Performing Department
(N/A)
Non Technical Summary
Heat increasingly threatens the US and world wheat production as climate change continues. In the Great Plains, heat is one of the two top yield killers, and heat tolerant cultivars are urgently needed. Breeding heat tolerance in wheat, however, is hindered by the lack of germplasm of satisfied heat tolerance and the complicated genetic mechanisms. Our goal is to improve heat tolerance of wheat via precision genome editing technology.Rubisco activase (Rca) emerged recently as a promising target for enhancing plant heat tolerance and photosynthesis. Several conserved amino acid substations in wheat Rca2 protein significantly increased temperature optimum of the protein. In this project, we will develop novel wheat germplasm with enhanced photosynthetic efficiency and heat tolerance by precise editing of TaRca2 gene as proof of concept with three specific objectives:1) Create novel Rca2 variations for enhanced thermostability.2) Evaluate mutation effect on photosynthetic efficiency and heat tolerance.3) Develop breeding-ready germplasm.These results are expected to have a positive impact on the improvement of wheat heat tolerance.
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
Goal:Improve productivity and sustainability of wheat via precision genome editing.Objectives:Create novel TaRca2 alleles for enhanced thermostability.Evaluate mutation effect on photosynthetic efficiency and heat tolerance.Develop breeding-ready germplasm.
Project Methods
Objective 1. Create novel TaRca2 alleles for enhanced thermostability. Ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco) activase (Rca) emerged recently as a promising target for enhancing plant heat tolerance and photosynthesis efficiency. The wheat genome harbors two genes, TaRca1 and TaRca2, encoding three isoforms, TaRca1-β, TaRca2-α, and TaRca2-β. In vitro analyses showed that M159I mutation increased the temperature optimum of TaRca2-β in vitro by 5ºC, and K161N and A392E mutation substitutions enhance the efficiency of Rubisco activation by TaRca2. We will install these mutations sequentially to the TaRca2 by precision gene editing using a tandem repeat-homology-directed repair (TR-HDR) strategy. To do so, we will design sgRNA to target TaRca2 sequences near the codons for M159 and A392. The sgRNA gene cassette will be mobilized into a binary vector carrying Ubi-Cas9 transgene as the CRISPR construct and synthesize 5?-phosphorylated double-stranded oligodeoxynucleotide (dsODN) containing the mutations as donor DNAs. The CRISPR construct and the donor DNA will be coated to gold particles and delivered into immature embryos of wheat cultivar Fielder by Biolistic transformation. The transgenic plants will be screened by mutation-specific PCR and validated by Sanger sequencing.Objective 2. Evaluate mutation effect on photosynthetic efficiency and heat tolerance. The homozygous mutants and wild type Fielder will phenotyped for photosynthetic efficiency and heat tolerance. To this end, ten biological replicates (pots) of each genotype will be moved into the growth rooms at growth stage Zadoks 3.0 when the first node appears. While the control room will be maintained at 20/15 ºC, the temperature in the treatment room will be raised from 20/15 °C to 36/30 °C with adequate moisture over a 48-h period and remained for duration of 10 d. During this period the leaf elongation, which positively correlates with Rubisco activation by Rca, will be examined by measuring leaf growth on a daily basis to estimate leaf elongation rate and leaf elongation duration. We will also measure leaf gas exchange to estimate photosynthetic parameters such as net photosynthesis and stomatal conductance and measure chlorophyll a fluorescence as an indirect method to assess thylakoid membrane damage. After the 10-day treatment, the plants will be moved back to the greenhouse room and allowed to mature. Spikes will be harvested separately and cataloged, grain number per spike will be recorded, and weight will be measured. The results will be compared between the genotypes in the control and treatment conditions.Objective 3: Develop breeding-ready germplasmThe TaRca mutations will be transferred into elite genetic background by crossing and backcrossing the mutants with spring wheat cultivars such as Prevail or Boost. The BC1F1 and BC1F2 will be genotyped for TaRca mutations against the transgenes. The homozygous plants will further be genotyped by genotyping-by-sequencing together with Prevail and Boost, and plants showing highest similarity to Prevail or Boost and dissimilarity to Fielder will be selected as germplasm to release to the wheat breeding community.