Abstract
Ms and Ms2 are restorer-of-fertility loci in onion (Allium cepa L.); additionally, Ms2 is responsible for unstable male fertility in some accessions. Although a candidate gene was previously reported for the Ms locus, the gene responsible for the Ms2 locus remains unidentified. A 12.5 Mb genomic region harboring the Ms2 locus was initially obtained from onion whole genome sequences using two flanking markers to identify candidates. This region was further delimited to 3.19 Mb via fine mapping using 12 recombinants and 11 additional markers. A gene coding for a pentatricopeptide repeat (PPR) protein was identified within the 3.19 Mb region and designated AcPPR876. Phylogenetic analysis showed that AcPPR876 and four homologs belonged to the Rf-like PPR gene family. Polymorphic sequences between male fertile (MF) and male sterile (MS) AcPPR876 alleles were concentrated in the 5' region of the gene. Among them, a 446 bp insertion was identified at the putative promoter region of the MF allele. Although overall AcPPR876 transcription levels were very low, transcription levels of the MF allele were generally higher than those of the MS allele. A simple PCR marker was developed using the 446 bp insertion to perform Ms2 locus genotyping in the diverse onion germplasm. Although the dominant Ms2 allele was not found in any of the 250 domestic breeding lines, 29 out of 108 exotic accessions were shown to contain the dominant Ms2 alleles. Overall, the AcPPR876 gene is proposed as a strong candidate for the Ms2 locus. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-025-01561-5.