Development and application of SSR markers based on transcriptome sequencing of turnip (Brassica rapa ssp. rapa)

doi:10.3969/j.issn.1004-1524.2023.02.09

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (2): 319-328.DOI: 10.3969/j.issn.1004-1524.2023.02.09

• Horticultural Science • Previous Articles Next Articles

Development and application of SSR markers based on transcriptome sequencing of turnip (Brassica rapa ssp. rapa)

LI Xiaojuan¹^,²(), ZHAO Wenju¹^,², ZHAO Mengliang¹^,², SHAO Dengkui¹^,², MA Yidong¹^,², REN Yanjing¹^,²^,^*()

1. Qinghai Academy of Agriculture and Forestry Sciences, Xining 810016, China
2. Qinghai Key Laboratory of Vegetable Genetics and Physiology, Xining 810016, China

Received:2021-11-02 Online:2023-02-25 Published:2023-03-14
Contact: REN Yanjing

Abstract

Abstract:

To investigate the level of diversity of turnips (Brassica rapa ssp. rapa) resources, transcriptome sequencing was used to explore the simple sequence repeat marker of turnip. The results showed that transcriptome sequencing analysis obtained 253 720 unigene and 13 247 unigene sequences contained 2 or more SSR sites. The frequency of SSR was 25.05%, with an average of 1 SSR per 3.15 kb, and a distribution frequency of 31.42%. All 30 primers eventually yielded high polymorphism and clear bands with a total of 126 polymorphism bands with an average of 4.2 pairs and 100% polymorphism. According to electrophoresis amplification results, 24 SSR primers with high polymorphism and clear bands were selected. Twenty-four SSR primers amplified 101 alleles in 50 turnip materials with an average of 4.21 alleles per locus. Analysis results showed that the average number of effective alleles (Ne) was 1.481 7 with an effective allele variation rate of 35.19%. These results showed that the selected primers could represent the genetic diversity of 50 turnips and played an important role in DNA fingerprint construction and hybrid identification of turnips.

Key words: turnip, molecular marker, transcriptome, genetic diversity

CLC Number:

S637.2

LI Xiaojuan, ZHAO Wenju, ZHAO Mengliang, SHAO Dengkui, MA Yidong, REN Yanjing. Development and application of SSR markers based on transcriptome sequencing of turnip (Brassica rapa ssp. rapa)[J]. Acta Agriculturae Zhejiangensis, 2023, 35(2): 319-328.

Figures/Tables 7

References 25

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编号No.	名称Name	产地Production area	编号No.	名称Name	产地Production area
1	1401	青海Qinghai	26	W29	青海Qinghai
2	1402	青海Qinghai	27	W30	青海Qinghai
3	1403	西藏Tibet	28	W31	青海Qinghai
4	1404	青海Qinghai	29	T1	河北Hebei
5	1405	青海Qinghai	30	T2	河北Hebei
6	1406	四川Sichuan	31	T4	黑龙江Heilongjiang
7	1407	四川Sichuan	32	T5	内蒙古Inner Mongolia
8	1408	新疆Xinjiang	33	T6	内蒙古Inner Mongolia
9	1409	新疆Xinjiang	34	T11	日本Japan
10	1410	青海Qinghai	35	T14	日本Japan
11	1411	西藏Tibet	36	T15	日本Japan
12	1412	云南Yunnan	37	T16	青海Qinghai
13	1413	云南Yunnan	38	T17	青海Qinghai
14	G1	青海Qinghai	39	T18	甘肃Gansu
15	G2	青海Qinghai	40	IC9	国家种质资源库The National Germplasm Bank
16	G3	青海Qinghai	41	IC22	国家种质资源库The National Germplasm Bank
17	G4	青海Qinghai	42	IC23	国家种质资源库The National Germplasm Bank
18	W21	北京Beijing	43	IC28	国家种质资源库The National Germplasm Bank
19	W22	河南Henan	44	IC39	国家种质资源库The National Germplasm Bank
20	W23	河北Hebei	45	IC40	国家种质资源库The National Germplasm Bank
21	W24	河北Hebei	46	IC43	国家种质资源库The National Germplasm Bank
22	W25	河北Hebei	47	IC44	国家种质资源库The National Germplasm Bank
23	W26	内蒙古Inner Mongolia	48	BLK	内蒙古Inner Mongolia
24	W27	北京Beijing	49	SK	黑龙江Heilongjiang
25	W28	内蒙古Inner Mongolia	50	NS1	内蒙古Inner Mongolia

编号No.	名称Name	产地Production area	编号No.	名称Name	产地Production area
1	1401	青海Qinghai	26	W29	青海Qinghai
2	1402	青海Qinghai	27	W30	青海Qinghai
3	1403	西藏Tibet	28	W31	青海Qinghai
4	1404	青海Qinghai	29	T1	河北Hebei
5	1405	青海Qinghai	30	T2	河北Hebei
6	1406	四川Sichuan	31	T4	黑龙江Heilongjiang
7	1407	四川Sichuan	32	T5	内蒙古Inner Mongolia
8	1408	新疆Xinjiang	33	T6	内蒙古Inner Mongolia
9	1409	新疆Xinjiang	34	T11	日本Japan
10	1410	青海Qinghai	35	T14	日本Japan
11	1411	西藏Tibet	36	T15	日本Japan
12	1412	云南Yunnan	37	T16	青海Qinghai
13	1413	云南Yunnan	38	T17	青海Qinghai
14	G1	青海Qinghai	39	T18	甘肃Gansu
15	G2	青海Qinghai	40	IC9	国家种质资源库The National Germplasm Bank
16	G3	青海Qinghai	41	IC22	国家种质资源库The National Germplasm Bank
17	G4	青海Qinghai	42	IC23	国家种质资源库The National Germplasm Bank
18	W21	北京Beijing	43	IC28	国家种质资源库The National Germplasm Bank
19	W22	河南Henan	44	IC39	国家种质资源库The National Germplasm Bank
20	W23	河北Hebei	45	IC40	国家种质资源库The National Germplasm Bank
21	W24	河北Hebei	46	IC43	国家种质资源库The National Germplasm Bank
22	W25	河北Hebei	47	IC44	国家种质资源库The National Germplasm Bank
23	W26	内蒙古Inner Mongolia	48	BLK	内蒙古Inner Mongolia
24	W27	北京Beijing	49	SK	黑龙江Heilongjiang
25	W28	内蒙古Inner Mongolia	50	NS1	内蒙古Inner Mongolia

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	>10	合计 Total	比例 Percentage/%
单核苷酸Mononucleotide	0	0	0	0	0	16 975	7 820	24 795	45.50
二核苷酸Dinucleotide	0	6 174	3 311	2 421	1 550	0	0	13 456	24.70
三核苷酸Trinucleotide	9 813	3 591	1 388	620	97	0	0	15 509	28.47
四核苷酸Tetranucleotide	342	94	12	7	2	0	6	461	0.85
五核苷酸Pentanucleotide	95	18	5	2	0	4	0	124	0.23
六核苷酸Hexanucleotide	77	32	17	6	1	0	5	137	0.25
合计Total	10 327	9 909	4 733	3 056	1 650	16 979	7 831	54 482	100
合计占比Total proportion/%	18.95	18.19	8.69	5.61	3.03	31.16	14.37

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	>10	合计 Total	比例 Percentage/%
单核苷酸Mononucleotide	0	0	0	0	0	16 975	7 820	24 795	45.50
二核苷酸Dinucleotide	0	6 174	3 311	2 421	1 550	0	0	13 456	24.70
三核苷酸Trinucleotide	9 813	3 591	1 388	620	97	0	0	15 509	28.47
四核苷酸Tetranucleotide	342	94	12	7	2	0	6	461	0.85
五核苷酸Pentanucleotide	95	18	5	2	0	4	0	124	0.23
六核苷酸Hexanucleotide	77	32	17	6	1	0	5	137	0.25
合计Total	10 327	9 909	4 733	3 056	1 650	16 979	7 831	54 482	100
合计占比Total proportion/%	18.95	18.19	8.69	5.61	3.03	31.16	14.37

重复类型 Repeat type	基序类型数 Number of motif type	重复基序 Repeat motif	数量 Number	比例 Percentage%	重复基序 Repeat motif	数量 Number	比例 Percentage%
单核苷酸Mononucleotide	2	A/T	24 649	45.24	C/G	146	0.27
二核苷酸Dinucleotide	4	AT/AT	2 585	4.74	AG/CT	9 288	17.05
		AC/GT	1 551	2.85	CG/CG	32	0.06
三核苷酸Trinucleotide	10	AAT/ATT	553	1.02	ACC/GGT	1 216	2.23
		AAC/GTT	1 535	2.82	ACG/CGT	517	0.95
		AAG/CTT	5 054	9.28	AGC/GCT	713	1.31
		ATC/GAT	1 381	2.53	AGG/CCT	2656	4.88
		ATG/CAT	1 511	2.77	CCG/TCC	464	0.85
四核苷酸Tetranucleotide	26	AAAC/GTTT	96	0.18	AAAG/CTTT	112	0.21
		AAAT/ATTT	80	0.15	其他Others	173	0.32
五核苷酸Pentanucleotide	26	AAAAT/ATTTT	24	0.04	AAAAG/CTTTT	13	0.02
		AACCA/TGGTT	16	0.03	其他Others	71	0.13
六核苷酸Hexanucleotide	44	GGCTGT/ACAGCC	14	0.03	CCAGCT/AGCTGG	13	0.02
		TTTTTG/CAAAAA	11	0.02	其他Others	99	0.18

Development and application of SSR markers based on transcriptome sequencing of turnip (Brassica rapa ssp. rapa)

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序号 No.	引物名称 Primer name	引物序列 Primer sequence (5'-3')	预期产物大小 Expected product size/bp	退火温度 Annealing temperature/ ℃	GC含量 GC content	扩增条带数 Number of amplification bands	多态性条带数 Number of polymorphism bands	多态性比例 Percentage of polymorphism/ %
1	Cluster-10000.0	TTAATCGATCTCCGGTGGCG	217	60.00	55.00	3	3	100
		TCTACGTATGGGACCAGCCA
2	Cluster-27140.0	GACTGATGAGATGCCTCGGG	209	59.97	60.00	3	3	100
		CCGGAGATCCAATGTACCCG
3	Cluster-30924.104122	CGGTTGTCTCCCCGGTTTAA	200	59.97	57.50	1	1	100
		CCCAGTAGATTCTCGCGTCC
4	Cluster-30924.109584	GGACGCACCAATCTCCTTGA	204	60.00	57.50	1	1	100
		GCCCAATCTACCGAGTCGAG
5	Cluster-30924.114983	AAGGAGACGTCTTGCGAAGG	208	60.04	52.50	6	6	100
		ATGCCGTTCCGAGTTTCCAT
6	Cluster-30924.120390	CCTTGTGTTGCAAAGAGGGC	211	59.97	55.00	1	1	100
		CTGGTGCGCATTTATCTGCC
7	Cluster-30924.126255	TGATGATGATGGCTGGGCTG	211	60.11	52.50	8	8	100
		GCGCTAGGCTTTGCTTTTGT
8	Cluster-30924.132245	TCGGCATATGCTAGCTGGTG	209	59.97	55.00	2	2	100
		GACCACGCTCCTTCAGACAA
9	Cluster-30924.138446	TCCGTTTGAACCACACCCAA	213	59.96	50.00	5	5	100
		TAACAGCAACCTCGTTGGCT
10	Cluster-30924.144894	CGGAGCGGATGCAAGACTAT	221	59.93	55.00	6	6	100
		TCCCTCAGGACCAAAAGTGC
11	Cluster-30924.151200	AAGGAGGCTGCAAGAGTGAC	221	59.96	57.50	6	6	100
		CCAGTGGGTGTCTCAGGTTC
12	Cluster-30924.157883	CATTCGACCCGCTCTCTCTC	206	59.97	60.00	2	2	100
		CACCCGACAGTATACGTCCG
13	Cluster-30924.164312	GGAGACTGAGGGGATGAGGT	219	60.03	57.50	4	4	100
		AGAAATCGGACCCGGGTTTC
14	Cluster-30924.171259	TGTGAACCCAAGCTCCGTAC	228	59.97	55.00	3	3	100
		GCCGTCTTCATCACATTCGC
15	Cluster-30924.22191	TGGCTATCATCCCCTGTCCA	207	60.03	55.00	1	1	100
		GGGGATCAAGAAACGCCTGA
16	Cluster-30924.28557	TCTTCATGCGGCTTCCTCTG	229	60.07	55.00	7	7	100
		AGCAAAGCTCCCATCAGACC
17	Cluster-30924.35340	AGAGAGGCTATTGGCGCATC	218	59.97	55.00	6	6	100
		TCGGACAAGACACGGTGAAG
18	Cluster-30924.41968	CGCAGTTGGTTGTCACACAG	203	59.97	55.00	3	3	100
		CGTCTCACTCGGTGTTCCAA
19	Cluster-30924.48364	GAAGGGTGGAGTCGACAAGG	206	59.97	60.00	1	1	100
		GGTGGCTCTAATCGGTGGAG
20	Cluster-30924.54426	CTGCTTCTCCTCTTGCACCA	257	59.97	55.00	3	3	100
		TGTCGGAGGAGCTGAAACAC
21	Cluster-30924.60140	AGGTGGGGATAAGCACAAGC	203	60.04	57.50	12	12	100
		GTTCTCCACTGCCTCTGTCC
22	Cluster-30924.65654	GCCATCTCCGACTTCCCAAA	224	60.04	55.00	2	2	100
		GTCCAGTTCTCGCCATTCCA
23	Cluster-30924.71367	GCTGTCGGTGGTGTTCAAAC	245	59.97	55.00	4	4	100
		TAACAGGGACCGGCAAAGAC
24	Cluster-30924.76636	AGAGATGGCTGTGTTGGTGG	203	60.00	55.00	13	13	100
		ATGAGCGTCTTCCTCCTCCT
25	Cluster-30924.82553	ATTCGAGTTGGTGCCTGAGG	241	60.04	55.00	2	2	100
		AACGGAAAGCTCGAGGTCTG
26	Cluster-30924.87846	GAGCAATCGGTTCCCTGGAA	217	60.03	55.00	2	2	100
		TCATCTGGTACCTCGGAGCA
27	Cluster-30924.93982	CCAACTTGACGGTCGAAGGA	218	60.00	55.00	1	1	100
		TCAGATAACTCGAGGGGGC
28	Cluster-30924.99449	GCTCTGCCTGTTTTGGAAGC	217	60.00	55.00	6	6	100
		TGCTCTCTTCAACAGCCTGG
29	Cluster-62597.0	TGGACAAGGTGAAGTCTCCG	212	59.57	55.00	6	6	100
		ATTCGGAGAGCGAGGTTGAC
30	Cluster-9993.0	GCGAGTCAGGCCTAAAGGTT	228	60.04	55.00	6	6	100
		TCAATTTCCCTGGCGTCTCC

引物 Primer	观测等位基因数 Na	有效等位基因数 Ne	Nei’s期望杂合度 h	多样性指数 I	多态性信息含量 PIC
Cluster-30924.35340	6	1.167 4	0.140 3	0.265 4	0.438 1
Cluster-30924.171259	3	1.408 7	0.277 3	0.443 3	0.698 6
Cluster-30924.151200	6	1.665 4	0.365 3	0.536 1	0.866 8
Cluster-30924.41968	3	1.701 0	0.377 6	0.551 1	0.689 3
Cluster-30924.28557	7	1.328 8	0.239 2	0.397 8	0.616 9
Cluster-30924.164312	4	1.856 3	0.455 1	0.646 0	0.737 0
Cluster-30924.157883	2	1.336 0	0.224 1	0.363 7	0.235 6
Cluster-9993.0	6	1.662 7	0.373 2	0.549 9	0.825 7
Cluster-62597.0	6	1.220 0	0.136 7	0.234 0	0.459 9
Cluster-30924.65654	2	1.456 3	0.265 6	0.407 9	0.312 4
Cluster-30924.71367	4	1.338 1	0.218 4	0.348 8	0.572 3
Cluster-30924.76636	6	1.761 2	0.428 9	0.619 4	0.858 5
Cluster-30924.99449	3	1.698 9	0.411 4	0.601 7	0.497 6
Cluster-30924.82553	2	1.423 5	0.255 4	0.397 3	0.289 0
Cluster-30924.87846	2	1.020 3	0.019 9	0.056 2	0.038 4
Cluster-30924.60140	6	1.742 9	0.422 9	0.613 1	0.639 7
Cluster-10000.0	3	1.366 2	0.198 8	0.306 1	0.439 9
Cluster-27140.0	3	1.796 7	0.442 2	0.633 9	0.575 0
Cluster-30924.54426	3	1.577 0	0.346 9	0.525 7	0.592 2
Cluster-30924.114983	6	1.181 4	0.111 0	0.190 9	0.310 7
Cluster-30924.126255	5	1.618 5	0.370 8	0.554 1	0.914 7
Cluster-30924.132245	2	1.197 9	0.160 5	0.292 7	0.404 7
Cluster-30924.138446	5	1.529 6	0.322 4	0.489 9	0.875 8
Cluster-30924.144894	6	1.505 7	0.322 9	0.498 8	0.769 0
平均值Mean	4.208 3	1.481 7	0.287 0	0.438 5	0.569 1
标准差Standard deviation	1.718 9	0.230 0	0.118 7	0.157 3	0.232 3

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