Choice of chloroplast regions
Based on the work of Shaw et al. (2007), the following nine non-coding chloroplast regions were chosen for initial screening of one accession each of C. lanatus , C. mucosospermus, C. amarus, and C. colocynthis :rpl 32-trn L, trn Q-5’rps 16, 3’trn V-ndh C, ndh F-rpl 32,psb D-trn T, psb J-pet A, 3’rps 16-5’trn K, atp I-atp H, andtrn T-trn L. For most of these regions, total levels of variation were low and exclusively inter-specific. However, forndh F-rpl 32 and trn T-trn L, polymorphisms were observed both within and among species; thus these two regions were selected for more in-depth investigation. These two regions of the chloroplast genome were amplified using the following primer pairs: 1)ndh F (5’-GAAAGGTATKATCAAYGMATATT-3’) and rpl 32-R (5’-CCAATATCCCTTYYTTTTCCAA-3’); and 2)trn L(UAG) (5’-CTGCTTCCTAAGAGCAGCCT-3’) andtrn T(GGU) (5’-CCCTTTTAACTCAGTGGTAG-3’).