[en] The Bioversity International Musa Germplasm Transit Centre (ITC) hosts more than 1,500 accessions covering the genetic diversity of the genus Musa. Its objective is to conserve this diversity and supply plant materials to users worldwide. These accessions can be distributed only if virus infection is not detected during indexing. The indexing is carried out by targeted IC-(RT)-PCR for 10 viruses, electron microscopy and symptom observation (De Clerck et al., 2017). One accession (ITC 0763) tested negative by IC-RT-PCR while viral particles were observed by electron microscopy, suggesting the infection by a new virus species or by a divergent viral strain from an existing species.
The accession was sequenced as follows: after RNA extraction, the sequencing library was prepared using the Ribo-Zero™ Plant Leaf Kit (Illumina) for ribodepletion followed by the TruSeq Stranded Total RNA Library Prep Kit (Illumina). The sample was sequenced on the Illumina Nextseq 500 platform (Liège University) and a total of 8,683,460 paired reads (2x150 nt) were obtained. Bioinformatics analysis was carried out using Geneious v9.1 software. The nearly complete genome sequence of a banana mild mosaic virus (BanMMV) isolate was identified.
The primers used for the detection of BanMMV during indexing at Gembloux Agro-Bio Tech are Poty1 and BanMMCP2 (De Clerck et al., 2017).
A careful analysis of the genome sequences complementary to the detection primers revealed four mismatches with BanMMCP2 primer. These mismatches could be the origin of the negative PCR results obtained. To confirm this hypothesis, a new primer was designed at the same genome location as BanMMCP2 but with two additional degenerate bases. Combined with Poty1, the new primer was able to detect the infection by BanMMV. Currently, a retrospective analysis is being carried out on all the banana accessions already indexed to validate the new degenerate primer for use in routine indexing.
