Résumé
Blast annotations of 44,164 CAP3-assembled sequences of public ESTs (NCBI) revealed the presence of a putative new macluravirus species infecting South-East Asian tropical yam species D. alata in Nigeria. Based on this new information, the diversity of macluraviruses was investigated in tropical yam germplasm using four complementary approaches: 1) Sanger sequencing of cloned yam-macluravirus-specific RT-PCR amplicons, 2) virion-associated nucleic acids (VANA), 3) dsRNA and 4) siRNA deep sequencing. Primers were designed in the cp gene of yam macluraviruses, based on the alignment of ESTs sequences and sequences of known viral species (Chinese yam necrotic mosaic virus and Yam chlorotic necrotic mosaic virus), and used to screen the worldwide yam germplasm collection maintained by Guadeloupe's Biological Resource Centre for Tropical Plants (CRB-PT), by RT-PCR performed on total nucleic acids. Additional samples from India and South Pacific Islands were also screened. Sanger sequencing of the RT-PCR amplicons revealed the existence of two novel macluravirus species in tropical yams: one species tentatively named Dioscorea alata macluravirus was identified in D. alata and D. nummularia, whereas a distinct species, tentatively named Dioscorea esculenta macluravirus, was identified in D. esculenta. These two species appear to be present in Nigeria, Guadeloupe, India and in some South Pacific Islands (Palau, Papua New Guinea, Tonga, Vanuatu), suggesting a worldwide distribution. siRNA sequences from a D. alata plant collected in India were assembled in contigs, leading, after Blast annotation, to the complete genomic sequence of a strain of Dioscorea alata macluravirus, which was confirmed by Sanger sequencing of RT-PCR and 3'-RACE amplicons. Using 454 pyrosequencing of VANA or cDNA synthesized from purified viral dsRNAs, RT-PCR and RACE analyses, the complete genomic sequence of another strain of Dioscorea alata macluravirus was obtained. (Résumé d'auteur)