Functional validation of Brassica napus genes related to clubroot resistance through high-throughput CRISPR/Cas9 genome editing
Term: 3 years, ending March 2025
Status: Ongoing
Researcher(s): Wei Xiao, Randy Kutcher, Yangdou Wei, Lipu Wang, University of Saskatchewan; Yu Chen, Cargill; Jin-Long Qiu, Gao Caixia, Chinese Academy of Sciences; Gary Peng and Rui Wen, Agriculture and Agri-Food Canada
SaskCanola Investment: $248,975
Total Project Cost: $433,000
Funding Partners: Western Grains Research Foundation
Project Description
Growing clubroot-resistant (CR) cultivars in appropriate rotations remains the most effective solution to the long-term management of clubroot disease. However, the domesticated B. napus has a relatively short evolutionary history, resulting in high gene redundancy and low genetic diversity. As a result, compared to other Brassica species, B. napus has limited sources of CR genes against clubroot. The limited genetic variation has reduced the potential to enhance resistance in B. napus. Although other Brassica species have good resistance sources, the introgression of such favorable alleles through conventional breeding is time consuming, labor intensive, and requires multiple rounds of crossing and selection to generate improved varieties. With imprecise control of the genetic material introgressed, this approach can also cause unpredictable effects such as the introduction of deleterious materials genetically linked to desirable traits via linkage drag. Most importantly, currently available CR varieties carry a single, race-specific, dominant R gene, which cannot provide durable resistance and are easily broken down with a shift in the pathogen population. As a result, CR cultivars have shown substantial resistance erosion in many Alberta fields in the past few years. Therefore, seeking new CR sources and developing novel breeding strategies are urgently required to keep pace with the need to mitigate this rapidly evolving pathogen.
Purpose
Unlike time consuming and labor-intensive traditional plant breeding, the CRISPR/Cas9 based genome editing technology has been showing great promise as a new breeding approach to introduce genetic variations to develop new traits by simple, inexpensive, fast, and precise methods that can directly modifying endogenous genes. This proposed project will explore the feasibility of this tool; if successful, this will provide a new breeding tool to expedite the breeding process and, consequently, reduce the investment cost by Saskatchewan agriculture industry on breeding new cultivars compared to traditional breeding approaches. Different from traditional strategies utilizing race-specific dominant R genes, this proposal focuses on race-independent genes, which will provide durable resistance against clubroot. This strategy will avoid the risk that CR varieties are easily broken down by rapid evolved pathogen and will reduce the cost to canola growers due to loss of resistance in CR varieties to newly evolved pathogen races. This proposed project will also develop a large-scale CRISPR/Cas9 genome editing system to edit and investigate hundreds of genes at one transformation event. This will accelerate the process of functional validation of CR genes and reduce breeding cycles.
Goal
To validate functions of clubroot resistance (CR) related genes and understand molecular mechanisms of clubroot resistance/susceptibility; To establish a high-throughput CRISPR/Cas9-based gene editing platform in B. napus. This platform can be applied to large-scale functional genomic study and functional validation of candidate genes identified from QTL fine mapping, genome-wide association studies and comparative genomics/transcriptomics.
Objectives
Select up to 150 candidate genes from RNA-Seq data and confirm their expression pattern by Droplet Digital PCR (ddPCR).
Design 2 sgRNAs per gene and construct CRISPR/Cas9 sgRNA plasmid library (300 plasmid constructs).
B. napus transformation using CRISPR/Cas9 sgRNA plasmid library.
Identify and phenotype gene-edited mutant lines.