基于大球盖菇基因组的SSR分子标记开发及指纹数据库应用

doi:10.3969/j.issn.1004-1524.20221149

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (1): 84-93.DOI: 10.3969/j.issn.1004-1524.20221149

基于大球盖菇基因组的SSR分子标记开发及指纹数据库应用

李雪松¹^,²(), 孙达锋¹^,², 刘绍雄², 张俊波¹, 岳万松¹, 李建英¹, 华蓉²^,^*()

1.中华全国供销合作总社昆明食用菌研究所,云南昆明 650221
2.云南省食用菌产业发展研究院,云南昆明, 650221

收稿日期:2022-08-05 出版日期:2024-01-25 发布日期:2024-02-18
作者简介:李雪松(1993—),男,云南峨山人,硕士,助理研究员,主要从事食药用菌驯化、遗传育种研究。E-mail: lixuesong1@qq.com
通讯作者: * 华蓉,E-mail: 626300629@qq.com
基金资助:
云南省科技人才与平台计划(202205AD160042);云南省乡村振兴科技专项(202204BP090012);云南省重大科技专项计划(202002AE320003)

Development of SSR molecular markers and application of fingerprint database based on whole genome of Stropharia rugosoannulata

LI Xuesong¹^,²(), SUN Dafeng¹^,², LIU Shaoxiong², ZHANG Junbo¹, YUE Wansong¹, LI Jianying¹, HUA Rong²^,^*()

1. Kunming Edible Fungi Institute of All China Federation of Supply and Marketing Cooperatives, Kunming 650221, China
2. Yunnan Edible Fungi Industry Development Research Institute, Kunming 650221, China

Received:2022-08-05 Online:2024-01-25 Published:2024-02-18

摘要/Abstract

摘要：

以13个大球盖菇菌株及其已公布的基因组为试材,采用生物信息学和试验验证相结合的方法,研究了大球盖菇基因组上的SSR类型及分布,设计了100对SSR引物开展试验验证,以期为大球盖菇的种质资源鉴定、品种选育、分子标记辅助育种等研究提供参考依据。结果表明:总计检索到6种SSR类型的2 124条序列,其中主要以三核苷酸类型为主,共计704,占SSR总数的33.15%;五、六碱基重复类型较多,但总和只占全部SSR数量的14.59%。由验证试验获得16对引物,其中引物DQGSSR020、DQGSSR031、DQGSSR077的多态性较高、特异性好、无杂峰、荧光亮度高、区分度好。同时,基于16对SSR引物进行大球盖菇DNA指纹编码构建,获得了13个大球盖菇菌株的32位数分子指纹数据库编码。

关键词: 大球盖菇, 基因组, SSR分子标记开发

Abstract:

Based on experimental validation and bioinformatics methods, SSR analysis result were exploited from the genomic of Stropharia rugosoannulata and 13 strains. In the meantime, 100 SSR were selected for primer design and experimental verification. In order to provide a theoretical basis for the germplasm resources identification, variety selection and molecular marker-assisted breeding of S. rugosoannulata. The results showed that a total of 2 124 sequences of 6 SSR types were retrieved. The main type of trinucleotide (P3) was 704, accounting for 33.15% of the total SSR number. Pentanucleotide and hexanucleotide repeat types were more, the total number accounted for 14.59% of the total SSR number. 16 pairs of primers were obtained by verification test, three primers with high pair property, repeatability and specificity were obtained from the validation experiment, which were DQGSSR020, DQGSSR031 and DQGSSR077. At the same time, the DNA fingerprint coding of 16 pairs of primers was constructed and the 32-digit molecular fingerprint database codes of 13 S. rugosoannulata strains were obtained.

Key words: Stropharia rugosoannulata, genome, SSR molecular markers development

中图分类号:

S646

李雪松, 孙达锋, 刘绍雄, 张俊波, 岳万松, 李建英, 华蓉. 基于大球盖菇基因组的SSR分子标记开发及指纹数据库应用[J]. 浙江农业学报, 2024, 36(1): 84-93.

LI Xuesong, SUN Dafeng, LIU Shaoxiong, ZHANG Junbo, YUE Wansong, LI Jianying, HUA Rong. Development of SSR molecular markers and application of fingerprint database based on whole genome of Stropharia rugosoannulata[J]. Acta Agriculturae Zhejiangensis, 2024, 36(1): 84-93.

图/表 8

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标记 Marker	上游引物 Forward primer (5'→3')	下游引物 Reverse primer (5'→3')	退火温度 Tm/℃	SSR类型 SSR type	SSR基元 SSR motif
DQGSSR006	GAGACGGATCGGACAGATGG	GACAATGGTAATGACACGGTGG	60	P4	(ACTA)4
DQGSSR007	CTAAGCATCGTCCAGCTCCC	TCATTCAGGACCAACAGAGCC	60	P3	(TGC)6
DQGSSR020	AGCTAGCGCGGATATTAGGG	TCTCGTTGCATCTCGTCTCG	60	P4	(GGTA)4
DQGSSR029	ATGAATAAGCGCTCGCAAGC	GAGCCAGGAAGCAAGGAACG	61	P3	(CGC)5
DQGSSR031	GCTAATGGCGCTTTGTAATTGC	CGCCATGGAACGTTGAACC	60	P4	(CACA)5
DQGSSR034	CAAAGTCAAGTGCGCATGCC	TCTTTGCAGACTTGGGTGGG	61	P3	(CAG)5
DQGSSR035	GTTTACTGGCCAGGGAAAGC	ACCTGAACAAGTCGACTGCG	60	P2	(AT)7
DQGSSR037	CCCGCTTTCTTCTGTTGTCC	GTGTCGCAAGGTTCAGTTCG	60	P4	(GAAT)5
DQGSSR039	GGTAGGTCCATTAGCCGGC	TACTCAAGTGCACCACCACC	60	P3	(GGT)5
DQGSSR052	TAAGATGGCTGTGGTGACGG	TAATCTGGTGCCTGTCGTGC	60	P3	(GGT)5
DQGSSR063	TGAGGAGAGAGAGAGCGAGC	CTCTTTGTCCAGCCTCGTCC	60	P2	(GA)15
DQGSSR065	GCACATACGCTGCATACTGC	GAGGAGGCATCATCAGCAGG	60	P3	(TGC)8
DQGSSR077	AGTGCTTTGATGCCTCCTCC	CTATTGCTGCTGTGACTGCG	60	P3	(GCA)9
DQGSSR081	ACTAATGTCGGGACCCATTCC	CAGAGTACTCAGCCGAACCC	60	P3	(GCT)8
DQGSSR088	ACCGTCATCGACTGCATCC	GGAGGCTCTTGTCCTTGTCC	60	P3	(CAG)6
DQGSSR097	ACAGTGCACACCTTAGGACG	GACAAGAGTCCTTCCTCCGG	60	P2	(AT)7

标记 Marker	上游引物 Forward primer (5'→3')	下游引物 Reverse primer (5'→3')	退火温度 Tm/℃	SSR类型 SSR type	SSR基元 SSR motif
DQGSSR006	GAGACGGATCGGACAGATGG	GACAATGGTAATGACACGGTGG	60	P4	(ACTA)4
DQGSSR007	CTAAGCATCGTCCAGCTCCC	TCATTCAGGACCAACAGAGCC	60	P3	(TGC)6
DQGSSR020	AGCTAGCGCGGATATTAGGG	TCTCGTTGCATCTCGTCTCG	60	P4	(GGTA)4
DQGSSR029	ATGAATAAGCGCTCGCAAGC	GAGCCAGGAAGCAAGGAACG	61	P3	(CGC)5
DQGSSR031	GCTAATGGCGCTTTGTAATTGC	CGCCATGGAACGTTGAACC	60	P4	(CACA)5
DQGSSR034	CAAAGTCAAGTGCGCATGCC	TCTTTGCAGACTTGGGTGGG	61	P3	(CAG)5
DQGSSR035	GTTTACTGGCCAGGGAAAGC	ACCTGAACAAGTCGACTGCG	60	P2	(AT)7
DQGSSR037	CCCGCTTTCTTCTGTTGTCC	GTGTCGCAAGGTTCAGTTCG	60	P4	(GAAT)5
DQGSSR039	GGTAGGTCCATTAGCCGGC	TACTCAAGTGCACCACCACC	60	P3	(GGT)5
DQGSSR052	TAAGATGGCTGTGGTGACGG	TAATCTGGTGCCTGTCGTGC	60	P3	(GGT)5
DQGSSR063	TGAGGAGAGAGAGAGCGAGC	CTCTTTGTCCAGCCTCGTCC	60	P2	(GA)15
DQGSSR065	GCACATACGCTGCATACTGC	GAGGAGGCATCATCAGCAGG	60	P3	(TGC)8
DQGSSR077	AGTGCTTTGATGCCTCCTCC	CTATTGCTGCTGTGACTGCG	60	P3	(GCA)9
DQGSSR081	ACTAATGTCGGGACCCATTCC	CAGAGTACTCAGCCGAACCC	60	P3	(GCT)8
DQGSSR088	ACCGTCATCGACTGCATCC	GGAGGCTCTTGTCCTTGTCC	60	P3	(CAG)6
DQGSSR097	ACAGTGCACACCTTAGGACG	GACAAGAGTCCTTCCTCCGG	60	P2	(AT)7

标记 Marker	荧光标记 Fluorescence labeling	扩增片段数 Fragment number	片段大小 Fragment size/bp
DQGSSR006	FAM	2	130 131
DQGSSR007	FAM	2	103 127
DQGSSR020	FAM	4	121 122 138 139
DQGSSR029	HEX	2	198 200
DQGSSR031	HEX	5	136 137 139 140 142
DQGSSR034	HEX	3	113 116 117
DQGSSR035	HEX	3	161 162 169
DQGSSR037	HEX	2	212 218
DQGSSR039	HEX	2	147 150
DQGSSR052	ROX	3	139 140 141
DQGSSR063	ROX	2	186 198
DQGSSR065	ROX	2	167 170
DQGSSR077	TAMRA	4	194 196 203 205
DQGSSR081	TAMRA	2	174 177
DQGSSR088	TAMRA	3	187 193 194
DQGSSR097	FAM	2	151 153

标记 Marker	荧光标记 Fluorescence labeling	扩增片段数 Fragment number	片段大小 Fragment size/bp
DQGSSR006	FAM	2	130 131
DQGSSR007	FAM	2	103 127
DQGSSR020	FAM	4	121 122 138 139
DQGSSR029	HEX	2	198 200
DQGSSR031	HEX	5	136 137 139 140 142
DQGSSR034	HEX	3	113 116 117
DQGSSR035	HEX	3	161 162 169
DQGSSR037	HEX	2	212 218
DQGSSR039	HEX	2	147 150
DQGSSR052	ROX	3	139 140 141
DQGSSR063	ROX	2	186 198
DQGSSR065	ROX	2	167 170
DQGSSR077	TAMRA	4	194 196 203 205
DQGSSR081	TAMRA	2	174 177
DQGSSR088	TAMRA	3	187 193 194
DQGSSR097	FAM	2	151 153

标记 Marker	观测等位基因 Observed number of alleles	有效等位基因 Effect number of alleles	香农指数 Shannon’s information index	多态信息指数 Polymorphism information content	观测杂合度 Observed heterozygosities	期望杂合度 Expected heterozygosities	哈迪温伯格平衡 Hardy- Weinberg’s equilibrium
DQGSSR006	2	1.17	0.27	0.13	0	0.15	0.000 001^***
DQGSSR007	2	1.99	0.69	0.37	0.92	0.52	0.003 176^**
DQGSSR020	4	3.80	1.36	0.69	1.00	0.77	0.000 002^***
DQGSSR029	5	2.52	1.10	0.52	1.00	0.63	0.000 057^***
DQGSSR031	3	2.50	1.00	0.53	0.92	0.62	0.033 507^*
DQGSSR034	3	2.09	0.86	0.44	0	0.54	<0.000 001^***
DQGSSR035	2	1.99	0.69	0.37	0.92	0.52	0.003 176^**
DQGSSR037	2	1.99	0.69	0.37	0.92	0.52	0.003 176^**
DQGSSR039	2	1.99	0.69	0.37	0.92	0.52	0.003 176^**
DQGSSR052	2	1.99	0.69	0.37	0.92	0.52	0.003 176^**
DQGSSR063	2	1.99	0.69	0.37	0.92	0.52	0.003 176^**
DQGSSR065	2	1.17	0.27	0.13	0	0.15	0.000 001^***
DQGSSR077	2	1.99	0.69	0.37	0.92	0.52	0.003 176^**
DQGSSR081	2	1.74	0.62	0.34	0	0.44	0.000 139^***
DQGSSR088	3	2.32	0.96	0.50	0	0.59	0.000 002^***
DQGSSR097	4	3.48	1.31	0.66	1.00	0.74	0.000 002^***
平均值Average value	2.63	2.17	0.79	0.41	0.65	0.52	0.003 496^**
标准差	0.96	0.69	0.31	—	0.45	0.17	—
Standard deviation

基于大球盖菇基因组的SSR分子标记开发及指纹数据库应用

Development of SSR molecular markers and application of fingerprint database based on whole genome of Stropharia rugosoannulata

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引物 Primer	赋值Assignment
引物 Primer	0	1	2	3	4	5
DQGSSR006	—	130	131	—	—	—
DQGSSR007	—	103	127	—	—	—
DQGSSR020	—	121	122	138	139	—
DQGSSR029	—	198	200	—	—	—
DQGSSR031	—	136	137	139	140	142
DQGSSR034	—	113	116	117	—	—
DQGSSR035	—	161	162	169	—	—
DQGSSR037	—	212	218	—	—	—
DQGSSR039	—	147	150	—	—	—
DQGSSR052	—	139	140	141	—	—
DQGSSR063	—	186	198	—	—	—
DQGSSR065	—	167	170	—	—	—
DQGSSR077	—	194	196	203	205	—
DQGSSR081	—	174	177	—	—	—
DQGSSR088	—	187	193	194	—	—
DQGSSR097	—	151	153	—	—	—

编号 Code	引物Primer																指纹编号 Fingerprint code
编号 Code	DQGS- SR006	DQGS- SR007	DQGS- SR020	DQGS- SR029	DQGS- SR031	DQGS- SR034	DQGS- SR035	DQGS- SR037	DQGS- SR039	DQGS- SR052	DQGS- SR063	DQGS- SR065	DQGS- SR077	DQGS- SR081	DQGS- SR088	DQGS- SR097	指纹编号 Fingerprint code
DQG1	02	12	24	01	24	13	02	12	12	03	12	12	13	12	03	12	02122401241302121-
																	203121213120312
DQG2	02	12	24	02	24	13	02	12	12	03	12	12	24	12	03	12	02122402241302121-
																	203121224120312
DQG3	02	12	24	02	24	13	02	12	12	02	12	12	24	12	03	12	02122402241302121-
																	202121224120312
DQG4	02	12	24	01	14	13	02	12	12	01	12	12	13	12	03	12	02122401141302121-
																	201121213120312
DQG5	02	12	24	01	24	13	01	12	12	00	12	12	13	12	03	12	02122401241301121-
																	200121213120312
DQG6	02	12	24	02	24	13	01	12	12	02	12	12	24	12	03	12	02122402241301121-
																	202121224120312
DQG7	02	12	24	02	24	13	01	12	12	02	12	12	24	02	03	12	02122402241301121-
																	202121224020312
DQG8	02	12	24	01	24	13	01	12	12	01	12	12	13	12	03	12	02122401241301121-
																	201121213120312
DQG9	02	12	13	01	24	12	02	12	12	02	12	12	13	12	02	12	02121301241202121-
																	202121213120212
DQG10	02	12	13	01	24	12	02	12	12	02	12	12	13	12	01	12	02121301241202121-
																	202121213120112
DQG11	02	12	13	01	24	12	02	12	12	03	12	12	13	12	02	12	02121301241202121-
																	203121213120212
DQG12	02	12	13	01	24	12	01	12	12	02	12	12	13	12	02	12	02121301241201121-
																	202121213120212
ZJJQG001	01	02	13	01	35	01	03	01	02	02	01	01	13	02	01	01	01021301350103010-
																	202010113020101

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