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Viruses of the genus Badnavirus (family Caulimoviridae) are double-stranded DNA-reverse transcribing (dsDNA-RT) plant viruses and have emerged as serious pathogens of tropical and temperate crops globally. Endogenous badnaviral sequences are found integrated in the genomes of several economically important plant species. Infection due to activation of replication-competent integrated copies of the genera Badnavirus, Petuvirus and Cavemovirus has been described. Such endogenous badnaviral elements pose challenges to the development of nucleic acid-based diagnostic methods for episomal virus infections and decisions on health certification for international movement of germplasm and seed. One major food security crop affected is yam (Dioscorea spp.). A diverse range of Dioscorea bacilliform viruses (DBVs), and endogenous DBV (eDBV) sequences have been found to be widespread in yams cultivated in West Africa and other parts of the world. This study outlines the development of multiplex PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE) to assist in the detection and analysis of eDBVs, through the example of analysing yam germplasm from Nigeria and Ghana. Primers targeting the three most prevalent DBV monophyletic species groups in West Africa were designed to improve DGGE resolution of complex eDBV sequence fingerprints. Multiplex PCR-DGGE with the addition of a tailor-made DGGE sequence marker enables rapid comparison of endogenous badnaviral sequence diversity across germplasm, as illustrated in this study for eDBV diversity in yam.

Badnaviruses (family Caulimoviridae, genus Badnavirus) have emerged as serious pathogens especially affecting the cultivation of tropical crops. Badnavirus sequences can be integrated in host genomes, complicating the detection of episomal infections and the assessment of viral genetic diversity in samples containing a complex mixture of sequences. Yam (Dioscorea spp.) plants are hosts to a diverse range of badnavirus species, and recent findings have suggested that mixed infections occur frequently in West African yam germplasm. Historically, the determination of the diversity of badnaviruses present in yam breeding lines has been achieved by cloning and sequencing of polymerase chain reaction (PCR) products. In this study, the molecular diversity of partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences from yam badnaviruses was analysed using PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE). This resulted in the identification of complex ‘fingerprints’ composed of multiple sequences of Dioscorea bacilliform viruses (DBVs). Many of these sequences show high nucleotide identities to endogenous DBV (eDBV) sequences deposited in GenBank, and fall into six monophyletic species groups. Our findings highlight PCR-DGGE as a powerful tool in badnavirus diversity studies enabling a rapid indication of sequence diversity as well as potential candidate integrated sequences revealed by their conserved nature across germplasm.