Fusarium species identification by HRM analysis

We selected the RPB2 and TEF-1α region for HRM analysis. In order to design primers flanking short and variable subsections of RPB2 and TEF-1α, we first amplified both regions for the eight reference strains of Fusarium (Table S1), as described by Lofgren et al. [59 (link)] for RPB2 and O’Donnell et al. [60 (link)] for TEF-1α. PCR products were purified and sent to Macrogen Europe for Sanger sequencing (Macrogen Europe B.V., Amsterdam, the Netherlands). The results were evaluated with Chromas version 2.6.6 (Technelysium Pty Ltd, South Brisbane, Australia). Multiple sequence alignment was then performed using ClustalW [61 (link)] in MEGA version 7.0.26 [62 (link)]. Alignments were processed in T-Coffee version 11.00y [63 (link)] and ESPript version 3.0 [64 (link)] (Figure S1). Two new primers suitable for HRM analysis were designed based on multiple gene alignments using the sequences of our eight reference strains (Table 1). Primer binding sites were conserved among species. We hereinafter refer to the selected subsections as sRPB2 and sTEF-1α. The amplicon length was 304 bp for sRPB2 and 247 bp for sTEF-1α (Table 1). Finally, a maximum likelihood analysis was conducted for sRPB2 (1000 bootstrap replications) and sTEF-1α (without bootstrapping due to low sample size (n = 3)) using MEGA 7.0.26.