A transposon insertion in CmKNAT2-like2 disrupts mottled rind formation in melon (Cucumis melo L.)

Abstract

The mottled rind is an important fruit external appearance trait that influences consumer preferences. Previous studies reported that CmMt1 and CmMt2 regulate rind mottling in melon, yet CmMt2 has not been cloned. In this study, we developed near-isogenic lines (NILs) using the nonmottled rind ‘13C’ as the recurrent parent and mottled ‘P114’ as the donor parent, and screened a mottled rind mutant ‘S249’ by ethyl methanesulfonate mutagenesis of ‘13C’. Combined with these genetic materials, CmMt2 was delimited to a 44-kb region on chromosome 2. Within this genetic interval, a CACTA-type TIR transposon insertion was detected in all nonmottled rind lines, and this insertion may lead to impaired nuclear localization and dimerization capability of CmKNAT2-like2 encoding a homeobox protein through the loss of conserved ELK and Homeodomain. Further, CRISPR/Cas9-mediated knockout of CmKNAT2-like2 confirmed its pivotal role in regulating mottled rind phenotype. In addition, transcriptome analysis suggested that the transposon insertion in CmKNAT2-like2 results in nonmottled rind by disrupting chloroplast development and altering the expression of chlorophyll biosynthesis-related genes, and population analysis revealed that the transposon associated with CmKNAT2-like2 has undergone selection in cultivated melons. Collectively, these results demonstrate that CmKNAT2-like2 is the causal gene underlying CmMt2, which regulates mottled rind in melon.

Acknowledgements

We thank associate professor Huimin Zhang (QAU) for providing genomic data from diverse germplasms. This work was supported by the National Natural Science Foundation of China (32472767 to S.C., 32102404 to S.C., 32225044 to Z.Z., 32130093 to Z.Z.,), the Natural Science Foundation of Shandong Province (ZR2024MC030), and the Qingdao Science and Technology for People’s Livelihood Demonstration Special Project (25-1-5-xdny-25-nsh).

Author contributions

Z.Z., S.C., and S.L. designed the research. S.C., S.L., J.F., Y.L.,and H.W. performed the experiments. X.C. and Y.X. performed the bioinformatics analyses. J.S. revised the manuscript. S.C. and S.L. analyzed the data. Z.Z., S.C., and S.L. wrote the manuscript. All authors participated in the research and approved the final manuscript.

Data availability

Relevant data can be found within the paper and its supporting materials. The sequencing data that support the findings of the Mutmap⁺ analysis and RNA-seq analysis have been deposited in the Sequence Read Archive under accession number PRJNA1284442. Other data of this study are available from https://zhanglab.qau.edu.cn/melow/index.php.

Conflict of interest statement

The authors declare no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Feder A, Burger J, Gao S. et al. A Kelch domain-containing F-box coding gene negatively regulates flavonoid accumulation in muskmelon. Plant Physiol. 2015; 169:1714-26

[2] Tadmor Y, Burger J, Yaakov I. et al. Genetics of flavonoid, carotenoid, and chlorophyll pigments in melon fruit rinds. J Agric Food Chem. 2010; 58:10722-8

[3] Kobayashi S, Goto-Yamamoto N, Hirochika H. Retrotransposon-induced mutations in grape skin color. Science. 2004; 304:982-2

[4] Zheng X, Zhu K, Sun Q. et al. Natural variation in CCD4 promoter underpins species-specific evolution of red coloration in citrus Peel. Mol Plant. 2019; 12:1294-307

[5] Powell ALT, Nguyen CV, Hill T. et al. Uniform ripening encodes a Golden 2-like transcription factor regulating tomato fruit chloro-plast development. Science. 2012; 336:1711-5

[6] Chuong EB, Elde NC, Feschotte C. Regulatory activities of trans-posable elements: from conflicts to benefits. Nat Rev Genet. 2017; 18:71-86

[7] Domínguez M, Dugas E, Benchouaia M. et al. The impact of transposable elements on tomato diversity. Nat Commun. 2020; 11:4058

[8] Sultana T, Zamborlini A, Cristofari G. et al. Integration site selec-tion by retroviruses and transposable elements in eukaryotes. Nat Rev Genet. 2017; 18:292-308

[9] Woodhouse MR, Cheng F, Pires JC. et al.Origin, inheritance, and gene regulatory consequences of genome dominance in polyploids. Proc Natl Acad Sci. 2014; 111:5283-8

[10] Alonge M, Wang X, Benoit M. et al. Major impacts of widespread structural variation on gene expression and crop improvement in tomato. Cell. 2020; 182:145-161.e23

[11] Hufford MB, Seetharam AS, Woodhouse MR. et al. De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes. Science. 2021; 373:655-62

[12] Liu Y, Du H, Li P. et al. Pan-genome of wild and cultivated soybeans. Cell. 2020; 182:162-176.e13

[13] Qin P, Lu H, du H. et al. Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations. Cell. 2021; 184:3542-3558.e16

[14] Song J-M, Guan Z, Hu J. et al. Eight high-quality genomes reveal pan-genome architecture and ecotype differentiation of Bras-sica napus. Nat Plants. 2020; 6:34-45

[15] 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

[16] Ganesan J. Genetic Studies on Certain Characters of Economic Impor-tance in Muskmelon (Cucumis melo L.). India: Annamalai Univer-sity; 1988:

[17] Liu L, Sun T, Liu X. et al. Genetic analysis and mapping of a stripedrindgene(st3) in melon (Cucumis melo L.). Euphytica. 2019; 215:20

[18] Lv J, Fu Q, Lai Y. et al. Inheritance and gene mapping of spotted to non-spotted trait gene CmSp-1 in melon (Cucumis melo L. var. chinensis Pangalo). Mol Breed. 2018; 38:105

[19] Pereira L, Ruggieri V, Pérez S. et al. QTL mapping of melon fruit quality traits using a high-density GBS-based genetic map. BMC Plant Biol. 2018; 18:324

[20] Périn C, Hagen L, de Conto V. et al. A reference map of Cucumis melo based on two recombinant inbred line populations. Theor Appl Genet. 2002; 104:1017-34

[21] Zhao G, Lian Q, Zhang Z. et al. A comprehensive genome varia-tion map of melon identifies multiple domestication events and loci influencing agronomic traits. Nat Genet. 2019; 51:1607-15

[22] Shen J, Xu X, Zhang Y. et al. Genetic mapping and identification of the candidate genes for mottled rind in Cucumis melo L. Front Plant Sci. 2021; 12:76998

[23] Zhang W, Liao S, Zhang J. et al. Recurrent excision of a hAT-like transposable element in CmAPRR2 leads to the “shooting star” melon phenotype. Plant J. 2024; 120:1206-20

[24] Oren E, Dafna A, Tzuri G. et al. Pan-genome and multi-parental framework for high-resolution trait dissection in melon (Cucumis melo). Plant J. 2022; 112:1525-42

[25] Cai X, Chang L, Zhang T. et al. Impacts of allopolyploidization and structural variation on intraspecific diversification in Bras-sica rapa. Genome Biol. 2021; 22:166

[26] Bhattacharyya MK, Smith AM, Ellis THN. et al. The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme. Cell. 1990; 60:115-22

[27] Butelli E, Licciardello C, Zhang Y. et al. Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. Plant Cell. 2012; 24:1242-55

[28] 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

[29] Li N, He Q, Wang J. et al. Super-pangenome analyses highlight genomic diversity and structural variation across wild and cul-tivated tomato species. Nat Genet. 2023; 55:852-60

[30] Shang L, Li X, He H. et al. A super pan-genomic landscape of rice. Cell Res. 2022; 32:878-96

[31] Tang D, Jia Y, Zhang J. et al. Genome evolution and diversity of wild and cultivated potatoes. Nature. 2022; 606:535-41

[32] Zhou Y, Zhang Z, Bao Z. et al. Graph pangenome captures missing heritability and empowers tomato breeding. Nature. 2022; 606:527-34

[33] Nadakuduti SS, Holdsworth WL, Klein CL. et al. KNOX genes influence a gradient of fruit chloroplast development through regulation of GOLDEN2-LIKE expression in tomato. Plant J. 2014; 78:1022-33

[34] Yue Z, Fu Y, Dai X. et al. The KNOX transcription factor ClSP activates ClAPRR2 to regulate dark green stripe formation in watermelon. Plant Biotechnol J. 2025; 23:3012-23

[35] Fekih R, Takagi H, Tamiru M. et al. MutMap+: genetic mapping and mutant identification without crossing in Rice. PLoS One. 2013; 8:e68529

[36] Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980; 8:4321-6

[37] Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25:1754-60

[38] Li H, Handsaker B, Wysoker A. et al. The sequence alignmen-t/map format and SAMtools. Bioinformatics. 2009; 25:2078-9

[39] McKenna A, Hanna M, Banks E. et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20:1297-303

[40] Cingolani P, Platts A, Wang LL. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms. Fly. 2012; 6:80-92

[41] Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32: 1792-7

[42] Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. MolBiolEvol. 2021; 38:3022-7

[43] Xia C, Yin S, Zhang J. et al. CAD1 enhances antioxidant activity to facilitate cold storage by regulating flavonoid synthesis in cucumber. Agric Commun. 2025; 3:100071

[44] Xing H-L, Dong L, Wang ZP. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014; 14:327

[45] 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

[46] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12:357-60

[47] Pertea M, Pertea GM, Antonescu CM. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015; 33:290-5

[48] Chen X, Li H, Dong Y. et al. A wild melon reference genome pro-vides novel insights into the domestication of a key gene respon-sible for melon fruit acidity. Theor Appl Genet. 2024; 137:144