Transposition of a non-autonomous DNA transposon in the gene coding for a bHLH transcription factor results in a white bulb color of onions (Allium cepa L.)

Key message

A DNA transposon was found in the gene encoding a bHLH transcription factor. Genotypes of the marker tagging this DNA transposon perfectly co-segregated with color phenotypes in large F2:3 populations


A combined approach of bulked segregant analysis and RNA-Seq was used to isolate causal gene for C locus controlling white bulb color in onions (Allium cepa L.). A total of 114 contigs containing homozygous single nucleotide polymorphisms (SNPs) between white and yellow bulked RNAs were identified. Four of them showed high homologies with loci clustered in the middle of chromosome 5. SNPs in 34 contigs were confirmed by sequencing of PCR products. One of these contigs showed perfect linkage to the C locus in F2:3 populations consisting of 2491 individuals. However, genotypes of molecular marker tagging this contig were inconsistent with color phenotypes of diverse breeding lines. A total of 146 contigs showed differential expression between yellow and white bulks. Among them, transcription levels of B2 gene encoding a bHLH transcription factor were significantly reduced in white RNA bulk and F2:3 individuals, although there was no SNP in the coding region. Phylogenetic analysis showed that onion B2 was orthologous to bHLH-coding genes regulating anthocyanin biosynthesis pathway in other plant species. Promoter regions of B2 gene were obtained by genome walking and a 577-bp non-autonomous DNA transposon designated as AcWHITE was found in the white allele. Molecular marker tagging AcWHITE showed perfect linkage with the C locus. Marker genotypes of the white allele were detected in some white accessions. However, none of tested red or yellow onions contained AcWHITE insertion, implying that B2 gene was likely to be a casual gene for the C locus.

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This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agriculture, Food and Rural Affairs Research Center Support Program (Vegetable Breeding Research Center) funded by the Ministry of Agriculture, Food and Rural Affairs (710011-03), Golden Seed Project (Center for Horticultural Seed Development, No 213007-05-3-SBB10), and a Grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ013400). The authors thank Ji-wha Hur, Jeong-Ahn Yoo, and Su-jung Kim for their dedicated technical assistance.

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CJ performed experiments and drafted the manuscript. SK organized and coordinated this research project and edited the final manuscript.

Correspondence to Sunggil Kim.

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Supplementary Fig. 1

Venn diagram showing the number of contigs containing homozygous SNPs and DEGs between white and yellow bulked RNAs. Reference-SNPs: the number of reference contigs containing homozygous SNPs. De novo assembly-SNPs: the number of de novo-assembled contigs containing homozygous SNPs. Reference-DEGs: the number of reference contigs showing differential expression. De novo assembly-DEGs: the number of de novo-assembled contigs showing differential expression (TIFF 87 kb)

Supplementary Fig. 2

Development of C9860, a molecular marker that is perfectly linked to the C locus. A. Structure of full-length yellow and white alleles of the contig, AC.Combine.Locus_9860.10. Exons and introns are shown as gray and empty boxes, respectively. Arrow-shaped boxes indicate 5′-to-3′ direction. A 53-bp indel is shown as a filled box in the yellow allele. Horizontal arrows indicate primer-binding sites of the C9860 marker. B. C9860 marker genotypes of white, yellow, and red breeding lines. A: homozygous dominant yellow F2:3 individuals; H: heterozygous yellow F2:3 individuals; B: homozygous recessive white F2:3 individuals (TIFF 358 kb)

Supplementary Fig. 3

Correlation of expression levels of contigs between white and yellow bulked RNAs. A. Reference transcriptome. B.De novo-assembled contigs (TIFF 65 kb)

Supplementary Fig. 4

Comparison of expression levels of structural genes in the anthocyanin biosynthesis pathway between white and yellow bulk RNAs. Detail information including GenBank accession numbers of structural genes is described in Baek et al. (2017). A. Reference transcriptome. B.De novo-assembled contigs (TIFF 124 kb)

Supplementary Fig. 5

Comparison of expression levels of onion MYB-coding genes between white and yellow bulk RNAs. Detail information of onion MYB-coding genes is described in Baek et al. (2017). A. Reference transcriptome. B.De novo-assembled contigs (TIFF 125 kb)

Supplementary Fig. 6

Comparison of expression levels of onion WD40-coding genes between white and yellow bulk RNAs. Detail information of onion WD40-coding genes is described in Baek et al. (2017). A. Reference transcriptome. B.De novo-assembled contigs (TIFF 96 kb)

Supplementary Fig. 7

Comparison of gene organizations of onion B2, petunia AN1, dahlia DvIVS, and maize IN1. Exons and introns are shown as gray and empty boxes, respectively. Arrow-shaped boxes indicate 5′-to-3′ direction. bHLH domains are shown as filled boxes. Hatched boxes in onion intron 5 indicate repeat sequences. (TIFF 124 kb)

Supplementary Fig. 8

PCR products of B2 marker. A. Band patterns of two different combination of primers. The eighth nucleotide ‘C’ in the B2-F1-1 primer is changed into ‘G’ in the B2-F1-2 primer. Primer biding sites are shown in Fig. 3. A: homozygous dominant yellow F2:3 individuals; H: heterozygous yellow F2:3 individuals; B: homozygous recessive white F2:3 individuals. B. B2 marker genotypes of white, yellow, and red breeding lines. A: homozygous dominant yellow F2:3 individuals; H: heterozygous yellow F2:3 individuals; B: homozygous recessive white F2:3 individuals (TIFF 335 kb)

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Jo, C., Kim, S. Transposition of a non-autonomous DNA transposon in the gene coding for a bHLH transcription factor results in a white bulb color of onions (Allium cepa L.). Theor Appl Genet 133, 317–328 (2020).

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  • Onion (Allium cepa L.)
  • White bulb color
  • bHLH transcription factor
  • RNA-Seq
  • Molecular marker