Characterization and Gene Mapping of a Glume Lesion Mimic Mutant glmm1 in Rice

doi:10.16819/j.1001-7216.2023.221201

Abstract

Abstract:

【Objective】 The aim of this study is to identify and map the glume lesion mimics mutant gene in rice, and to lay a foundation for gene cloning and molecular mechanism research. 【Method】 Agronomic traits analysis, scanning electron microscopy observation, DAB staining, and total silicon content determination were performed on the wild-type material LR005 and the glume lesion mimics mutant glmm1 obtained through EMS mutagenesis. The F₂ population derived from glmm1 and L422 was used for genetic analysis, and gene mapping by map-based cloning and BSA-seq method. 【Result】 The mutant gradually showed brown spots on the glumes and leaves 10 days after heading, and the glumes were completely brown at mature stage. Compared with the wild type, the mutant showed highly significant reductions in plant height, panicle length, total number of grains per panicle, seed setting rate and 1000-grain weight. DAB staining showed that the glmm1 glumes and leaves had increased ROS(reactive oxygen species) content; Scanning electron microscopy observation showed that the siliceous cells on the surface of the mutant glumes and leaves wrinkled. The genetic analysis showed that the glume lesion mimics phenotype of glmm1 was controlled by a pair of recessive genes. Using the F₂ segregating population of glmm1 and L422, GLMM1 was localized to a 68-kb interval containing 10 genes on chromosome 2 by map-based cloning and BSA-seq. Sequence analysis revealed only one SNP in this interval, which located in the fifth exon of the gene Lsi1 (LOC_Os02g51110), resulting in a substitution of Ile with Thr. The reduced accumulation of glmm1 silicon was found by measuring total silicon content of the glumes and leaves, suggesting that glmm1 may be a mutant allele of Lsi1. 【Conclusion】 glmm1 is a new mutant allele of Lsi1, which causes a reduction in silicon content and accumulation of reactive oxygen species in plants, resulting in brown spots on glumes and leaves.

Key words: rice, mutant, lesion mimic, silicon transporters, gene mapping

摘要：

【目的】 鉴定水稻颖壳类病斑突变体，并进行基因定位，为基因克隆及其分子机制研究奠定基础。【方法】 对野生型材料LR005和经EMS诱变得到的颖壳类病斑突变体glmm1（glume lesion mimics mutant 1）进行农艺性状分析、扫描电镜分析、DAB染色和全硅含量测定。glmm1与广亲和材料L422杂交获得的F₂群体用于遗传分析，利用图位克隆和BSA-seq方法进行基因定位。【结果】 突变体glmm1在抽穗10 d后颖壳和叶片逐渐出现褐色斑点，成熟后颖壳完全呈现褐色。与野生型相比，突变体的株高、穗长、每穗总粒数、结实率和千粒重等都极显著降低。DAB染色表明glmm1颖壳和叶片的活性氧含量增多；扫描电镜显示突变体颖壳和叶片表面硅质细胞皱缩。遗传分析结果表明，突变体glmm1的颖壳类病斑表型受到一对隐性基因控制。利用glmm1与L422的F₂分离群体，通过图位克隆和BSA-seq等策略将glmm1定位在水稻第2染色体上68 kb的区间内。该区间内有10个候选基因。序列分析发现该区间仅有一个SNP位点，位于基因Lsi1（LOC_Os02g51110）的第5个外显子上，导致第238位氨基酸由异亮氨酸变为苏氨酸。对颖壳和叶片全硅含量的测定表明，glmm1突变体中硅的积累减少，说明GLMM1可能是Lsi1的等位突变。【结论】 GLMM1是Lsi1新的等位突变基因，该突变造成植株硅含量的降低和活性氧的积累，致使颖壳和叶片产生褐色类病斑。

关键词: 水稻, 突变体, 类病斑, 硅转运蛋白, 基因定位

XU Huan, ZHOU Tao, SUN Yue, WANG Mumei, YANG Yachun, MA Hui, LI Hao, XU Dawei, ZHOU Hai, YANG Jianbo, NI Jinlong. Characterization and Gene Mapping of a Glume Lesion Mimic Mutant glmm1 in Rice[J]. Chinese Journal OF Rice Science, 2023, 37(5): 497-506.

徐欢, 周涛, 孙悦, 王木妹, 杨亚春, 马卉, 李浩, 徐大伟, 周海, 杨剑波, 倪金龙. 水稻颖壳类病斑突变体glmm1的鉴定与基因定位[J]. 中国水稻科学, 2023, 37(5): 497-506.

Figures/Tables 9

References 44

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引物名称 Primer name	正向引物 Forward primer (5'→3')	反向引物 Reverse primer (5'→3')
X1	ACAACTGACCTACTCACCTAC	CAATGCAGGTGACATATTTTCAA
X2	TGCCTCATACAAATGAGAGAC	ATAAAAGTAGGACGGCTCTTC
X3	ACAGGTCACGAAGTACTGAA	GGGCTATTTATGTGTGCCTT
X4	CCTTATCTTGGTTTGGTCTTTT	CATCTGCCATCCTACCTGAA
X5	TTTTGACCGAGGGAGTACG	TCAGGTGACCCCACGACT
X6	AGAGGTGGCTTCGCTATTG	TTTGGGACGGAGGGAGTA
Y1	GGGCCGTCTGTCTCTAAA	CACGAAAAACCTTGACCA
Y2	AGGCCCAAGTCATTCAAC	CGCAAAGCATGAGACAAT
Y3	CCTACGTAAGAAGCCATCG	TCGGTTTAGAGGGGATTG
Y4	AGGCTCCATCAAACCAAC	ATTTCACATGCCCCTCAT
Y5	CGCTCACACAAGTTAATTGGA	GCGTTAAAACGTATGGACAG
Y6	GTAACTGGGAGTACAGTGGTAA	AAATTAAAGCGACAGAGATTTCC

引物名称 Primer name	正向引物 Forward primer (5'→3')	反向引物 Reverse primer (5'→3')
X1	ACAACTGACCTACTCACCTAC	CAATGCAGGTGACATATTTTCAA
X2	TGCCTCATACAAATGAGAGAC	ATAAAAGTAGGACGGCTCTTC
X3	ACAGGTCACGAAGTACTGAA	GGGCTATTTATGTGTGCCTT
X4	CCTTATCTTGGTTTGGTCTTTT	CATCTGCCATCCTACCTGAA
X5	TTTTGACCGAGGGAGTACG	TCAGGTGACCCCACGACT
X6	AGAGGTGGCTTCGCTATTG	TTTGGGACGGAGGGAGTA
Y1	GGGCCGTCTGTCTCTAAA	CACGAAAAACCTTGACCA
Y2	AGGCCCAAGTCATTCAAC	CGCAAAGCATGAGACAAT
Y3	CCTACGTAAGAAGCCATCG	TCGGTTTAGAGGGGATTG
Y4	AGGCTCCATCAAACCAAC	ATTTCACATGCCCCTCAT
Y5	CGCTCACACAAGTTAATTGGA	GCGTTAAAACGTATGGACAG
Y6	GTAACTGGGAGTACAGTGGTAA	AAATTAAAGCGACAGAGATTTCC

材料 Material	株高 Plant height / cm	穗长 Panicle length/cm	有效穗数 No. of effective panicles	每穗总粒数 No. of spikelets per panicle	结实率 Seed-setting rate / %	千粒重 1000-grain weight / g
野生型WT	92.06±4.24	20.59±1.43	5.6±2.2	535.8±62.9	42.07±8.41	20.23±0.14
glmm1	85.45±3.02**	18.86±1.41**	5.2±1.2	408.2±72.4**	29.21±9.50**	16.84±0.27**

材料 Material	株高 Plant height / cm	穗长 Panicle length/cm	有效穗数 No. of effective panicles	每穗总粒数 No. of spikelets per panicle	结实率 Seed-setting rate / %	千粒重 1000-grain weight / g
野生型WT	92.06±4.24	20.59±1.43	5.6±2.2	535.8±62.9	42.07±8.41	20.23±0.14
glmm1	85.45±3.02**	18.86±1.41**	5.2±1.2	408.2±72.4**	29.21±9.50**	16.84±0.27**

基因注释号	功能注释	基因符号
基因注释号 Gene annotation	功能注释 Function note	基因名称 Gene name
LOC_Os02g51100	砷泵驱动ATP酶 Arsenical pump-driving ATPase
LOC_Os02g51110	水通道蛋白 Aquaporin protein	Lsi1
LOC_Os02g51119	表达蛋白 Expressed protein
LOC_Os02g51130	含有糖基转移酶8结构域的蛋白质 Glycosyl transferase 8 domain containing protein
LOC_Os02g51140	富氮蛋白 N-rich protein
LOC_Os02g51150	鞘脂C4-羟化酶SUR2 Sphingolipid C4-hydroxylase SUR2
LOC_Os02g51160	水解酶，作用于C-N Hydrolase, acting on carbon-nitrogen
LOC_Os02g51164	表达蛋白Expressed protein
LOC_Os02g51170	表达蛋白Expressed protein
LOC_Os02g51180	Cullin蛋白 Cullin protein	OsCUL3a