Sequencing of the internal transcribed spacer region ITS1 as a molecular tool detecting variation in the Stylosanthes guianensis species complex |
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Authors: | J Vander Stappen S Van Campenhout S Gama Lopez G Volckaert |
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Institution: | (1) K.U. Leuven, Laboratory of Gene Technology, Kardinaal Mercierlaan 92, B-3001 Leuven, Belgium Fax: +3216321965 E-mail: guido.volckaert@agr.kuleuven.ac.be, BE;(2) UNAM, Instituto de Biologia, Apartado Postal 70-233, Delegacion Coyacan, 04510 Mexico, D.F., MX |
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Abstract: | The internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA from Stylosanthes guianensis CIAT 1283 and cv ‘Schofield’ were amplified by polymerase chain reaction using conserved ITS primers from the 18S, 5.8S and
26S ribosomal genes flanking those regions. The entire region of 683 bp long was cloned, and seven clones were sequenced.
Comparison of the ITS spacer regions with published DNA sequences of other plant species revealed limited homology only; this
was in contrast to their comparison with the 5.8S rDNA sequences. The ITS1 region of 45 S. guianensis accessions was amplified by PCR and sequenced on both strands using the conserved primers ITS2-ITS5. These sequences, ranging
from 201 to 204 bp, were aligned to each other to assess intra-specific polymorphism. Within the S. guianensis (Aubl.) Sw. species complex, 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 each individual, reminiscent of an incomplete homogenization of the repeat
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 PCR.
Received: 24 June 1997 / Accepted: 31 October 1997 |
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Keywords: | Stylosanthes Internal transcribed spacer DNA sequence Molecular markers |
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