Characterisation of badnavirus sequences in West African yams (Dioscorea spp.)

Yam (Dioscorea spp.) is an important staple food crop in Sub-Saharan Africa and is vegetatively-propagated. 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 (177 breeding lines, 78 landraces), using an optimised CTAB-extraction method and then screened using degenerate badnavirus-specific PCR primers targeting a 579 bp RT-RNaseH region. All 255 yam samples (100%) tested badnavirus PCR-positive. Denaturation gradient gel electrophoresis (DGGE) analysis of these PCR products revealed 24 discrete bands in total. Sequence analysis of the bands confirmed they were typical of the genus Badnavirus and a nucleotide diversity of 1-37% in this partial RT-RNaseH region representative nine of badnavirus species group. To determine which sequences were from episomal infections, rolling circle amplification (RCA) was performed on samples, and three complete genome sequences of yam badnaviruses were amplified, cloned and sequenced. Two of these full viral genome sequences (7258 and 7538 bp) of D. rotundata origin represent new species in the genus Badnavirus and the third (7529 bp) from D. alata represented an isolate of Dioscorea bacilliform AL virus. The three new genomes shared nucleotide identities of 68.3-70.5% and demonstrated a typical size and organisation of yam badnaviruses. PCR-based assays were developed for the detection of the five yam badnavirus genomes, and for the detection of three putative badnavirus species groups (K08, K09 and U12) that contain integrated sequences. Southern hybridisation results using individual DGGE band partial RT-RNaseH sequences (NGb4_Dr, NGb5_Dr and NGb6_Dr), supported integration of badnavirus sequences in genomes of D. rotundata breeding lines. Fluorescent in situ hybridisation (FISH) results using badnavirus complete and partial cloned genome sequences as probes were inconclusive for the yam samples tested. The consequences of the integrated and episomal badnavirus sequences for yam improvement programmes in West Africa are discussed.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Greenwich

Year dc:date.issued

2014

Author dc:creator

• Turaki, Aliyu Abdullahi

Advisors dc:contributor.advisor

• Seal, Susan
• Maruthi, Midatharahally

• SB Plant culture