State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Maize Research Institute, Sichuan Agricultural University, No. 211 Huimin Road, Chengdu, 611130, Wenjiang, China.
Chengdu Normal University, Chengdu, 611130, Wenjiang, China.
Theor Appl Genet. 2023 Oct 25;136(11):234. doi: 10.1007/s00122-023-04480-1.
A novel strong fertility restorer gene Rf12 for C-type cytoplasmic male sterility of maize was finely mapped on chromosome 2. Its best candidate gene Zm00001d007531 is predicted to encode a p-type PPR protein. The lack of strong restorer gene of maize CMS-C greatly limits its application in hybrid seed production. Therefore, the cloning of maize CMS-C novel strong restorer genes is necessary. In this study, a strong restorer line ZH91 for maize CMS-C was found, and the novel restorer gene named Rf12 in ZH91 had been mapped in a 146 kb physical interval on maize chromosome 2. Using the third-generation high-throughput sequencing (ONT), the whole genome sequence of ZH91 was got, and with integrating the annotation information of the reference genome B73_RefGen_v4 and B73_RefGen_v5, four candidate genes were predicted in ZH91 within the mapping region. Then using gene cloning, stranded specific RNA sequencing, qRT-PCR analysis and subcellular localization, Zm00001d007531 was identified as the most likely candidate gene of Rf12. Zm00001d007531 encodes a p-type PPR protein with 19 PPR motifs and targets mitochondria and chloroplast. Stranded specific RNA sequencing and qRT-PCR results both show that the expression of Zm00001d007531 between anthers of near-isogenic lines C478 and C478 was significantly difference in pollen mother cell stage. And the result of sequence alignment for Zm00001d007531 gene in 60 materials showed that there are twelve SNPs in CDS region of Zm00001d007531 were tightly linked to the fertility. The finding of a novel strong restorer germplasm resource ZH91 for maize CMS-C can greatly promote the application of maize CMS-C line in maize hybrid seeds production, and the identification of candidate gene Zm00001d007531 can accelerate the backcrossing process of maize CMS-C strong restorer gene Rf12 to some extent.
一个新的强育性恢复基因 Rf12 被精细定位到玉米 C 型细胞质雄性不育的 2 号染色体上。其最佳候选基因 Zm00001d007531 预测编码一个 p 型 PPR 蛋白。玉米 CMS-C 缺乏强恢复基因极大地限制了其在杂交种子生产中的应用。因此，克隆玉米 CMS-C 的新型强恢复基因是必要的。本研究发现了一个玉米 CMS-C 的强恢复系 ZH91，新的恢复基因 Rf12 已被精细定位到玉米 2 号染色体上的 146kb 物理区间内。利用第三代高通量测序（ONT）获得了 ZH91 的全基因组序列，并整合参考基因组 B73_RefGen_v4 和 B73_RefGen_v5 的注释信息，在定位区域内预测到 ZH91 中的四个候选基因。然后通过基因克隆、链特异性 RNA 测序、qRT-PCR 分析和亚细胞定位，鉴定 Zm00001d007531 为 Rf12 的最可能候选基因。Zm00001d007531 编码一个 p 型 PPR 蛋白，含有 19 个 PPR 基序，靶向线粒体和叶绿体。链特异性 RNA 测序和 qRT-PCR 结果均表明，在近等基因系 C478 和 C478 的花粉母细胞阶段，其花药中 Zm00001d007531 的表达存在显著差异。对 60 个材料的 Zm00001d007531 基因序列比对结果表明，在 Zm00001d007531 的 CDS 区有 12 个 SNP 与育性紧密连锁。玉米 CMS-C 新型强恢复种质资源 ZH91 的发现，极大地促进了玉米 CMS-C 系在玉米杂交种子生产中的应用，候选基因 Zm00001d007531 的鉴定，在一定程度上加速了玉米 CMS-C 强恢复基因 Rf12 的回交进程。
