Computational protocol: The influence of the mating type on virulence of Mucor irregularis

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[…] To obtain the putative MAT locus (sex) genes in M. irregularis, the published sequences of the SexM/P genes in Phycomyces blakesleeanus (EU009461, EU009462), M. circinelloides (FJ009106, FJ009107 and HM565940), Rhizopus oryzae (HQ450316) and M. mucedo (JN587498) from NCBI were used as query sequences to search against the assembled M. irregularis genome (B50p, accession No. AZYI00000000.1) by BLASTP and TBLASTN programs (e-value < 1e-10). Then the MAT locus sequence derived from the genomic analysis of B50p was used as a template for primer design to target the SexM/P gene loci in 17 tested isolates. Specific primers were designed targeting between the 5’ of tpt and the 5’ of RNA helicase using MacVector 11 (Accelrys, USA) (listed in Supplementary Table ).The genomic DNA of the 17 isolates’ was extracted as previously described. The PCR mix contained (25 μl total volume): 12.5 ng genomic DNA, 10 pmol primers, 12.5 μl 2 × Vazyme LAmp Master Mix (P312, Vazyme, China). The temperature profile was: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 3 min. The amplified fragments were isolated from an gel purified and sequenced. FGENESH was used to predict the putative transcription start site (TSS) and number of exons. The sense of genes in the MAT locus was determined in ORF Finder ( and displayed with a dot plot diagram. Finally, the ORF of SexM/P was cloned using gene specific primers (listed in Supplementary Table ), sequenced and verified in each tested isolate.The sequences of MAT genes from 17 M. irregularis isolates were aligned using MacVector 11 with manual adjustment. The phylogenetic relationship among these isolations was inferred with MEGA 6.0 using neighbor-joining (NJ) algorithms with the following parameters: P distance, pairwise deletion and bootstrap (1,000 replicates). Also the amino acid sequences of SexM/P were aligned with SexM/P sequences reported from other Mucorales species (listed in Supplementary Table ). A phylogenetic tree was constructed by NJ with 500 bootstrap replications using MEGA 6.0 as described above. These newly determined MAT sequences of M. irregularis isolates have been deposited in GenBank under accession numbers KY434081 to KY434097. […]

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