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.