Molecular Marker Characterization of Commercially Used Cytoplasmic Male Sterilities in Onion

Hybrid-onion (Allium cepa) seed is produced using systems of cytoplasmic male sterility (CMS) and two different CMS systems have been genetically characterized. S cytoplasm was the first source of onion CMS identified in the 1920s, followed by T cytoplasm that was described in the 1960s. Numerous studies have documented polymorphisms in the organellar DNAs differentiating S and T cytoplasms from the normal male-fertile cytoplasm of onion. There may be additional source(s) of onion CMS that have been described as “T-like” and appear to be more similar to N and T cytoplasms than S cytoplasm. In this study, onion breeding lines from commercial entities were evaluated for molecular markers distinguishing sources of onion CMS. Our results reveal that bona fide T cytoplasm is rarely used commercially to produce hybrid-onion seed, and both S cytoplasm and “T-like” cytoplasm are widely used. We propose that this “T-like” cytoplasm be labeled as “R” cytoplasm because it may have originated from population(s) of ‘Rijnsburger’ onion in the Netherlands. The results of this study also help to clarify inconsistent reports regarding nuclear male-fertility restoration for different sources of onion CMS.

Identification of Ms2, a Novel Locus Controlling Male-fertility Restoration of Cytoplasmic Male-sterility in Onion (Allium Cepa L.), and Development of Tightly Linked Molecular Markers

Cytoplasmic male-sterility (CMS) has been exclusively used to produce F1 hybrid seeds of onion (Allium cepa L.). A single nuclear locus, Ms, is known to restore male-fertility of CMS in onions. Unstable male-sterile onions producing a small amount of pollen grains have been identified in a previous study. When such unstable male-sterile onions were crossed with stable male-sterile onions containing CMS-T cytoplasm, male-fertility was completely restored, although genotypes of the Ms locus were homozygous recessive. Inheritance patterns indicated that male-fertility restoration was controlled by a single locus designated as Ms2. A combined approach of bulked segregant analysis and RNA-seq was used to identify candidate genes for the Ms2 locus. High resolution melting (HRM) markers were developed based on single nucleotide polymorphisms (SNPs) detected by RNA-Seq. Comparative mapping of the Ms2 locus showed that Ms2 was positioned at the end of chromosome 2 with a distance of approximately 70 cM away from the Ms locus. Although 38 contigs containing reliable SNPs were analyzed using recombinants selected from 1,344 individuals, no contig showed perfect linkage to Ms2. Interestingly, transcription levels of orf725, a CMS-associated gene in onions, were significantly reduced in male-fertile individuals of segregating populations. However, no significant change in its transcription level was observed in individuals of a segregating population with male-fertility phenotypes determined by the Ms locus, suggesting that male-fertility restoration mechanism of Ms2 might be different from that of the Ms locus.

Genetic architecture of male fertility restoration in a hybrid breeding system of rye (Secale cereale L.)

The ‘Gülzow’ (G) type cytoplasmic male sterility (CMS) system in hybrid rye (Secale cereale L.) breeding exhibits a strong and environmentally stable restoration of male fertility (Rf). While having received little scientific attention, three G-type Rf genes had been identified on 4RL (Rfg1) and two minor genes on 3R (Rfg2) and 6R (Rfg3) chromosome. Here, we report a comprehensive investigation of the genetics underlying restoration of male fertility in a large G-type CMS breeding system using a palette of complementing forward and reverse genetic analysis. This includes (i) genome wide association studies (GWAS) on a G-type germplasm, (ii) GWAS on a biparental mapping population, (iii) in silico identification of Rf-like pentatricopeptide repeat (RFL-PPR) genes and their expressed in G-type rye hybrids, and (iv) mining patterns in linkage disequilibrium. Our findings provide compelling evidence of a novel major G-type non-PPR Rf gene on the 3RL chromosome. In the in silico analysis, we identified 22 RFL-PPR of which 15 were expressed in the transcriptome of G-type hybrids. Our findings provides a novel insight into the underlying genetics of male fertility restoration in a G-type CMS system in rye. The discovery made in this study is distinct to known P- and C-type systems in rye in addition to known CMS systems in barley and wheat. This study constitutes a steppingstone towards understanding the restoration of male fertility in G-type CMS system and a potential resources for addressing the inherent issues of the P-type system.

Variable Penetrance among Different Sources of the Male Fertility Restoration Allele of Onion

Hybrid onion (Allium cepa) seed is produced using cytoplasmic male sterility (CMS). For the most commonly used source of onion CMS, male fertile plants possess male sterile (S) cytoplasm and dominant allele(s) at one nuclear male fertility locus (Ms). Because male fertility restoration is not necessary for bulb production, it is desirable to purge dominant alleles at Ms from populations and breeding lines to facilitate the development of male sterile inbreds for hybrid production. In this research, we used molecular markers to establish the cytoplasms and genotypes at Ms in progenies from testcrosses of male sterile lines with plants from three populations [B2354, Ailsa Craig (AC), and Sapporo-Ki (Ski)] possessing the dominant Ms allele. We scored male fertility of testcross progenies by visual examination of flowers and acetocarmine staining of pollen. Different sources of the dominant Ms allele showed significantly different amounts of male fertility restoration and proportions of stainable pollen, complicating visual selection against the dominant Ms allele. For AC and Ski, molecular markers correctly predicted male sterility vs. male fertility of progenies in the greenhouse and field. However, for B2354, male fertility restoration was less clear and especially difficult to score under field conditions, consistent with reduced penetrance of male fertility restoration for this source of the dominant Ms allele. These results will be of interest to onion breeders selecting S-cytoplasmic male sterile lines for hybrid onion development.