DNA was extracted from ear or liver tissue preserved in ethanol using the QIAamp DNA Mini Kit (QIAGEN Inc .. Extracted DNA was used as template in PCR using primers defined in Matthee et al. [ 40, 46] and Eick et al. [ 60].
The coding sequences were aligned and oligonucleotide primers were defined in conserved regions as distant as possible.
Based on 100 ITS sequences previously published [ 5, 22, 33], we designed new primer pairs specifically defined in Ficus using the Oligo4 software.
Six specific primer pairs were defined in divergent parts of 3'-untranslated regions to test whether the corresponding RGA08 genes (i.e. RGA08E-1, RGA08K-1, RGA08M-1, RGA08L-1, RGA08M-1, RGA08O-1, Figure 2) are expressed in planta.
The underlined sequence in the REP1-N primer defines EcoRI and SacI restriction sites, whereas the underlined sequence in the REP1-C primer defines a KpnI restriction site.
Thus, the molecular assays used for diagnostic purposes should rely on primers and probes defined in the most conserved regions of the HDV genome to avoid false-negative results (5, 14, 15 ).
An additional reverse primer ITSF5 (CGGAGGTTACGCTGGGGTC) was defined in the zone at the junction between ITS2 and 26S conferring a high specificity in Ficus species.
A forward primer (nadpme-491-for; AYGAGAGGCTBTTCTACAAG) was defined in the fourth exon and a reverse primer (nadpme-1606-rev; GGGAARATGTAGGCRTTGTT) in the 17th exon (fig. 1).
A total of 409 primer pairs (Supplementary Table S2) were defined in the 100 larger non-anchored scaffolds using Primer3 software and screened for their ability to segregate in the UPA402 × UF676 progeny.
In the next steps, a software script based on a selection algorithm developed by our group [29] was used to select primers from defined primer regions of each target based on criteria defined in previous studies [18].
