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Phylogenetic Biodiversity of Yeasts

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Phylogenetic Biodiversity of Yeasts Eco. Env. & Cons. 26 (4) : 2020; pp. (1527-1530) Copyright@ EM International ISSN 0971–765X Phylogenetic biodiversity of Yeasts Alan Makarovich Hoziev1*, Boris Georgievich Tsugkiev1, Valentina Batyrbekovna Tsugkieva1, Andrey Georgievich Petrukovich1, Zalina Alimbekovna Kubatieva1, Susanna Konstantinovna Cherchesova2 and Soslan Anatolievich Siukaev1 1Gorsky State Agrarian University, 37 Kirov Street, Vladikavkaz, Republic of North Ossetia-Alania, 362040, Russia 2North Ossetian State University named after Kosta Levanovich Khetagurov, 44-46 Vatutina Street, Vladikavkaz, Republic of North Ossetia-Alania, 362025, Russia (Received 17 July, 2020; accepted 2 September, 2020) ABSTRACT The authors identified yeast strains from the following grape cultivars: Rhein Riesling -DEN1- (7-2018); Cabernet -ZH-3 (9-2018) and L-3 (6-2018); Flora -DEN-5 (10-2018). According to the analysis results of nucleotide sequence that codes part of rRNA genes, performed in BRC VKPM NRC “Kurchatov Institute” – State Research Institute of Genetics, it was found that DEN-1 (7-2018) strain belongs to the species Metschnikowia pulcherrima; ZH-1 (9-2018) strain is closest to the species Pichia kluyveri; L-3 (6-2018) strain is the representative of the species Metschnikowia pulcherrima and DEN-5 (10-2018) belongs to the species Pichia kudriavzevii. The studies confirm that the grape cultivars – Rhein Riesling, Cabernet, Flora, introduced in the Republic of North Ossetia-Alania, are promising natural resource of systematic yeast diversity. Key words : Yeasts, Strain, Gene encoding 18S rRNA, DNA, PCR mode Introduction Currently, biotechnology plays an important role in the fermentation of various substrates and in the Yeast cells are microbial eukaryotes that belong to production of ethanol, as well as in the production ascomycetes, which are a good source of vitamin B of biodiesel and many other biofuels (Madigan and and protein (Madigan, 2003). Yeasts are unicellular Martinko, 2006). Advances in biotechnology have fungi. As a living organism, fungi require heat, wa- shed the new light on ancient and well-proven tech- ter, protein, nitrogenous substances, and sugar to nologies, having provided humanity for both a new, stay alive (the Artisan, The Yeast Treatise, 2002). clean, sustainable biofuel and bright future (Orelli, The yeast cell averages about 8 µm in diameter 2006). (Ando, 2006). Each cell has a two-layer wall that is The yeasts strain – “Baker’s Yeasts” is scientifi- porous and semi-permeable to certain substances cally known as Saccharomyces cerevisiae, 5-10 µm- and thus through the wall nutrients enter the cell sized in diameter (Halasz, 1988). Saccharomyces and metabolites leave it (Madigan, 2003). cerevisiae is the most famous and popular yeast The use of yeasts (Saccharomyces cerevisiae) in the species, as it is used to make alcohol and bread. It is production of wine and beer, as well as bread has known for being the most commonly used as an al- been known since the Bible (Cocolin, 2001). coholic beverages fermenter and the most com- 1528 Eco. Env. & Cons. 26 (4) : 2020 monly used for making bread (Halasz, 1991). The genetic affinity of similar species. In the course of yeast species Saccharomyces cerevisiae is also safe for the study the following results were obtained: humans as it is not pathogenic and does not nega- GATCTCGCTTATTGATATGCGCAGGTT tively affect the environment. CACCTACGGAMTCCTCCGCTTATTGATATG Under certain conditions some yeast fungi can CGCASGWTCACCKACSRAWYCSKMCGYTTRW show their negative for human health properties KRRTATRYATMGGTYACCCWMS CAAAWY (Nasseri, 2011). CCTGG GGAAWACCCCGGGGCGCAATGTG CGTTCAAAGATTCAATGATTCACGTCTG Experimental CAAG TCATATTACGTATCGCAATTCGCTG The research material was yeast strains: DEN-1 (7- CGTTCTTCATCGATGCGAGAACCAAGAG 2018), ZH-3 (9-2018), L-3 (6-2018), DEN-5 (10-2018) ATCCGTTGTTGAAAGTTTTTKRATTGWGTTA isolated from berries of Rhein Riesling, Cabernet, TTGAMRRAAAAGATTYAGWTGTTTTTYCYWA and Flora grape cultivars. AAG GGGGWAAWA WKATT TTTWAWGAWY The primary identification of the selected strains CYTCCSCARGGTCMCCYWMSGGAGGKGG was performed in the Research Institute of Biotech- Phylogenetic analysis, performed by using nology of Gorsky State Agrarian University, using closely related strains, shows that yeast species traditional research methods. (Babyeva, 1979). Metschnikowia pulcherrima is the closest to L-3 (6- For the final identification based on the analysis 2018) strain. of ribosomal genes sequence, the strains DEN-1 (7- Identification of DEN-1 (7-2018) strain 2018), ZH-3 (9-2018), L-3 (6-2018), DEN-5 (10-2018) were sent to the Bioresource Centre – All-Russian b) Analysis of the gene sequences coding 18S rRNA. Collection of Industrial Microorganisms (BRC The similarity of the nucleotide sequence of the VKPM) National Research Centre “Kurchatov Insti- gene coding 18S rDNA of the studied strain was tute” - State Research Institute of Genetics. BRC analyzed using BLAST server [http:// VKPM NRC “Kurchatov Institute” – State Research www.ncbi.nlm.nih.gov/blast]. Institute of Genetics conducted standard genetic Results studies. Primary screening on the GenBank database shows Results and Discussion that the studied DEN-1 (7-2018) strain belongs to the following systematic group: Eukaryota; Fungi; Determining the taxonomic affiliation of L-3 (6- Dikarya; Ascomycota; Saccharomycotina; 2018) strain Saccharomycetes; Saccharomycetales; b) Analysis of the gene sequences coding 18S Metschnikowiaceae; Metschnikowia. rRNA of the studied strain was performed using Homology of at least 97% is considered to be the BLAST server [http://www.ncbi.nlm.nih.gov/ criterion for classifying the studied strain to a par- blast]. ticular species. In this regard the studied strain be- longs to several species. Results The method to compare nucleotide sequences Primary screening on the GenBank database shows coding 5,8 S rRNA gene and internal transcribed that the studied strain belongs to the following sys- spacers ITS1 and ITS2 was also used to determine tematic group: Eukaryota; Fungi; Dikarya; phylogenetic affinity of similar species. Ascomycota; Saccharomycotina; Saccharomycetes; Sac- When sequencing the DNA section coding 5,8 S charomycetales; Metschnikowiaceae; Metschnikowia. rRNA gene and internal transcribed spacers ITS1 Homology of at least 97% is considered to be the and ITS2, the following sequence was obtained: criterion for classifying the studied strain to a par- TCGGTCTCCGCTTATTGATATGCGCAGGTT ticular species. CACCTACGGAWTCCTCCGCTTATTGATATG The studied strain of the microorganism can be CGCAGGTTCACCTACSGAWTCCTMCGYT referred to a number of species. The method to com- TAWTRRTATRYATCMGSTYACCCWCS pare nucleotide sequences coding 5,8 S rRNA gene CAAAAYCCTGGGGAATACCCCGGGGCG and internal transcribed spacers ITS1 and ITS2 was CATGTGCGTTCAAAGATTCAATGATTCACGT used to determine taxonomic affiliation and phylo- CTGCAAGTCATATTACGTATCGCATTCGCT HOZIEV ET AL 1529 GCGTTCTTCATCGATGCGAGAACCAAGAGAT Pichia kudriavzevii is the closest to DEN-5 (10- CCGTTGTTGAAAGTTTTTTWATTGWGTGW 2018) strain. WTGAMRAWAA AKATTTG GAGTTT GTTTCTS Identification of ZH-3 (9-2018) strain CMARAG WGTGW GAAWW ATTA TTA TGAAT GATCCYCCCGYA CGCCCA CCGAAG GAA. (b) Analysis of the gene sequences coding 18S Phylogenetic analysis, performed by using rRNA of the studied strain was performed using closely related strains, shows that yeast species BLAST server [http://www.ncbi.nlm.nih.gov/ Metschnikowia pulcherrima is the closest to DEN-1 (7- blast]. 2018) strain. Results Identification of DEN-5 (10-2018) strain Primary screening on the GenBank database shows b) Analysis of the gene sequences coding 18S rRNA. that the studied strain belongs to the following sys- The similarity of the nucleotide sequence of the tematic group: Eukaryota; Fungi; Dikarya; gene coding 18S rDNA was analyzed using BLAST Ascomycota; Saccharomycotina; Saccharomycetes; Sac- server [http://www.ncbi.nlm.nih.gov/blast]. charomycetales; Pichiaceae; Pichia. Homology of at least 97% is considered to be the Results criterion for classifying the studied strain to one or Primary screening on the GenBank database shows another species. The analyzed strain can be referred that the studied strain belongs to the following sys- to several species. tematic group: Eukaryota; Fungi; Dikarya; The method to compare nucleotide sequences Ascomycota; Saccharomycotina; Saccharomycetes; Sac- coding 5,8 S rRNA gene and internal transcribed charomycetales; Pichiaceae; Pichia. spacers ITS1 and ITS2 was also used to determine Homology of at least 97% is considered to be the phylogenetic affinity of similar species. criterion for classifying the studied strain to a par- When sequencing the DNA section coding 5,8 S ticular species. In this regard the studied strain be- rRNA gene and internal transcribed spacers ITS1 longs to several species. and ITS2, the following sequence was obtained: The method to compare nucleotide sequences CCGTATCCTCCKCTTATTGATRTGCGCAGGTTCA coding 5,8 S rRNA gene and internal transcribed CCTACGRARTCCTCCGCTTATTGATATRMTKC spacers ITS1 and ITS2 was used to determine phy- ARGTTCACYTMSYSCWTCAYCCTCYTTTCGWA logenetic affinity of similar species. TAAGGSTAGCCYGTTCTCAACTCTGCTTGCGC When sequencing the DNA section coding 5,8 S AAGAAGGAACGACGCTSAGACGGCATGCCC rRNA gene and internal transcribed spacers ITS1 CATGGAATACCATGGGGCGCAATGTGCG and ITS2, the following sequence was obtained: TTSAAGAACTCGATGATTCACGATGGCT TCGCATCTCCGCTTATTGATATGCGCAGGTTCA GCCATTCACAMTAGGTATCGCATTTCGCTG CCTACGRAMTCCTCCGCTTATYGRTMKGCGC CGCTCTTCATCGATGCGAGAACCAAGAGAT
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