Design and validation of a robust multiplex polymerase chain reaction assay for <i>MAT</i> idiomorph within the <i>Fusarium fujikuroi</i> species complex

We discovered that published polymerase chain reaction (PCR) assays for determining mating type (<i>MAT</i>) idiomorph failed to genotype some of the <i>Fusarium fujikuroi</i> species complex (FFSC) isolates recovered from <i>Mangifera indica</i> (mango), <i>Swietenia macrophylla</i> (big-leaf mahogany), <i>Annona muricata</i> (soursop), <i>Bursera</i> sp., and <i>Tabebuia</i> sp. in Mexico. Thus, the primary objective of this study was to design and validate a robust multiplex PCR–based diagnostic for typing <i>MAT</i> within the FFSC. To accomplish this objective, we mined the <i>MAT1-1</i> or <i>MAT1-2</i> locus from the genomes of 60 FFSC isolates, representing 56 phylospecies, and from four species in its sister group, the <i>F. nisikadoi</i> species complex (FNSC). Bioinformatic searches were facilitated by targeting DNA lyase (<i>SLA2</i>) and apurinic endonuclease (<i>APN1</i>), the genes that flank the <i>MAT</i> locus in <i>Fusarium</i>. As expected, three genes were identified within <i>MAT1-1</i> (<i>MAT1-1-1, MAT1-1-2</i>, and <i>MAT1-1-3</i>) and two in <i>MAT1-2</i> (<i>MAT1-2-1</i> and <i>MAT1-2-9</i>), using the <i>ab initio</i> prediction tool AUGUSTUS. Of the three multiplex PCR assays we designed and tested, the one targeting <i>MAT1-1-2</i> and <i>MAT1-2-1</i> 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).