水稻雄性不育突变体oss125的遗传分析及基因定位

doi:10.3864/j.issn.0578-1752.2015.04.01

摘要/Abstract

摘要： 【目的】对水稻甲磺酸乙酯（EMS）诱变产生的雄性不育突变体oss125进行遗传分析，并利用改进的MutMap方法克隆突变基因，为进一步探讨该基因功能及在农业生产上的应用奠定基础。【方法】用化学诱变剂EMS处理籼稻品种黄华占，通过观察表型，从突变体库中筛选出一株雄性不育突变体，记为oss125。将oss125与野生型黄华占进行杂交，调查F₁的育性和F_{2^{群体的育性分离情况。随机挑取F}2}中30个雄性不育表型的株系，提取DNA后等量混合形成DNA池，采用Illumina Hiseq 2000进行高通量测序。利用改进的MutMap方法分析测序数据获得候选突变位点，并进一步采用高分辨率溶解（HRM）方法确定突变基因与不育表型的连锁关系。对候选基因进行序列分析，同时利用RT-PCR分析该候选基因的表达模式。【结果】oss125突变体在营养生长期表型与野生型黄华占相同，进入生殖生长后，花粉经1% I₂-KI染色显示，以碘败为主（85%），15%能正常染色，但植株表现为完全雄性不育。oss125作为花粉受体与野生型黄华占杂交能够正常结实，F₁表现为可育，F₂群体的可育植株与不育植株分离比为3﹕1，表明雄性不育表型由1对隐性核基因控制。利用改进的MutMap方法分析突变体测序数据，得到4个候选位点，其中3个位于基因间区，1个位于OsRPA1a的第二个外显子区，编码区A663位点突变为C，导致其编码的氨基酸从谷氨酰胺（Q）突变成脯氨酸（P），HRM分析显示该突变与雄性不育性状紧密连锁。【结论】OsRPA1a是控制突变体oss125表型的基因，OsRPA1a编码区A663位点突变为C，导致花粉发育异常，植株表现为雄性全不育，但雌性发育正常。OsRPA1a参与水稻雄配子和雌配子发育过程，为水稻减数分裂和体细胞DNA修复所必需。前人报道OsRPA1a的T-DNA插入突变体表现为雌性全不育而雄性半不育，但oss125突变体表现为雄性全不育而雌性可育，说明该基因控制雄性发育和雌性发育的功能可能分布在蛋白质的不同区域，oss125突变体中的OsRPA1a点突变可能坐落于雄性发育功能区，不影响雌性发育功能。

关键词: 水稻, 突变体, 雄性不育, HRM, OsRPA1a

Abstract: 【Objective】This study is to conduct genetic analysis on the rice male sterility mutant oss125 and clone the mutant gene using modified MutMap method, laying a foundation for further study of the gene function and its application in agricultural production. 【Method】 A male sterility mutant, designated as oss125, was isolated from a mutant library of the indica rice Huanghuazhan (HHZ) generated with ethyl methane sulfonate (EMS) treatment. Phenotype of the oss125 mutant was observed. The oss125 mutant was crossed with the wild-type HHZ, fertility of the F₁DNA of 30 F₂plants showing the male sterile phenotype were isolated and bulked in equal amount. Bulked DNA was subjected to whole-genome re-sequencing on Hiseq 2000 platform. A modified MutMap method was utilized to identify genomic regions harboring the causal mutation. Linkage of candidate genes with the mutant phenotype was validated with HRM analysis. Sequence analysis of the candidate gene and the expression of the candidate gene in various rice tissues were studied using real-time PCR. 【Result】 Compared with the wild type HHZ, oss125 mutant had normal vegetative growth, inflorescence and flower morphology. 1% I₂-KI staining revealed 85% abnormal pollen and 15% pollen of normal phenotype. But the plant displayed complete male sterility. All F₁plants derived from the cross of oss125 mutant with the wild-type HHZ were fertile, and the F₂ plants displayed 3:1 segregation of fertile to male sterile plants, indicating that oss125 is controlled by a single recessive gene. Using a modified MutMap method, four candidate SNPs were identified, three in intergenic region, and one in the second exon of OsRPA1a. A single nucleotide mutation (A663C) occured in the coding region of the OsRPA1a gene, resulting in a nonsynonymous mutation (Q221P) in protein sequence. Linkage of the mutation with the male sterile phenotype was validated using HRM analysis. 【Conclusion】 Mutation of the OsRPA1a gene is responsible for the oss125 mutant phenotype. A663C mutation causes abnormal pollen development, leading to complete male sterility. OsRPA1a is essential for meiosis and DNA repair in somatic cells. It was previously reported that a T-DNA insertion mutant of OsRPA1a was reduced in male fertility but completely female sterile. However, oss125 mutant is completely male sterile, and the female fertility appears not affected, suggesting that OsRPA1a involves distinct regions in controlling male and female gametophyte development. The mutation in the oss125 mutant may be in the region governing male gametophyte development but not affecting the function of the female gametophyte development.and F2 plants was investigated.

Key words: rice, mutant, male sterility, HRM, OsRPA1a

张文辉，严维，陈竹锋，谢刚，卢嘉威，刘东风，唐晓艳. 水稻雄性不育突变体oss125的遗传分析及基因定位[J]. 中国农业科学, 2015, 48(4): 621-629.

ZHANG Wen-hui, YAN Wei, CHEN Zhu-feng, XIE Gang, LU Jia-wei, LIU Dong-feng, TANG Xiao-yan. Genetic Analysis and Mapping of the Rice Male Sterile Mutant oss125[J]. Scientia Agricultura Sinica, 2015, 48(4): 621-629.

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