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Molecular Characterization and New Reports of Two Green Algae from Bangladesh

MD. ALMUJADDADE ALFASANE
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

MD. MIRAJ KOBAD CHOWDHURY
Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000, Bangladesh.

MALIHA MEHNAZ
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

This communication portrays the molecular characterization and confirms the new reports of two fresh water green algae namely, Pithophora polymorpha Wittrock and Spirogyra maxima (Hassall) Wittrock from Bangladesh. The samples of these algal species were cultured and partial 18S rDNA was sequenced and analysed for their molecular identification. It was found that the primers reported here could sufficiently identify these algae as P. polymorpha and S. maxima. Furthermore, the Neighbourjoining (NJ) tree generated from 18s rDNA sequences suggested that Spirogyra maxima of Bangladesh is distantly related to the cluster of S. juergensii and S. platensis. Pithophora polymorpha along with P. roettleri, P. sano and Pithophora sp. seems to form a strongly supported monophyletic group. The alga AP1 clusters with Pithophora and the alga AS1 clusters with Spirogyra. This study is the first-time report of molecular identification of Bangladeshi algae and a landmark towards the future exploration of the algal biodiversity of Bangladesh.

Keywords: Spirogyra maxima, Pithophora polymorpha, Molecular characterization, New reports, 18S rDNA

Location: Bandarban district, Bangladesh

Start Date: 00-00-2017

End Date: 00-00-2017

Program Area: Conservation and Biodiversity

Commodity: Algae

Objectives:

To confirm the identification of two green algae from Bangladesh and their molecular characterization using partial sequencing of 18S rDNA.

Methodology:

Collection and morphological characterization: Fresh filaments of Pithophora were collected from the Shoilo Propat fall, Bandarban (22°10'48" N, 092°13'48" E), and fresh filaments of Spirogyra were collected from the Sangu river, Bandarban (22°08'60" N, 92°12'36" E), Bangladesh on 13 March 2017. These specimens were kept in source water and were transferred to the Limnology Laboratory of the Department of Botany, University of Dhaka within 24 hours of collection. As soon as the specimens arrived at the laboratory, they were examined under a light microscope as wet mounts and photomicrographs were taken using Nikon Eclipse E200. The cellular length, width, number and shape of chloroplasts as well as the number of granules in each filament were recorded for morphological characterization and taxonomic identification. Culture of algae: Algae were cultured with the method as described before with some modifications (Sulfahri et al., 2017). Briefly, Bold’s Basal Medium (BBM) was prepared before the collection of algae and was stored at 4^oC after sterilization using 0.22 µm filter. The BBM was warmed at 37^oC before the culture of the algae. Individual filaments were transferred immediately to warmed BBM after microscopic observation for rescuing the algae and for purification of unialgal culture of the collected samples. The culture was incubated at 25oC for five to seven days in an orbital shaker at 125 rpm under 150 µE/m2 /s intensity of light with 12:12 light/dark cycle, growth of filaments was observed time to time and the filaments were sub-cultured following the technique mentioned above to purify the algae. DNA isolation and PCR: For molecular characterization and identification of species, partial sequences of 18S rDNA was amplified using the primers: Forward 5′-AGGGC AAGTC TGGTG CCAGCAG-3′ and Reverse 5′-GTTGA GTCAA ATTAA GCCGC-3′. Genomic DNA was extracted using modified phenol-chloroform-isoamyl alcohol method (Tabrejee et al., 2018). Briefly, individual algal culture was rinsed with distilled water followed by with 70% ethanol. Then the filaments were airdried to remove excess ethanol. The filaments were grinded to fine powder using liquid nitrogen. About 100 mg homogenized tissue was mixed with 500 µl of CTAB extraction buffer (2% cetyltrimethylammonium bromide, 1% polyvinyl pyrrolidone,100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA) and was vortexed thoroughly. The homogenate was transferred to a 60°C water bath for 30 minutes. Then the homogenate was centrifuged for 5 minutes at 14,000 x g and the supernatant was collected for DNA extraction. The DNA was estimated using NanoDropTM and was used for PCR. The PCR condition includes an initial step of 5 min at 95 °C, then 40 cycles of 30 seconds at 95°C, 30 seconds at 57°C, and 1 min at 72°C, followed by 10 min at 72°C. The PCR products were visualized on 1% agarose gel under UV-transilluminator and photomicrograph was taken. Sequencing and phylogenetic analysis To design the primers, multiples sequences of 18S rDNA genes were aligned using Clustal Omega. From the alignment, two conserved regions spacing 600-800 bp were selected. Sequences from these regions were used for primer design. Next, these primers were verified using the Primer-BLAST tool. The PCR products were purified using a PureLinkTM PCR purification kit and sequenced at the Macrogen, South Korea by Sanger sequencing. All the raw sequences were processed using FinchTV and aligned using CLC workbench. These sequences were aligned using Basic Local Alignment Search Tool (BLAST) with the 18S rDNA sequences database of the National Center for Biotechnology Information (NCBI) for molecular identification and were submitted to GenBank with referred accession numbers (MH894274- MH894275). Nucleotide compositions of the processed sequences were analyzed using Mega v5.05. Neighbor-joining (NJ) tree based on K2P distances was created to illustrate molecular phylogeny using Mega v5.05. For this, Vampyrella lateritia was used as an outgroup.

Source of Information: Bangladesh J. Plant Taxon. 26(1): 39–45, 2019 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Here, two different algae from Bangladesh were studied in classical morphology method as well as molecular method (Barsanti and Gualtieri, 2014). These algae were collected and were successfully cultured using BBM. While studied under the light microscope, it was observed that the collected algal filaments were either of Pithophora genus or of Spirogyra genus. The filaments of Pithophora were green to dark brown in color, freely but sparsely branched, and containing intercalary and terminal akinetes. Cells of Pithophora were slender and cylindrical with 1100-1450 µm length and 50-120 µm width comprising thin cell wall without layers. Each cell contained one reticulated chloroplast with numerous pyrinoids. Terminal cells are conical and rounded. These data were consistent with previous reports (Manoylov, 2014; MouraJúnior et al., 2016). Filaments of Spirogyra were light green to green and unbranched. Vegetative cells of Spirogyra were 70–100 µm wide and 120–230 µm long. Cell wall plane was transverse, and each cell contained 5–7 spiral chloroplasts with numerous pyrinoids. Brown, multilayered, and reticulated mesospores were present in the filament. Often, lenticular zygospores were present in the filaments of Spirogyra. Such observations were similar to previous findings (Manoylov, 2014; Volkova et al., 2018). However, confirming the species of these algae-based on such morphological observations was difficult. Hence, molecular characterization of these algae was applied based on partial 18S rDNA typing to confirm the genus and to identify the species. This study accounts for the first report on molecular characterization of algae found in Bangladesh. Both the Pithophora polymorpha and the Spirogyra maxima were successfully identified by analyzing partial 18S rDNA sequences and these two algae are also new reports for Bangladesh. The development of a complete dataset on 18S rDNA sequences of all the algae found in Bangladesh is required for future identification of algal species and for the conservation of algal biodiversity of Bangladesh.

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