Improwing winter survival and quality of Norwegian cultivated strawberry

Last year and in this year we have developed two F2-populations from Fragaria vesca (diploid) parents diverging in low temperature (LT) tolerance. Based on results from our Planta paper (Davik et al 2013) we selected the genotypes Alta, CGR1363, and FDP81 7 to parent two not-robust x robust segregating populations (namely NCGR1363 x Alta and FDP817 x Alta). One plant from each cross - proven by microsatellites to be a hybrid plant - was selfed and gave rise to the two F2-populations. Phenotyping this mater ial was done with controlled LT-exposure. Based on LT-tests of the parents, the two F2-populations, the F1-hybrids, and the parents have been subsequently subjected to freezing tests. These tests include six weeks of acclimation at 2 °C and a subsequent f reezing exposure at -9.5 °C. We planned to perform two LT-experiments with each population and are about to finalize the last one these very days. For genotyping we have extracted DNA from these two populations (~ 200 samples) and shipped them to Instit ute for Genomic Diversity, US, for single nucleotide polymorphism (SNP) detection. They have run into problems working with strawberry and only 75% of the genotypes have given results. Their issues are still not resolved. However, they are working on it o n their own costs. In an additional acclimations experiment (three parents x 8 time points) crown and leaf tissues are investigated for candidate gene expression using qPCR and two dimensional protein separation. In this direction we have studied the rel ative expression levels of 13 transcripts to cold treatment using quantitative RT-PCR in the 3 p2x diploid cultivars. So far, our results have shown that XERO2, COR47 and ADH are up-regulated (late expression) in cold treatment in the 3 cultivars in both crowns and leaves. The CBFs have shown an early up regulated expression in all the cultivars in the leaves but shown a down regulation in Alta and FDP 187 Crowns. The ABREs were highly up regulated with cold treatment in the 3 cultivars in leaves. FvRHA11 showed a down regulation in the 3 cultivar in crowns. FvPP2C16, FvPP2C27 and FvGEM showed a down regulation expression using Actin as a reference but showed an up regulation using PP2A as reference in the 3 cultivars in Crown. Moreover, this material wil l be subjected to RNA-sequencing for deep transcriptomic analyses. A whole transcriptomic experiment has been performed using the two commercial octaploid cultivars Elsanta and Jonsok (F. × ananassa). Our own investigations (Koehler et al 2012) and ane cdotal knowledge has indicated that these cultivars show differential responses to low temperature stress. Crown tissues were sampled in triplicate at five time points during acclimation and total RNA was extracted, shipped to the sequencing facility at Oslo University for. Very deep RNA sequencing was performed and typically ~150 mill paired end 100bp reads from each experimental cell was retrieved. Initially, we performed in silico normalization the 150 mill read fastq files in order to get rid of red undant information. Typically ~30 mill reads from each experimental cell and replicate was retained in the normalized files. Since no reference genome exist for the commercial strawberry, a de novo assembly was performed using the software package Trinity . A total of 417 431 isoform transcripts were found having a GC-content of 43.2%. (Referring to the enclosed heatmap the separation between cultivars and time points is very clear). Our downstream analyses is aiming at identifying genes/metabolic pathw ays accounting for the differences in the LT robustness in these cultivars and possibly identifying polymorphisms usable in the applied selection work. The number of reads of a particular type is generally regarded as a direct reflection of gene expressio n. Hence, the counting of reads from the original sequencing files that map back to the various transcripts in the de novo assembly was done using RSEM yielding a 417 431 x 30 counts matrix, which has been imported into the edgeR for in-depth differential expression analyses. This is currently being performed.

Important characteristics for strawberry consumers are aroma, flavor, texture, health benefits, and indirectly, shelf life. In addition, a crucial consideration for producers is the winter survival/freezing damage of strawberry. A long-term goal of the st rawberry breeder is to increase winter hardiness while maintaining or even improving fruit quality. Due to the complexity involved in the regulation and enhancing freezing tolerance and fruit quality, the progress in the improvement of cultivars using tra ditional screening methods have had limited success. Therefore, the goals of this proposal are to identify molecular markers which associate with winter survival and superior quality (taste, health compounds, and shelf life) for accurate selection and pro vide an integrative understanding of the genetics and the metabolic pathways that underlie these characteristics. Strawberry production yield relies mainly on the plants cold acclimation, winter survival, and re-growth/recovery in spring time (de-acclimat ion). For the last three years, Graminor AS and collaborators have developed and adopted state-of-art molecular tools to investigate cold response in strawberry plants during the acclimation phase resulting in identifying a large number of genes, proteins , and distinct metabolites that correspond to cold/freezing tolerance in strawberry. However, the ability of plants to withstand freezing temperature also depends on their re-growth ability after winter. In this proposal we will employ cutting edge appro aches to investigate the strawberry response during the de-acclimation phase in order to optimize the chances of success in obtaining suitable markers. At the same time we will begin to characterize fruit quality at the metabolomic, proteomic, and transcr iptomic levels. This multi-faceted project will expand and further develop molecular tools for strawberry breeding, focusing on two major traits important to Norwegian producers; winter hardiness and fruit quality.