Purifying genotypes of Plasmodiophora brassicae and developing SNP markers linked to races of P. brassicae populations collected in western Canada

Project Summary

Background

The clubroot casual pathogen Plasmodiophora brassicae exists as mixtures of pathotypes in the soil. A recent study showed that most of single galls collected in the canola fields in Alberta consisted of more than one strains or pathotypes.  Results from resistance tests can lack consistency based on mixed pathotypes in breeding programs, which can cause confusion in resistance labeling.  Many attempts have been made to produce single spore derived isolates to obtain pure genotypes of P. brassicae. However, the practicability of the methods is poor and their infection rates are very low and unreliable due to the small size of the resting spores.  

To overcome the issues, the researchers will be experimenting to use resting spores derived from single root cells through protoplast isolation or single calluses through protoplast culture of single cortical cells to produce near pure genotype isolates (NPGI) and then performing a two-step selection to obtain pure genotype isolates.

Plasmodiophora brassicae  has been classified into various pathotypes in Canada using the Canadian Clubroot Differential (CCD) set. Most of the CCD lines are non-canola crops, which belong to vegetable or fodder Brassicas with unknown genetic information on clubroot resistance (CR). Alternately, a set of near isogenic lines (NILs) containing single identified CR genes will become available to replace the CCD set. However, pathotying with the CCD or race profiling with the NILs is a phenotyping based approach, which can be tedious and time consuming.

Purpose

A genotyping based method could be an ultimate solution for race profiling. One prerequisite for this is to determine the presence of  avirulence (Avr) genes in P. brassicae corresponding to CR genes. Association mapping is a method that exploits the variation in a collection of genetically diverse materials. It can be used for the identification of single nucleotide polymorphisms (SNP) that are associated with casual genes. The researchers will perform the identification of Avr genes in P. brassicae through an association mapping approach after the phenotypic data from the interactions of the canola NILs and NPGIs of P. brassicae are collected.  Genome sequencing of NPGIs will be performed and SNP markers associated with races of P. brassicae will be developed. In addition:

• Genotyping based race profiling will be used to characterize P. brassicae population in western Canada, generating information on pathogen population dynamics for industry and producers in use of developing and growing cultivars for effective control of clubroot. This could lead to a breakthrough in monitoring changes in race structure of the pathogen, providing critical insights into pathogen race dynamics.
• Inconsistency and variation in canola disease assessment caused by mixture of pathogen populations will be minimized, which will provide more accurate information on CR spectrum for each cultivar, ensuring canola producers have effective CR cultivars for clubroot management.  
• The genetic resources developed from the project will make the identification and fine mapping of novel CR genes much more efficient, so canola breeders and geneticists can obtain more precise and richer genetic information from their routine screening and assessment.
• It will open up new opportunities to better understand the interaction between the host and the pathogen, so more robust resistance strategies could be developed for sustainable management of the disease.  

Objectives

1. Develop an efficient method to produce NPGI
2. Produce diverse NPGIs from clubroot galls collected in western Canada
3. Determine race profile for each NPGI
4. Carry out genome sequencing of selected NPGIs 
5. Develop SNP markers tightly linked to each Avr gene and obtain pure genotype isolates of P. brassicae