Plant Biotechnology Reports

, Volume 13, Issue 6, pp 579–590 | Cite as

De novo assembly and transcriptome analysis of differentially expressed genes relevant to variegation in hawthorn flowers

  • Wei JiEmail author
  • Wei Zhao
  • Rong-Chen Liu
  • Xiao-Bo Jiao
  • Kai Han
  • Zhong-Yi Yang
  • Mei-Ying Gao
  • Rui Ren
  • Xiu-Juan Fan
  • Ming-Xia YangEmail author
Original Article


Flower color variegation has been observed in many plant species. However, pink flowers on the white-blooming hawthorn trees found by our group earlier have never been reported. To better understand the differentially expressed genes (DEGs) in variegated hawthorn flowers, white and pink flowers at different developmental stages (S1 and S2) underwent transcriptome sequencing separately. Approximately 34.28 Gb of high-quality data were obtained and assembled into 100,013 unigenes with an average length of 706.93 bp. These unigenes were further subjected to functional annotation and biochemical pathway analysis, and DEGs of two types of hawthorn flowers at different developmental stages were studied. Based on the enrichment analysis of DEGs, eight anthocyanin-modified enzyme genes or other enzyme genes that indirectly affect anthocyanin synthesis (5AT, 3GGT, and AI, β-Glu, two Aux/IAAs, two PODs), eight structural genes (UFGT, DFR, CHI, two F3Hs, and three PALs), and three transcription factors (one MYB and two bHLHs) were also identified. We randomly selected 15 genes, and the trends in the expression levels of these genes in the organs of white and pink flowers at different developmental stages were verified by quantitative real-time PCR. Mass sequence data obtained by RNA-seq of variegated hawthorn flowers provided basic sequence information and a unique opportunity to uncover the genetic mechanisms underlying flower color variegation.


Hawthorn Variegation De novo assembly Transcriptome analysis DEGs qRT-PCR 



Differentially expressed genes


Quantitative real-time PCR


Non-redundant protein database


Gene ontology


Cluster of orthologous groups of proteins database


Kyoto Encyclopedia of Genes and Genomes database




Anthocyanin 3-O-glucoside-2″-O-glucosyltransferase


Acid invertase


Auxin/indoleacetic acid






UDP-flavonoid 3-O-glucosyltransferase


Dihydroflavonol 4-reductase


Flavonoid 3-hydroxylase


Phenylalanine ammonia-lyase


Chalcone isomerase



This research was financially supported by Modern and Key Technology of Traditional Chinese Medicine in Shanxi Province (no. 2016ZD0109); Breeding of New Early Maturing Hawthorn Varieties (no. 201703D221014-1); Shanxi Youth Talent Support Program (2018); Shanxi Province Outstanding Young Academic Leaders (2017); and Program for the Top Young Innovative Talents of Shanxi Agricultural University (no. TYIT201401).

Supplementary material

11816_2019_551_MOESM1_ESM.docx (39 kb)
Supplementary material 1 (DOCX 39 kb)
11816_2019_551_MOESM2_ESM.tiff (437 kb)
Supplementary material 2 (TIFF 437 kb)
11816_2019_551_MOESM3_ESM.tiff (3.5 mb)
Supplementary material 3 (TIFF 3608 kb)
11816_2019_551_MOESM4_ESM.tiff (13.5 mb)
Supplementary material 4 (TIFF 13861 kb)
11816_2019_551_MOESM5_ESM.tiff (11.6 mb)
Supplementary material 5 (TIFF 11858 kb)
11816_2019_551_MOESM6_ESM.tiff (64 kb)
Supplementary material 6 (TIFF 63 kb)
11816_2019_551_MOESM7_ESM.tiff (57 kb)
Supplementary material 7 (TIFF 57 kb)
11816_2019_551_MOESM8_ESM.tiff (66 kb)
Supplementary material 8 (TIFF 66 kb)
11816_2019_551_MOESM9_ESM.tiff (68 kb)
Supplementary material 9 (TIFF 67 kb)


  1. Antonio BTJ, Margarita CR, Daniel MI (2015) Biological properties and antioxidant activity of hawthorn Crataegus mexicana. J Pharmacogenom Pharmacoproteom 6:1Google Scholar
  2. Auler PA, Benitez LC, Do Amaral MN, Vighi IL, Dos Rodrigues GS, da Maia LC, Braga EJ (2017) Evaluation of stability and validation of reference genes for RT-qPCR expression studies in rice plants under water deficit. J Appl Genet 58:163–177. CrossRefPubMedGoogle Scholar
  3. Ban Y, Honda C, Hatsuyama Y, Igarashi M, Bessho H, Moriguchi T (2007) Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol 48:958–970. CrossRefPubMedGoogle Scholar
  4. Brunetti C, Fini A, Sebastiani F, Gori A, Tattini M (2018) Modulation of phytohormone signaling: a primary function of flavonoids in plant–environment interactions. Front Plant Sci 9:1042. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chen Y, Mao Y, Liu H, Yu F, Li S, Yin T (2014) Transcriptome analysis of differentially expressed genes relevant to variegation in peach flowers. PLoS One 9:e90842. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cheng J, Wei G, Zhou H, Gu C, Vimolmangkang S, Liao L, Han Y (2014) Unraveling the mechanism underlying the glycosylation and methylation of anthocyanins in peach. Plant Physiol 166:1044–1058. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chen J, Zheng W, Wang T, Wang W, Xu X (2017) Transcriptome analysis of Gerbera delavayi based on high-throughput sequencing technology and differential expression analysis. Acta Bot Boreal Occident Sin 37:470–477Google Scholar
  8. Everaert C, Luypaert M, Maag JLV, Cheng QX, Dinger ME, Hellemans J, Mestdagh P (2017) Benchmarking of RNA-sequencing analysis workflows using whole-transcriptome RT-qPCR expression data. Sci Rep 7:1559. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Hu DG, Li YY, Zhang QY, Li M, Sun CH, Yu JQ, Hao YJ (2017) The R2R3-MYB transcription factor MdMYB73 is involved in malate accumulation and vacuolar acidification in apple. Plant J 91:443–454. CrossRefPubMedGoogle Scholar
  11. Jia Z, Ma P, Bian X, Yang Q, Guo X, Xie Y (2014) Biosynthesis metabolic pathway and molecular regulation of plants anthocyanin. Acta Bot Boreal Occident Sin 34:1496–1506Google Scholar
  12. Jia H, Wang JA, Yang Y, Liu G, Bao Y, Cui H (2015) Changes in flavonol content and transcript levels of genes in the flavonoid pathway in tobacco under phosphorus deficiency. Plant Growth Regul 76:225–231. CrossRefGoogle Scholar
  13. Jiao Y, Shu Q, Liu Z, Zhang J (2015) Research progress of anthocyanin biosynthesis and accumulation in blood-flesh peach. J Anhui Agric Sci 43:64–68Google Scholar
  14. Kwon Y, Oh JE, Noh H, Hong SW, Bhoo SH, Lee H (2011) The ethylene signaling pathway has a negative impact on sucrose-induced anthocyanin accumulation in Arabidopsis. J Plant Res 124:193–200. CrossRefPubMedGoogle Scholar
  15. Li J, Lv X, Wang L, Qiu Z, Song X, Lin J, Chen W (2017) Transcriptome analysis reveals the accumulation mechanism of anthocyanins in ‘Zijuan’ tea (Camellia sinensis var. asssamica (Masters) kitamura) leaves. Plant Growth Regul 81:51–61. CrossRefGoogle Scholar
  16. Li Y, Luo X, Wu C, Cao S, Zhou Y, Jie B, Cao Y, Meng H, Wu G (2018) Comparative transcriptome analysis of genes involved in anthocyanin biosynthesis in red and green walnut (Juglans regia L.). Molecules 23:25. CrossRefGoogle Scholar
  17. Lin Q, Zhong Q, Zhang Z (2019) Comparative transcriptome analysis of genes involved in anthocyanin biosynthesis in the pink-white and red fruits of Chinese bayberry (Morella rubra). Sci Hortic 250:278. CrossRefGoogle Scholar
  18. Liu XJ, An XH, Liu X, Hu DG, Wang XF, You CX, Hao YJ (2017) MdSnRK1.1 interacts with MdJAZ18 to regulate sucrose-induced anthocyanin and proanthocyanidin accumulation in apple. J Exp Bot 68:2977–2990. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T (2017) Transcriptomics technologies. PLoS Comput Biol 13:e1005457. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628. CrossRefPubMedGoogle Scholar
  21. Mu C, Wang L, Jia X, Wang K, Peng F, Guo L, Wang L (2013) RNA interference of peroxidase gene Rsprx1 increased anthocyanidin accumulation in Chinese red radish. Chin J Biochem Mol Bio 29:867–872Google Scholar
  22. Nicole S, Barcaccia G, Erickson DL, Kress JW, Lucchin M (2013) The coding region of the UFGT gene is a source of diagnostic SNP markers that allow single-locus DNA genotyping for the assessment of cultivar identity and ancestry in grapevine (Vitis vinifera L.). BMC Res Notes 6:502. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Onik JC, Hu X, Lin Q, Wang Z (2018) Comparative transcriptomic profiling to understand pre- and post-ripening hormonal regulations and anthocyanin biosynthesis in early ripening apple fruit. Molecules 23:E1908. CrossRefPubMedGoogle Scholar
  24. Oren-Shamir M (2009) Does anthocyanin degradation play a significant role in determining pigment concentration in plants? Plant Sci 177:310–316. CrossRefGoogle Scholar
  25. Sagaradze V, Babaeva E, Kalenikova E (2017) HPLC-UV method for determining flavonoids in hawthorn flowers and leaves. Pharm Chem J 51:277–280. CrossRefGoogle Scholar
  26. Saito K, Yonekura-Sakakibara K, Nakabayashi R, Higashi Y, Yamazaki M, Tohge T, Fernie AR (2013) The flavonoid biosynthetic pathway in Arabidopsis: structural and genetic diversity. Plant Physiol Biochem 72:21–34. CrossRefPubMedGoogle Scholar
  27. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108. CrossRefPubMedGoogle Scholar
  28. Shi Q, Li L, Zhang X, Luo J, Li X, Zhai L, He L, Zhang Y (2017) Biochemical and comparative transcriptomic analyses identify candidate genes related to variegation formation in Paeonia rockii. Molecules 22:E1364. CrossRefPubMedGoogle Scholar
  29. Soubeyrand E, Basteau C, Hilbert G, Leeuwen C, Delrot S, Gomès E (2014) Nitrogen supply affects anthocyanin biosynthetic and regulatory genes in grapevine cv. Cabernet–Sauvignon berries. Phytochemistry 103:38–49. CrossRefPubMedGoogle Scholar
  30. Tiwari SB, Hagen G, Guilfoyle T (2003) The roles of auxin response factor domains in auxin-responsive transcription. Plant Cell 15:533–543. CrossRefPubMedPubMedCentralGoogle Scholar
  31. To KY, Wang CK (2006) Molecular breeding of flower color. In: Teixeira da Silva JA (ed) Floriculture, ornamental and plant biotechnology: advances and topical issues, vol I. Isleworth, England, pp 300–310Google Scholar
  32. Van den Berge K, Soneson C, Robinson MD, Clement L (2017) stageR: a general stage-wise method for controlling the gene-level false discovery rate in differential expression and differential transcript usage. Genome Biol 18:151. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Wang L (2010) Advances on the studies of invertase on sucrose metabolism in higher plant. J Agric Sci 31:70–75CrossRefGoogle Scholar
  34. Wang X, Yamagishi M (2019) Mechanisms suppressing carotenoid accumulation in flowers differ depending on the hybrid groups of lilies (Lilium spp.). Sci Hortic 243:159–168. CrossRefGoogle Scholar
  35. Wang Y, Huang H, Ma Y, Fu J, Wang L, Dai S (2014) Construction and de novo characterization of a transcriptome of Chrysanthemum lavandulifolium: analysis of gene expression patterns in floral bud emergence. Plant Cell Tissue Organ Cult 116:297–309. CrossRefGoogle Scholar
  36. Wang H, Wang C, Fan W, Yang J, Appelhagen I, Wu Y, Zhang P (2018a) UDP-glucose: anthocyanidin 3-O-glucoside-2″-O-glucosyltransferase catalyzes glycosyl extension of anthocyanins in purple Ipomoea batatas. J Exp Bot. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Wang YC, Wang N, Xu HF, Jiang SH, Fang HC, Su MY, Zhang ZY, Zhang TL, Chen XS (2018b) Auxin regulates anthocyanin biosynthesis through the Aux/IAA–ARF signaling pathway in apple. Hortic Res 5:59. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Wen PF, Ji W, Gao MY, Niu TQ, Xing YF, Niu XY (2015) Accumulation of flavanols and expression of leucoanthocyanidin reductase induced by postharvest UV-C irradiation in grape berry. Genet Mol Res 14:7687–7695. CrossRefPubMedGoogle Scholar
  39. Yamazaki M, Shibata M, Nishiyama Y, Springob K, Kitayama M, Shimada N, Aoki T, Ayabe S, Saito K (2008) Differential gene expression profiles of red and green forms of Perilla frutescens leading to comprehensive identification of anthocyanin biosynthetic genes. FEBS J 275:3494–3502. CrossRefPubMedGoogle Scholar
  40. Yang Q, Yuan T, Sun X (2015) Preliminary studies on the changes of flower color during the flowering period in two tree peony cultivars. Acta Hortic Sin 42:930–938Google Scholar
  41. Yue J, Zhu C, Zhou Y, Niu X, Miao M, Tang X, Chen F, Zhao W, Liu Y (2018) Transcriptome analysis of differentially expressed unigenes involved in flavonoid biosynthesis during flower development of Chrysanthemum morifolium ‘Chuju’. Sci Rep 8:13414. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Zhang Y, Chu G, Hu Z, Gao Q, Cui B, Tian S, Wang B, Chen G (2016) Genetically engineered anthocyanin pathway for high health-promoting pigment production in eggplant. Mol Breeding 36:54. CrossRefGoogle Scholar
  43. Zhao J (2016) Research progresses on molecular mechanism of hormone regulation of plant anthocyanin biosynthesis. Mol Plant Breeding 14:1884–1891Google Scholar

Copyright information

© Korean Society for Plant Biotechnology 2019

Authors and Affiliations

  • Wei Ji
    • 1
    • 2
    • 3
    Email author
  • Wei Zhao
    • 1
    • 2
    • 3
  • Rong-Chen Liu
    • 1
    • 2
    • 3
  • Xiao-Bo Jiao
    • 1
    • 2
    • 3
  • Kai Han
    • 1
    • 2
    • 3
  • Zhong-Yi Yang
    • 1
    • 2
    • 3
  • Mei-Ying Gao
    • 1
    • 2
    • 3
  • Rui Ren
    • 2
    • 4
  • Xiu-Juan Fan
    • 5
  • Ming-Xia Yang
    • 2
    • 4
    Email author
  1. 1.College of HorticultureShanxi Agricultural UniversityTaiguChina
  2. 2.Province Key Laboratory of Fruit Germplasm Development and UtilizationTaiyuanChina
  3. 3.Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess PlateauMinistry of AgricultureTaiyuanChina
  4. 4.Pomology InstituteShanxi Academy of Agricultural SciencesTaiguChina
  5. 5.Shanxi Forestry Seedling Management StationTaiyuanChina

Personalised recommendations