A study of endogenous transposon activity in grapevine (Vitis vinifera L.)

Transposable elements (TEs) are recognised as a significant and ubiquitous component of eukaryotic genomes. This thesis contributes to the current knowledge of these elements by describing the stimulated mobilisation of multiple class I and class II elements from eight TE superfamilies in grapevine somatic embryo cultures following stress treatments and tissue culture, leading to the production of new vegetative material. An in silico analysis of class I TEs in the grapevine genome revealed that although the majority of the 137 defined retrotransposon families exist mainly as eroded fragments, several families show evidence of recent mobility or appear in transcript databases. Based on these results, a high-resolution S-SAP technique was used to identify insertion polymorphisms of three Ty1-Copia TE families (Edel, Noble and Cremant) and one Ty3-Gypsy family (Gret1) across 32 grapevine genotypes, demonstrating the contribution of these elements to genetic diversity in Vitis. By supplementing bacterial suspensions with an organosilicone surfactant, the efficiency of Agrobacterium-mediated transient transformation of grapevine leaf tissue was improved by an average of 72-fold. This protocol was used to show that of the above four TE families, only the long-terminal repeat (LTR) sequence of Edel is able to drive expression of reporter genes in grapevine leaf tissue, but all four are capable of stimulating expression in the model plant N. tabaccum. After two generations, the LTR sequences of Gret1 and Edel no longer induced reporter gene expression in stable N. tabaccum transgenic plants, but the LTR sequences of Cremant and Noble retained a wound-responsive expression pattern. Due to their sessile lifestyle, plants are forced to endure and adapt to environmental challenges. Biotic (e.g. pathogen attack) and abiotic (e.g. wounding / drought) stress events have previously been shown to stimulate the activity of certain TEs in plants. In this study, transcripts all four TE families (Gret1, Edel, Cremant and Noble) were found to increase when Pinot noir embryogenic callus (EC) cultures were co-cultivated with live yeast species endemic to New Zealand vineyards. Abiotic stresses and fungal extracts did not elicit the same response. A total of 24 new TE polymorphisms, relating to all four of the TE families analysed, were detected by S-SAP in a population of 183 vines regenerated from EC cultures. The majority (14) of these polymorphisms were found in vines regenerated from yeast-stressed EC tissue. The regenerated vines also displayed a variety of phenotypic abnormalities. Finally, whole-genome sequence data from twenty of the regenerated vines revealed that vines passaged through somatic embryogenesis experienced a general activation of the mobilome, resulting in an average of 64 new TE insertions per plant from both TE classes. Yeast stress at the embryogenic callus stage increased the number of new TE insertions identified by 63%. Despite a strong general bias against coding DNA sequence (CDS) insertions, approximately 2 insertions were found in this context per plant. These data are discussed with regards to the biological implications of endogenous TE mobilisation and the potential use of these elements for saturation mutagenesis in grapevine