RAD-Seq Analysis of Peppermint Varieties

Both the quality and quantity of genomic DNA were evaluated by agarose gel electrophoresis and by UV/Vis spectrophotometry (NanoDrop 2000c, Thermo Fisher Scientific, Pittsburgh, PA, USA). RAD seq was performed as reported [32 (link)] with the exception that the restriction enzyme ApeKI (New England Biolabs, Ipswich, MA, USA) was used. Adapter-ligated DNA fragments were pooled and sheared to a mean size of 500 bp. The RAD-Seq libraries were enriched by polymerase chain reaction amplification and sequenced on an Illumina HiSeq 2000 (BGI, Shenzhen, China) using single-ended reads (50 bp) for each peppermint variety. For SNP calling and data analysis, the Illumina sequence reads were quality-filtered by removing the adapter sequences and reads containing greater than 50% low-quality bases. These processed reads were mapped on the reference peppermint genome using BWA-MEM (version 2, GitHub, Hobro, Denmark). Then, the comparison results filtered and SNPs were called by SAMTOOLS (version 1.16, Genome Research, Cambridgeshire, UK). High quality SNPs were used for the estimate of pairwise genetic distances and the reconstruction of phylogenetic trees based on maximum parsimony using MEGA 11 (Mega Software Technologies, Philadelphia, PA, USA) [33 (link)].