The first de novo transcriptome of pepino (Solanum muricatum): assembly, comprehensive analysis and comparison with the closely related species S. caripense, potato and tomato

[EN] Background Solanum sect. Basarthrum is phylogenetically very close to potatoes (Solanum sect. Petota) and tomatoes (Solanum sect. Lycopersicon), two groups with great economic importance, and for which Solanum sect. Basarthrum represents a tertiary gene pool for breeding. This section includes the important regional cultigen, the pepino (Solanum muricatum), and several wild species. Among the wild species, S. caripense is prominent due to its major involvement in the origin of pepino and its wide geographical distribution. Despite the value of the pepino as an emerging crop, and the potential for gene transfer from both the pepino and S. caripense to potatoes and tomatoes, there has been virtually no genomic study of these species. Results Using Illumina HiSeq 2000, RNA-Seq was performed with a pool of three tissues (young leaf, flowers in pre-anthesis and mature fruits) from S. muricatum and S. caripense, generating almost 111,000,000 reads among the two species. A high quality de novo transcriptome was assembled from S. muricatum clean reads resulting in 75,832 unigenes with an average length of 704 bp. These unigenes were functionally annotated based on similarity of public databases. We used Blast2GO, to conduct an exhaustive study of the gene ontology, including GO terms, EC numbers and KEGG pathways. Pepino unigenes were compared to both potato and tomato genomes in order to determine their estimated relative position, and to infer gene prediction models. Candidate genes related to traits of interest in other Solanaceae were evaluated by presence or absence and compared with S. caripense transcripts. In addition, by studying five genes, the phylogeny of pepino and five other members of the family, Solanaceae, were studied. The comparison of S. caripense reads against S. muricatum assembled transcripts resulted in thousands of intra- and interspecific nucleotide-level variants. In addition, more than 1000 SSRs were identified in the pepino transcriptome. Conclusions This study represents the first genomic resource for the pepino. We suggest that the data will be useful not only for improvement of the pepino, but also for potato and tomato breeding and gene transfer. The high quality of the transcriptome presented here also facilitates comparative studies in the genus Solanum. The accurate transcript annotation will enable us to figure out the gene function of particular traits of interest. The high number of markers (SSR and nucleotide-level variants) obtained will be useful for breeding programs, as well as studies of synteny, diversity evolution, and phylogeny.Herraiz García, FJ.; Blanca Postigo, JM.; Ziarsolo Areitioaurtena, P.; Gramazio, P.; Plazas Ávila, MDLO.; Anderson, GJ.; Prohens Tomás, J.... (2016). The first de novo transcriptome of pepino (Solanum muricatum): assembly, comprehensive analysis and comparison with the closely related species S. caripense, potato and tomato. BMC Genomics. 17(321). doi:10.1186/s12864-016-2656-817321Anderson GJ, Jansen RK, Kim Y. The origin and relationships of the pepino, Solanum muricatum (Solanaceae): DNA restriction fragment evidence. Econ Bot. 1996;50:369–80.Anderson GJ, Martine CT, Prohens J, Nuez F. Solanum perlongistylum and S. catilliflorum, new endemic Peruvian species of Solanum, Section Basarthrum, are close relatives of the domesticated pepino, S. muricatum. Novon. 2006;16:161–7.Rodríguez-Burruezo A, Prohens J, Fita AM. Breeding strategies for improving the performance and fruit quality of the pepino (Solanum muricatum): A model for the enhancement of underutilized exotic fruits. Food Res Int. 2011;44:1927–35.Yalçin H. Effect of ripening period on composition of pepino (Solanum muricatum) fruit grown in Turkey. Afr J Biotechnol. 2010;9:3901–3.Abouelnasr H, Li Y-Y, Zhang Z-Y, Liu J-Y, Li S-F, Li D-W, Yu J-L, McBeath JH, Han C-G. First Report of Potato Virus H on Solanum muricatum in China. Plant Dis. 2014;98:1016.Spooner DM, Anderson GJ, Jansen RK. Chloroplast DNA evidence for the interrelationships of tomatoes, potatoes, and pepinos (Solanaceae). Am J Bot. 1993;80:676–88.Sarkinen T, Bohs L, Olmstead RG, Knapp S. A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree. BMC Evol Biol. 2013;13:214.Nakitandwe J, Trognitz FCH, Trognitz BR. Genetic mapping of Solanum caripense, a wild relative of pepino dulce, tomato and potato, and a genetic resource for resistance to potato late blight. In: VI International Solanaceae Conference: Genomics Meets Biodiversity 745. 2006. p. 333–42.Sakomoto K, Taguchi T. Regeneration of intergeneric somatic hybrid plants between Lycopersicon esculentum and Solanum muricatum. Theor Appl Genet. 1991;81:509–13.Bernardello LM, Anderson GJ. Karyotypic studies in Solanum section Basarthrum (Solanaceae). Am J Bot. 1990;77:420–31.Arumuganathan K, Earle ED. Nuclear DNA content of some important plant species. Plant Mol Biol Report. 2004;9:208–18.Spooner DM, Rodríguez F, Polgár Z, Ballard HE, Jansky SH. Genomic origins of potato polyploids: GBSSI gene sequencing data. Crop Sci. 2008;48(Supplement to crop science):27–36.Herraiz FJ, Vilanova S, Andújar I, Torrent D, Plazas M, Gramazio P, Prohens J. Morphological and molecular characterization of local varieties, modern cultivars and wild relatives of an emerging vegetable crop, the pepino (Solanum muricatum), provides insight into its diversity, relationships and breeding history. Euphytica. 2015;206:301–18.Trognitz FC, Trognitz BR. Survey of resistance gene analogs in Solanum caripense, a relative of potato and tomato, and update on R gene genealogy. Mol Genet Genomics. 2005;274:595–605.Hajjar R, Hodgkin T. The use of wild relatives in crop improvement: a survey of developments over the last 20 years. Euphytica. 2007;156:1–13.Doebley JF, Gaut BS, Smith BD. The molecular genetics of crop domestication. Cell. 2006;127:1309–21.Blanca JM, Prohens J, Anderson GJ, Zuriaga E, Canizares J, Nuez F. AFLP and DNA sequence variation in an Andean domesticate, pepino (Solanum muricatum, Solanaceae): implications for evolution and domestication. Am J Bot. 2007;94:1219–29.Rodríguez-Burruezo A, Prohens J, Nuez F. Wild relatives can contribute to the improvement of fruit quality in pepino (Solanum muricatum). Euphytica. 2003;129:311–8.Herraiz FJ, Villaño D, Plazas M, Vilanova S, Ferreres F, Prohens J, Moreno DA. Phenolic profile and biological activities of the pepino (Solanum muricatum) fruit and its wild relative S. caripense. Int J Mol Sci. 2016;17:394.Leiva-Brondo M, Prohens J, Nuez F. Characterization of pepino accessions and hybrids resistant to Tomato mosaic virus (ToMV). J Food Agric Env. 2006;4:138.Nakitandwe J, Trognitz F, Trognitz B. Reliable allele detection using SNP-based PCR primers containing Locked Nucleic Acid: application in genetic mapping. Plant Methods. 2007;3:2.Andrivon D. The origin of Phytophthora infestans populations present in Europe in the 1840s: a critical review of historical and scientific evidence. Plant Pathol. 1996;45:1027–35.Prohens J, Ruiz JJ, Nuez F. The pepino (Solanum muricatum, Solanaceae): A “new” crop with a history. Econ Bot. 1996;50:355–68.Heiser CB. Origin and Variability of the Pepino (Solanum Muricatum). In: Preliminary Report. 1964.Ahmad H, Khan A, Muhammad K, Nadeem MS, Ahmad W, Iqbal S, Nosheen A, Akbar N, Ahmad I, Que Y. Morphogenetic study of pepino and other members of solanaceae family. Am J Plant Sci. 2014;5:3761.Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. 2013;8:1494–512.Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644–52.Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10:57–63.McKain MR, Wickett N, Zhang Y, Ayyampalayam S, McCombie WR, Chase MW, Pires JC, de Pamphilis CW, Leebens-Mack J. Phylogenomic analysis of transcriptome data elucidates co-occurrence of a paleopolyploid event and the origin of bimodal karyotypes in Agavoideae (Asparagaceae). Am J Bot. 2012;99:397–406.Barker MS, Vogel H, Schranz ME. Paleopolyploidy in the Brassicales: analyses of the Cleome transcriptome elucidate the history of genome duplications in Arabidopsis and other Brassicales. Genome Biol Evol. 2009;1:391–9.Rensink W, Lee Y, Liu J, Iobst S, Ouyang S, Buell CR. Comparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts. BMC Genomics. 2005;6:124.Koenig D, Jimenez-Gomez JM, Kimura S, Fulop D, Chitwood DH, Headland LR, Kumar R, Covington MF, Devisetty UK, Tat A V, Tohge T, Bolger A, Schneeberger K, Ossowski S, Lanz C, Xiong G, Taylor-Teeples M, Brady SM, Pauly M, Weigel D, Usadel B, Fernie AR, Peng J, Sinha NR, Maloof JN. Comparative transcriptomics reveals patterns of selection in domesticated and wild tomato. Proc Natl Acad Sci U S A. 2013;110:E2655–62.Blanca JM, Cañizares J, Ziarsolo P, Esteras C, Mir G, Nuez F, Garcia-Mas J, Picó MB. Melon transcriptome characterization: Simple sequence repeats and single nucleotide polymorphisms discovery for high throughput genotyping across the species. Plant Genome. 2011;4:118–31.Blanca J, Canizares J, Roig C, Ziarsolo P, Nuez F, Pico B. Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genomics. 2011;12:104.Howe GT, Yu J, Knaus B, Cronn R, Kolpak S, Dolan P, Lorenz WW, Dean JF. A SNP resource for Douglas-fir: de novo transcriptome assembly and SNP detection and validation. BMC Genomics. 2013;14:137.Consortium TG. The tomato genome sequence provides insights into fleshy fruit evolution. Nature. 2012;485:635–41.Potato Genome Sequencing Consortium. Genome sequence and analysis of the tuber crop potato. Nature. 2011;475:189–95.Anderson GJ, Jansen RK. Biosystematic and molecular systematic studies of Solanum section Basarthrum and the origin and relationships of the pepino (S. muricatum). In: Proceedings of the VI Congreso Latinoamericano de botanica: Mar del Plata, Argentina. 1994. p. 2–8.Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–402.Swiss Prot [ http://web.expasy.org/docs/swiss-prot_guideline.html ]. Accessed 29 Apr 2016.SGN release versionITAG2.4 [ ftp://ftp.sgn.cornell.edu/tomato_genome/annotation/ ]. Accessed 29 Apr 2016.Uniref [ http://www.ebi.ac.uk/uniprot/database/download.html ]. Accessed 29 Apr 2016.Wei D-D, Chen E-H, Ding T-B, Chen S-C, Dou W, Wang J-J. De novo assembly, gene annotation, and marker discovery in stored-product pest Liposcelis entomophila (Enderlein) using transcriptome sequences. PLoS One. 2013;8:e80046.Li D, Deng Z, Qin B, Liu X, Men Z. De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.). BMC Genomics. 2012;13:192.Lulin H, Xiao Y, Pei S, Wen T, Shangqin H. The first Illumina-based de novo transcriptome sequencing and analysis of safflower flowers. PLoS One. 2012;7:e38653.Mitraki A, Barge A, Chroboczek J, Andrieu JP, Gagnon J, Ruigrok RWH. Nomenclature committee of the international union of biochemistry and molecular biology (NC-IUBMB). Eur J Biochem. 1999;264:610–50.Sierro N, Battey JN, Ouadi S, Bovet L, Goepfert S, Bakaher N, Peitsch MC, Ivanov N V. Reference genomes and transcriptomes of Nicotiana sylvestris and Nicotiana tomentosiformis. Genome Biol. 2013;14:R60.Garzon-Martinez GA, Zhu ZI, Landsman D, Barrero LS, Marino-Ramirez L. The Physalis peruviana leaf transcriptome: assembly, annotation and gene model prediction. BMC Genomics. 2012;13:151.Wang L, Li J, Zhao J, He C. Evolutionary developmental genetics of fruit morphological variation within the Solanaceae. Front Plant Sci. 2015;6:248.Iseli C, Jongeneel CV, Bucher P. ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. Proc Int Conf Intell Syst Mol Biol. 1999;99:138–48.Peralta IE, Spooner DM. Granule-bound starch synthase (GBSSI) gene phylogeny of wild tomatoes (Solanum L. section Lycopersicon [Mill.] Wettst. subsection Lycopersicon). Am J Bot. 2001;88:1888–902.Martins TR, Barkman TJ, Smith JF. Reconstruction of Solanaceae phylogeny using the nuclear gene SAMT. Syst Bot. 2005;30:435–47.Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013;30:2725–9.Wang Y, Diehl A, Wu F, Vrebalov J, Giovannoni J, Siepel A, Tanksley SD. Sequencing and comparative analysis of a conserved syntenic segment in the Solanaceae. Genetics. 2008;180:391–408.Garrison E. FreeBayes. In: Marth Lab. 2010.Collins DW, Jukes TH. Rates of transition and transversion in coding sequences since the human-rodent divergence. Genomics. 1994;20:386–96.Xie F, Burklew CE, Yang Y, Liu M, Xiao P, Zhang B, Qiu D. De novo sequencing and a comprehensive analysis of purple sweet potato (Ipomoea batatas L.) transcriptome. Planta. 2012;236:101–13.Mooers AØ, Holmes EC. The evolution of base composition and phylogenetic inference. Trends Ecol Evol. 2000;15:365–9.Aoki K, Yano K, Suzuki A, Kawamura S, Sakurai N, Suda K, Kurabayashi A, Suzuki T, Tsugane T, Watanabe M, Ooga K, Torii M, Narita T, Shin-I T, Kohara Y, Yamamoto N, Takahashi H, Watanabe Y, Egusa M, Kodama M, Ichinose Y, Kikuchi M, Fukushima S, Okabe A, Arie T, Sato Y, Yazawa K, Satoh S, Omura T, Ezura H, et al. Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics. BMC Genomics. 2010;11:210.Crookshanks M, Emmersen J, Welinder KG, Nielsen KL. The potato tuber transcriptome: analysis of 6077 expressed sequence tags. FEBS Lett. 2001;506:123–6.Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30.Lester RN. Evolutionary relationships of tomato, potato, pepino, and wild species of Lycopersicon and Solanum. In: Hawkes JG, Lester RN, Nee M, Estrad N, editors. Solanaceae III Taxonomy, Chem Evol Kew Linn Soc London. 1991. p. 283–301.Butelli E, Titta L, Giorgio M, Mock H-P, Matros A, Peterek S, Schijlen EGWM, Hall RD, Bovy AG, Luo J, Martin C. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotech. 2008;26:1301–8.Clé C, Hill LM, Niggeweg R, Martin CR, Guisez Y, Prinsen E, Jansen MAK. Modulation of chlorogenic acid biosynthesis in Solanum lycopersicum; consequences for phenolic accumulation and UV-tolerance. Phytochemistry. 2008;69:2149–56.Niggeweg R, Michael AJ, Martin C. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat Biotechnol. 2004;22:746–54.Prohens J, Sánchez MC, Rodríguez-Burruezo A, Cámara M, Torija E, Nuez F. Morphological and physico-chemical characteristics of fruits of pepino (Solanum muricatum), wild relatives (S. caripense and S. tabanoense) and interspecific hybrids. Implications in pepino breeding. Eur J Hortic Sci. 2005;70:224.Blanca J, Montero-Pau J, Sauvage C, Bauchet G, Illa E, D’iez MJ, Francis D, Causse M, van der Knaap E, Cañizares J. Genomic variation in tomato, from wild ancestors to contemporary breeding accessions. BMC Genomics. 2015;16:1–19.Rong J, Lammers Y, Strasburg JL, Schidlo NS, Ariyurek Y, de Jong TJ, Klinkhamer PGL, Smulders MJM, Vrieling K. New insights into domestication of carrot from root transcriptome analyses. BMC Genomics. 2014;15:895.Swanson-Wagner R, Briskine R, Schaefer R, Hufford MB, Ross-Ibarra J, Myers CL, Tiffin P, Springer NM. Reshaping of the maize transcriptome by domestication. Proc Natl Acad Sci. 2012;109(29):11878–83.Feng Z, Zhang B, Ding W, Liu X, Yang D-L, Wei P, Cao F, Zhu S, Zhang F, Mao Y. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. 2013;23:1229–32.Park T, Vleeshouwers V, Jacobsen E, Van Der Vossen E, Visser RGF. Molecular breeding for resistance to Phytophthora infestans (Mont.) de Bary in potato (Solanum tuberosum L.): a perspective of cisgenesis. Plant Breed. 2009;128:109–17.Hedges SB, Dudley J, Kumar S. TimeTree: a public knowledge-base of divergence times among organisms. Bioinformatics. 2006;22:2971–2.Zhai L, Xu L, Wang Y, Cheng H, Chen Y, Gong Y, Liu L. Novel and useful genic-SSR markers from de novo transcriptome sequencing of radish (Raphanus sativus L.). Mol Breed. 2014;33:611–24.Ahn Y-K, Tripathi S, Kim J-H, Cho Y-I, Lee H-E, Kim D-S, Woo J-G, Yoon M-K. Microsatellite marker information from high-throughput next-generation sequence data of Capsicum annuum varieties Mandarin and Blackcluster. Sci Hortic. 2014;170:123–30.Metzgar D, Bytof J, Wills C. Selection against frameshift mutations limits microsatellite expansion in coding DNA. Genome Res. 2000;10:72–80.Li Y, Korol AB, Fahima T, Beiles A, Nevo E. Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review. Mol Ecol. 2002;11:2453–65.Varshney RK, Graner A, Sorrells ME. Genic microsatellite markers in plants: features and applications. Trends Biotechnol. 2005;23:48–55.Anderson GJ. The variation and evolution of selected species of Solanum section Basarthrum. Brittonia. 1975;27:209–22.Murray BG, Hammett KRW, Grigg FDW. Seed set and breeding system in the pepino Solanum muricatum Ait., Solanaceae. Sci Hortic (Amsterdam). 1992;49:83–92.Perez-de-Castro AM, Vilanova S, Canizares J, Pascual L, Blanca JM, Diez MJ, Prohens J, Pico B. Application of genomic tools in plant breeding. Curr Genomics. 2012;13:179–95.Ruiz JJ, Prohens J, Nuez F. “Sweet Round” and “Sweet Long”: Two pepino cultivars for Mediterranean, climates. HortSci. 1997;32:751–2.FASTAQC [ http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ]. Accessed 29 Apr 2016.Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9.Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323.Blanca JM, Pascual L, Ziarsolo P, Nuez F, Cañizares J. ngs_backbone: a pipeline for read cleaning, mapping and SNP calling using Next Generation Sequence. BMC Genomics. 2011;12:1–8.Conesa A, Gotz S. Blast2GO: A comprehensive suite for functional analysis in plant genomics. Int J Plant Genomics. 2008;2008:619832.Lippman ZB, Cohen O, Alvarez JP, Abu-Abied M, Pekker I, Paran I, Eshed Y, Zamir D. The making of a compound inflorescence in tomato and related nightshades. PLoS Biol. 2008;6:e288.Zhang Y, Hu Z, Chu G, Huang C, Tian S, Zhao Z, Chen G. Anthocyanin accumulation and molecular analysis of anthocyanin biosynthesis-associated genes in eggplant (Solanum melongena L.). J Agric Food Chem. 2014;62:2906–12.Kohara A, Nakajima C, Hashimoto K, Ikenaga T, Tanaka H, Shoyama Y, Yoshida S, Muranaka T. A novel glucosyltransferase involved in steroid saponin biosynthesis in Solanum aculeatissimum. Plant Mol Biol. 2005;57:225–39.Gramazio P, Prohens J, Plazas M, Andujar I, Herraiz FJ, Castillo E, Knapp S, Meyer RS, Vilanova S. Location of chlorogenic acid biosynthesis pathway and polyphenol oxidase genes in a new interspecific anchored linkage map of eggplant. BMC Plant Biol. 2014;14:350–014–0350–z.Klann E, Yelle S, Bennett AB. Tomato fruit Acid invertase complementary DNA: nucleotide and deduced amino Acid sequences. Plant Physiol. 1992;99:351–3.Lam Cheng KL. Golden2--like (GLK2) Transcription Factor: Developmental Control of Tomato Fruit Photosynthesis and Its Contribution to Ripe Fruit Characteristics. Davis: University of California; 2013.Mott R. EST_GENOME: A program to align spliced DNA sequences to unspliced genomic DNA. Comput Appl Biosci. 1997;13:477–8.EMBOSS [ http://www.bioinformatics.nl/emboss-explorer/ ]. Accessed 29 Apr 2016.Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19:1639–45.Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–8.Abajian C. Sputnik. University of Washington Department of Molecular Biotechnology. 1994.[ http://wheat.pw.usda.gov/ITMI/EST-SSR/LaRota ]. Accessed 29 Apr 2016

GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology

[EN] Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.This work was supported by the Spanish Ministry of Economy and Competitiveness (grant no. BIO2010-15384), by a Research Personnel in Training fellowship to A.S.-P., and by a Junta de Ampliacion de Estudios fellowship to M.V.-V.Sarrion-Perdigones, A.; Vázquez Vilar, M.; Palací Bataller, J.; Castelijns, B.; Forment Millet, JJ.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2013). GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiology. 162(3):1618-1631. https://doi.org/10.1104/pp.113.217661S16181631162

Transcriptome sequencing for SNP discovery across Cucumis melo

Background: Melon (Cucumis melo L.) is a highly diverse species that is cultivated worldwide. Recent advances in massively parallel sequencing have begun to allow the study of nucleotide diversity in this species. The Sanger method combined with medium-throughput 454 technology were used in a previous study to analyze the genetic diversity of germplasm representing 3 botanical varieties, yielding a collection of about 40,000 SNPs distributed in 14,000 unigenes. However, the usefulness of this resource is limited as the sequenced genotypes do not represent the whole diversity of the species, which is divided into two subspecies with many botanical varieties variable in plant, flowering, and fruit traits, as well as in stress response. As a first step to extensively document levels and patterns of nucleotide variability across the species, we used the high-throughput SOLiD¿ system to resequence the transcriptomes of a set of 67 genotypes that had previously been selected from a core collection representing the extant variation of the entire species.Results: The deep transcriptome resequencing of all of the genotypes, grouped into 8 pools (wild African agrestis, Asian agrestis and acidulus, exotic Far Eastern conomon, Indian momordica and Asian dudaim and flexuosus, commercial cantalupensis, subsp. melo Asian and European landraces, Spanish inodorus landraces, and Piel de Sapo breeding lines) yielded about 300 M reads. Short reads were mapped to the recently generated draft genome assembly of the DHL line Piel de Sapo (inodorus) x Songwhan Charmi (conomon) and to a new version of melon transcriptome. Regions with at least 6X coverage were used in SNV calling, generating a melon collection with 303,883 variants. These SNVs were dispersed across the entire C. melo genome, and distributed in 15,064 annotated genes. The number and variability of in silico SNVs differed considerably between pools. Our finding of higher genomic diversity in wild and exotic agrestis melons from India and Africa as compared to commercial cultivars, cultigens and landraces from Eastern Europe, Western Asia and the Mediterranean basin is consistent with the evolutionary history proposed for the species. Group-specific SNVs that will be useful in introgression programs were also detected. In a sample of 143 selected putative SNPs, we verified 93% of the polymorphisms in a panel of 78 genotypes.Conclusions: This study provides the first comprehensive resequencing data for wild, exotic, and cultivated (landraces and commercial) melon transcriptomes, yielding the largest melon SNP collection available to date and representing a notable sample of the species diversity. This data provides a valuable resource for creating a catalog of allelic variants of melon genes and it will aid in future in-depth studies of population genetics, marker-assisted breeding, and gene identification aimed at developing improved varieties. © 2012 Blanca et al.; licensee BioMed Central Ltd.This project was carried out in the frame of the MELONOMICS project (2009-2012) of the Fundacion Genoma Espana.Blanca Postigo, JM.; Esteras Gómez, C.; Ziarsolo Areitioaurtena, P.; Perez, D.; Fernández-Pedrosa, V.; Collado, C.; Rodríguez De Pablos, R.... (2012). Transcriptome sequencing for SNP discovery across Cucumis melo. BMC Genomics. 13(280):1-18. doi:10.1186/1471-2164-13-280S1181328

ABCC transporters mediate insect resistance to multiple Bt toxins revealed by bulk segregant analysis

[EN] Background: Relatively recent evidence indicates that ABCC2 transporters play a main role in the mode of action of Bacillus thuringiensis (Bt) Cry1A-type proteins. Mapping of major Cry1A resistance genes has linked resistance to the ABCC2 locus in Heliothis virescens, Plutella xylostella, Trichoplusia ni and Bombyx mori, and mutations in this gene have been found in three of these Bt-resistant strains. Results: We have used a colony of Spodoptera exigua (Xen-R) highly resistant to a Bt commercial bioinsecticide to identify regions in the S. exigua genome containing loci for major resistance genes by using bulk segregant analysis (BSA). Results reveal a region containing three genes from the ABCC family (ABBC1, ABBC2 and ABBC3) and a mutation in one of them (ABBC2) as responsible for the resistance of S. exigua to the Bt commercial product and to its key Spodoptera-active ingredients, Cry1Ca. In contrast to all previously described mutations in ABCC2 genes that directly or indirectly affect the extracellular domains of the membrane protein, the ABCC2 mutation found in S. exigua affects an intracellular domain involved in ATP binding. Functional analyses of ABBC2 and ABBC3 support the role of both proteins in the mode of action of Bt toxins in S. exigua. Partial silencing of these genes with dsRNA decreased the susceptibility of wild type larvae to both Cry1Ac and Cry1Ca. In addition, reduction of ABBC2 and ABBC3 expression negatively affected some fitness components and induced up-regulation of arylphorin and repat5, genes that respond to Bt intoxication and that are found constitutively up-regulated in the Xen-R strain. Conclusions: The current results show the involvement of different members of the ABCC family in the mode of action of B. thuringiensis proteins and expand the role of the ABCC2 transporter in B. thuringiensis resistance beyond the Cry1A family of proteins to include Cry1Ca.We want to thank C. S. Hern ndez-Rodr guez for her comments on binding assays, Ismael Mingarro for his help in determination of ABCC domains and William Moar (Auburn University, Auburn, AL) for his comments on the manuscript and generating the Xen-R colony. This research was partially supported by IPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry of Agriculture, Food and Rural Affairs to YK. Research at the University of Valencia was supported by Generalitat Valenciana (prometeo/2011/044) and Ministry of Science and Innovation (AGL2011-30352-C02-02 and AGL2012-39946-C02-01).Park, Y.; González Martínez, RM.; Navarro Cerrillo, G.; Chakroun, M.; Kim, Y.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2014). ABCC transporters mediate insect resistance to multiple Bt toxins revealed by bulk segregant analysis. BMC Biology. 12(46):1-15. https://doi.org/10.1186/1741-7007-12-46S115124

La tomata ‘Valenciana’ del Perelló: comparació de les seues característiques genètiques i fenotípiques amb el conjunt de tomates tradicionals europees.

[CA] La tomata ‘Valenciana del Perelló’, originaria de l’horta de València, representa una de les més importants i apreciades varietats tradicional valencianes de consum en fresc. A pesar d’açò manquem d’una tipificació i caracterització en profunditat, el que dificulta identificar de manera especifica i objectiva característiques distintives d’aquest tipus de tomata tant especial. En aquest treball comparem les característiques genètiques i fenotípiques de la tomata la ‘Valenciana’ i, concretament la ‘Valenciana del Perelló’ amb un conjunt de mes de 1000 varietats de tomates tradicionals Europees amb la finalitat de proporcionar les bases per distingir la tomata ‘Valenciana’ del Perelló’ d’aquelles pertanyents a altres varietats tradicionals anomenades també valencianes o aquelles que són similars dins del conjunt tradicional europeu. La caracterització morfològica, basada en 10 caràcters morfològics i qualitatius ens indica que encara que hi ha diferències poblacionals, no són suficients per discriminar entre tipus varietals. El genotipat de les varietats tradicionals europees revela una estructura genètica ben definida per a la varietat de tomata ‘Valenciana del Perelló’ i respecte a altres tomates valencianes o tomates europees de característiques similars. En particular, 18 variants de seqüència SNPs en llocs específics del genoma de la tomata són suficients per distingir clarament la tomata ‘Valenciana’ tipus ‘ el Perelló’ de les altres varietats de tomata ‘Valenciana’ i la tomata ‘Valenciana’ del conjunt de tomata tradicional europeu. Tenint en compte açò, els nostres resultats proporcionen l’empremta genètica de la tomata ‘Valenciana del Perelló’, punt de partida per a la valorització d’aquesta varietat local i per a la seua utilització en programes de millora o el seu us certificat en mercats de productes d’alta qualitat.[EN] The ‘Valenciana d’El Perelló’ tomato, originating from the Spanish region l’Horta de València, represents one of the most important and appreciated Valencian landraces for fresh market. Despite this, we still lack a detailed typification and characterization of this variety which is a prerequisite for identifying specific and objective distinctive characteristics of this type of tomato. In this work we compared genetic and phenotypic traits of the tomato variety ‘Valenciana’, in particular the ‘Valenciana d’El Perelló’, against more than 1000 traditional varieties in order to provide the basis to distinguish the tomato ‘Valenciana d’El Perelló’ from a number of other landraces named ‘Valenciana’ or to those similar within the traditional European tomato collection. Morphological characterization, based in 10 fruit morphological and qualitative traits indicated that despite differences between populations, these traits do not discriminate varietal types. The genotyping of European traditional varieties reveal a well defined genetic structure of ‘Valenciana d’El Perelló’ with respect to other Valencian or European tomatoes with similar characteristics. In particular, 18 sequence SNP variants are sufficient to distinguish clearly the tomato ‘Valenciana d’El Perelló’ type from other ‘Valenciana’ varieties and the ‘Valenciana’ variety from the rest of European traditional tomato. Taking this into account, our results provide the ‘Valenciana d’El Perelló’ tomato genetic fingerprint, starting point for the valorization of this landrace and for its use in breeding programs or its certified use in high quality markets.Aquest projecte ha rebut financiació del programa Horizon 2020 de la Unió Europea a través del projecte No 634561 [This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 634561].Pons Puig, C.; Monforte Gilabert, AJ.; Figás Moreno, MDR.; Soler Aleixandre, S.; Blanca Postigo, JM.; Ziarsolo Areitioaurtena, P.; Cañizares Sales, J.... (2020). La tomata ‘Valenciana’ del Perelló: comparació de les seues característiques genètiques i fenotípiques amb el conjunt de tomates tradicionals europees. En I Congrés de la Tomaca Valenciana: La Tomaca Valenciana d'El Perelló. Editorial Universitat Politècnica de València. 141-152. https://doi.org/10.4995/TOMAVAL2017.2017.6196OCS14115

Analysis of the coding-complete genomic sequence of groundnut ringspot virus suggests a common ancestor with tomato chlorotic spot virus

[EN] Groundnut ringspot virus (GRSV) and tomato chlorotic spot virus (TCSV) share biological and serological properties, so their identification is carried out by molecular methods. Their genomes consist of three segmented RNAs: L, M and S. The finding of a reassortant between these two viruses may complicate correct virus identification and requires the characterization of the complete genome. Therefore, we present for the first time the complete sequences of all the genes encoded by a GRSV isolate. The high level of sequence similarity between GRSV and TCSV (over 90 % identity) observed in the genes and proteins encoded in the M RNA support previous results indicating that these viruses probably have a common ancestor.This work was supported by Fundacion Mani Argentino and the PNIND PE 1108072 project of Instituto Nacional de Tecnologia Agropecuaria (INTA).De Breuil, S.; Cañizares Sales, J.; Blanca Postigo, JM.; Bejerman, N.; Trucco, V.; Giolitti, F.; Ziarsolo Areitioaurtena, P.... (2016). Analysis of the coding-complete genomic sequence of groundnut ringspot virus suggests a common ancestor with tomato chlorotic spot virus. Archives of Virology. 161(8):2311-2316. https://doi.org/10.1007/s00705-016-2912-xS231123161618Almeida MMS, Orílio AF, Melo FL, Rodriguez R, Feliz A, Cayetano X, Martínez RT, Resende RO (2014) The first report of tomato chlorotic spot virus (TCSV) infecting long beans and chili peppers in the dominican republic. Plant Dis 98:1285Ananthakrishnan TN, Annadurai RS (2007) Thrips–tospovirus interactions: biological and molecular implications. Curr Sci 92:1083–1086Blanca JM, Pascual L, Ziarsolo P, Nuez F, Cañizares J (2011) ngs_backbone: a pipeline for read cleaning, mapping and snp calling using next generation sequence. BMC Genom 12:285Boari AJ, Maciel-Zambolim E, Lau DD, Lima GSA, Kitajima EW, Brommonschenkel SH, Zerbini FM (2002) Detection and partial characterization of an isolate of groundnut ringspot virus in Solanum sessiliflorum. Fitopatol Bras 27:249–253Briese T, Calisher CH, Higgs S (2013) Viruses of the family Bunyaviridae: are all available isolates reassortants? Virology 446:207–216Hagen C, Frizzi A, Kao J, Jia L, Huang M, Zhang Y, Huang S (2011) Using small RNA sequences to diagnose, sequence, and investigate the infectivity characteristics of vegetable-infecting viruses. Arch Virol 156:1209–1216Hogenhout SA, Ammar E-D, Whitfield AE, Redinbaugh MG (2008) Insect vector interactions with persistently transmitted viruses. Annu Rev Phytopathol 46:327–359Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388:1–7Law MD, Moyer JW (1990) A tomato spotted wilt-like virus with a serologically distinct N protein. J Gen Virol 71:933–938Lewandowskia DJ, Adkins S (2005) The tubule-forming NSm protein from tomato spotted wilt virus complements cell-to-cell and long-distance movement of Tobacco mosaic virus hybrids. Virology 342:26–37Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth GT, Abecasis GR, Durbin R (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079Lian S, Lee JS, Cho WK, Yu J, Kim MK, Choi HS, Kim KH (2013) Phylogeneticand recombination analysis of tomato spotted wilt virus. PLOS ONE 8:e63380. doi: 10.1371/journal.pone.0063380Londoño A, Capobianco H, Zhang S, Polston JE (2012) First record of tomato chlorotic spot virus in the USA. Trop Plant Pathol 37:333–338Lovato FA, Nagata T, de Oliveira Resende R, de Avila AC, Inoue-Nagata AK (2004) Sequence analysis of the glycoproteins of tomato chlorotic spot virus and groundnut ringspot virus and comparison with other tospoviruses. Virus Genes 29:321–328Margaria P, Ciuffo M, Rosa C, Turina M (2015) Evidence of a tomato spotted wilt virus resistance-breaking strain originated through natural reassortment between two evolutionary-distinct isolates. Virus Res 196:157–161Milne I, Stephen G, Bayer M, Cock PJA, Pritchard L, Cardle L, Shaw PD, Marshall D (2013) Using tablet for visual exploration of second-generation sequencing data. Brief Bioinform 14:193–202Pappu HR, Jones RAC, Jain RK (2009) Global status of tospovirus epidemics in diverse cropping systems: successes achieved and challenges ahead. Virus Res 141:219–236Plyusnin A, Beaty BJ, Elliott RM, Goldbach R, Kormelink R, Lundkvist A, Schmaljohn CS, Tesh RB (2012) Family Bunyaviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz (eds) Virus taxonomy: ninth report of the international committee on taxonomy of viruses. Elsevier Inc, London, pp 725–741Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborSilva MS, Martins CRF, Bezerra IC, Nagata T, de Ávila AC, Resende RO (2001) Sequence diversity of NSm movement proteins of tospoviruses. Arch Virol 146:1267–1281Sin SH, McNulty BC, Kennedy GG, Moyer JW (2005) Viral genetic determinants for thrips transmission of Tomato spotted wilt virus. Proc Natl Acad Sci USA 102:5168–5173Soellick T-R, Uhrig JF, Bucher GL, Kellmann J-W, Schreier PH (2000) The movement protein NSm of tomato spotted wilt topovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting proteins. Proc Natl Acad Sci USA 97:2373–2378Sundaraj S, Srinivasan R, Culbreath AK, Riley DG, Pappu HR (2014) Host plant resistance against Tomato spotted wilt virus in peanut (Arachis hypogaea) and its impact on susceptibility to the virus, virus population genetics, and vector feeding behavior and survival. Phytopathology 104:202–210Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729Tentchev D, Verdin E, Marchal C, Jacquet M, Aguilar JM, Moury B (2011) Evolution and structure of tomato spotted wilt virus populations: evidence of extensive reassortment and insights into emergence processes. J Gen Virol 92:961–973Timmerman-Vaughan GM, Lister R, Cooper R, Tang J (2014) Phylogenetic analysis of New Zealand tomato spotted wilt virus isolates suggests likely incursion history scenarios and mechanisms for population evolution. Arch Virol 159:993–1003Tsompana M, Moyer JW (2008) Tospoviruses. In: Mahy BWJ, Van Regenmortel MHV (eds) Encyclopedia of virology, vol 5, 3rd edn. Elsevier Ltd, Oxford, pp 157–162Webster CG, Reitz SR, Perry KL, Adkins S (2011) A natural M RNA reassortant arising from two species of plant- and insect-infecting bunyaviruses and comparison of its sequence and biological properties to parental species. Virology 413:216–225Webster CG, Frantz G, Reitz SR, Funderburk JE, Mellinger HC, McAvoy E, Turechek WW, Marshall SH, Tantiwanich Y, McGrath MT, Daughtrey ML, Adkins S (2015) Emergence of groundnut ringspot virus and tomato chlorotic spot virus in vegetables in Florida and the Southeastern United States. Phytopatology 105:388–398Wu Q, Luo Y, Lu R, Lau N, Lai EC, Li W-X, Ding S-W (2010) Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs. Proc Natl Acad Sci USA 107:1606–1611Zerbino DR, Birney E (2008) Velvet: algorithms for de novo read assembly using de Bruijn graphs. Genome Res 18:821–82

Transcriptome analysis and molecular marker discovery in Solanum incanum and S. aethiopicum, two close relatives of the common eggplant (Solanum melongena) with interest for breeding

[EN] Background: Solanum incanum is a close wild relative of S. melongena with high contents of bioactive phenolics and drought tolerance. S. aethiopicum is a cultivated African eggplant cross-compatible with S. melongena. Despite their great interest in S. melongena breeding programs, the genomic resources for these species are scarce. Results: RNA-Seq was performed with NGS from pooled RNA of young leaf, floral bud and young fruit tissues, generating more than one hundred millions raw reads per species. The transcriptomes were assembled in 83,905 unigenes for S. incanum and in 87,084 unigenes for S. aethiopicum with an average length of 696 and 722 bp, respectively. The unigenes were structurally and functionally annotated based on comparison with public databases by using bioinformatic tools. The single nucleotide variant calling analysis (SNPs and INDELs) was performed by mapping our S. incanum and S. aethiopicum reads, as well as reads from S. melongena and S. torvum available on NCBI database (National Center for Biotechnology Information), against the eggplant genome. Both intraspecific and interspecific polymorphisms were identified and subsets of molecular markers were created for all species combinations. 36 SNVs were selected for validation in the S. incanum and S. aethiopicum accessions and 96 % were correctly amplified confirming the polymorphisms. In addition, 976 and 1,278 SSRs were identified in S. incanum and S. aethiopicum transcriptomes respectively, and a set of them were validated. Conclusions: This work provides a broad insight into gene sequences and allelic variation in S. incanum and S. aethiopicum. This work is a first step toward better understanding of target genes involved in metabolic pathways relevant for eggplant breeding. The molecular markers detected in this study could be used across all the eggplant genepool, which is of interest for breeding programs as well as to perform marker-trait association and QTL analysis studies.This work has been partially funded by Spanish Ministerio de Economia y Competitividad and FEDER (grant AGL2015-64755-450 R).Gramazio, P.; Blanca Postigo, JM.; Ziarsolo Areitioaurtena, P.; Herraiz García, FJ.; Plazas Ávila, MDLO.; Prohens Tomás, J.; Vilanova Navarro, S. (2016). Transcriptome analysis and molecular marker discovery in Solanum incanum and S. aethiopicum, two close relatives of the common eggplant (Solanum melongena) with interest for breeding. BMC Genomics. 17(300). doi:10.1186/s12864-016-2631-4S1730

Melon transcriptome characterization: Simple Sequence Repeats and Single Nucleotide Polymorphisms discovery for high throughput genotyping across the species

Melon (Cucumis melo L.) ranks among the highest-valued fruit crops worldwide. Some genomic tools are available for this crop, including a Sanger transcriptome. We report the generation of 689,054 C. melo high-quality expressed sequence tags (ESTs) from two 454 sequencing runs, using normalized and nonnormalized complementary DNA (cDNA) libraries prepared from four genotypes belonging to the two C. melo subspecies and the main commercial types. 454 ESTs were combined with the Sanger available ESTs and de novo assembled into 53,252 unigenes. Over 63% of the unigenes were functionally annotated with Gene Ontology (GO) terms and 21% had known orthologs of Arabidopsis thaliana (L.) Heynh. Annotation distribution followed similar tendencies than that reported for Arabidopsis thaliana, suggesting that the dataset represents a fairly complete melon transcriptome. Furthermore, we identified a set of 3298 unigenes with microsatellite motifs and 14,417 sequences with single nucleotide variants of which 11,655 single nucleotide polymorphism met criteria for use with high-throughput genotyping platforms, and 453 could be detected as cleaved amplified polymorphic sequence (CAPS). A set of markers were validated, 90% of them being polymorphic in a number of variable C. melo accessions. This transcriptome provides an invaluable new tool for biological research, more so when it includes transcripts not described previously. It is being used for genome annotation and has provided a large collection of markers that will allow speeding up the process of breeding new melon varieties.Blanca Postigo, JM.; Cañizares Sales, J.; Ziarsolo Areitioaurtena, P.; Esteras Gómez, C.; Mir, G.; Nuez Viñals, F.; Garcia-Mas, J.... (2011). Melon transcriptome characterization: Simple Sequence Repeats and Single Nucleotide Polymorphisms discovery for high throughput genotyping across the species. Plant Genome, The. 4(2):118-131. doi:10.3835/plantgenome2011.01.0003S1181314

The genome of melon (Cucumis melo L.)

We report the genome sequence of melon, an important horticultural crop worldwide. We assembled 375 Mb of the double-haploid line DHL92, representing 83.3%of the estimatedmelon genome.We predicted 27,427 protein-coding genes, which we analyzed by reconstructing 22,218 phylogenetic trees, allowing mapping of the orthology and paralogy relationships of sequenced plant genomes. We observed the absence of recent whole-genome duplications in the melon lineage since the ancient eudicot triplication, and our data suggest that transposon amplification may in part explain the increased size of the melon genome compared with the close relative cucumber. A low number of nucleotide-binding site leucinerich repeat disease resistance genes were annotated, suggesting the existence of specific defense mechanisms in this species. The DHL92 genome was compared with that of its parental lines allowing the quantification of sequence variability in the species. The use of the genome sequence in future investigations will facilitate the understanding of evolution of cucurbits and the improvement of breeding strategies.We thank Marc Oliver (Syngenta) for the recombinant inbred line genetic map. The cucumber Gy14 genome was produced by the Joint Genome Institute (http://www.jgi.doe.gov/). We acknowledge funding from Fundacion Genoma Espana; Semillas Fito; Syngenta Seeds; the governments of Catalunya, Andalucia, Madrid, Castilla-La Mancha, and Murcia; Savia Biotech; Roche Diagnostics; and Sistemas Genomicos. P. P. and J.G.-M. were funded by the Spanish Ministry of Science and Innovation (CSD2007-00036) and the Xarxa de Referencia d'R+D+I en Biotecnologia (Generalitat de Catalunya). R. G. and A.N. acknowledge the Spanish National Bioinformatics Institute for funding. T.M.-B. is supported by European Research Council Starting Grant StG_20091118.Garcia-Mas, J.; Benjak, A.; Sanseverino, W.; Bourgeois, M.; Mir, G.; Gonzalez, VM.; Henaff, E.... (2012). The genome of melon (Cucumis melo L.). Proceedings of the National Academy of Sciences. 109(29):11872-11877. https://doi.org/10.1073/pnas.1205415109S11872118771092