基于转录组序列的叶用芥菜奶奶青菜EST-SSR标记开发与遗传多样性分析

doi:10.3969/j.issn.1004-1524.2021.09.08

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (9): 1640-1649.DOI: 10.3969/j.issn.1004-1524.2021.09.08

基于转录组序列的叶用芥菜奶奶青菜EST-SSR标记开发与遗传多样性分析

马杰¹^,²(), 屈雯², 陈春艳¹, 王磊², 马俊¹, 刘针杉², 马维¹, 周平¹, 何远宽¹, 孙勃²^,^*()

1.毕节市农业科学研究所,贵州毕节 551700
2.四川农业大学园艺学院,四川成都 611130

收稿日期:2020-11-05 出版日期:2021-09-25 发布日期:2021-10-09
通讯作者: 孙勃
作者简介:* 孙勃,E-mail: bsun@sicau.edu.cn
马杰(1981—),男,山西大同人,硕士,高级农艺师,研究方向为芥菜种质资源与育种研究。E-mail: 115705717@qq.com
基金资助:
贵州省科技厅科技支撑计划(黔科合支撑〔2018〕2372-1);贵州省科技厅科技支撑计划(黔科合支撑〔2018〕2372-2);贵州省人才办项目(RCJD2020-7);贵州省科技厅高层次创新型人才项目(毕科人才合字〔2021〕02号)

Development of SSR markers based on transcriptome sequencing and genetic diversity analysis of Nainaiqingcai leaf mustard

MA Jie¹^,²(), QU Wen², CHEN Chunyan¹, WANG Lei², MA Jun¹, LIU Zhenshan², MA Wei¹, ZHOU Ping¹, HE Yuankuan¹, SUN Bo²^,^*()

1. Bijie Institute of Agricultural Science, Bijie 551700, China
2. College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China

Received:2020-11-05 Online:2021-09-25 Published:2021-10-09
Contact: SUN Bo

摘要/Abstract

摘要：

为了探究叶用芥菜奶奶青菜资源的多样性水平,基于转录组数据研究了奶奶青菜简单重复序列标记(EST-SSR)特征,筛选了适合奶奶青菜的EST-SSR引物,并分析了奶奶青菜的遗传多样性。转录组测序分析共获得46 386条非冗余基因(unigene),其中13 544条unigene序列中含有18 720个简单重复序列(simple sequence repeats, SSR)位点,SSR的发生频率为29.20%,平均每2.73 kb出现1个SSR,分布频率为40.36%。优势重复序列为单核苷酸,占总SSR数量的49.39%,其次为三核苷酸和二核苷酸,分别占总SSR数量的25.44%和24.13%。AT、AGCT和AAG/CTT分别是单核苷酸、二核苷酸以及三核苷酸的优势重复基元。以30份奶奶青菜和5份其他芸薹属蔬菜为材料,从37对引物中筛选出17对多态性引物,扩增得到135条多态性条带,多态性比例达88.2%。遗传多样性分析结果显示,平均等位基因数(N_a)为5.705 9,平均有效等位基因数(N_e)为2.397 8,平均多样性指数(I)为1.036 1,平均观察杂合度(H_o)为0.312 1,平均期望杂合度(H_e)为0.530 0,平均Nei’s期望杂合度为0.521 9,表明筛选出的17对SSR引物具有较好的遗传多样性。通过非加权组平均法(unweighted pair-group method with arithmetic means, UPGMA)聚类分析,在相似系数为0.677处可将30份叶用芥菜奶奶青菜材料分为3类。试验结果可为叶用芥菜奶奶青菜的种质资源鉴定、亲缘关系分析和分子标记辅助育种等研究提供引物支持和技术参考。

关键词: 奶奶青菜, 分子标记, 转录组, 遗传多样性

Abstract:

In order to explore the diversity level of leaf mustard Nainaiqingcai (Brassica juncea L.), this study investigated the characteristics of simple sequence repeat markers (EST-SSR) based on transcriptome, and screened suitable EST-SSR primers for Nainaiqingcai mustard, and then analyzed the genetic diversity of Nainaiqingcai mustard. The results showed that a total of 46 386 unigenes were obtained based on the transcriptome sequencing data of the leaf mustard Nainaiqingcai. It was found that 18 720 SSR loci were in 13 544 unigene sequences, the frequency of SSR was 29.20%, and the average distribution was 2.73 kb, and the average distribution frequency was 40.36%. Single nucleotide SSR was the abundant, which accounted for 49.39% of the total SSR, followed by tri-nucleotide and di-nucleotide, which accounted for 25.44% and 24.13%, respectively. A/T, AG/CT and AAG/CTT were the dominant repeat types in single nucleotide, di-nucleotide and tri-nucleotide, respectively. Thirty varieties of Nainaiqingcai mustard and five other Brassica vegetables were collected as materials, and 17 pairs of polymorphic primers were selected from a total of 37 primers. A total of 135 polymorphic bands were obtained, and the polymorphism rate was 88.2%. The genetic diversity analysis showed: average number of alleles (N_a) was 5.705 9, average effective allele number (N_e) was 2.397 8, average diversity index (I) average was 1.036 1, average observed heterozygosity (H_o) was 0.312 1, average expected heterozygosity (H_e) was 0.530 0, and average Nei’s gene diversity was 0.521 9, indicating that the selected 17 pairs of SSR primers had good genetic diversity. Unweighted pair-group method with arithmetic means (UPGMA) cluster analysis showed that 30 varieties of Nainaiqingcai mustard could be divided into three groups at the genetic similarity coefficient of 0.677. In summary, the results provided primer supporting and technical reference for germplasm resource identification, genetic relationship analysis and molecular marker assisted breeding of leaf mustard Nainaiqingcai.

Key words: Nainaiqingcai mustard, molecular marker, transcriptome, genetic diversity

中图分类号:

S637.2

马杰, 屈雯, 陈春艳, 王磊, 马俊, 刘针杉, 马维, 周平, 何远宽, 孙勃. 基于转录组序列的叶用芥菜奶奶青菜EST-SSR标记开发与遗传多样性分析[J]. 浙江农业学报, 2021, 33(9): 1640-1649.

MA Jie, QU Wen, CHEN Chunyan, WANG Lei, MA Jun, LIU Zhenshan, MA Wei, ZHOU Ping, HE Yuankuan, SUN Bo. Development of SSR markers based on transcriptome sequencing and genetic diversity analysis of Nainaiqingcai leaf mustard[J]. Acta Agriculturae Zhejiangensis, 2021, 33(9): 1640-1649.

图/表 7

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编号 No.	名称 Name	产地 Producing area	编号 No.	名称 Name	产地 Producing area
1	DG-1	贵州省毕节市 Bijie City, Guizhou Province	19	BD-2	贵州省毕节市 Bijie City, Guizhou Province
2	DG-2	贵州省毕节市 Bijie City, Guizhou Province	20	CCB-1	贵州省毕节市 Bijie City, Guizhou Province
3	DG-3	贵州省毕节市 Bijie City, Guizhou Province	21	CCB-2	贵州省毕节市 Bijie City, Guizhou Province
4	DG-4	贵州省毕节市 Bijie City, Guizhou Province	22	CCB-3	贵州省毕节市 Bijie City, Guizhou Province
5	DG-5	贵州省毕节市 Bijie City, Guizhou Province	23	CCB-4	贵州省毕节市 Bijie City, Guizhou Province
6	XJ-1	贵州省毕节市 Bijie City, Guizhou Province	24	CCB-5	贵州省毕节市 Bijie City, Guizhou Province
7	XJ-2	贵州省毕节市 Bijie City, Guizhou Province	25	CCB-6	贵州省毕节市 Bijie City, Guizhou Province
8	XJ-3	贵州省毕节市 Bijie City, Guizhou Province	26	ZC-1	贵州省毕节市 Bijie City, Guizhou Province
9	XJ-4	贵州省毕节市 Bijie City, Guizhou Province	27	ZC-2	贵州省毕节市 Bijie City, Guizhou Province
10	XJ-5	贵州省毕节市 Bijie City, Guizhou Province	28	JS-1	贵州省毕节市 Bijie City, Guizhou Province
11	DXQ-1	贵州省毕节市 Bijie City, Guizhou Province	29	YB-1	四川省宜宾市 Bijie City, Sichuan Province
12	DXQ-2	贵州省毕节市 Bijie City, Guizhou Province	30	YB-2	四川省宜宾市 Bijie City, Sichuan Province
13	DXQ-3	贵州省毕节市 Bijie City, Guizhou Province	31	香港竹芥菜(叶用芥菜) Hong Kong Bamboo Mustard (leaf mustard)	澳大利亚 Australia
14	DXQ-4	贵州省毕节市 Bijie City, Guizhou Province	32	改良榨菜(茎用芥菜) Gailiang Tuber Mustard (stem mustard)	四川省绵阳市 Mianyang City, Sichuan Province
15	GYQ-1	贵州省毕节市 Bijie City, Guizhou Province	33	日本光头芥菜(根用芥菜) Japanese Guangtou Mustard (root mustard)	甘肃省 Gansu Province
16	GYQ-2	贵州省毕节市 Bijie City, Guizhou Province	34	早熟五号(大白菜) Zaoshu 5 (Chinese cabbage)	河南省 Henan Province
17	GYQ-3	贵州省毕节市 Bijie City, Guizhou Province	35	绿球三号(甘蓝) Green ball 3 (cabbage)	日本 Japan
18	BD-1	贵州省毕节市 Bijie City, Guizhou Province

编号 No.	名称 Name	产地 Producing area	编号 No.	名称 Name	产地 Producing area
1	DG-1	贵州省毕节市 Bijie City, Guizhou Province	19	BD-2	贵州省毕节市 Bijie City, Guizhou Province
2	DG-2	贵州省毕节市 Bijie City, Guizhou Province	20	CCB-1	贵州省毕节市 Bijie City, Guizhou Province
3	DG-3	贵州省毕节市 Bijie City, Guizhou Province	21	CCB-2	贵州省毕节市 Bijie City, Guizhou Province
4	DG-4	贵州省毕节市 Bijie City, Guizhou Province	22	CCB-3	贵州省毕节市 Bijie City, Guizhou Province
5	DG-5	贵州省毕节市 Bijie City, Guizhou Province	23	CCB-4	贵州省毕节市 Bijie City, Guizhou Province
6	XJ-1	贵州省毕节市 Bijie City, Guizhou Province	24	CCB-5	贵州省毕节市 Bijie City, Guizhou Province
7	XJ-2	贵州省毕节市 Bijie City, Guizhou Province	25	CCB-6	贵州省毕节市 Bijie City, Guizhou Province
8	XJ-3	贵州省毕节市 Bijie City, Guizhou Province	26	ZC-1	贵州省毕节市 Bijie City, Guizhou Province
9	XJ-4	贵州省毕节市 Bijie City, Guizhou Province	27	ZC-2	贵州省毕节市 Bijie City, Guizhou Province
10	XJ-5	贵州省毕节市 Bijie City, Guizhou Province	28	JS-1	贵州省毕节市 Bijie City, Guizhou Province
11	DXQ-1	贵州省毕节市 Bijie City, Guizhou Province	29	YB-1	四川省宜宾市 Bijie City, Sichuan Province
12	DXQ-2	贵州省毕节市 Bijie City, Guizhou Province	30	YB-2	四川省宜宾市 Bijie City, Sichuan Province
13	DXQ-3	贵州省毕节市 Bijie City, Guizhou Province	31	香港竹芥菜(叶用芥菜) Hong Kong Bamboo Mustard (leaf mustard)	澳大利亚 Australia
14	DXQ-4	贵州省毕节市 Bijie City, Guizhou Province	32	改良榨菜(茎用芥菜) Gailiang Tuber Mustard (stem mustard)	四川省绵阳市 Mianyang City, Sichuan Province
15	GYQ-1	贵州省毕节市 Bijie City, Guizhou Province	33	日本光头芥菜(根用芥菜) Japanese Guangtou Mustard (root mustard)	甘肃省 Gansu Province
16	GYQ-2	贵州省毕节市 Bijie City, Guizhou Province	34	早熟五号(大白菜) Zaoshu 5 (Chinese cabbage)	河南省 Henan Province
17	GYQ-3	贵州省毕节市 Bijie City, Guizhou Province	35	绿球三号(甘蓝) Green ball 3 (cabbage)	日本 Japan
18	BD-1	贵州省毕节市 Bijie City, Guizhou Province

重复基元 Repeat motif length	重复次数Repeat number								合计 Total	比例 Percentage/%
重复基元 Repeat motif length	5	6	7	8	9	10	11~20	>20	合计 Total	比例 Percentage/%
单核苷酸Mononucleotide	0	0	0	0	0	3 677	5 263	306	9 246	49.39
二核苷酸Dinucleotide	0	1 625	975	651	398	249	554	66	4 518	24.13
三核苷酸Trinucleotide	2 898	1 117	391	159	97	62	36	2	4 762	25.44
四核苷酸Tetranucleotide	66	21	9	3	1	0	1	0	101	0.54
五核苷酸Pentanucleotide	27	9	5	2	0	1	1	0	45	0.24
六核苷酸Hexanucleotide	38	2	5	1	2	0	0	0	48	0.26
合计Total	3 029	2 774	1 385	816	498	3 989	5 855	374	18 720	100.00
合计占比Total proportion/%	16.18	14.82	7.40	4.36	2.66	21.31	31.28	2.00	100.00

重复基元 Repeat motif length	重复次数Repeat number								合计 Total	比例 Percentage/%
重复基元 Repeat motif length	5	6	7	8	9	10	11~20	>20	合计 Total	比例 Percentage/%
单核苷酸Mononucleotide	0	0	0	0	0	3 677	5 263	306	9 246	49.39
二核苷酸Dinucleotide	0	1 625	975	651	398	249	554	66	4 518	24.13
三核苷酸Trinucleotide	2 898	1 117	391	159	97	62	36	2	4 762	25.44
四核苷酸Tetranucleotide	66	21	9	3	1	0	1	0	101	0.54
五核苷酸Pentanucleotide	27	9	5	2	0	1	1	0	45	0.24
六核苷酸Hexanucleotide	38	2	5	1	2	0	0	0	48	0.26
合计Total	3 029	2 774	1 385	816	498	3 989	5 855	374	18 720	100.00
合计占比Total proportion/%	16.18	14.82	7.40	4.36	2.66	21.31	31.28	2.00	100.00

重复基元 Repeat motif	重复次数Repeat number								合计 Total	比例 Percentage/%
重复基元 Repeat motif	5	6	7	8	9	10	11~20	>20	合计 Total	比例 Percentage/%
A/T	0	0	0	0	0	3 650	5 218	304	9 172	49.00
C/G	0	0	0	0	0	27	45	2	74	0.40
AC/GT	0	178	100	62	28	13	21	0	402	2.15
AG/CT	0	1 206	762	507	309	202	428	58	3 472	18.55
AT/AT	0	241	109	82	61	34	105	8	640	3.42
CG/CG	0	0	4	0	0	0	0	0	4	0.02
AAC/GTT	267	114	27	17	6	3	2	0	436	2.33
AAG/CTT	973	375	153	65	37	34	19	2	1 658	8.86
AAT/ATT	65	26	12	7	4	0	3	0	117	0.63
ACC/GGT	251	115	31	5	5	1	1	0	409	2.18
ACG/CGT	69	17	3	0	0	0	0	0	89	0.48
ACT/AGT	52	16	5	2	2	1	1	0	79	0.42
AGC/CTG	186	56	25	3	4	1	0	0	275	1.47
AGG/CCT	510	182	65	22	13	9	0	0	801	4.28
ATC/ATG	434	186	65	37	26	13	10	0	771	4.12
CCG/CGG	91	30	5	1	0	0	0	0	127	0.68
合计Total	2 898	2742	1 366	810	495	3 988	5 853	374	18 526	98.96
合计频率Total frequency/%	15.48	14.65	7.30	4.33	2.64	21.30	31.27	2.00	98.96

基于转录组序列的叶用芥菜奶奶青菜EST-SSR标记开发与遗传多样性分析

Development of SSR markers based on transcriptome sequencing and genetic diversity analysis of Nainaiqingcai leaf mustard

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序号 No.	引物编号 Primer No.	引物序列 Primer sequence(5'→3')	重复单元 Repeat motif	退火温度 Temperature/ ℃	预期产物大小 Expected product size/bp	扩增条带数 Number of amplification bands	多态性条带数 Number of polymorphi bands	多态性比例 Percentage of polymorphism/ %
1	M2-1	TCTCATTGTGCGTGTGAGCA	(TA)21	56.14	211	12	9	75.00
		ACGGCAATGGCAGATCAAGA
2	M2-5	ACTTGATCTACTGCACCTGCT	(AG)22	55.06	197	7	6	85.70
		ACAAGGCAGTTTCTAGACGGA
3	M2-8	GACAACGTCGTCGGAGAGTT	(AG)23	55.96	177	15	13	86.70
		AACCGCCTCTTTGCCCTAAA
4	M2-9	CCGTCTCTTAGCTGCTAGCC	(AG)21	56.11	246	11	10	90.90
		AACTCGGTCCCAAGACTTGC
5	M2-11	ATACAGTGTGTGTGGCAGCA	(TA)20	56.11	223	6	5	83.30
		CGTGCTAAGTCTTGGGGCTT
6	M2-13	ATTGCTCTCATCACGACACA	(CT)22	54.61	276	6	6	100.00
		TTGTTGGCTAACGCAACAGC
7	M2-15	GGTGCACCAGCCTGTACTTA	(AT)28	55.75	258	10	10	100.00
		TCGGGAAGCTCGATGTTCTG
8	M2-16	CCAAACTCACATCCACATTTCA	(AG)22	54.56	232	12	7	58.30
9	M2-17	TCAGGTGGTGAGAGAGATCGT	(TC)32	56.02	256	11	9	81.80
		ACAAGATCCAGACAAGTCAATAGCT
10	M2-18	AGAAGGCTTTTCTATTGGCCA	(TC)21	54.75	272	15	14	93.30
		CAGAGGCAGAGTTGAAGGCA
11	M2-20	TCTCGTTTTCACCTGAGGAAA	(CT)20	54.64	218	6	6	100.00
		GCCACTCCCTAAAGTCGCAT
12	M2-21	GGTTGGTCTCAGCTGCTGAA	(AG)21	55.45	234	13	13	100.00
		ACTCCATCTCTGTGGCTGTT
13	M3-1	TGATGGTACTGCTTCGAGGG	(TTC)17	55.36	152	4	4	100.00
		CCCCTTCTCGCGAGATCTTT
14	M3-2	GCGGTTTGGAATCTCAAGGC	(TCT)14	55.90	277	9	9	100.00
		CAACAGCAAAACAGGAGGGC
15	M5-2	AACACCCAGGCCTATGTGTG	(GTTTT)7	56.00	244	3	3	100.00
		AAGCCTCCACCATGCTTCTC
16	M5-4	TCATCCTCCTCTGGCTCGAA	(GAATC)7	56.03	164	6	6	100.00
		AGAAGAAGAAGCTTCCGCCC
17	M6-1	CAATGGCAGCGCATTCCTTC	(CCAAGG)9	55.76	278	7	5	71.40
		TCTGCGTCTCTCTTTCTCTCTC

引物编号 Primer No.	观测等位基因数 N_a	有效等位基因数 N_e	多样性指数 I	观察杂合度 H_o	期望杂合度 H_e	Nei’s期望杂合度 Nei	多态性信息含量 PIC
M2-1	7	1.929 1	0.974 1	0.171 4	0.488 6	0.481 6	0.436 1
M2-5	5	1.755 0	0.910 4	0.151 5	0.436 8	0.430 2	0.409 5
M2-8	4	1.891 9	0.890 0	0.085 7	0.478 3	0.471 4	0.432 9
M2-9	8	2.203 2	1.091 8	0.371 4	0.554 0	0.546 1	0.488 0
M2-11	9	3.058 2	1.408 0	0.931 0	0.684 8	0.673 0	0.617 7
M2-13	4	1.467 9	0.634 2	0.314 3	0.323 4	0.318 8	0.294 6
M2-15	5	2.245 8	0.936 9	0.769 2	0.565 6	0.554 7	0.455 3
M2-16	4	1.373 3	0.585 2	0	0.276 0	0.271 8	0.259 8
M2-17	7	1.809 5	0.972 8	0.057 1	0.453 8	0.447 3	0.423 9
M2-18	5	2.442 7	1.044 4	0.085 7	0.599 2	0.590 6	0.505 2
M2-20	6	4.257 8	1.594 3	0.176 5	0.776 6	0.765 1	0.733 3
M2-21	8	2.991 8	1.369 5	0.969 7	0.676 0	0.665 7	0.608 9
M3-1	4	2.342 5	1.030 1	0.176 5	0.581 7	0.573 1	0.514 8
M3-2	6	4.905 4	1.674 5	0.636 4	0.808 4	0.796 1	0.765 7
M5-2	3	1.102 7	0.224 5	0.032 3	0.094 7	0.093 1	0.090 8
M5-4	6	2.660 2	1.176 7	0.200 0	0.633 1	0.624 1	0.552 1
M6-1	6	2.326 0	1.096 9	0.176 5	0.578 6	0.570 1	0.511 1
平均值Mean	5.705 9	2.397 8	1.036 1	0.312 1	0.530 0	0.521 9	0.476 4
标准差Standard deviation	1.686 9	0.984 4	0.360 0	0.315 2	0.180 5	0.177 7	0.160 2

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