谷子黄叶色突变体ylm-1的精细定位与功能分析

doi:10.16035/j.issn.1001-7283.2022.03.008

摘要/Abstract

摘要：

谷子叶色突变体在研究C₄光能利用效率和叶绿素代谢机制方面具有重要作用。为探究谷子叶色突变的分子机制,利用甲基磺酸乙酯（EMS）处理谷子品种豫谷1号,获得1个稳定遗传的黄叶色突变体ylm-1。通过表型和遗传分析及遗传背景检测,同时利用突变位点图谱（MutMap）技术对突变基因进行精细定位。结果表明,ylm-1整个生育期叶色均为淡黄色;ylm-1与野生型遗传背景相同且受1对隐性核基因控制;MutMap精细定位到第9号染色体关联区间内共13个非同义突变基因,其中,Seita.9G249900是一个编码与红叶绿素代谢还原酶（RCCR）相关的基因,该基因位于第9号染色体19 937 988与19 940 620bp之间,含有2个外显子和1个内含子,在第2外显子361bp处发生A/T碱基突变,导致1个谷氨酸（E）变成天冬氨酸（D）。根据突变碱基设计了dCAPS标记,进一步验证了ylm-1候选基因突变位点。黄叶色突变体ylm-1的鉴定与基因功能分析将为该基因的有效利用、功能验证及作用机制研究奠定理论与技术基础。

关键词: 谷子, 黄叶色突变体, 精细定位, 功能分析

Abstract:

Leaf color mutants play an important role in investigating the utilization of C₄ light energy and the mechanism of chlorophyll metabolism in foxtail millet. To study the molecular mechanism of the leaf color mutant in foxtail millet, we identified a stable mutant ylm-1 from the library generated by treating Yugu 1 with ethyl methanesulfonate (EMS). The mutant was studied by phenotype identification, genetic analysis and genetic background identification. At the same time, the mutated gene was mapped quickly and precisely using a mutation map (MutMap). The results showed that ylm-1 displayed a yellow leaf phenotype throughout the growth phase. ylm-1 had the same genetic background as the wild type controlled by a recessive nuclear gene with genetic analysis. The association interval contained 13 non-synonymous mutant genes in the ninth chromosome by the MutMap procedure. Seita.9G249900 was one of the genes encoding a chloroplast associated red chlorophyll catabolite reductase whose physical position was between 19 937 988 and 19 940 620bp of the ninth chromosome and contains two exons and one intron. An A/T base mutation occurred at 361bp in the second exon, resulting in a glutamate (E) changing to an aspartic acid (R). The mutation site of the candidate gene in ylm-1 was further verified by the dCAPS marker. The identification and gene functional analysis of a new yellow leaf mutant ylm-1, which will provide a theoretical and technical basis for the effective use, the study of the functional evidence and the mechanism of action of the gene in foxtail millet.

Key words: Foxtail millet, Yellow leaf mutant, Fine mapping, Functional analysis

秦娜, 朱灿灿, 代书桃, 宋迎辉, 李君霞, 王春义. 谷子黄叶色突变体ylm-1的精细定位与功能分析[J]. 作物杂志, 2022 (3): 55–62

Qin Na, Zhu Cancan, Dai Shutao, Song Yinghui, Li Junxia, Wang Chunyi. Fine Mapping and Functional Analysis of Yellow Leaf Mutant ylm-1 in Foxtail Millet[J]. Crops, 2022(3): 55–62

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引物名称Primer name	引物序列（5′-3′）Primer sequence (5′-3′)	用途Purpose
SICAAS1008	F：GAGACGAAGAATGAAAGGACGA	突变体遗传背景检测
	R：CGGGGCACCGTTAAAACTA
SICAAS1010	F：TGTTTATTTTCCCAGCCTCA	突变体遗传背景检测
	R：AAAAGTCAATGGCATCATCG
SICAAS5055	F：TGTTTTGGGGTCCCTGGACTTGGCT	突变体遗传背景检测
	R：TCTGCCTTCTGTCCACATCGCACAT
SICAAS9036	F：CGCCGCTCATCCTCTTCCACAC	突变体遗传背景检测
	R：GTGCCCATGAACGGATCGCACT
dCAPS标记	F：CCCGCCGACGCGCGCATA	突变位点的dCAPS标记检测
dCAPS marker	R：GGGGCGAACCGTCAAGCT

染色体ID Chromosome ID	起始位点 Start	终止位点 End	大小 Size (Mb)	基因数量 Gene number
scaffold_9	19424328	19832057	0.41	22
scaffold_9	19849585	19876789	0.03	3
scaffold_9	19932905	19941829	0.01	1
总计Total				26

基因ID Gene ID	物理位置 Physical position	基因型 Genotype	质控 QUAL	功能注释 Functional annotation
Seita.9G249500	19824287,19825523,19827781	C/T,T/C,A/C	809.62,800.62,83.19	C末端LisH蛋白
Seita.9G249400	19805657,19805736,19806419	G/A,T/C,G/T	562.47,632.42,489.52	羟基苯丙酮酸还原酶
Seita.9G249100	19699956,19700068	G/A,T/C	413.17,211.62	类atherin蛋白
Seita.9G249600	19856418,19856498	T/C,A/T	865.52,880.47	核糖体构成蛋白
Seita.9G248700	19601573,19602304	C/T,G/A	689.42,698.43	叶绿体中碳运输与代谢蛋白
Seita.9G249900	19939508	A/T	553.52,394.42	红叶绿素代谢还原酶
Seita.9G247700	19493068	C/T	1532.44	热激蛋白转录因子A-2c
Seita.9G247500	19461470,19462867	G/T,T/A	550.42,732.42	嘧啶还原酶
Seita.9G249300	19766023	G/A	349.53	色胺羟基肉桂酰转移酶2
Seita.9G247600	19483769,19484142	C/G,T/C	356.52,573.17	锌指双链RNA结构域
Seita.9G248800	19652152	C/G	466.43	核糖体蛋白S7
Seita.9G247400	19430677	G/A	440.81	假设蛋白SETIT_039055mg
Seita.9G249200	19731658,19731749,19731779	G/A,T/C,T/C	529.81,364.43,432.43	色胺羟基肉桂酰转移酶2