Genetic Resources and Crop Evolution 00: 1–0, 2001. 1  2001 Kluwer Academic Publishers. Printed in the Netherlands. Evidence of rapid evolution and incipient speciation in Vicia sativa species complex based on nuclear and organellar RFLPs and PCR analysis 1,2 1, * Behrouz Shiran and S.N. Raina 1 Laboratory of Cellular and Molecular Cytogenetics, Department of Botany, University of Delhi, Delhi 110 2 007, India; Current address: Plant Breeding Division, Agriculture Department, Shahr-e-Kord University, * Shahr-e-Kord, Iran; Author for correspondence (e-mail: snraina@satyam.net.in; soomr@yahoo.com; phone: 191 -11 -7666402; fax: 191 -11 -7666541) Received 16 May 2000; accepted in revised form 19 January 2001 Key words: Chloroplast DNA, Evolution, Mitochondrial DNA, Nuclear DNA, RAPD, RFLPs, Vicia sativa species complex Abstract Seventy three accessions of the seven Vicia species belonging to Sativa species complex were screened for nuclear and organellar restriction fragment length polymorphic (RFLP), and random amplified polymorphic DNA (RAPD) markers. Total genomic DNAs of 73 accessions was restricted with three enzymes, and the restriction fragments were hybridized to the wheat rDNA probe pTa71 (containing 18S, 5.8S and 25S rDNA genes, and spacers), and faba bean probesVer 6-5 (entire intergenic spacer flanked by small part of 25S and 18S fragments) and Ver 18-6 (part of the coding region of the gene and internal transcribed spacers). In XbaI digests, 16 repeat unit length classes in 24 combinations were identified. Digestion with EcoRI and DraI gave 2–4 and 1–3 fragments, respectively, with detectable hybridization to the probes, indicating the existence of internal XbaI sites. All the accessions produced 3.5 kb EcoRI fragment arising from coding region of the repeat unit. Four hundred and eighteen RAPD markers among 45 accessions were identified with 14 arbitrary 10-base primers. The percentage of polymorphic bands within species ranged from 20 in V. angustifolia to 98% in V. nigra. Both RFLP and RAPD markers were unable to assess the relationships between accessions within species as there was often much closer resemblances between certain accessions of different species rather than between accessions within each taxon. This analysis supports the view based on morphological, cytogenetical and crossability data that it is not possible to classify Sativa species complex into a small finite number of taxa which are clearly circumscribed, and that the complex represents a unique case of rapid evolution and incipient speciation. A study of chloroplast and mitochondrial RFLPs was undertaken to analyze phylogeny through maternal lineage. Chloroplast DNA restriction fragment patterns, using 13 restriction endonucleases, revealed 92.6 to 99% homology between the seven species. Twelve enzyme-probe combinations yielded identical fragment patterns for all the seven species. The molecular sizes of the chloroplast DNAs obtained were similar (121.5–123.5 kb), indicating that they had all lost one of the inverted repeats. Total DNAs digested with three restriction enzymes and hybridized to six heterologous probes of mitochondrial origin yielded monomorphic bands in five enzyme—probe combinations across all the 73 accessions. In other combinations as well, 40–66 accessions yielded monomorphic profiles. The small variation in the remaining accessions was not species-specific since the same profiles were present in more than one species. These results i) strongly suggest that the seven species within the complex share a common ancestor, or direct lineage and, ii) indicate that these species should be relegated to a rank, perhaps of subspecies, within V. sativa species complex. ICPC - XPS 30297 (GRES) - product element 334372 - Tue Aug 28 18:24:47 2001