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Funding Information: The project is funded by the European Commission as well as the following national/regional bodies: Formas—the Swedish Research Council for Sustainable Development (grant no: 2020–02376), Academy of Finland (grant no. 344726), Research Foundation—Flanders (grant no. FWO ERANET G0H6520N), and Agencia Estatal de Investigación (grant no. PCI2020-120719-2). Publisher Copyright: Copyright © 2022 De-la-Cruz, Batsleer, Bonte, Diller, Hytönen, Muola, Osorio, Posé, Vandegehuchte and Stenberg. Aboveground plant-arthropod interactions are typically complex, involving herbivores, predators, pollinators, and various other guilds that can strongly affect plant fitness, directly or indirectly, and individually, synergistically, or antagonistically. However, little is known about how ongoing natural selection by these interacting guilds shapes the evolution of plants, i.e., how they affect the differential survival and reproduction of genotypes due to differences in phenotypes in an environment. Recent technological advances, including next-generation sequencing, metabolomics, and gene-editing technologies along with traditional experimental approaches (e.g., quantitative genetics experiments), have enabled far more comprehensive exploration of the genes and traits involved in complex ecological interactions. Connecting different levels of biological organization (genes to communities) will enhance the understanding of evolutionary interactions in complex communities, but this requires a multidisciplinary approach. Here, we review traditional and modern methods and concepts, then highlight future avenues for studying the evolution of plant-arthropod interactions (e.g., plant-herbivore-pollinator interactions). Besides promoting a fundamental understanding of plant-associated arthropod communities’ genetic background and evolution, such knowledge can also help address many current global environmental challenges.

The epidermis is the external cell layer in direct contact with the environment, and it plays essential biological roles. Transcriptome analysis (RNA-seq) of Fragaria vesca fruit receptacles at four ripening stages (green, white, turning and red) and of different tissue types of receptacles (pith, vascular bundles, cortex and epidermis) at two ripening stages (green and red) allowed us to infer tissue- and stage-specific Gene Regulatory Networks (GRN). Due to the potential role of the epidermis in defense and in the differential anthocyanin accumulation pattern that shows F. vesca fruits at the ripe stage (the skin is red, while the inner part is white), we have focused on the GRN of the epidermis of red receptacles. In this study, we aim at the functional characterization of two transcription factors (TFs) that constituted the main hubs of this GRN: a MYB-like gene, and a member of the NAC family of TFs. MapMan analysis of the genes constituting the GRN in ripe epidermis showed that wax and flavonoid biosynthesis were significantly overrepresented functions in the epidermis of red receptacles. Using the Luciferase/Renilla (Luc/Ren) system, the interaction of the MYB and NAC TFs with their wax-related putative targets was validated. To experimentally validate the target genes of these two TFs, their binding sites were mapped genome-wise using DAP-seq analyses. Consistently, MYB bound to a set of genes involved in cuticle formation and flavonoid biosynthesis, while a number of genes involved in solute transport were enriched among the NAC direct targets. Finally, protein interaction assays showed that MYB physically interacts with two members of the bHLH TF family specifically expressed in the red epidermis of receptacles, leading to the subcellular relocalization of one of them from the cytoplasm to the nucleus. Currently, we are phenotyping transgenic overexpressing lines of MYB and generating CRISPR/Cas9 mutant lines to functionally characterize these two TFs. Plan Estatal de Investigación Científica y Técnica y de Innovación RTI2018-097309-A-I00 and the ERC Starting Grant ERC-2014-StG 638134

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 (TF) involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. Among them, we have identified two TFs belonging to the NAC (FaRIF) and the BLH9 (FaRPL) family. Functional analyses establishing stable silencing and overexpression lines support that both TFs play a critical role in the regulation of fruit ripening and development. Furthermore, using a stage- and tissue-specific transcriptome analysis, we have identified TFs specifically expressed in the external layer of ripe receptacles of F. vesca fruits, which are involved in the regulation of wax and cuticle formation. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect to open opportunities for engineering this species to improve traits of economic importance.

Climate factors such as temperature and precipitation vary significantly over continental scales, strongly structuring biomes along latitudinal gradients, and resulting in species differently adapted either genetically or plastically to cope with their local climate. However, climate change will likely alter these biomes. Thus, it is expected that Nordic regions, historically colder and rainier, will tend to have higher temperatures and less rainfall, which might lead to changes in the distribution of plants leading to novel patterns of local adaptation and maladaptation. In this study we aim to study how plant traits vary with latitude and in response to different temperature and drought conditions in order to find genetic determinants of climate adaptation. Our group is focused in determining the role of the metabolic profiling to that adaptation. For that purpose, we use the woodland strawberry (Fragaria vesca) as the model organism. In particular, we have analyzed 16 different genotypes that have been grown in five common gardens located in Belgium, Sweden, Finland, and Spain, in which drought treatments were also performed. Here, we present the chemical analysis (primary metabolism) in leaves of these genotypes in order to better understand how environmental factors can alter the primary metabolic profiles of F. vesca accessions grown in different locations. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. BiodivERsA project PlantCline -(PCI2020-120719-2) from “Programación Conjunta Internacional” program, Ministerio de Ciencia e Innovación.

Strawberry is becoming a model for studying the molecular mechanism of ripening in non-climacteric fruits. However, a limited number of transcriptional regulators of this process have been identified so far. In this study, we have identified and characterized a gene encoding for a NAC transcription factor (TF), named as FaRIF (Ripening Inducing Factor). FaRIF expression presents a fruit-specific pattern, which is upregulated during ripening. In order to functionally characterize this TF, we have generated silencing and overexpressing stable transgenic lines. While the RNAi lines showed an apparent delay of fruit ripening, the overexpressing lines displayed an acceleration of this process. Transcriptomic analysis of the silenced lines showed a significantly altered expression of genes involved in development, hormone metabolism, flavonoid pathway, and cell-wall disassembly, being many of these confirmed by phenotypical and metabolomics analysis. Our results support a main role of FaRIF in the control of relevant ripening-associated processes in strawberry fruit.

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.

In contrast to climacteric fruits such as tomato, the knowledge on key regulatory genes controlling the ripening of strawberry, a non-climacteric fruit, is still limited. NAC transcription factors are proteins that mediate different developmental processes in plants. In this work, we have identified and characterized FaRIF (Ripening Inducing Factor), a novel NAC transcription factor which is highly expressed and induced in strawberry receptacles during ripening. Functional analysis establishing stable transgenic lines with RNAi, driven by either the constitutive 35S or the ripe receptacle-specific EXP2 promoters, and overexpression constructs showed that FaRIF controls critical ripening-related processes such as fruit softening and pigment and sugars accumulation. Physiological, metabolomic and transcriptomic analyses of receptacles of FaRIFsilenced and overexpression lines point to FaRIF as a key regulator of strawberry fruit ripening from early developmental stages, controlling ABA biosynthesis and signaling, cell wall degradation and modification, the phenylpropanoid pathway, and the balance of the aerobic/anaerobic metabolism, being therefore a target to be modified/edited to control the quality of strawberry fruits. ERC Starting Grant ERC-2014-StG 638134

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.

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, and resistance to pathogens. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect to open opportunities for engineering this species to improve traits of economic importance. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in Rosaceae. APETALA3 (AP3), a member of this class, controls petal and stamen identities in Arabidopsis. In this study, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria x ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using CRISPR/Cas9 system is becoming a robust tool for targeted and stable DNA mutagenesis. However, whether it can be efficiently used in an octoploid species such as F. x ananassa is not yet established. Here we report the application of CRISPR/Cas9 to characterize the function of FaTM6 in strawberry flower development. An analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a highly efficient genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in anther development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene functional analysis in F. x ananassa as an octoploid species, and they offer new opportunities for engineering strawberry to improve traits of interest in breeding programs. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

El contenido del poster presentado está desarrollado en: doi:10.1093/jxb/ery400 y Publicado por Oxford University press The B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in the Rosaceae family. APETALA3 (AP3), a member of this class, controls the identity of petals and stamens in Arabidopsis thaliana. In this work, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria × ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA. However, whether it can be efficiently used in an octoploid species such as F. × ananassa is not known. In our study, we report the application of the CRISPR/Cas9 in F. × ananassa to characterize the function of FaTM6 in flower development. An exhaustive analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a high efficiency genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in petal and especially in anther development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene functional analysis in an octoploid species such as F. × ananassa, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

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

Anthocyanins are the pigments responsible for the red color of strawberries. Their biosynthesis is controlled at the transcriptional level by a ternary complex consisting of R2R3-MYB and bHLH transcription factors associated with a WD40-repeat protein. In order to map the genetic factors involved in fruit coloring we generated a mapping population crossing a F. vesca accession bearing white fruits (ESP138.596) with the red-fruited ‘Reine des Vallées’. DNA from white- or red-fruited F2 individuals was pooled to perform a bulk segregant analysis (BSA) linked with high-throughput genome sequencing. This analysis revealed the presence of a gypsy-like retrotransposon inserted in the third exon of FvMYB10. The presence of this retroelement in homozygosis co-segregated with white fruits in the complete F2 population. We further extended this analysis to other white-fruited F. vesca accessions but none of them harbored this retroelement in FvMYB10. Instead we identified two additional polymorphisms affecting FvMYB10, (1) a single nucleotide insertion, which generates a truncated protein, and (2) a large deletion of ~100 Kb spanning a genomic region that contains 7 genes, one of them being FvMYB10. The three newly identified polymorphisms on FvMYB10 differ from the previously described single nucleotide mutation, responsible for the lack of anthocyanins in other F. vesca white/yellow fruited accessions. We next analyzed QTL for fruit color in a segregating population derived from the red-fruited F. x ananassa ‘Senga Sengana’ and a F. chiloensis accession with white flesh. A major QTL controlling 45.7 - 54.7% of variance in internal flesh color was detected on LG I-3. The confidence interval spans the orthologous region where FvMYB10 is located. Furthermore, transient overexpression on FvMYB10 on different F. chiloensis accessions resulted in red sectors both in the epidermis and fruit flesh.

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.

External and internal fruit color are important traits in strawberry (Fragaria spp.) breeding programs, where different preferences are sought depending on whether the fruits are produced for fresh consumption or processing. Therefore, there is a great interest in the development of predictive markers that effectively speed the development of new cultivars with increased consumer acceptance and/or which address processed fruit industry´s preferences. In order to identify loci controlling fruit color variation, two mapping populations were generated: one crossing diploid F. vesca parentals and another interspecific population between two octoploid species: the cultivated and the Chilean strawberry, F. x ananassa and F. chiloensis. Both populations allowed the detection of a QTL spanning a region of the F. vesca linkage group 1 (LG I) that includes the MYB10 gene, a known key regulator of anthocyanin biosynthesis. Mapping by sequencing in the F. vesca population revealed an LTR retrotransposon inserted in the third exon of FvMYB10, which produces a premature stop codon, and co-segregates with white fruits in the entire population. Genotyping by Sanger sequencing of additional white-fruited F. vesca accessions resulted in the identification of another three independent mutations in MYB10, two of them not previously described1. In octoploid strawberry, a mayor QTL on LG I-3 controls about 55% variation in internal flesh color and is associated with an insertion in the promoter region of FcMYB10. Similar insertions have been detected in other F. chiloensis accessions bearing white fruits. In all cases, transient over-expression of FvMYB10 restored anthocyanin biosynthesis and red color in fruit flesh and skin, indicating that lack of function of MYB10 was the underlying cause of white fruits in all analyzed cases.

La maduración del fruto es un proceso de desarrollo altamente regulado en el que se producen una serie de cambios fisiológicos, bioquímicos y estructurales que lo hace más atractivo para la dispersión de las semillas. La fresa silvestre (Fragaria vesca) se ha convertido por sus características en el modelo para el estudio de la maduración de frutos no climatéricos. En este trabajo se ha realizado la validación experimental de un modelo de red génica regulatoria en la que se infiere interacciones entre genes específicos de epidermis de frutos de F. vesca en estadio inicial de maduración (estadio verde). Entre estos genes, hemos analizado tres factores de transcripción (FTs), FvREMX, FvREM7 y FvWRKY38, y un posible gen diana de estos FTs que codifica para la enzima FvCAD9, implicada en la ruta de biosíntesis de lignina. Para validar el modelo se clonaron las secuencias codificantes de los tres FTs y de los cuatro promotores, y se llevó a cabo un ensayo de actividad de promotores con luciferasa como gen reportero. Este trabajo ha confirmado experimentalmente regulaciones directas predichas en la red génica regulatoria de epidermis verdes, abriendo la posibilidad de realizar estudios en un futuro para determinar la función de estos genes en dicho tejido y estadio de maduración. European Research Council TRANSFR-Q: ERC Starting Grant ERC-2014-StG 638134

Strawberry is becoming a model for studying the molecular mechanism of ripening in non-climacteric fruits. However, a limited number of transcriptional regulators of this process have been identified so far. In this study, we have identified and characterized a gene encoding for a NAC transcription factor (TF), named as FaRIF (Ripening Inducing Factor). FaRIF expression presents a fruit-specific pattern, which is upregulated during ripening. In order to functionally characterize this TF, we have generated silencing (35S::RIF-RNAi) and overexpressing (35S::RIF-GFP) stable transgenic lines. While the RNAi lines showed an apparent delay of fruit ripening, the overexpressing lines displayed an acceleration of this process. Transcriptomic analysis, by RNA-seq, of the silenced lines showed a significantly altered expression of genes involved in the flavonoids pathway, as well as genes of the metabolism of the main sugars of the fruit. Metabolomics analysis confirmed these changes in the transgenic fruits. Both, transcriptomic and metabolomics data, were in agreement with the general phenotype observed in the fruits of the FaRIF-silenced lines. All together, our results support a main role of FaRIF in the control of relevant ripening-associated processes in strawberry fruit. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

During ripening, fruits undergo a number of metabolic and physiological changes leading to softening and improvement of characters such as flavor and palatability. Insights into transcriptome changes during strawberry fruit ripening have been reported, but always using either complete fruits in the analysis or separating achenes and the fleshy part (receptacle). However, the receptacle is composed of heterogeneous cell types, each of them with different characteristics and functions. Hence, transcriptomic studies performed so far may have lost important regulatory elements which expression is low but important in a specific cell-type specific. In our study, we use Laser Capture Microdissection (LCM) technique for the isolation of cells from specific tissue types such as the epidermis, vascular bundles, cortex, and pith. Transcriptome profiling of these tissue types was performed by RNAseq. A gene co-expression analysis was performed by Weighted Correlation Network Analysis (WGCNA). Ontology analysis of each module showed wax biosynthesis as the main biological pathway enriched at the red epidermis specific module. In order to elucidate the putative regulatory elements that control the synthesis of waxes in this tissue, a Gene Regulatory Network (GRN) was generated using GENIST (de Luis Balaguer, 2017). As a result, we have identified a set of transcription factors that might regulate the expression of eceriferum genes and a fatty acid elongase necessary for wax biosynthesis in ripe epidermis. Ultimately, our results open the possibility of implementing novel targeted breeding approaches. Moreover, this work shows that LCM followed by RNAseq is a powerful tool that can be used to clarify the regulatory scenario of tissue-specific biological processes during strawberry ripening. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Ponencia invitada 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. Also, we are using a collection of around two hundred wild strawberry (Fragaria vesca) accessions to identify loci involved in important traits such as aroma, size or resistance to pathogens. Finally, we are implementing 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.

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 (TRANSFR-Q, Starting Grant-ERC), 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, and resistance to pathogens. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect to open opportunities for engineering this species to improve traits of economic importance. ERC Starting Grant ERC-2014-StG 638134

The B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in the Rosaceae family. APETALA3 (AP3), a member of this class, controls the identity of petals and stamens in Arabidopsis thaliana. In this work, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria × ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA. However, whether it can be efficiently used in an octoploid species such as F. × ananassa is not known. In our study, we report the application of the CRISPR/Cas9 in F. × ananassa to characterize the function of FaTM6 in flower development. An exhaustive analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a high efficiency genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in petal and especially in anther development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene functional analysis in an octoploid species such as F. × ananassa, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.