Single Nucleotide Polymorphisms (SNPs) of Beauveria species
Fungi are rich in complexes of sibling species that need combinations of approaches to be delimited, including with genomic information. Beauveria (Cordycipitaceae, Hypocreales) is a well-known genus of entomopathogenic fungi, used as a biocontrol agent. We present in this study a polyphasic taxonomy regarding two widely distributed complexes of Beauveria: B. asiatica and B. bassiana sensu lato. Some of the genetic groups as previously detected within both taxa were either confirmed or fused using population genomics. High levels of divergence were found between two clades in B. asiatica and between three clades in B. bassiana, supporting their subdivision as distinct species. Fungal culture and DNA extraction. Seventy-eight strains of presumably 12 Beauveria species from BIOTEC culture collection (BCC) and ARS collection of entomopathogenic fungal cultures (ARSEF), including the type strains of known species and strains previously identified as B. bassiana or B. asiatica as well as some unidentified strains. The strains were cultured in 50 ml of potato dextrose broth and incubated at 25 °C for a week. Fungal mycelia were harvested by filtering with a sterilized nylon mesh and washed with ethylene diamine tetraacetic acid (EDTA) and distilled water. DNA extraction was done using a cetrimonium bromide (CTAB)-based method; the DNAs were purified using the high pure PCR template preparation kit (Roche). DNA library construction and sequencing For whole genome shotgun sequencing of 78 strains, approximately 300 ng of each DNA sample was used for a library construction following the protocol in the MGIEazy FS library prep kit (MGI Tech, Shenzhen, China). The samples were pooled and sequenced in a single lane. Paired-end (150 bp) sequencing was performed on the MGISEQ-2000RS (MGI Tech, Shenzhen, China) according to the manufacturer’s instructions. Data processing and detection of single nucleotide polymorphisms (SNPs) Using the MegaBOLT V126.96.36.199 software package, raw reads were de-multiplexed according to their barcodes and the adapter/barcode sequences were removed. After removing the low-quality regions, clean reads were mapped to the B. bassiana reference genome ARSEF8028 (Valero-Jiménez et al. 2016) using the MegaBOLT V188.8.131.52 alignment software (Minimap2 version 2.11-r797-v03) and the variants were called using GATK HaplotypeCaller version 3.8. SNP markers with poor quality data were filtered out using the following criteria: (a) a minor allele frequency <0.1; (b) depth coverage less than 10x; (c) more than 20% missing data. The ploidy was set as haploid as all the strains were collected from asexual mycelia, i.e. putatively haploid for this ascomycete fungus.