基于BSA-seq技术对豌豆花色基因的精细定位

doi:10.3724/SP.J.1006.2023.24055

作物学报 ›› 2023, Vol. 49 ›› Issue (4): 1006-1015.doi: 10.3724/SP.J.1006.2023.24055

基于BSA-seq技术对豌豆花色基因的精细定位

严昕^1,**(), 项超^2,**(), 刘荣¹, 李冠¹, 李孟伟¹, 李正丽³, 宗绪晓¹^,^*(), 杨涛¹^,^*()

¹中国农业科学院作物科学研究所, 北京 100081
²四川省农业科学院作物研究所, 四川成都 610066
³贵州省农业科学院园艺研究所, 贵州贵阳 550006

收稿日期:2022-03-10 接受日期:2022-07-21 出版日期:2023-04-12 网络出版日期:2022-08-22
通讯作者: *杨涛, E-mail: yangtao02@caas.cn;宗绪晓, E-mail: zongxuxiao@caas.cn
作者简介:严昕, E-mail: yanxin5290@163.com;
项超, E-mail: xc2011cib@163.com
^**同等贡献
基金资助:
国家作物种质资源库-食用豆资源整合与共享项目(NCGRC-2021-07);普查收集食用豆资源鉴定评价与繁殖编目入库项目(19210867);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-08-G11)

Fine mapping of flower colour gene in pea (Pisum sativum L.) based on BSA-seq technique

YAN Xin^1,**(), XIANG Chao^2,**(), LIU Rong¹, LI Guan¹, LI Meng-Wei¹, LI Zheng-Li³, ZONG Xu-Xiao¹^,^*(), YANG Tao¹^,^*()

¹Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
²Institute of Crop Sciences, Sichuan Academy of Agricultural Sciences, Chengdu 610066, Sichuan, China
³Institute of Horticulture, Guizhou Academy of Agricultural Sciences, Guiyang 550006, Guizhou, China

Received:2022-03-10 Accepted:2022-07-21 Published:2023-04-12 Published online:2022-08-22
Contact: *E-mail: yangtao02@caas.cn;E-mail: zongxuxiao@caas.cn
About author:^**Contributed equally to this work
Supported by:
National Infrastructure for Crop Germplasm Resources Project from the Ministry of Science and Technology of China(NCGRC-2021-07);Project of Identification, Evaluation, Replication, and Preservation of Food Legumes Collected by the Survey and Collection Action on Crop Germplasm Resources(19210867);China Agriculture Research System of MOF and MARA(CARS-08-G11)

摘要/Abstract

摘要：

BSA-seq技术在挖掘农艺性状相关的新基因中已被广泛应用, 随着豌豆首个参考基因组问世, 将BSA-seq技术结合豌豆基因组的基因定位策略势在必行。本研究利用紫花亲本G0004562、白花亲本G0002930以及F₂群体, 通过BSA-seq技术对豌豆花色基因进行初步定位, 获得31.42 Mb定位区间, 再通过设计InDel分子标记分析进一步缩小定位区间, 最终将目标基因定位在包含19个基因的0.99 Mb区间内, 通过基因注释信息推测出Psat6g060480.1为豌豆花色候选基因。本研究结果验证了BSA-seq技术快速高效定位豌豆花色基因的可行性, 为利用该技术挖掘豌豆其他重要农艺性状相关基因奠定了基础。

关键词: 豌豆, BSA-seq, InDel, 基因定位

Abstract:

In recent years, BSA-seq technology has been widely used in the mining of new genes related to agronomic traits. With the development of the first reference genome of pea, it is imperative to combine BSA-seq method with genome-wide sequencing for gene mapping. In this study, we used purple flower parent G0004562, white flower parent G0002930, and F₂ populations for preliminarily locate the target genes controlling flower color by BSA-seq technology, and a mapping region of 31.42 Mb was obtained. Then, the InDel molecular markers were designed to further narrow the mapping interval, and finally the target gene was located in the range of 0.99 Mb with 19 genes. Based on gene annotation, Psat6g060480.1 was considered as the candidate gene that controled the flower color. The results of this study verified the feasibility of gene mapping by BSA-seq technology in pea.

Key words: pea, BSA-seq, InDel, gene mapping

严昕, 项超, 刘荣, 李冠, 李孟伟, 李正丽, 宗绪晓, 杨涛. 基于BSA-seq技术对豌豆花色基因的精细定位[J]. 作物学报, 2023, 49(4): 1006-1015.

YAN Xin, XIANG Chao, LIU Rong, LI Guan, LI Meng-Wei, LI Zheng-Li, ZONG Xu-Xiao, YANG Tao. Fine mapping of flower colour gene in pea (Pisum sativum L.) based on BSA-seq technique[J]. Acta Agronomica Sinica, 2023, 49(4): 1006-1015.

图/表 9

表1

表2

图1

图2

表3

表4

精细定位所用多态性分子标记"

位置 Position	引物名称 Primer name	正向引物 Forward sequence (5-3°)	反向引物 Reverse sequence (5-3°)
62,185,053	WDBH141-1	TGATTCCAAGGGCCAATGACA	GCACGAAGCTAGCTAGTGAATAA
62,390,612	WDBH144-1	TTGTTCCACCACTGCAACCT	TGGATTTACCCGCAGTTGGT
62,698,646	WDBH147-1	TGAAACGCCCCTTATTGAAGT	TCTTCGAGTTCGGCTTTGCT
位置 Position	引物名称 Primer name	正向引物 Forward sequence (5-3°)	反向引物 Reverse sequence (5-3°)
64,421,149	WDBH172-1	ACCCAAAGCCACCAAAGAGAA	TGACACAACTGTTTGACACCA
64,707,966	WDBH008-3	ATGAGGTGCCCCTATGTGAG	TAATTGGTGGTGGTGAGATGGG
65,304,246	WDBH028-1	AGTCACGGCAATGGATGGAA	ACATGTGCAAGCGACCTTCA
65,627,591	WDBH179-2	GGTGGGATGTATCTGCTGACC	TCTGGTAGGAACACAAGTGGTTA
66,874,335	WDBH183-1	AACTGTGAGGAATGTCTATCCCA	AGGTTTGCTCCCATTTGTCAC
66,960,275	WDBH013-2	GTTCGGTCTAGTGTGCTCGT	TGCCAGCTGTACCATAGCAAA
67,036,795	WDBH194-1	ACTGTGTACACCCTTGCACT	TCCATATTCTAGCCAACACCACC
67,579,583	WDBH187-1	CGGTCCAACTGAGATGGCAA	TGGCTAGGGCTATAGGTATGAAGT
67,645,017	WDBH209-1	TGGGAGTTTGAAGTTGAGGGG	TCCCTAAAATGAGGGAACTCAAA
67,998,027	WDBH084-1	GTGCAGGCTTGAGGGTTTTG	TGTGTTCCCCAGTGTGTATCTC
68,240,556	WDBH200-1	TGTGGTGAGGAAATCGACGAA	CCGAAACATGAGCGCGTAAC
68,307,829	WDBH113-1	TTTTCCTGTCCTGGCGTGAC	AGGGTTGGGCGGTAATTCAA
68,309,277	WDBH090-1	GCTTCTTTGGCAGCGTTTGT	TACACAATCCCGAGTCATGGT
68,551,555	WDBH029-1	TAGGGTTGCATGGGTTGCTT	CACAAGTTGCTGCACACATTTC
68,566,772	WDBH220-1	CCATAGACTAAAGTTGAGACGAGC	GATTGTGCCCCTGGAAGTGA
68,673,374	WDBH133-1	TCCCACAAGCCATGTCCAAA	CGTGCACGAGAACGAAGGTT
69,494,713	WDBH129-1	CAGAACATGGCCAGGATCAATG	TGCAGAGGCGTGTCTTTCAA
69,965,980	WDBH051-1	GAGATGCAGTGAAATCGCCG	CAATTGGGGTGATGCACTCG
70,060,557	WDBH104-1	TCACCTCCATCATTTAAGCGGA	ACATCGCGCGTCTCAATCTA
70,411,248	WDBH053-1	CCGATCCGCAGGTTGGAAAA	GTGTGCAGACGAACCGATTT
72,140,477	WDBH059-1	AGTCCAATGGTCTGATAGCGT	TTGTGCGAACCTCTTTCTGC
73,347,184	WDBH063-1	GAAAAACGCGAGCAGAGAGC	TCCGAGTGGTCCGAGTATGT
75,496,657	WDBH032-1	ACGGAGGTCAGAGGCTACAA	CGGCATAGGTTGACACATACG
75,976,029	WDBH080-1	TGGCCAATTCCCAAGCTCAA	TTTGGGCTAGATGCGGAGAC
84,516,982	WDBH109-1	ACACGTTGATGAAAATGGGCG	ATTTTGGTCTCCGACAGCGT
84,765,375	WDBH037-1	TTGCCCCTAGACGGTAGTGA	TTTCCTGAGCCCACACATGG
86,084,668	WDBH011-2	CGGGTACCACGGGAATATACG	GTTCGGCGGCGAATATGTTT
92,203,258	WDBH044-1	GCCACCTATTCGTCCATCGT	AAGGGGACAAAGCACCCTTC

表4

图3

附表1

InDel标记检测到的交换单株统计结果"

InDel标记 InDel markers	F₂交换单株F₂ exchange individual plant
InDel标记 InDel markers	003	013	014	034	038	044	049	057	079	082	089	094	099	115	122	123	131	148	186	198	206	216	240	242	250	254	279	285	300	302	320
WDBH141-1	+	+	-	+	-	+	-	-	+	+	+	+	+	-	+	+	-	-	-	-	+	-	-	+	-	-	-	-	-	+	-
WDBH144-1	+	+	-	+	-	+	-	-	+	+		+	+	-	+	+	-	-	-	-	+	-	-	+	-	-	-	-	-	+	-
WDBH147-1	+	+	-	+	-	+	-	-	+	+	+	+	+	-	+	+	-	-	-	-	+	-	-	+	-	-	-	-	-	+	-
WDBH172-1	+	-	-	+	-	-	-	-	-	-	-	+	-	-	+	+	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-
WDBH008-3	+	-	-	+	-	-	-	-	-	-	-	+	-	-	+	+	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-
WDBH028-1	+	-	-	+	-	-	-	-	-	-	-	+	-	-	+	+	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-
WDBH179-2	+	-	-	-	-	-	-	-	-	-	-	+	-	-	-	+	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-
WDBH183-1	+	-	-	-	-	-	-	-	-	-	-	+	-	-	-	+	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-
WDBH013-2	+	-	-	-	-	-	-	-	-	-	-	+	-	-	-	+	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-
WDBH194-1	+	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-
WDBH187-1	+	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-
WDBH209-1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
WDBH084-1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
WDBH200-1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
WDBH113-1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
WDBH090-1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
WDBH029-1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
WDBH220-1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-	-	-	-	-
WDBH133-1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	+	-	-	-	-	-	-	-	-	-	-	-	-	-	-
WDBH129-1	-	-	+	-	-	-	-	+	-	-	-	-	-	-	-	-	+	+	-	-	-	-	+	-	+	-	-	-	-	-	-
WDBH051-1	-	-	+	-	-	-	-	+	-	-	-	-	-	-	-	-	+	+	-	-	-	-	+	-	+	-	-	-	-	-	-
WDBH104-1	-	-	+	-	-	-	-	+	-	-	-	-	-	-	-	-	+	+	-	+	-	-	+	-	+	-	-	-	+	-	-
WDBH053-1	-	-	+	-	-	-	-	+	-	-	-	-	-	-	-	-	+	+	-	+	-	-	+	-	+	-	-	-	+	-	-
WDBH059-1	-	-	+	-	-	-	+	+	-	-	-	-	-	+	-	-	+	+	-	+	-	-	+	-	+	-	-	+	+	-	-
WDBH063-1	-	-	+	-	-	-	+	+	-	-	-	-	-	+	-	-	+	+	-	+	-	-	+	-	+	-	-	+	+	-	-
WDBH032-1	-	-	+	-	+	-	+	+	-	-	-	-	-	+	-	-	+	+	-	+	-	+	+	-	+	-	+	+	+	-	-
WDBH080-1	-	-	+	-	+	-	+	+	-	-	-	-	-	+	-	-	+	+	-	+	-	+	+	-	+	-	+	+	+	-	-
WDBH109-1	-	-	+	-	+	-	+	+	-	-	-	-	-	+	-	-	+	+	+	+	-	+	+	-	+	-	+	+	+	-	-
WDBH037-1	-	-	+	-	+	-	+	+	-	-	-	-	-	+	-	-	+	+	+	+	-	+	+	-	+	-	+	+	+	-	+
WDBH011-2	-	-	+	-	+	-	+	+	-	-	-	-	-	+	-	-	+	+	+	+	-	+	+	-	+	+	+	+	+	-	+
WDBH044-1	-	-	+	-	+	-	+	+	-	-	-	-	-	+	-	-	+	+	+	+	-	+	+	-	+	+	+	+	+	-	+

附表1

表5

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样品编号 Sample ID	全部reads数 Total_reads	与参考基因组比对 Mapped (%)	平均覆盖深度 Ave_depth	基因组覆盖度 Cov_ratio_1× (%)	碱基数质量值大于30 Q30 (%)
R125	584,524,436	99.78	21	83.02	92.96
R127	558,380,728	99.64	20	79.03	93.28
R135	882,730,682	99.73	30	86.34	92.70
R138	880,248,082	99.75	30	86.58	92.94

染色体位置 Chromosome ID	关联区域始位置 Start	关联区域终止位置 End	关联区域大小 Size (Mb)	关联区域内基因数量 Gene number
Chr6LG2	60,560,000	91,060,000	30.50	434
Chr6LG2	91,090,000	91,150,000	0.060	2
Chr6LG2	91,320,000	91,630,000	0.310	7
Chr6LG2	91,690,000	91,750,000	0.060	1
Chr6LG2	92,190,000	92,680,000	0.490	5
总共Total	—	—	31.42	449

基因ID Gene ID	起始位置 Start	终止位置 End	基因功能注释 Gene function annotation
Psat6g060000.1	67,641,138	67,657,547	—
Psat6g060040.1	67,824,817	67,825,137	—
Psat6g060080.1	67,833,124	67,835,423	41 kD的叶绿体茎环结合蛋白 Chloroplast stem-loop binding protein of 41 kD
Psat6g060120.1	67,836,103	67,837,783	RAN GTP酶激活蛋白1 RAN GTPase-activating protein 1
Psat6g060160.1	67,921,631	67,923,302	—
Psat6g060200.1	67,934,766	67,936,280	—
Psat6g060240.1	68,020,488	68,022,587	不活跃Patatin类蛋9 Probable inactive patatin-like protein 9
Psat6g060280.1	68,235,164	68,237,023	保卫细胞S型阴离子通道SLAC1 Guard cell S-type anion channel SLAC1
Psat6g060320.1	68,259,311	68,271,214	类Rac GTP结合蛋白RAC9 (前体) Rac-like GTP-binding protein RAC9 (precursor)
Psat6g060360.1	68,302,270	68,302,889	—
Psat6g060400.1	68,305,575	68,309,198	五肽重复含蛋白 Pentatricopeptide repeat-containing protein
Psat6g060440.1	68,328,642	68,331,221	—
Psat6g060480.1	68,330,158	68,340,923	碱性螺旋-环-螺旋蛋白A Basic helix-loop-helix protein A
Psat6g060560.1	68,369,085	68,372,125	F-box蛋白 F-box protein
Psat6g060600.1	68,422,110	68,426,447	Sm类蛋白 LSM4 Sm-like protein LSM4
Psat6g060640.1	68,458,147	68,461,838	核糖体循环因子, 叶绿体(前体) Ribosome-recycling factor, chloroplastic (precursor)
Psat6g060680.1	68,511,345	68,517,001	TIME FOR COFFEE蛋白 Protein TIME FOR COFFEE
Psat6g060720.1	68,549,094	68,551,796	Mut11蛋白 Protein Mut11
Psat6g060760.1	68,554,342	68,554,618	—

基于BSA-seq技术对豌豆花色基因的精细定位

Fine mapping of flower colour gene in pea (Pisum sativum L.) based on BSA-seq technique

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