Precision breeding in wheat mediated by engineered haploid inducers

Funding Partners: Alberta Wheat Commission (AWC)

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Project Description

The main focus of this project is to develop a simple and reliable method of gene editing in wheat and to improve on the current method being used by wheat breeders to produce doubled haploids.

In Wheat breeding, one method that currently takes years off the breeding process is doubled haploid (DH) production through the maize pollination method, in which the immature embryo has to be rescued to allow further development and recovery. The goal of the proposed research is to improve seed viability following crossing by attempting to remove the endosperm block and to bring gene editing to the wheat DH production pipeline.

Research Results

The rate of wheat improvement through conventional breeding has plateaued and novel approaches are needed to achieve the gains required for population growth and to meet the challenges imposed by climate change. Creating genetic diversity through gene editing offers a means to accelerate wheat breeding. This is because natural genetic variation in crops and wild relatives is being revealed through comparative genomics and gene editing can bring this variation into wheat without the need for wide crosses. In this project we used the wheat x maize haploid induction system for performing gene editing in wheat. This allowed us to take advantage of a procedure that is currently being utilized by most wheat breeding programs for their doubled haploid production. Our project was designed to perform gene editing in wheat through engineered maize lines and to explore RNAi as a means to restore endosperm growth. We created maize haploid inducer lines expressing the Cas9 enzyme and guide RNAs for gene editing of circadian clock genes in wheat. We tested RNAi against two genes (RNA Polymerase Four – RNA Pol IV, and Fertilization-Independent Endosperm – FIE) with known roles in endosperm development. Mutations in these genes can facilitate endosperm growth in other species. The first challenge we faced in this project was getting our editing vectors into our maize haploid induction line. This took a considerable amount of time to optimize, but after careful selection of morphogenic genes and appropriate promoters to drive gene expression, a successful protocol was achieved. We are now one of a few labs in Canada to perform maize transformation routinely and at a relatively high efficiency (~10%). Our transformation pipeline then allowed us to proceed with haploid induction in wheat. Over the course of the project, we performed around 4000 hand pollinations, with one of our vectors achieving a gene editing efficiency of 4.29%. This is about a four times higher rate than that reported by Syngenta scientists. These edited wheat lines gave us several mutations in the circadian clock gene LHY (Late Elongated Hypocotyl). The advantage to this approach is that because the maize genome is naturally eliminated, these lines are non-transgenic and thus could be readily commercialized. Since bread wheat is hexaploid, mutations in any of the six gene copies allows us to fine tune gene expression. We are exploring mutations in circadian clock genes given their role in governing daily metabolic transitions that affect yield. Our ultimate goal is to enhance and extend the period of active photosynthesis.

We learned many lessons during this project, including the importance of morphogenic regulators and tissue culture conditions for maize transformation. Our success in maize transformation allowed us to address all our objectives and places us in a unique position to continue making advancements in gene editing via wheat x maize haploid induction. We also gained some insights into endosperm growth through our RNAi experiments. We saw that wheat haploid embryos grow to a more advanced developmental state in seeds pollinated from maize expressing RNAi towards the wheat RNA Pol IV gene when an early endosperm specific promoter was used. This was a landmark success because it showed that we could make improvements in haploid seed development using a maize line expressing RNAi towards wheat genes. This lesson demonstrated to us the possibility for utilizing RNAi for improving haploid induction. It also told us that more work is needed to understand early endosperm development in wheat in order to make further increases in haploid plant recovery efficiency. Since we are likely the only lab in Canada that can perform gene editing through haploid induction, we will continue this work in close collaboration with breeders to see that non-transgenic wheat lines with improved genetics can be produced for accelerating wheat improvement. Our next steps are to build maize lines that express gene editing machinery other than Cas9, to avoid potential commercialization issues. We also plan to improve our editing efficiency by using stronger zygote/early embryo-specific promoters to drive our gene editing machinery.

Introducing targeted genetic variation provides the industry with a much more efficient and rapid way for enabling breeders to develop higher yielding varieties. Such varieties would be better adapted to respond to the impacts of climate change on wheat yield.