A modification to the SCAR (Sequence Characterized Amplified Region) method provides phylogenetic insights within Ceratozamia (Zamiaceae)

Abstract

Phylogenetic relationships among closely related plant species is still problematic. DNA intergenic regions often are insufficiently variable to provide desired resolution or support. In this study, a modification to the Sequence Characterized Amplified Region (SCAR) method was used to find polymorphic loci for phylogenetic analyses within Ceratozamia . RAPD markers were first used to detect variation in five species. Then, equal length fragments in two or more species were excised from the gel, purified and digested with frequent cutter restriction enzymes for isolating both ends, which have the same primer site. Later, digested fragments were sequenced with the RAPD primer. Last, variable sequences were used to design specific primers for amplifying and sequencing all species for phylogenetic analyses. Our results confirmed the previously known high genome sequence resemblance within this genus that contrasts with its high morphological variation. Only six parsimony informative characters were generated with this approach. Nonetheless, the SCAR method provided some phylogenetic insights. Three main clades consistent with distribution ranges of the species were detected. The approach presented here was effective to solve some relationships within the genus and can be implemented in other organisms to find polymorphic loci for phylogenetic studies.