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Transcriptional RegulAtory Network controlling Strawberry Fruit Ripening and Quality

Periodic Reporting for period 3 - TRANSFR-Q (Transcriptional RegulAtory Network controlling Strawberry Fruit Ripening and Quality)

Reporting period: 2018-06-01 to 2019-11-30

Ripening is the critical step for the development of flavour quality in fruit. This character has significantly declined in many fleshy fruits over recent decades, primarily due to the focus of current breeding programs on agronomic traits such as production, firmness, and postharvest shelf life. This strategy has caused a tunnelling effect on genetic variability in many crops. This is particularly significant in strawberry, where current cultivars are derived from a narrow germplasm stock. Moreover, strawberry fruits posses two notable features: i) the fleshy part of the strawberry fruit is the result of the enlargement of the flower receptacle, while the true fruits are the dry structures called achenes, anchored on the surface of the receptacle; ii) while the hormone ethylene is essential for the ripening of climacteric fruits such as tomato, ethylene only plays a marginal role in strawberry fruits, and as such is considered non-climacteric. Therefore, improving fruit flavour in present strawberry varieties requires two important breakthroughs: 1) a precise understanding of non-climacteric and receptacle ripening regulation that will allow the targeting of relevant quality genes, and 2) the identification of unexploited allelic variants from wild germplasm to be introgressed through the generation of novel breeding lines. In our project, we are interested in generating information and tools that will help to achieve these goals. Specifically, we are interested in the following specific aims:
1) To characterize molecularly the strawberry fruit ripening performing transcriptomics and metabolomics analyses
2) To identify and investigate the role played by key transcription factors (TFs) regulating fruit ripening
3) To uncover the gene regulatory network for these key TFs, and to study their impact on fruit quality characters
4) To identify novel genes controlling fruit quality characters of the ripe fruit using a germplasm collection of woodland strawberry (Fragaria vesca) accessions.
Overall, TRANSFR-Q project is aiming to incorporate quality characters into new breeding lines. Moreover, we plan to establish comprehensive genomic resources not only for strawberry but also for the Rosaceae community, an aspect of utmost importance since this family contains many economically important fruit trees, including apple, peach, cherry, apricot and almond.
During the first period of the project we have dedicated our effort mainly to: i) characterize strawberry fruit ripening by studying the gene expression and metabolite changes that occur during this process; ii) to identify genes that may play a key role in the regulation of ripening; iii) to optimize a cutting-edge genome editing tool in order to mutagenize genes of interest; and iv) to establish a collection of Fragaria vesca coming from all across Europe in order to identify genes involved in fruit quality.

In more detail, we have achieved the following results during this period of the project:

1) We have studied the global gene expression in different tissues and ripening stages of the woodland strawberry Fragaria vesca fruit. Furthermore, we have characterized the metabolic changes (primary and secondary metabolites) that occur in receptacles (fleshy part) at these stages of ripening. These dataset has provided a complete transcriptomic and metabolomic overview during woodland strawberry fruit ripening. Analysis of the transcriptome has allowed us to identify candidate transcription factors (TFs) that may act as master regulators of the ripening process.
2) Besides the previous transcriptomic study, we have performed a more specific expression analysis by capturing different tissue types of the receptacle with a laser coupled to a microscope. This tissue- and stage-specific transcriptomic study has allowed us to identify genes that work specifically in each tissue type, providing more accurate information than the study previously mentioned. Moreover, we have generated Gene Regulatory Networks (GRNs) combining spatial and temporal data. These GRNs show several interesting TFs that may play a key role in their respective tissues and stages, functioning as hubs of the networks.
3) We have identified candidate TFs with a putative key role in the control of the receptacle ripening process. These TFs has been studied, cloned and transformed into the cultivated strawberry species Fragaria x ananassa in order to study their role in ripening.
4) We have studied the viability of the genome edition system CRISPR/Cas9 in Fragaria x ananassa. We have successfully mutated a target gene, obtaining an impaired fruit development as a result. This result positions CRISPR/Cas9 as a feasible alternative to the sometimes inefficient post-transcriptional gene silencing approaches, which are the most common strategies used in strawberry for gene functional analysis.
5) We have sequenced the genome and started the phenotypic characterization of a Fragaria vesca germplasm collection. This will allow us in the near future to perform genome-wide association studies (GWAs) that will provide candidate genes responsible for quality traits of interest (size, flavour, aroma, resistance to Powdery Mildew, etc).
The central aspect of our project is dedicated to understand how fruit ripening is controlled in strawberry fruits and to identify genes involved in fruit quality in order to apply this knowledge into new breeding programs. In order to accomplish these objectives, I will summarize the progress achieved so far, and the results we expect until the end of the project:
1) We have characterized the global gene expression and metabolic changes that occur during strawberry fruit ripening, and moreover, we have obtained a tissue-specific transcriptome. The latter analysis will provide a much more reliable and realistic gene expression atlas for strawberry fruits than any transcriptomic study performed so far.
2) We will identify TFs that control the ripening process of the strawberry fruit, with a main emphasis in the fleshy receptacle. We will analyse the roles of these TFs using molecular, genetic, genomic and metabolomic approaches and gain, for the first time, a deep understanding of the gene regulatory pathways leading to strawberry fruit ripening. We are planning to identify the direct targets of these key TFs. This study is based on the Chromatin Immunoprecipitation (ChIP) methodology, which is essential for the characterization of TFs and that has never been applied in strawberry plants.
3) We are opening for the first time the possibility of using a genome-editing tool (CRISPR/Cas9) in strawberry plants. This methodology will be of great value, not only to the scientific community interested in this species but also to strawberry breeding companies.
4) Finally, we will use the underexploited genetic diversity present in the woodland species Fragaria vesca to identify genes involved in fruit quality traits in order to proceed to the generation of new breeding lines in the future.

In summary, TRANSFR-Q aims to uncover key regulatory genes that govern the transcriptional networks leading to the quality characters of strawberry fruits. In addition to providing an essential understanding of fruit ripening, it will set the basis for breeding quality characters in current strawberry cultivars using state-of-the-art molecular techniques.