甘蓝型油菜角果数突变体基因的定位及候选基因分析

doi:10.3724/SP.J.1006.2022.04281

作物学报 ›› 2022, Vol. 48 ›› Issue (1): 27-39.doi: 10.3724/SP.J.1006.2022.04281

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

甘蓝型油菜角果数突变体基因的定位及候选基因分析

赵改会¹(), 李书宇², 詹杰鹏¹, 李晏斌³, 师家勤¹^,^*(), 王新发¹, 王汉中¹

¹中国农业科学院油料作物研究所, 湖北武汉 430062
²江西省农业科学院作物研究所, 江西南昌 330200
³武汉市农业技术推广中心, 湖北武汉 430400

收稿日期:2020-12-27 接受日期:2021-04-14 出版日期:2022-01-12 网络出版日期:2021-06-15
通讯作者: 师家勤
作者简介:E-mail: 3468382960@qq.com第一联系人：^**同等贡献
基金资助:
江西省自然科学基金项目(20202BABL205016);湖北省自然科学基金重点(杰青)项目(2018CFA075);优质高产多抗油菜新品种选育项目(2018ABA087);国家现代农业产业技术体系建设专项资助(CARS-13)

Mapping and candidate gene analysis of silique number mutant in Brassica napus L.

ZHAO Gai-Hui¹(), LI Shu-Yu², ZHAN Jie-Peng¹, LI Yan-Bin³, SHI Jia-Qin¹^,^*(), WANG Xin-Fa¹, WANG Han-Zhong¹

¹Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, Hubei, China
²Crop Research Institute, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, Jiangxi, China
³Wuhan Agricultural Technology Extension Center, Wuhan 430400, Hubei, China

Received:2020-12-27 Accepted:2021-04-14 Published:2022-01-12 Published online:2021-06-15
Contact: SHI Jia-Qin
About author:First author contact:^** Contributed equally to this work
Supported by:
Natural Science Foundation of Jiangxi Province(20202BABL205016);Hubei Provincial Natural Science Foundation (Outstanding Youth)(2018CFA075);Breeding of New Rapeseed Varieties with High Quality, High Yield and Multiple Resistance(2018ABA087);China Agriculture Research System(CARS-13)

摘要/Abstract

摘要：

角果数是油菜单株产量重要的构成因子之一, 其优异等位基因的发掘和利用对产量的提高至关重要。油菜中已定位到上百个角果数QTL, 但大多数效应不大且不稳定, 难以进行精细定位或克隆。本研究前期发掘到一个油菜突变体(No.7931), 其花序顶端在分化出约十朵花后即停止生长, 因而成熟期角果极少。利用该少角果突变体和多角果品系No.73290构建F₂分离群体, 从中挑选角果数极端单株各30株进行BSA-seq, 在C02染色体检测到3个关联区间: 0~1.1 Mb、4.7~6.2 Mb、11.5~12.4 Mb。该候选区间在油菜参考基因组DarmorV8.1中有522个注释基因, 存在SNP或Indel差异且有同源注释的基因235个。在花芽分化初期, 选取两亲本(No.73290和No.7931)的茎尖分生组织进行RNA-seq, 总共鉴定到8958个差异表达基因(DEGs)。这些DEGs显著富集于20个生物学通路, 包括碳代谢、翻译、氨基酸代谢(和花芽分化高度相关)等, 其中99个位于关联区间。结合基因功能注释以及序列和表达差异分析确定了9个候选基因(BnaC02g00490.1D2、BnaC02g01030.1D2、BnaC02g01120.1D2、BnaC02g00270.1D2、BnaC02g02670. 1D2、BnaC02g08680.1D2、BnaC02g08890.1D2、BnaC02g09480.1D2和BnaC02g10490.1D2), 它们主要参与花序分生组织特性的维持和花器官的发育。上述研究结果为后续油菜角果数基因的精细定位和克隆奠定了坚实的基础。

关键词: 油菜, 油菜, 角果数突变体, 角果数突变体, 花芽分化, 花芽分化, BSA-seq, BSA-seq, RNA-seq, RNA-seq

Abstract:

The silique number is one of the important components of yield per plant in oilseed rape (Brassica napus L.) and the exploitation and utilization of its excellent alleles are essential to increase yield. More than hundreds of silique number QTLs have been mapped in oilseed rape, but they are difficult to be fine-mapped or cloned because of their moderate and unstable effects. A oilseed rape mutant (No.7931) was detected in previous study and it had few siliques at mature stage due to the stop growth after differentiation about 10 flowers on the top of inflorescence. A F₂ segregating population consisting of 3400 individuals was constructed using this mutant and another more-silique lines No.73290. Among them, we performed BSA-seq on 30 individuals with extreme more- or less-siliques and detected three associated intervals of 0-1.1 Mb, 4.7-6.2 Mb, and 11.5-12.4 Mb on the C02 chromosome. These genomic intervals contained a total of 522 annotated genes in the reference genome DarmorV8.1, among which 235 genes had functional annotation and SNP/InDel variation. At the early stage of flower bud differentiation, the shoot apical meristems of two parents were subjected to RNA-seq, and a total of 8958 differentially expressed genes (DEGs) were detected. These DEGs were significantly enriched into 20 pathways, including carbohydrate metabolism, translation, and amino acid metabolism (highly associated with flower bud differentiation) and so on, among which 99 were located in the associated intervals. By the integration of gene functional annotation as well as sequence and expression variation analysis, a total of nine candidate genes (BnaC02g00490.1D2, BnaC02g01030.1D2, BnaC02g01120.1D2, BnaC02g00270.1D2, BnaC02g02670.1D2, BnaC02g08680.1D2, BnaC02g08890.1D2, BnaC02g09480.1D2, and BnaC02g10490.1D2) were identified, which were mainly involved in the maintenance of inflorescence meristems and the regulation of flower development. The above results lay the foundation for the following fine-mapping and cloning of the silique number mutant gene in oilseed rape.

Key words: Brassica napus L., Brassica napus L., silique number mutant, silique number mutant, flower bud differentiation, flower bud differentiation, BSA-seq, BSA-seq, RNA-seq, RNA-seq

赵改会, 李书宇, 詹杰鹏, 李晏斌, 师家勤, 王新发, 王汉中. 甘蓝型油菜角果数突变体基因的定位及候选基因分析[J]. 作物学报, 2022, 48(1): 27-39.

ZHAO Gai-Hui, LI Shu-Yu, ZHAN Jie-Peng, LI Yan-Bin, SHI Jia-Qin, WANG Xin-Fa, WANG Han-Zhong. Mapping and candidate gene analysis of silique number mutant in Brassica napus L.[J]. Acta Agronomica Sinica, 2022, 48(1): 27-39.

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亲本 Parent	主花序花器官数目 FNm	主花序角果数 SNm	株高 PH
No.7931	8.33±1.24	7.67±0.94	154.3±2.06
No.73290	199.3±1.24	93.67±2.49	150.4±1.57
t检验值t-test value	5.45E-09	6.91E-07	0.0170

亲本 Parent	主花序花器官数目 FNm	主花序角果数 SNm	株高 PH
No.7931	8.33±1.24	7.67±0.94	154.3±2.06
No.73290	199.3±1.24	93.67±2.49	150.4±1.57
t检验值t-test value	5.45E-09	6.91E-07	0.0170

基因 Gene	正向引物 Forward primer (5°-3°)	反向引物 Reverse primer (5°-3°)
BnaA02G0314300ZS	CCGACACGCTTCAGAAGGTC	AGAGATCCCGCTTCGACTCC
BnaC03G0027000ZS	TGAGAACACGCCGGTCAAAG	CCTCGCGTTCTCTCTCTCCA
BnaC03G0100100ZS	CTGATGGCCGTAGAGCATGC	AGCCATCTCCTGTTGTTGCA
BnaC03G0091000ZS	TCCCACTCTACCCTGCACTG	GACGTTGTTCACATTCGCGC
BnaC02G0283700ZS	ACAACATGGCCCTGGAAACTG	CTTGGAGGCGGATGATCGTT
BnaC02G0139700ZS	CGGCGGAGGTACAGACATTT	TCGTCATGATGAGCCTCTCCT
BnaC02G0175400ZS	GCGCCTCGTATCCATTCTCG	CCTGAAGTTGTGGCGAGCTT
BnaA01G0077800ZS	CCACCGTCATGTCTTCCTTCC	ACGAGTTGGAAGTGTGCGTT
BnaA01G0061200ZS	GGGAACGGCTTAGATGGTGC	TTGCTAGTACCAGGGCTGCT
BnaC02G0013900ZS	CATTGGCTCGTCATGAACATC	TTCACGAGTGTTGAACTGATCC
BnaC02G0159100ZS	CTGTCACTGGAAACCACCCG	TCAATCGACCATGGCAAGCA
BnaA01G0009100ZS	TCTGCTCTGAACGCGACCAA	ACCAGCCAAAGAACCAGGGT
BnaA01G0037200ZS	TCCTCAACTGTGCCGACATGT	AAAGCCGTCGTCAATCTCGC
BnaC05G0266700ZS	AGACTACGTGAAGCAGCCGA	CTCCAGCTTCCGACCAGTCT
BraActin	CTGGAATTGCTGACCGTATGAG	ATCTGTTGGAAAGTGCTGAGGG

基因 Gene	正向引物 Forward primer (5°-3°)	反向引物 Reverse primer (5°-3°)
BnaA02G0314300ZS	CCGACACGCTTCAGAAGGTC	AGAGATCCCGCTTCGACTCC
BnaC03G0027000ZS	TGAGAACACGCCGGTCAAAG	CCTCGCGTTCTCTCTCTCCA
BnaC03G0100100ZS	CTGATGGCCGTAGAGCATGC	AGCCATCTCCTGTTGTTGCA
BnaC03G0091000ZS	TCCCACTCTACCCTGCACTG	GACGTTGTTCACATTCGCGC
BnaC02G0283700ZS	ACAACATGGCCCTGGAAACTG	CTTGGAGGCGGATGATCGTT
BnaC02G0139700ZS	CGGCGGAGGTACAGACATTT	TCGTCATGATGAGCCTCTCCT
BnaC02G0175400ZS	GCGCCTCGTATCCATTCTCG	CCTGAAGTTGTGGCGAGCTT
BnaA01G0077800ZS	CCACCGTCATGTCTTCCTTCC	ACGAGTTGGAAGTGTGCGTT
BnaA01G0061200ZS	GGGAACGGCTTAGATGGTGC	TTGCTAGTACCAGGGCTGCT
BnaC02G0013900ZS	CATTGGCTCGTCATGAACATC	TTCACGAGTGTTGAACTGATCC
BnaC02G0159100ZS	CTGTCACTGGAAACCACCCG	TCAATCGACCATGGCAAGCA
BnaA01G0009100ZS	TCTGCTCTGAACGCGACCAA	ACCAGCCAAAGAACCAGGGT
BnaA01G0037200ZS	TCCTCAACTGTGCCGACATGT	AAAGCCGTCGTCAATCTCGC
BnaC05G0266700ZS	AGACTACGTGAAGCAGCCGA	CTCCAGCTTCCGACCAGTCT
BraActin	CTGGAATTGCTGACCGTATGAG	ATCTGTTGGAAAGTGCTGAGGG

油菜基因编号 B. napus ID	拟南芥基因名 Name of A. thaliana	拟南芥基因编号 A. thaliana ID	位置Position (Mb)	基因注释 Gene annotation	序列差异 Sequence difference	差异表达基因 Differential expressed genes
BnaC02g00490.1D2	FLC	AT5g10140	0.208	K盒区和MADS转录因子家族蛋白 K-box region and MADS-box transcription factor family protein	有Yes	是No
BnaC02g01030.1D2	TPR1	AT1G80490	0.465	TPL相关的基因1 TOPLESS-related 1	无No	否No
BnaC02g01120.1D2	AGAL2	AT5G08370	0.493	α-半乳糖苷酶2 Alpha-galactosidase 2	无No	否No
BnaC02g00270.1D2	AIL6	AT5G10510	0.996	类ANT基因6 AINTEGUMENTA-LIKE 6	有Yes	否No
BnaC02g02670.1D2	TFL1	AT5G03840	1.10	磷脂酰乙醇胺结合蛋白 Phosphatidylethanolamine-binding-protein	无No	是Yes
BnaC02g08680.1D2	COL1	AT5G15850	4.83	类CO基因1 CONSTANS-like 1	有Yes	否No
BnaC02g08890.1D2	ELF9	AT5G16260	4.96	RNA结合(RRM/RBD/RNP基序)家族蛋白RNA binding (RRM/RBD/RNP motifs) family protein	有Yes	否No
BnaC02g09480.1D2	AIM1	AT4G29010	5.23	烯酰辅酶A水合酶/异构酶家族 Enoyl-CoA hydratase/isomerase family	有Yes	否No
BnaC02g10490.1D2	CHR17	AT5G18620	5.97	染色质重塑因子17 Chromatin remodeling factor 17	无No	是Yes

甘蓝型油菜角果数突变体基因的定位及候选基因分析

Mapping and candidate gene analysis of silique number mutant in Brassica napus L.

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