Species identification of red and brown seaweeds using ITS ribosomal DNA amplification and RFLP patterns

Abstract

The identity of five macroalgae used as sea vegetables or food ingredients has been determined by amplification of the ribosomal DNA ITS region and RFLP analysis. This allows the detection of specificity patterns for each species and provides an alternative method, when morphological or biochemical methods fail, for control of their use as food ingredients. Alga-specific PCR primers have been used to determine the ITS rDNA sequences of DNAs extracted from dried and ground algae up to 5 years old. The seaweeds studied were the red algae Palmaria palmata (dulse), Porphyra umbilicalis (nori), and Chondrus crispus (pioca) and the brown algae Himanthalia elongata (sea spaghetti) and Laminaria sp (konbu). Total genomic DNA suitable for amplification was extracted from the alga powder following the CTAB method. This methodology allowed the sequencing of the amplified product and the drawing of theoretical restriction maps useful in the choice of restriction enzymes for RFLP analysis. Enzymes that appeared useful included Mbo I and Alu I. Cutting with a single enzyme was sufficient to obtain characteristic patterns for the red algae P palmata, P umbilicalis and C crispus. For the two brown algae H elongata and Laminaria sp the ITS rDNA sequence showed a lack of suitable restriction site, contrary to other species for which characteristic patterns were obtained.

Simultaneous extraction of proteins and DNA by an enzymatic treatment of the cell wall of Palmaria palmata (Rhodophyta)

“…Our results revealed a three-to five-fold increase in DNA yield within the range of 800 μg g −1 of DNA. Our DNA yield appeared high compared with those reported by Wattier et al (2000) and Antoine and Fleurence (2003), which were 83 and 250 μg g −1 respectively for Palmaria palmata. DNA purity, monitored by using A 260 /A 280 ratios, is within the range of 1.8-1.9, indicating a suitable quality of DNA for further molecular analysis.…”

“…DNA purity, monitored by using A 260 /A 280 ratios, is within the range of 1.8-1.9, indicating a suitable quality of DNA for further molecular analysis. These values were higher than those reported (1.6) by Antoine and Fleurence (2003). As previously published by Joubert and Fleurence (2005), our procedure led to a DNA of high quality, a prerequisite for molecular analyses such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and sequencing.…”

“…In the case of the red alga P. palmata, the cetyltrimethylammonium bromide (CTAB) process is generally used to remove contaminants such as polysaccharides (Doyle and Doyle 1987) with a DNA recovery in the range 83 μg g −1 (Wattier et al 2000) to 250 μg g −1 wet weight (Antoine and Fleurence 2003). DNA purity estimated by the absorbance ratio A 260 /A 280 was about 1.60 (Antoine and Fleurence 2003). We examined DNA extraction yield following the same enzymatic procedure mentioned above.…”

“…DNA extraction from red seaweeds for amplification and analysis of the internal transcribed spacer region of the nuclear ribosomal unit (ITSs) has previously been reported (Goff and Moon 1993;Van Oppen et al 1995;Hughey et al 2002;Antoine and Fleurence 2003;Joubert and Fleurence 2005). In the case of the red alga P. palmata, the cetyltrimethylammonium bromide (CTAB) process is generally used to remove contaminants such as polysaccharides (Doyle and Doyle 1987) with a DNA recovery in the range 83 μg g −1 (Wattier et al 2000) to 250 μg g −1 wet weight (Antoine and Fleurence 2003). DNA purity estimated by the absorbance ratio A 260 /A 280 was about 1.60 (Antoine and Fleurence 2003).…”

Genetic Identification and Mass Propagation of Economically Important Seaweeds

“…To date, the application of different genes for the genetic identification of seaweed species is widely carried out, where the targeted DNA regions are the nuclear, plastid, and mitochondrial DNA (mtDNA). Most molecular characterization targets seaweeds with economic value such as Palmaria palmate (Dulse), Porphyra umbilicalis (Nori), Gracilaria changii, Kappaphycus alvare-zii, K. striatus, Eucheuma denticulatum (carrageenophytes), and many more [19][20][21][22][23]. Currently, large-scale DNA barcoding such as Red Algal Tree of Life Initiatives (RedToL) is analyzing phylogenetic relationship of 471 red seaweeds using two nuclear, four plastid, and two mitochondrial encoded gene markers [24].…”