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. 2008 Apr 16;82(13):6697–6710. doi: 10.1128/JVI.00212-08

FIG. 7.

FIG. 7.

Genotyping of BSGfV EPRV-7 and EPRV-9 (A, B, and C) and detection of recombinant EPRV (C). (A) PCR DifGf F/R-RFLP to genotype BSGfV EPRVs in cv. PKW. Endonuclease TaaI discriminates between EPRV-7 and EPRV-9; amplification products carry two versus one restriction sites, respectively. Lane M, DNA ladder (low molecular weight; Invitrogen). Digestion of PCR product DifGfF/R on clone MBP_94I16 carrying EPRV-9 (lane 1), on clone MBP_71C19 carrying EPRV-7 (lane 2), and on M. balbisiana cv. PKW carrying both EPRVs (lane 3) was performed. No amplification product was seen on M. acuminata cv. IDN 110 4x (data not shown). (B) Multiplex PCR with primers VV3 and VV5 for BSGfV EPRV genotyping. Primers VV3F/R amplify a 376-bp product in both EPRVs, primers VV5F/R amplify a 628-bp product in EPRV-9 only, and primers VV5F/R amplify a 1,012-bp product on both EPRVs and the BSGfV circular genome. Lane M, 1-kb ladder (Invitrogen). Lane 1, BAC MBP_94I16; lane 2, BAC MBP_71C19; lane 3, DNA of M. balbisiana cv. PKW; lane 4, DNA of M. acuminata infected by BSGfV; lane 5, PCR negative control. (C) PCR detection of recombination between the two BSGfV EPRVs. PCR results with Spe7F/R (top) specific to EPRV-7 and Spe9bisF/R (bottom) specific to EPRV-9. Lane M, 1-kb ladder (Invitrogen). Lane 1, negative PCR control (water); lane 2, M. acuminata cv. IDN 110 4x genomic DNA; lane 3, M. balbisiana cv. PKW genomic DNA; lane 4, BAC MBP_94I16; lane 5, BAC MBP_71C19.

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