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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

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.

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References

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

  2. 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

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

  4. 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

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

  6. 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

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

  8. 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

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

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

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

  12. 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

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

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

  15. 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

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

  17. 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

  18. 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

  19. 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

  20. 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

  21. 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

  22. 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

  23. 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

  24. 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

  25. 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

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

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

  28. 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

  29. 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

  30. 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

  31. 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

  32. 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

  33. 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

  34. 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

  35. 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

  36. 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

  37. 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

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

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

  40. 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

  41. 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

  42. 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

  43. 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

  44. 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

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

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

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

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

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

  50. 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

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

  52. 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

  53. 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

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

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

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

  57. 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

  58. 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

  59. 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

  60. 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

  61. 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

  62. 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

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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.

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

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Communicated by Alan H. Schulman.

Electronic supplementary material

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

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Keywords

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