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.
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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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
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
Ayabe S, Akashi T (2006) Cytochrome P450s in flavonoid metabolism. Phytochem Rev 5:271–282
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
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120
Broman K, Sen S (2009) A guide to QTL mapping with R/qtl. Springer, New York
Broman KW, Wu H, Sen Ś, Churchill GA (2003) R/qtl: QTL mapping in experimental crosses. Bioinformatics 19:889–890
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
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
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
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
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
Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15
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
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
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
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
He J, Giusti MM (2010) Anthocyanins: natural colorants with health-promoting properties. Annu Rev Food Sci Technol 1:163–187
Herrmann KM, Weaver LM (1999) The shikimate pathway. Annu Rev Plant Physiol Plant Mol Biol 50:473–503
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
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
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
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
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
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
Koes RE, Quattrocchio F, Mol JNM (1993) The flavonoid biosynthetic pathway in plants: function and evolution. BioEssays 16:123–132
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
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
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359
Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform 12:323
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
Martens S, Mithöfer A (2005) Flavones and flavone synthases. Phytochemistry 66:2399–2407
Mazza G, Miniati E (1993) Anthocyanins in fruits, vegetables, and grains. CRC Press, Boca Raton, p 265
Mazza G, Cacace JE, Kay CD (2004) Methods of analysis for anthocyanins in plants and biological fluids. J AOAC Int 87:29–45
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
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
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
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
Prior RL, Wu X (2006) Anthocyanins: structural characteristics that result in unique metabolic patterns and biological activities. Free Radic Res 40:1014–1028
Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11:R25
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140
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
Shirley BW (1996) Flavonoid biosynthesis: “New” functions for an “old” pathway. Trends Plant Sci 1:377–382
Simon PW (1996) Inheritance and expression of purple and yellow storage root color in carrot. J Hered 87:63–66
Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456
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
Van Ooijen JW (2006) JoinMap 4.0: software for the calculation of genetic linkage maps in experimental populations. Kyazma BV, Wageningen
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
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
Xu S (2008) Quantitative trait locus mapping can benefit from segregation distortion. Genetics 180:2201–2208
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
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
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
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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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