title: The use of SLAF-seq as a cost effective method of genetic resource characterization in Medicago Polymorpha
design: Specific-locus amplified fragment sequencing (SLAF-seq) is an efficient method of large-scale genotyping, which is based on Reduced Representation Libraries (RRL) and high-throughput sequencing. The SLAF-seq method was described previously with slight modifications. First, we performed a SLAF pre-design experiment. The reference genome of Medicago truncatula (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA_000219495.2_MedtrA17_4.0) was used. A simulated restriction enzyme digestion was carried out to establish conditions to optimize SLAF yield, avoid repetitive SLAFs, and obtain an even distribution of SLAFs for maximum SLAF-seq efﬁciency. Based on the result of the preliminary experiment, RsaI and HaeIII restriction enzyme were selected. Second, genomic DNA samples of ten accessions were digested with restriction enzyme RsaI (NEB, Ipswich, MA, USA) and ligated to RsaI adapter, and then digested for additional enzyme HaeIII (NEB). Polymerase chain reactions (PCR) were carried out using the diluted restriction–ligation samples, dNTP, Taq DNA polymerase (NEB), and RsaI-primer containing barcode 1. After puriﬁcation, the PCR products were incubated at 37 °C with RsaI, T4 DNA ligase, adenosine triphosphate, and Solexa adapter (Illumina, San Diego, CA, USA). The samples were puriﬁed using a Quick Spin column (Qiagen), then run on a 2% agarose gel; fragments of 550–600 bp (with indices and adaptors) were isolated using a Gel Extraction Kit (Qiagen) and subjected to PCR ampliﬁcation with Phusion Master Mix (NEB) and Solexa Ampliﬁcation primer mix (Illumina) to add barcode 2 according to the Illumina sample preparation guide. Finally, the puriﬁed PCR products of 314–414 bp were sequenced with the pair-end sequencing method on an Illumina High-seq 2500 sequencing platform (Illumina) at Biomarker Technologies Corporation (Beijing, China).