Cheol-Woo Kim

Identification of candidate genes associated with fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.) using a combination of bulked segregant analysis and RNA-seq

Key messageA combination of BSA and RNA-seq was performed to identify candidates for the restorer-of-fertility gene in onion. The AcPMS1 involved in DNA mismatch repair was identified as the best candidate.AbstractTo identify candidate genes of the restorer-of-fertility gene (Ms) responsible for fertility restoration of onion cytoplasmic male-sterility, a combined approach of bulked segregant analysis and RNA-seq was employed. From 32,674 de novo assembled contigs, 430 perfectly homozygous SNPs between male-fertile (MF) and male-sterile (MS) bulks were identified in 141 contigs. After verifying the homozygosity of the SNPs by PCR amplification and sequencing, the SNPs on 139 of the contigs were genotypes for the two recombinants which contained crossover events between the Ms locus and two tightly linked molecular markers. As a result, 30 contigs showing perfect linkage with the Ms locus in the large-sized segregating population were identified. Among them, 14 showed perfect linkage disequilibrium (LD) with the Ms locus, as determined by genotyping 251 domestic breeding lines. Furthermore, molecular markers tagging the 14 contigs also showed almost perfect LD with each other in 124 exotic accessions introduced from 21 countries, except for one accession which contained a crossover event by which the 14 markers were divided into two groups. After sequencing of the full-length cDNA of the 14 contigs showing perfect LD, the deduced amino acids sequences of the MF and MS alleles were compared. Four genes were shown to harbor putative critical amino acid changes in the known domains. Among them, the gene encoding PMS1, involved in the DNA mismatch repair pathway, was assumed to be the best candidate gene responsible for fertility restoration of male-sterility in onion.

At least nine independent natural mutations of the DFR-A gene are responsible for appearance of yellow onions (Allium cepa L.) from red progenitors

Inactivation of the gene (DFR-A) coding for dihydroflavonol 4-reductase (DFR) involved in the anthocyanin biosynthesis pathway results in a yellow bulb color in onion (Allium cepa L.). Three inactive alleles have previously been identified in onion, and in this study we identified three active and six inactive novel DFR-A alleles from extensive analyses of diverse onion germplasms. Of the germplasms analyzed, we identified a yellow mutant containing a 171-bp deletion in the promoter region, which we designated DFR-APD. Critically reduced transcription of this mutant allele and perfect co-segregation with color phenotypes in segregating populations were observed. A second yellow mutant (DFR-A5′DEL) which we identified contained a 518-bp deletion covering exons 1 and 2, which played important roles in DFR function. Both 2- and 4-bp insertions in the coding region leading to the creation of pre-mature stop codons were also identified and designated DFR-AGT and DFR-A2AT, respectively. A 1-bp substitution mutation (DFR-AK48N) which changed a positively charged lysine residue into a neutral asparagine was identified. This lysine residue, a NADPH binding site, was strictly conserved in other species. In addition, insertion of a leucine residue around substrate binding sites and catalytic triad was identified in several yellow accessions and designated DFR-ATTA. Phylogenetic analysis of DFR-A alleles showed that all inactive alleles were independently derived from four different active alleles. In addition, the close relatedness and diversity of DFR-A mutants implied that all these mutations might have occurred after domestication of onions and had probably been maintained by artificial selection.

Construction of a draft reference transcripts of onion (Allium cepa) using long-read sequencing

To obtain intact and full-length RNA transcripts of onion (Allium cepa), long-read sequencing technology was first applied. Total RNAs extracted from four tissues; flowers, leaves, bulbs and roots, of red–purple and yellow-colored onions (A. cepa) were sequenced using long-read sequencing (RSII platform, P4-C2 chemistry). The 99,247 polished high-quality isoforms were produced by sequence correction processes of consensus calling, quality filtering, orientation verification, misread-nucleotide correction and dot-matrix view. The dot-matrix view was subsequently used to remove artificial inverted repeats (IRs), and resultantly 421 IRs were removed. The remaining 98,826 isoforms were condensed to 35,505 through the removal process of redundant isoforms. To assess the completeness of the 35,505 isoforms, the ratio of full-length isoforms, short-read mapping to the isoforms, and differentially expressed genes among the four tissues were analyzed along with the gene ontology across the tissues. As a result, the 35,505 isoforms were verified as a collection of isoforms with high completeness, and designated as draft reference transcripts (DRTs, ver 1.0) constructed by long-read sequencing.

Identification of two novel mutant ANS alleles responsible for inactivation of anthocyanidin synthase and failure of anthocyanin production in onion (Allium cepa L.)

Two genes (DFR-A and ANS) encoding dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) enzymes in the anthocyanin biosynthesis pathway, respectively, are complementarily involved in anthocyanin production in onion (Allium cepa L.). Eleven inactive DFR-A alleles have been reported, with only a single inactive ANS allele previously identified. Two additional inactive ANS alleles are reported in this study. A mutant ANS allele containing a 4-bp insertion at the end of exon1 was identified from yellow bulbs of the F2 population in which the DFR-A genotype was homozygous for an active allele. The 4-bp insertion caused a frame-shift mutation and resulted in creation of a premature stop codon at the start of exon2. This mutant ANS allele was designated ANSPS allele. RT-PCR results showed that transcripts of the ANSPS allele were almost undetectable in yellow F2 bulbs, implying the involvement of nonsense-mediated mRNA decay. A cleaved amplified polymorphic sequence marker was developed for detection of the ANSPS allele. Another inactive ANS allele was identified from the light-red F1 populations showing complementation between DFR-A and ANS genes. A critical amino acid change of the strictly conserved serine residue into leucine was found in this mutant allele designated ANSS188L. In addition, seven variants of active ANS alleles were identified from diverse onion germplasm. A stepwise process consisting of PCR amplification and sequencing of PCR products was devised to identify three inactive (ANSPS, ANSS188L, and ANSG229R), one leaky (ANSP), and two active ANS alleles (ANSL and ANSh1).

Development of a simple PCR marker tagging the Allium roylei fragment harboring resistance to downy mildew (Peronospora destructor) in onion (Allium cepa L.)

For the efficient introgression of the downy mildew resistance gene from a resistant cultivar into domestic breeding lines, molecular markers used for marker-assisted backcrossing (MAB) were developed in onion (Allium cepa L.). The resistance gene was originally introgressed from the wild species Allium roylei using interspecific hybridization, and the resistant gene was known to be positioned at the end of chromosome 3. Therefore, the cDNA sequences of the loci located at the ends of chromosome 3 of two linkage maps were obtained from a transcriptome database. Primer pairs were designed on the exon sequences of eight loci; among them, the PCR products of the i25255 locus showed length polymorphism between the A. roylei and onions, and both large- and small-sized PCR products were observed in the resistant cultivar. A sequence analysis showed that a 67-bp indel existed in the intron sequences. Based on this indel polymorphism, a simple PCR marker, designated “DMR1”, was developed. An analysis of diverse onion accessions showed that, with the exception of the resistant cultivar, none of the accessions contained the A. roylei-specific marker genotype. These results indicate that the DMR1 marker was successfully tagging the A. roylei fragment harboring the downy mildew resistance gene. After further analysis of multiple loci positioned at chromosome 3, a range of the A. roylei fragment introgressed in the resistant cultivar was determined in two linkage maps. On the basis of the range of the A. roylei fragment, three molecular markers used for recombinant selection in MAB were also developed.

Distribution of various types of repetitive DNAs in Allium cepa L. based on dual color FISH

Fluorescence in situ hybridization (FISH) is a powerful tool for the detection of DNA sequences in a specific region of a chromosome as well as for integrated physical mapping. The detailed karyotypes of two onion cultivars (‘Eumjinara’ and ‘Sinseonhwang’), which are resources for the onion genome sequencing project were constructed based on dual color FISH using 5S and 45S rDNAs, telomeric tandem repeats, and Cot-1 DNA. All materials showed 2n = 2x = 16. Four loci of 5S rDNAs were located on the interstitial regions of the short arms of one pair of chromosomes in both onion cultivars. One loci of 45S rDNA signal was distally detected on each short arm of the two pairs of chromosomes in ‘Eumjinara’ and ‘Sinseonhwang’, but the latter possessed another locus of 45S rDNA on the distal part of the long arm in one homolog of a chromosome pair. Co-localization of telomeric tandem repeats and 45S rDNA signals was observed in ‘Eumjinara’ and ‘Sinseonhwang’. A difference in the distribution of 45S rDNA sites and the co-localization of signals observed between the two cultivars are indicators of recent activities in the nuclear genome that may involve homologous recombination or transposition of certain repeats. Cot-1 DNA signals are distributed throughout the chromosomes and show stronger signals in the terminal regions. The elucidation of Cot-1 DNA through in-situ hybridization would only show a large amount of tandemly repeating, non-coding and dispersedly repetitive DNAs in onion genome.

Inheritance of fertility restoration of male-sterility conferred by cytotype Y and identification of instability of male fertility phenotypes in onion (Allium cepa L.)

ABSTRACT A novel onion (Allium cepa L.) cytoplasm, cytotype Y, was found in a previous study. Cytotype Y contained unique stoichiometry of coxI and orf725, a candidate gene responsible for male-sterility induction in onions. A S1 segregating population was produced from a single plant selected from PI273626. Although male-fertility segregated in this population, the ratio significantly deviated from single-gene inheritance. However, genotypes of RF31446 marker perfectly linked to Ms locus-controlling fertility restoration completely matched with male-fertility phenotypes, indicating that male-fertility restoration of male-sterility conferred by cytotype Y might be determined by the Ms locus. One plant derived from the S1 population showed discrepancy between male-fertility phenotype and RF31446 genotype. Although the RF31446 genotype was homozygous recessive, reduced amount of pollen grains were observed in anthers. Many pollen grains of the unstable male-sterile plant were deformed. Analysis of 13 molecular markers flanking the Ms locus showed no crossover between the Ms locus and the RF31446 marker. Ten more unstable male-sterile plants were identified from open-pollinated progenies of the unstable male-sterile plant. Viable seeds were successfully produced from unstable male-sterile plants, indicating that pollen grains of the unstable male-sterile plants were partially viable. In addition, an umbel containing unstable male-fertile flowers was identified from one of maintainer lines, although both male and female organs might be sterile in these flowers.