Triallelic SNP-mediated genotyping of regenerated protoplasts of the heterokaryotic fungus Rhizoctonia solani.
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.
Department of Plant Pathology, NC State University, Raleigh, NC 27695, United States. ethomas@ncsu.edu
Elizabeth
E
Thomas
Suman
S
Pakala
Natalie D
ND
Fedorova
William C
WC
Nierman
Marc A
MA
Cubeta
D000483:Alleles; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics* / Q000378:metabolism; D016681:Genome, Fungal; D060005:Genotyping Techniques; D020641:Polymorphism, Single Nucleotide; D012232:Rhizoctonia / Q000166:cytology / Q000235:genetics* / Q000254:growth & development