2.6. Primer Design and PCR Amplification

Primers for the development of ccsA-ndhD (forward (F): ACACATAGAAATTTGCGGGGTGC; reverse (R): TCGATGGCTTCCCTTGCATTACCA) and trnL-trnF (F: ATCGAAGAAATTCCCCGGCT; R: GCGCACATTACTTAGACGGGTT) molecular markers were designed from assembled plastomes by using the MacVector software (MacVector Inc., Apex, NC, USA). PCR amplifications were carried out, using Taq DNA polymerase according to the manufacturer’s instructions (Invitrogen, Paisley, UK), on 25 ng of total DNA or cpDNA of the following genotypes: C. baccatum subsp. baccatum (bac.b, Table 1), C. baccatum subsp. pendulum (bac.p, Table 1; bac.p2, {"type":"entrez-protein","attrs":{"text":"CGN17015","term_id":"813305762","term_text":"CGN17015"}}CGN17015; bac.p3, {"type":"entrez-protein","attrs":{"text":"CGN22181","term_id":"877692920","term_text":"CGN22181"}}CGN22181; bac.p4, {"type":"entrez-protein","attrs":{"text":"CGN17174","term_id":"813522662","term_text":"CGN17174"}}CGN17174), C. praetermissum (pra, Table 1), C. pubescens (pub, Table 1; pub2, {"type":"entrez-protein","attrs":{"text":"CGN22796","term_id":"876081188","term_text":"CGN22796"}}CGN22796; pub3, CAP1486), C. chacoense (cha, Table 1; cha2, CAP1445; cha3, CAP499; cha4, CAP501), C. annuum (ann2, Table 1; ann4, {"type":"entrez-protein","attrs":{"text":"CGN17175","term_id":"874771595","term_text":"CGN17175"}}CGN17175; ann5, {"type":"entrez-protein","attrs":{"text":"CGN21490","term_id":"877302986","term_text":"CGN21490"}}CGN21490; ann6, {"type":"entrez-protein","attrs":{"text":"CGN24355","term_id":"877378062","term_text":"CGN24355"}}CGN24355; ann7, {"type":"entrez-protein","attrs":{"text":"CGN23249","term_id":"877678210","term_text":"CGN23249"}}CGN23249), C. chinense (chi2, {"type":"entrez-protein","attrs":{"text":"CGN17220","term_id":"813463522","term_text":"CGN17220"}}CGN17220; chi3, {"type":"entrez-protein","attrs":{"text":"CGN23565","term_id":"876081207","term_text":"CGN23565"}}CGN23565; chi4, {"type":"entrez-protein","attrs":{"text":"CGN17219","term_id":"812326368","term_text":"CGN17219"}}CGN17219), and C. frutescens (fru2, {"type":"entrez-protein","attrs":{"text":"CGN22792","term_id":"813180627","term_text":"CGN22792"}}CGN22792; fru3, RCAT077650; fru4, {"type":"entrez-protein","attrs":{"text":"CGN21546","term_id":"874700228","term_text":"CGN21546"}}CGN21546). The reaction conditions for all amplifications were as follows: denaturation at 94 °C for 3 min, then 30 cycles (94 °C, 30 s; annealing temperature, 30 s; 72 °C, 1 min/kb), followed by 5 min final extension at 72 °C.