基于BSA-Seq谷子落粒主效基因的定位与候选基因分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-1249

摘要/Abstract

摘要：

目的落粒性是作物驯化过程中关键的人工选择性状。定位并克隆调控落粒性的主效基因，为阐明谷子驯化的遗传基础、开发分子标记辅助育种工具提供理论依据和应用价值。方法以易落粒种质资源YLL5和抗落粒现代育成品种冀谷42（Si42）为研究对象，运用石蜡切片比较两亲本离层（abscission zone）细胞结构与细胞壁成分差异。为定位控制落粒性基因，构建F₄遗传群体。采用BSA-Seq（Bulked segregant analysis sequencing），并结合ED、G′ value、SNP-loess和SNP-index等连锁分析方法初步定位相关性状位点。对定位区间内注释基因进行筛选，通过实时荧光定量PCR（quantitative reverse transcription PCR, RT-qPCR）验证候选基因在两亲本多组织中的表达差异。结果石蜡切片结果显示，YLL5和Si42在细胞排列、组织结构及细胞壁成分上存在显著差异。经关联分析，初步将控制目标性状的位点定位在第5（3.3 Mb）和第8（1.1 Mb）染色体。利用InDel标记将目标区间精细定位至第5染色体约0.3 Mb的区域，该区间共注释25个基因，从中筛选出在穗部高表达的基因。RT-qPCR分析表明，候选基因Seita.5G087200在易落粒亲本YLL5的穗和穗码组织中的表达量显著高于Si42，其表达模式与落粒表型高度一致。Sanger测序进一步揭示，该基因第2个外显子存在一处转座子插入变异；基于该变异开发的分子标记能够有效区分不同落粒表型，具备分子标记辅助选择的应用潜力。结论 Seita.5G087200可能是调控谷子落粒性状的关键候选基因。

关键词: 谷子, 驯化性状, BSA-Seq技术, 落粒性, 基因定位

Abstract:

Objective Seed shattering is a crucial artificially - selected trait in the process of crop domestication. Mapping and cloning the major gene regulating seed shattering provides a theoretical basis and application value for clarifying the genetic basis of foxtail millet domestication and developing molecular marker - assisted breeding tools. Method The easily-shattering germplasm resource YLL5 and the shattering - resistant modern cultivar Jigu 42 (Si42) were taken as the research objects. Paraffin sectioning was used to compare the differences in the cell structure and cell-wall components of the abscission zone between the two parents. To map the gene controlling seed shattering, an F₄ genetic population was constructed. BSA-Seq (Bulked segregant analysis sequencing) was carried out, and in combination with linkage analysis methods such as ED, G′ value, SNP-loess, and SNP-index, the loci related to the trait were preliminarily mapped. The annotated genes in the mapped interval were screened, and quantitative reverse-transcription PCR (RT-qPCR) was used to verify the expression differences of candidate genes in multiple tissues of the two parents. Result The results of paraffin sectioning showed significant differences in cell arrangement, tissue structure, and cell wall components between YLL5 and Si42. Through association analysis, the locus controlling the target trait was preliminarily mapped to chromosome 5 (3.3 Mb) and chromosome 8 (1.1 Mb). Using InDel markers, the target interval was finely mapped to a region of approximately 0.3 Mb on chromosome 5. A total of 25 genes were annotated in this interval, and genes with high expression in the panicle were screened out. RT-qPCR analysis indicated that the expression level of the candidate gene Seita.5G087200 in the panicle and spikelet tissues of the easily-shattering parent YLL5 was significantly higher than that in Si42, and its expression pattern was highly consistent with the shattering phenotype. Sanger sequencing further revealed a transposon-insertion variation in the second exon of this gene. A molecular marker developed based on this variation could effectively distinguish different shattering phenotypes, showing potential for application in molecular marker-assisted selection. Conclusion Seita.5G087200 is likely a key candidate gene regulating the seed-shattering trait in foxtail millet.

Key words: Setaria italica, domestication traits, BSA-Seq technology, seed shattering, genetic mapping

刘倩, 王宗燕, 罗浚豪, 吴张璋, 胡振, 吕建珍, 姜亮. 基于BSA-Seq谷子落粒主效基因的定位与候选基因分析[J]. 生物技术通报, 2026, 42(7): 138-147.

LIU Qian, WANG Zong-yan, LUO Jun-hao, WU Zhang-zhang, HU Zhen, LYU Jian-zhen, JIANG Liang. Mapping and Candidate Gene Analysis of the Major Gene Controlling Seed Shattering in Foxtail Millet Based on BSA-Seq[J]. Biotechnology Bulletin, 2026, 42(7): 138-147.

图/表 7

表1 样本测序数据质控统计分析

Table 1 Statistical analysis of sample sequencing data quality control

样本名称 Sample name	质控后Clean reads	过滤后的碱基数 Clean bases	GC（%）	Q30（%）	比对效率 Mapping efficiency （%）	平均测序深度 Average sequencing depth （×）	覆盖率Coverage（≥1×）（%）
Si42	21 809 827	6 531 588 068	43.71	96.55	98.13	16	95.82
YLL5	21 479 290	6 431 795 254	43.84	96.58	98.24	16	90.46
NSH	61 192 923	18 322 677 641	43.69	96.88	98.59	43	99.46
SH	54 118 595	16 205 874 945	43.52	97.23	98.83	38	98.33

图1 杂交群体落粒性状调查A：亲本离层区甲苯胺蓝染色石蜡切片；B：亲本极端落粒（YLL5）和极端不落粒（Si42）穗部表型图；C：684份F4分离群体落粒率与单穗籽粒数二维分布密度图

Fig. 1 Investigation on the shattering trait of hybrid populationA: Toluidine blue-stained paraffin sections of the parental abscission zone (AZ). B: Panicle phenotypes of the parental lines with extreme shattering in YLL5 and extreme non-shattering in Si42. C: Two-dimensional density distribution plot of shattering rate versus grain number per panicle for the 684 F₄ segregating population

表2 BSA-Seq检测到SNP的数量与类型

Table 2 Number and types of SNPs detected by BSA-Seq

样本名称 Sample name	SNP数量 No. of SNPs	转换 Transition	颠换 Transversion	转换颠换比 Ti/Tv	杂合SNP数 Number of heterozygous SNPs	纯合SNP数Number of homozygous SNPs
Si42	956 457	705 022	251 957	2.80	144 654	4 603 689
YLL5	3 224 385	2 380 056	846 130	2.81	407 128	4 293 680
NSH	3 199 121	2 350 746	851 613	2.76	2 968 098	1 971 634
SH	3 394 412	2 499 872	898 351	2.78	2 920 233	1 985 825

图2 ED、G′ value、SNP-loess及SNP-index定位区间图

Fig. 2 Plots of the mapping intervals of ED, G′ value, SNP-loess and SNP-index

图3 落粒性状相关基因的精细定位图BL32-BL664：极端不落粒后代；L26-L557：极端落粒后代；黑色、白色和灰色格分别代表Si42、YLL5和杂合体基因型；Si为极端不落粒性状，YLL为极端落粒性状；红色虚线之间为精细定位候选区间

Fig. 3 Fine mapping of the candidate gene for the seed shattering traitBL32-BL664: Extreme non-shattering progeny. L26-L557: Extreme shattering progeny. Black, white, and gray cells represent the genotypes of Si42, YLL5, and heterozygotes, respectively; Si represents the extreme non-shattering trait, YLL the extreme shattering trait. The region between the red dashed lines is the candidate interval for fine mapping

表 3 候选区间内基因的注释信息

Table 3 Annotation information of genes in candidate intervals

基因编号 Gene_ID	基因功能注释 Annotation of gene function
Seita.5G086900	类受体蛋白激酶（与拟南芥At4g00960同源）
Seita.5G087000	甘露糖-6-磷酸异构酶1
Seita.5G087200	R2R3-MYB转录因子家族（含保守MYB结构域）
Seita.5G087300	核酸内切酶2
Seita.5G087400	核酸内切酶2
Seita.5G087500	叶绿体定位的MAO HUZI 4蛋白
Seita.5G087700	突触结合蛋白-5
Seita.5G087800	泛素相关蛋白家族（含泛素结合结构域）
Seita.5G088000	含LOB结构域蛋白42
Seita.5G088100	WD40重复结构域蛋白家族（含WD40保守结构域）
Seita.5G088300	金属耐受蛋白7同工型X1
Seita.5G088500	α-木糖苷酶1
Seita.5G088700	丝氨酸蛋白酶家族（含丝氨酸蛋白酶保守结构域）
Seita.5G088900	跨膜蛋白家族（含7次跨膜结构域）
Seita.5G089100	乙烯响应转录因子ERF118
Seita.5G089200	信号肽酶复合物亚基3
Seita.5G089300	Bowman-Birk型创伤诱导蛋白酶抑制剂WIP1

图4 YLL5与Si42中Seita.5G087200/Sevir.5G085400基因座转座子插入A：候选基因在各组织中的最高表达量；B：Setia.5G087200与Sevir.5G085400基因结构对比图；C：YLL5与Si42中Seita.5G087200/Sevir.5G085400基因座转座子插入PCR验证图；D：YLL5与Si42内不同组织部位Seita.5G087200的表达分析。*和***分别表示在P<0.05和P<0.001水平差异显著；E：Sv插入基因型与落粒表型共分离分析叠加统计；F：极端不落粒P1、P2、P3基因型与表型检测；G：极端落粒P1、P2、P3基因型与表型检测。P1、P2、P3分别为3对特异性引物（P1：跨越转座子插入位点，预期Si42产物7 000 bp、YLL5产物294 bp；P2：F-转座子左侧基因组，R-转座子内部，预期Si42产物750 bp；P3：F-转座子内部，R-转座子右侧基因组，预期Si42产物750 bp）

Fig. 4 Transposon insertion at the Seita.5G087200/Sevir.5G085400 locus in Si42 and YLL5A: Maximum expression levels of candidate genes across different tissues. B: Comparison of gene structures between Setia.5G087200 and Sevir.5G085400. C: PCR validation of transposon insertion at the Seita.5G087200/Sevir.5G085400 locus in YLL5 and Si42. D: Expression analysis of Seita.5G087200 in different tissues of YLL5 and Si42. * and *** indicate significant differences at P<0.05 and P<0.001 levels, respectively. E: Overlay statistical analysis of co-segregation between Sv insertion genotype and shattering phenotype. F: Detection of genotype and phenotype in extremely non-shattering P1, P2, P3. G: Detection of genotype and phenotype in extremely shattering P1, P2, P3. P1, P2, and P3 represent three pairs of specific primers (P1: spanning the transposon insertion site, expected product sizes: 7 000 bp in Si42 and 294 bp in YLL5; P2: F-genomic region left of the transposon, R-internal region of the transposon, expected product: 750 bp in Si42; P3: F-internal region of the transposon, R-genomic region right of the transposon, expected product: 750 bp in Si42)

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