Design and validation of a robust multiplex polymerase chain reaction assay for idiomorph within the species complex.

We discovered that published polymerase chain reaction (PCR) assays for determining mating type () idiomorph failed to genotype some of the species complex (FFSC) isolates recovered from (mango), (big-leaf mahogany), (soursop), sp., and sp. in Mexico. Thus, the primary objective of this study was to design and validate a robust multiplex PCR-based diagnostic for typing within the FFSC. To accomplish this objective, we mined the or locus from the genomes of 60 FFSC isolates, representing 56 phylospecies, and from four species in its sister group, the species complex (FNSC). Bioinformatic searches were facilitated by targeting DNA lyase () and apurinic endonuclease (), the genes that flank the locus in . As expected, three genes were identified within (, and ) and two in ( and ), using the prediction tool AUGUSTUS. Of the three multiplex PCR assays we designed and tested, the one targeting and successfully genotyped the entire 71-isolate validation panel, which included 56 FFSC and 4 FNSC phylospecies. By contrast, the published PCR assays we tested produced positive genotypes for only 46.5-59% of the 71-isolate validation panel, but only when they were run as a uniplex assay. Although only one-fifth of the FFSC/FNSC are known to reproduce sexually, our results suggest that if they possess a sexual cycle, it is heterothallic (self-sterile).