Periodic Reporting for period 1 - SomaGrapeGenome (Fast-forward genomics of somatic variants adaptable to climate change for cultivar innovation in grapevine) Reporting period: 2018-09-01 to 2020-08-31 Summary of the context and overall objectives of the project Grapevine, with its derived products constitutes, as measured by cultivated area and economic volume, the most relevant fruit crop in the world (7.6 million ha of vineyard and 30.4 billion Euro trade wine value worldwide in 2017). Viti-viniculture is particularly important for the economy of Europe that hosts 50% of the worldwide vineyard. By the time being, winemakers face unprecedented challenges: they must meet a growing demand for quality and recognizable wines, and at the same time, there is a need to make viticulture more sustainable by decreasing the waste of precious resources. In addition, cultivation and production has to become better adapted to current and future climates. Global warming is threatening the production of quality wine in classical winegrowing regions. Increasing temperature and drought episodes alter grape fruit ripening and composition, leading to the production of excessively alcoholic wines that are unbalanced in colour and flavour. Adaptation of the plant material would be a suitable strategy to overcome these challenges. However, this strategy is limited as both conventional breeding and genetic modification result in cultivars that are unlikely to become widely accepted by the wine market in the short-term. A viable alternative is the use of somatic variants that emerge from spontaneous somatic mutations throughout long periods (in many cases even centuries) of clonal propagation of the same grapevine cultivar. The general goal of this project was to improve our understanding of the origin of somatic variation, so that it can be more effectively exploited as a natural source of diversity for the improvement of elite wine cultivars.In this MSCA action we focused on the clonal improvement of Tempranillo, a Spanish cultivar appreciated worldwide for the production of full-bodied red wines. Tempranillo is the most widespread red wine cultivar in the Iberian Peninsula and the third in the world. Natural somatic variants of Tempranillo with ripening features adaptable to quality wine production under predicted future climate have been selected. For this innovation to become feasible, it is necessary to identify the genetic origin of this variation. Knowledge of the causal somatic mutations will enable the development of reliable molecular markers for the identification and protection of selected improved variants. To this end, we proposed the following aims:1. Produce a de novo-assembled reference genome for Tempranillo.2. Characterize at the molecular level the somatic variation of Tempranillo clones with ripening phenotypes suitable for quality wine production under warmer climate.3. Locate the somatic mutations responsible for climate change adaptable phenotypes in selected clones. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We sequenced the genome of Tempranillo with last generation long-read sequencing Pacific Biosciences (PacBio) and Oxford Nanopore Technology (ONT). For the genome assembly we followed cutting-edge trio approach to disentangle the genetic diversity hosted in the maternally and paternally inherited chromosome copies or haplo-phases. We sequenced DNA from Albillo and Benedicto, the parental cultivars that, through a sexual cross, gave rise to the ancestral Tempranillo seed. Small stretches of parent specific sequences were detected, which served to classify the Tempranillo long-reads in bins of sequence from maternal or paternal origin that were assembled independently. Contiguous and complete assemblies (~480 Mb size) were obtained for the two Tempranillo haplo-phases. We also used PacBio long-reads for the sequencing of full-length gene transcripts to obtain accurate annotations of the assembly with more than 35,000 genes detected per haplo-phase.Based on the de novo assembly, we searched for genetic variation in 10 re-sequenced clones of Tempranillo that were selected for relevant agronomic traits, including increased fruit colour or low fruit sugar accumulation (convenient for quality wine production in warmer climates) or loose bunches that are less prone to fungal diseases affecting fruits. We detected thousands of somatic mutations in each clone, indicating for a high impact of somatic variation throughout long cycles of clonal propagation. Combined with the de novo gene annotations, putative functional variants were identified as candidate responsible mutations related with improved features in different Tempranillo clones. Gene expression studies in fruits identified altered metabolic processes that could be in line with the putative effects of the candidate genes. These results open possibilities for further research to prove the effect of these mutations and to exploit them for the traceability and commercialisation of the improved Tempranillo clones.Results of this action have been presented in two international scientific conferences. We are preparing two manuscripts that will be published open-access to make our final findings widely available. The progress of the action had an extensive dissemination through social media. Results were presented in one online and one on-site seminar engaging with international researchers and the industry sector. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We combined two cutting-edge sequencing technologies as an optimized input for de novo genome assembly. We used an innovative pipeline that leveraged cultivar pedigree information for phased genome assembly. With this pipeline we were not only able to fully phase a genome assembly for the first time in grapevine, but also produced the most contiguous assembly (near chromosome-arm level) reported in this species. We also included a last-generation approach for full-length and high-fidelity sequencing of gene transcripts to be collapsed in assembly annotations. Collectively, we show that a combination of long-read sequencing with trio binning is useful to produce high-quality genome assemblies in highly heterozygous organisms of known pedigree, as it is the case in many grapevine cultivars.We used the Tempranillo de novo genome assembly as a reference to identify somatic mutations leading to features suitable for quality production under climate change conditions. With 10 selected somatic variants, our study involved the largest genomic characterization of cultivar clones selected for improved agronomic features so far. Thousands of somatic mutations per clone were called, showing the potential of somatic variation to produce genetic diversity. This large-scale genomic comparison enabled the detection mutations with putative functional effects leading to the specific improved productive features. These findings show how de novo genome assemblies can be exploited to detect genetic variation useful for cultivar improvement and clonal tracking. In conclusion, findings in this MSCA action demonstrate that extensive somatic genetic variation accumulates in grapevine cultivars. We also show that this diversity includes natural genetic variants that can be selected to enable a quality and sustainable production in viticulture and specifically, under warmer conditions related with climate change. Our project highlights a suitable way for a sustainable adaptation of viticulture to current and upcoming scenarios and challenges. The identification of causal somatic mutations enables the technological traceability of the improved germplasm through molecular markers, ensuring the establishment and commercialization of this valuable germplasm resource.