The haplotype-resolved T2T reference genome highlights structural variation underlying agronomic traits of melon

Abstract

Melon (Cucumis melo L.) is an important vegetable crop that has an extensive history of cultivation. However, the genome of wild and semi-wild melon types that can be used for the analysis of agronomic traits is not yet available. Here we report a chromosome-level T2T genome assembly for 821 (C. melo ssp. agrestis var. acidulus), a semi-wild melon with two haplotypes of ∼373 Mb and ∼364 Mb, respectively. Comparative genome analysis discovered a significant number of structural variants (SVs) between melo (C. melo ssp. melo) and agrestis (C. melo ssp. agrestis) genomes, including a copy number variation located in the ToLCNDV resistance locus on chromosome 11. Genome-wide association studies detected a significant signal associated with climacteric ripening and identified one candidate gene CM_ac12g14720.1 (CmABA2), encoding a cytoplasmic short chain dehydrogenase/reductase, which controls the biosynthesis of abscisic acid. This study provides valuable genetic resources for future research on melon breeding.

Acknowledgements

This work was supported by funding from the Agricultural Science and Technology Innovation Program (CAAS-ASTIP- 2016-ZFRI-06), the China Agriculture Research System (CARS- 25-2023-G6), the Key Research and Development Program of Hainan (ZDYF2021XDNY164), the European Research Council (ERC-NectarGland, 101095736), the 111 Project (B17043) and Henan Province Science and Technology Research Project (232102110185).

Author contributions

G.Z. and T.L. conceived and designed the study. Y.H. and Y.X. planted and prepared the materials. G.L. performed the bioinformatics analysis. L.T. designed and performed molecular experiments. G.L. and L.T. wrote the manuscript. G.Z., T.L., A.B. and J.G-M. edited and improved the manuscript. All authors approved the final manuscript.

Data availability

The original sequencing data for genome assembly have been deposited in the Genome Sequence Archive (GSA) database at BIG Data Center (https://ngdc.cncb.ac.cn/gsa/) with Accession Number CRA010716. The assembled genome 821 was deposited in the Genome Warehouse (GWH) database of the Big Data Center (https://bigd.big.ac.cn/gwh/) under the accession number GWH- DOOI00000000. The assembled genome 821 and annotations are available on figshare (10.6084/m9.figshare.23701680).

Conflict of interest statement

The authors declare no competing interest.

References

[1] Jeffrey C. A review of the Cucurbitaceae. Bot J Linn Soc. 1980; 81: 233-47

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

[3] Yang JH, Deng G, Lian J et al. The chromosome-scale genome of melon dissects genetic architecture of important agronomic traits. iScience. 2020; 23: 101422

[4] Zhang H, Li X, Yu H et al. A high-quality melon genome assembly provides insights into genetic basis of fruit trait improvement. iScience. 2019; 22: 16-27

[5] Pichot C, Djari A, Tran J et al. Cantaloupe melon genome reveals 3D chromatin features and structural relationship with the ancestral cucurbitaceae karyotype. iScience. 2022; 25: 103696

[6] Yano R, Ariizumi T, Nonaka S et al. Comparative genomics of muskmelon reveals a potential role for retrotransposons in the modification of gene expression. Commun Biol. 2020; 3: 432

[7] Ling J, Xie X, Gu X et al. High-quality chromosome-level genomes of Cucumis metuliferus and Cucumis melo provide insight into Cucumis genome evolution . Plant J. 2021; 107: 136-48

[8] Castanera R, Ruggieri V, Pujol M et al. An improved melon reference genome with single-molecule sequencing uncovers a recent burst of transposable elements with potential impact on genes. Front Plant Sci. 2019; 10: 1815

[9] Nurk S, Koren S, Rhie A et al. The complete sequence of a human genome. Science. 2022; 376: 44-53

[10] Deng Y, Liu S, Zhang Y et al. A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding. Mol Plant. 2022; 15: 1268-84

[11] Lin T, Zhu G, Zhang J et al. Genomic analyses provide insights into the history of tomato breeding. Nat Genet. 2014; 46: 1220-6

[12] Yu H, Lin T, Meng X et al. A route to de novo domestication of wild allotetraploid rice. Cell. 2021; 184: 1156-1170.e14

[13] Tian J, Wang C, Xia J et al. Teosinte ligule allele narrows plant architecture and enhances high-density maize yields. Science. 2019; 365: 658-64

[14] Pitrat M. Phenotypic diversity in wild and cultivated melons (Cucumis melo). Plant Biotechnol. 2013; 30: 273-8

[15] McCreight JD, Wintermantel WM . Genetic resistance in melon PI 313970 to cucurbit yellow stunting disorder virus. HortScience. 2011; 46: 1582-7

[16] Martin-Hernandez AM, Pico B . Natural resistances to viruses in cucurbits. Agronomy. 2021; 11: 23

[17] Romay G, Pitrat M, Lecoq H et al. Resistance against melon chlorotic mosaic virus and tomato leaf curl New Delhi virus in melon. Plant Dis. 2019; 103: 2913-9

[18] Ezura H, Owino WO . Melon, an alternative model plant for elucidating fruit ripening. Plant Sci. 2008; 175: 121-9

[19] Pereira L, Santo Domingo M, Ruggieri V et al. Genetic dissection of climacteric fruit ripening in a melon population segregating for ripening behavior. Hortic Res. 2020; 7: 187

[20] Giordano A, Santo Domingo M, Quadrana L et al. CRISPR/Cas9 gene editing uncovers the roles of CONSTITUTIVE TRIPLE RESPONSE 1 and REPRESSOR OF SILENCING 1 in melon fruit ripening and epigenetic regulation. J Exp Bot. 2022; 73: 4022-33

[21] Liu B, Santo Domingo M, Mayobre C et al. Knock-out of CmNAC-NOR affects melon climacteric fruit ripening. Front Plant Sci. 2022; 13: 878037

[22] Rios P, Argyris J, Vegas J et al. ETHQV6.3 is involved in melon climacteric fruit ripening and is encoded by a NAC domain transcription factor. Plant J. 2017; 91: 671-83

[23] Santo Domingo M, Areco L, Mayobre C et al. Modulating climacteric intensity in melon through QTL stacking. Hortic Res. 2022; 9: uhac131

[24] Santo Domingo M, Mayobre C, Pereira L et al. Fruit morphology and ripening-related QTLs in a newly developed introgression line collection of the elite varieties ’Vedrantais’ and ’Piel de Sapo’. Plants (Basel). 2022; 11: 3120

[25] Pereira L, Santo Domingo M, Argyris J et al. A novel introgression line collection to unravel the genetics of climacteric ripening and fruit quality in melon. Sci Rep. 2021; 11: 11364

[26] Li BJ, Grierson D, Shi Y et al. Roles of abscisic acid in regulating ripening and quality of strawberry, a model non-climacteric fruit. Hortic Res. 2022; 9: 878037

[27] Fenn MA, Giovannoni JJ . Phytohormones in fruit development and maturation. Plant J. 2021; 105: 446-58

[28] Jia HF, Xie Z, Wang C et al. Abscisic acid, sucrose, and auxin coordinately regulate berry ripening process of the Fujiminori grape. Funct Integr Genomic. 2017; 17: 441-57

[29] Tijero V, Teribia N, Muñoz P et al. Implication of abscisic acid on ripening and quality in sweet cherries: differential effects during pre- and post-harvest. Front Plant Sci. 2016; 7: 00602

[30] Saez C, Martínez C, Ferriol M et al. Resistance to tomato leaf curl New Delhi virus in Cucurbita spp. Ann Appl Biol. 2016; 169: 91-105

[31] Cui L, Siskos L, Wang C et al. Breeding melon (Cucumis melo) with resistance to powdery mildew and downy mildew . Hortic Plant J. 2022; 8: 545-61

[32] McCreight JD . Genes for resistance to powdery mildew races 1 and 2U.S. In melon PI 313970. HortScience. 2003; 38: 591-4

[33] Cheng HY, Concepcion GT, Feng X et al. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods. 2021; 18: 170

[34] Lin T, Li GL, Wang BA et al. C&RAP annotation software (V1.0). China Copyright Protection Center, 2023R11L0028447 (2022).

[35] Manni M, Berkeley MR, Seppey M et al. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol. 2021; 38: 4647-54

[36] Manni M, Berkeley MR, Seppey M et al. BUSCO: assessing genomic data quality and beyond. Curr Protoc. 2021; 1: e323

[37] Rhie A, Walenz BP, Koren S et al. Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies. Genome Biol. 2020; 21: 245

[38] Ou S, Chen J, Jiang N . Assessing genome assembly quality using the LTR assembly index (LAI). Nucleic Acids Res. 2018; 46: e126

[39] Ou S, Jiang N . LTR_retriever: a highly accurate and sensitive program for identification of long terminal repeat retrotransposons. Plant Physiol. 2018; 176: 1410-22

[40] Garcia-Mas J, Benjak A, Sanseverino W et al. The genome of melon (Cucumis melo L.). Proc Natl Acad Sci U S A. 2012; 109: 11872-7

[41] Chovelon V, Feriche-Linares R, Barreau G et al. Building a cluster of NLR genes conferring resistance to pests and pathogens: the story of the vat gene cluster in cucurbits. Hortic Res. 2021; 8: 72

[42] McHale LK, Haun WJ, Xu WW et al. Structural variants in the soybean genome localize to clusters of biotic stress-response genes. Plant Physiol. 2012; 159: 1295-308

[43] Haider MS, Tahir M, Latif S et al. First report of tomato leaf curl New Delhi virus infecting Eclipta prostrata in Pakistan . Plant Pathol. 2006; 55: 285-5

[44] Mizutani T, Daryono BS, Ikegami M et al. First report of tomato leaf curl New Delhi virus infecting cucumber in Central Java, Indonesia. Plant Dis. 2011; 95: 1485-5

[45] Juarez M, Tovar R, Fiallo-Olivé E et al. First detection of tomato leaf curl New Delhi virus infecting zucchini in Spain. Plant Dis. 2014; 98: 857

[46] Ito T, Sharma P, Kittipakorn K et al. Complete nucleotide sequence of a new isolate of tomato leaf curl New Delhi virus infecting cucumber, bottle gourd and muskmelon in Thailand. Arch Virol. 2008; 153: 611-3

[47] Yazdani-Khameneh S, Aboutorabi S, Shoori M et al. Natural occurrence of tomato leaf curl New Delhi virus in Iranian cucurbit crops. Plant Pathol J. 2016; 32: 201-8

[48] Li R, Liu Y, Yin C et al. Occurrence of tomato leaf curl New Delhi virus in tomato (Lycopersicon esculentum) in China . Plant Dis. 2023; 107: 2561.

[49] Moriones E, Praveen S, Chakraborty S . Tomato leaf curl New Delhi virus: an emerging virus complex threatening vegetable and fiber crops. Viruses. 2017; 9: 264

[50] Saez C, Esteras C, Martínez C et al. Resistance to tomato leaf curl New Delhi virus in melon is controlled by a major QTL located in chromosome 11. Plant Cell Rep. 2017; 36: 1571-84

[51] Wang YP, Wu YY, Tang DZ . The autophagy gene, ATG18a, plays a negative role in powdery mildew resistance and mildew-induced cell death in Arabidopsis. Plant Signal Behav. 2011; 6: 1408-10

[52] Prasad A, Prasad M . Interaction of ToLCNDV TrAP with SlATG8f marks it susceptible to degradation by autophagy. Cell Mol Life Sci. 2022; 79: 241

[53] Mayobre C, Pereira L, Eltahiri A et al. Genetic dissection of aroma biosynthesis in melon and its relationship with climacteric ripening. Food Chem. 2021; 353: 129484

[54] Chen K, Li GJ, Bressan RA et al. Abscisic acid dynamics, signaling, and functions in plants. J Integr Plant Biol. 2020; 62: 25-54

[55] Mou W, Li D, Bu J et al. Comprehensive analysis of ABA effects on ethylene biosynthesis and signaling during tomato fruit ripening. PLoS One. 2016; 11: e0154072

[56] Leng P, Zhang GL, Li XX et al. Cloning of 9-cis-epoxycarotenoid dioxygenase (NCED) gene encoding a key enzyme during abscisic acid (ABA) biosynthesis and ABA-regulated ethylene production in detached young persimmon calyx. Chin Sci Bull. 2009; 54: 2830-8

[57] Wang Y, Wang Y, Ji K et al. The role of abscisic acid in regulating cucumber fruit development and ripening and its transcriptional regulation. Plant Physiol Biochem. 2013; 64: 70-9

[58] Pitrat M. Disease Resistance in Melon and Its Modification by Molecular Breeding Techniques. In: Functional Genomics and Biotechnology in Solanaceae and Cucurbitaceae Crops. 2016: 175-97.

[59] Saez C, Ambrosio LGM, Miguel SM et al. Resistant sources and genetic control of resistance to ToLCNDV in cucumber. Microorganisms. 2021; 9: 913

[60] Roman B, Gómez P, Picó B et al. Candidate gene analysis of tomato leaf curl New Delhi virus resistance in Cucumis melo. Sci Hortic. 2019; 243: 12-20

[61] Maruthi MN, Rekha AR, Cork A et al. First report of tomato leaf curl New Delhi virus infecting tomato in Bangladesh. Plant Dis. 2005; 89: 1011

[62] Saez C, Martínez C, Montero-Pau J et al. A major QTL located in chromosome 8 of Cucurbita moschata is responsible for resistance to tomato leaf curl New Delhi virus . Front Plant Sci. 2020; 11: 207

[63] Sáez C, Flores-León A, Montero-Pau J et al. RNA-Seq transcriptome analysis provides candidate genes for resistance to tomato leaf curl New Delhi virus in melon. Front Plant Sci. 2021; 12: 798858

[64] Zhang M, Leng P, Zhang G et al. Cloning and functional analysis of 9-cis-epoxycarotenoid dioxygenase (NCED) genes encoding a key enzyme during abscisic acid biosynthesis from peach and grape fruits. J Plant Physiol. 2009; 166: 1241-52

[65] Clayton-Cuch D, Yu L, Shirley N et al. Auxin treatment enhances anthocyanin production in the non-climacteric sweet cherry (Prunus avium L.). Int J Mol Sci. 2021; 22: 798858

[66] Ji K, Chen P, Sun L et al. Non-climacteric ripening in strawberry fruit is linked to ABA, FaNCED2 and FaCYP707A1. Funct Plant Biol. 2012; 39: 351-7

[67] Bai Q, Huang Y, Shen Y . The physiological and molecular mechanism of abscisic acid in regulation of fleshy fruit ripening. Front Plant Sci. 2020; 11: 619953

[68] Leng P, Yuan B, Guo Y . The role of abscisic acid in fruit ripening and responses to abiotic stress. J Exp Bot. 2014; 65: 4577-88

[69] Zhang M, Yuan B, Leng P . The role of ABA in triggering ethylene biosynthesis and ripening of tomato fruit. J Exp Bot. 2009; 60: 1579-88

[70] Durand NC, Shamim MS, Machol I et al. Juicer provides a one-click system for analyzing loop-resolution hi-C experiments. Cell Syst. 2016; 3: 95-8

[71] Durand NC, Robinson JT, Shamim MS et al. Juicebox provides a visualization system for hi-C contact maps with unlimited zoom. Cell Syst. 2016; 3: 99-101

[72] Dudchenko O, Batra SS, Omer AD et al. De novo assembly of the Aedes aegypti genome using hi-C yields chromosome-length scaffolds. Science. 2017; 356: 92-5

[73] Roach MJ, Schmidt SA, Borneman AR . Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies. BMC Bioinformatics. 2018; 19: 460

[74] Ranallo-Benavidez TR, Jaron KS, Schatz MC . GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes. Nat Commun. 2020; 11: 1432

[75] Li H . Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018; 34: 3094-100

[76] Delcher AL, Salzberg SL, Phillippy AM . Using MUMmer to identify similar regions in large sequence sets. Curr Protoc Bioinformatics. 2003; 10: 10.3

[77] Hao Z, Lv D, Ge Y et al. RIdeogram: drawing SVG graphics to visualize and map genome-wide data on the idiograms. PeerJ Comput Sci. 2020; 6: e251

[78] Flynn JM, Hubley R, Goubert C et al. RepeatModeler2 for automated genomic discovery of transposable element families. Proc Natl Acad Sci U S A. 2020; 117: 9451-7

[79] Tarailo-Graovac M, Chen N . Using RepeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioinformatics. 2009; 4: 4.10.1-14

[80] Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999; 27: 573-80

[81] Stanke M, Diekhans M, Baertsch R et al. Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics. 2008; 24: 637-44

[82] Holt C, Yandell M . MAKER2: an annotation pipeline and genome-database management tool for second-generation genome projects. BMC Bioinformatics. 2011; 12: 491

[83] Keilwagen J, Hartung F, Grau J . GeMoMa: homology-based gene prediction utilizing intron position conservation and RNA-seq data. Methods Mol Biol. 2019; 1962: 161-77

[84] Kim D, Pertea G, Trapnell C et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013; 14: R36

[85] Buchfink B, Reuter K, Drost HG . Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods. 2021; 18: 366-8

[86] Jones P, Binns D, Chang HY et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014; 30: 1236-40

[87] Kanehisa M, Sato Y, Morishima K . BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol. 2016; 428: 726-31

[88] Calle Garcia J, Guadagno A, Paytuvi-Gallart A et al. PRGdb 4.0: an updated database dedicated to genes involved in plant disease resistance process. Nucleic Acids Res. 2022; 50: D1483-90

[89] Goel M, Sun H, Jiao WB et al. SyRI: finding genomic rearrangements and local sequence differences from whole-genome assemblies. Genome Biol. 2019; 20: 277

[90] Cingolani P, Platts A, Wang LL et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w(1118); iso-2; iso-3. Fly. 2012; 6: 80-92

[91] Wang Y, Tang H, DeBarry JD et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012; 40: e49

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

[93] 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

[94] Purcell S, Neale B, Todd-Brown K et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007; 81: 559-75

[95] Dong SS, He WM, Ji JJ et al. LDBlockShow: a fast and convenient tool for visualizing linkage disequilibrium and haplotype blocks based on variant call format files. Brief Bioinform. 2021; 22: bbaa227