2.2 | Genotyping
To examine chloroplast (cp) DNA haplotypes, we selected 7–8 individuals from each of 17 out of the 24 populations (130 individuals in total; Table 1). We determined nucleotide sequences in four cpDNA regions: 3’ to rps 2, trn T(UGU)–trn L(UAA) 5’ exon, rps 16 intron, and rp L32–trn L(UAG) using a BigDye Terminator Sequencing Kit and a 3100 Genetic Analyzer (Thermo Fisher Scientific, Waltham, USA). Obtained sequences were assembled and manually edited using Sequencher 10.4.1 (Gene Codes Corporation, Ann Arbor, USA).
To examine nuclear (nc) microsatellite genotypes, we selected 32 expressed sequence tagged (EST) SSR loci (Ueno et al. 2008; Ueno and Tsumura 2008; Ueno et al. 2009a; Ueno et al. 2009b) (Data S1 in Supporting Information). We amplified these ncEST-SSR loci using a multiplex PCR Kit (Qiagen) and genotyped them using a 3100 Genetic Analyzer and Genotyper 3.2 software (Thermo Fisher Scientific).
Detailed methods of cpDNA sequencing and ncEST-SSR genotyping were described in our previous study (San Jose-Maldia et al. 2017).