Skip to main content

Advertisement

Springer Nature Link
Log in

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.)

  • Original Article
  • Published:
Theoretical and Applied Genetics Aims and scope Submit manuscript

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

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

References

  • Amawi H, Ashby CR Jr, Tiwari AK (2017) Cancer chemoprevention through dietary flavonoids: What’s limiting? Chin J Cancer 36:50

    Article  PubMed  PubMed Central  Google Scholar 

  • Antonescu C, Antonescu V, Sultana R, Quackenbush J (2010) Using the DFCI gene index batabases for biological discovery. Curr Protoc Bioinform 29:1.6.1–1.6.36

    Article  Google Scholar 

  • Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–218

    Article  CAS  Google Scholar 

  • Baek G, Kim C, Kim S (2017) Development of a molecular marker tightly linked to the C locus conferring a white bulb color in onion (Allium cepa L.) using bulked segregant analysis and RNA-seq. Mol Breed 37:94

    Article  CAS  Google Scholar 

  • Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cao J, Chen W, Zhang Y, Zhang Y, Zhao X (2010) Content of selected flavonoids in 100 edible vegetables and fruits. Food Sci Technol Res 16:395–402

    Article  CAS  Google Scholar 

  • Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552

    Article  CAS  PubMed  Google Scholar 

  • Chang Y, Yan M, Yu J, Zhu D, Zhao Q (2017) The 5′ untranslated region of potato SBgLR gene contributes to pollen-specific expression. Planta 246:389–403

    Article  CAS  PubMed  Google Scholar 

  • Chin S, Behm CA, Mathesius U (2018) Functions of flavonoids in plant-nematode interactions. Plants 7:85

    Article  CAS  PubMed Central  Google Scholar 

  • Clarke AE, Jones HA, Little TM (1944) Inheritance of bulb color in the onion. Genetics 29:569–575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Czemmel S, Heppel SC, Bogs J (2012) R2R3 MYB transcription factors: key regulators of the flavonoid biosynthetic pathway in grapevine. Protoplasma 249:S109–S118

    Article  PubMed  CAS  Google Scholar 

  • Davies KM, Albert NW, Schwinn KE (2012) From landing lights to mimicry: the molecular regulation of flower colouration and mechanisms for pigmentation patterning. Funct Plant Biol 39:619–638

    Article  CAS  PubMed  Google Scholar 

  • Dixon RA, Pasinetti GM (2010) Flavonoids and isoflavonoids: from plant biology to agriculture and neuroscience. Plant Physiol 154:453–457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Duangjit J, Bohanec B, Chan AP, Town CD, Havey MJ (2013) Transcriptome sequencing to produce SNP-based genetic maps of onion. Theor Appl Genet 126:2093–2101

    Article  CAS  PubMed  Google Scholar 

  • El-Shafie MW, Davis GN (1967) Inheritance of bulb color in the onion (Allium cepa L.). Hilgardia 38:607–622

    Article  Google Scholar 

  • Farhadi F, Khameneh B, Iranshahi M, Iranshahy M (2018) Antibacterial activity of flavonoids and their structure–activity relationship: an update review. Phytother Res 33:13–40

    Article  PubMed  CAS  Google Scholar 

  • Feller A, Machemer K, Braun EL, Grotewold E (2011) Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J 66:94–116

    Article  CAS  PubMed  Google Scholar 

  • Fernández-Rojas B, Gutiérrez-Venegas G (2018) Flavonoids exert multiple periodontic benefits including anti-inflammatory, periodontal ligament-supporting, and alveolar bone-preserving effects. Life Sci 209:435–454

    Article  PubMed  CAS  Google Scholar 

  • Fini A, Brunetti C, Di Ferdinando M, Ferrini F, Tattini M (2011) Stress-induced flavonoid biosynthesis and the antioxidant machinery of plants. Plant Signal Behav 6:709–711

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, Macmanes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, Leduc RD, Friedman N, Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512

    Article  CAS  PubMed  Google Scholar 

  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for window 95/98/NT. Nucl Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Han Y, Qin S, Wessler SR (2013) Comparison of class 2 transposable elements at superfamily resolution reveals conserved and distinct features in cereal grass genomes. BMC Genom 14:71

    Article  CAS  Google Scholar 

  • Heim MA, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey PC (2003) The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol 20:735–747

    Article  CAS  PubMed  Google Scholar 

  • Hichri I, Barrieu F, Bogs J, Kappel C, Delrot S, Lauvergeat V (2011) Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J Exp Bot 62:2465–2483

    Article  CAS  PubMed  Google Scholar 

  • Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1070–1083

    Article  Google Scholar 

  • Jaakola L (2013) New insights into the regulation of anthocyanin biosynthesis in fruits. Trends Plant Sci 18:477–483

    Article  CAS  PubMed  Google Scholar 

  • Kim S, Jones R, Yoo K, Pike LM (2005a) The L locus, one of complementary genes required for anthocyanin production in onions (Allium cepa), encodes anthocyanidin synthase. Theor Appl Genet 111:120–127

    Article  CAS  PubMed  Google Scholar 

  • Kim S, Yoo K, Pike LM (2005b) The basic color factor, the C locus, encodes a regulatory gene controlling transcription of chalcone synthase genes in onions (Allium cepa). Euphytica 142:273–282

    Article  CAS  Google Scholar 

  • Kim S, Yoo K, Pike LM (2005c) Development of a PCR-based marker utilizing a deletion mutation in the DFR (dihydroflavonol 4-reductase) gene responsible for the lack of anthocyanin production in yellow onions (Allium cepa). Theor Appl Genet 110:588–595

    Article  CAS  PubMed  Google Scholar 

  • Kim S, Lee E, Kim C, Yoon M (2009) Distribution of three cytoplasm types in onion (Allium cepa L.) cultivars bred in Korea and Japan. Kor J Hortic Sci Technol 27:275–279

    CAS  Google Scholar 

  • Kim S, Kim M, Kim Y, Yeom S, Cheong K, Kim K, Jeon J, Kim S, Kim D, Sohn S, Lee Y, Choi D (2015a) Integrative structural annotation of de novo RNA-Seq provides an accurate reference gene set of the enormous genome of the onion (Allium cepa L.). DNA Res 22:19–27

    Article  CAS  PubMed  Google Scholar 

  • Kim S, Park JY, Yang T (2015b) Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the Pink allele of the ANS gene in onion (Allium cepa L.). Mol Genet Genomics 290:1027–1037

    Article  CAS  PubMed  Google Scholar 

  • Kim B, Kim K, Yang T, Kim S (2016a) Completion of the mitochondrial genome sequence of onion (Allium cepa L.) containing the CMS-S male-sterile cytoplasm and identification of an independent event of the ccmF N gene split. Curr Genet 62:873–885

    Article  CAS  PubMed  Google Scholar 

  • Kim E, Kim C, Kim S (2016b) Identification of two novel mutant ANS alleles responsible for inactivation of anthocyanidin synthase and failure of anthocyanin production in onion (Allium cepa L.). Euphytica 212:427–437

    Article  CAS  Google Scholar 

  • Kim B, Cho Y, Kim S (2017) Identification of a novel DFR-A mutant allele determining the bulb color difference between red and yellow onions (Allium cepa L.). Plant Breed Biotechnol 5:45–53

    Article  CAS  Google Scholar 

  • Kim B, Yang T, Kim S (2019) Identification of a gene responsible for cytoplasmic male-sterility in onions (Allium cepa L.) using comparative analysis of mitochondrial genome sequences of two recently diverged cytoplasms. Theor Appl Genet 132:313–322

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform 12:323

    Article  CAS  Google Scholar 

  • Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics 25:2078–2079

    Article  PubMed  PubMed Central  Google Scholar 

  • Li Y, Shan X, Gao R, Yang S, Wang S, Gao X, Wang L (2016) Two IIIf clade-bHLHs from Freesia hybrida play divergent roles in flavonoid biosynthesis and trichome formation when ectopically expressed in Arabidopsis. Sci Rep 6:30514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nakatsuka A, Yamagishi M, Nakano M, Tasaki K, Kobayashi N (2009) Light-induced expression of basic helix-loop-helix genes involved in anthocyanin biosynthesis in flowers and leaves of Asiatic hybrid lily. Sci Hortic 121:84–91

    Article  CAS  Google Scholar 

  • Ohno S, Hosokawa M, Hoshino A, Kitamura Y, Morita Y, Park K, Nakashima A, Deguchi A, Tatsuzawa F, Doi M, Iida S, Yazawa S (2011) A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis). J Exp Bot 62:5105–5116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Passeri V, Koes R, Quattrocchio FM (2016) New challenges for the design of high value plant products: stabilization of anthocyanins in plant vacuoles. Front Plant Sci 7:153

    Article  PubMed  PubMed Central  Google Scholar 

  • Petroni K, Tonelli C (2011) Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant Sci 181:219–229

    Article  CAS  PubMed  Google Scholar 

  • Pires N, Dolan L (2010) Origin and diversification of basic-helix-loop-helix proteins in plants. Mol Biol Evol 27:862–874

    Article  CAS  PubMed  Google Scholar 

  • Ramsay NA, Glover BJ (2005) MYB-bHLH-WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci 10:63–70

    Article  CAS  PubMed  Google Scholar 

  • Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scarano A, Chieppa M, Santino A (2018) Looking at flavonoid biodiversity in horticultural crops: a colored mine with nutritional benefits. Plants 7:98

    Article  CAS  PubMed Central  Google Scholar 

  • Schwinn KE, Ngo H, Kenel F, Brummell DA, Albert NW, McCallum JA, Pither-Joyce M, Crowhurst RN, Eady C, Davies KM (2016) The onion (Allium cepa L.) R2R3-MYB gene MYB1 regulates anthocyanin biosynthesis. Front Plant Sci 7:1865

    Article  PubMed  PubMed Central  Google Scholar 

  • Slimestad R, Fossen T, Vågen IM (2007) Onions: a source of unique dietary flavonoids. J Agric Food Chem 55:10067–10080

    Article  CAS  PubMed  Google Scholar 

  • Song S, Kim C, Moon JS, Kim S (2014) At least nine independent natural mutations of the DFR-A gene are responsible for appearance of yellow onions (Allium cepa L.) from red progenitors. Mol Breed 33:173–186

    Article  CAS  Google Scholar 

  • Spelt C, Quattrocchio F, Mol JN, Koes RE (2000) anthocyanin1 of Petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes. Plant Cell 12:1619–1631

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Veitch NC, Grayer RJ (2011) Flavonoids and their glycosides, including anthocyanins. Nat Prod Rep 28:1626–1695

    Article  CAS  PubMed  Google Scholar 

  • Voorrips RE (2002) MapChart: software for the graphical presentation of linkage maps and QTLs. J Hered 93:77–78

    Article  CAS  PubMed  Google Scholar 

  • Wallace TC (2015) Anthocyanins. Adv Nutr 6:620–622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O, Paux E, SanMiguel P, Schulman AH (2007) A unified classification system for eukaryotic transposable elements. Nat Rev Genet 8:973–982

    Article  CAS  PubMed  Google Scholar 

  • Xu Z, Feng K, Que F, Wang F, Xiong A (2017) A MYB transcription factor, DcMYB6, is involved in regulating anthocyanin biosynthesis in purple carrot taproots. Sci Rep 7:45324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yamasaki S, Sanada Y, Imase R, Matsuura H, Ueno D, Demura T, Kato K (2018) Arabidopsis thaliana cold-regulated 47 gene 5′-untranslated region enables stable high-level expression of transgenes. J Biosci Bioeng 125:124–130

    Article  CAS  PubMed  Google Scholar 

  • Yamazaki M, Makita Y, Springob K, Saito K (2003) Regulatory mechanisms for anthocyanin biosynthesis in chemotypes of Perilla frutescens var. crispa. Biochem Eng J 14:191–197

    Article  CAS  Google Scholar 

  • Zaynab M, Fatima M, Abbas S, Sharif Y, Umair M, Zafar MH, Bahadar K (2018) Role of secondary metabolites in plant defense against pathogens. Microb Pathog 124:198–202

    Article  CAS  PubMed  Google Scholar 

  • Zhang C, Feng L, Tian XS (2018) Alterations in the 5′ untranslated region of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene influence EPSPS overexpression in glyphosate-resistant Eleusine indica. Pest Manag Sci 74:2561–2568

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

  1. Department of Horticulture, Biotechnology Research Institute, Chonnam National University, Gwangju, 500-757, Korea

    Changyeong Jo & Sunggil Kim

Authors
  1. Changyeong Jo
    View author publications

    Search author on:PubMed Google Scholar

  2. Sunggil Kim
    View author publications

    Search author on:PubMed Google Scholar

Contributions

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

Corresponding author

Correspondence to Sunggil Kim.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All experiments performed in this study were in compliance with current laws of the Republic of Korea.

Additional information

Communicated by Alan H. Schulman.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

Supplementary material 9 (XLSX 10 kb)

Supplementary material 10 (DOCX 12 kb)

Supplementary material 11 (DOCX 13 kb)

Supplementary material 12 (DOCX 13 kb)

Supplementary material 13 (DOCX 14 kb)

Supplementary material 14 (XLSX 68 kb)

Supplementary material 15 (XLSX 58 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1007/s00122-019-03460-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00122-019-03460-8

Keywords