水稻开颖半不育突变体的观察、遗传分析和基因定位

Observation, Genetic Analysis and Gene Mapping of an Open Hull Semi-Sterility Mutant in Rice(Oryza sativa)

【目的】通过对一份航天诱变水稻（Oryza sativa L.）开颖半不育突变体ohss（open-hull semi-sterility）进行形态特性调查、遗传分析和基因定位,筛选候选基因,为下一步基因克隆和功能分析奠定基础。【方法】以籼稻品种航恢七号为材料,通过＂神舟八号＂飞船搭载,诱变获得一份水稻开颖半不育突变体ohss。对其进行形态特征解剖观察,分析颖花器官发育突变特点。调查突变体和野生型的花粉可育率、自然结实率和套袋自交结实率,对其育性进行鉴定。随机选取5个成熟单株,考察穗部谷粒相关性状并进行统计分析。通过覆盖全基因组的SSR分子标记检测,解析空间诱变的分子变异效应。以航恢七号、Francis和02428与突变体ohss配制杂交组合,观察F1和F2植株的花器官表型,进行χ~2测验,对突变性状进行遗传分析。以02428/ohss的F_2分离群体作为目标基因定位群体,同时利用SSR标记以及新开发的多个In Del分子标记开展基因定位研究。利用RAP水稻基因组注释数据库对定位区间的候选基因进行预测,通过序列比对和基因表达分析筛选候选基因。【结果】开颖半不育花器官突变体ohss与野生型相比,抽穗期穗部明显包茎,颖花发育出现异常,内外稃片瘪弱、扭曲变形且开裂不抱合,颖花内部发育类似内稃状的器官,部分颖花没有内稃的分化。ohss发育异常颖花中可育花粉率58.74%,导致单株结实率、穗重、穗实粒数与野生型相比极显著降低。全基因组SSR标记检测表明突变体ohss总变异频率为0.0336,除了第7、12染色体未检测到突变位点,其他染色体上检测到突变频率范围为0.0143—0.0889。遗传分析结果显示ohss的开颖半不育表型受单隐性核基因ohss（t）控制,并将ohss（t）定位在水稻第3染色体上2个In Del标记In Del6043和In Del6070之间约27.6 kb的物理距离内。该区域有3个预测注释基因,序列比对和表达分析表明突变体ohss的Os MADS1编码区及启动子序列未发生突变,但是表达模式发生强烈改变。【结论】开颖半不育的花器官发育突变体ohss受单隐性核基因ohss（t）控制,ohss（t）定位在水稻第3染色体上In Del6043和In Del6070标记之间约27.6 kb的物理距离内,其Os MADS1的编码序列及5′UTR区未发生碱基突变但表达受到强烈抑制。

【Objective】The paper is order to carry out a morphological characteristics investigation and genetic analysis of an open-hull semi-sterility mutant（ohss） of rice（Oryza sativa L.）, induced by spaceflight, laid the groundwork for further gene cloning and function analysis by fine mapping and preliminary screening of the candidate gene responsible for the mutated trait.【Method】The ohss mutant was derived from rice variety Hanghui 7, which was induced on Spaceship ＂Shenzhou 8＂. The morphological characteristics of ohss were anatomically observed to analyze the mutagenic features of floral organ development. Subsequently,pollen fertility, natural seed setting rate and bagged seed setting rate were investigated for fertility evaluation. Five plants of ohss mutation and WT were random selected to survey the panicle and grain related traits. SSR markers covering the whole genome were used to detect the mutagenic effect of ohss. Moreover, genetic analysis was conducted using the crosses between ohss and three wild type varieties, Hanghui 7, Francis and 02428 where the phenotypes of the F_1 and F_2 were surveyed and χ~2 test was performed. A population from the cross of 02428/ohss was used to map the ohss（t） gene using SSR markers and newly developed In Del markers. The candidate gene was predicted based on the RAP gene annotation database of the mapping region and screened through sequences alignment and expression of candidate genes.【Result】Compared with wild-type, panicles of mutant ohss were enclosed and florets showed abnormalities at the reproductive stage, and the palea and lemma were weak, distorted and dehiscent, with organ similar to the palea in the floret, while some of florets had no palea differentiation. Sterility testing showed that pollen grain rate of the abnormal spikelet of ohss was 58.7%, leading to significantly lower seed set, less panicle weight per plant and filled grain number per panicle compared with the wild-type. Mutation survey based on SSR markers revealed a total of 0.0336 of variation frequency was caused between ohss and WT, and variation frequency of different chromosome varied from 0.0143 to 0.0889, except chromosome 7 and 12. Genetic analysis indicated that the mutant phenotype in ohss was controlled by a single recessive nuclear gene, namely ohss（t）, which had been fine mapped to a ~27.6 kb physical distance between two InD el markers, In Del6043 and In Del6070 on chromosome 3, where three annotated genes were predicted. Based on the result of sequencing, semi-quantitative RT-PCR and real time quantitative PCR, there was no mutation occurring in the coding and promoter sequence of OsM ADS1 of ohss, but strong changes on gene expression pattern【.Conclusion】The mutated trait of ohss was controlled by a single recessive nuclear gene ohss（t）, which was fine mapped to a ~27.6 kb physical distance between In Del6043 and InD el6070 on chromosome 3. No nucleotide sequence mutation was found to occur in the coding sequence or the 5′UTR of Os MADS1, but expression of OsM ADS1 was strongly inhibited in ohss（t）.