Bisht Deepak Singh, Kumar Nitin, Watts Anshul, Chamola Rohit, Kumar Vajinder, Jain Priyanka, Kumar Manish, Adhikari Balwant Singh, Savadi Siddanna, Bhat Shripad Ramachandra
ICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi, 110012, India.
AIMMSCR, Amity University Uttar Pradesh, Sector 125, Noida, 201313, Uttar Pradesh, India.
Mol Genet Genomics. 2025 Sep 2;300(1):83. doi: 10.1007/s00438-025-02291-1.
An introgression from Moricandia arvensis is known to restore male fertility to Brassica juncea cytoplasmic male sterile lines carrying M. arvensis, Diplotaxis berthautii, D. catholica or D. erucoides cytoplasm. We have previously mapped the fertility-restorer gene (Rfm) to the distal end of A09 chromosome of B. juncea but the restorer gene remains to be discovered. This study was undertaken to identify and clone the restorer gene(s) using next-generation sequencing approach, leveraging its known chromosomal location and flanking markers. We assembled the draft genome of the B. juncea fertility restorer line (MRS15), carrying the M. arvensis introgression. Alignment of the MRS15 genomic scaffolds to B. juncea reference genome identified six scaffolds aligned to the terminal region of chromosome A09 (between 51 and 58.5 Mb) harbouring the Rfm. The high-density linkage map of Rfm locus confirmed the correct orientation of these scaffolds. Based on segregation of tightly linked flanking markers, namely, the earlier reported BjESSR06 and a newly identified SRB17 marker, the Rfm gene was assigned to Scaffold-547. In silico analysis revealed six pentatricopeptide repeat (PPR)-encoding Restorer-of-Fertility-Like (RFL) genes in the ~ 300 kb region delimited by the above stated markers. Based on the expression profiles of these genes in CMS and fertility restorer lines, and in a segregating population, PPR-640 was identified as the Rfm gene. Further, we designed a gene-based, co-dominant marker perfectly co-segregating with fertility restorer trait through collinearity analysis of the genomic region spanning PPR-640 and the B. juncea genome. The Rfm gene and the marker reported here are critical for utilising this CMS system in hybrid breeding and to clone and study evolution of restorer genes in other Brassicaceae members.
已知来自田野碎米荠（Moricandia arvensis）的渐渗能够恢复携带田野碎米荠、贝氏双角果芥（Diplotaxis berthautii）、卡氏双角果芥（D. catholica）或细叶双角果芥（D. erucoides）细胞质的芥菜型油菜细胞质雄性不育系的雄性育性。我们之前已将育性恢复基因（Rfm）定位到芥菜型油菜A09染色体的远端，但恢复基因仍有待发现。本研究旨在利用下一代测序方法，借助其已知的染色体位置和侧翼标记来鉴定和克隆恢复基因。我们组装了携带田野碎米荠渐渗的芥菜型油菜育性恢复系（MRS15）的基因组草图。将MRS15基因组支架与芥菜型油菜参考基因组进行比对，确定了六个与A09染色体末端区域（51至58.5 Mb之间）对齐的支架，该区域含有Rfm。Rfm位点的高密度连锁图谱证实了这些支架的正确方向。基于紧密连锁的侧翼标记（即先前报道的BjESSR06和新鉴定的SRB17标记）的分离情况，Rfm基因被定位到Scaffold-547。电子分析显示，在上述标记界定的约300 kb区域内有六个编码五肽重复序列（PPR）的类育性恢复基因（RFL）。根据这些基因在细胞质雄性不育系和育性恢复系以及分离群体中的表达谱，PPR-640被鉴定为Rfm基因。此外，通过对跨越PPR-640的基因组区域与芥菜型油菜基因组的共线性分析，我们设计了一个与育性恢复性状完全共分离的基于基因的共显性标记。本文报道的Rfm基因和标记对于在杂交育种中利用该细胞质雄性不育系统以及克隆和研究其他十字花科成员中恢复基因的进化至关重要。
