Figure 2

PCR amplifications on cDNAs to reveal anther-specific NATs in olive. A. RT-PCR analysis in different tissues using primers for the start and stop codons, OeSLG_full-length_FOR and OeSLG_full-length_REV, respectively, to amplify the target gene, OeSLG , and using primers OeEF_FOR and OeEF_REV to amplify the housekeeping gene, OeEF. Ctrl-: negative control. A lower band (indicated by an arrow) is present only in anthers. B. Final PCR amplifications of the OeSLG gene were performed using primer pairs suitable to detect either the sense transcripts or the antisense transcripts. The housekeeping gene OeEF was amplified using specific primers. Sense transcripts are detected in all of the tissues, whereas the antisense transcripts are found only in anthers and are present by two different forms, corresponding to the medium and short forms, as expected. C. Final PCR amplifications were performed after the synthesis of the complementary strands as follows: FOR, OeSLG_full-length_5’-tag_FOR; REV, OeSLG_full-length_5’-tag_REV; NP, no primers were added to reactions. PCR analysis was performed using the following combination of primers: sense, primer_TAG and OeSLG_internal_5’_REV; antisense, primer_TAG and OeSLG_internal_3’_FOR; OeEF , primer_TAG and OeEF_REV; Ctrl-, negative control. Sense transcripts are detected in all tissues, whereas antisense transcripts are found only in the anthers. The negative control showed that the possible complementary strands synthesised in a primer-independent manner were not amplified. The antisense transcripts resolved as single band because the internal primers used were external to the introns and they were not able to discriminate between the medium and short forms.