Characterization of the cytochrome b gene fragment of Puccinia species responsible for the binding site of QoI fungicides

The fragment of the cytochrome b (cyt b) gene responsible for the binding site of QoI fungicides was sequenced for different Puccinia species by using DNA and RNA as template for PCR and RT-PCR, respectively. Degenerated primers for the cyt b gene amplified in P. recondita f.sp. tritici a 450 bp fragment, which was cloned and sequenced. At cDNA level, several Thermal Asymmetric InterLaced (TAIL)-PCR cycles were needed to produce a 996 bp long fragment, which corresponded to almost the whole cyt b gene (about 1160-1180 bp, without introns). This fragment was sequenced and specific primers were designed. Amplification with cyt b specific primers using genomic DNA as template revealed the presence of an intron of about 1500 bp length after the codon for glycine at amino acid position 143. By using the same primer pair, the cyt b gene fragment was amplified and sequenced both at cDNA and genomic DNA level also for other rust species, including P. graminis f.sp. tritici (length: 506 bp), P. striiformis f.sp. tritici (755 bp), P. coronata f.sp. avenae (644 bp), P. hordei (660 bp), P. recondita f.sp. secalis (687 bp), P. sorghi (709 bp), and P. horiana (478 bp). At the same position as for P. recondita f.sp. tritici, an intron of about 1500-1600 bp length was detected also in all other Puccinia species. High homologies were observed among all Puccinia species for both the exonic and intronic fragments of the cyt b gene. Specific primers for the cyt b gene of all eight Puccinia species were developed, which easily amplified the fragment of the gene including all possible mutations known to confer resistance to QoIs in several plant pathogens. However, in all tested isolates of the Puccinia species included in this study, the sequence of cyt b gene fragment did not contain any point mutations.