Sequencing Australian wine grapevine germplasm
Summary
Objective
This is the first phase of a project that aims to sequence and analyse all of the commercial grapevine germplasm in Australia. This will provide a complete foundational dataset that encompasses the breadth of genetic diversity in grapevine that is available to Australian grapegrowers and winemakers and will provide the knowledge base required for cultivar and clone identification services.
Background
The identification of grapevine cultivars is complex due to the legacy of many centuries of cultivation in many countries and the significant movement of material between regions and countries. While grapevines can be unequivocally typed to a variety level using morphological differences (traditionally ampelography), clonal identification currently depends on tracing supply records back to the point of origin. Such records are not always available or reliable, particularly for older accessions. Genome sequencing technology provides the most highly detailed methodology for defining grapevine cultivars and, more importantly, clones of those cultivars.
Clone identification based on genetic differences has yet to be fully explored, either here or overseas. Previous work by the AWRI (AWR 1701-4.3.1) was able to define specific Chardonnay clones with the use of single nucleotide polymorphisms (SNPs) that had accumulated during the history of Chardonnay propagation, in comparison to a reference genome sequence. Similar work in a current project (AWR 2001) aims to develop a genetic database of Pinot Noir clones, which will enable key source blocks lost in the Cudlee Creek bushfires to be replaced with verified, true-to-type clonal material.
A compilation of holdings by vine improvement groups and commercial nurseries suggests that there are more than 150 cultivars of wine grapes in Australia, with more than 850 unique clones. Many cultivars exist as singletons. As clones are available from more than one supplier, this project aims to sequence clones from several independent suppliers. When this is considered, the degenerate clone set approaches 1500 samples (including singletons). There is also considerable variation in the number of clones available for each cultivar, such that the top five cultivars (by degenerate clone number) account for around 40 per cent of the total number of clones. It is these cultivars that will be represented the first tranche of the project, which will sequence up to 500 clones.
Research approach
Given their importance to the Australian wine sector, combined with the large number of available clones, sequencing efforts will be initially directed at cultivars Chardonnay, Pinot, Shiraz, Riesling and Cabernet Sauvignon. The project will:
- Collect samples of grapevine germplasm representing the complete catalogue of available cultivars and clones from key industry sources, including vine improvement associations and independent nurseries. All clones will be tagged, and sampling recorded using GPS coordinates.
- Generate high-quality reference genomes for the cultivars Shiraz and Riesling, using a selected individual DNA sample that represents a commonly-used clone originating from high-provenance material for Oxford nanopore sequencing and de novo assembly.
- Sequence samples of DNA from around 500 individual clones using standard Illumina short-read sequencing. These will be aligned to a relevant reference sequence for that particular clone in order for single nucleotide variants (SNPs) relative to the reference sequence to be determined, using pipelines already established at the AWRI. Sequence data for Chardonnay and Pinot Noir clones will be added to existing data sets.
- Align clonal sequences to the relevant cultivar reference genome in order to identify differences between the clones. Establish sets of single nucleotide polymorphisms (SNPs) to form a cultivar-specific clonal phylogeny and as a basis of a diagnostic molecular tool to distinguish between grapevine clones. Clustering of results from samples assumed to be from the same clone will be assessed to investigate inter-clone variation and to formulate a genetic-identity-by-consensus.
- The data produced will be combined with a projected future project that will sequence a further 500 accessions across several additional varieties.
Sector benefits
The project will initiate a foundational dataset that encompasses the breadth of genetic diversity in grapevines in Australia. This will provide a framework around which new material can be assessed before importation and form the basis for development of a commercial service to identify grapevine clones, an analytical process that currently is not possible. As Australia also has some of the oldest grapevine plantings globally, this work will also provide the means to compare the genetics of these historical grapevine plantings with modern commercial clones.