The Rosaceae contains many important commercially grown fruit crops. No comprehensive genomics platform is currently under development for fruit crops, giving functional genomics studies with wild strawberry (Fragaria vesca L.) the potential of identifying genes important in fruit crops. Fragaria vesca has a small genome size compared to the cultivated strawberry, Fragaria à ananassa Duch. (164 vs. 600 Mbp per 1C nucleus). This feature, in addition to a short life cycle (12-16 weeks) and small plant size make F. vesca a good candidate for a model plant for genetic and molecular studies. The specific objective of this work was to develop an efficient high-throughput Agrobacterium-mediated transformation protocol to generate an insertional mutant population to support the justification of F. vesca as a model organism for rosaceous crops. The transformation techniques described by Alsheikh et al. (2002) and Oosumi et al. (2005) were modified and applied to a range of germplasm obtained from the USDA National Germplasm Repository. We found that the modifications made to the Alsheikh protocol were unsuccessful when applied to our germplasm. With the Oosumi et al. (2005) protocol, transformation efficiencies ranging from 11 to 100% were obtained for two accessions when explants were exposed to varying durations on TDZ containing medium during shoot regeneration. The transformation efficiency was given as the mean number of GFP+ plants obtained per primary explant cultured. Multiplex PCR, for amplification of the hptII and GFP genes, was performed on a random sample of GFP+ plants to verify insertion of the T-DNA. The statistical power of our experiment was insufficient to detect treatment effect but based on our findings the transformation efficiencies were high enough to justify PI 551572 for use in the high throughput transformations that are required to generate a population of insertional mutants large enough for gene discovery in F. vesca. / Master of Science

We investigated the transferability of 20 Fragaria vesca microsatellite primer pairs to 13 Fragaria vesca accessions, six Fragaria species and ten commercially important species in Rosaceae. Genetic diversity studies were carried among 16 diploid Fragaria accessions using these polymorphic microsatellites. The average number of alleles amplified for a polymorphic locus was 4.7 with maximum being 8.0 and minimum being 3.0. Observed heterozygosity ranged from 0.00 to 0.84 with an average of 0.28. Expected heterozygosity ranged from 0.33 to 0.91 with an average of 0.76. Power of discrimination varied from 0.43 to 0.92 with an average of 0.78. Transferability of microsatellites to F. orientalis (4x) and F. Ã ananassa (8x) was high, i.e., 18 (90%) primers produced amplicons. Cross species amplification within Rosaceae using these primers showed limited transference. Four microsatellites showed amplification for different species in Rosaceae. Products generated by UDF-003 and UDF-018 primers were sequenced. Sequencing results for UDF-018 showed that three species, i.e., Pyrus calleryana, Prunus persica and Rubus idaeus contained the expected microsatellite whereas another four, i.e., Cotoneaster salicifolius, Rosa rugosa, Amelanchier arborea and Potentilla fruticosa had conserved regions resulting in generation of amplicons. For UDF 003, Spirea xbumalda and Prunus persica did not contain a microsatellite although there was some sequence similarity with Fragaria. Size homoplasy, i.e., alleles of identical size with different numbers of repeats within the SSR was observed among Fragaria and Rosaceae species for primer UDF-018, suggesting a need for caution when interpreting SSR variation from band migration in the absence of DNA sequences. / Master of Science

Tyrimo metodai: Spektrofotometrinis flavonoidų nustatymas naudojant aliuminio chloridą, fenolinių junginių nustatymas Folin-Ciocalteu metodu, antioksidacinio aktyvumo nustatymas ABTS ir DPPH metodais, chlorogeno rūgšties nustatymas efektyviąja skysčių chromatografija. Tyrimo tikslas: palyginti flavonoidų, fenolinių junginių kiekio ir antioksidacinio aktyvumo skirtumus kelių Rosaceae Juss. šeimos rūšių lapų žaliavose. Tyrimo uždaviniai: spektrofotometriškai nustatyti flavonoidų ir fenolinių junginių kiekį, chromatografiškai - chlorogeno rūgšties kiekį kelių Rosaceae Juss. augalų šeimos rūšių lapuose iš skirtingų Kauno apskr. augaviečių; spektrofotometriškai DPPH ir ABTS metodais nustatyti tirtų žaliavų antioksidantines savybes; palyginti kelių aviečių lapų arbatų iš vaistinės ėminių sudėtį ir antioksidacines savybes. Išvados: kiekybiškai turtinga sudėtimi pasižymėjo žemuogių lapai, nustatyta stipri bendro flavonoidų kiekio ir antioksidacinio aktyvumo koreliacija. Vidutiniškai fenolinių junginių gudobelių lapų žaliavose nustatyta 1,18 karto daugiau, nei žemuogių lapų žaliavose ir 3,18 karto daugiau, nei aviečių lapų žaliavose. Nustatyti antioksidantinio aktyvumo skirtumai: žemuogių lapų DPPH laisvųjų radikalų surišimo geba buvo 1,81 karto didesnė nei aviečių lapų ir 1,67 karto didesnis, nei gudobelių lapų. Aviečių lapų ABTS trolokso ekvivalentas buvo 1,01 karto didesnis nei žemuogių ir gudobelių lapų. Didžiausias vidutinis chlorogeno rūgšties kiekis nustatytas gudobelių... [toliau žr. visą tekstą] / Research methods: spectrophotometric determination of flavonoids using aluminum chloride, determination of phenolic compounds using Folin - Ciocalteu method, determination of antioxidant activity using ABTS and DPPH tests, determination of chlorogenic acid using high performance liquid chromatography. The aim: to compare flavonoids, phenolic compounds, chlorogenic acid contents and antioxidant activity in raw leaf materials of some Rosaceae Juss. family species. Objectives of the study: to determine flavonoids, phenolic compounds and chlorogenic acid content in leaves of some Rosaceae Juss. plant species from different locations of Kaunas county; to determine antioxidant properties of tested raw leaf materials with DPPH and ABTS methods; to compare a composition and antioxidant properties of some different raspberry leaf teas from the drugstore. Conclusions: The highest total flavonoid content average was measured in strawberry leaves, with strong correlation between total flavonoid content and antioxidant activity. The phenolic compounds content average of hawthorn leaves was 1.18 times higher than average of strawberry leaves and 3.18 times higher than average of raspberry leaves. The antioxidant activity differences depended on the method: DPPH free radical scavenging ability of strawberry leaves was 1.81 times higher than ability of raspberry leaves and 1.67 times higher than ability of hawthorn leaves. The ABTS trolox equivalent average of raspberry leaves was 1.01... [to full text]

Le fraisier diploïde (Fragaria vesca, 2x=14) comporte deux modes de reproduction : la reproduction sexuée via la floraison, et la multiplication végétative via la production de stolons. Cette espèce présente également deux types de floraison, non-remontants ou remontants, selon la période d’initiation florale du génotype. Chez le fraisier diploïde, le gène responsable de la remontée florale a été identifié comme étant l’homologue du répresseur floral TFL1 et celui du stolonnage reste inconnu. L’objectif de cette thèse est d’identifier le gène responsable du stolonnage et de mieux comprendre la balance entre les deux modes de reproduction, sexuée et asexuée, chez le fraisier diploïde. Une approche de cartographie fine associée à du NGS a permis d’identifier le gène responsable du stolonnage, FvGA20ox4. Ce gène intervient dans la voie de biosynthèse des gibbérellines. Les génotypes non-stolonnants présentent une délétion de neuf bases qui ne modifie pas le cadre de lecture du gène. L’observation cytologique des méristèmes axillaires montre que les génotypes mutés présentent un allongement des axillaires comme les génotypes sauvages mais que ces bourgeons nécrosent par la suite. Une étude de l’activité enzymatique de la protéine délétée de trois acides aminés a montré qu’elle était inactive et ne permettrait pas de transformer les précurseurs de GA (GA12) en GA intermédiaires, qui seront convertis en GA actives via d’autres enzymes. Chez les génotypes mutés, un apport de GA3 permet de restaurer l’émergence de stolons. L’étude de la balance entre les deux modes de reproduction a été étudiée au sein d’une population en ségrégation pour le stolonnage et la remontée florale. Une approche QTL a permis de confirmer le rôle des gènes TFL1 et GA20ox4 dans le devenir des méristèmes en floral ou stolon respectivement et d’identifier deux loci présents sur le LG3 et LG4 impliqués dans la variation quantitative de la floraison ou du stolonnage respectivement. Pour le stolonnage, une interaction forte entre TFL1 et GA20ox4 a été mise en évidence par ANOVA. De plus, le suivi hebdomadaire de cette population pendant deux ans a montré que le processus de remontée florale prédominait celui de stolonnage. En conclusion, les données obtenues permettent une meilleure compréhension du stolonnage et de la balance entre les deux modes de reproduction chez le fraisier diploïde. / The diploid woodland strawberry (2n=2x=14) is a good model for genetic and genomic studies since it presents a small genome completely sequenced and its generation period is short. This species displays two modes of reproduction, sexual with flowering and vegetative through runnering, which is the capacity to form stolon. In addition, this species displays contrasted genotypes for their variation of flowering length because of difference in the period of floral initiation. This trait is under the control of TFL1. However, the genetic and molecular control of vegetative reproduction is poorly understood as the one of the balance between the two modes of reproduction. The objective of this work was to characterize the gene responsible of runnering and to decipher genetically and physiologically the balance between the two modes of reproduction. By developing a strategy combining linkage map, bulks and NGS, we found that runnering is controlled by the gibberellin (GA) oxidase, FvGA20ox4. The loss of runnering function is caused by a deletion of nine nucleotides, which does not change the open reading frame of the RNA and leads to a protein shortened by three amino acids. The GA oxidase activity was confirmed in enzymatic activity assays with recombinant protein. In addition, the loss-of-function can be restored by exogenous GA3 application. The genetic architecture of the balance between two reproductive modes, sexual and vegetative, was deciphered based on QTL approach. Results showed that, beside the known roles of TFL1 and GA20ox4 genes in promoting perpetual flowering and runnering processes respectively, two loci localized on LG4 and LG3 were linked to the quantitative responses of these processes. In addition, TFL1 and GA20ox4 genes showed epistatic interaction, and the process of flowering was maintained as priority over runnering, which decreased. These results provided new insight in the genetic control of the relationship between sexual and vegetative reproduction.

The world faces severe challenges in providing food security for a growing world population during climate change. This puts pressure on modern agriculture, including adapting crops to new environments and cultivation on less acreage. The tools for adapting crops exist within a species' genetic diversity.  Crop wild relatives (CWR) are wild taxa with a close genetic relationship to our crops. CWRs contain a breadth of genetic adaption for various habitats due to their wide geographical distribution. This invaluable diversity of genes is essential for improving breeding of crops and therefore needs to be sustainably conserved in situ to prevent the loss of the future crop adaptation.  Fragaria vesca appears on the list of priority CWRs for conservation within the Nordic region.  To create an efficient conservation strategy for F. vesca, knowledge about the genetic differences between populations within the Nordic region must be obtained. Therefore, this study aimed to optimise PCR protocol for 10 microsatellite primers in F. vasca. The annealing temperature was successfully optimised for all 10 primer pairs. Two of the primer pairs revealed intra-specific diversity. The study also found support for the earlier discovered genetic divergence between Icelandic and other European populations.

Abiotic stresses - drought, salinity or extreme temperatures - cause morphological and physiological changes, affect growth, development and plant productivity. In crops, these limitations often reduce yields up to 50%. Plants have developed various mechanisms ensuring some degree of abiotic stress tolerance, which undoubtedly include changes in carbohydrate metabolism. The nature of many defense mechanisms is not yet fully understood, moreover, most findings were obtained by studying model plants and little is known about their validity in crops. This work was therefore focused on woodland strawberry (Fragaria vesca L.) from economically important family Rosaceae. Controlled conditions and targeted exposure to stress factors was obtained through the cultivation in vitro. Strawberry plants and callus cultures were exposed to various stresses and their combinations, better reflecting the natural conditions. At various stages of stress reaction, growth characteristics, changes in the content and the spectrum of soluble carbohydrates and oxidative stress levels were monitored and compared with plants growing under optimal conditions. Abiotic stress responses of strawberry plants were very inhomogeneous, which complicated the obtaining of statistically significant results. The only significant response...

The aim of this study is to provide a further insight into influence of environment on clonal plants. The study focuses particularly on effects of fertilization level and light availability on production and growth of clonal organs. Three experiments were carried out within the study, targeted to elicit influence of these environmental conditions or clonal interactions on six species of clonal plants. Interspecies dependencies on these conditions was compared, regarding habitat occurrence of these species. A comparison was made also between species producing rhizomes and stolons. The experiments revealed that five of six studied species show significant relationship among at least one environmental condition and parameters of clonal reproduction. Most of the species showed higher elongation and production of clonal organs in relation to fertilization level. On the contrary, only three species reacted significantly to the light availability level by alternation of at least one parameter of clonal reproduction and the light availability level affected each species differently. A strong influence on production and elongation of clonal organs had also a size of a plant. There was not found significant difference in influence of environmental conditions on clonal reproduction among plant families. It...

Indiana University-Purdue University Indianapolis (IUPUI) / Strawberry (Fragaria spp.) is an agricultural crop grown often in temperate regions that has high variability in its susceptibility to freezing injury. To breed cultivars for frost and freezing tolerance, identification of molecular markers associated with low temperature tolerance is advantageous. In this work, I investigated a high-throughput method for protein assays and western blotting. Success in streamlining these processes saves an immense amount of time and allows for the processing of more samples and obtaining larger datasets. Thirty-three octoploid varieties were tested for their accumulation of five different potential biomarkers in response to cold exposure. It was found that total dehydrin content, has the strongest potential to be reliable biomarkers for breeding programs. Previous work identified seven putative dehydrins in Fragaria, where two were purified and positively identified by mass spectrometry and determined to be COR47-like (SKn) and XERO2-like (YnSKn). This work demonstrated that cold tolerance positively correlated with dehydrin protein expression levels. To understand the cold-regulated expression of dehydrins as a function of cold exposure time, the levels of transcripts and corresponding proteins were examined in strongly cold tolerant (Alta) and lesser cold tolerant (FDP817, NCGR1363) Fragaria diploid genotypes. The COR47-like (SKn) and XERO2-like (YnSKn) dehydrins both had higher transcript accumulation and protein levels in the more cold tolerant line in comparison to the two less cold tolerant lines. Lack of correlation between transcript and resulting COR47 protein level in Alta were observed at several different timepoints, where protein accumulation preceded an increase in RNA. This trend was not seen with XERO2. This initiated an investigation to discover at what level COR47 is being regulated. First, the COR47 coding region was sequenced for all the genotypes to confirm against the predicted sequence. In addition, since two isoforms of the COR47 gene exist, and could possibly explain the discrepancy in transcript counts, primers were designed for both isoforms and RT-qPCR was performed to examine the transcripts of COR47 more closely. Through examination of the non-congruence of COR47 transcripts and protein, it was found that transcriptional mechanisms of regulation are not involved, and that post transcriptional and post-RNA splicing mechanisms are likely to be responsible for the observed trend in Alta. Conclusions from this work demonstrate that dehydrin transcripts and dehydrin protein accumulations are strong potential biomarkers for identifying low temperature tolerance in diploid strawberry.