Discrimination of Tulipa species using AFLP analysis

The recently introduced PCR- based DNA fingerprinting technique AFLP (Amplified Fragment Length Polymorphism ) has been used for multiple purposes such as construction of linkage maps, marker satura tion at specific genomic regions, analysis of genetic diversity and molecular phyloge ny and cultivar identification. AFLP can be tailored by varying the number of selective nucleotides added to core primers and can allow accurate amplification, even in complex template mixtures generated from plant species with very large genomes. In this study Tulipa , a plant species with a very large genome, was tested for ad apting the AFLP protocol. The AFLP method was performed as described by Vos et al . (1995 ) with some minor modifications. 300-500 ng DNA was restricted at 37° C for 1.5 hours, with 5 U EcoRI and 5 U MseI in 4 µl of NEB4 buffer in a total volume of 40 µl. Ligation was done at 37° C overnight in the same so lution after adding 5 pmol EcoRI adapter and 5pmol MseI adapter together with 0.01mmol ATP, 2 U T4DNA ligase and 1 µl NEB4 buffer in a total reaction volume of 50 µl. Digestion product was diluted 5 times of which 10 µl was used for AFLP. The general protocol included four steps: restricti on of genomic DNA with EcoRI and MseI, and ligation of adaptor sequences t o the restriction fragments in order to generate the primary template; selective p reamplifications of this primary template with AFLP primers having varios additional 3' selective nucleotides; selective amplification with 33 P- labeled EcoRI primers having three 3' selective nucleotides and MseI primers with four 3' selective nucleotides(M52G/E33 and M52C/E37); separation of labeled amplification products on a denaturing polyacrylamide sequencing gel. The results indicated reproducible AFLP patterns, c lear and labor saving fingerprints, when seven selective nucleotides were used. REFERENCES: