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Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars ('MoneyMaker' and 'San Marzano') and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 'MoneyMaker' gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health., This work was supported in part by the PID2019-108203RB-I00 project of the Spanish Plan Nacional I+D, Spanish Ministry of Science and Innovation and by HARNESSTOM Innovation Action EU-H2020-SFS-2020-1 (Contract number: 101000716). Finally, this work was supported by COST Actions ROXy CA18210 and EUROcarotene CA15136 for networking.

[EN] The present invention relates to the use of the esters acetate, propionate and (Z)-3-hexenyl butyrate, or of a composition comprising these compounds, to protect plants, in particular agricultural crops, against pests, by stimulating the defense mechanisms of the plants themselves. The invention also relates to a method for protecting plants against pests by bringing said esters into contact with the plants, [ES] La presente invención se refiere al uso de los ésteres acetato, propionato y butirato de (Z)-3-hexenüo, o de una composición que comprende dichos compuestos, para proteger plantas, en particular de cultivos agrícolas, frente a plagas, mediante la estimulación de los mecanismos de defensa que tienen las propias plantas. La invención también se refiere a un método para proteger plantas frente a plagas poniendo los mencionados ésteres en contacto con las plantas., [FR] La presente invención se refiere al uso de los ésteres acetato, propionato y butirato de (Z)-3-hexenüo, o de una composición que comprende dichos compuestos, para proteger plantas, en particular de cultivos agrícolas, frente a plagas, mediante la estimulación de los mecanismos de defensa que tienen las propias plantas. La invención también se refiere a un método para proteger plantas frente a plagas poniendo los mencionados ésteres en contacto con las plantas, Universitat Politècnica de València, Instituto Valenciano de Investigaciones Agrarias (IVIA), Consejo Superior de Investigaciones Científicas (España), A1 Solicitud de patente con informe sobre el estado de la técnica

Woodland strawberry (Fragaria vesca, 2x) is the diploid closest ancestor of the cultivated strawberry (Fragaria x annassa, 8x) and the model species for genetic studies in the Fragaria genus. It is naturally distributed all across Europe and it is appreciated for their delicate aroma and flavor. Methylketones are compounds with demonstrated insect repellent effects. Its synthesis, reported to take place in glandular trichomes of wild tomato (Solanum habrochaites), is mediated by a thioesterase (ShMKS2) and a decarboxylase (ShMKS1) (Ben Israel et al. 2009; Yu et al. 2010). A natural population of 199 accessions of F. vesca that represents the European diversity was resequenced (>1.8M SNPs) and characterized for the accumulation of methylketones and their derived alcohols. By genome wide association analysis (GWAS), we have detected a stable series of polymorphisms associated to the accumulation of C7, C9 and C11 methylketones and their respective alcohols. Three different haplotypes in the candidate region with different patterns of methylketones were found, suggesting differences in enzymatic activity and/or substrate affinity. The candidate region includes two genes annotated as thioesterases that are homolog to ShMKS2, FvMKS2A, FvMKS2B. Functional validation of both genes has revealed methyl ketones synthesis ability in transient expression in Nicotiana benthamiana. The function and specificity of these candidate genes is currently under study through several approaches, including transient expression in different hosts and enzymatic function characterization. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Methyl ketones are compounds with demonstrated insect repellent effects. They are highly abundant in the glandular trichomes of wild tomato (Solanum habrochaites), where their pathway was first described, but not in the cultivated species (S. lycopersicum). Their synthesis derives from fatty acids in a two-step process mediated by a thioesterase (ShMKS2) and a decarboxylase (ShMKS1). Higher diversity and quantity of methyl ketones are present in the volatilome of woodland strawberry ripe fruits than in those of F. × ananassa. The aim of this study is to reveal the genetic basis of methyl ketone production in strawberry fruit. We quantified methyl ketones (2-heptanone, 2-nonanone, 2-undecanone), their secondary alcohols (2-heptanol, 2-nonanol, 2-undecanol) and the methyl esters of their fatty acid precursors (methyl octanoate, methyl decanoate, methyl dodecanoate) by GC-MS in a natural collection of European woodland strawberry, that comprises 199 accessions fully genotyped with >1.8 M SNPs representing the continental diversity. Conducting a Genome-Wide Association Study (GWAS), we identified a candidate region linked to methyl ketones accumulation harbouring three homologues of ShMKS2: FvMKS2A, FvMKS2B and FvMKS2C. Interestingly, FvMKS2A, which presented two alleles in the European collection (FvMKS2A-1 and FvMKS2A-2), is the only FvMKS2 paralog expressed in woodland strawberry fruit, being up-regulated during ripening. Functional validation of all candidate genes and alleles by transient over-expression and silencing in both Nicotiana benthamiana leaves and F. vesca fruits has revealed that FvMKS2A and FvMKS2B, but not FvMKS2C, are capable of synthesizing methyl ketones, and point to a single SNP in FvMKS2A as responsible for the enzymatic substrate specificity, supporting FvMKS2A as the main MKS2 paralog responsible for methyl ketones in woodland strawberries. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

La presente invención se refiere al uso de los ésteres acetato, propionato y butirato de (Z)-3-hexenüo, o de una composición que comprende dichos compuestos, para proteger plantas, en particular de cultivos agrícolas, frente a plagas, mediante la estimulación de los mecanismos de defensa que tienen las propias plantas. La invención también se refiere a un método para proteger plantas frente a plagas poniendo los mencionados ésteres en contacto con las plantas. [ES], The present invention relates to the use of the esters acetate, propionate and (Z)-3-hexenyl butyrate, or of a composition comprising these compounds, to protect plants, in particular agricultural crops, against pests, by stimulating the defense mechanisms of the plants themselves. The invention also relates to a method for protecting plants against pests by bringing said esters into contact with the plants. [EN], Universitat Politècnica de València, Instituto Valenciano de Investigaciones Agrarias (IVIA), Consejo Superior de Investigaciones Científicas (CSIC), A1 Solicitud de patente con informe sobre el estado de la técnica

Woodland strawberry (Fragaria vesca, 2x) is the diploid closest ancestor of the cultivated strawberry (Fragaria ´ annassa, 8x) and the model species for genetic studies in the Fragaria genus. It is naturally distributed all across Europe and it is appreciated for their delicate aroma and flavor. Aiming to describe the genetic and organoleptic diversity of European woodland strawberry and decipher the genetic control of its characteristic volatile compounds, we have sequenced and metabolically-phenotyped a diverse collection of 199 geographically distant European accessions. The metabolic profiling of the lines includes a set of 100 unambiguosly identified volatiles. This study has revealed genetic and metabolic differences between subpopulations with different geographical origin. In addition, Genome Wide Association Analysis points to several candidate genetic regions controlling the accumulation of volatiles compounds sharing common biosynthetic pathways. Specifically, we have detected SNPs associated to the accumulation of methyl ketones and their corresponding alcohols mapping to a small region of chromosome 4 with a reduced set of candidate genes. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

La fresa silvestre (Fragaria vesca, 2x), es el ancestro más cercano de la fresa cultivada (Fragaria x ananassa, 8x) y la especie modelo para estudios genéticos dentro del género Fragaria. Se distribuye naturalmente a lo largo de toda Europa y es muy apreciada por su aroma y sabor. Con el objetivo de describir la diversidad genética y organolétpica de la fresa silvestre europea y descifrar el control genético de los compuestos volátiles característicos de su aroma, hemos re-secuenciado y fenotipado el volatiloma de una colección de 199 accesiones que representan la diversidad genética Europea. Este estudio ha proporcionado un set de >1.8M de polimorfismos simples (SNPs) y la cuantificación relativa de 100 compuestos inequivocamente identificados mediante GCMS en el fruto maduro. Los resultados revelan diferencias genéticas y metabólicas entre subpoblaciones con diferente origen geográfico. Además, el estudio de asociación de genoma completo (GWAS) de los compuestos volátiles, indica varias regiones candidatas (significativamente asociadas) para controlar la accumulación de uno o varios compuestos volátiles que comparten rutas biosintéticas. Más espeficamente, hemos detectado una región del cromosoma 4 (

Ripening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project, we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. On the other hand, we are carrying out a Genome-Wide Association Study using a germplasm collection of the woodland strawberry (Fragaria vesca) in order to identify loci involved in important traits such as aroma, fruit size or resistance to pathogens. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect it might open opportunities for engineering this species to improve traits of economic importance. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Póster presentado en el XIII Congresso Luso-Espanhol de Fisiologia Vegetal, celebrado en Lisboa del 24 al 28 de julio de 2013.