Evidence of rapid evolution and incipient speciation in Vicia sativa species complex based on nuclear and organellar RFLPs and PCR analysis

Abstract

Seventy three accessions of the sevenVicia species belonging to Sativa speciescomplex were screened for nuclear and organellar restriction fragmentlength polymorphic (RFLP), and random amplified polymorphicDNA (RAPD) markers. Total genomic DNAs of 73 accessions wasrestricted with three enzymes, and the restriction fragments werehybridized to the wheat rDNA probe pTa71 (containing 18S, 5.8Sand 25S rDNA genes, and spacers), and faba bean probes Ver6-5 (entire intergenic spacer flanked by small part of25S and 18S fragments) and Ver 18-6 (part of thecoding region of the gene and internal transcribed spacers). InXbaI digests, 16 repeat unit length classes in24 combinations were identified. Digestion withEcoRI and DraI gave2–4 and 1–3 fragments, respectively, with detectablehybridization to the probes, indicating the existence of internalXbaI sites. All the accessions produced 3.5 EcoRI fragment arising fromcoding region of the repeat unit. Four hundred and eighteen RAPDmarkers among 45 accessions were identified with 14 arbitrary10-base primers. The percentage of polymorphic bands withinspecies ranged from 20 in V.angustifolia to 98% inV. nigra. Both RFLP andRAPD markers were unable to assess the relationships betweenaccessions within species as there was often much closer resemblancesbetween certain accessions of different species rather than betweenaccessions within each taxon. This analysis supports the view basedon morphological, cytogenetical and crossability data that it is notpossible to classify Sativa species complex into a small finitenumber of taxa which are clearly circumscribed, and that the complexrepresents a unique case of rapid evolution and incipient speciation.A study of chloroplast and mitochondrial RFLPs was undertaken toanalyze phylogeny through maternal lineage. Chloroplast DNArestriction fragment patterns, using 13 restriction endonucleases,revealed 92.6 to 99% homology between the seven species.Twelve enzyme-probe combinations yielded identical fragmentpatterns for all the seven species. The molecular sizes of thechloroplast DNAs obtained were similar (121.5–123.5), indicating that they had all lost one of theinverted repeats. Total DNAs digested with three restriction enzymesand hybridized to six heterologous probes of mitochondrial originyielded monomorphic bands in five enzyme—probe combinationsacross all the 73 accessions. In other combinations as well,40–66 accessions yielded monomorphic profiles. The smallvariation in the remaining accessions was not species-specificsince the same profiles were present in more than one species. Theseresults i) strongly suggest that the seven species within thecomplex share a common ancestor, or direct lineage and, ii)indicate that these species should be relegated to a rank, perhaps ofsubspecies, within V.sativa species complex.