Saccharomyces cerevisiae DNA Barkodunun Belirlenmesi ve Veri Tabanının Oluşturulması
Kurban , Mithat
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The aim of this project was to isolate and identify DNA barcodes of S. cerevisiae , which a model microorganism used in many traditional products, biotechnological processes and many researches, by using DNA barcoding technology used in the identification of living things. In this study; soil and grape samples were collected from the vineyards in different cities of Central Anatolia and the isolations were made. Also strains of S. cerevisiae which were used for barcoding research obtained from national and international yeast producers and from research laboratories of national universities, the strains which decided to be used as a control obtained from national and international culture collection organizations, ((S. cerevisiae ATCC® 9763 ™, S. cerevisiae ATCC®). 6328 ™, S. cerevisiae RSK08022 (Refik Saydam Culture Colllection)), and comparatively analysis of universal DNA barcodes was performed. All these strains were identified by morphological (macroscopic, microscopic) and biochemical tests, the universal DNA barcode sequences for these strains comparatively analyzed and revealed. For this purpose, 287 isolates were obtained and 229 of them were found to be suitable for the characteristics of S. cerevisiae according to macroscopic and microscopic definitions. To increase the identification level of species to the strain level in addition to classical validation studies on these strains the MALDI-TOF MS (Matrix Assisted Laser Desorption / Ionization, Time-Of-Flight Mass Spectrometry) method has also used. When MALDI TOF MS and molecular definitions were taken into consideration, 152 isolation were determined as S. cerevisae species. Following the identification of the isolations obtained, for choosing gene regions to be used in DNA barcoding research, according to the literature ITS “Internal Transcription Spacer” , LSU “Long Sub Unit”, SSU “Short Sub Unit” RPB1 “RNA polymerase subunit1”, RPB2 “RNA polymerase subunit2”, TOP1 “DNA Topoisomerase gene regions” pre-PCR “polymerase chain reaction” experiments were performed. According to the preliminary experiments, it was decided to use the appropriate gene regions (ITS, LSU, RPB2) to identify effective DNA barcode structures. PCR amplifications were performed on these 120 isolates of 2016-2017 years, whose DNA isolation and pre-definition have already done and DNA barcodes were investigated on these 120 isolates. Gel electrophoresis differentiations for PCR amplifications of isolates in the ITS gene region can be seen by due to taxonomic groups, while for the LSU gene region, single size band separation was observed for all isolations so separations could not be seen by due to taxonomic groups. Since for the amplification of RPB2 gene region gel separation that taxon distinctions could not be made on the gel images, because multiple band formation was observed for each isolate. This multi-band formation problem observed in the RPB2 gene region was determined that could be effectively overcome by extraction from the gel and doing sequence analysis. Sequence analysis was performed by Sanger Sequencing method on isolations whose DNA extraction and related gene regions PCR amplification has been done already. An average of 820 base pairs for the ITS gene region, 1100 base pairs for the LSU gene region, and 1200 base pairs for the RPB2 gene region were used for DNA barcode-based identification. The raw data obtained from the strains were cleaned with FinchTv (https://digitalworldbiology.com/FinchTV) and the raw data were subjected to necessary pre-treatment with BioEdit program for the preparation of advanced data analysis (http://www.mbio.ncsu.edu/ BioEdit / bioedit.html). Then, molecular level based comparison were made with NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) on data. Considering all the definitions made, phylogenetic trees have been formed which reveal the inter-species and intra-species relationship by ClustalW program (https://www.genome.jp/tools-bin/clustalw). With the help of these created phylogenetic trees, it was tried to determine the barcode regions that best describe S. cerevisiae species. The MEME-suit program (http://meme-suite.org/) was used to determine the motif regions to be used as barcode structure. The resulting sequence data were evaluated separately as isolation gene regions sequences and barcode sequences. As a result of the evaluations, according the phylogenetic distinction the success of the isolation sequences of the RPB2 gene region for S.cerevisiae species has been determined as % 98.24, success of the the ITS gene region determined as % 95.83, success of the the LSU gene region determined as % 92.3, and success of the unified region (ITS_LSU_RPB2) determined as % 95.16. In the phylogenetic distinction using barcode sequence structures, the succession of RPB2 gene region barcode structures for S. cerevisiae species is % 100, the separation success of the barcode structures of the ITS gene region is % 95.83, the separation success of the barcode structures of the LSU gene region is % 96.153, and the separation success of the barcode structures of the unified region (ITS_LSU_RPB2) determined as % 95.45. The dissociation coefficients of gene regions for isolation sequences and barcode sequence are respectively (ITS, LSU, RPB, ITS_LSU_RPB2) 0.848 ± 0.01, 0.157 ± 0.002, 0.427 ± 0.011, 0.412 ± 0.008 and 0.873 ± 0.01, 0.725 ± 0.017, 0.7636 ± 0.0136, 0.412 ± 0.008. While the use of the barcode sequence did not create a large difference in discrimination power for the ITS region, it was observed that the difference between the LSU and RPB2 gene regions was effective. As a result, it was observed that the ITS, LSU and RPB2 gene regions could be used successfully in the identification of S. cerevisiae yeast species and the barcode structures obtained by the MEME-suite program could provide this distinction more successfully. However, it was determined that the obtained barcode structures did not make a meaningful homogenous distinction in terms of isolation source and year for S. cerevisiae species. All research information obtained from the isolations is recorded in a database (“www.dnabarcodefoodomics.com”). As a result, it is thought that the creation and recording of DNA barcode information and thus providing quick access to the information in this field has contributed to the competitiveness for our country which is an important international yeast producer.