First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding.Financial support was provided by the Spanish Project INIA (Instituto Nacional de Investigacion y Tecnologia Agraria y Almentaria) RTA2011-00044C02-01, the ANR MELODY (ANR-11-BSV7-0024), the European Research Council (ERCSEXYPARTH), FEDER, and FSE funds. NVD has been awarded a grant by the Andalusian Institute of Agronomy Research IFAPA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Vicente-Dolera, N.; Troadec, C.; Moya, M.; Río-Celestino, MD.; Pomares-Viciana, T.; Bendahmane, A.; Picó Sirvent, MB.... (2014). First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement. PLoS ONE. 9(11):112743-112743. https://doi.org/10.1371/journal.pone.0112743S112743112743911Paris, H. S., Yonash, N., Portnoy, V., Mozes-Daube, N., Tzuri, G., & Katzir, N. (2002). Assessment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using DNA markers. Theoretical and Applied Genetics, 106(6), 971-978. doi:10.1007/s00122-002-1157-0Parry, M. A. J., Madgwick, P. J., Bayon, C., Tearall, K., Hernandez-Lopez, A., Baudo, M., … Phillips, A. L. (2009). Mutation discovery for crop improvement. Journal of Experimental Botany, 60(10), 2817-2825. doi:10.1093/jxb/erp189Gilchrist, E., & Haughn, G. (2010). Reverse genetics techniques: engineering loss and gain of gene function in plants. Briefings in Functional Genomics, 9(2), 103-110. doi:10.1093/bfgp/elp059McCallum, C. M., Comai, L., Greene, E. A., & Henikoff, S. (2000). Targeting Induced LocalLesions IN Genomes (TILLING) for Plant Functional Genomics. Plant Physiology, 123(2), 439-442. doi:10.1104/pp.123.2.439Colbert, T., Till, B. J., Tompa, R., Reynolds, S., Steine, M. N., Yeung, A. T., … Henikoff, S. (2001). High-Throughput Screening for Induced Point Mutations. Plant Physiology, 126(2), 480-484. doi:10.1104/pp.126.2.480Wang, T. L., Uauy, C., Robson, F., & Till, B. (2012). TILLINGin extremis. Plant Biotechnology Journal, 10(7), 761-772. doi:10.1111/j.1467-7652.2012.00708.xDong, C., Dalton-Morgan, J., Vincent, K., & Sharp, P. (2009). A Modified TILLING Method for Wheat Breeding. The Plant Genome Journal, 2(1), 39. doi:10.3835/plantgenome2008.10.0012Uauy, C., Paraiso, F., Colasuonno, P., Tran, R. K., Tsai, H., Berardi, S., … Dubcovsky, J. (2009). A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biology, 9(1), 115. doi:10.1186/1471-2229-9-115Kumar, A. P., Boualem, A., Bhattacharya, A., Parikh, S., Desai, N., Zambelli, A., … Bendahmane, A. (2013). SMART -- Sunflower Mutant population And Reverse genetic Tool for crop improvement. BMC Plant Biology, 13(1), 38. doi:10.1186/1471-2229-13-38Kurowska, M., Daszkowska-Golec, A., Gruszka, D., Marzec, M., Szurman, M., Szarejko, I., & Maluszynski, M. (2011). TILLING - a shortcut in functional genomics. Journal of Applied Genetics, 52(4), 371-390. doi:10.1007/s13353-011-0061-1Rigola, D., van Oeveren, J., Janssen, A., Bonné, A., Schneiders, H., van der Poel, H. J. A., … van Eijk, M. J. T. (2009). High-Throughput Detection of Induced Mutations and Natural Variation Using KeyPoint™ Technology. PLoS ONE, 4(3), e4761. doi:10.1371/journal.pone.0004761González, M., Xu, M., Esteras, C., Roig, C., Monforte, A. J., Troadec, C., … Picó, B. (2011). Towards a TILLING platform for functional genomics in Piel de Sapo melons. BMC Research Notes, 4(1). doi:10.1186/1756-0500-4-289Elias, R., Till, B. J., Mba, C., & Al-Safadi, B. (2009). Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L). BMC Research Notes, 2(1), 141. doi:10.1186/1756-0500-2-141Dahmani-Mardas, F., Troadec, C., Boualem, A., Lévêque, S., Alsadon, A. A., Aldoss, A. A., … Bendahmane, A. (2010). Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach. PLoS ONE, 5(12), e15776. doi:10.1371/journal.pone.0015776Boualem, A., Fleurier, S., Troadec, C., Audigier, P., Kumar, A. P. K., Chatterjee, M., … Bendahmane, A. (2014). Development of a Cucumis sativus TILLinG Platform for Forward and Reverse Genetics. PLoS ONE, 9(5), e97963. doi:10.1371/journal.pone.0097963Blanca, J., Cañizares, J., Roig, C., Ziarsolo, P., Nuez, F., & Picó, B. (2011). Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genomics, 12(1). doi:10.1186/1471-2164-12-104Esteras, C., Gomez, P., Monforte, A. J., Blanca, J., Vicente-Dolera, N., Roig, C., … Pico, B. (2012). High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping. BMC Genomics, 13(1), 80. doi:10.1186/1471-2164-13-80Vicente-Dólera, N., Pinillos, V., Moya, M., Del Río-Celestino, M., Pomares-Viciana, T., Román, B., & Gómez, P. (2014). An improved method to obtain novel mutants in Cucurbita pepo by pollen viability. Scientia Horticulturae, 169, 14-19. doi:10.1016/j.scienta.2014.01.045Martín, B., Ramiro, M., Martínez-Zapater, J. M., & Alonso-Blanco, C. (2009). A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis. BMC Plant Biology, 9(1), 147. doi:10.1186/1471-2229-9-147Wienholds, E. (2003). Efficient Target-Selected Mutagenesis in Zebrafish. Genome Research, 13(12), 2700-2707. doi:10.1101/gr.1725103Dalmais, M., Schmidt, J., Le Signor, C., Moussy, F., Burstin, J., Savois, V., … Bendahmane, A. (2008). UTILLdb, a Pisum sativum in silico forward and reverse genetics tool. Genome Biology, 9(2), R43. doi:10.1186/gb-2008-9-2-r43Triques, K., Sturbois, B., Gallais, S., Dalmais, M., Chauvin, S., Clepet, C., … Bendahmane, A. (2007). Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea. The Plant Journal, 51(6), 1116-1125. doi:10.1111/j.1365-313x.2007.03201.xTaylor, N. E. (2003). PARSESNP: a tool for the analysis of nucleotide polymorphisms. Nucleic Acids Research, 31(13), 3808-3811. doi:10.1093/nar/gkg574Ng, P. C. (2003). SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Research, 31(13), 3812-3814. doi:10.1093/nar/gkg509Obrero, Á., González-Verdejo, C. I., Die, J. V., Gómez, P., Del Río-Celestino, M., & Román, B. (2013). Carotenogenic Gene Expression and Carotenoid Accumulation in Three Varieties of Cucurbita pepo during Fruit Development. Journal of Agricultural and Food Chemistry, 61(26), 6393-6403. doi:10.1021/jf4004576Cosio, C., Vuillemin, L., De Meyer, M., Kevers, C., Penel, C., & Dunand, C. (2009). An anionic class III peroxidase from zucchini may regulate hypocotyl elongation through its auxin oxidase activity. Planta, 229(4), 823-836. doi:10.1007/s00425-008-0876-0Sisko, M. (2003). Genome size analysis in the genus Cucurbita and its use for determination of interspecific hybrids obtained using the embryo-rescue technique. Plant Science, 165(3), 663-669. doi:10.1016/s0168-9452(03)00256-5Campa A (1991) Biological roles of plant peroxidases: known and potential function. In Peroxidases in Chemistry and Biology Vol. II. (Everse, J., Everse, K.E. & Grisham, M.B., eds), pp. 25–50, CRC Press, Boca Raton, FL.Schuller, D. J., Ban, N., van Huystee, R. B., McPherson, A., & Poulos, T. L. (1996). The crystal structure of peanut peroxidase. Structure, 4(3), 311-321. doi:10.1016/s0969-2126(96)00035-4Stephenson, P., Baker, D., Girin, T., Perez, A., Amoah, S., King, G. J., & Østergaard, L. (2010). A rich TILLING resource for studying gene function in Brassica rapa. BMC Plant Biology, 10(1), 62. doi:10.1186/1471-2229-10-62Suzuki, T., Eiguchi, M., Kumamaru, T., Satoh, H., Matsusaka, H., Moriguchi, K., … Kurata, N. (2007). MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Molecular Genetics and Genomics, 279(3), 213-223. doi:10.1007/s00438-007-0293-2Chantreau, M., Grec, S., Gutierrez, L., Dalmais, M., Pineau, C., Demailly, H., … Hawkins, S. (2013). PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics. BMC Plant Biology, 13(1), 159. doi:10.1186/1471-2229-13-159Rawat, N., Sehgal, S. K., Joshi, A., Rothe, N., Wilson, D. L., McGraw, N., … Gill, B. S. (2012). A diploid wheat TILLING resource for wheat functional genomics. BMC Plant Biology, 12(1), 205. doi:10.1186/1471-2229-12-205Minoia, S., Petrozza, A., D’Onofrio, O., Piron, F., Mosca, G., Sozio, G., … Carriero, F. (2010). A new mutant genetic resource for tomato crop improvement by TILLING technology. BMC Research Notes, 3(1). doi:10.1186/1756-0500-3-69Markiewicz, P., Kleina, L. G., Cruz, C., Ehret, S., & Miller, J. H. (1994). Genetic Studies of the lac Repressor. XIV. Analysis of 4000 Altered Escherichia coli lac Repressors Reveals Essential and Non-essential Residues, as well as «Spacers» which do not Require a Specific Sequence. Journal of Molecular Biology, 240(5), 421-433. doi:10.1006/jmbi.1994.1458Carpin, S., Crèvecoeur, M., Greppin, H., & Penel, C. (1999). Molecular Cloning and Tissue-Specific Expression of an Anionic Peroxidase in Zucchini. Plant Physiology, 120(3), 799-810. doi:10.1104/pp.120.3.799Welinder, K. G., Justesen, A. F., Kjaersgård, I. V. H., Jensen, R. B., Rasmussen, S. K., Jespersen, H. M., & Duroux, L. (2002). Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana. European Journal of Biochemistry, 269(24), 6063-6081. doi:10.1046/j.1432-1033.2002.03311.

Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits

[EN] Cucurbita pepo contains two cultivated subspecies, each of which encompasses four fruit-shape morphotypes (cultivar groups). The Pumpkin, Vegetable Marrow, Cocozelle, and Zucchini Groups are of subsp. pepo and the Acorn, Crookneck, Scallop, and Straightneck Groups are of subsp. ovifera. Recently, a de novo assembly of the C. pepo subsp. pepo Zucchini genome was published, providing insights into its evolution. To expand our knowledge of evolutionary processes within C. pepo and to identify variants associated with particular morphotypes, we performed whole-genome resequencing of seven of these eight C. pepo morphotypes. We report for the first time whole-genome resequencing of the four subsp. pepo (Pumpkin, Vegetable Marrow, Cocozelle, green Zucchini, and yellow Zucchini) morphotypes and three of the subsp. ovifera (Acorn, Crookneck, and Scallop) morphotypes. A high-depth resequencing approach was followed, using the BGISEQ-500 platform that enables the identification of rare variants, with an average of 33.5X. Approximately 94.5% of the clean reads were mapped against the reference Zucchini genome. In total, 3,823,977 high confidence single-nucleotide polymorphisms (SNPs) were identified. Within each accession, SNPs varied from 636,918 in green Zucchini to 2,656,513 in Crookneck, and were distributed homogeneously along the chromosomes. Clear differences between subspecies pepo and ovifera in genetic variation and linkage disequilibrium are highlighted. In fact, comparison between subspecies pepo and ovifera indicated 5710 genes (22.5%) with Fst > 0.80 and 1059 genes (4.1%) with Fst = 1.00 as potential candidate genes that were fixed during the independent evolution and domestication of the two subspecies. Linkage disequilibrium was greater in subsp. ovifera than in subsp. pepo, perhaps reflective of the earlier differentiation of morphotypes within subsp. ovifera. Some morphotype-specific genes have been localized. Our results offer new clues that may provide an improved understanding of the underlying genomic regions involved in the independent evolution and domestication of the two subspecies. Comparisons among SNPs unique to particular subspecies or morphotypes may provide candidate genes responsible for traits of high economic importance.This work has been supported by Hellenic Agricultural Organization (ELGO) Demeter. Furthermore, we thank the Conselleria de Educacio, Investigacio, Cultura i Esport (Generalitat Valenciana) for funding Project Prometeo 2017/078 "Seleccion de Variedades Tradicionales y Desarrollo de Nuevas Variedades de Cucurbitaceas Adaptadas a la Produccion Ecologica". Also, this work was supported by Chiang Mai University.Xanthopoulou, A.; Montero-Pau, J.; Mellidou, I.; Kissoudis, C.; Blanca Postigo, JM.; Picó Sirvent, MB.; Tsaballa, A.... (2019). Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits. Horticulture Research. 6:1-17. https://doi.org/10.1038/s41438-019-0176-9S1176Maynard, D. & Paris, H. in The Encyclopedia of Fruits & Nuts (eds Paull, R. E. & Janick, J.) 276–313 (CABI, New Jersey, U.S.A., 2018).Paris, H. S. in Genetics and Genomics of Cucurbitaceae, Grumet, Rebecca, Katzir, Nurit, Garcia-Mas, Jordi (Eds.) 111–154 (Springer, New York, U.S.A., 2016).Whitaker, T. W. & Davis, G. N. Cucurbits (Leonard Hill (Books) Ltd., London, and Interscience Publishers Inc., New York, 1962).Paris, H. S. History of the cultivar-groups of Cucurbita pepo. Hortic. Rev. 25, 71–170 (2001).Paris, H. S. A proposed subspecific classifiaction for Cucurbita pepo. Phytologia (USA) 61, 133–138 (1986).Lira, R., Andres, T. C. & Nee, M. in Systematic and Ecogeographic Studies on Crop Genepools, Vol. 9, 1–115 (International Plant Genetic Resources Institute, Roma, Italia, 1995).Castellanos-Morales, G. Historical biogeography and phylogeny of Cucurbita: insights from ancestral area reconstruction and niche evolution. Mol. Phylogenet. Evol. 128, 38–54 (2018).Paris, H. S., Lebeda, A., Křistkova, E., Andres, T. C. & Nee, M. H. Parallel evolution under domestication and phenotypic differentiation of the cultivated subspecies of Cucurbita pepo (Cucurbitaceae). Econ. Bot. 66, 71–90 (2012).Dong, W., Wu, D., Li, G., Wu, D. & Wang, Z. Next-generation sequencing from bulked segregant analysis identifies a dwarfism gene in watermelon. Sci. Rep. 8, 2908 (2018).Galpaz, N. et al. Deciphering genetic factors that determine melon fruit‐quality traits using RNA‐Seq‐based high‐resolution QTL and eQTL mapping. Plant J. 94, 169–191 (2018).Gur, A. et al. Genome-wide linkage-disequilibrium mapping to the candidate gene level in melon (Cucumis melo). Sci. Rep. 7, 9770 (2017).Blanca, J. et al. Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genom. 12, 104 (2011).Esteras, C. et al. High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping. BMC Genom. 13, 80 (2012).Montero-Pau, J. et al. An SNP-based saturated genetic map and QTL analysis of fruit-related traits in Zucchini using genotyping-by-sequencing. BMC Genom. 18, 94 (2017).Vicente-Dólera, N. et al. First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement. PLoS ONE 9, e112743 (2014).Wyatt, L. E., Strickler, S. R., Mueller, L. A. & Mazourek, M. An acorn squash (Cucurbita pepo ssp. ovifera) fruit and seed transcriptome as a resource for the study of fruit traits in Cucurbita. Hortic. Res. 2, 14070 (2015).Xanthopoulou, A. et al. De novo comparative transcriptome analysis of genes involved in fruit morphology of pumpkin cultivars with extreme size difference and development of EST-SSR markers. Gene 622, 50–66 (2017).Montero‐Pau, J. et al. De novo assembly of the zucchini genome reveals a whole‐genome duplication associated with the origin of the Cucurbita genus. Plant Biotechnol. J. 16, 1161–1171 (2018).Garcia-Mas, J. et al. Cloning and mapping of resistance gene homologues in melon. Plant Sci. 161, 165–172 (2001).Xanthopoulou, A. et al. Comparative analysis of genetic diversity in Greek Genebank collection of summer squash (‘Cucurbita pepo’) landraces using start codon targeted (SCoT) polymorphism and ISSR markers. Aust. J. Crop Sci. 9, 14 (2015).Huang, J. et al. A reference human genome dataset of the BGISEQ-500 sequencer. Gigascience 6, gix024 (2017).Natarajan, K. N. et al. Comparative analysis of sequencing technologies for single-cell transcriptomics. Genome Biol. 20, 70 (2019).Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).Tian, L. et al. Transcript and proteomic analysis of developing white lupin (Lupinus albus L.) roots. BMC Plant Biol. 9, 1 (2009).Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).Bradbury, P. J. et al. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635 (2007).Chang, C. C. et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience 4, 7 (2015).Team, R. C. (2015). http://www.r-project.org/ .Krzywinski, M. I. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).Kosman, E. & Leonard, K. J. Similarity coefficients for molecular markers in studies of genetic relationships between individuals for haploid, diploid, and polyploid species. Mol. Ecol. 14, 415–424 (2005).Huson, D. H. & Bryant, D. Estimating Phylogenetic Trees and Networks Using SplitsTree 4. www.splitstree.org (2005).Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strainw1118; iso-2; iso-3. Fly 6, 80–92 (2012).Wu, S. et al. A common genetic mechanism underlies morphological diversity in fruits and other plant organs. Nat. Commun. 9, 4734 (2018).Drevensek, S. et al. The Arabidopsis TRM1–TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes. Plant Cell 24, 178–191 (2012).Sievers, F. et al. Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2014).Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587 (2017).Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2017).Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).Leida, C. et al. Variability of candidate genes, genetic structure and association with sugar accumulation and climacteric behavior in a broad germplasm collection of melon (Cucumis melo L.). BMC Genet. 16, 28 (2015).Esteras, C. et al. SNP genotyping in melons: genetic variation, population structure, and linkage disequilibrium. Theor. Appl. Genet. 126, 1285–1303 (2013).Maria José Gonzalo et al. Re-evaluation of the role of Indian germplasm as center of melon diversification based on genotyping-by-sequencing analysis. BMC Genom. 20, p. 448 (2019).Nimmakayala, P. et al. Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity, linkage disequilibrium, and selective sweeps in cultivated watermelon. BMC Genom. 15, 767 (2014).Gonzalo, M. J. & Monforte, A. J. in Genetics and Genomics of Cucurbitaceae, Grumet, Rebecca, Katzir, Nurit, Garcia-Mas, Jordi (Eds.) 269–290 (Springer, New York, U.S.A., 2016).Pomares-Viciana, T. et al. First RNA-seq approach to study fruit set and parthenocarpy in zucchini (Cucurbita pepo L.). BMC Plant Biol. 19, 61 (2019).Lu, S. et al. The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of β-carotene accumulation. Plant Cell 18, 3594–3605 (2006).Jin, B., Kim, J., Jung, J., Kim, D. & Park, Y. Characterization of IQ domain gene homologs as common candidate genes for elongated fruit shape in cucurbits. Hortic. Sci. Technol. 36, 85–97 (2018).van der Knaap, E. et al. What lies beyond the eye: the molecular mechanisms regulating tomato fruit weight and shape. Front. Plant Sci. 5, 227 (2014).Xiao, H., Jiang, N., Schaffner, E., Stockinger, E. J. & Van Der Knaap, E. A retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit. Science 319, 1527–1530 (2008).Dou, J. et al. Genetic mapping reveals a candidate gene (ClFS1) for fruit shape in watermelon (Citrullus lanatus L.). Theor. Appl. Genet. 131, 947–958 (2018).Pan, Y. et al. Round fruit shape in WI7239 cucumber is controlled by two interacting quantitative trait loci with one putatively encoding a tomato SUN homolog. Theor. Appl. Genet. 130, 573–586 (2017).Liu, J. et al. Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening. PLoS ONE 10, e0123870 (2015).Liu, J. et al. Mu MADS 1 and Ma OFP 1 regulate fruit quality in a tomato ovate mutant. Plant Biotechnol. J. 16, 989–1001 (2018).Cong, B., Barrero, L. S. & Tanksley, S. D. Regulatory change in YABBY-like transcription factor led to evolution of extreme fruit size during tomato domestication. Nat. Genet. 40, 800 (2008).Huang, Z., Van Houten, J., Gonzalez, G., Xiao, H. & van der Knaap, E. Genome-wide identification, phylogeny and expression analysis of SUN, OFP and YABBY gene family in tomato. Mol. Genet. Genom. 288, 111–129 (2013).Bowman, J. L. The YABBY gene family and abaxial cell fate. Curr. Opin. Plant Biol. 3, 17–22 (2000).Liu, J., Van Eck, J., Cong, B. & Tanksley, S. D. A new class of regulatory genes underlying the cause of pear-shaped tomato fruit. Proc. Natl Acad. Sci. USA 99, 13302–13306 (2002).Tsaballa, A., Pasentsis, K., Darzentas, N. & Tsaftaris, A. S. Multiple evidence for the role of an Ovate-like gene in determining fruit shape in pepper. BMC Plant Biol. 11, 46 (2011).Wang, S., Chang, Y., Guo, J. & Chen, J. G. Arabidopsis Ovate family protein 1 is a transcriptional repressor that suppresses cell elongation. Plant J. 50, 858–872 (2007).Lazzaro, M. D., Wu, S., Snouffer, A., Wang, Y. & Van Der Knaap, E. Plant organ shapes are regulated by protein interactions and associations with microtubules. Front. Plant Sci. 9, 1766 (2018)