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Updated: Jan 20, 2026
PCR: Principle, Instrumentation, and Applications
Published on: April 30, 2023
Design and validation of a robust multiplex polymerase chain reaction assay for MAT idiomorph within the Fusarium
Amelia C Montoya-Martínez1, Gerardo Rodríguez-Alvarado1, Sylvia P Fernández-Pavía1
1Laboratorio de Patología Vegetal, IIAF, Universidad Michoacana de San Nicolás de Hidalgo , Km. 9.5 Carr. Morelia-Zinapécuaro, Michoacán 58880 , Mexico.
Abstract:
We discovered that published polymerase chain reaction (PCR) assays for determining mating type (MAT) idiomorph failed to genotype some of the Fusarium fujikuroi species complex (FFSC) isolates recovered from Mangifera indica (mango), Swietenia macrophylla (big-leaf mahogany), Annona muricata (soursop), Bursera sp., and Tabebuia sp. in Mexico. Thus, the primary objective of this study was to design and validate a robust multiplex PCR-based diagnostic for typing MAT within the FFSC. To accomplish this objective, we mined the MAT1-1 or MAT1-2 locus from the genomes of 60 FFSC isolates, representing 56 phylospecies, and from four species in its sister group, the F. nisikadoi species complex (FNSC). Bioinformatic searches were facilitated by targeting DNA lyase (SLA2) and apurinic endonuclease (APN1), the genes that flank the MAT locus in Fusarium. As expected, three genes were identified within MAT1-1 (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and two in MAT1-2 (MAT1-2-1 and MAT1-2-9), using the ab initio prediction tool AUGUSTUS. Of the three multiplex PCR assays we designed and tested, the one targeting MAT1-1-2 and MAT1-2-1 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).
Insights
Published PCR assays failed to genotype mating types in Fusarium species. We developed a new multiplex PCR assay that accurately genotypes mating types in the Fusarium fujikuroi species complex, suggesting heterothallism in these fungi.
Area of Science:
- Mycology and Molecular Biology
- Fungal genetics and genomics
- Plant pathology diagnostics
Background:
- Existing polymerase chain reaction (PCR) assays for mating type (MAT) idiomorph genotyping are unreliable for certain Fusarium fujikuroi species complex (FFSC) isolates.
- Accurate mating type determination is crucial for understanding the reproductive biology and population genetics of FFSC, which includes important plant pathogens.
Purpose of the Study:
- To design and validate a robust multiplex PCR-based diagnostic assay for accurate mating type (MAT) idiomorph determination within the Fusarium fujikuroi species complex (FFSC).
- To address the limitations of previously published PCR methods that failed to genotype FFSC isolates from various plant hosts in Mexico.
Main Methods:
- Mined MAT1-1 and MAT1-2 loci from FFSC and FNSC genomes, using flanking genes SLA2 and APN1 for bioinformatic searches.
- Employed the AUGUSTUS prediction tool to identify specific mating type genes (MAT1-1-1, MAT1-1-2, MAT1-1-3, MAT1-2-1, MAT1-2-9).
- Designed and tested three multiplex PCR assays, with the most effective targeting MAT1-1-2 and MAT1-2-1.
Main Results:
- The developed multiplex PCR assay targeting MAT1-1-2 and MAT1-2-1 successfully genotyped all 71 isolates in the validation panel (56 FFSC and 4 FNSC phylospecies).
- In contrast, previously published uniplex PCR assays only achieved 46.5-59% genotyping success rates on the same panel.
- The findings suggest that FFSC/FNSC, even those with a potential sexual cycle, are likely heterothallic (self-sterile).
Conclusions:
- A novel, highly accurate multiplex PCR assay for FFSC mating type determination has been developed and validated.
- This new diagnostic tool overcomes the limitations of previous methods, enabling reliable genetic analysis of FFSC populations.
- The study provides evidence supporting heterothallism in the FFSC/FNSC, impacting our understanding of their evolution and epidemiology.
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