利用改进的MutMap方法克隆水稻雄性不育基因

doi:10.3724/SP.J.1005.2014.00085

遗传 ›› 2014, Vol. 36 ›› Issue (1): 85-93.doi: 10.3724/SP.J.1005.2014.00085

• 技术与方法 • 上一篇

利用改进的MutMap方法克隆水稻雄性不育基因

陈竹锋¹, 严维², 王娜¹, 张文辉¹, 谢刚¹, 卢嘉威¹, 简智华¹, 刘东风¹, 唐晓艳^1,2

1. 深圳市作物分子设计育种研究院, 深圳 518107;
2. 首都师范大学生命科学学院, 北京 100089

收稿日期:2013-10-18 修回日期:2013-11-04 出版日期:2014-01-20 发布日期:2013-12-20
通讯作者: 唐晓艳, 博士, 研究员, 研究方向：植物分子生物学。E-mail: txy@frontier-ag.com E-mail:txy@frontier-ag.com
作者简介:陈竹锋, 博士, 助理研究员, 研究方向：作物分子遗传育种。E-mail: czf@frontier-ag.com
基金资助:
国家自然科学基金项目(编号：31110103917), 广东省引进创新科研团队计划资助项目(编号：201001S0104725509)和深圳市科创委基础研究重点项目(编号：JC201005280655A)资助

Cloning of a rice male sterility gene by a modified MutMap method

Zhufeng Chen¹, Wei Yan², Na Wang¹, Wenhui Zhang¹, Gang Xie¹, Jiawei Lu¹, Zhihua Jian¹, Dongfeng Liu¹, Xiaoyan Tang^1,2

1. Shenzhen Institute of Molecular Crop Design, Shenzhen 518107 , China;
2. College of Life Sciences, Capital Normal University, Beijing 100089, China

Received:2013-10-18 Revised:2013-11-04 Online:2014-01-20 Published:2013-12-20

摘要/Abstract

摘要：

通过对籼稻黄华占EMS(甲磺酸乙酯)诱变, 筛选得到一隐性核不育的水稻雄性不育突变体osms55, 遗传分析表明该突变体为单基因控制的隐性核不育, 采用高通量的Illumina Infinium iSelect SNP(50 K)芯片检测技术鉴定该突变体的遗传背景, 确认该突变体的遗传背景与黄华占一致。文章利用改进的MutMap方法成功克隆该雄性不育基因, 突变位点与突变表型的共分离分析表明LOC_Os02g40450(MER3)是控制osms55突变体雄性不育的基因, 该基因的剪切识别位点发生变异后导致剪切异常, 造成第5外显子缺失15个碱基, 从而产生雄性不育。改进的MutMap方法无需精确组装的野生型基因组序列作对照, 而是通过将定位群体中有突变表型植株的DNA pool和野生型植株DNA的重测序结果分别与日本晴参考基因组进行比对, 然后再比较突变体和野生型的差异SNP来确定候选基因, 该方法大大降低了野生型基因组测序和组装成本, 进一步扩大了MutMap方法的应用范围。

关键词: 水稻(Oryza sativa L.), 突变体, 雄性不育, MutMap, 剪切识别位点

Abstract:

A pollen defective male sterile rice mutant, osms55, was isolated from an elite indica cultivar HHZ using EMS (ethyl methanesulfonate) mutagenesis strategy. Genetic analysis showed that osms55 was controlled by a single recessive gene. Genome-wide SNP analysis using the high-throughput Illumina Infinium iSelect SNP (50 K) microarray technology indicated that the genetic makeup of osms55 is the same as wild type (WT) HHZ. Using a modified MutMap method, we successfully identified a mutation in the LOC_Os02g40450 (MER3) gene that is co-segregated with the male sterility phenotype. The mutation is located at the intron splice-recognition site, leading to a 15 nucleotide deletion in the fifth exon. Different from the published MutMap method that aligns the mutant pool DNA sequence with the assembled WT genome, the method used in this study was to align the re-sequencing data of the mutant pool DNA and WT HHZ with the Nipponbare reference genome. The resulting SNPs of mutant/Nipponbare and WT HHZ/Nipponbare were further compared to determine the candidate mutant gene. This modified method does not need an assembled WT genome as reference and thus is more cost-effective and widely applicable.

Key words: rice, mutant, male sterility, MutMap, splice-recognition site

陈竹锋, 严维, 王娜, 张文辉, 谢刚, 卢嘉威, 简智华, 刘东风, 唐晓艳. 利用改进的MutMap方法克隆水稻雄性不育基因[J]. 遗传, 2014, 36(1): 85-93.

Zhufeng Chen, Wei Yan, Na Wang, Wenhui Zhang, Gang Xie, Jiawei Lu, Zhihua Jian, Dongfeng Liu, Xiaoyan Tang. Cloning of a rice male sterility gene by a modified MutMap method[J]. HEREDITAS, 2014, 36(1): 85-93.

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利用改进的MutMap方法克隆水稻雄性不育基因

Cloning of a rice male sterility gene by a modified MutMap method

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