biotechnology and meiotic recombination
Three most commonly used gene editing systems. All have a DNA scanning domain and an associated nuclease capable of making single or double stranded breaks.
Jones (2015) Nature Plants 1: 14011
A SNP in the 5’ regulatory region of qSH1has been shown to cause loss of seed shattering in rice. Konishi et al. (2006) Science
The scutellum tissue of immature seed embryos (top) and protoplasts isolated from leaf mesophyll (lower pics) are ideal cell types to rapidly test gene expression of functional genes. Here showing expression of a nuclear-target GFP.
Phymocyces blakesleeanus mycelia network treated with 5 µM Cupid-GFP for 90 minutes. Imaged by phase contract (left) and or green fluorescence (right).
The objective is to develop and apply methodologies to generate novel genetic and phenotypic variation in forage grasses and clovers. We focus here on seed-shedding and control of meiotic recombination; characteristics that underpin breeding and commercial resilience in these forage crops.
Approach: To develop and apply biotechnologies such as tissue culture, transient and stable transformation, gene editing and protoplast isolation in forage crops that are highly recalcitrant to these techniques.
Potential impact: To contribute to the development of a robust biotechnology pipeline including the ability to gene edit forage crops to benefit research, and ultimately produce superior crop varieties.
Key research insights and findings: Genetics and genomics knowledge in forage crops is continuing to expand however it cannot be fully exploited because these crops lack some of the in-vitro cellular and molecular technologies necessary to underpin reverse genetics. The generation of stable transformants is a time consuming process and is currently the principal bottleneck in the development of an efficient genome-editing pipeline. To serve as a high-throughput gRNA screen for editing we have developed novel methods for isolation and transformation of Lolium protoplasts (Davis et al 2019 in press) and for transient transformation of Lolium and Brachypodium zygotic embryos.
Although robust stable transformation protocols are challenging in grass species, they are needed for many reverse genetic applications. We have established a pipeline for Brachypodium transformation (a model grass species) and re-establishing one for Lolium perenne.
Brachypodium is an model grass ideal for investigating so called ‘domestication genes’ we have identified the orthologues of the rice qSH1 gene in Brachypodium and Lolium perenne with the aim of editing functional knockouts with lower levels of seed shattering.
Because tissue culture and transformation of forage species are more challenging than for many crops, we are investigating novel methods of DNA delivery including a novel cell membrane penetrating peptide CUPID (Cellular Permeability factor in Dictyostelium). We have demonstrated that, at relatively low concentrations (1, 5 or 10 µM), CUPID-GFP can permeate cell membranes in a non-toxic and time-dependent manner. We are working on conjugating nucleic acids to CUPID with the aim of developing novel gene-delivery systems.
One of the other targets for genome editing will be key genes that underpin meiotic recombination. Genetic recombination is largely confined to the ends of the chromosomes of Lolium, a characteristic shared with other members of the Triticeae. This localisation consigns many genes to recombination backwaters, which effectively limits the genotypic and phenotypic variation available for selection by breeders. Genes that have been shown to modulate the recombination landscape, such as RECQ4 and HEI10, will be targeted, along with an exploration of factors that shown to alter patterns of recombination, such as nutrient levels
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