Molecular cytogenetic characterisation and phylogenetic analysis of the seven cultivated Vigna species (Fabaceae)

The genomic organisation of the seven cultivated Vigna species, V.unguiculata, V.subterranea, V.angularis, V.umbellata, V.radiata, V.mungo and V.aconitifolia, was determined using sequential combined PI and DAPI (CPD) staining and dual-colour fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. For phylogenetic analyses, comparative genomic in situ hybridisation (cGISH) onto somatic chromosomes and sequence analysis of the internal transcribed spacer (ITS) of 45S rDNA were used. Quantitative karyotypes were established using chromosome measurements, fluorochrome bands and rDNA FISH signals. All species had symmetrical karyotypes composed of only metacentric or metacentric and submetacentric chromosomes. Distinct heterochromatin differentiation was revealed by CPD staining and DAPI counterstaining after FISH. The rDNA sites among all species differed in their number, location and size. cGISH of V.umbellata genomic DNA to the chromosomes of all species produced strong signals in all centromeric regions of V.umbellata and V.angularis, weak signals in all pericentromeric regions of V.aconitifolia, and CPD-banded proximal regions of V.mungo var. mungo. Molecular phylogenetic trees showed that V.angularis and V.umbellata were the closest relatives, and V.mungo and V.aconitifolia were relatively closely related; these species formed a group that was separated from another group comprising V.radiata, V.unguiculata ssp. sesquipedalis and V.subterranea. This result was consistent with the phylogenetic relationships inferred from the heterochromatin and cGISH patterns; thus, fluorochrome banding and cGISH are efficient tools for the phylogenetic analysis of Vigna species.