Identification of a novel chimeric gene, orf725, and its use in development of a molecular marker for distinguishing among three cytoplasm types in onion (Allium cepa L.)

Abstract

A novel chimeric gene with a 5' end containing the nearly complete sequence of the coxI gene and a 3' end showing homology with chive orfA501 was isolated by genome walking from two cytoplasm types: CMS-S and CMS-T, both of which induce male-sterility in onion (Allium cepa L.). In addition, the normal active and variant inactive coxI genes were also isolated from onions containing the normal and CMS-S cytoplasms, respectively. The chimeric gene, designated as orf725, was nearly undetectable in normal cytoplasm, and the copy number of the normal coxI gene was significantly reduced in CMS-S cytoplasm. RT-PCR results showed that orf725 was not transcribed in normal cytoplasm. Meanwhile, the normal coxI gene, which is essential for normal mitochondrial function, was not expressed in CMS-S cytoplasm. However, both orf725 and coxI were transcribed in CMS-T cytoplasm. The expression of orf725, a putative male-sterility-inducing gene, was not affected by the presence of nuclear restorer-of-fertility gene(s) in male-fertility segregating populations originating from the cross between a male-sterile plant containing either CMS-T or CMS-S and a male-fertile plant whose genotypes of nuclear restorer gene(s) might be heterozygous. The specific stoichiometry of orf725 and coxI in the mtDNA of the three cytoplasm types was consistent among diverse germplasm. Therefore, a molecular marker based on the relative copy numbers of orf725 and coxI was designed for distinguishing among the three cytoplasm types by one simple PCR. The reliability and applicability of the molecular marker was shown by testing diverse onion germplasm.

Inheritance mode of male sterility in bunching onion (Allium fistulosum L.) accessions

“…Since the fertility restoring gene for Zhangqiu-and Guangzhou-derived CMS has not been discovered in this study, we have compared the nucleotide sequences of three mitochondrial genes, cob, coxI and V7 region of srRNA among 'Zhangqiu', 'Guangzhou' and four F 1 commercial cultivars, 'Natsuougi-2', 'Fuyuougi-2', 'Ryuushou' and 'White Tower'. The primer sequences and PCR conditions for amplification of these genes were according to the reports of Engelke et al (2003) for cob, Kim et al (2009) for coxI and Buiteveld et al (1998) for V7 region of srRNA. In the three regions, sequence polymorphism was not detected among the six materials (data not shown).…”

The Application of Molecular Markers to Accelerate the Recovery of Cytoplasmic and Nuclear Male Sterility in South African Onion (Allium cepa L.) Hybrid Parental Lines

“…Specific primers for normal cox1 (cox1-F and cox1-R) yields an 833bp fragment for N-cytoplasm, but primers for orf725, expected in S and T-cytoplasm, yields a 628bp fragment (Kim et al, 2009). N-fragments were observed in all onion samples ( The smaller S-cytoplasm IGS fragment (299bp; Havey, 1995;Kim, 2014) was produced from all onion samples designated as S (Figure 1; IGS; line 1: all samples except 5 and 7; line 2: all samples; lines 3 and 4: only samples 6 and 7, respectively).…”

Application of the Molecular Marker in Linkage Disequilibrium with Ms, a Restorer-of-fertility Locus, for Improvement of Onion Breeding Efficiency

“…For identification of the cytoplasm types, a previously developed molecular marker was employed (Kim et al, 2009a). This marker was developed on the basis of polymorphic mitochondrial genome organizations.…”

“…Since the jnurf13 marker was found to be in complete linkage disequilibrium with the Ms locus, it would be very versatile for use in onion F 1 hybrid breeding in combination with the molecular marker developed for the distinction of cytoplasm types (Kim et al, 2009a). These two molecular markers were applied to accomplish four main purposes in onion breeding programs.…”

“…Since onion is a biennial crop, it generally takes at least four years to identify the cytoplasm types and Ms genotypes through conventional progeny tests. Several molecular markers have been developed for the identification of cytoplasm type based on polymorphic sequences of the chloroplast and mitochondrial genomes (Havey, 1995;Sato, 1998;Engelke et al, 2003;Kim et al, 2009a). As for the nuclear Ms locus, although a couple of molecular makers have been developed (Gökçe et al, 2002;Bang et al, 2013, they have experienced limitations in predicting the Ms genotypes of diverse breeding lines.…”