1.School of Life Sciences, Anhui Agricultural University, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences;2.Institute of Crop Sciences, Chinese Academy of Agricultural Sciences
The National Key Research and Development Program of China (2022YFD1200802), The Agricultural Science and Technology Innovation Program (CAAS-ZDRW202004), The National Engineering Research Center of Crop Molecular Breeding
玉米突变体male-sterile 20s2(ms20s2)是在玉米自交系KWS49中发现的一个无花粉型雄性不育突变体。与野生型相比,ms20s2突变体花药细小且颜色偏浅,花药内未观察到花粉,而在株高、穗位等农艺性状上突变体与野生型无显著差异。扫描电镜观察表明,与野生型相比,9叶期突变体ms20s2的花药中未观察到正在减数分裂的花粉母细胞；抽雄后突变体花药壁外部角质层形成异常,内部未观察到乌式体结构。观察不同发育阶段花药的石蜡切片发现,在S6-S7时期,与野生型相比,ms20s2突变体花药部分中间层和绒毡层细胞发生异常分裂,导致花药壁萎缩,花粉母细胞无法正常进行减数分裂,最终造成花粉母细胞死亡,产生雄性不育表型。遗传分析表明,突变体ms20s2的雄性不育表型受单个隐性核基因控制。利用玉米10K SNP芯片对F2定位群体中60株不育株进行基因型分析,初步将该突变位点定位在玉米2号染色体长臂上6.21Mb区段内。进一步增设InDel标记,扩大定位群体,最终将基因精细定位在了590kb区间内,该区间包含一个已知的蛋白编码基因MS32(Zm00001eb106620)。对该基因进行测序分析,在4号外显子上发现了一段3166bp的大片段插入,该插入可能影响了MS32蛋白功能,进而造成ms20s2的花药发育异常和雄性不育的表型。等位测验结果表明,突变体ms20s2是雄性不育基因MS32的新等位突变。基因的组织表达分析结果表明,该基因在玉米花药中特异表达,且仅在花药发育的S6和S7时期表达量较高,进一步验证了该基因在玉米花药绒毡层和中间层细胞发育过程中的重要作用。
The maize male-sterile 20s2 (ms20s2) is a pollen-free genic male sterility mutant that we identified in maize inbred line KWS49. Compared with wild type (WT), the mutant anthers were small and whitish, lacking pollen grains. However, there were no significant differences in agronomic traits such as plant height and ear height between the WT and ms20s2 mutant. The scanning electron microscopy (SEM) observations showed that, compared to WT, no pollen mother cells undergoing meiosis were observed in the anthers of the ms20s2 at V9 stage; and the anther cuticular was abnormal, and no ubisch bodies were observed on the inner surface of the anther wall of ms20s2 at tasseling stage. Analysis of paraffin sections of anthers from different developmental stages showed that some middle layer cells and tapetum cells of the ms20s2 anther underwent abnormal division from S6 to S7 stages compared to WT, causing the anther wall shrinking, pollen mother cells cannot perform meiosis normally, and leading to the death of the pollen mother cells and male sterile phenotype. The genetic analysis revealed that the male sterile phenotype of the ms20s2 mutant was controlled by a single recessive nuclear gene. By analyzing the genotypes of 60 sterile plants in the F2 mapping population with the maize 10K SNP chip, the mutation was preliminarily mapped to the 6.21Mb region on the long arm of chromosome 2 of maize. After developing new InDel markers and expanding the mapping population, the gene was fine mapped to the 590kb region, which contains a known protein-coding gene MS32 (Zm00001eb106620). Sequencing analysis of MS32 gene revealed that there is a 3166bp insertion in the exon 4 of ms20s2, which may affect the protein function of MS32, resulting in abnormal anther development and male sterility phenotype of ms20s2 mutant. Allelism test showed that the ms20s2 was a new allelic variation of maize male sterile gene MS32. The tissue specific expression analysis showed that the gene was expressed in maize anthers at S6 and S7 stages, which further verified that the gene played an important role in regulating the development of tapetum and middle layer of maize anthers.