Fig. 4.

INO gene fragments could not be amplified from Ts but were readily amplified from wild-type A. squamosa and five other species. (A) Primers pairs (Table S1) were designed to the A. cherimola INO sequence to enable amplification of the indicated regions. (B) Primer pairs for regions delineated in A, as indicated by corresponding letters at the tops of the gels, were used to amplify sequences from genomic DNA from the species/genotypes indicated below the gels. All primer pairs amplified the expected fragments from wild-type (WT) A. squamosa and from five other species spanning the diversity of this genus, the only exception being the “a” fragment consisting only of 5′-flanking sequences that failed to amplify from A. muricata, one of the species most divergent from A. squamosa (18). These same INO-specific primer pairs failed to amplify fragments from Ts DNA, whereas primers specific to an Annona 1-aminocyclopropane-1-carboxylate synthase gene (ACC) readily amplified the expected fragment from the Ts DNA sample (lane ACC) even when in a reaction that included primers that failed to amplify an INO fragment (Fig. S2). No amplification products were produced when genomic DNA was omitted (lanes No DNA). Rollinia indicates Rollinia emarginata that is phylogenetically embedded within the genus Annona and has been reclassified as a member of this genus (18).