Identification and Fine Mapping of a Grain Width Mutant gw4 in Rice

doi:10.16819/j.1001-7216.2021.01204

Abstract

Abstract:

【Objective】Grain size is an important agronomic trait that determines yield and appearance quality in rice. Screening and identification of new grain size mutants will lay a foundation for further study of the regulation mechanism of grain development. 【Method】A grain width mutant gw4（grain width on chromosome 4）was obtained from 1% EMS mutagenized population of Zhonghua 11. The scanning electron microscope analysis of the outer and inner surfaces cells of glumes was carried out, and grain size and other agronomic traits were measured at the heading and maturity stages, respectively. With the map-based cloning, the mutant individuals derived from F₂ population of gw4 and TN1 were used for fine mapping. The candidate gene was determined by gene sequencing based on bioinformatics analysis, and expression analysis of grain size-related genes was carried out by the RT-PCR. 【Result】Besides narrow grains, gw4 also showed decreased phenotype values in plant height, grain length, thousand grain weight, grain number, number of primary and secondary rachis branches. Scanning electron microscope observation presented that both cell length and width of inner and outer surfaces cells of glumes were smaller than the wild type. Genetic analysis showed that the narrow grain phenotype was regulated by a single recessive nuclear gene, which was fine mapped to a region of 31.74 kb between the two markers BS6 and EX49 on chromosome 4. Sequence analyses revealed a single base substitution of G to A was found in LOC_Os04g01590 which resulted in a substitution of glycine to aspartic acid, and RT-PCR analysis indicated that it was mainly expressed in young panicles with declined expression level in mutant as compared with the wild type. 【Conclusion】GW4 is mainly involved in grain width development, and its candidate gene should be LOC_Os04g01590, which lays a foundation for further perfecting the genetic regulatory network of grain size.

Key words: rice, grain size, grain width, gene mapping, appearance quality

摘要：

【目的】水稻粒形是影响水稻产量和决定稻米外观品质的主要性状之一。筛选和鉴定新的粒形突变材料,可为研究水稻籽粒发育的调控机制奠定基础。【方法】粳稻品种中花11经1%的EMS处理,在诱变群体中获得一份窄粒突变体gw4(grain width on chromosome 4);分析粒形和其他主要农艺性状,在扫描电镜下观察颖壳细胞变化;利用突变体与籼稻品种台中本地1号配组的F₂分离群体,选择隐性个体完成基因的精细定位;开展生物信息和测序分析,确定定位区间的候选基因;采用RT-PCR分析该基因在根、茎、叶、鞘、穗等组织中的表达模式及其他粒形相关基因的表达水平。【结果】与野生型相比,除了表现窄粒外,gw4的粒长、千粒重、每穗粒数、一次枝梗数和二次枝梗数等显著下降;扫描电镜发现gw4的颖壳内外表皮细胞均小于野生型;遗传分析表明该窄粒表型受一对单隐性核基因控制;通过开发的新标记最终将该基因定位在第4染色体BS6与EX49两个标记之间约31.74 kb的范围内;测序结果发现在LOC_Os04g01590基因编码区发生了一个由G至A的单碱基突变,导致原来编码的甘氨酸变成了天冬氨酸;qRT-PCR结果表明,LOC_Os04g01590主要在幼穗中表达,且在突变体中表达显著下降。【结论】GW4主要调控水稻粒宽的发育,预测LOC_Os04g01590为其候选基因。这为进一步丰富粒形的遗传调控网络打下了基础。

关键词: 水稻, 粒形, 粒宽, 基因定位, 外观品质

Xuemei DENG, Peng HU, Yueying WANG, Yi WEN, Yiqing TAN, Hao WU, Kaixiong WU, Junge WANG, Linlin HOU, Lixin ZHU, Li ZHU, Guang CHEN, Dali ZENG, Guangheng ZHANG, Longbiao GUO, Zhenyu GAO, Deyong REN, Qian QIAN, Jiang HU. Identification and Fine Mapping of a Grain Width Mutant gw4 in Rice[J]. Chinese Journal OF Rice Science, 2021, 35(3): 259-268.

邓雪梅, 胡鹏, 王月影, 文艺, 谭义青, 伍豪, 吴凯雄, 王俊格, 侯琳琳, 朱黎欣, 朱丽, 陈光, 曾大力, 张光恒, 郭龙彪, 高振宇, 任德勇, 钱前, 胡江. 水稻粒宽突变体gw4的鉴定与基因定位[J]. 中国水稻科学, 2021, 35(3): 259-268.

Figures/Tables 8

References 41

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分子标记	正向引物序列（5'-3'）	反向引物序列（5'-3'）
Marker	Forward primer sequence (5'-3')	Reverse primer sequence (5'-3')	Purpose
BS6	AGTACACATGGGGAGTAGTTGG	GATGGCAATTGAAAAGTGACC	定位Mapping
BS11	TGCAGGTACACACACATACCC	GGAAGTAGCATCCATGCAACTA	定位Mapping
BS15	TTTGCAGCTGGCTAGCTTA	ACTTCCATAATTACGGGTCCT	定位Mapping
BS16	GGCAAATAATTAATAGCATGGTCC	TGGATGATTTCTGTTAGTGGTTCA	定位Mapping
BS18	TGGTTACAACGACACAACTGC	TCACTCATACCAACCTTGCG	定位Mapping
EX49	GTGCCTCCTTTTCTTGGAGAT	CACAGTCATCTCACTTGCAATC	定位Mapping
BS19	ACGTGTCTTGGGGCTGTGTC	TCCCTACACTTATTGCCCCTCT	测序Sequencing
BS20	TCCTCATTACATGTCCCCATCC	CCCTATCTGAACGCAAGTGCT	测序Sequencing
BS21	GTGGTAGCATGTGCTCTCCCT	AGGGTTGGCAGAGTTGTCAGA	测序Sequencing
BS22	TACCATGTTGCACCTGAATACCT	CAACCCTCCTGGTGATTCCC	测序Sequencing
BS23	CCAGTTGTTAGGGCTGTGTCTG	AGATTAGGGAGCCAATGCGA	测序Sequencing
BS24	GATGCTCTGTTTGGACAGCCT	TGTCGTCAGCATGTTGCCTG	测序Sequencing
BS25	ACCAACCAACCCCTTGAGTCT	TGGAAATCGGAGAGATTATACGTAC	测序Sequencing
Histon	GGTCAACTTGTTGATTCCCCTCT	AACCGCAAAATCCAAAGAACG	RT-PCR
RT-GW2	ATCGTGGACAAGGGCACCTGC	CCGTCCCGCTCGCGAGGCATG	RT-PCR
RT-GW5	AATCCAGACGGCATTCAGAG	CTTCACCAGCGCCTTGAG	RT-PCR
RT-GW7	CCCTAGCATCGACACCAAGT	TCGAGGACAGAGATGGGACT	RT-PCR
RT-GW8	ACAGCCAGATCCCATGAACT	GCGTGTAGTATGGGCTCTCC	RT-PCR
RT-GS2	TGCGTCCCTTCTTTGATGAGT	ACAGTTGGGTGCCTGAGAATG	RT-PCR
RT-GS3	CGGAAGAACTCCTGATCCATTC	CACTTGCTCTGCACAAACAGC	RT-PCR
RT-GS5	GTTCTCGGTACTGCGTGGAAG	ACTCCACAAACCTCCCAGCA	RT-PCR
RT-GL6	TCCAAACCAGATGTTGGTGA	GCCGTCGACTACATCTCGTT	RT-PCR
RT-LOC_Os04g01590	TTGGTGATGTCCCCATACAA	ACCAAATCTCTTCCCCTGCT	RT-PCR

分子标记	正向引物序列（5'-3'）	反向引物序列（5'-3'）
Marker	Forward primer sequence (5'-3')	Reverse primer sequence (5'-3')	Purpose
BS6	AGTACACATGGGGAGTAGTTGG	GATGGCAATTGAAAAGTGACC	定位Mapping
BS11	TGCAGGTACACACACATACCC	GGAAGTAGCATCCATGCAACTA	定位Mapping
BS15	TTTGCAGCTGGCTAGCTTA	ACTTCCATAATTACGGGTCCT	定位Mapping
BS16	GGCAAATAATTAATAGCATGGTCC	TGGATGATTTCTGTTAGTGGTTCA	定位Mapping
BS18	TGGTTACAACGACACAACTGC	TCACTCATACCAACCTTGCG	定位Mapping
EX49	GTGCCTCCTTTTCTTGGAGAT	CACAGTCATCTCACTTGCAATC	定位Mapping
BS19	ACGTGTCTTGGGGCTGTGTC	TCCCTACACTTATTGCCCCTCT	测序Sequencing
BS20	TCCTCATTACATGTCCCCATCC	CCCTATCTGAACGCAAGTGCT	测序Sequencing
BS21	GTGGTAGCATGTGCTCTCCCT	AGGGTTGGCAGAGTTGTCAGA	测序Sequencing
BS22	TACCATGTTGCACCTGAATACCT	CAACCCTCCTGGTGATTCCC	测序Sequencing
BS23	CCAGTTGTTAGGGCTGTGTCTG	AGATTAGGGAGCCAATGCGA	测序Sequencing
BS24	GATGCTCTGTTTGGACAGCCT	TGTCGTCAGCATGTTGCCTG	测序Sequencing
BS25	ACCAACCAACCCCTTGAGTCT	TGGAAATCGGAGAGATTATACGTAC	测序Sequencing
Histon	GGTCAACTTGTTGATTCCCCTCT	AACCGCAAAATCCAAAGAACG	RT-PCR
RT-GW2	ATCGTGGACAAGGGCACCTGC	CCGTCCCGCTCGCGAGGCATG	RT-PCR
RT-GW5	AATCCAGACGGCATTCAGAG	CTTCACCAGCGCCTTGAG	RT-PCR
RT-GW7	CCCTAGCATCGACACCAAGT	TCGAGGACAGAGATGGGACT	RT-PCR
RT-GW8	ACAGCCAGATCCCATGAACT	GCGTGTAGTATGGGCTCTCC	RT-PCR
RT-GS2	TGCGTCCCTTCTTTGATGAGT	ACAGTTGGGTGCCTGAGAATG	RT-PCR
RT-GS3	CGGAAGAACTCCTGATCCATTC	CACTTGCTCTGCACAAACAGC	RT-PCR
RT-GS5	GTTCTCGGTACTGCGTGGAAG	ACTCCACAAACCTCCCAGCA	RT-PCR
RT-GL6	TCCAAACCAGATGTTGGTGA	GCCGTCGACTACATCTCGTT	RT-PCR
RT-LOC_Os04g01590	TTGGTGATGTCCCCATACAA	ACCAAATCTCTTCCCCTGCT	RT-PCR

基因 Gene	功能注释 Function annotation
LOC_Os04g01560	表达蛋白Expressed protein
LOC_Os04g01570	蔗糖酶/果胶甲基酯酶抑制剂家族蛋白Invertase/Pectin methylesterase inhibitor family protein
LOC_Os04g01580	表达蛋白Expressed protein
LOC_Os04g01590	精氨酸酶Arginase
LOC_Os04g01600	赤藓酸-4-磷酸脱氢酶结构域蛋白Erythroate-4-phosphate dehydrogenase domain protein
LOC_Os04g01610	表达蛋白Expressed protein

基因 Gene	功能注释 Function annotation
LOC_Os04g01560	表达蛋白Expressed protein
LOC_Os04g01570	蔗糖酶/果胶甲基酯酶抑制剂家族蛋白Invertase/Pectin methylesterase inhibitor family protein
LOC_Os04g01580	表达蛋白Expressed protein
LOC_Os04g01590	精氨酸酶Arginase
LOC_Os04g01600	赤藓酸-4-磷酸脱氢酶结构域蛋白Erythroate-4-phosphate dehydrogenase domain protein
LOC_Os04g01610	表达蛋白Expressed protein