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

Thomas E, Pakala S, Fedorova ND, Nierman WC, Cubeta MA.

Citation

Thomas E, Pakala S, Fedorova ND, Nierman WC, Cubeta MA. (2012) Triallelic SNP-mediated genotyping of regenerated protoplasts of the heterokaryotic fungus Rhizoctonia solani. Journal of Biotechnology

Abstract

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 24h culture of R. solani increased the proportion of protoplasts with a diameter of �7.5μm 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.