Moo-Kyoung Yoon

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.)

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.

Discovery of a novel cytoplasmic male-sterility and its restorer lines in radish (Raphanus sativus L.)

A male-sterile (MS) radish (Raphanus sativus L.) was found in an accession collected from Uzbekistan. Unlike Ogura MS radishes in which no pollen grain is typically visible during anthesis, a small number of pollen grains stuck together in the dehiscing anthers was observed in the newly identified MS radish. Fluorescein diacetate tests and scanning electron micrographs showed that pollen grains in the new MS radish were severely deformed and non-viable. Cytological examination of pollen development stages showed a clear difference in the defective stage from that seen in Ogura male-sterility. Reciprocal cross-pollination with diverse male-fertile lines indicated that pollen grains of the new MS radish were completely sterile, and the female organs were fully fertile. When the new MS radish and Ogura MS lines were cross-pollinated with a set of eight breeding lines, all F1 progeny originating from crosses with the new MS radish were male-sterile. In contrast, most of the F1 progeny resulting from crosses with Ogura MS lines were male-fertile. These results demonstrated that factors associated with induction of the newly identified male-sterility are different from those of Ogura male-sterility. The lack of restorer lines for the newly identified male-sterility led us to predict that it might be a complete cytoplasmic male-sterility without restorer-of-fertility genes in nuclear genomes. However, cross-pollination with more diverse radish germplasm identified one accession introduced from Russia that could completely restore fertility, proving the existence of restorer-of-fertility gene(s) for the new male-sterility. Meanwhile, the PCR amplification profile of molecular markers for the classification of radish mitochondrial genome types revealed that the new MS radish contained a novel mitotype.

Comparison of Mitochondrial and Chloroplast Genome Segments from Three Onion (Allium cepa L.) Cytoplasm Types and Identification of a Trans-splicing Intron of Cox2

To study genetic relatedness of two male sterility-inducing cytotypes, the phylogenetic relationship among three cytotypes of onions (Allium cepa L.) was assessed by analyzing polymorphisms of the mitochondrial DNA organization and chloroplast sequences. The atp6 gene and a small open reading frame, orf22, did not differ between the normal and CMS-T cytotypes, but two SNPs and one 4-bp insertion were identified in CMS-S cytotype. Partial sequences of the chloroplast ycf2 gene were integrated in the upstream sequence of the cob gene via short repeat sequence-mediated recombination. However, this chloroplast DNA-integrated organization was detected only in CMS-S. Interestingly, disruption of a group II intron of cox2 was identified for the first time in this study. Like other trans-splicing group II introns in mitochondrial genomes, fragmentation of the intron occurred in domain IV. Two variants of each exon1 and exon2 flanking sequences were identified. The predominant types of four variants were identical in both the normal and the CMS-T cytotypes. These predominant types existed as sublimons in CMS-S cytotypes. Altogether, no differences were identified between normal and CMS-T, but significant differences in gene organization and nucleotide sequences were identified in CMS-S, suggesting recent origin of CMS-T male-sterility from the normal cytotype.

Identification of two novel inactive DFR-A alleles responsible for failure to produce anthocyanin and development of a simple PCR-based molecular marker for bulb color selection in onion (Allium cepa L.)

Two novel inactive alleles of Dihydroflavonol 4-reductase-A (DFR-A) were identified in yellow onion (Allium cepa L.) cultivars and breeding lines from Korea and Japan. Unlike the previously reported inactive yellow DFR-A allele, designated as DFR-ATRN, in which the 3′ portion of the coding sequences was deleted, an allele containing a premature stop codon, DFR-APS, was isolated from the majority of cultivars. Co-segregation of DFR-APS and color phenotypes in the F2 population from a cross between yellow and red parents showed that inactivation of DFR-A was responsible for lack of anthocyanin in these yellow onions. In addition, RT-PCR analysis of F2 population showed that the transcription level of the DFR-APS allele was significantly reduced owing to non-sense-mediated mRNA decay. A 20-bp deletion of a simple sequence repeat in the promoter region of the DFR-APS allele was used to develop a simple PCR-based molecular marker for selection of the DFR-APS allele. All genotypes of 138 F2 individuals were clearly distinguished by this molecular marker. In addition to the DFR-APS allele, another DFR-A allele, DFR-ADEL, was identified in some cultivars. In case of the DFR-ADEL allele, no PCR products were amplified throughout DFR-A sequences including promoter regions, suggesting deletion of the entire DFR-A gene. Co-segregation of the absence of DFR-A and color phenotypes was confirmed in another F2 population. Furthermore, RT-PCR results showed that no DFR-A transcript was detected in any yellow F2 individuals.

Construction of high-resolution linkage map of the Ms locus, a restorer-of-fertility gene in onion (Allium cepa L.)

For the purpose of developing closely-linked molecular markers to the Ms locus, a restorer-of-fertility gene in onions (Allium cepa L.), bulked segregant analysis and randomly amplified polymorphic DNA (RAPD) analyses were utilized. Five RAPD markers polymorphic between male-fertile and male-sterile bulks were identified. These RAPD markers were converted into a simple PCR marker or cleaved amplified polymorphic sequence (CAPS) markers after sequencing the RAPD products and obtaining flanking sequences of the RAPD markers by genome walking. A linkage map was constructed with the Ms locus and flanking markers using a F2 population. There was no recombinant between the Ms locus and two CAPS markers, jnurf05 and jnurf17. To increase resolution among these closely linked molecular markers and the Ms locus, a total of 1,346 F2:3 and 2,927 F2:4 plants were analyzed with two flanking markers for detection of recombinants. Segregation of male-fertility phenotypes in large-sized populations confirmed allelic segregation distortion in favor of the recessive Ms allele. Analysis of the recombinants with closely linked markers revealed only two recombinants between the Ms locus and the jnurf05 markers among 4,273 segregating plants, showing very tight linkage between the two loci. However, linkage disequilibrium between the two loci was not too strong among the breeding lines. Despite weak linkage disequilibrium, these tightly linked markers are useful in accurate marker-assisted selection of the Ms alleles and ultimate isolation of the Ms gene by map-based cloning approach.

Development of an Efficient Agrobacterium- Mediated Transformation System and Production of Herbicide-Resistant Transgenic Plants in Garlic (Allium sativum L.)

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.