Improwing winter survival and quality of Norwegian cultivated strawberry

This project will complete this year, 2014, Dec. The project was very successful in terms of research and commercial results and strengthen collaboration with national and international groups. Strawberry has complicated genetics, thus the project team has applied for further funding to continue investigate candidate molecular markers at the functional level and determine their usefulness for identifying low temperature tolerant genotypes and assess cultivars and advanced breeding accessions in Graminor?s collection for their low temperature tolerance using field and laboratory tests. The project team will deliver final report by March 2014 summarizing all results. However, in the last period an experiment was performed in order to study the changes in expression profiles during acclimation in the cultivated strawberry (8X RNA-Seq). Two cultivars, Jonsok and Elsanta, were chosen based on their differing low temperature tolerance. After propagation the plants were put under acclimation temperature at 2 °C and samples were taken of crowns in triplicate at 0hs, 1hs, 5hs, 48hs, and 240hs post experiment start. RNA was isolated, quality checked, and shipped to the sequencing facility at Oslo University Hospital, where RNA-sequencing was performed on Illumina HiSeq2000 sequencers. Each experimental cell (cultivar x time point x biological replicate) was sequenced to 80 mill paired end reads (on average), giving a total of 2.4 billion PE reads. This experiment was performed in early 2014. However the data was processed in the last six months. The read data was trimmed using Trimmomatic, normalized using the in silico normalization tool in the Trinity suite, assembled de novo using the Trinity assembler, and finally the counts matrix was produced by RSEM. 417431 transcripts were produced and filtered down to 66095 transcripts based on a relatively relaxed expression condition using edgeR. The 66095-transcriptome has been annotated using Blast2GO and further downstream analyses is currently being performed including SNP detection in differentially expressed transcripts and identification of differentially expressed transcription factors in early acclimation stages. We expect to submit a paper based on this experiment to peer review in 2015. In relation to the diploid 2X freezing experiment that was performed in 2013, DNA has been extracted from two low temperature susceptible (NCGR1363 and FDP812) and one robust (Alta) Fragaria vesca genotypes. DNA was quality checked from the entire material and is ready to be shipped to the laboratory for reduced genomic sequencing and SNP marker detection. The purpose is to find SNPs co-segregating with low temperature tolerance and possibly use them in the applied selection. Depending on the sequencing results, we expect to publish a peer reviewed paper on this material. From protein research activities, the focus was in the last six month is to understand the induction of Fragaria dehydrins in response to cold, particularly investigating the apparent discrepancy between the time of mRNA accumulation and that of protein expression during acclimation. This work was the core activity of Msc research of Nelson Uchechukwu Osuagwu, HiHM, title ? Molecular characterization of novel proteins associated with low temperature stress tolerance in diploid strawberry (Fragaria vesca)?. Nelson is expected to defend his thesis on the 9 Dec 2014. The following are key parts of that investigation. A. and b. are mostly complete, significant work needed on part c. (a) Analysis of dehydrin mRNA expression by RT-QPCR as function of time during acclimation. (b) Analysis of dehydrin protein expression by quantitative western blots. (c) Purification and identification of the dehydrin species visualized on western blots. Purification and identification by mass spectroscopy is required to connect the identities of the mRNA species quantitated and dehydrin bands on western blots. This work still in progress. A technique to isolate and separate the proteins has been worked out. We need to now do this with crowns from the same species (Fragaria vesca, alta) in which the experiments a. and b. were performed. There may be material limitation issues but the hope is we can obtain enough purified dehydrins to identify by MS. In addition, analysis of proteins that respond during de-acclimation of fragaria crowns following a acclimation and freezing period. All gels have been run. We now need to quantitate spots, and identify the protein by MS. These latter items cannot be accomplished without further influx of funds. Presently writing a paper to be submitted to Frontier in Plant Biology in January ? Koehler et al.): Proteomic and metabolomic analysis of cold acclimation in strawberry, Fragaria x ananassa ?Korona?

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.