Identification of a candidate gene responsible for male sterility conferred by CMS-T cytoplasm in onion (Allium cepa L.) and development of molecular markers for detection of CMS-T cytoplasm

Cytoplasmic male sterility (CMS) has been exclusively used as an emasculation tool in seed production of hybrids in onion (Allium cepa L.).Three types of onion CMS (CMS-S, CMS-R, and CMS-T) have been used in hybrid onion breeding. Male sterility conferred by both CMS-S and CMS-R is likely induced by open reading frame (orf) 725, a chimeric gene. However, this gene was not detected in onion accessions containing CMS-T cytoplasm. Instead, a region showing high homology with orfA501, which is located in the 3' end of orf725, was detected in CMS-T. A novel chimeric gene, which we named orf219, was identified by genome walking PCR amplification based on the orfA501 homolog. The 684-bp open reading frame of orf219 consisted of an 128-bp of exon 1 of atp1 and a 556-bp sequence of an orfA501 homolog. The high-copy-number orf219 was detected only in the CMS-T cytoplasm (T cytotype). Analysis of RT-PCR products showed normal transcription of orf219 and eight RNA editing sites, one of which created a stop codon, resulting in a shorter amino acid sequence upon translation. Sequences of four hypervariable regions in the chloroplast genome and the organization of syntenic blocks in mitochondrial genome indicated that CMS-T was very closely related to the normal (N) male-fertile and CMS-R cytotypes. Based on these findings, new molecular markers were developed for the identification of cytotypes CMS-T, -R, -S, and -N. Cytotypes of 424 diverse onion accessions were identified using six molecular markers. The CMS-S and CMS-R cytotypes predominated among the analyzed accessions. In contrast, only five accessions possessed CMS-T, supporting earlier reports that CMS-T is rarely used in hybrid-onion breeding.