Triallelic SNP-mediated genotyping of regenerated protoplasts of the heterokaryotic fungus Rhizoctonia solani

J Biotechnol. 2012 Apr 15;158(3):144-50. doi: 10.1016/j.jbiotec.2012.01.024. Epub 2012 Feb 1.


The aneuploid and heterokaryotic nuclear condition of the soil fungus Rhizoctonia solani have provided challenges in obtaining a complete genome sequence. To better aid in the assembly and annotation process, a protoplast and single nucleotide polymorphism (SNP)-based method was developed to identify regenerated protoplasts with a reduced nuclear genome. Protocol optimization experiments showed that enzymatic digestion of mycelium from a 24 h culture of R. solani increased the proportion of protoplasts with a diameter of ≤7.5 μm and 1-4 nuclei. To determine whether strains regenerated from protoplasts with a reduced number of nuclei were genetically different from the parental strain, triallelic SNPs identified from variance records of the genomic DNA sequence reads of R. solani were used in PCR-based genotyping assays. Results from 16 of the 24 SNP-based PCR assays provided evidence that one of the three alleles was missing in the 11 regenerated protoplast strains, suggesting that these strains represent a reduced genomic complement of the parental strain. The protoplast and triallelic SNP-based method used in this study may be useful in strain development and analysis of other basidiomycete fungi with complex nuclear genomes.

MeSH terms

  • Alleles*
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • Cytoplasm / genetics*
  • Cytoplasm / metabolism
  • Genome, Fungal*
  • Genotyping Techniques*
  • Polymorphism, Single Nucleotide*
  • Rhizoctonia / cytology
  • Rhizoctonia / genetics*
  • Rhizoctonia / growth & development