大豆长片段插入/缺失标记的开发与应用

doi:10.7668/hbnxb.20192459

华北农学报 ›› 2022, Vol. 37 ›› Issue (1): 18-26. doi: 10.7668/hbnxb.20192459

所属专题： 油料作物；生物技术；

• 作物遗传育种·种质资源·生物技术 • 上一篇下一篇

大豆长片段插入/缺失标记的开发与应用

李曼¹^,², 史晓蕾², 邸锐², 刘志芳³, 孟庆民², 付才⁴, 杨春燕², 王冬梅¹, 张孟臣², 张洁¹, 闫龙²

1.华北作物改良与调控国家重点实验室,河北省植物生理与分子病理学重点实验室,河北农业大学生命科学学院,河北保定 071001
2.河北省农林科学院粮油作物研究所,国家大豆改良中心石家庄分中心,农业农村部黄淮海大豆生物学与遗传育种重点实验室,河北省作物遗传育种实验室,河北石家庄 050035
3.河北省种子总站,河北石家庄 050031
4.河北省农林科学院遗传生理研究所,河北石家庄 050051

收稿日期:2021-06-23 出版日期:2022-02-28
作者简介:
作者简介:李曼(1994—),女,河北唐山人,在读博士,主要从事分子育种研究。李曼、史晓蕾为同等贡献作者。
基金资助:
河北省农林科学院创新工程项目(2019-4-3-2)

Development and Application of Large-size Insertion/Deletion Markers in Soybean

LI Man¹^,², SHI Xiaolei², DI Rui², LIU Zhifang³, MENG Qingmin², FU Cai⁴, YANG Chunyan², WANG Dongmei¹, ZHANG Mengchen², ZHANG Jie¹, YAN Long²

1. State Key Laboratory of North China Crop Improvement and Regulation,Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology,College of Life Sciences,Hebei Agricultural University,Baoding 071001,China
2. Institute of Cereal and Oil Crops,Hebei Academy of Agriculture and Forestry Sciences,Shijiazhuang Branch Center of National Center for Soybean Improvement, Huang-Huai-Hai Key Laboratory of Biology and Genetic Improvement of Soybean,Ministry of Agriculture and Rural Affairs,Laboratory of Crop Genetics and Breeding in Hebei,Shijiazhuang 050035,China
3. Hebei Seed Station,Shijiazhuang 050031,China
4. Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences,Shijiazhuang 050051,China

Received:2021-06-23 Published:2022-02-28

摘要/Abstract

摘要：

为了开发稳定可靠、操作简便的大豆分子标记,以12份地理来源不同的大豆种质资源为材料,通过基因组10倍深度重测序开发长片段插入/缺失(InDel)标记,并应用2018年黄淮海大豆多点鉴定96份参试品系DNA指纹图谱构建。结果表明,在参试材料中,共检测到插入/缺失片段长度大于20 bp的InDel标记66 561个,平均每条染色体InDel标记的数量为3 262个;位于内含子的InDel占比12.35%,位于基因上游序列和下游序列的InDel占比分别为25.83%,20.44%,位于基因间隔区的InDel占比34.39%,位于外显子的InDel占比0.19%,位于5'-UTR和3'-UTR的InDel占比分别为0.93%和1.51%;片段长度介于20~40 bp的InDel数量为42 453个,41~60 bp为13 044个,61~80 bp为5 034个,81~100 bp为2 285个,大于100 bp为2 413个;从检测到的66 561个InDel位点中,根据插入/缺失片段长度,在基因组中随机选区160个InDel位点,利用引物设计、PCR和琼脂糖凝胶电泳等技术,在55 ℃的退火温度条件下,开发出32个有且仅有2个等位变异、基于1%琼脂糖凝胶电泳技术易于分辨的长片段插入/缺失标记。应用新开发的32个标记,构建了2018年黄淮海大豆多点鉴定96个参试品系DNA指纹图谱,参试的大豆材料纯度为96.84%,没有同物异名现象发生。研究开发的InDel标记稳定可靠、操作简便,可应用于大豆DNA指纹图谱构建、种子纯度检测等工作。

关键词: 大豆, 分子标记, 长片段插入/缺失标记, 遗传多样性分析

Abstract:

The objective of this work was to develop novel,convenient,and reliable molecular marker in soybean.Twelve soybean germplasm accessions from different geography region were used to develop large-size InDel markers based on 10 depth genomic re-sequencing.Developed InDel markers were used to construct DNA fingerprint of 96 varieties in Huanghuaihai Uniform Test in 2018.The results showed that a total of 66 561 InDel markers,with insertion/deletion fragment size larger than 20 bp,were discovered among the 12 accessions.The distribution pattern among the genome of these InDel markers were also illustrated.The ratio of the InDel in intron,upstream of the gene,downstream of the gene,intergenic region,intragenic region,5'-UTR and 3'-UTR were 12.35%,25.83%,20.44%,34.39%,0.19%,0.93%,1.51%,respectively.There were 42 453 InDels yield insertion/deletion fragment size between 20 to 40 bp,13 044 InDels between 41—60 bp,5 034 InDels between 61—80 bp,2 285 InDels between 81—100 bp,and 2 413 InDels larger than 100 bp.Then,160 InDel markers were randomly selected according to the insertion/deletion fragment size throughout the whole genome.As results,thirty-two InDel markers,that only yield 2 alleles in for each locus and could be distinguished easily on agarose gel,were developed and validated through primer design,PCR and agarose gel electrophoresis.Additionally,DNA fingerprint of 96 varieties in Huanghuaihai Uniform Test in 2018,were constructed using the 32 developed InDel markers.As results,the average purity of the tested soybean varieties was 96.84%,and no varieties shared the same name.The InDel markers developed in this study were stable,reliable and user friendly.They could be used to construct DNA fingerprint and test seed purity of soybean.

Key words: Soybean, Molecular markers, Large size insertion/deletion marker, Genetic diversity analysis

李曼, 史晓蕾, 邸锐, 刘志芳, 孟庆民, 付才, 杨春燕, 王冬梅, 张孟臣, 张洁, 闫龙. 大豆长片段插入/缺失标记的开发与应用[J]. 华北农学报, 2022, 37(1): 18-26. doi: 10.7668/hbnxb.20192459.

LI Man, SHI Xiaolei, DI Rui, LIU Zhifang, MENG Qingmin, FU Cai, YANG Chunyan, WANG Dongmei, ZHANG Mengchen, ZHANG Jie, YAN Long. Development and Application of Large-size Insertion/Deletion Markers in Soybean[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 18-26. doi: 10.7668/hbnxb.20192459.

http://www.hbnxb.net/CN/Y2022/V37/I1/18

图/表 8

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类型 Type	插入/缺失数目/个 InDel No.
基因间隔区(无转录本信息)Intergenic region	22 893
外显子Exon	125
内含子Intron	8 222
基因上游区域Upstream	17 190
基因下游区域Downstream	13 602
5'非翻译区 5'-UTR	617
3'非翻译区 3'-UTR	1 004

类型 Type	插入/缺失数目/个 InDel No.
基因间隔区(无转录本信息)Intergenic region	22 893
外显子Exon	125
内含子Intron	8 222
基因上游区域Upstream	17 190
基因下游区域Downstream	13 602
5'非翻译区 5'-UTR	617
3'非翻译区 3'-UTR	1 004

名称 Name	染色体 Chromosome	位置 Position	上游引物序列 Forward sequence	下游引物序列 Reverse sequence	多态性信息含量 PIC
InDel-1-7	1	38601796	ATTCTCAAGGTAAGGGATGG	ACCAAATTGTTCACCTCACG	0.354
InDel-1-8	1	50513481	AAACACTCACTCTCCACAAC	ATTTCACTTCCCCCTGCGAT	0.305
InDel-2-1	2	22242181	GCAATGTCCAACCTTTTCTT	CTAGGTTGTGGGTCAGGTCA	0.325
InDel-2-8	2	2237743	GTTGCTTTCTTTCACTATGC	GGTCAGTAGCACAACAATAG	0.370
InDel-3-4	3	30874756	GGCAGTTATCCAACACACTC	GGTATCGGGTCCTTTCTAAA	0.280
InDel-4-4	4	48358162	CGTCGCATAACCAATGTAGT	CCTTCCCTTTCTATTTGCAA	0.280
InDel-5-2	5	34356071	ATAAGAATGCCCAAACGAAG	GCAACAAAAGAAAGGAAGTG	0.370
InDel-5-5	5	39857792	CGAACCATGTAGAGGAAAAT	GAAAGACAACTTTCATCCAC	0.370
InDel-6-1	6	20039692	GTTTGCACCATCTCCCGCTT	GCCATTAGGGACAAAGTCTT	0.354
InDel-6-4	6	10896943	AGTCTCTTCTCCAATCGTTG	GAGAACCTACACCTCAAACG	0.341
InDel-7-3	7	17704210	ATAGGGGCAAATGGTCATTC	GGCCGCTAAATGACAACGTA	0.363
InDel-8-1	8	47711883	CCAACGAAAATAGGGAGCAG	CAGGCAGTATGCTGAGTGAA	0.215
InDel-9-4	9	49917537	TAGTGTTAAATGGGAGAGCC	CTTTATGCTCGGTAACCTGT	0.280
InDel-9-8	9	29913558	TGCTTCTTATCCTTCCCATC	GGGTGTTAGAGCTTGTATTA	0.280
InDel-10-5	10	49646363	ACCATTTTCACCATGCCTAG	GGTTGTACGTACTCCCTTTA	0.370
InDel-10-6	10	11287611	TCAACACTTCTCAGGTATTG	GTACTAAATCCTCCCTAACC	0.354
InDel-11-7	11	15581830	TCAGACAGCCATTTTGCTAG	CAATTGTGATGTAGGGTTCA	0.215
InDel-12-7	12	11949731	CGAGCAACGGTCTTCTGCAT	CATCTCCATAAGCAACAGTG	0.325
InDel-13-1	13	31358622	TGTGGAAAAGCTCAAAATCC	AAAATCCGTAATGGAATGGG	0.325
InDel-13-4	13	37748838	CCCTGCTAACAAAAACTAAC	CAGACACACTATTCATTTGC	0.354
InDel-14-1	14	4424007	GCAATAACATCACCAGCGAG	CATGGAAGTCTAATACACAG	0.375
InDel-14-3	14	43544210	ACCCCTCAGGTGTGTAAAAG	CTTAGCCTTAGATAACGACA	0.325
InDel-15-4	15	15360421	AACTGCCCTGTTATTTTCCT	GTGTTGGGTAGTAAATTGGG	0.370
InDel-16-2	16	25311303	TATGACAAAGGGTGGGCGAT	GGGGCTCTCCAATCAGGTTT	0.354
InDel-16-3	16	156095	ACACAGCCTTAGTAGTTAGT	ATCAAACCACACTTAGGATG	0.354
InDel-16-5	16	31016663	TACTGCTGGGGAACAATGTG	CTGATTTGAAGTTGCTGAAC	0.354
InDel-16-7	16	29753588	CTATGCTATGCTAACTACAC	GACCTCATCCAATGCGATCC	0.325
InDel-17-1	17	39593964	GTTCTTCTTTGGTTGTGTTG	GGATATTTTGACCTTACAGC	0.215
InDel-18-3	18	51025175	GAAACTACCCAATCGAGATC	GGACTTTGGTTAGACTGTTT	0.354
InDel-19-4	19	41727589	TGCTATTTCAAGGAGAGAAG	CCGGAGAGGAAAAGAATAAA	0.215
InDel-19-8	19	8157745	AGCACACTCTAACACATTCT	AATGGCCTGATACAATAGCT	0.250
InDel-20-1	20	36027621	AAATAAGCCATACACAGTGC	AGAGCTATCACAAAAACCAG	0.305

名称 Name	染色体 Chromosome	位置 Position	上游引物序列 Forward sequence	下游引物序列 Reverse sequence	多态性信息含量 PIC
InDel-1-7	1	38601796	ATTCTCAAGGTAAGGGATGG	ACCAAATTGTTCACCTCACG	0.354
InDel-1-8	1	50513481	AAACACTCACTCTCCACAAC	ATTTCACTTCCCCCTGCGAT	0.305
InDel-2-1	2	22242181	GCAATGTCCAACCTTTTCTT	CTAGGTTGTGGGTCAGGTCA	0.325
InDel-2-8	2	2237743	GTTGCTTTCTTTCACTATGC	GGTCAGTAGCACAACAATAG	0.370
InDel-3-4	3	30874756	GGCAGTTATCCAACACACTC	GGTATCGGGTCCTTTCTAAA	0.280
InDel-4-4	4	48358162	CGTCGCATAACCAATGTAGT	CCTTCCCTTTCTATTTGCAA	0.280
InDel-5-2	5	34356071	ATAAGAATGCCCAAACGAAG	GCAACAAAAGAAAGGAAGTG	0.370
InDel-5-5	5	39857792	CGAACCATGTAGAGGAAAAT	GAAAGACAACTTTCATCCAC	0.370
InDel-6-1	6	20039692	GTTTGCACCATCTCCCGCTT	GCCATTAGGGACAAAGTCTT	0.354
InDel-6-4	6	10896943	AGTCTCTTCTCCAATCGTTG	GAGAACCTACACCTCAAACG	0.341
InDel-7-3	7	17704210	ATAGGGGCAAATGGTCATTC	GGCCGCTAAATGACAACGTA	0.363
InDel-8-1	8	47711883	CCAACGAAAATAGGGAGCAG	CAGGCAGTATGCTGAGTGAA	0.215
InDel-9-4	9	49917537	TAGTGTTAAATGGGAGAGCC	CTTTATGCTCGGTAACCTGT	0.280
InDel-9-8	9	29913558	TGCTTCTTATCCTTCCCATC	GGGTGTTAGAGCTTGTATTA	0.280
InDel-10-5	10	49646363	ACCATTTTCACCATGCCTAG	GGTTGTACGTACTCCCTTTA	0.370
InDel-10-6	10	11287611	TCAACACTTCTCAGGTATTG	GTACTAAATCCTCCCTAACC	0.354
InDel-11-7	11	15581830	TCAGACAGCCATTTTGCTAG	CAATTGTGATGTAGGGTTCA	0.215
InDel-12-7	12	11949731	CGAGCAACGGTCTTCTGCAT	CATCTCCATAAGCAACAGTG	0.325
InDel-13-1	13	31358622	TGTGGAAAAGCTCAAAATCC	AAAATCCGTAATGGAATGGG	0.325
InDel-13-4	13	37748838	CCCTGCTAACAAAAACTAAC	CAGACACACTATTCATTTGC	0.354
InDel-14-1	14	4424007	GCAATAACATCACCAGCGAG	CATGGAAGTCTAATACACAG	0.375
InDel-14-3	14	43544210	ACCCCTCAGGTGTGTAAAAG	CTTAGCCTTAGATAACGACA	0.325
InDel-15-4	15	15360421	AACTGCCCTGTTATTTTCCT	GTGTTGGGTAGTAAATTGGG	0.370
InDel-16-2	16	25311303	TATGACAAAGGGTGGGCGAT	GGGGCTCTCCAATCAGGTTT	0.354
InDel-16-3	16	156095	ACACAGCCTTAGTAGTTAGT	ATCAAACCACACTTAGGATG	0.354
InDel-16-5	16	31016663	TACTGCTGGGGAACAATGTG	CTGATTTGAAGTTGCTGAAC	0.354
InDel-16-7	16	29753588	CTATGCTATGCTAACTACAC	GACCTCATCCAATGCGATCC	0.325
InDel-17-1	17	39593964	GTTCTTCTTTGGTTGTGTTG	GGATATTTTGACCTTACAGC	0.215
InDel-18-3	18	51025175	GAAACTACCCAATCGAGATC	GGACTTTGGTTAGACTGTTT	0.354
InDel-19-4	19	41727589	TGCTATTTCAAGGAGAGAAG	CCGGAGAGGAAAAGAATAAA	0.215
InDel-19-8	19	8157745	AGCACACTCTAACACATTCT	AATGGCCTGATACAATAGCT	0.250
InDel-20-1	20	36027621	AAATAAGCCATACACAGTGC	AGAGCTATCACAAAAACCAG	0.305

材料名称 Material name	指纹代码 Fingerprint code	材料名称 Material name	指纹代码 Fingerprint code
洛1408 Luo 1408	00011001010100000101101001100001	洛1337 Luo 1337	00101110010101100011011011100011
洛1407 Luo 1407	01111000011110000001110111011010	洛1330 Luo 1330	00111100001109100100111111010111
陕豆1号Shandou 1	00111101001110000111001119000101	中黄104 Zhonghuang 104	00011001011110100000101000101119
中品16067 Zhongpin 16067	00911001010110000011100990011100	中黄105 Zhonghuang 105	00011100010111100010011000011110
中品1184 Zhongpin 1184	00111001000111000101111110090010	中作11-11 Zhongzuo 11-11	00111110110111011001911111101011
廊轮选4号Langlunxuan 4	00111010011111100111000011101111	中作11-54 Zhongzuo 11-54	00011101001111000110001111001111
廊轮选2号Langlunxuan 2	01111190011101000911010001110001	潍豆1897 Weidou 1897	00110110001111000111111111010000
HN2018	00111110000111100011111001111000	石16S379 Shi 16S379	00111110010101010011001010111101
中作J161087 Zhongzuo J161087	00011001010110100000101001111010	安豆6023 Andou 6023	111111119101111011111111110111001
科豆26 Kedou 26	01111019000110000010010009111011	濉科57 Suike 57	00011011010111100010010001010111
中作12-1251 Zhongzuo 12-1251	00111011010110100000110009011010	邯15-685 Han 15-685	00011001000111000010110111000101
沧豆1123 Cangdou 1123	00011001010100100100000110000110	邯豆5号Handou 5	00111001000111000010011111100001
沧豆1312 Cangdou 1312	01111110010111100111011110010110	邯13-109 Han 13-109	00111110110111110011001111101011
冀1708 Ji 1708	00101110110111100111010001111010	冀1707 Ji 1707	00111001010110000100001111100101
冀豆12 Jidou 12	01119000010110000110010001010110	沧豆11 Cangdou 11	00111010010111000111011111100000
冀1702 Ji 1702	10111111111111111001111001111000	沧豆09Y1 Cangdou 09Y1	00111111001111000110011000111110
冀1701 Ji 1701	00111000001111000101010000011110	周12102-4 Zhou 12102-4	00110101011100000100001001111010
邯16-139 Han 16-139	11111001010101000111110111100010	商豆1310 Shangdou 1310	00011001010101100111000000111010
邯15-324 Han 15-324	00101111100001100111111000011110	商豆161 Shangdou 161	01101110010111000911101111110001
濉科56 Suike 56	00101100001111000110111111100001	秦豆2017 Qindou 2017	00111110011101000119011001110111
濉科55 Suike 55	00111110011191000010011000010010	秦豆220 Qindou 220	00111111011910100000001111000101
安豆6223 Andou 6223	00111110011011111000011111110101	中黄320 Zhonghuang 320	00110010011111110190101111000000
安豆331 Andou 331	00111110111101100100011000010111	菏豆44 Hedou 44	00110100001110000110101000011190
晋遗59 Jinyi 59	00011010010101119011001111100010	濮豆5136 Pudou 5136	11109111010111000111911111000101
晋遗57 Jinyi 57	00010110010101110011001000101111	科豆18 Kedou 18	00111110010111100100110110000010
科豆31 Kedou 31	01911110010111010191010000011110	中作11-518 Zhongzuo 11-518	10111110110019010011111111110001
中黄103 Zhonghuang 103	01111111011111110191111000011101	中作11-719 Zhongzuo 11-719	00110000011100100110000001010111
中作08-08 Zhongzuo 08-08	00100110010111111111011111100000	郑1677 Zheng 1677	00011111010111101110011000101011
中作11-58 Zhongzuo 11-58	11001110011111100111111111100011	郑16175 Zheng 16175	00011000001001100010911111000010
潍豆1897 Weidou 1897	00100119010001100110011111111011	济J14187 JiJ14187	00110001000110100010000100010001
材料名称 Material name	指纹代码 Fingerprint code	材料名称 Material name	指纹代码 Fingerprint code
潍豆12 Weidou 12	10110111110100110110111000010101	汾豆104 Fendou 104	00111110110100001000011111001110
石12503 Shi 12503	00110110001100010011011111010101	济G15073 JiG15073	11919110001110110111910000101101
石11893 Shi 11893	00100100111111100101011111900000	汾豆103 Fendou 103	00111101911111000010000000111110
圣豆102 Shengdou 102	00011000010110000110000000011001	永育1号Yongyu 1	10111000010101111111111111000009
圣豆103 Shengdou 103	11101111010101111111111001111000	鲁0540-4 Lu 0540-4	01101110010111000011101110001010
华豆30 Huadou 30	01111110111111010101111010111110	HN1030	00011101001001100111111111000101
道秋37 Daoqiu 37	00111910011111001001001001111010	晋科3号Jinke 3	11111001011191000911110000101011
漯豆4904 Luodou 4904	11111110110111110001011111100010	晋黄11号Jinhuang 11	0010101000000100011010100001111
漯豆7901 Luodou 7901	00001100011111009110111111101010	晋黄12号Jinhuang 12	11111110011001100110011111000111
华豆21 Huadou 21	11111100111111111110111111001010	安豆109 Andou 109	11111111011011110110111000101000
圣育5号Shengyu 5	00110110100111911001009101110101	皖豆920 Wandou 920	00111000000011000010100111010101
圣育6号Shengyu 6	00111110010100000119111010011110	濉科58 Suike 58	00111100010901010111011000011101
华育2号Huayu 2	00111110010100090110011000100111	商豆18 Shangdou 18	00111000000110000010001111110000
道秋6号Daoqiu 6	00011001011110100000901111001111	陕豆3号Shandou 3	00111000011901000110001111000011
泛豆13 Fandou 13	00111001000111000010010001011100	鲁0126 Lu 0126	99111110110111100111011111000101
泛豆19 Fandou 19	01110011010110900010101111000900	邯617 Han 617	01101110110110100100111000101110
鲁Lh284 LuLh284	00911110010190000111111111000001	安豆115 Andou 115	11111011010110100111001111000001
陕豆2号Shandou 2	10101110110111100110011100101011	安豆5451 Andou 5451	00011000001101000010000900001111

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