Homing in on Endogenous Badnaviral Elements: Development of Multiplex PCR-DGGE for Detection and Rapid Identification of Badnavirus Sequences in Yam Germplasm

Abstract

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.

Viruses of Yams (Dioscorea spp.): Current Gaps in Knowledge and Future Research Directions to Improve Disease Management

“…For example, in the case of DMaV, multiple isolates can be detected [55], which is relevant for diversity studies. However, many of the other molecular tests detect yam viruses of the same genus but do not discriminate them at the species level [8,24,57,74]. Cloning followed by sequencing or the use of a multiplex PCR test based on specific primers is then needed for species identification and diversity studies.…”

“…Yams host a wide range of endogenous viral elements (EVEs) originating from badnaviruses [22,62,77] and geminiviruses [31,126], whose study helped refine the taxonomy of both genera [18,19,22,62] and improve molecular diagnostic tools [8,62,74,77]. However, the potential for infectious status EVEs in yams is not widely supported [22,62] and remains unknown, although vertical transmission of yam viruses through the activation of infectious EVEs would have major consequences on yam germplasm conservation and exchange as well as yam breeding strategies, as is the case for bananas [127].…”

“…PCR-denaturing gradient gel electrophoresis (PCR-DGGE) [57,73,74] has been proposed to overcome these limitations, but it is not well suited for routine diagnosis of large numbers of samples. Furthermore, DGGE is also limited by the finding that it is unlikely to detect any target DNA less than 1 % of the total target pool [75].…”