Microsatellite primers for the Green Toad, Bufotes viridis

Microsatellite library for the gReen toad, Bufotes viridis.Methods: In brief, genomic DNA was extracted from the tissue samples with a Qiagen Blood and Tissue Kit (Qiagen, Hilden, Germany), digested in three separate reactions with the restriction enzymes AluI, RsaI, and Hpy166II, and combined in equal amounts after heat inactivation of the restriction enzymes. The blunt ends were adenylated (+A) with Klenow (exo-) and dATP, and after heat inactivation of the Klenow (exo-), the reactions were supplemented with ATP to 1 mM and an Illumina Y-adaptor was ligated with T4 DNA ligase. The fragments were enriched for microsatellites by hybridisation to and magnetic capture of biotinylated repeat probes (representing two unique dimers, five unique trimers, seven unique tetramers and two unique pentamers), amplified and barcoded by PCR, and sequenced on an Illumina MiSeq instrument (2 × 250 bp paired reads). The raw reads were assembled in SeqMan NGen (version 11). The assembly was scanned for microsatellite loci and automatically designed primer pairs with the program msatcommander 1.0.8_beta (for Mac OSX). A library was constructed with minimum consecutive perfect repeat lengths of at least six (12 bp) for any dimer and at least five for any trimer, tetramer, or pentamer and PCR product size of 150–450 bp. The full library is available as supplementary information (Supplementary Table S1). Out of this library, we chose 12 loci based on following criteria (Perl et al. 2018): (i) tetrameric, (ii) repeat motif between 10 and 15, (iii) less than 1000 reads, as deep coverage could indicate multiple copies and (iv) GC content of 50 (Table 1).