Fig. 4

Putative circular Helitrons detected by PCR. a Schematic representation of FoHeli1 in the genome. The grey line represents FoHeli1 and the 5’ and 3’ terminal sequences are indicated above. The black thick lines represent the flanking genomic region. The arrows indicate the positions of the primers. For each subgroup, FoHeli1 to FoHeli5, we designed four specific PCR primers (Additional file 1: Table S4). Primer pairs 1 + 2 and 3 + 4 are specific to FoHeli 5’ and 3’ ends and their flanking sequences, respectively. Primers 2 + 3 anneal close to FoHeli ends and are directed outwards; these are expected to amplify a PCR product only from molecules that contain nearby or joined FoHeli ends. b Schematic representation of a FoHeli1 circle with joined ends (possible template for the amplification of a PCR product using primers 2 + 3) c PCR experiment showing amplification of PCR products using primer pair A (primers 2 + 3), B (primers 1 + 2) and C (primers 3 + 4) specific for FoHeli1 – FoHeli5. The template for the PCR reaction was genomic DNA isolated from Fol4287. We used two sets of primers for FoHeli4, because this subgroup is more divergent than the others. Note that there is ~400 bp PCR product of FoHeli5 using outward directed primers. However, the sequence of FoHeli5 with joined ends between these primers is 570 bp. Moreover, the sequence of this amplicon did not show any similarity to a FoHeli. Hence we concluded that this amplicon does not correspond to a FoHeli5 with joined ends. d Structure of FoHeli1 joined ends. The terminal sequences are shown in bold