Large-scale analysis of MYB genes in Cucurbitaceae identifies a novel gene regulating plant height

Abstract

The MYB transcription factor (TF) family, which is involved in plant growth and development, is large and diverse. Previous studies on MYB family in Cucurbitaceae were mostly based on a single genome or focused on the R2R3 subfamily. Here, we analyzed 91 genomes of Cucurbitaceae and identified a total of 15 858 MYB genes. According to phylogenetic relationships, these genes were divided into 27 subgroups. The identified MYB genes were further classified into 121 MYB orthologous gene groups (OGGs), including 25 core, 57 softcore, 19 shell and 20 line-specific/cloud groups. Whole-genome duplication was the most common mechanism of MYB genes expansion. In core group, the higher proportions of MYB genes were found to be in the coexpression network constructed by the RNA-seq data. Through the comprehensive analysis including phylogeny and gene expression profile of cucumber MYB genes, as well as genetic variations in 103 cucumber germplasms, we identified a MYB gene CsRAX5, which may be related to cucumber plant height. We used gene editing technology to knockout and overexpress CsRAX5. In the knockout lines, Csrax5, the height was significantly increased compared with wild type (WT), whereas after overexpression the height of CsRAX5-OE plants was significantly decreased compared with WT. These results indicated that MYB gene CsRAX5 negatively regulated cucumber plant height. The large-scale analysis of MYB genes in Cucurbitaceae in this study provides insights for further investigating the evolution and function of MYB genes in Cucurbitaceae crops.

Acknowledgments

This work is supported by Natural Science Foundation of Shandong Province (ZR2024MC015 and ZR2023QC104), the Taishan Program of Shandong Province (Grant tsqn202306246), the National Natural Science Foundation of China (32472771 and 32302553), the National Natural Science Union Fund (U24A20419), and the ‘Pioneer’ and ‘Leading Goose’ R&D Program of Zhejiang (2022C02051).

Author contributions

C.Y., H.W., and Z.Z. designed the research; W.Z., J.C., and J.W. performed the experiments; H.Y. conducted bioinformatics analysis; X.H. and J.C. provided experimental assistance; W.Z. wrote the paper; C.Y. and J.C. revised the manuscript. All authors read and approved the contents of this the manuscript.

Data availability

All data supporting the results of this study are included in the article and additional files.

Conflict of interest statement

The authors declare no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Christenhusz MJM, Byng JW. The number of known plants species in the world and its annual increase. Phytotaxa. 2016; 261: 201-17

[2] Esteras C, Gómez P, Monforte AJ. et al. High-throughput SNP genotyping in Cucurbita pepo for map construction and quanti-tative trait loci mapping. BMC Genomics. 2012; 13:1-21

[3] Kaźmińska K, Sobieszek K, Targońska-Karasek M. et al. Genetic diversity assessment of a winter squash and pumpkin (Cucurbita maxima Duchesne) germplasm collection based on genomic Cucurbita-conserved SSR markers. Sci Hortic. 2017; 219:37-44

[4] Borecka M, Karaś M. A comprehensive review of the nutritional and health-promoting properties of edible parts of selected Cucurbitaceae plants. Foods. 2025; 14:1200

[5] Du H, Yang SS, Liang Z. et al. Genome-wide analysis of the MYB transcription factor superfamily in soybean. BMC Plant Biol. 2012; 12:1-22

[6] Pabo CO, Sauer RT. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992; 61: 1053-95

[7] Lipsick JS. One billion years of Myb. Oncogene. 1996; 13:223-35

[8] Feng G, Burleigh JG, Braun EL. et al. Evolution of the 3R-MYB gene family in plants. Genome Biol Evol. 2017; 9:1013-29

[9] Zhu Y, Deng Y, Yao Y. et al. Characteristics and expression analysis of the MYB-related subfamily gene in Rosa chinensis. Int JMolSci. 2024; 25:12854

[10] Ogata K, Morikawa S, Nakamura H. et al. Comparison of the free and DNA-complexed forms of the DMA-binding domain from c-Myb. Nat Struct Biol. 1995; 2:309-20

[11] Ogata K, Kanei-Ishii C, Sasaki M. et al. The cavity in the hydropho-bic core of Myb DNA-binding domain is reserved for DNA recog-nition and trans-activation. Nat Struct Biol. 1996; 3:178-87

[12] Liu J, Osbourn A, Ma P. MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol Plant. 2015; 8: 689-708

[13] Stracke R, Holtgräwe D, Schneider J. et al. Genome-wide identi-fication and characterisation of R2R3-MYB genes in sugar beet (Beta vulgaris). BMC Plant Biol. 2014; 14:1-17

[14] Grotewold E, Sainz MB, Tagliani L. et al. Identification of the residues in the Myb domain of maize C1 that specify the interaction with the bHLH cofactor R. Proc Natl Acad Sci. 2000; 97: 13579-84

[15] Qi J, Liu X, Shen DI. et al. A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Nat Genet. 2013; 45:1510-5

[16] Li H, Wang S, Chai S. et al. Graph-based pan-genome reveals structural and sequence variations related to agronomic traits and domestication in cucumber. Nat Commun. 2022; 13:682

[17] Wu S, Sun H, Gao L. et al. A Citrullus genus super-pangenome reveals extensive variations in wild and cultivated watermelons and sheds light on watermelon evolution and domestication. Plant Biotechnol J. 2023; 21:1926-8

[18] Zhang Y, Zhao M, Tan J. et al. Telomere-to-telomere Citrullus super-pangenome provides direction for watermelon breeding. Nat Genet. 2024; 56:1750-61

[19] Mo C, Wang H, Wei M. et al. Complete genome assembly provides a high-quality skeleton for pan-NLRome construction in melon. Plant J. 2024; 118:2249-68

[20] Wei M, Huang Y, Mo C. et al. Telomere-to-telomere genome assembly of melon (Cucumis melo L. var.inodorus) provides a high-quality reference for meta-QTL analysis of important traits. Hortic Res. 2023;10:uhad189

[21] Matthews CA, Watson-Haigh NS, Burton RA. et al.A gentle introduction to pangenomics. Brief Bioinform. 2024;25:bbae588

[22] Shigita G, Tanaka K, Kato K. Current and future pangenomic research in cucurbit crops. Breed Sci. 2025; 75:34-50

[23] Jiang C, Gu X, Peterson T. Identification of conserved gene structures and carboxy-terminal motifs in the Myb gene family of Arabidopsis and Oryza sativa L. ssp. indica. Genome Biol. 2004; 5: 1-11

[24] Xu M, Fu J, Ni Y. et al. Genome-wide analysis of the MYB gene family in pumpkin. PeerJ. 2024; 12:e17304

[25] Qing XU, Jie HE, Jianhui D. et al. Genomic survey and expression profiling of the MYB gene family in watermelon. Hortic Plant J. 2018; 4:1-15

[26] Romano JM, Dubos C, Prouse MB. et al. AtMYB61, an R2R3-MYB transcription factor, functions as a pleiotropic regulator via a small gene network. New Phytol. 2012; 195:774-86

[27] Huang H, Gao H, Liu B. et al. Arabidopsis MYB24 regu-lates jasmonate-mediated stamen development. Front Plant Sci. 2017; 8:1525

[28] Wan J, Wang R, Zhang P. et al. MYB70 modulates seed ger-mination and root system development in Arabidopsis. Iscience. 2021; 24:103228

[29] Keller T, Abbott J, Moritz T. et al. Arabidopsis REGULATOR OF AXILLARY MERISTEMS1 controls a leaf axil stem cell niche and modulates vegetative development. Plant Cell. 2006; 18:598-611

[30] Wang T, Jin Y, Deng L. et al. The transcription factor MYB110 regulates plant height, lodging resistance, and grain yield in rice. Plant Cell. 2024; 36:298-323

[31] Pan J, Sohail H, Sharif R. et al. Cucumber JASMONATE ZIM-DOMAIN 8 interaction with transcription factor MYB6 impairs waterlogging-triggered adventitious rooting. Plant Phys-iol. 2025;197:kiae351

[32] Chen J, Liu L, Chen G. et al. CsRAXs negatively regulate leaf size and fruiting ability through auxin glycosylation in cucumber. J Integr Plant Biol. 2024; 66:1024-37

[33] Mu Q, Wei J, Longest HK. et al. A MYB transcription factor underlying plant height in sorghum qHT7.1 and maize Brachytic 1 loci. Plant J. 2024; 120:2172-92

[34] Jing F, Shi S, Kang W. et al. The physiological basis of alfalfa plant height establishment. Plants. 2024; 13:679

[35] Zhao H, Sun P, Tong C. et al. CsIREH1 phosphorylation regulates DELLA protein affecting plant height in cucumber (Cucumis sativus). New Phytol. 2025; 245:1528-46

[36] Zhang M, Yan W, Yan M. et al. Development of near-isogenic line of dwarf gene Cldw-1 and transcriptome analysis in watermelon (Citrullus lanatus). Sci Hortic. 2025; 343:114065

[37] Ferrero-Serrano Á, Cantos C, Assmann SM. The role of dwarfing traits in historical and modern agriculture with a focus on rice. Cold Spring Harb Perspect Biol. 2019;11: a034645

[38] Lu Z, Yu H, Xiong G. et al. Genome-wide binding analysis of the transcription activator ideal plant architecture1 reveals a complex network regulating rice plant architecture. Plant Cell. 2013; 25:3743-59

[39] LiuW, ChenG, HeM. et al. ABI5 promotes heat stress-induced chlorophyll degradation by modulating the stability of MYB44 in cucumber. Hortic Res. 2023;10:uhad089

[40] Wang B, Wang G, Wang Y. et al. A cold-inducible MYB like transcription factor, CsHHO2, positively regulates chilling tol-erance of cucumber fruit by enhancing CsGR-RBP3 expression. Postharvest Biol Technol. 2024; 218:113172

[41] Li Z, Zhang Z, Yan P. et al. RNA-Seq improves annotation of protein-coding genes in the cucumber genome. BMC Genomics. 2011; 12:1-11

[42] Yano R, Nonaka S, Ezura H. Melonet-DB, a grand RNA-Seq gene expression atlas in melon (Cucumis melo L.). Plant Cell Physiol. 2018; 59:e4-4

[43] Xue Z, Liu L, Zhang C. Regulation of shoot apical meristem and axillary meristem development in plants. Int J Mol Sci. 2020; 21: 2917

[44] Yao F, Jiang MY, Yang YS. et al. Bioinformatics and expression analysis on MYB-related family in Angelica dahurica var. for-mosana. China J Chinese Mater Med. 2022; 47:1831-46

[45] Hao N, Du Y, Li H. et al. CsMYB36 is involved in the formation of yellow green peel in cucumber (Cucumis sativus L.). Theor Appl Genet. 2018; 131:1659-69

[46] Wang C, Yao H, Wang C. et al. Transcription factor CsMYB36 regu-lates fruit neck length via mediating cell expansion in cucumber. Plant Physiol. 2024; 195:958-69

[47] Qi X, Zhu Y, Li S. et al. Identification of genes related to mesocarp development in cucumber. Hortic Plant J. 2020; 6:293-300

[48] Li Q, Zhang C, Li J. et al. Genome-wide identification and char-acterization of R2R3MYB family in Cucumis sativus. PLoS One. 2012;7:e47f576

[49] Shi J, Tian Z, Lai J. et al. Plant pan-genomics and its applications. Mol Plant. 2023; 16:168-86

[50] Liu Z, Zhang C, He J. et al. plantGIR: a genomic database of plants. Hortic Res. 2024;11:uhae342

[51] Liu Z, Shen S, Li C. et al. SoIR: a comprehensive Solanaceae information resource for comparative and functional genomic study. Nucleic Acids Res. 2025;53:D1623-32

[52] Liu J, Huang C, Xing D. et al. The genomic database of fruits: a comprehensive fruit information database for compara-tive and functional genomic studies. Agric Commun. 2024; 2: 100041

[53] Feng S, Liu Z, Chen H. et al. PHGD: an integrative and user-friendly database for plant hormone-related genes. Imeta. 2024; 3:e164

[54] Tong C, Jia Y, Hu H. et al. Pangenome and pantranscriptome as the new reference for gene-family characterization: a case study of basic helix-loop-helix (bHLH) genes in barley. Plant Commun. 2025; 6:101190

[55] Yu J, Chen Q, Yuan L. et al. Phylogenomic and super-pangenome analyses unveil the genetic landscape of tomato evolution and domestication. Plant Biotechnol J. 2025;

[56] Zhang Y, Yu C, Lin J. et al. OsMPH1 regulates plant height and improves grain yield in rice. PLoS One. 2017; 12:e0180825

[57] Yang X, Li X, Shan J. et al. Overexpression of GmGAMYB accel-erates the transition to flowering and increases plant height in soybean. Front Plant Sci. 2021; 12:667242

[58] Ren R, Wang H, Guo C. et al. Widespread whole genome duplica-tions contribute to genome complexity and species diversity in angiosperms. Mol Plant. 2018; 11:414-28

[59] Wu J, Zhang L, Ma X. et al. The evolutionary significance of whole genome duplications in oil biosynthesis of oil crops. Hortic Res. 2024;11:uhae156

[60] Pottier M, Le Thi VA, Primard-Brisset C. et al. Duplica-tion of NRAMP3 gene in poplars generated two homologous transporters with distinct functions. MolBiolEvo.l 2022;39: msac129

[61] Song X, Hu J, Wu T. et al. Comparative analysis of long noncoding RNAs in angiosperms and characterization of long noncoding RNAs in response to heat stress in Chinese cabbage. Hortic Res. 2021; 8:48

[62] Schmitz G, Tillmann E, Carriero F. et al. The tomato Blind gene encodes a MYB transcription factor that controls the formation of lateral meristems. Proc Natl Acad Sci. 2002; 99:1064-9

[63] Muller D, Schmitz G, Theres K. Blind homologous R2R3 Myb genes control the pattern of lateral meristem initiation in Arabidopsis. Plant Cell. 2006; 18:586-97

[64] Li Y, Wang N, Feng J. et al. Enhancing genetic transformation efficiency in cucurbit crops through AtGRF5 overexpression: mechanistic insights and applications. J Integr Plant Biol. 2025; 67: 1843-60

[65] Emms DM, Kelly S. OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol. 2015; 16:1-14

[66] Qiao X, Li Q, Yin H. et al. Gene duplication and evolution in recur-ring polyploidization-diploidization cycles in plants. Genome Biol. 2019; 20:1-23

[67] Zhang Z, Xiao J, Wu J. et al. ParaAT: a parallel tool for constructing multiple protein-coding DNA alignments. Biochem Biophys Res Commun. 2012; 419:779-81

[68] Hu B, Li D, Liu X. et al. Engineering non-transgenic gynoecious cucumber using an improved transformation protocol and opti-mized CRISPR/Cas 9 system. Mol Plant. 2017; 10:1575-8