The Genome of African Native Yam (D. rotundata) Contain Several Endogenous Pararetroviral Sequences

Cultivated yams (Dioscorea spp.) are propagated vegetatively through their tubers, which results in the accumulation of tuber-borne virus infections, and their perpetuation from one cropping season to the next. This had led to the accumulation of viruses decreasing yam production and hindering international movement of selected germplasm.  This study was to determine the prevalence and diversity of yam badnaviruses, as well as determine if badnavirus sequences are also integrated in the genomes of West African yam breeding lines.  DNAs were extracted from Nigerian yam leaf samples (125 breeding lines), using an optimised CTAB-extraction method and then screened using degenerate badnavirus-specific PCR primers targeting a 579 bp RT-RNaseH region.  All 125 yam samples (100%) tested badnavirus PCR-positive.  Analysis of the PCR-amplified badnavirus RT-RNaseH sequences has grouped them into 12 species clusters each sharing <80% nucleotide identity to each other. As such species differences were not identifiable from their single RT-RNaseH PCR products, denaturing gradient gel electrophoresis (DGGE) was evaluated for its usefulness to discriminate these sequences. Seventeen RT-RNaseH PCR products from West African yam breeding lines produced 11 discrete DGGE bands that represented sequence variants. DGGE was found to be a successful technique for rapid identification of the true diversity of yam badnavirus sequences in a given sample. The existence of badnavirus PCR-positive, but rolling circle amplification (RCA) negative results indicated that some breeding lines contain integrated badnavirus sequences, and this has been supported by nucleic acid hybridization studies. In search to determine which of these sequences represent dead integrants, and which are activatable sequences that will pose a serious threat to yam germplasm health and movement, Fluorescent in situ hybridisation (FISH) was conducted using complete and partial virus genome sequences as probes, the results were inconclusive for the yam samples tested.