利用SSR标记分析茄镰孢豌豆专化型的遗传多样性

doi:10.3724/SP.J.1003.2012.10062

生物多样性 ›› 2012, Vol. 20 ›› Issue (6): 693-702. DOI: 10.3724/SP.J.1003.2012.10062

利用SSR标记分析茄镰孢豌豆专化型的遗传多样性

向妮¹^,², 肖炎农², 段灿星¹, 王晓鸣¹, 朱振东¹^,^*()

1 中国农业科学院作物科学研究所, 农作物基因资源与基因改良国家重大科学工程, 北京 100081
2 华中农业大学植物科技学院, 武汉 430070

收稿日期:2012-03-02 接受日期:2012-07-16 出版日期:2012-11-20 发布日期:2013-01-04
通讯作者: 朱振东
作者简介:* E-mail: zhuzd115@caas.net.cn
基金资助:
现代农业产业技术体系建设专项资金(CARS-09);作物种质资源保护项目(NB2010-2130135-25-14)

Genetic diversity in Fusarium solani f. sp. pisi based on SSR markers

Ni Xiang¹^,², Yannong Xiao², Canxing Duan¹, Xiaoming Wang¹, Zhendong Zhu¹^,^*()

1 Institute of Crop Science, Chinese Academy of Agricultural Sciences/The National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing 100081
2 College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070

Received:2012-03-02 Accepted:2012-07-16 Online:2012-11-20 Published:2013-01-04
Contact: Zhendong Zhu

摘要/Abstract

摘要：

由茄镰孢豌豆专化型(Fusarium solani f. sp. pisi, Fsp)引起的根腐病是豌豆(Pisum sativum)最重要的病害之一。研究不同地理来源Fsp的遗传多样性, 对了解该菌的遗传背景及防治病害具有重要意义。本研究从血红丛赤壳交配群VI(Nectria haematococca MPVI)全基因组序列中筛选SSR位点, 选取107个SSR位点设计引物, 获得24对多态性引物。用24对多态性引物对不同地理来源的96个Fsp分离物进行遗传多样性分析, 结果表明, 24对引物共扩增出132个等位基因, 变异范围为3-15, 平均为5.5。基因多样性指数范围为0.4855-0.8264, 平均为0.7038。供试的96个Fsp分离物可分为93个基因型。聚类分析表明, 在相似性系数为0.8时, 96个Fsp分离物被划分为10个组群。Fsp分离物的地理来源或致病性与SSR聚类结果无关。分子方差分析(AMOVA)结果表明, Fsp遗传变异主要存在于群体内, 地理条件和生态区环境对Fsp遗传分化有显著影响。

关键词: 豌豆根腐病, 豌豆, Fusarium solani f. sp. pisi, SSR标记, 遗传变异

Abstract

Pea root rot, caused by Fusarium solani f. sp. pisi (Fsp), is one of the most important diseases on pea (Pisum sativum). Assessing the genetic diversity of the pathogen isolates from different geographical regions is crucially important for understanding of the genetic background of this pathogen and intelligently deploying host resistance. We screened SSRs in complete genome sequence of Nectria haematococca MPVI, and 107 SSR loci were selected for designing markers, from which 24 polymorphic primer pairs were developed. The 24 primer pairs were used to assess genetic diversity of 96 Fsp isolates from different geographical regions. Among 24 SSR markers, a total of 132 alleles were detected among the 96 Fsp isolates, the number of alleles for each of the loci ranged from 3 to 15 with an average of 5.5. The genetic diversity was estimated to range from 0.4855 to 0.8264 with the average value of 0.738. Using these markers, 93 genotypes were detected. When the genetic similarity coefficient was 0.8, 96 Fsp isolates were clustered into 10 groups by phylogenetic analysis. There was no correlation between SSR profile and either geographic origin or pathogenicity. Analysis of AMOVA revealed that variation mainly presented within Fsp populations (86.14%), and genetic differentiation of Fsp was significantly affected by geographical conditions and ecological environment.

Key words: pea root rot, Pisum sativum, Fusarium solani f. sp. pisi, SSR marker, genetic variation

向妮, 肖炎农, 段灿星, 王晓鸣, 朱振东 (2012) 利用SSR标记分析茄镰孢豌豆专化型的遗传多样性. 生物多样性, 20, 693-702. DOI: 10.3724/SP.J.1003.2012.10062.

Ni Xiang, Yannong Xiao, Canxing Duan, Xiaoming Wang, Zhendong Zhu (2012) Genetic diversity in Fusarium solani f. sp. pisi based on SSR markers. Biodiversity Science, 20, 693-702. DOI: 10.3724/SP.J.1003.2012.10062.

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链接本文: https://www.biodiversity-science.net/CN/10.3724/SP.J.1003.2012.10062

https://www.biodiversity-science.net/CN/Y2012/V20/I6/693

图/表 7

参考文献 41

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来源 Location	数量 Amount	分离物 Isolates
安徽 Anhui	17	AH-1^a、AH-2、AH-3、AH-4、AH-5、AH-6、AH-7、AH-8、AH-9、AH-10、AH-11、AH-12、AH-13、AH-14^b、AH-15^a、AH-16、AH-17
北京 Beijing	13	BJ-1、BJ-2、BJ-3、BJ-4、BJ-5、BJ-6、BJ-7、BJ-8、BJ-9、BJ-10、BJ-11、BJ-12、BJ-13
福建 Fujian	4	FJ-1^b、FJ-2^a、FJ-3^b、FJ-4^a
甘肃 Gansu	20	LZ-1、LZ-2、LZ-3^b、LZ-4、LZ-5、LZ-6、DX-1、DX-2、DX-3、DX-4^a、DX-5、DX-6、DX-7、DX-8、DX-9、DX-10、DX-11、DX-12、DX-13、DX-14
河北 Hebei	10	ZB-1、ZB-2、ZB-3、ZB-4、ZB-5、ZB-6、ZB-7、ZB-8、ZB-9、ZB-10
内蒙古 Inner Mongolia	7	NM-1、NM-2、NM-3、NM-4^b、NM-5、NM-6^b、NM-7
吉林 Jilin	1	BC-1^a
宁夏 Ningxia	12	SB-1、SB-2、SB-3、SB-4、SB-5、SB-6、SB-7、SB-8、XB-1、XB-2、PC-1、PC-2
青海 Qinghai	5	QH-1、QH-2、QH-3^b、QH-4^b、QH-5^b
四川 Sichuan	3	SC-1、SC-2、SC-3
云南 Yunnan	4	YN-1^a、YN-2^a、YN-3、YN-4

来源 Location	数量 Amount	分离物 Isolates
安徽 Anhui	17	AH-1^a、AH-2、AH-3、AH-4、AH-5、AH-6、AH-7、AH-8、AH-9、AH-10、AH-11、AH-12、AH-13、AH-14^b、AH-15^a、AH-16、AH-17
北京 Beijing	13	BJ-1、BJ-2、BJ-3、BJ-4、BJ-5、BJ-6、BJ-7、BJ-8、BJ-9、BJ-10、BJ-11、BJ-12、BJ-13
福建 Fujian	4	FJ-1^b、FJ-2^a、FJ-3^b、FJ-4^a
甘肃 Gansu	20	LZ-1、LZ-2、LZ-3^b、LZ-4、LZ-5、LZ-6、DX-1、DX-2、DX-3、DX-4^a、DX-5、DX-6、DX-7、DX-8、DX-9、DX-10、DX-11、DX-12、DX-13、DX-14
河北 Hebei	10	ZB-1、ZB-2、ZB-3、ZB-4、ZB-5、ZB-6、ZB-7、ZB-8、ZB-9、ZB-10
内蒙古 Inner Mongolia	7	NM-1、NM-2、NM-3、NM-4^b、NM-5、NM-6^b、NM-7
吉林 Jilin	1	BC-1^a
宁夏 Ningxia	12	SB-1、SB-2、SB-3、SB-4、SB-5、SB-6、SB-7、SB-8、XB-1、XB-2、PC-1、PC-2
青海 Qinghai	5	QH-1、QH-2、QH-3^b、QH-4^b、QH-5^b
四川 Sichuan	3	SC-1、SC-2、SC-3
云南 Yunnan	4	YN-1^a、YN-2^a、YN-3、YN-4

位点^a Locus^a	引物序列 Primer sequence(5'-3')	重复基元 Repeat motif	基因组位置^b Location in the genome ^b	期望大小 Expected size (bp)	片段大小 Allele size range (bp)	退火温度 Annealing tem- perature (℃)
Fsp1.7	F: TGTTCCCATAGTCCGATTCCG	(AC)₁₇	Contig 1: 3457867-3458072	206	180-220	54
	R: GCCGCTTGACTTGCTAACCA
Fsp1.10	F: CGATTCGTTCTTCCCAACACC	(TCT)₁₄	Contig 1/: 709046-709225	180	147-242	54
	R: AAACCTGCCTCAAGCCCTGA
Fsp1.17	F: AATCCCATTGTTTCGGTGCG	(GT)₂₁	Contig 1/: 245108-245324	217	165-217	54
	R: CCGTTGAGGAATAGAGAGGCA
Fsp2.2	F: GCTTTGGGTCCAGGTATTACG	(AC)₁₁	Contig 15/: 2408632-2409004	373	600-622	54
	R: TTCTCTCTCTCCCGTCACCA
Fsp2.3	F: TGCTCCTACACCTCCGTCTC	(TG)₁₂	Contig 15: 1878221-1878425	205	160-209	54
	R: CACGTTTTATCTGCCTTGCTG
Fsp2.4	F: GATGATTGTGCTGGTAGCCTG	(TTTC)₈	Contig 15: 802092-802323	232	195-238	54
	R: CGCTTGACGCCATAATTGAG
Fsp3.1	F: AACAAGGAGGGTGACGGGAT	(AC)₁₃	Contig 2/: 1062296-1062559	264	210-290	54
	R: AGTGACAAATAGGGAGGTGGG
Fsp3.6	F: CTTGCCCAAACGCATTGAAG	(TG)₁₆	Contig 2/: 2066491-2066782	292	260-320	54
	R: ACTAACACACACCCAGCCTC
Fsp3.7	F: CCATTTCCATTCCACCTCACC	(AGC)₉	Contig 2/: 1170545-1170752	208	180-217	52
	R: CAGTCTGCTGTTATGAAGGGC
Fsp4.2	F: TTGACCTCTGCACACCATCAG	(AC)₁₅	Contig 3/: 2330520-2330703	184	160-200	54
	R: CTCACTCTCGGCTCTCGTTAT
Fsp4.4	F: CGGTCACAGGCAAAGCAAT	(TG)₁₅	Contig 3/: 2288923-2289149	227	190-250	54
	R: GGGATAGAGCAAGCAGCGTT
Fsp4.7	F: ACACACACACAGGCACAAG	(AC)₁₀	Contig 4/: 1510008-1510245	238	201-260	54
	R: TGAGTAAGGTAGGCTGGGTCT
Fsp5.3	F: TACACTCCCGTTCCTGGCTA	(TC)₁₅	Contig 4/: 161679-161833	155	90-147	54
	R: CCAAATCTCAACCGTCGTGG
Fsp5.4	F: TGAATCTGACTGTGGCTGGAG	(TG)11	Contig 4/: 330399-330579	181	160-210	54
	R: ATGGTTCGCCTTGGCTCTGA
Fsp5.8	F: CAGTTAGGTTCCACTCCAGGT	(ACT)₁₄	Contig 4/: 216245-216574	330	260-375	54
	R: TGGGCGGTCATTGTGTAGGT
Fsp6.3	F: AGCACGAGGAACGAGTCTA	(AC)₁₅	Contig 18/: 1864740-1864874	135	110-150	52
	R: CGCTACTTGTTTGTGTCCG
Fsp6.7	F: TGACCGAGAACTGAAGCCA	(TTC)₉	Contig 18/: 2280272-2280530	259	250-350	52
	R: GACGACGAAACCTTTGAAGAG
Fsp7.3	F: CTCTCATTATGTAGCGACGAC	(TCG)₁₀	Contig 7/: 808299-808470	173	150-250	52
	R: TCGATACCCCTGAGTTCTGTG
Fsp9.6	F: GCATCCAATGTCTTTCCCAAT	(TG)₁₇	Contig 5/: 189772--190021	250	230-275	52
	R: GACTCGATATTCATCAACACCA
Fsp9.8	F: TTGGTTTCCCTGCCTTGT	(TG)₂₀	Contig 5/: 1802083-1802348	266	240-300	52
	R: TGTTGGGTCATCTTGGTTTC
Fsp9.9	F: ACGCCACTCTTCCATACTCAG	(AAC)₁₅	Contig 5/: 764399-764632	234	200-280	54
	R: GTTGACGGTCTTGTGGCATAC
Fsp9.10	F: TCAACAACACCAACCACCTG	(AGC)₉	Contig 5/: 509965-510211	247	200-300	54
	R: TTCGTCCGAGTCGCCTTCTA
Fsp9.15	F: GCTTGTGCTGGAGTTGACCT	(GAAT)₈	Contig 5/: 168328-168492	165	123-155	52
	R: CAGGAGCATAGGAGGAATAC
Fsp10.2	F: CGAGAAAGGTGAGGAAGGGA	(GGA)₈	Contig 11/: 258852-259033	182	175-200	52
	R: TATCTGTGAGGTGTGGCGA

位点^a Locus^a	引物序列 Primer sequence(5'-3')	重复基元 Repeat motif	基因组位置^b Location in the genome ^b	期望大小 Expected size (bp)	片段大小 Allele size range (bp)	退火温度 Annealing tem- perature (℃)
Fsp1.7	F: TGTTCCCATAGTCCGATTCCG	(AC)₁₇	Contig 1: 3457867-3458072	206	180-220	54
	R: GCCGCTTGACTTGCTAACCA
Fsp1.10	F: CGATTCGTTCTTCCCAACACC	(TCT)₁₄	Contig 1/: 709046-709225	180	147-242	54
	R: AAACCTGCCTCAAGCCCTGA
Fsp1.17	F: AATCCCATTGTTTCGGTGCG	(GT)₂₁	Contig 1/: 245108-245324	217	165-217	54
	R: CCGTTGAGGAATAGAGAGGCA
Fsp2.2	F: GCTTTGGGTCCAGGTATTACG	(AC)₁₁	Contig 15/: 2408632-2409004	373	600-622	54
	R: TTCTCTCTCTCCCGTCACCA
Fsp2.3	F: TGCTCCTACACCTCCGTCTC	(TG)₁₂	Contig 15: 1878221-1878425	205	160-209	54
	R: CACGTTTTATCTGCCTTGCTG
Fsp2.4	F: GATGATTGTGCTGGTAGCCTG	(TTTC)₈	Contig 15: 802092-802323	232	195-238	54
	R: CGCTTGACGCCATAATTGAG
Fsp3.1	F: AACAAGGAGGGTGACGGGAT	(AC)₁₃	Contig 2/: 1062296-1062559	264	210-290	54
	R: AGTGACAAATAGGGAGGTGGG
Fsp3.6	F: CTTGCCCAAACGCATTGAAG	(TG)₁₆	Contig 2/: 2066491-2066782	292	260-320	54
	R: ACTAACACACACCCAGCCTC
Fsp3.7	F: CCATTTCCATTCCACCTCACC	(AGC)₉	Contig 2/: 1170545-1170752	208	180-217	52
	R: CAGTCTGCTGTTATGAAGGGC
Fsp4.2	F: TTGACCTCTGCACACCATCAG	(AC)₁₅	Contig 3/: 2330520-2330703	184	160-200	54
	R: CTCACTCTCGGCTCTCGTTAT
Fsp4.4	F: CGGTCACAGGCAAAGCAAT	(TG)₁₅	Contig 3/: 2288923-2289149	227	190-250	54
	R: GGGATAGAGCAAGCAGCGTT
Fsp4.7	F: ACACACACACAGGCACAAG	(AC)₁₀	Contig 4/: 1510008-1510245	238	201-260	54
	R: TGAGTAAGGTAGGCTGGGTCT
Fsp5.3	F: TACACTCCCGTTCCTGGCTA	(TC)₁₅	Contig 4/: 161679-161833	155	90-147	54
	R: CCAAATCTCAACCGTCGTGG
Fsp5.4	F: TGAATCTGACTGTGGCTGGAG	(TG)11	Contig 4/: 330399-330579	181	160-210	54
	R: ATGGTTCGCCTTGGCTCTGA
Fsp5.8	F: CAGTTAGGTTCCACTCCAGGT	(ACT)₁₄	Contig 4/: 216245-216574	330	260-375	54
	R: TGGGCGGTCATTGTGTAGGT
Fsp6.3	F: AGCACGAGGAACGAGTCTA	(AC)₁₅	Contig 18/: 1864740-1864874	135	110-150	52
	R: CGCTACTTGTTTGTGTCCG
Fsp6.7	F: TGACCGAGAACTGAAGCCA	(TTC)₉	Contig 18/: 2280272-2280530	259	250-350	52
	R: GACGACGAAACCTTTGAAGAG
Fsp7.3	F: CTCTCATTATGTAGCGACGAC	(TCG)₁₀	Contig 7/: 808299-808470	173	150-250	52
	R: TCGATACCCCTGAGTTCTGTG
Fsp9.6	F: GCATCCAATGTCTTTCCCAAT	(TG)₁₇	Contig 5/: 189772--190021	250	230-275	52
	R: GACTCGATATTCATCAACACCA
Fsp9.8	F: TTGGTTTCCCTGCCTTGT	(TG)₂₀	Contig 5/: 1802083-1802348	266	240-300	52
	R: TGTTGGGTCATCTTGGTTTC
Fsp9.9	F: ACGCCACTCTTCCATACTCAG	(AAC)₁₅	Contig 5/: 764399-764632	234	200-280	54
	R: GTTGACGGTCTTGTGGCATAC
Fsp9.10	F: TCAACAACACCAACCACCTG	(AGC)₉	Contig 5/: 509965-510211	247	200-300	54
	R: TTCGTCCGAGTCGCCTTCTA
Fsp9.15	F: GCTTGTGCTGGAGTTGACCT	(GAAT)₈	Contig 5/: 168328-168492	165	123-155	52
	R: CAGGAGCATAGGAGGAATAC
Fsp10.2	F: CGAGAAAGGTGAGGAAGGGA	(GGA)₈	Contig 11/: 258852-259033	182	175-200	52
	R: TATCTGTGAGGTGTGGCGA

SSR位点 SSR locus	观测等位基因数 Observed number of alleles (Na)	有效等位基因数 Effective number of alleles (Ne)	基因多样性指数 Gene diversity index (H)	Shannon指数 Shannon index (I)
Fsp1.7	5	2.8447	0.6485	1.2162
Fsp1.10	15	4.0502	0.7531	1.9836
Fsp1.17	5	3.6560	0.7265	1.4019
Fsp2.2	6	4.8578	0.7941	1.6700
Fsp2.3	5	3.3457	0.7011	1.3637
Fsp2.4	5	2.1525	0.5354	0.9979
Fsp3.1	4	2.3027	0.5657	1.0762
Fsp3.6	3	2.7466	0.6359	1.0521
Fsp3.7	5	4.4211	0.7738	1.5415
Fsp4.2	4	1.9438	0.4855	0.8826
Fsp4.4	4	3.5333	0.7170	1.3187
Fsp4.7	4	2.5645	0.6101	1.0413
Fsp5.3	6	4.3986	0.7727	1.5779
Fsp5.4	5	4.2793	0.7663	1.5211
Fsp5.8	6	5.1658	0.8064	1.7170
Fsp6.3	4	2.5908	0.6140	1.1040
Fsp6.7	7	5.2721	0.8103	1.7429
Fsp7.3	5	2.7909	0.6417	1.2545
Fsp9.6	5	4.0180	0.7511	1.4874
Fsp9.8	4	3.1232	0.6798	1.2192
Fsp9.9	8	5.7601	0.8264	1.8905
Fsp9.10	7	5.3629	0.8135	1.7745
Fsp9.15	4	3.1419	0.6817	1.2351
Fsp10.2	6	4.5338	0.7794	1.6062
平均 Mean	5.5	3.7023	0.7038	1.4031

利用SSR标记分析茄镰孢豌豆专化型的遗传多样性

Genetic diversity in Fusarium solani f. sp. pisi based on SSR markers

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群体 Population	菌株数 No. of isolates	观测等位基因数 Observed number of alleles (Na)	有效等位基因数 Effective number of alleles (Ne)	基因多样性指数 Gene diversity Index (H)	Shannon指数 Shannon index (I)
河北 Hebei	10	2.8750	2.5377	0.5925	0.9599
宁夏 Ningxia	12	3.3750	2.5005	0.5593	0.9905
甘肃 Gansu	20	3.9583	2.9460	0.6353	1.1590
安徽 Anhui	17	3.8750	2.7750	0.6040	1.1058
北京 Beijing	13	2.8750	2.2937	0.5262	0.8820
内蒙古 Inner Mongolia	7	2.1250	1.7474	0.4007	0.6140
青海 Qinghai	5	1.5000	1.3867	0.2144	0.3091

变异来源 Source of variation	自由度 df	平方和 Sum of squares	方差分量 Variance component	变异率 Percentage of variation (%)	F_ST	P
群体间 Among populations	6	134.733	1.25595	13.86	0.13865	<0.001
群体内 Within population	77	600.814	7.80278	86.14	-	<0.001
合计 Total	83	735.548	9.05873	100	-	-

群体 Population	河北 Hebei	宁夏 Ningxia	甘肃 Gansu	安徽 Anhui	北京 Beijing	内蒙古 Inner Mongolia	青海Qinghai
河北 Heibei	****	0.6780	0.7550	0.6943	0.5416	0.5859	0.5435
宁夏 Ningxia	0.3886	****	0.8536	0.6432	0.4899	0.4528	0.6226
甘肃 Gansu	0.2810	0.1583	****	0.7515	0.5628	0.5260	0.5931
安徽 Anhui	0.3648	0.4413	0.2857	****	0.5917	0.5285	0.4808
北京 Beijing	0.6133	0.7135	0.5748	0.5248	****	0.6442	0.4073
内蒙古 Inner Mongolia	0.5346	0.7922	0.6425	0.6377	0.4398	****	0.4802
青海 Qinghai	0.6097	0.4738	0.5224	0.7324	0.8983	0.7336	****

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