利用芥菜转录组信息挖掘SSR标记及用于种质分析

李永平; 张双照; 薛珠政; 温庆放

doi:10.19303/j.issn.1008-0384.2020.02.007

利用芥菜转录组信息挖掘SSR标记及用于种质分析

doi: 10.19303/j.issn.1008-0384.2020.02.007

李永平^{1, 2,},
张双照³,
薛珠政^{1, 2, ,},
温庆放^{1, 2}

1.
福建省农业科学院蔬菜研究中心，福州　350013
2.
福建省农业科学院作物研究所，福州　350013
3.
龙岩市农业科学研究所，龙岩　36400

基金项目: 国家特色蔬菜产业技术体系项目（CARS-24-G-07）；福建省科技厅农业引导性（重点）项目（2017N0048）；福建省农业科学院10、福建省财政专项－福建省农业科学院科技创新团队建设项目蔬菜科技创新团队项目（STIT2017-1-2）

详细信息

作者简介:
李永平（1974−），女，硕士，副研究员，研究方向：蔬菜分子育种（E-mail：248937256@qq.com）

通讯作者:
薛珠政（1971−），男，研究员，研究方向：蔬菜育种（E-mail：369654817@qq.com）

中图分类号: S 649
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出版历程
- 收稿日期: 2019-10-23
- 修回日期: 2019-12-19
- 刊出日期: 2020-02-01

Using SSR Markers from Brassica juncea Transcriptome for Germplasm Analysis

LI Yong-ping^{1, 2
,},
ZHANG Shuang-zhao³,
XUE Zhu-zheng^{1, 2
, ,},
WEN Qing-fan^{1, 2}

1.
Vegetable Research Center, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
2.
Crops Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
3.
Longyan Agricultural Science Institute, Longyan, Fujian　36400, China

摘要

摘要: 目的为开发高效的芥菜Brassicajuncea.分子标记。方法本研究通过对芥菜转录组测序的信息搜索SSR位点并分析其分布特点，应用Primer 3.0软件设计SSR引物，随机选择 50 对引物扩增44 份芥菜种质，检测其多态性。结果芥菜转录组测序共获得55 636条unigene，全部序列有48 193 376 bp；有7 834条unigene包含SSR的序列，从中鉴定出9 526个SSR位点，其中有1 371条序列包含1个以上SSR，复合SSR有572个，SSR发生频率为14.08%。优势重复基序为三核苷酸和二核苷酸，分别占总SSR的51.12%和41.91%。二核苷酸重复基元中以AG/CT为优势重复基元，占总位点的34.74%，三核苷酸重复基元以AAG/CTT为主，占总位点的18.30%。共设计出21 282对SSR引物，随机选择 50 对引物进行 PCR 扩增，其中41对扩增出清晰可重复的预期条带，17对（占34%）在 44 份芥菜种质中表现出多态性。应用UPGMA得到将44份供试材料分为4大类聚类图，可准确地体现了芥菜种质材料的关系。结论根据芥菜转录组数据能开发出类型丰富、效率较高的SSR标记，为芥菜亲缘关系分析和遗传图谱构建等提供更可靠的标记。
- 芥菜 /
- 转录组 /
- SSR /
- 多态性
Abstract: Objective To search for applicable molecular markers for studying Brassica juncea. Method SSR sites were searched from transcriptional sequencing of B. juncea. Potential primers were designed using Primer 3.0, and polymorphisms of 44 B. juncea germplasms detected from randomly selected 50 pairs of primers for the investigation. Result Totally, 55,636 unigenes were obtained from the transcriptome sequencing. The entire sequence was 48,193,376bp with 9,526 SSR loci identified from 7,834 unigenes at a rate of 14.08%. Of the loci, 1,371 contained more than one SSR locus and 572 compound loci. Of the major repeat types, 51.12% were trinucleotide dominated by AGA/TCT (18.30%), and 41.91% dinucleotide dominated by AG/CT (34.74%). Using Primer 3.0, from 21,282 pairs of SSR primers, 50 were randomly selected for the PCR amplification. Out of 44 amplifications, 41 showed clear and reproducible bands, and 17 with polymorphisms. The 44 germplasms were classified into 4 distinctive groups by UPGMA. Conclusion The SSR markers with high frequency and polymorphism could be acquired from the transcriptome to be used for the genetic diversity analysis and mapping construction on B. juncea.
- Brassica juncea /
- transcriptome /
- simple sequence repeat /
- polymorphism

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图 1 引物BaSSR046（A）和BaSSR007（B）在44份芥菜种质资源（1-44）中的多态性

注： M：DNA marker；1-44：芥菜种质资源。

Figure 1. Polymorphisms in 44 B. juncea germplasms (1–44) as shown by primer BaSSR046 (A) and BaSSR007 (B)

Note: M: DNA marker; 1-44: Brassicajuncea germplasms.

下载: 全尺寸图片幻灯片

图 2 芥菜种质资源的UPGMA聚类图

Figure 2. B. juncea germplasms classified by UPGMA

下载: 全尺寸图片幻灯片

表 1 44份芥菜种质材料主要性状

Table 1. Major properties of 44 B. juncea germplasms

编号 Code	品种名称 Cultivar	品种来源 Source	株型 Plant type	叶特征（叶形、叶面光滑与否、有无刺毛、蜡粉） Leaf characteristics（leaf shape, smooth or not, with or without bristles, wax powder）	抽䑓性 Bolting property	结球性（是/否） Heading ability（Y/N）	食用器官 Edible organ
1	龙岩青皮芥菜 Longyan Green Mustard	福建龙岩市 Fujian Longyan	直立 Upright	卵圆形、皱、无刺毛、无蜡粉 Ovoid, wrinkled, spineless, no wax powder	中 Medium	否 No	茎 Stem
2	连城紫芥菜 Liancheng Pulse Purple Mustard	福建龙岩市 Fujian Longyan	开展 Expansion	阔椭圆、微皱、无刺毛、有蜡粉 Broad oval, slightly wrinkled, spineless, wax powder	中 Medium	否 No	叶 Leaf
3	抗热四季 ok甜芥菜 Hot Resistant Four Seasons OK Sweet Mustard	广西横县 Guangxi Hengxian	直立 Upright	阔椭圆、平滑、无刺毛、有蜡粉 Broad oval, smooth, spineless, wax powder	早 Early	否 No	叶 Leaf
4	华517 Hua517	湖北武汉市 Hubei Wuhan	直立 Upright	倒卵、微皱、无刺毛、无蜡粉 Obovate, slightly wrinkled, spineless, no wax powder	中 Medium	否 No	茎 Stem
5	厦门芥菜 Xiamen Mustard	福建龙岩市 Fujian Longyan	开展 Expansion	扇形、皱、无刺毛、有蜡粉 Fan-shaped, wrinkled, spineless, wax powder	中 Medium	否 No	叶 Leaf
6	华515 Hua515	华中农大 Central China Agricultural University	开展 Expansion	阔椭圆、皱、无刺毛、无蜡粉 Broad oval, wrinkled, spineless, no wax powder	中 Medium	否 No	叶 Leaf
7	平和芥菜 Pinghe Mustard	福建龙岩市 Fujian Longyan	直立 Upright	椭圆、微皱、无刺毛、有蜡粉 Oval, slightly wrinkled, spineless, wax powder	中 Medium	否 No	叶 Leaf
8	特选竹芥菜 Selected Bamboo Mustard	广东广州市 Guangdong Guangzhou	直立 Upright	阔椭圆、平滑、无刺毛、有蜡粉 Broad oval, smooth, spineless, wax powder	早 Early	否 No	叶 Leaf
9	龙岩长茎芥菜2 Longyan Long stem Mustard 2	北京市 Beijing	直立 Upright	卵圆形、皱、无刺毛、无蜡粉 Ovoid, wrinkled, spineless, no wax powder	中 Medium	否 No	茎 Stem
10	细叶雪里蕻 Spire Potherb Mustard	福建龙岩市 Fujian Longyan	半直立 Semi-upright	倒披针、平滑、无刺毛、有蜡粉 Inverted lanceolate, smooth, spineless, wax powder	中 Medium	否 No	叶 Leaf
11	龙岩芥菜3 Longyan Mustard 3	福建龙岩市 Fujian Longyan	半直立 Semi-upright	倒卵、皱、无刺毛、有蜡粉 Obovate, wrinkled, spineless, wax powder	中 Medium	否 No	茎 Stem
12	客家甜芥菜 Kejia Mustard	广东兴宁市 Guangdong Xingning	半直立 Semi-upright	阔椭圆、平滑、无刺毛、有蜡粉 Broad oval, smooth, spineless, wax powder	早 Early	否 No	叶 Leaf
13	四季甜客家芥 Four-season sweet hakka Mustard	江西宜春市 Jiangxi Yichun	直立 Upright	阔椭圆、平滑、无刺毛、有蜡粉 Broad oval, smooth, spineless, wax powder	早 Early	否 No	叶 Leaf
14	本地大菜 Local Mustard	广东兴宁市 Guangdong Xingning	直立 Upright	倒卵、皱、无刺毛、有蜡粉 Obovate, wrinkled, spineless, wax powder	早 Early	否 No	叶 Leaf
15	张白土茎用芥菜 Zhangbaitu stem Mustard	福建龙岩市 Fujian Longyan	直立 Upright	卵圆形、皱、无刺毛、无蜡粉 Ovoid, wrinkled, spineless, no wax powder	中 Medium	否 No	茎 Stem
16	漳州芥菜2 Zhangzhou Mustard	福建龙岩市 Fujian Longyan	直立 Upright	倒卵、皱、无刺毛、有蜡粉 Obovate, wrinkled, spineless, wax powder	早 Early	否 No	叶 Leaf
17	特选大坪埔大肉包心芥菜 Specially selected Dapingpu big meat stuffed mustard	广西横县 Guangxi Hengxian	开展 Expansion	扁圆、皱、无刺毛、无蜡粉 Oval, wrinkled, spineless, no wax powder	中 Medium	是 Yes	叶球 Leaf Bulb
18	广东鸡那菜 Guangdong jina Mustard	福建龙岩市 Fujian Longyan	直立 Upright	倒披针、微皱、无刺毛、有蜡粉 Inverted lanceolate, slightly wrinkled, spineless, wax powder	早 Early	否 No	叶 Leaf
19	美口宽杆芥菜 Meikou Mustard	甘肃省武威市 Gansu Wuwei	开展 Expansion	卵圆、皱、无刺毛、有蜡粉 Ovoid, wrinkled, spineless, wax powder	晚 Later	否 No	叶 Leaf
20	闽清大芥菜 Minqin big Mustard	福建福州市 Fujian Fuzhou	半直 Upright	倒卵、微皱、无刺毛、无蜡粉 Obovate, slightly wrinkled, spineless, no wax powder	中 Medium	否 No	叶 Leaf
21	精品大肉大坪包心芥 Premium Dapingpu big meat stuffed Mustard	江西宜春市 Jiangxi Yichun	开展 Expansion	扁圆、皱、无刺毛、有蜡粉 Oval, wrinkled, spineless, wax powder	中 Medium	是 Yes	叶球 Leaf Bulb
22	台湾芥菜1 Taiwan Mustard	福建龙岩市 Fujian Longyan	半直立 Semi-upright	长倒卵、平滑、无刺毛、无蜡粉 Long obovate, smooth, spineless, no wax powder	中 Medium	否 No	叶 Leaf
23	宽帮青菜 Wide petiole Mustard	四川省绵阳市 Sichuan Mianyang	开展 Expansion	阔椭圆、微皱、无刺毛、有蜡粉 Broad oval, slightly wrinkled, spineless, wax powder	中 Medium	否 No	叶 Leaf
24	四川大肉青 Sichuan big meat Mustard	四川省广汉市 Sichuan Guanghan	开展 Expansion	阔倒卵、皱、无刺毛、有蜡粉 Broad oval, wrinkled, spineless, wax powder	中 Medium	否 No	叶 Leaf
25	华513 Hua513	湖北武汉市 Hubei Wuhan	开展 Expansion	倒卵、微皱、有刺毛、无蜡粉 Obovate, slightly wrinkled, spine, no wax powder	中 Medium	否 No	叶 Leaf
26	华512 Hua512	湖北武汉市 Hubei Wuhan	开展 Expansion	阔椭圆、微皱、有刺毛、无蜡粉 Broad oval, slightly wrinkled, spine, no wax powder	中 Medium	否 No	叶 Leaf
27	华516 Hua516	湖北武汉市 Hubei Wuhan	开展 Expansion	阔椭圆、微皱、有刺毛、有蜡粉 Broad oval, slightly wrinkled, spine, wax powder	中 Medium	否 No	叶 Leaf
28	上杭毛芥菜 Shanghang Hairy Mustard	福建龙岩市 Fujian Longyan	半直立 Semi-upright	卵圆形、微皱、有刺毛、无蜡粉 Ovoid, slightly wrinkled, spine, no wax powder	中 Medium	否 No	叶球 Leaf Bulb
29	宽叶厚肉高脚芥 Broadleaf Thick Meat Tall Mustard	广东兴宁市 Guangdong Xingning	直立 Upright	倒卵、微皱、无刺毛、有蜡粉 Obovate, slightly wrinkled, spine, wax powder	中 Medium	否 No	叶 Leaf
30	连城芥菜 Liancheng Mustard	福建龙岩市 Fujian Longyan	直立 Upright	卵圆形、皱、无刺毛、有蜡粉 Ovoid, wrinkled, spineless, wax powder	中 Medium	否 No	茎 Stem
31	香港客家甜芥菜 Hong Kong Hakka Sweet Mustard	广东兴宁市 Guangdong Xingning	半直立 Semi-upright	阔椭圆、平滑、无刺毛、有蜡粉 Broad oval, smooth, spineless, wax powder	极早 Very Early	否 No	叶 Leaf
32	福州宽杆芥菜 Fuzhou Wide pole Mustard	福建福州 Fujian Fuzhou	半直立 Semi-upright	阔椭圆、皱、无刺毛、有蜡粉 Broad oval, slightly wrinkled, spineless, wax powder	中 Medium	否 No	叶 Leaf
33	闽清大挂菜 Minqing Large Mustard	福建福州 Fujian Fuzhou	半直立 Semi-upright	倒卵、微皱、无刺毛、有蜡粉 Obovate, slightly wrinkled, spine, wax powder	中 Medium	否 No	叶 Leaf
34	特选甜脆客家芥菜 Special sweet and crisp Hakka Mustard	广东深圳市 Guangdong Shenzhen	半直立 Semi-upright	阔椭圆、平滑、无刺毛、有蜡粉 Broad oval, smooth, spineless, wax powder	极早 Very Early	否 No	叶 Leaf
35	皱叶芥 Crinkle Mustard	福建龙岩市 Fujian Longyan	直立 Upright	阔椭圆、皱、无刺毛、无蜡粉 Broad oval, smooth, spineless, wax powder	中 Medium	是 Yes	叶球 Leaf Bulb
36	永定客家芥 Yongding Mustard	福建龙岩市 Fujian Longyan	直立 Upright	卵圆形、皱、无刺毛、有蜡粉 Ovoid, wrinkled, spineless, wax powder	中 Medium	否 No	茎 Stem
37	万安客家芥菜 Wanan Mustard	福建龙岩市 Fujian Longyan	直立 Upright	倒卵、微皱、无刺毛、有蜡粉 Obovate, slightly wrinkled, spine, wax powder	中 Medium	否 No	茎 Stem
38	桂林甜芥菜薹 Guilin Mustard Stalk	广西横县 Guangxi Hengxian	开展 Expansion	阔椭圆、平滑、无刺毛、无蜡粉 Broad oval, smooth, spineless, no wax powder	中 Medium	是 Yes	叶、花 Leaf and Flower 薹 Bolting
39	绿欣芥菜 Luxin Mustard	福建龙岩市 Fujian Longyan	直立 Upright	倒卵、皱、无刺毛、有蜡粉 Obovate, wrinkled, spine, wax powder	中 Medium	否 No	茎 Stem
40	龙芥1号 Long Mustard 1	福建龙岩市 Fujian Longyan	直立 Upright	卵圆形、皱、无刺毛、有蜡粉 Ovoid, wrinkled, spineless, wax powder	中 Medium	否 No	茎 Stem
41	大排芥菜 Dapai Mustard	福建龙岩市 Fujian Longyan	直立 Upright	阔椭圆、微皱、无刺毛、有蜡粉 Broad oval, slightly wrinkled, spineless, wax powder	中 Medium	是 Yes	叶球 Leaf Bulb
42	紫脉芥 Purple vein Mustard	福建龙岩市 Fujian Longyan	开展 Expansion	倒卵、皱、无刺毛、有蜡粉 Obovate, wrinkled, spine, wax powder	中 Medium	否 No	叶 Leaf
43	台湾芥菜2 Taiwan Mustard 2	福建龙岩市 Fujian Longyan	开展 Expansion	阔倒卵、微皱、无刺毛、有蜡粉 Broad oval, slightly wrinkled, spineless, wax powder	中 Medium	是 Yes	叶球 Leaf Bulb
44	龙岩长茎芥菜 Longyan Long stem Mustard	福建龙岩市 Fujian Longyan	直立 Upright	卵圆形、皱、无刺毛、有蜡粉 Ovoid, wrinkled, spineless, wax powder	中 Medium	否 No	茎 Stem

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表 2 芥菜SSR的类型分布

Table 2. Distribution of SSR types in B. juncea

SSR类型 SSR type	重复次数 Repeat number									总计 Total	比例/% Ratio
SSR类型 SSR type	4	5	6	7	8	9	10	11～14	≥ 15	总计 Total	比例/% Ratio
二核苷酸 Di	0	0	1 510	1 011	645	362	226	134	104	3 992	41.91
三核苷酸 Tri	0	3 108	1 185	403	78	20	32	29	15	4 870	51.12
四核苷酸 Tetra	273	55	17	5	1	0	1	0	0	308	3.70
五核苷酸 Penta	99	24	6	1	2	0	0	1	0	122	1.40
六核苷酸 Hexa	133	34	4	4	2	0	0	2	0	183	1.87
总计 Total	505	3 221	2 722	1 424	728	382	259	166	119	9 526
比例% Ratio	5.30	33.82	28.57	14.95	7.64	4.01	2.72	1.74	1.25		100

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表 3 芥菜转录中不同SSR重复基序出现的频率

Table 3. Frequency of different SSR repeat motifs in B. juncea transcriptome

SSR类型 SSR type	重复基序种类数 Repeat species	重复基序类型 Repeat motif type	数量 Number	发生频率/% Frequency	比例/% Proportion
二核苷酸 Di	4	AG/CT	3 309	34.74	82.89
		AC/GT	388	4.07	9.72
		AT/AT	291	3.05	7.29
		CG/CG	4	0.04	0..10
三核苷酸 Tri	10	AAG/CTT	1743	18.30	35.79
		AGG/CCT	827	8.68	16.98
		ATC/ATG	740	7.77	15.20
		AAC/GTT	461	4.84	9.47
		ACC/GGT	390	4.09	8.00
		AGC/CTG	320	3.36	6.57
		CCG/CGG	138	1.45	2.83
		ACG/CGT	94	0.99	1.93
		ACT/AGT	83	0.87	1.70
		AAT/ATT	74	0.78	1.52
四核苷酸 Tetra	25	AAAG/CTTT	74	0.78	24.02
		AAAC/GTTT	68	0.71	22.08
		AAGG/CCTT	41	0.43	12.99
		AATC/ATTG	40	0.43	12.99
		其他 Other	85	0.89	27.60
五核苷酸 Penta	41	AAGAG/CTCTT	14	0.15	11.48
		AATCG/ATTCG	13	0.14	10.66
		AAAAC/GTTTT	12	0.13	9.84
		其他 Other	83	0.87	68.03
六核苷酸 Hexa	92	AAGGAG/CCTTCT	15	0.16	8.20
		AAGAGG/CCTCTT	12	0.13	6.56
		AAAAAC/GTTTTT	10	0.10	5.46
		其他 Other	146	1.53	79.78

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表 4 芥菜17对SSR多态性引物信息

Table 4. Information on 17 pairs of SSR primers for studying polymorphism of B. juncea

引物名称 Primer name	引物序列（5′→3′） Primer sequence	SSR基元 SSR motif	产物长度/bp Length of product	多态条带数 Number of polymorphic bands	多态信息量 PIC
BaSSR002	TCACCGAGCTTCTTCGTCTT/CTCCTTTCTATTCGGTTGCG	（AG）9	235	3	0.54
BaSSR003	TGACTTGGGCCAAAGCTTAC/CACTTCTCTTTCGGGTCCAG	（AG）20	247	3	0.63
BaSSR007	CCTCTCCGAATCAAGTCTCG/TGTTAAACCTCAACCTCGCA	（AG）9	286	1	0.43
BaSSR009	GGGAACATACATATCAGATTGTGG/TTCCCACAGAAGCCATTCTC	（AG）23	225	3	0.67
BaSSR012	ACGAAACCCCAGAAGAAAGC/GATCATTCCTCCCCTCTTCC	（AGA）7	181	3	0.71
BaSSR013	AAAGTGGAACCCTGTGAACG/AATCGGCGGTAACTCCTTCT	（GAA）8	264	2	0.58
BaSSR015	TGGATAAGGGAACAAGGCTG/GAAGATTCACGAGCTCCAGG	（GCT）8	241	3	0.58
BaSSR016	AGCACCTCACCAAACTGGAC/CAACAGTTCCAGCAGCTTCA	（TGC）8	169	2	0.53
BaSSR019	GAAGTCTTTGCGATTGAGAAAAA/TCCCATTGTACACGACCCTT	（GAA）15	280	4	0.72
BaSSR020	CAAAAAGTGGGTTCGAGGAG/CGCAAAACTCTTCCTTCCAC	（AAG）7	238	3	0.71
BaSSR022	CATCACCTAACCGATTGCCT/TCTTGTTTCTGTGGCGTGAG	（AACC）6	131	3	0.70
BaSSR023	GTCGAGAGAACCTTTCGTGC/GCCCATATCAGCCTCGTTTA	（TTTC）7	257	2	0.51
BaSSR032	TAAGGAAGGCGAAAGTTGGA/AGAGAATCAGCTTCCCGTCA	（TTGGT）7	249	2	0.53
BaSSR037	GATTCTCTTTCGCGATTCGT/ATCTTGTTTTCGCCCTGACA	（TTTGG）6	278	2	0.53
BaSSR043	TAGCTGCAGCAACAATCGTC/GATGTGCAAGCAAGGATTGA	（AAAAG）5	217	2	0.47
BaSSR045	TCCATCTGAACGGTTCTTTG/CATAGATCCATTGCTTAGCGG	（CTGTTA）6	185	2	0.50
BaSSR046	CACGAATGTGAAGCAGCAGT/GTAGCCAGGTCGTGGTTGTT	（CACCCA）5	277	6	0.82

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参考文献(35)

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