基于BSA-Seq和蛋白组学筛选辣椒核雄性不育基因

doi:10.3864/j.issn.0578-1752.2026.03.012

摘要/Abstract

摘要：

【目的】辣椒雄性不育系是实现辣椒杂种优势和开展辣椒遗传育种的主要方式之一，研究辣椒核雄性不育基因的精确定位，为后续基因克隆、功能验证、分子机制解析和新种质创制提供相关基因资源，为创制辣椒雄性不育系奠定基础。【方法】以田间发现的自然雄性不育突变体pby-1与野生型PBY-1为亲本进行杂交，获得F₁后代，并自交至F₂代，选取F₂群体中表现不育与正常个体各30株，构建2个极端池。通过混池测序（bulked segregant analysis sequencing，BSA-Seq）分析，鉴定雄性不育候选区间，挖掘区间内候选基因，并进行GO（Gene Ontology）和KEGG（Kyoto Encyclopedia of Genes and Genomes）富集分析。进一步将候选基因与亲本pby-1和PBY-1不同发育时期的差异表达蛋白进行联合分析，缩小辣椒核雄性不育相关候选基因的范围。运用实时荧光定量PCR技术检测候选基因在辣椒花器官中的表达水平，验证候选基因调控辣椒雄性不育的潜力。【结果】通过BSA-Seq测序分析，在Chr.07染色体上鉴定到12个与雄性不育相关联的区域，总长度约70.02 Mb，包含343个候选基因，与已知msc-1、msc-2、msc-3不育基因区间无重叠。GO富集和KEGG富集分析结果表明，碳水化合物代谢、脂质运输代谢和植物激素信号传导等途径与辣椒雄性不育性密切相关。结合蛋白组学数据综合分析，筛选到12个候选基因（Capana07g000676、Capana07g000956、Capana07g000979、Capana07g000993、Capana07g001228、Capana07g001239、Capana07g001241、Capana07g001254、Capana07g001294、Capana07g001295、Capana07g001312和Capana07g001315），经qRT-PCR检测，Capana07g000676、Capana07g000979、Capana07g000993、Capana07g001228、Capana07g001241和Capana07g001294为雄性不育关键候选基因。【结论】结合BSA-Seq与蛋白质组学，在辣椒第7染色体上定位到核雄性不育功能区段，并筛选出6个核心候选基因。

关键词: 辣椒, 核雄性不育, BSA-Seq, 蛋白组学, 多组学联合分析

Abstract:

【Objective】Male-sterile lines constitute a principal means of exploiting heterosis and advancing genetic breeding in pepper (Capsicum annuum L.). Fine mapping of the Genic male sterility (GMS) genes can provide relevant gene resources for subsequent gene cloning, functional verification, molecular mechanism analysis, and creation of new germplasm, laying the foundation for the development of nuclear male sterile lines in pepper. 【Method】A natural GMS mutant pby-1 and its wild type PBY-1 were used as parents for hybridization to obtain F₁ progeny, which were then self-pollinated to F₂ generation. Thirty plants showing sterile and normal phenotypes were selected from the F₂ population to construct two extreme pools. Through bulked segregant analysis sequencing (BSA-Seq), candidate regions associated with male sterility were identified, and candidate genes within the regions were mined. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were conducted. Further, the candidate genes were jointly analyzed with the differentially expressed proteins of the parents pby-1 and PBY-1 at different developmental stages to narrow down the range of candidate genes related to nuclear male sterility in pepper. The expression levels of the candidate genes in pepper flower organs were detected by real-time fluorescence quantitative PCR to verify their potential in regulating male sterility in pepper. 【Result】Through BSA-Seq sequencing analysis, 12 regions associated with male sterility were identified on chromosome Chr.07, with a total length of approximately 70.02 Mb, containing 343 candidate genes, which did not overlap with the known ms-1, ms-2, and ms-3 sterility gene regions. The results of GO enrichment and KEGG enrichment analysis indicated that carbohydrate metabolism, lipid transport metabolism, and plant hormone signal transduction pathways were closely related to male sterility in pepper. Through comprehensive analysis combining proteomics data, 12 candidate genes (Capana07g000676, Capana07g000956, Capana07g000979, Capana07g000993, Capana07g001228, Capana07g001239, Capana07g001241, Capana07g001254, Capana07g001294, Capana07g001295, Capana07g001312, and Capana07g001315) were screened. Through qRT-PCR detection, Capana07g000676, Capana07g000979, Capana07g000993, Capana07g001228, Capana07g001241, and Capana07g001294 were identified as key candidate genes for male sterility. 【Conclusion】By combining BSA-Seq with proteomics, the functional region of nuclear male sterility was located on chromosome 7 of the pepper, and six core candidate genes were screened.

Key words: Capsicum spp., genic male sterility, BSA-Seq, proteomics, integrated multi-omics analysis

裴红霞, 汪露瑶, 蒋雅苹, 李生梅, 高晶霞. 基于BSA-Seq和蛋白组学筛选辣椒核雄性不育基因[J]. 中国农业科学, 2026, 59(3): 637-654.

PEI HongXia, WANG LuYao, JIANG YaPing, LI ShengMei, GAO JingXia. Identification of Nuclear Male Sterility Genes in Pepper Based on BSA-Seq and Proteomics[J]. Scientia Agricultura Sinica, 2026, 59(3): 637-654.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2026.03.012

https://www.chinaagrisci.com/CN/Y2026/V59/I3/637

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雄性不育候选基因注释及蛋白表达"

基因 Gene_IDs	Pfam号 Pfam_IDs	Pfam注释 Pfam_description	染色体位置 Chromosome location	蛋白编号 Protein accession	蛋白功能注释 Protein description	比率 Ratio	调控类型 Regulated type	分子质量 MW (kDa)
Capana07g000676	PF12697	α/β水解酶家族 Alpha/beta hydrolase family	Chr.07:63427965:63430386:-	A0A2G2ZK80	含AB水解酶-1结构域蛋白 AB hydrolase-1 domain-containing protein	1.515	上调 Up	36.1
	PF00561	α/β水解酶折叠 Alpha/beta hydrolase fold		A0A1U8FW28	含AB水解酶-1结构域蛋白 AB hydrolase-1 domain-containing protein	0.751	下调 Down	36.8
	PF00561	α/β水解酶折叠 Alpha/beta hydrolase fold		A0A2G2ZK45	含AB水解酶-1结构域蛋白 AB hydrolase-1 domain-containing protein	1.624	上调 Up	36.5
Capana07g000956	PF00571	胱硫醚β-合成酶结构域CBS domain	Chr.07:124819375:124819884:+	A0A2G3A5F5	含CBS结构域蛋白 CBS domain-containing protein	1.304	上调 Up	26.0
Capana07g000979	PF00128	α-淀粉酶 Alpha amylase	Chr.07:132522155:132527031:+
Capana07g000979	PF00128	催化结构域 Catalytic domain	Chr.07:132522155:132527031:+
Capana07g000993	PF00082	枯草杆菌蛋白酶家族Subtilase family	Chr.07:135830482:135833179:-	A0A2G3AE94	枯草杆菌蛋白酶样蛋白酶 Subtilisin-like protease	0.702	下调 Down	79.0
Capana07g000993	PF05922	肽酶抑制剂I9家族Peptidase inhibitor I9	Chr.07:135830482:135833179:-	A0A2G3AF34	含抑制剂I9结构域蛋白 Inhibitor I9 domain-containing protein	0.683	下调 Down	24.1
Capana07g001228	PF00646	F-box结构域 F-box domain	Chr.07:165750151:165751491:+	A0A1U8H680	含F-box结构域蛋白 F-box domain-containing protein	1.319	上调 Up	43.0
Capana07g001239	PF02518	组氨酸激酶-, DNA旋转酶B-, 热休克蛋白90样ATP酶结构域 Histidine kinase-, DNA gyrase B-, and HSP90- like ATPase	Chr.07:166068142:166074467:-	A0A2G2Z950	热休克蛋白82 Heat shock protein 82	1.349	上调 Up	80.3
Capana07g001239	PF02518		Chr.07:166068142:166074467:-	A0A1U8GAJ4	内质网样蛋白 Endoplasmin-like protein	0.74	下调 Down	92.9
Capana07g001241	PF00106	短链脱氢酶 Short chain dehydrogenase	Chr.07:166153425:166159029:-
Capana07g001241	PF13561	烯酰-（酰基载体蛋白）还原酶 Enoyl-(Acyl carrier protein) reductase	Chr.07:166153425:166159029:-	A0A2G2YR93	托品酮还原酶2 Tropinone reductase 2	0.763	下调 Down	29.5
Capana07g001254	PF00069	蛋白激酶结构域 Protein kinase domain	Chr.07:167320450:167335317:-	A0A1U8GXD8	酪蛋白激酶1δ Casein kinase I isoform delta	1.483	上调 Up	53.1
	PF07714	蛋白酪氨酸激酶 Protein tyrosine kinase		A0A2G2YZW4	含蛋白激酶结构域蛋白 Protein kinase domain-containing protein	3.431	上调 Up	48.5
	PF07714	蛋白酪氨酸激酶 Protein tyrosine kinase		A0A2G2YBJ3	假定丝氨酸/苏氨酸蛋白激酶 Putative serine/threonine-protein kinase	1.32	上调 Up	54.2
Capana07g001294	PF00651	BTB/POZ结构域BTB/POZ domain	Chr.07:170187515:170190321:-
Capana07g001295	PF00564	PB1结构域 PB1 domain	Chr.07:170302463:170304671:+
Capana07g001312	PF07714	蛋白酪氨酸激酶 Protein tyrosine kinase	Chr.07:171800459:171817027:+	A0A2G3APD0	丝氨酸/苏氨酸蛋白激酶CTR1 Serine/threonine-protein kinase CTR1	1.429	上调 Up	80.9
	PF00069	蛋白激酶结构域 Protein kinase domain
	PF01476	LysM结构域 LysM domain
Capana07g001315	PF02902	Ulp1蛋白酶家族 Ulp1 protease family	Chr.07:171946489:171949534:+
Capana07g001315	PF02902	C-末端催化结构域 C-terminal catalytic domain	Chr.07:171946489:171949534:+

表7

图7

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引物ID Primer ID	引物序列 Primer sequence (5′-3′)
Capana07g000676-qPCR-F	GAGCACAAAATGGTAGTTGCGA
Capana07g000676-qPCR-R	AAGTAGACGATTTGCTGGCG
Capana07g000956-qPCR-F	CATCGACTGTGGTGGTCCTC
Capana07g000956-qPCR-R	TGTTGAAGACGACGCTGGAA
Capana07g000979-qPCR-F	AAGATACGCATCCGCTGG
Capana07g000979-qPCR-R	TAAAACACACCGCCGTCAA
Capana07g000993-qPCR-F	TGGCGAGGGGAATGTGAATC
Capana07g000993-qPCR-R	CGATTCAGGCTGTCTGGGTT
Capana07g001228-qPCR-F	GCAGGAGCAGTGGTAATGGT
Capana07g001228-qPCR-R	CTCAGCAACACTATCCGGCA
Capana07g001239-qPCR-F	TCCGACAGAAGCCAAGAAGG
Capana07g001239-qPCR-R	TCAGAAATGGGAAAGACGACAGT
Capana07g001241-qPCR-F	TACTTGGTACCTGGTGCTGG
Capana07g001241-qPCR-R	GCCAAATAACGAAGGCACCC
Capana07g001254-qPCR-F	TCGGAGATCGCGTTGTTGAA
Capana07g001254-qPCR-R	CGCCTTGATGGAAACGTGAC
Capana07g001294-qPCR-F	CTTGAACCAGGTTGTGCTGC
Capana07g001294-qPCR-R	AGGCAAGTGACTCGATGCAA
Capana07g001295-qPCR-F	GCTGAGGAAGCTGAAGAGCA
Capana07g001295-qPCR-R	TCCATTGCGACCTCCAAACA
Capana07g001312-qPCR-F	TAGTCTTGCTGTTGGCTGGG
Capana07g001312-qPCR-R	GCTTTAACTGTCGTGCTGCC
Capana07g001315-qPCR-F	AGGGGTGCACTGATTTGCAT
Capana07g001315-qPCR-R	CTGCTCCTCCGTACCAACAT
CaREV05-qPCR-F	GGACCAGCAAAGGTTGATTT
CaREV05-qPCR-R	CAGATGGAGGGTTGATTCCT

组合 Combination	世代 Generations	可育株 Fertile strain	不育株 Sterile strain	总数 Total	期望比例 Expectation ratio	实际比例 Actual proportion	χ²
pby-1×PBY-1	F₁	255	0	255	全可育 Fully fertile	全可育 Fully fertile	/
	F₂	770	264	1034	3F﹕1S	2.917﹕1	0.156（P=0.69）
	BC₁	799	808	1607	1F﹕1S	1﹕1.011	0.0504（P=0.82）

编号 ID	原始读段 Raw reads	接头比例 Adapter percent (%)	有效读段 Clean_reads	有效碱基数 Clean_base	Q30比例 Q30 (%)	GC含量 GC (%)	比对率 Properly- mapped (%)	覆盖深度 Ave_depth	覆盖比例 Cov_ratio (%)
R01	121331436	0.02	121312127	36345286212	92.60	35.52	95.49	11×	88.36
R02	118143280	0.04	118091227	35365642094	93.55	39.12	93.72	10×	85.47
R03	240816363	0.01	240773371	72130680194	92.92	34.80	95.85	22×	89.37
R04	230255180	0.03	230183074	68955090602	92.42	34.74	95.69	21×	89.68

染色体 Chromosome	开始 Start (bp)	结束 End (bp)	区间大小 Size (Mb)	基因数 Gene number
Chr.07	100650000	101180000	0.53	1
Chr.07	102340000	110650000	8.31	33
Chr.07	117910000	137750000	19.84	52
Chr.07	160200000	172740000	12.54	137
Chr.07	51230000	61780000	10.55	49
Chr.07	62420000	65490000	3.07	21
Chr.07	69800000	91530000	21.73	60
Chr.07	98710000	100300000	1.59	6
总计Total	-	-	78.16	359

染色体 Chromosome	开始 Start (bp)	结束 End (bp)	区间大小 Size (Mb)	基因数 Gene number
Chr.07	102730000	110510000	7.78	32
Chr.07	118520000	137650000	19.13	52
Chr.07	149840000	150650000	0.81	6
Chr.07	152260000	152980000	0.72	1
Chr.07	160550000	176010000	15.46	171
Chr.07	176540000	176600000	0.06	1
Chr.07	49410000	50610000	1.20	1
Chr.07	51160000	65660000	14.50	71
Chr.07	69440000	74810000	5.37	24
Chr.07	75590000	78070000	2.48	5
Chr.07	78150000	79000000	0.85	5
Chr.07	81480000	82020000	0.54	3
Chr.07	84940000	91280000	6.34	13
Chr.07	98750000	101240000	2.49	7
总计Total	-	-	77.73	392