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Dissecting the genetic control of root and leaf tissue-specific anthocyanin pigmentation in carrot (Daucus carota L.)

Theoretical and Applied Genetics volume 132, pages 2485–2507 (2019)Cite this article

Abstract

Key message

Inheritance, QTL mapping, phylogenetic, and transcriptome (RNA-Seq) analyses provide insight into the genetic control underlying carrot root and leaf tissue-specific anthocyanin pigmentation and identify candidate genes for root phloem pigmentation.

Abstract

Purple carrots can accumulate large quantities of anthocyanins in their root tissues, as well as in other plant parts. This work investigated the genetic control underlying tissue-specific anthocyanin pigmentation in the carrot root phloem and xylem, and in leaf petioles. Inheritance of anthocyanin pigmentation in these three tissues was first studied in segregating F2 and F4 populations, followed by QTL mapping of phloem and xylem anthocyanin pigments (independently) onto two genotyping by sequencing-based linkage maps, to reveal two regions in chromosome 3, namely P1 and P3, controlling pigmentation in these three tissues. Both P1 and P3 condition pigmentation in the phloem, with P3 also conditioning pigmentation in the xylem and petioles. By means of linkage mapping, phylogenetic analysis, and comparative transcriptome (RNA-Seq) analysis among carrot roots with differing purple pigmentation phenotypes, we identified candidate genes conditioning pigmentation in the phloem, the main tissue influencing total anthocyanin levels in the root. Among them, a MYB transcription factor, DcMYB7, and two cytochrome CYP450 genes with putative flavone synthase activity were identified as candidates regulating both the presence/absence of pigmentation and the concentration of anthocyanins in the root phloem. Concomitant expression patterns of DcMYB7 and eight anthocyanin structural genes were found, suggesting that DcMYB7 regulates transcription levels in the latter. Another MYB, DcMYB6, was upregulated in specific purple-rooted samples, suggesting a genotype-specific regulatory activity for this gene. These data contribute to the understanding of anthocyanin regulation in the carrot root at a tissue-specific level and maybe instrumental for improving carrot nutritional value.

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References

  • Akashi T, Aoki T, Ayabe S (1998) Identification of a cytochrome P450 cDNA encoding (2S)-flavanone 2-hydroxylase of licorice (Glycyrrhiza echinata L.; Fabaceae) which represents licodione synthase and flavone synthase II. FEBS Lett 431:287–290

    Article  CAS  PubMed  Google Scholar 

  • Andrews S (2010) FastQC a quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

  • Arbizu CI, Ellison SL, Senalik D, Simon PW, Spooner DM (2016) Genotyping-by-sequencing provides the discriminating power to investigate the subspecies of Daucus carota (Apiaceae). BMC Evol Biol 16:1–16

    Article  Google Scholar 

  • Ayabe S, Akashi T (2006) Cytochrome P450s in flavonoid metabolism. Phytochem Rev 5:271–282

    Article  CAS  Google Scholar 

  • Boiteux L, Fonseca MEN, Simon PW (1999) Effects of plant tissue and DNA purification method on randomly amplified polymorphic DNA-based genetic fingerprinting analysis in carrot. J Am Soc Hortic Sci 124:32–38

    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 

  • Broman K, Sen S (2009) A guide to QTL mapping with R/qtl. Springer, New York

    Book  Google Scholar 

  • Broman KW, Wu H, Sen Ś, Churchill GA (2003) R/qtl: QTL mapping in experimental crosses. Bioinformatics 19:889–890

    Article  CAS  PubMed  Google Scholar 

  • Cavagnaro PF, Iorizzo M, Yildiz M, Senalik D, Parsons J, Ellison S, Simon PW (2014) A gene-derived SNP-based high resolution linkage map of carrot including the location of QTL conditioning root and leaf anthocyanin pigmentation. BMC Genom 15:1–17

    Article  CAS  Google Scholar 

  • Charron CS, Kurilich AC, Clevidence BA, Simon PW, Harrison DJ, Britz SJ, Baer DJ, Novotny JA (2009) Bioavailability of anthocyanins from purple carrot juice: effects of acylation and plant matrix. J Agric Food Chem 57:1226–1230

    Article  CAS  PubMed  Google Scholar 

  • Dai X, Sinharoy S, Udvardi M, Xuechun Zhao P (2013) PlantTFcat: an online plant transcription factor and transcriptional regulator categorization and analysis tool. BMC Bioinform 14:321

    Article  CAS  Google Scholar 

  • Danecek P, Auton A, Abecasis G, Albers CA, Banks E, De Pristo MA, Handsaker RE, Lunter G, Marth GT, Sherry ST, McVean G, Durbin R, 1000 Genomes Project Analysis Group (2011) The variant call format and VCF tools. Bioinformatics 27:2156–2158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Deguchi A, Ohno S, Hosokawa M, Tatsuzawa F, Doi M (2013) Endogenous post-transcriptional gene silencing of flavone synthase resulting in high accumulation of anthocyanins in black dahlia cultivars. Planta 237:1325–1335

    Article  CAS  PubMed  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Du Y, Chu H, Wang M, Chu IK, Lo C (2010) Identification of flavone phytoalexins and a pathogen-inducible flavone synthase II gene (SbFNSII) in sorghum. J Exp Bot 61:983–994

    Article  CAS  PubMed  Google Scholar 

  • Du H, Ran F, Dong HL, Wen J, Li JN, Liang Z (2016) Genome-wide analysis, classification, evolution, and expression analysis of the cytochrome P450 93 family in land plants. PLoS ONE 11:e0165020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, Qureshi M, Richardson LJ, Salazar GA, Smart A, Sonnhammer ELL, Hirsh L, Paladin L, Piovesan D, Tosatto SCE, Finn RD (2018) The Pfam protein families database in 2019. Nucleic Acids Res 47:427–432

    Article  CAS  Google Scholar 

  • Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE 6:1–10

    Article  CAS  Google Scholar 

  • He J, Giusti MM (2010) Anthocyanins: natural colorants with health-promoting properties. Annu Rev Food Sci Technol 1:163–187

    Article  CAS  PubMed  Google Scholar 

  • Herrmann KM, Weaver LM (1999) The shikimate pathway. Annu Rev Plant Physiol Plant Mol Biol 50:473–503

    Article  CAS  PubMed  Google Scholar 

  • Iorizzo M, Ellison S, Senalik D, Zeng P, Satapoomin P et al (2016) A high-quality carrot genome assembly provides new insights into carotenoid accumulation and asterid genome evolution. Nat Genet 48:657–666

    Article  CAS  PubMed  Google Scholar 

  • Iorizzo M, Cavagnaro P, Bostan H, Zhao Y, Zhang J, Simon PW (2018) A cluster of MYB transcription factors regulates anthocyanin biosynthesis in carrot (Daucus carota L.) root and petiole. Front Plant Sci 9:1927

    Article  PubMed  Google Scholar 

  • Jaakola L, Poole M, Jones MO, Kamarainen-Karppinen T, Koskimaki JJ, Hohtola A et al (2010) A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits. Plant Physiol 153:1619–1629

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jing P, Bomser J, Schwartz SJ, He J, Magnuson B, Giusti MM (2008) Structure-function relationships of anthocyanins from various anthocyanin-rich extracts on the inhibition of colon cancer cell growth. J Agric Food Chem 56:9391–9398

    Article  CAS  PubMed  Google Scholar 

  • Kammerer D, Carle R, Schieber A (2003) Detection of peonidin and pelargonidin glycosides in black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) by high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 17:2407–2412

    Article  CAS  PubMed  Google Scholar 

  • Kammerer D, Carle R, Schieber A (2004) Quantification of anthocyanins in black carrot extracts (Daucus carota ssp. sativus var atrorubens Alef.) and evaluation of their color properties. Eur Food Res Technol 219:479–486

    Article  CAS  Google Scholar 

  • Koes RE, Quattrocchio F, Mol JNM (1993) The flavonoid biosynthetic pathway in plants: function and evolution. BioEssays 16:123–132

    Article  Google Scholar 

  • Kurilich AC, Clevidence B, Britz SJ, Simon PW, Novotny JA (2005) Plasma and urine responses are lower for acylated vs nonacylated anthocyanins from raw and cooked purple carrots. J Agric Food Chem 53:6537–6542

    Article  CAS  PubMed  Google Scholar 

  • Lalusin AG, Nishita K, Kim SH, Ohta M, Fujimura T (2006) A new MADS-box gene (IbMADS10) from sweet potato (Ipomoea batatas (L.) Lam) is involved in the accumulation of anthocyanin. Mol Gen Genom 275:44–54

    Article  CAS  Google Scholar 

  • Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359

    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 

  • Lin-Wang K, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie TK et al (2010) An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biol 10:50

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Martens S, Mithöfer A (2005) Flavones and flavone synthases. Phytochemistry 66:2399–2407

    Article  CAS  PubMed  Google Scholar 

  • Mazza G, Miniati E (1993) Anthocyanins in fruits, vegetables, and grains. CRC Press, Boca Raton, p 265

    Google Scholar 

  • Mazza G, Cacace JE, Kay CD (2004) Methods of analysis for anthocyanins in plants and biological fluids. J AOAC Int 87:29–45

    Google Scholar 

  • McCarthy DJ, Chen Y, Smyth GK (2012) Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res 40:4288–4297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Montilla EC, Arzaba MR, Hillebrand S, Winterhalter P (2011) Anthocyanin composition of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) cultivars antonina, beta sweet, deep purple, and purple haze. J Agric Food Chem 59:3385–3390

    Article  CAS  PubMed  Google Scholar 

  • Nakatsuka T, Nishihara M, Mishiba K, Yamamura S (2006) Heterologous expression of two gentian cytochrome P450 genes can modulate the intensity of flower pigmentation in transgenic tobacco plants. Mol Breed 17:91–99

    Article  CAS  Google Scholar 

  • Nesi N, Debeaujon I, Jond C, Stewart AJ, Jenkins GI, Caboche M, Lepiniec L (2002) The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS domain protein and is required for proper development and pigmentation of the seed coat. Plant Cell 14:2463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Prior RL, Wu X (2006) Anthocyanins: structural characteristics that result in unique metabolic patterns and biological activities. Free Radic Res 40:1014–1028

    Article  CAS  PubMed  Google Scholar 

  • Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11:R25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140

    Article  CAS  PubMed  Google Scholar 

  • Schopfer CR, Kochs G, Lottspeich F, Ebel J (1998) Molecular characterization and functional expression of dihydroxypterocarpan 6a-hydroxylase, an enzyme specific for pterocarpanoid phytoalexin biosynthesis in soybean (Glycine max L.). FEBS Lett 432:182–186

    Article  CAS  PubMed  Google Scholar 

  • Shirley BW (1996) Flavonoid biosynthesis: “New” functions for an “old” pathway. Trends Plant Sci 1:377–382

    Google Scholar 

  • Simon PW (1996) Inheritance and expression of purple and yellow storage root color in carrot. J Hered 87:63–66

    Article  Google Scholar 

  • Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456

    Article  CAS  PubMed  Google Scholar 

  • Tan GF, Ma J, Zhang XY, Xu ZS, Xiong AS (2017) AgFNS overexpression increase apigenin and decrease anthocyanins in petioles of transgenic celery. Plant Sci 263:31–38

    Article  CAS  PubMed  Google Scholar 

  • Van Ooijen JW (2006) JoinMap 4.0: software for the calculation of genetic linkage maps in experimental populations. Kyazma BV, Wageningen

    Google Scholar 

  • Vivek BS, Simon PW (1999) Linkage relationships among molecular markers and storage root traits of carrot (Daucus carota L. ssp. sativus). Theor Appl Genet 99:58–64

    Article  CAS  Google Scholar 

  • Wang R, Ming M, Li J, Shi D, Qiao X, Li L, Zhang S, Wu J (2017) Genome-wide identification of the MADS-box transcription factor family in pear (Pyrus bretschneideri) reveals evolution and functional divergence. PeerJ 5:e3776

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu S (2008) Quantitative trait locus mapping can benefit from segregation distortion. Genetics 180:2201–2208

    Article  PubMed  PubMed Central  Google Scholar 

  • Xu ZS, Huang Y, Wang F, Song X, Wang GL, Xiong AS (2014) Transcript profiling of structural genes involved in cyanidin-based anthocyanin biosynthesis between purple and non-purple carrot (Daucus carota L.) cultivars reveals distinct patterns. BMC Plant Biol 14:262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu ZS, Feng K, Que F, Wang F, Xiong AS (2017) A MYB transcription factor, DcMYB6, is involved in regulating anthocyanin biosynthesis in purple carrot taproots. Sci Rep 7:1–9

    Article  CAS  Google Scholar 

  • Yildiz M, Willis DK, Cavagnaro PF, Iorizzo M, Abak K, Simon PW (2013) Expression and mapping of anthocyanin biosynthesis genes in carrot. Theor Appl Genet 126:1689–1702

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors acknowledge the BEC.AR Scholarship Program of the Argentine Ministry of Education. FB and MP were supported by the Argentine National Council of Scientific and Technical Research (CONICET) PhD and postdoctoral fellowships, respectively. This work was partly funded by the ‘Agencia Nacional de Promoción Científica y Tecnológica’ through grant ‘Préstamo BID PICT-2015-1625.’ SE was supported by the National Institute of Food and Agriculture, the USA Department of Agriculture (NIFA-USDA), under Award Number 2016-51181-25400. MI was supported by the USA Department of Agriculture National Institute of Food and Agriculture, Hatch Project 1008691.

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Authors and Affiliations

  1. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. Rivadavia 1917, C1033AAJ, Buenos Aires, Argentina

    Florencia Bannoud, Marcos Paolinelli & Pablo F. Cavagnaro

  2. USDA-Agricultural Research Service, Vegetable Crops Research Unit, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI, 53706-1590, USA

    Shelby Ellison, Thomas Horejsi, Douglas Senalik & Philipp W. Simon

  3. Instituto Nacional de Tecnología Agropecuaria INTA EEA Mendoza, San Martin 3853, Luján de Cuyo, 5507, Mendoza, Argentina

    Marcos Paolinelli & Martín Fanzone

  4. Plants for Human Health Institute, North Carolina State University, Kannapolis, USA

    Massimo Iorizzo

  5. Department of Horticultural Science, North Carolina State University, Raleigh, NC, USA

    Massimo Iorizzo

  6. Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI, 53706-1590, USA

    Philipp W. Simon

  7. Instituto de Horticultura, Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo, Mendoza, Argentina

    Pablo F. Cavagnaro

  8. Instituto Nacional de Tecnología Agropecuaria (INTA) - E.E.A. La Consulta, Ex Ruta 40 km 96, La Consulta, 5567, San Carlos, Mendoza, Argentina

    Pablo F. Cavagnaro

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  1. Florencia Bannoud
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  2. Shelby Ellison
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  3. Marcos Paolinelli
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  4. Thomas Horejsi
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  5. Douglas Senalik
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  9. Pablo F. Cavagnaro
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Correspondence to Philipp W. Simon or Pablo F. Cavagnaro.

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Communicated by Herman J. van Eck.

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Bannoud, F., Ellison, S., Paolinelli, M. et al. Dissecting the genetic control of root and leaf tissue-specific anthocyanin pigmentation in carrot (Daucus carota L.). Theor Appl Genet 132, 2485–2507 (2019). https://doi.org/10.1007/s00122-019-03366-5

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