Assessing the use of mitochondrial cox1 gene and cox2-3 spacer for genetic diversity study of Malaysian Gracilaria changii (Gracilariaceae, Rhodophyta) from Peninsular Malaysia

Advances in DNA-based genetic markers provide the essential tools in measurement of genetic diversity relating to the evolution, biogeography, and systematics of red algae by exploiting genetic variation in the entire genome of organisms. The understanding of genetic diversity in Gracilaria changii (Gracilariaceae, Rhodophyta) will provide valuable information for conservation, plant breeding management, and strain selection for cultivation. However, information of intraspecific genetic variation is still rudimentary. In this study, two mitochondrial encoded markers, cytochrome oxidase subunit 1 (cox1) and intergenic spacer between the cytochrome oxidase subunits 2 and 3 (cox2-3 spacer) were used to investigate genetic diversity in 40 individuals of G. changii collected from 11 different geographically distinct populations from Peninsular Malaysia. Seven distinct mitochondrial haplotypes were identified with the cox1 gene and three mitochondrial haplotypes with the cox2-3 spacer. Intraspecific nucleotide differences ranged from 0 to 6 bp for the cox1 and 0–4 bp for the cox2-3 spacer, respectively. This is the first report comparing the suitability of mitochondrial markers of the cox1 gene and the cox2-3 spacer for genetic diversity studies on G. changii. The present study showed that the cox1 gene is a potential molecular marker to infer intraspecific genetic variation in red macroalgae. The cox1 marker is more variable compared to the cox2-3 spacer and revealed genetic variation and phylogeographic structure for this ecologically and economically important species.     

Molecular characterisation of Sargassum polycystum and S. siliquosum (Phaeophyta) by polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers

Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10) tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships between species within a genus and in developing individual fingerprints for individual samples.     

Genetic diversity of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariaceae,Rhodophyta) from west coast,Peninsular Malaysia based on mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 gene analysis

Mitochondrial cytochrome c oxidase subunit I (cox1) was employed to investigate the intraspecific genetic diversity of Gracilaria changii collected from various localities distributed along the west coast of Peninsular Malaysia. Gracilaria changii is an agarophyte with potential for commercialization in Malaysia as it has high yields of good quality agar with high gel strength for the production of food grade agar and agarose. The phylogeographic aspect of G. changii has not been studied despite its abundance and potential commercialization. In this study, six mitochondrial haplotypes (C1–C6) were revealed from 62 specimens varying by 0–3 bp over 923 bp. Results indicate that haplotype C1 is the common ancestor and the most widespread haplotype due to its prevalence in Morib, Gua Tanah, Middle Banks, Batu Besar, Batu Tengah, Sungai Pulai, and Kuala Sungai Merbok. In this study, Morib was suggested as contributing the highest intra-population diversity with the identification of three haplotypes. The mitochondrial marker cox1 is a highly divergent mitochondrial marker and is applicable for studies on species identification and assessment of genetic diversity of G. changii.     

Radiation of the Red Algal Parasite Congracilaria babae onto a Secondary Host Species,Hydropuntia sp. (Gracilariaceae,Rhodophyta)

Congracilaria babae was first reported as a red alga parasitic on the thallus of Gracilaria salicornia based on Japanese materials. It was circumscribed to have deep spermatangial cavities, coloration similar to its host and the absence of rhizoids. We observed a parasitic red alga with morphological and anatomical features suggestive of C. babae on a Hydropuntia species collected from Sabah, East Malaysia. We addressed the taxonomic affinities of the parasite growing on Hydropuntia sp. based on the DNA sequence of molecular markers from the nuclear, mitochondrial and plastid genomes (nuclear ITS region, mitochondrial cox1 gene and plastid rbcL gene). Phylogenetic analyses based on all genetic markers also implied the monophyly of the parasite from Hydropuntia sp. and C. babae, suggesting their conspecificity. The parasite from Hydropuntia sp. has a DNA signature characteristic to C. babae in having plastid rbcL gene sequence identical to G. salicornia. C. babae is likely to have evolved directly from G. salicornia and subsequently radiated onto a secondary host Hydropuntia sp. We also recommend the transfer of C. babae to the genus Gracilaria and propose a new combination, G. babae, based on the anatomical observations and molecular data.     

Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65–1.14 g g^–1 fresh weight) with high quality (A_260:280 ratio 1.80–2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies.     

Use of an algal consortium of five algae in the treatment of landfill leachate using the high-rate algal pond system

Five species of microalgae Chlorella vulgaris, Scenedesmus quadricauda, Euglena gracilis, Ankistrodesmus convolutus and Chlorococcum oviforme, were screened for their ability to grow in treated landfill leachate (TL) using shake flask cultures. The treated leachate had undergone previous treatment through mechanical aeration in treatment ponds at the landfill site. The five algae, except for C. oviforme, were able to grow in medium containing up to 50% TL. Two high-rate algal ponds (HRAP) equipped with paddlewheel were used for the semi-continuous cultures. A mixture of the five algae was used to inoculate one of two HRAPs for secondary treatment of TL. The other HRAP was filled with natural lake water containing mixed populations of algae. A volume of 400?mL (1%) from both ponds were removed daily and replaced with TL. The leachate loading rate was increased to 2% (0.8?L?day^?1) on day?197 and then to 4% (1.6?L?day^?1) on day?309, providing hydraulic retention time of 100, 50 and 25?days, respectively. Although higher biomass was obtained in the HRAP containing the consortium of five algae, there was no significant difference in reduction of pollutants between the two ponds. The HRAPs produced algal biomass ranging from 2.00 to 5.54?g dry weight?L^?1 with significant reduction in chemical oxygen demand (91.0%), ammoniacal nitrogen (99.9%) and orthophosphate (86.0%) contents. The HRAP offers a potential treatment system for TL which is simple, low cost, flexible in use and requiring low maintenance.     

Application of polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers in the molecular identification of selected Sargassum species (Phaeophyta,Fucales)

The random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used for the molecular characterisation and identification of Sargassum spp. A total of 17 samples of Sargassum (Sargassaceae, Fucales) was obtained from various localities around Peninsular Malaysia and Singapore. On the basis of morphological characteristics, the samples were tentatively grouped into five species: Sargassum baccularia, S. glaucescens, S. oligocystum, S. polycystum and S. siliquosum. By RAPD-PCR, five of 31 random primers tested generated reproducible amplification products, and polymorphic loci were detected by four of them (OPA02, OPA03, OPA04, OPA13). The RAPD-PCR profiles did not correlate with the morphological grouping into five species and extensive variation was detected between different isolates of the same species. A 450 base pair fragment generated using OPA13 was detected in 12 of 17 samples of Sargassum. This fragment was also present in profiles from Turbinaria (Sargassaceae). This study suggests that RAPD-PCR is useful in discriminating individual samples of the genus Sargassum and in developing fingerprints for them.     

Studies on the production of useful chemicals,especially fatty acids in the marine diatom Nitzschia conspicua Grunow

A local marine diatom, Nitzschia conspicua Grunow, was cultured in enriched synthetic seawater using flasks (agitated by magnetic stirring) and a 1.2 l fermenter. Lipids, fatty acids, proteins, carbohydrates and ash of the flask cultures were determined at various stages of growth (day 3, 5, 7, 10, 13, 15 and 17). The fermenter culture was harvested during the stationary phase for similar chemical analyses. N. conspicua attained a higher biomass concentration during the stationary phase when cultured in the fermenter (188 mg dry weight l^–1) than in flasks (140–151 mg dry weight l^–1). However, both systems showed similar specific growth rates based on chlorophyll-a concentration. Appreciable amounts of the essential fatty acids 20:4 (0.6–4.7% total fatty acids) and 20:5 (1.9–4.7% total fatty acids) are present in this diatom. Maximal amounts of these fatty acids were produced after 7 days' growth (i.e. 2 days after the end of the exponential phase). Lipids, fatty acids, proteins, carbohydrates and ash varied with culture age in N. conspicua.author for correspondence