Single-cell sequencing reveals distinct populations of Plasmodiophora brassicae in a single clubbed root.


McDonald, M.R., Sedaghatkish, A., and Gossen, B.D. 2023. Single-cell sequencing reveals distinct populations of Plasmodiophora brassicae in a single clubbed root. Presentation at Clubroot Workshop, September 24, 2023 at the 16th Intern Rapeseed Congress, Sydney, Australia.

Résumé en langage clair



Plasmodiophora brassicae is an obligate Chromist causing abnormal cell growth and club formation in the roots of brassica crops. The use of resistant cultivars for clubroot management is effective but resistance can be overcome within a few years. The pathogen exists as many different pathotypes and it is hypothesized that several are present in a field or even a single club.

To evaluate the genotypes of single resting spores extracted from a single clubbed root to determine the genetic diversity of P. brassicae within a club.

Single-cell sequencing technology was used to determine the genetic diversity of single spores or P. brassicae in a clubbed root. A single club of highly virulent pathotype 5 grown in the growth room was used. Single resting spores were extracted and cells were barcoded and sequenced. Four thousand single spores were captured from the spore suspension and sequenced using 10X genomics. Five hundred cells that had the highest sequencing quality were selected for the analysis. De novo genome assembly was performed using SPAdes genome assembler. Python api and vireoSNP were used to both cluster the reads and produce plots for the cell assignment probability, the mean allelic ratio, and the ELBO plots

Both ELBO plots and principal component analysis demonstrated that there were at least seven distinct genotypes present, and one genotype was much more prevalent (roughly 75% of the cells) than the other six. The seven genotypes differed in hundreds or thousands of SNPs, so were not the result of one or a few mutations.

The results demonstrated that many genotypes are present within a single club, and that the pathogen population is genetically (and likely pathogenically) very diverse. This could happen as the result of many individual zoospores infecting in a single plant, each contributing to club formation. The presence of multiple genetically-distinct genotypes in a single club strongly supports the hypothesis of balancing selection in P. brassicae populations, in which selection works to maintain genetic polymorphisms (or multiple alleles) within a population. This diversity, in turn, explains how the pathogen population can shift rapidly when single-gene host resistance is applied, since a range of pathotypes is already present in the population.

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