜 Rape	湖北武汉 Wuhan, Hubei			74		Let-11	莴苣 Lettuce	湖北宜昌 Yichang, Hubei
67	Cor-10	芫荽 Coriander	湖北宜昌 Yichang, Hubei			75		Can02-22	油菜 Rape	湖北武汉 Wuhan, Hubei
68	Rad-30-2	萝卜 Radish	湖北神农架 Shennongjia, Hubei			76		Let-26	莴苣 Lettuce	加拿大 Canada

编号 No.	菌株名称 Isolate	寄主 Host		来源 Origin	编号 No.		菌株名称 Isolate		寄主 Host	来源 Origin
1	RSC-1	油菜 Rape		陕西勉县 Mianxian, Shaanxi	31		F5-219-S2 clone 39		大豆 Soybean	加拿大 Canada
2	RSC-2	油菜 Rape		陕西咸阳 Xianyang, Shaanxi	32		F5-138-L4 clone 321		大豆 Soybean	加拿大 Canada
3	RSC-3	油菜 Rape		陕西大荔 Dali, Shaanxi	33		LMK211 clone 2		油菜 Rape	加拿大 Canada
4	RSC-4	油菜 Rape		湖北武汉 Wuhan, Hubei	34		CW312 clone 1		大豆 Soybean	加拿大 Canada
5	RSC-5	油菜 Rape		湖北武汉 Wuhan, Hubei	35		CW317 clone 2		大豆 Soybean	加拿大 Canada
6	RSC-6	油菜 Rape		湖北武汉 Wuhan, Hubei	36		SC-13 Sweden		油菜 Rape	瑞典 Sweden
7	RSC-7	油菜 Rape		湖北襄樊 Xiangfan, Hubei	37		IC16		油菜 Rape	挪威 Norway
8	RSC-8	油菜 Rape		河南确山 Queshan, Henan	38		IC6		油菜 Rape	挪威 Norway
9	RSC-9	油菜 Rape		湖南汉寿 Hanshou, Hunan	39		824 clone 1		油菜 Rape	加拿大 Canada
10	RSC-10	油菜 Rape		河南遂平 Suiping, Henan	40		Xbz-4		小白菜 Pakchoi	湖北罗田 Luotian, Hubei
11	RSC-11	油菜 Rape		河南信阳 Xinyang, Henan	41		Xbz-5		小白菜 Pakchoi	湖北十堰 Shiyan, Hubei
12	RSC-12	油菜 Rape		河南郑州 Zhengzhou, Henan	42		Xbz-9		小白菜 Pakchoi	湖北洪湖 Honghu, Hubei
13	RSC-13	油菜 Rape		河南郑州 Zhengzhou, Henan	43		Sb-2		大豆 Soybean	黑龙江 Heilongjiang
14	RSC-14	油菜 Rape		江西宜春 Yichun, Jiangxi	44		Sb-4		大豆 Soybean	美国 USA
15	RSC-15	油菜 Rape		浙江杭州 Hangzhou, Zhejiang	45		Sb-6		大豆 Soybean	加拿大 Canada
16	RSC-16	油菜 Rape		浙江杭州 Hangzhou, Zhejiang	46		Cor-3		芫荽 Coriander	湖北神农架 Shennongjia, Hubei
17	RSC-17	油菜 Rape		贵州思南 Sinan, Guizhou	47		Cor-6		芫荽 Coriander	湖北通城 Tongcheng, Hubei
18	RSC-18	油菜 Rape		湖北罗田 Luotian, Hubei	48		Brb-1		蚕豆 Horsebean	湖北武汉 Wuhan, Hubei
19	RSC-19	油菜 Rape		湖北英山 Yingshan, Hubei	49		Brb-9		蚕豆 Horsebean	湖北通城 Tongcheng, Hubei
20	RSC-20	油菜 Rape		江苏南京 Nanjing, Jiangsu	50		Rap-1		油菜 Rape	湖北云梦 Yunmeng, Hubei
21	RSC-21	油菜 Rape		江苏南京 Nanjing, Jiangsu	51		Rap-2		油菜 Rape	湖北沙市 Shashi, Hubei
22	RSC-22	油菜 Rape		江西南昌 Nanchang, Jiangxi	52		Rap-6		油菜 Rape	四川雅安 Yaan, Sichuan
23	RSC-23	油菜 Rape		四川绵阳 Mianyang, Sichuan	53		Rap-20		油菜 Rape	湖北罗田 Luotian, Hubei
24	RSC-24	油菜 Rape		陕西杨凌 Yangling, Shaanxi	54		Rap-21		油菜 Rape	湖北宣恩 Xuanen, Hubei
25	RSC-25	油菜 Rape		湖北武汉 Wuhan, Hubei	55		Rap-31		油菜 Rape	湖北宜昌 Yichang, Hubei
26	RSC-26	油菜 Rape		黑龙江佳木斯 Jiamusi, Heilongjiang	56		Rap-35		油菜 Rape	湖北五峰 Wufeng, Hubei
27	RSC-27	油菜 Rape		青海西宁 Xining, Qinghai	57		Rap-36		油菜 Rape	英国 Britain
28	RSC-28	油菜 Rape		青海西宁 Xining, Qinghai	58		Rap-37		油菜 Rape	加拿大 Canada
29	RSC-29	油菜 Rape		青海西宁 Xining, Qinghai	59		Hzd-3		小白菜 Pakchoi	湖北仙桃 Xiantao, Hubei
30	RSC-30	油菜 Rape		青海西宁 Xining, Qinghai	60		Hzd-4		小白菜 Pakchoi	湖北黄梅 Huangmei, Hubei
编号 No.	菌株名称 Isolate	寄主 Host	来源 Origin			编号 No.		菌株名称 Isolate	寄主 Host	来源 Origin
61	Nic-1	烟草 Tobacco	湖北咸丰 Xianfeng, Hubei			69		Let-46	莴苣 Lettuce	湖北神农架 Shennongjia, Hubei
62	Zxd-1	红豆 Red bean	内蒙古扎兰屯 Zalanten, Inner Monglia			70		Brb-8	蚕豆 Horsebean	湖北通山 Tongshan, Hubei
63	Rad-1	萝卜 Radish	湖北竹山 Zhushan, Hubei			71		Let-9	莴苣 Lettuce	湖北五峰 Wufeng, Hubei
64	clone-22	油菜 Rape	加拿大 Canada			72		Let-3	莴苣 Lettuce	湖北武汉 Wuhan, Hubei
65	UK-2	不清 Unknown	湖北武汉 Wuhan, Hubei			73		Let-27	莴苣 Lettuce	台湾 Taiwan
66	Can02-6	油菜 Rape	湖北武汉 Wuhan, Hubei			74		Let-11	莴苣 Lettuce	湖北宜昌 Yichang, Hubei
67	Cor-10	芫荽 Coriander	湖北宜昌 Yichang, Hubei			75		Can02-22	油菜 Rape	湖北武汉 Wuhan, Hubei
68	Rad-30-2	萝卜 Radish	湖北神农架 Shennongjia, Hubei			76		Let-26	莴苣 Lettuce	加拿大 Canada

上游引物 Forward primers		下游引物 Reverse primers
em1	5′-GACTGCGTACGAATTAAT-3′	me1	5′-TGAGTCCAAACCGGATA-3′
em2	5′-GACTGCGTACGAATTTGC-3′	me2	5′-TGAGTCCAAACCGGAGC-3′
em3	5′-GACTGCGTACGAATTGAC-3′	me3	5′-TGAGTCCAAACCGGAAT-3′
em4	5′-GACTGCGTACGAATTTGA-3′	me4	5′-TGAGTCCAAACCGGACC-3′
em5	5′-GACTGCGTACGAATTAAC-3′	me5	5′-TGAGTCCAAACCGGAAG-3′
em6	5′-GACTGCGTACGAATTGCA-3′

上游引物 Forward primers		下游引物 Reverse primers
em1	5′-GACTGCGTACGAATTAAT-3′	me1	5′-TGAGTCCAAACCGGATA-3′
em2	5′-GACTGCGTACGAATTTGC-3′	me2	5′-TGAGTCCAAACCGGAGC-3′
em3	5′-GACTGCGTACGAATTGAC-3′	me3	5′-TGAGTCCAAACCGGAAT-3′
em4	5′-GACTGCGTACGAATTTGA-3′	me4	5′-TGAGTCCAAACCGGACC-3′
em5	5′-GACTGCGTACGAATTAAC-3′	me5	5′-TGAGTCCAAACCGGAAG-3′
em6	5′-GACTGCGTACGAATTGCA-3′

引物组合 Primer combination	位点总数 Total fragments	多态位点数 Number of polymorphic loci	多态位点比例 Percentage of polymorphic loci (%)
em1-me3	43	16	37.21
em2-me2	35	12	34.29
em2-me3	38	14	36.84
em1-me4	51	26	50.98
em6-me5	32	18	56.25
em4-me1	28	15	53.57
em1-me2	33	13	39.39
总计 Total	260	114	43.85
平均 Mean	37.1	16.3	43.85

利用SRAP分析核盘菌遗传多样性

Genetic diversity in Sclerotinia sclerotiorum assessed with SRAP markers

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组 Group	份数 No.	Shannon指数 Shannon index	Simpson指数 Simpson index
寄主 Host plant
油菜 Rape	47	0.3822	0.2417
大豆 Soybean	7	0.3083	0.2034
莴苣 Lettuce	6	0.2870	0.1901
小白菜 Pakchoi	5	0.2340	0.1614
芫荽 Coriander	3	0.1898	0.1326
蚕豆 Horsebean	3	0.1731	0.1236
萝卜 Radish	2	0.1702	0.1228
地区 Geographical region
中国 China	61	0.3516	0.2239
美洲 America	11	0.3278	0.2175
欧洲 Europe	4	0.3368	0.2216
油菜生态区 Rape ecological region
春 Spring	21	0.4050	0.2628
冬 Winter	55	0.3401	0.2175
总计 Total	76	0.4002	0.2550

变异来源 Source of variation	自由度 df	均方 Mean square	方差分量 Variance component	方差分量比率 Percentage of variation (%)	P
2个油菜生态区 Two rape ecological regions
群体间 Among populations	1	108.034	3.111	18.8	<0.001
群体内 Within population	74	13.467	13.467	81.2	<0.001
7个寄主来源 Seven plant hosts
群体间 Among populations	6	26.784	0.334	2.40	0.8673
群体内 Within population	66	13.590	13.590	97.6	<0.001
3个地理来源 Three geographical regions
群体间 Among populations	2	68.315	4.363	24.8	<0.001
群体内 Within population	73	13.260	13.260	75.2	<0.001

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