A GENERAL-APPROACH FOR PCR-AMPLIFICATION AND SEQUENCING OF CHLOROPLAST DNA FROM CRUDE VASCULAR PLANT AND ALGAL TISSUE

A universal and reliable sequencing approach suitable for evolutionary and phylogenetic studies of most plant species has been developed. Th e initial step in the approach is PCR with primers from conserved regi ons in the chloroplast genome. Prior to PCR a simple DNA extraction is carried out where lysed tissue is embedded in agarose and dialyzed. D ue to the use of one biotinylated primer in the PCR, the PCR products can be bound to magnetic streptavidin-coated beads and then sequenced directly by the solid-phase approach. The regions used for sequencing and subsequent analyses are the intron of the trnL gene and the interg enic spacer between. the trnL and the trnF genes in the chloroplast DN A. Comparisons of the sequences obtained from closely and distantly re lated species show that the different chloroplast DNA regions harbor v ariations suitable for phylogenetic inference from the subspecific to the interfamiliar level. The strategy can be used both for manual and automated sequencing.