2.3 DNA extraction, genotyping by sequencing (GBS), SNP calling and quality filtering
Tender leaves of C. chuniana and C. chingii were sampled and placed into centrifuge tubes, which were instantly immersed in liquid nitrogen and stored at -80℃. Leaf tissue was ground in tubes with glass beads with the tissue homogenizer Tissuelyser-96 (Shanghai Jingxin Industrial Development Co., Ltd). Total genomic DNA was extracted with the modified cetyl trimethyl ammonium bromide (CTAB) method (Doyle & Doyle, 1986). DNA concentration was quantified with a Nanodrop spectrophotometer (Thermo Scientific, Carlsbad, CA, USA), and a final DNA concentration of > 30 ng/µL was used.
Genotyping by sequencing (GBS) is a streamlined workflow for generating reduced representation libraries for Illumina sequencing (Heffelfinger et al. , 2014; Ilut, Nydam, & Hare, 2014; Melo, Bartaula, & Hale, 2016). The genomic DNA was digested with a combination ofMse I and Nla III enzymes. Subsequent ligation to barcodes after multiplex amplification was constructed and the desired fragments were selected for GBS library construction in Novogene Co. Ltd. The Illumina HiSeq sequencing platform (Illumina, San Diego, CA, USA) was used for paired-end (PE) 150 sequencing. Further advanced analyses and DNA library assembly were performed to remove low-quality reads. Reads in fastq format were assembled by using STACKS v2.2 (Catchen, Hohenlohe, Bassham, Amores, & Cresko, 2013) with one individual of Cercis glabra Pamp. as reference. BWA v0.7.8 (Li & Durbin, 2009) and SAMtools v1.3.1 (Li et al., 2009) were used for sequence mapping and sorting. We used 132 individuals for SNP calling with Stacks. For population analysis, we extracted SNPs with a minor allele frequency (MAF) of at least 0.05 and a genotyping rate of at least 80% of individuals within populations. We also specified a maximum observed heterozygosity of 50% and a minimum number of five populations per locus.