Development of a molecular method for the rapid discrimination of red seaweeds used for agar production

Abstract In Tunisia, agar is produced from the species Gracilaria verrucosa . Other species, such as Gracilariopsis sp. which have a very similar morphology to Gracilaria , can be harvested in a mixture with Gracilaria with the result that the quantity and quality of agar extracted is different than initially expected. In this study, we tested the use of ITS sequences from 5.8s rDNA for species discrimination between G. verrucosa and Gracilariopsis harvested from two production sites (Bizerte and Tunis Lagoons). The amplification of genomic ITS sequences and sequencing shows that the samples collected from Tunis can be identified by PCR fragment of 1124 bp whereas the samples from Bizerte are characterized by the presence of two fragments of 1124 and 983 bp, respectively. A BLAST investigation in Genbank shows that the sequence similarities between the fragment of 1124 pb and G. verrucosa was 85% and between the fragment of 983 pb and Gracilariopsis sp. Plymoutn was 94%. Gracilariopsis sp. possesses a PCR fragment with a length of 983 bp, which discriminates it from G. verrucosa when these two species are collected in a mixture as in the lagoon of Bizerte. The RFLP method after Eco RI application provides a characteristic pattern for G. verrucosa , which is composed of two main fragments with respective sizes of 823 and 301 bp whereas amplified DNA of Gracilariopsis was uncut under these conditions. The molecular method studied here could also be used for the identification of G. verrucosa , which is the only agarophyte authorized in Europe as a sea vegetable.