Crops ›› 2022, Vol. 38 ›› Issue (3): 55-62.doi: 10.16035/j.issn.1001-7283.2022.03.008

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Fine Mapping and Functional Analysis of Yellow Leaf Mutant ylm-1 in Foxtail Millet

Qin Na(), Zhu Cancan, Dai Shutao, Song Yinghui, Li Junxia(), Wang Chunyi   

  1. Cereal Crops Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
  • Received:2021-12-13 Revised:2022-03-30 Online:2022-06-15 Published:2022-06-20
  • Contact: Li Junxia E-mail:qinna2004@126.com;lijunxia@126.com

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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

Table 1

Name and sequence of primers"

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

Fig.1

Leaf color of the wild type (WT), mutant ylm-1 and F1 at seedling stage"

Fig.2

Leaf color of the wild type （WT）, mutant ylm-1 and F1 at flowering stage"

Fig.3

Genetic background detection of mutant ylm-1 a-d are electrophoretic images of partial markers SICAAS1008, SICAAS1010, SICAAS5055 and SICAAS9036 used to detect the genetic background of ylm-1, respectively; 1-6 gel strips are wild type Yugu 1, ylm-1, Yugu 17, Jigu 41, Yugu 28 and Jinmiaohongjiugu"

Fig.4

Distribution map of SNP-index values on chromosomes"

Table 2

Statistical table of association region information"

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

Table 3

Analysis of non-synonymous mutation genes for the mutant ylm-1"

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

Fig.5

FPKM values of thirteen non-synonymous mutation genes in different parts"

Fig.6

Analysis of electrophoretic images of dCAPS marker for wild type×ylm-1 of F2 populations 1: wild type Yugu 1, 2: mutant ylm-1, 3~23: partial F2 populations of wild×ylm-1, M: DNA marker"

Fig.7

The structure diagram of Seita.9G249900 coding region (a) and analysis of peptide sequence (b)"

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