Molecular analysis of banana streak virus integrants in the nuclear genome of Musa balbisiana

Recently, it was demonstrated that Banana streak virus (BSV) sequences are integrated into the nuclear genome of #Musa balbisiana#. There is strong experimental evidence that some of these BSV integrated sequences, called BSV endogenous pararetroviruses (BSV EPRVs), can give rise to infectious episomal BSV genomes upon stress conditions. Such stress include interspecific genetic crosses, therefore this phenomenon represents a serious limitation to the creation of novel #Musa# hydrids between A and B genomes and to the diffusion of such hybrids. As part of an international effort within the #Musa# Genomics Consortium aimed at defining the integration patterns of BSV EPRV sequences into the nuclear genome of #Musa# species and the mechanisms leading to the activation of such BSV EPRV sequences, we have constructed and characterised three BAC libraries from #M. acuminata# 'Cavendish' (AAA), #M. acuminata# 'Calcutta 4' (AA) and #M. balbisiana# PKW (BB) nuclear genomes, respectively. Complete genomes of four different BSV strains (BSV-Ol "Obino L'Ewai", BSV-Gf "Gold Finger", BSV-Im "Imove", BSV-Mys "Mysore") were used as probes to screen the BAC libraries and FISH experiments were carried out to test for the presence of integrated viral sequences within #Musa# A and B chromosomes. Upon screening we identified 10 BAC clones positive for BSV-Ol, 9 BAC clones positive for BSV-Gf and 24 BAC clones positive for BSV-Im from the #M. balbisiana# PKW BAC library. On the other hand, screening of either #M. acuminata# 'Calcutta 4' and #M. acuminata# 'Cavendish' BAC libraries with the four complete viral sequences revealed that none of these BAC clones contained any BSV sequences. BAC clones found positive upon screening were further characterized by BAC-end sequencing and RFLP-fingerprinting, and selected BACs containing BSV-Ol or BSV-Gf EPRV sequences were completely sequenced. Detailed analysis of the nucleotide sequences and chromosomal organisation of BSV-Ol and BSV-Gf EPRV sequences within these BAC clones will be presented and discussed. This study represents the first step towards the characterization of mechanisms leading to the activation of BSV EPRV sequences in #Musa# and the implementation of novel strategies to counteract this phenomenon. (texte intégral)