DNA BARCODING AS A TOOL FOR IDENTIFYING THE DIVERSITY OF DIATOMS ORIGINATING FROM THE WATERS OF LAMPUNG BAY AND SERIBU ISLANDS

• Main Author: Rosa Pane, Elfira
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/75033
• Institution: Institut Teknologi Bandung

Indonesia is an archipelagic country with a percentage of its territory consisting of water reaching 70%. Various types of organisms thrive in Indonesian waters. The diversity of organisms found in Indonesian waters is a natural treasure that needs to be protected and identified by their species. Diatoms are one of the important organisms in aquatic environments as they serve as the foundation of the food chain and are capable of photosynthesis. Diatoms are single-celled microorganisms, and to observe the morphology of diatom cells, a light microscope is required. Observation under a light microscope can reveal the diversity of shapes, sizes, and colors of diatom cells. However, it is not sufficient to determine their identity because the morphology of different diatom species can appear similar under a light microscope. Therefore, alternative methods, such as genetic identification, are needed for species identification. Several genes, including COX1, ITS1, ITS2, 5.8S, 28S, 18S, rbcL, and rbcS, have been utilized as genetic markers for identification purposes. However, as of now, no gene has been definitively established for diatom identification. The rbcL gene is responsible for encoding the rubisco protein, which is crucial for carbon dioxide fixation during the dark phase of photosynthesis. Diatoms, being photosynthetic organisms, possess the rbcL gene and can synthesize the rubisco protein. Therefore, in this research, the rbcL gene, along with the 18S V4rRNA gene, was employed to identify diatoms originating from tropical marine environments, specifically in Lampung Bay and Seribu Islands. The research methodology involved the collection of diatom samples from Indonesian marine waters, specifically in Lampung Bay and Seribu Islands, specifically in the cluster of Pari Islands. Seawater samples were observed under a light microscope, and individual diatoms found in the seawater samples were carefully selected and cultivated in reaction tubes containing modified seawater media. Not all the collected diatoms were able to grow successfully. Only those that could adapt were able to grow and gradually propagate until a sufficient number of cells were obtained for DNA genome isolation. The isolated DNA was used as a PCR template to amplify the 18S rRNA V4 and rbcL genes. The nucleotide sequences of both genes were determined and analyzed using the Blast application (NCBI) to identify the diatom samples. A phylogenetic tree based on the 18S V4 rRNA and rbcL genes was constructed using 20 diatom genera. The nucleotide sequences of the rbcL gene from these 20 genera were analyzed to identify nucleotide markers that differentiate between clades and genera. The effects of these nucleotide markers on the deduced amino acid residues in the rubisco protein were analyzed. Barcode sequences were created from short rbcL fragments that contain the nucleotide markers for clades and genera. The results of this study revealed the presence of 18 diatoms from the waters of Lampung Bay and 28 diatoms from Pari Island. Out of all the diatoms collected, only 13 were able to grow, be cultivated in the laboratory, and have their DNA genomes isolated. Based on the morphology and nucleotide sequences of the 18S V4 rRNA and rbcL genes, these 13 diatoms were identified as the following genera: Serratifera (SELBA1 isolate), Lauderia (LLBA2 isolate), Actinocyclus (ALBA1 isolate), Cyclotella (TMA2 isolate), Nitzschia (TMGS1 and PBI2 isolates), Psammodyction (TMA3 isolate), Entomoneis (EPI1 isolate), Navicula (NLA1, PBI1, and NLBA2 isolates), and Halamphora (HLBA1 and NLBA1 isolates). The phylogenetic tree constructed based on the rbcL gene exhibited similar branching patterns and clustering of Clades and genera as the tree based on the 18S V4 rRNA gene. This indicates that rbcL possesses the same capability as the 18S V4 rRNA gene in distinguishing clades and genera of diatoms. Analysis of the rbcL sequences from the 20 diatom genera resulted in a total of 120 nucleotide markers, including 17 nucleotide markers for Clades and 113 nucleotide markers for genera. To facilitate identification using the rbcL short chain gene sequence, the rbcL gene sequence is divided into four regions, each capable of identifying 20 analyzed genera. These four regions are as follows: region 1, spanning 228 bp starting from nucleotide 177 to 405; region 2, spanning 336 bp starting from nucleotide 513 to 849; region 3, spanning 237 bp starting from nucleotide 909 to 1146; and region 4, spanning 207 bp starting from nucleotide 1182 to 1389. Nucleotide variations at clade marker positions and genus have led to varying amino acid residues within the rubisco protein, although these variations do not affect the catalytic residues. The positions of the four regions that are capable of identification in rbcL were observed: regions 1 and 3 are involved in interactions with their neighboring RbcL in rubisco, while regions 2 and 4 are involved in interactions with RbcS in rubisco. These four regions can be used as a DNA barcode for rapid identification of diatom genera. The barcode DNA sequence is a short DNA chain sequence that can be used for identification purposes. The rbcL gene has 1473 nucleotides, which is too long to be used as a barcode DNA sequence. Four regions within the rbcL gene that have been analyzed are capable of identifying 3 clades and 20 genera, each of which can be used as a barcode DNA sequence. Among the four regions in the rbcL gene that we have mapped, based on the minimum number of nucleotides required to identify clades and genera, region 1 > region 3 > region 2 > region 4. Region 1 has a length of 250 base pairs and is located at nucleotide positions 177 to 405 in the rbcL gene. Based on these results, primers for amplifying the rbcL gene in region 1 can be designed, and rapid identification of diatom clades and genera can be achieved.