Abstract
Key message
Transposon insertion and point mutation independently activated the BoMYB2 gene in three purple cultivars of Brassica oleracea including kale, kohlrabi, and cabbage.
Abstract
Several varieties of B. oleracea have both green and purple cultivars. In this study, the causal genes for the purple traits in kale, kohlrabi and cabbage were cloned using map-based cloning approach. The purple traits in all three varieties were mapped to the same locus as the BoMYB2 gene in cauliflower. Surprisingly, the insertion of Harbinger transposon of BoMYB2 in cauliflower was not found in purple kale, kohlrabi and cabbage. Sequencing of the BoMYB2 gene in purple kale and purple kohlrabi discovered a 7606 bp CACTA-like transposon in its promoter region. Transient assay and promoter activity study showed that the insertion upregulated the expression of the BoMYB2 gene. On the other hand, the activation of the BoMYB2 gene in purple cabbage was caused by point mutation and/or 1-bp insertion in its promoter region. Sequence analysis of the BoMYB2 gene in different varieties suggested that the activating events most likely occurred independently after the divergence of cabbage, cauliflower, and kale/kohlrabi. Our results not only contribute to a better understanding of anthocyanin inheritance in B. oleracea, but also provide useful information for future hybrid breeding of purple cultivars through combination of different functional alleles of the BoMYB2 gene.
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References
Allan AC, Hellens RP, Laing WA (2008) MYB transcription factors that colour our fruit. Trends Plant Sci 13:99–102
Amaya I, Pillet J, Folta KM (2016) Identification of genes responsible for natural variation in volatile content using next-generation sequencing technology. Methods Mol Biol 1363:37–45
Baudry A, Heim MA, Dubreucq B et al (2004) TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J 39:366–380
Borevitz JO, Xia Y, Blount J et al (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12:2383–2394
Butelli E, Licciardello C, Zhang Y et al (2012) Retro-transposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. Plant Cell 24:1242–1255
Chiu LW, Li L (2012) Characterization of the regulatory network of BoMYB2 in controlling anthocyanin biosynthesis in purple cauliflower. Planta 236:1153–1164
Chiu LW, Zhou XJ, Burke S et al (2010) The purple cauliflower arises from activation of a MYB transcription factor. Plant Physiol 154:1470–1480
Clough SJ, Bent AF (1998) Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743
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
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797
Espley RV, Hellens RP, Putterill J et al (2007) Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49:414–427
Feder A, Burger J, Gao S et al (2015) A Kelch domain-containing F-Box coding gene negatively regulates flavonoid accumulation in muskmelon. Plant Physiol 169:1714–1726
Goff SA, Cone KC, Chandler VL (1992) Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins. Genes Dev 6:864–875
Gould KS (2004) Nature’s Swiss army knife: the diverse protective roles of anthocyanins in leaves. J Biomed Biotechnol 2004:314–320
He JA, Giusti MM (2010) Anthocyanins: natural colorants with health-promoting properties. Annu Rev Food Sci Technol 1:163–187
Hellens RP, Allan AC, Friel EN et al (2005) Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1:13
Izzah NK, Lee J, Perumal S et al (2013) Microsatellite-based analysis of genetic diversity in 91 commercial Brassica oleracea L. cultivars belonging to six varietal groups. Genet Resour Crop Evol 60:1967–1986
Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci 10:236–242
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874
Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079
Li P, Chen B, Zhang G et al (2016) Regulation of anthocyanin and proanthocyanidin biosynthesis by Medicago truncatula bHLH transcription factor MtTT8. New Phytol 210:905–921
Liu S, Liu Y, Yang X et al (2014) The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat Commun 5:3930
Liu S, Yeh CT, Tang HM et al (2012) Gene mapping via bulked segregant RNA-Seq (BSR-Seq). PLoS ONE 7:e36406
Liu XP, Gao BZ, Han FQ et al (2017) Genetics and fine mapping of a purple leaf gene, BoPr, in ornamental kale (Brassica oleracea L. var. acephala). BMC Genom 18:230
Lu S, Zhang Y, Zheng X et al (2016) Isolation and functional characterization of a lycopene beta-cyclase gene promoter from citrus. Front Plant Sci 7:1367
Lyi SM, Zhou X, Kochian LV et al (2007) Biochemical and molecular characterization of the homocysteine S-methyltransferase from broccoli (Brassica oleracea var. italica). Phytochemistry 68:1112–1119
Mathews H, Clendennen SK, Caldwell CG et al (2003) Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15:1689–1703
Neill SO, Gould KS (2003) Anthocyanins in leaves: light attenuators or antioxidants? Funct Plant Biol 30:865–873
Nesi N, Jond C, Debeaujon I et al (2001) The arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed. Plant Cell 13:2099–2114
Noda K, Glover BJ, Linstead P et al (1994) Flower color intensity depends on specialized cell-shape controlled by a MYB-related transcription factor. Nature 369:661–664
Parkin IA, Koh C, Tang H et al (2014) Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea. Genome Biol 15:R77
Quattrocchio F, Wing J, van der Woude K et al (1999) Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color. Plant Cell 11:1433–1444
Ramsay NA, Glover BJ (2005) MYB-bHLH-WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci 10:63–70
Schwinn K, Venail J, Shang YJ et al (2006) A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell 18:831–851
Song H, Yi H, Lee M et al (2018) Purple Brassica oleracea var. capitata F-rubra is due to the loss of BoMYBL2-1 expression. BMC Plant Biol 18:82
Stuurman J, Hoballah ME, Broger L et al (2004) Dissection of floral pollination syndromes in Petunia. Genetics 168:1585–1599
Sweeney MT, Thomson MJ, Pfeil BE et al (2006) Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice. Plant Cell 18:283–294
Takagi H, Abe A, Yoshida K et al (2013) QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J 74:174–183
Tang HM, Liu SZ, Hill-Skinner S et al (2014) The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation. Plant J 77:380–392
Tang Q, Tian M, An G et al (2017) Rapid identification of the purple stem (Ps) gene of Chinese kale (Brassica oleracea var. alboglabra) in a segregation distortion population by bulked segregant analysis and RNA sequencing. Mol Breed 37:153
Tsuda T, Horio F, Uchida K et al (2003) Dietary cyanidin 3-O-beta-D-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. J Nutr 133:2125–2130
Udvardi MK, Czechowski T, Scheible WR (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20:1736–1737
Van den Ende W, El-Esawe SK (2014) Sucrose signaling pathways leading to fructan and anthocyanin accumulation: a dual function in abiotic and biotic stress responses? Environ Exp Bot 108:4–13
Walker AR, Lee E, Bogs J et al (2007) White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J 49:772–785
Wang LS, Stoner GD (2008) Anthocyanins and their role in cancer prevention. Cancer Lett 269:281–290
Wu FH, Shen SC, Lee LY et al (2009) Tape-arabidopsis sandwich—a simpler arabidopsis protoplast isolation method. Plant Methods 5:16
Xie QL, Hu ZL, Zhang YJ et al (2014) Accumulation and molecular regulation of anthocyanin in purple tumorous stem mustard (Brassica juncea var. tumida Tsen et Lee). J Agric Food Chem 62:7813–7821
Yin XR, Allan AC, Chen KS et al (2010) Kiwifruit EIL and ERF genes involved in regulating fruit ripening. Plant Physiol 153:1280–1292
Zhang B, Hu Z, Zhang Y et al (2012) A putative functional MYB transcription factor induced by low temperature regulates anthocyanin biosynthesis in purple kale (Brassica Oleracea var. acephala f. tricolor). Plant Cell Rep 31:281–289
Zhang Y, Butelli E, Martin C (2014a) Engineering anthocyanin biosynthesis in plants. Curr Opin Plant Biol 19:81–90
Zhang YJ, Chen GP, Dong TT et al (2014b) Anthocyanin accumulation and transcriptional regulation of anthocyanin biosynthesis in purple bok-choy (Brassica rapa var. chinensis). J Agric Food Chem 62:12366–12376
Zhang YJ, Hu ZL, Zhu MK et al (2015) Anthocyanin accumulation and molecular analysis of correlated genes in purple kohlrabi (Brassica oleracea var. gongylodes L.). J Agric Food Chem 63:4160–4169
Acknowledgements
We thank Dr. Xiuxin Deng (Huazhong Agricultural University) for providing the vector of pGreenII0800 and pCAMBIA1301.
Funding
This work was supported by National Natural Science Foundation of China [31471162] and [31572131] and the Fundamental Research Funds for the Central Universities [2662015PY084].
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Communicated by Carlos F. Quiros.
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Yan, C., An, G., Zhu, T. et al. Independent activation of the BoMYB2 gene leading to purple traits in Brassica oleracea. Theor Appl Genet 132, 895–906 (2019). https://doi.org/10.1007/s00122-018-3245-9
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DOI: https://doi.org/10.1007/s00122-018-3245-9