Abstract
Key message
This study found that three paralogous R2R3-MYB transcription factors exhibit functional divergence among different subspecies and cultivated types in radish.
Abstract
Cultivated radish taproots exhibit a wide range of color variations due to unique anthocyanin accumulation patterns in various tissues. This study investigated the universal principles of taproot color regulation that developed during domestication of different subspecies and cultivated types. The key candidate genes RsMYB1 and RsMYB2, which control anthocyanin accumulation in radish taproots, were identified using bulked segregant analysis in two genetic populations. We introduced the RsMYB1-RsF3′H-RsMYB1Met genetic model to elucidate the complex and unstable genetic regulation of taproot flesh color in Xinlimei radish. Furthermore, we analyzed the expression patterns of three R2R3-MYB transcription factors in lines with different taproot colors and investigated the relationship between RsMYB haplotypes and anthocyanin accumulation in a natural population of 56 germplasms. The results revealed that three paralogous RsMYBs underwent functional divergence during radish domestication, with RsMYB1 regulating the red flesh of Xinlimei radish, and RsMYB2 and RsMYB3 regulating the red skin of East Asian big long radish (R. sativus var. hortensis) and European small radish (R. sativus var. sativus), respectively. Moreover, RsMYB1-H1, RsMYB2-H10, and RsMYB3-H6 were identified as the primary haplotypes exerting regulatory functions on anthocyanin synthesis. These findings provide an understanding of the genetic mechanisms regulating anthocyanin synthesis in radish and offer a potential strategy for early prediction of color variations in breeding programs.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Asako Y, Owaki Y, Ozeki Y, Sasaki N, Abe Y, Momose T, Shimomura K (2011) Parental line ‘“Inuidani”’ of Japanese radish (Raphanus sativus L.) accumulating pelargonidin as a major anthocyanidin entirely within its underground part and preliminary genetic analysis. Breed Res 13(3):65–73. https://doi.org/10.1270/jsbbr.13.65
Bendokas V, Stanys V, Maeikien I, Trumbeckaite S, Liobikas J (2020) Anthocyanins: from the field to the antioxidants in the body. Antioxidants 9(9):819. https://doi.org/10.3390/antiox9090819
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):1118. https://doi.org/10.1186/1471-2164-15-1118
Duan AQ, Deng YJ, Tan SS, Xu ZS, Xiong AS (2023) A MYB activator, DcMYB11c, regulates carrot anthocyanins accumulation in petiole but not taproot. Plant Cell Environ 46(9):2794–2809. https://doi.org/10.1111/pce.14653
Dwiningsih Y, Alkahtani J (2022) Genetics, biochemistry and biophysical analysis of anthocyanin in Rice (Oryza sativa L.). Adv Sustain Sci Eng Technol 4(1):0220103. https://doi.org/10.26877/asset.v4i1.11659
Espley RV, Brendolise C, Chagné D, Kutty-Amma S, Green S, Volz R, Putterill J, Schouten HJ, Gardiner SE, Hellens RP, Allan AC (2009) Multiple repeats of a promoter segment cause transcription factor autoregulation in red apples. Plant Cell 21(1):168–183. https://doi.org/10.1105/tpc.108.059329
Fan LX, Wang Y, Xu L, Tang M, Zhang X, Ying J, Li C, Dong J, Liu L (2020) A genome-wide association study uncovers a critical role of the RsPAP2 gene in red-skinned Raphanus sativus L. Hortic Res 7(1):164. https://doi.org/10.1038/s41438-020-00385-y
He J, Giusti MM (2010) Anthocyanins: natural colorants with health-promoting properties. Annu Rev Food Sci Technol 1:163–187. https://doi.org/10.1146/annurev.food.080708.100754
Huang D, Wang X, Tang Z, Yuan Y, Xu Y, He J, Jiang X, Peng SA, Li L, Butelli E, Deng X, Xu Q (2018) Subfunctionalization of the Ruby2–Ruby1 gene cluster during the domestication of citrus. Nat Plants 4(11):930–941. https://doi.org/10.1038/s41477-018-0287-6
Inukai T, Kim H, Matsunaga W, Masuta C (2023) Battle for control of anthocyanin biosynthesis in two Brassicaceae species infected with turnip mosaic virus. J Exp Bot 74(5):1659–1674. https://doi.org/10.1093/jxb/erac502
Iorizzo M, Cavagnaro PF, Bostan H, Zhao Y, Zhang J, Simon PW (2019) A cluster of MYB transcription factors regulates anthocyanin biosynthesis in carrot (Daucus carota L.) root and petiole. Front Plant Sci 9:1927. https://doi.org/10.3389/fpls.2018.01927
Jeong YM, Kim N, Ahn BO, Oh M, Chung WH, Chung H, Jeong S, Lim KB, Hwang YJ, Kim GB, Baek S, Choi SB, Hyung DJ, Lee SW, Sohn SH, Kwon SJ, Jin M, Seol YJ, Chae WB, Choi KJ, Park BS, Yu HJ, Mun JH (2016) Elucidating the triplicated ancestral genome structure of radish based on chromosome-level comparison with the Brassica genomes. Theor Appl Genet 129(7):1357–1372. https://doi.org/10.1007/s00122-016-2708-0
Kim S, Song H, Hur Y (2021a) Intron-retained radish (Raphanus sativus L.) RsMYB1 transcripts found in colored-taproot lines enhance anthocyanin accumulation in transgenic Arabidopsis plants. Plant Cell Rep 40:1735–1749. https://doi.org/10.1007/s00299-021-02735-z
Kim DH, Lee J, Rhee J, Lee JY, Lim SH (2021b) Loss of the R2R3 MYB transcription factor RsMYB1 shapes anthocyanin biosynthesis and accumulation in Raphanus sativus. Int J Mol Sci 22(20):10927. https://doi.org/10.3390/ijms222010927
Kline RA, Becker RF, Belluscio L (1981) The heirloom vegetable garden: gardening in the 19th century. Produced by Cornell University Media Services for Life Science, at Cornell University. https://hdl.handle.net/1813/3715
Kobayashi H, Shirasawa K, Fukino N, Hirakawa H, Akanuma T, Kitashiba H (2020) Identification of genome-wide single-nucleotide polymorphisms among geographically diverse radish accessions. DNA Res 27(1):dsaa001. https://doi.org/10.1093/dnares/dsaa001
Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci 10(5):236–242. https://doi.org/10.1016/j.tplants.2005.03.002
Lai B, Cheng Y, Liu H, Wang Q, Wang Q, Wang C, Su R, Chen F, Wang H, Du L (2020) Differential anthocyanin accumulation in radish taproot: importance of RsMYB1 gene structure. Plant Cell Rep 39(2):217–226. https://doi.org/10.1007/s00299-019-02485-z
Leigh JW, Bryant D (2015) PopART: full-feature software for haplotype network construction. Methods Ecol Evol 6(9):1110–1116. https://doi.org/10.1111/2041-210X.12410
Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25(14):1754–1760. https://doi.org/10.1093/bioinformatics/btp324
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R et al (2009a) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352
Li R, Yu C, Li Y, Lam TW, Yiu SM, Kristiansen K, Wang J (2009b) SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics 25(15):1966–1967. https://doi.org/10.1093/bioinformatics/btp336
Lim SH, Song JH, Kim DH, Kim JK, Lee JY, Kim YM, Ha SH (2016) Activation of anthocyanin biosynthesis by expression of the radish R2R3-MYB transcription factor gene RsMYB1. Plant Cell Rep 35(3):641–653. https://doi.org/10.1007/s00299-015-1909-3
Liu T, Wang J, Wu C, Zhang Y, Zhang X, Li X, Wang H, Song J, Li X (2019) Combined QTL-Seq and traditional linkage analysis to identify candidate genes for purple skin of radish fleshy taproots. Front Genet 10:808. https://doi.org/10.3389/fgene.2019.00808
Lü N, Yamane K, Ohnishi O (2008) Genetic diversity of cultivated and wild radish and phylogenetic relationships among Raphanus and Brassica species revealed by the analysis of trnK/matK sequence. Breed Sci 58(1):15–22. https://doi.org/10.1270/jsbbs.58.15
Luo X, Xu L, Wang Y, Dong J, Chen Y, Tang M, Fan L, Zhu Y, Liu L (2020) An ultra-high-density genetic map provides insights into genome synteny, recombination landscape and taproot skin colour in radish (Raphanus sativus L.). Plant Biotechnol J 18(1):274–286. https://doi.org/10.1111/pbi.13195
Luo X, Plunkert M, Teng Z, Mackenzie K, Guo L, Luo Y, Hytönen T, Liu Z (2023) Two MYB activators of anthocyanin biosynthesis exhibit specialized activities in petiole and fruit of diploid strawberry. J Exp Bot 74(5):1517–1531. https://doi.org/10.1093/jxb/erac507
Masukawa T, Cheon KS, Mizuta D, Nakatsuka A, Kobayashi N (2018a) Insertion of a retrotransposon into a flavonoid 30-hydroxylase homolog confers the red root character in the radish (Raphanus sativus L. var. longipinnatus L. H. Bailey). Hortic J 87(1):89–96. https://doi.org/10.2503/hortj.OKD-075
Masukawa T, Kadowaki M, Matsumoto T, Nakatsuka A, Cheon KS, Kato K, Tatsuzawa F, Kobayashi N (2018b) Enhancement of food functionality of a local pungent radish ‘“Izumo orochi daikon”’ ‘Susanoo’ by introduction of a colored root character. Hortic J 87(3):356–363. https://doi.org/10.2503/hortj.OKD-132
Masukawa T, Cheon KS, Mizuta D, Kadowaki M, Nakatsuka A, Kobayashi N (2019) Development of mutant RsF3’H allele-based marker for selection of purple and red root in radish (Raphanus sativus L. var. longipinnatus L. H. Bailey). Euphytica. https://doi.org/10.1007/s10681-019-2442-1
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA (2010) The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20(9):1297–1303. https://doi.org/10.1101/gr.107524.110
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321–4325. https://doi.org/10.1093/nar/8.19.4321
Nishio T, Kitashiba H (2017) The radish genome. Springer, Cham, pp 11–30
Oikawa T, Maeda H, Oguchi T, Yamaguchi T, Tanabe N, Ebana K, Yano M, Ebitani T, Izawa T (2015) The birth of a black rice gene and its local spread by introgression. Plant Cell 27(9):2401–2414. https://doi.org/10.1105/tpc.15.00310
Petroni K, Tonelli C (2011) Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant Sci 181(3):219–229. https://doi.org/10.1016/j.plantsci.2011.05.009
Saxena RK, Penmetsa RV, Upadhyaya HD, Kumar A, Carrasquilla-Garcia N, Schlueter JA, Farmer A, Whaley AM, Sarma BK, May GD, Cook DR, Varshney RK (2012) Large-scale development of cost-effective single-nucleotide polymorphism marker assays for genetic mapping in pigeonpea and comparative mapping in legumes. DNA Res 19(6):449–461. https://doi.org/10.1093/dnares/dss025
Shi MZ, Xie DY (2014) Biosynthesis and metabolic engineering of anthocyanins in Arabidopsis thaliana. Recent Pat Biotechnol 8(1):47–60. https://doi.org/10.2174/1872208307666131218123538
So M, Imai Y, Terasawa Y (1919) On the non-Mendelian inheritance of Raphanus sativus. Bot Mag Tokyo 33:21–33 (in Japanese)
Stintzing FC, Carle R (2004) Functional properties of anthocyanins and betalains in plants, food, and in human nutrition. Trends Food Sci Technol 15(1):19–38. https://doi.org/10.1016/j.tifs.2003.07.004
Sun X, Zhang Z, Chen C, Wu W, Ren N, Jiang C, Yu J, Zhao Y, Zheng X, Yang Q, Zhang H, Li J, Li Z (2018) The C-S-A gene system regulates hull pigmentation and reveals evolution of anthocyanin biosynthesis pathway in rice. J Exp Bot 69(7):1485–1498. https://doi.org/10.1093/jxb/ery001
Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, Uemura A, Utsushi H, Tamiru M, Takuno S, Innan H, Cano LM, Kamoun S, Terauchi R (2013) QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J 74(1):174–183. https://doi.org/10.1111/tpj.12105
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729. https://doi.org/10.1093/molbev/mst197
Tao J, Li S, Wang Q, Yuan Y, Ma J, Xu M, Yang Y, Zhang C, Chen L, Sun Y (2022) Construction of a high-density genetic map based on specific-locus amplified fragment sequencing and identification of loci controlling anthocyanin pigmentation in Yunnan red radish. Hortic Res 9:uhab031. https://doi.org/10.1093/hr/uhab031
Tatebe T (1940) Studies on the inheritance of color in the Japanese and Chinese radish (II). J Japan Soc Hortic Sci 11(3):300–316. https://doi.org/10.2503/jjshs.11.300
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):4876–4882. https://doi.org/10.1093/nar/25.24.4876
Tian Y, Thrimawithana A, Ding T, Guo J, Gleave A, Chagné D, Ampomah-Dwamena C, Ireland HS, Schaffer RJ, Luo Z, Wang M, An X, Wang D, Gao Y, Wang K, Zhang H, Zhang R, Zhou Z, Yan Z, Zhang L, Zhang C, Cong P, Deng CH, Yao JL (2022) Transposon insertions regulate genome-wide allele-specific expression and underpin flower colour variations in apple (Malus spp.). Plant Biotechnol J 20(7):1285–1297. https://doi.org/10.1111/pbi.13806
Wang H, Zhang H, Yang Y, Li M, Zhang Y, Liu J, Dong J, Li J, Butelli E, Xue Z, Wang A, Wang G, Martin C, Jin W (2020a) The control of red colour by a family of MYB transcription factors in octoploid strawberry (Fragaria × ananassa) fruits. Plant Biotechnol J 18(5):1169–1184. https://doi.org/10.1111/pbi.13282
Wang Q, Wang Y, Sun H, Sun L, Zhang L (2020b) Transposon-induced methylation of the RsMYB1 promoter disturbs anthocyanin accumulation in red-fleshed radish. J Exp Bot 71(9):2537–2550. https://doi.org/10.1093/jxb/eraa010
Wittmeyer K, Cui J, Chatterjee D, Lee TF, Tan Q, Xue W, Jiao Y, Wang PH, Gaffoor I, Ware D, Meyers BC, Chopra S (2018) The dominant and poorly penetrant phenotypes of maize unstable factor for orange1 are caused by DNA methylation changes at a linked transposon. Plant Cell 30(12):3006–3023. https://doi.org/10.1105/tpc.18.00546
Xia D, Zhou H, Wang Y, Li P, Fu P, Wu B, He Y (2021) How rice organs are colored: the genetic basis of anthocyanin biosynthesis in rice. Crop J 9(3):598–608. https://doi.org/10.1016/j.cj.2021.03.013
Xu Y, Zhu X, Gong Y, Xu L, Wang Y, Liu L (2012) Evaluation of reference genes for gene expression studies in Radish (Raphanus Sativus L.) using quantitative real-time PCR. Biochem Biophys Res Commun 424(3):398–403. https://doi.org/10.1016/j.bbrc.2012.06.119
Xu ZS, Yang QQ, Feng K, Xiong AS (2019) Changing carrot color: insertions in DcMYB7 alter the regulation of anthocyanin biosynthesis and modification. Plant Physiol 181(1):195–207. https://doi.org/10.1104/pp.19.00523
Xu ZS, Yang QQ, Feng K, Yu X, Xiong AS (2020) DcMYB113, a root-specific R2R3-MYB, conditions anthocyanin biosynthesis and modification in carrot. Plant Biotechnol J 18(7):1585–1597. https://doi.org/10.1111/pbi.13325
Yamagishi H (2017) Speciation and diversification of radish. In: Nishio T, Kitashiba H (eds) The radish genome. Springer, Cham, pp 11–30
Yan H, Pei X, Zhang H, Li X, Zhang X, Zhao M, Chiang VL, Sederoff RR, Zhao X (2021) MYB-mediated regulation of anthocyanin biosynthesis. Int J Mol Sci 22(6):3103. https://doi.org/10.3390/ijms22063103
Yi G, Kim JS, Park JE, Shin H, Yu SH, Park S, Huh JH (2018) MYB1 transcription factor is a candidate responsible for red root skin in radish (Raphanus sativus L). PLoS ONE 13(9):e0204241. https://doi.org/10.1371/journal.pone.0204241
Zhang X, Yue Z, Mei S, Qiu Y, Yang X, Chen C et al (2015) A de novo genome of a Chinese radish cultivar. Hortic Plant J 1(3):155–164. https://doi.org/10.16420/j.issn.2095-9885.2016-0028
Zhang L, Hu J, Han X, Li J, Gao Y, Richards CM, Zhang C, Tian Y, Liu G, Gul H, Wang D, Tian Y, Yang C, Meng M, Yuan G, Kang G, Wu Y, Wang K, Zhang H, Wang D, Cong P (2019) A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour. Nat Commun 10(1):1494. https://doi.org/10.1038/s41467-019-09518-x
Zhang X, Liu T, Wang J, Wang P, Qiu Y, Zhao W, Pang S, Li X, Wang H, Song J, Zhang W, Yang W, Sun Y, Li X (2021) Pan-genome of Raphanus highlights genetic variation and introgression among domesticated, wild, and weedy radishes. Mol Plant 14(12):2032–2055. https://doi.org/10.1016/j.molp.2021.08.005
Zheng J, Wu H, Zhu H, Huang C, Liu C, Chang Y, Kong Z, Zhou Z, Wang G, Lin Y, Chen H (2019) Determining factors, regulation system, and domestication of anthocyanin biosynthesis in rice leaves. New Phytol 223(2):705–721. https://doi.org/10.1111/nph.15807
Funding
This work was supported by grants from the Collaborative innovation program of the Beijing Vegetable Research Center (XTCX202302), Innovation and Development Program of Beijing Vegetable Research Center (KYCX202302, KYCX20230126), and Natural Science Foundation of China (32372721).
Author information
Authors and Affiliations
Contributions
LZ, QW and YW designed experiments; QW, YW, XW, WS, NC and YP performed experiments; and QW and LZ wrote the manuscript. All authors edited the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
Additional information
Communicated by Yiqun Weng.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Wang, Q., Wang, Y., Wu, X. et al. Sequence and epigenetic variations of R2R3-MYB transcription factors determine the diversity of taproot skin and flesh colors in different cultivated types of radish (Raphanus sativus L.). Theor Appl Genet 137, 133 (2024). https://doi.org/10.1007/s00122-024-04631-y
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00122-024-04631-y