SEQUENCING OF THE INTERNAL TRANSCRIBED SPACER REGION ITS1 AS A MOLECULAR TOOL DETECTING VARIATION IN THE STYLOSANTHES-GUIANENSIS SPECIES COMPLEX

The internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA from Stylosanthes guianensis CIAT 1283 and cv 'Schofield' were am plified by polymerase chain reaction using conserved ITS primers from the 18S, 5.8S and 26S ribosomal genes flanking those regions. The enti re region of 683 bp long was cloned, and seven clones were sequenced. Comparison of the ITS spacer regions with published DNA sequences of o ther plant species revealed limited homology only; this was in contras t to their comparison with the 5.8S rDNA sequences. The ITS1 region of 45 S. guianensis accessions was amplified by PCR and sequenced on bot h strands using the conserved primers ITS2-ITS5. These sequences, rang ing from 201 to 204 bp, were aligned to each other to assess intraspec ific polymorphism. Within the S. guianensis (Aubl.) Sw. species comple x, 11 DNA sequence types could be distinguished based on an insertion/ deletion (indel) event and 15 single base-pair substitutions. In 1 of the S. guianensis types, two kinds of ITS1 sequence were observed in e ach individual, reminiscent of an incomplete homogenization of the rep eat structure in this type. Polymorphisms in the sequence of the ITS1 region were used to define molecular markers for S. guianensis on the basis of PCR-restriction fragment length polymorphism and selective PC R.