Elevated transcription of transposable elements is accompanied by het-siRNA-driven de novo DNA methylation in grapevine embryogenic callus

Background: Somatic variation is a valuable source of trait diversity in clonally propagated crops. In grapevine, which has been clonally propagated worldwide for centuries, important phenotypes such as white berry colour are the result of genetic changes caused by transposable elements. Additionally, epiallele formation may play a role in determining geo-specific (‘terroir’) differences in grapes and thus ultimately in wine. This genomic plasticity might be co-opted for crop improvement via somatic embryogenesis, but that depends on a species-specific understanding of the epigenetic regulation of transposable element (TE) expression and silencing in these cultures. For this reason, we used whole-genome bisulphite sequencing, mRNA sequencing and small RNA sequencing to study the epigenetic status and expression of TEs in embryogenic callus, in comparison with leaf tissue. Results: We found that compared with leaf tissue, grapevine embryogenic callus cultures accumulate relatively high genome-wide CHH methylation, particularly across heterochromatic regions. This de novo methylation is associated with an abundance of transcripts from highly replicated TE families, as well as corresponding 24 nt heterochromatic siRNAs. Methylation in the TE-specific CHG context was relatively low over TEs located within genes, and the expression of TE loci within genes was highly correlated with the expression of those genes. Conclusions: This multi-‘omics analysis of grapevine embryogenic callus in comparison with leaf tissues reveals a high level of genome-wide transcription of TEs accompanied by RNA-dependent DNA methylation of these sequences in trans. This provides insight into the genomic conditions underlying somaclonal variation and epiallele formation in plants regenerated from embryogenic cultures, which is an important consideration when using these tissues for plant propagation and genetic improvement

Using mobile DNA to increase grapevine diversity

Mobile DNA sequences harbour the potential to generate new genetic variations, through mutations caused by their occasional rearrangement of the genome. By exposing grapevine cells to stress conditions such as temperature shock and microbial populations, we have demonstrated that the rate at which this change occurs can be artificially modulated. By increasing the activity of mobile elements, we can effectively accelerate the natural genetic diversification of a plant population. This provides a mean to access genetic and physical traits not currently extant in the crop species

A brief history of DNA testing in vines

Although DNA testing has been a commercial reality since the 1980s, varietal identification in grapevine was only developed in 1996 and uptake of this technology has been slow - particularly in New Zealand. A key reason for this is the traditional reliance on ampelography (identification based on leaf shape) and high initial prices per test (in excess of US$1,000). However, using data obtained from the sequencing of the entire grapevine genome, we have been able to develop genetic tests that are able to discern grapevines at the clone, as well as variety level. Adoption of new, automatable technologies has increased sample throughput, allowing samples to be tested for as little as$60. These advances make DNA testing a compelling and economical tool for the New Zealand Industry

Using mobile DNA to increase grapevine diversity

This paper was the winning entrant for the David Jackson Prize for 2013, awarded by the Center for Viticulture and Oenology, Lincoln University. The paper focuses on the molecular mechanisms by which genetic diversity naturally accumulated within grapevines and the affect that environmental stress events have on the rate at which this occurs. It goes on to report on research being done at Lincoln University that seeks to use this knowledge to produce novel elite clones

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

Watching vines evolve: Genome-wide methylation changes and small RNA accumulation that accompanies the production of new grapevine clones

A presentation about the research carried out at Lincoln University's Grapevine Genetic Improvement Program

Accelerated clone evolution

A presentation on potential use of genetics techniques for the development of new grapevine clones for the New Zealand wine industr

Viticulture genetics in New Zealand

Invited speaker at a meeting of the NZW governing board. Presented an overview of genetics technologies applicable to crop improvement, including breeding, mutagenesis and transgenesis. Reviewed how these apply to the international wine industry, and the challenges and opportunities facing the New Zealand wine industry in particular

New horizons in research: Viticulture genetics in New Zealand

Modern biotechnologies are enabling an unprecedented understanding of the genetic differences among grapevine varieties and the natural interactions between the environment and a vine’s DNA. This talk will review the impact that various genetic technologies have in the unique context of the local wine industry and the New Zealand legislation on genetic modification (GM). It also aims to explain how next-generation DNA sequencing technologies are being used locally to better understand the elite grapevine varieties grown in New Zealand and accelerate the production of novel ‘NZ-native’ clones

Harnessing the value of bud-sport mutations

Despite a low contribution by volume to the global market, the New Zealand wine industry has managed to build a respected international reputation. This has largely been achieved by exporting wines that present flavour profiles that are both interesting and sometimes unusual for their variety. By taking advantage of the diversity of flavours that can be obtained from established varieties, the local wine industry has been able to place itself at the high-value end of the market. However, in order to maintain this reputation as the industry matures, a continued effort to offer unique and diverse wine styles will be essential