Cleavase Fragment Length Polymorphism (CFLP): A Methodology to Further Exploit Polymorphisms from PCR Products of Plastid DNA (ptDNA) in Phaseolus

Abstract

The CFLP methodology was applied for Cleavase I site detection within ptDNA intergenic regions (atpB-rbcL and rps14-psaB) at both interspecific and intraspecific levels in the genus Phaseolus. Optimal Cleavase I reaction temperature was 55 °C and the semi-dry electrophoretic transfer was more efficient than the original capillary one. Cleavase reactions yield a high number of fragments as compared to PCR-RFLP and allowed differentiation within and between landraces and wild forms of the Lima bean (Phaseolus lunatus L.) originating from Andean and Mesoamerican regions of Latin America. From sequencing data and using stemloop program (GCG, Madison), congruent numbers of hairpins/fragments were identified within the sequences, highlighting the robustness of the Cleavase I. Our results pointed out the ubiquity of short conserved motifs amongst a geographically localized group of species. In the vicinity of these motifs, synapomorphic-like substitutions were frequently observed. A phylogenetic tree based on these sequences is congruent with the CFLP pattern as well as with the widely accepted phylogeny of the genus. The usefulness of this new tool as alternative and/or complementary to PCR-RFLP technology on ptDNA is suggested and discussed.