A pentatricopeptide repeat protein restores fertility in Moricandia arvensis based cytoplasmic male sterility system in Brassica juncea.

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.