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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.

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

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.

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