A contamination-reduced genome of Plasmodiophora brassicae with comprehensive functional annotation.

Plasmodiophora brassicae causes clubroot disease in brassica crops worldwide but cannot be cultured outside its host, complicating its genome sequencing. Previous efforts relied on infected plant tissues and subtracting host DNA, which left microbial contamination and reduced the accuracy of the genome sequence. Single-cell genome sequencing (SCS) offers a promising solution by isolating individual cells free of host of microbial DNA and improving assembly accuracy. We applied a single-cell genome sequencing approach to ~ 4,000 protoplasts isolated from a highly virulent P. brassicae pathotype 5X population collected in Canada. The de novo assembly yielded a 23.3 Mb genome containing 8,758 predicted genes. Functional annotation using InterProScan identified 176,410 total domain hits, representing 6,809 distinct domain annotations and covering nearly all predicted genes, a substantial improvement compared to earlier annotations that covered only 34–57% of genes. We also identified 22 genes associated with virulence-related domains. A comparison with the previous reference genome revealed the de novo assembly of single cells was ~ 8% smaller, suggesting the removal of 1.9 Mb of contaminant sequences. This study demonstrates the value of SCS for overcoming contamination challenges in obligate, soil-borne pathogens, and establishes a framework to apply similar approaches to other non-culturable microbes.