Comparison of Rdna Regions (ITS, LSU, and SSU) of Some Aspergillus, Penicillium, and Talaromyces Spp
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Turkish Journal of Botany Turk J Bot (2016) 40: 576-583 http://journals.tubitak.gov.tr/botany/ © TÜBİTAK Research Article doi:10.3906/bot-1603-12 Comparison of rDNA regions (ITS, LSU, and SSU) of some Aspergillus, Penicillium, and Talaromyces spp. Rasime DEMİREL* Department of Biology, Faculty of Science, Anadolu University, Eskişehir, Turkey Received: 07.03.2016 Accepted/Published Online: 28.06.2016 Final Version: 06.12.2016 Abstract: Aspergillus, Penicillium, and Talaromyces spp. are commonly found worldwide and include industrially and medicinally important members. These genera are deeply related with life from soil to health hazards via their industrial products, surface proteins, biodiversity, food spoilage, mycotoxins, etc. These three genera progressively encounter important taxonomic revisions, such as the one fungus-one name concept, and various methods are available to establish their evolutionary and phylogenetic relationships. Besides morphological, microscopic, and extrolite analyses, nucleotide sequence analysis is the most preferred method. In particular, rDNA regions, such as the internal transcribed spacer (ITS), large subunit (LSU), and small subunit (SSU), are commonly used for taxonomic and phylogenetic studies. Although the ITS locus is accepted as the barcode gene for fungal identification, it has some limitations. Therefore, the aim of the present study was to compare ITS, LSU, and SSU loci in terms of ease of application, discrimination of species, and phylogeny by using 43 strains belonging to the above-mentioned three genera of fungi. The results indicated that the ITS and LSU loci were the most effective, exhibiting identical topology and species discrimination, while the SSU locus was the least effective, demonstrating lower species discrimination and the worst topology. Furthermore, the long and high-quality ITS, LSU, and SSU sequences obtained in the present study, which belong to the above-mentioned three genera of fungi, have been deposited in the NCBI database. Key words: ITS, LSU, SSU, Aspergillus, Penicillium, Talaromyces 1. Introduction clade with Talaromyces (Samson et al., 2011; Yılmaz et Aspergillus and Penicillium spp. are well known and the al., 2014). The genus Talaromyces includes medically most common fungi found in a range of habitats, such as important members, fungal antagonists, and enzyme soil, indoor and outdoor air, and various food products and soluble pigment producers. Following the recent (Krijgsheld et al., 2013; Houbraken et al., 2014; Samson et development in this genus, particularly through multilocus al., 2014; Visagie et al., 2014). In addition, these two genera gene sequencing and phylogenetic studies, taxonomy has have worldwide distribution and cause major economic gained more importance in mycology (Yılmaz et al., 2014). and health effects on human, animal, and plant life (Asan, To achieve accurate and quick results, various 2004; Demirel et al., 2013). They have many functions, identification strategies are available that consider including decomposition of organic materials, pre- and morphological properties and molecular structures of postharvest pathogenesis of food products, production of extrolites. Morphological identification techniques are important mycotoxins (Demirel and Sariozlu, 2014), and one of the traditional identification strategies based on the causing some health effects, such as aspergillosis (Samson investigation of morphological and microscopic properties et al., 2014; Visagie et al., 2014). Owing to their numerous of the fungi on different culture media and conditions. positive and negative effects, studies on the identification However, the findings obtained through these techniques of these genera are extremely important. are affected by the experiences and decisions of the In 1955, the genus Talaromyces was described by researchers (Pitt, 2000; Klich, 2002; Samson et al., 2010). Benjamin as a sexual stage of Penicillium. Subsequently, Extrolite profile analyses are based on the determination of based on polyphasic species studies and the one fungus- secondary metabolites and are very useful with ultrahigh- one name concept, Penicillium subgenus Biverticillum was performance equipment and well-developed databases. reassigned to this genus because it forms a monophyletic Although these analyses, together with morphological * Correspondence: [email protected] 576 DEMİREL / Turk J Bot identification studies, have been very successful, and LS266-R, 5’-GCATTCCCAAACAACTCGACTC-3’; identification purely based on secondary metabolites LR0R, 5’-ACCCGCTGAACTTAAGC-3’ and is not feasible (Frisvad et al., 2007; Visagie et al., 2014). LR5,5’-TCCTGAGGGAAACTTCG-3’; and NS1, Although many biochemical and physiological techniques 5’-GTAGTCATATGCTTGTCTC-3’ and NS4, provide useful information for the identification of fungi, 5’-CTTCCGTCAATTCCTTTAAG-3’ (Samson et al., the nucleotide sequence is the most accurate and sensitive 2010), respectively. identification tool to discriminate between organisms and PCRs were performed using a Veriti 96-Well Thermal accurately determine their evolutionary and phylogenetic Cycler (Applied Biosystems, Foster City, CA, USA) in relationships (White et al., 1990; Hibbet et al., 2007; 25 mL of solution containing 1 µL of genomic DNA, 2.5 Samson et al., 2010). µL of 2.5 µM forward and reverse primers, 2.5 µL of Taq Numerous gene regions, such as rDNA regions, buffer + KCl-MgCl2 (Fermentas), 2.5 µL of 25 mM MgCl2 mitochondrial regions, and some protein-expressing (Fermentas), 2 µL of 2.5 mM dNTP mix, 0.25 µL of 5 U/µL genes, can be used for sequencing and phylogenetic Taq DNA polymerase (Fermentas), and 11.75 µL of sterile analyses (White et al., 1990; Hibbet et al., 2007; Peterson, deionized water. The amplification conditions consisted of 2012; Schoch et al., 2012). For molecular identification denaturation at 94 °C for 5 min (ITS)/10 min (LSU and based on sequencing, rDNA regions, such as the internal SSU), followed by 35 cycles at 94 °C for 45 s (ITS)/15 s (LSU transcribed spacer (ITS), large subunit (LSU), and small and SSU), 56 °C for 30 s (ITS)/48 °C for 30 s (LSU)/52 °C subunit (SSU) regions, are very effective. In particular, the for 30 s (SSU), and 72 °C for 2 min (ITS)/1.5 min (LSU and ITS locus is considered to be the official DNA barcode SSU) and a final extension at 72 °C for 6 min (ITS)/7 min for fungi (Seifert, 2009; Schoch et al., 2012; Yılmaz et al., (LSU and SSU) (Samson et al., 2010). The PCR products 2014; Das and Deb, 2015). Therefore, the purpose of the were separated by agarose gel electrophoresis (1% w/v in present study was to compare the performance of ITS, 1X TAE), visualized by GelRed staining, cleaned using LSU, and SSU loci of the genera Aspergillus, Penicillium, EXOSAP-IT (Affymetrix, Santa Clara, CA, USA), and and Talaromyces in terms of ease of application, species used for sequencing. discrimination, and phylogeny using maximum likelihood Fragments containing the ITS locus, which were (ML) analysis. amplified using the primers V9G and LS266, were sequenced using ITS1– 5’-TCCGTAGGTGAACCTGCGG-3’ and 2. Material and methods ITS4–5’-TCCTCCGCTTATTGATATGC-3’ (White et 2.1. Fungal strains al., 1990). Similarly, fragments containing the LSU locus A total of 43 strains belonging to Aspergillus, Penicillium, were sequenced using the LR0R and LR5 primers, while and Talaromyces were used in this study. The strains were those containing the SSU locus were sequenced using obtained from 25 different cereal-based flour samples (12 the NR1 and NR4 primers. All the sequencing reactions wheats, 3 whole wheats, 3 corns, 3 rusks, 1 rice, 1 soy, were performed using the CEQ DTCS Quick Start Kit 1 gluten-free, and 1 rye) from Thrace (Tekirdağ, three (Beckman Coulter, Brea, CA, USA) with the CEQ 8000 samples), Northwest Anatolia (İstanbul, six samples; Genetic Analysis System. Bilecik, two samples), West Anatolia (Aydın, five samples; 2.3. Data analyses İzmir, one sample), and Central Anatolia (Ankara, six For the DNA sequence analyses, all the sequences were samples; Konya, two samples) of Turkey. Additional compared with GenBank sequences using BLAST, and information on these and related strains can be found alignments were performed using Muscle in the MEGA 6.0 elsewhere (Demirel and Sariozlu, 2014). The strains were software package, together with the barcode genes for ITS maintained at 4 °C on malt extract agar (MEA). and the closest BLAST results for LSU and SSU (Tamura 2.2. DNA extraction, PCR amplification, and sequencing et al., 2013). The aligned datasets were examined using DNA extraction was performed with strains grown on MEA ML analysis based on the Tamura–Nei model (Tamura for 7 days at 25 °C using the Ultraclean Microbial DNA and Nei, 1993) as implemented in MEGA 6.0 with 1000 Isolation Kit (MoBio, Solana Beach, CA, USA) following bootstrap replications. All the positions containing gaps the manufacturer’s protocols, and the extracted DNA was and missing data were eliminated. Paecilomyces variotii stored at –20 °C. To study the phylogenetic relationships (GU968674, JN938878 and JN939029), not a member of among the three genera of fungi, the ITS, LSU, and SSU the investigated three genera, was used as the outgroup. regions of the rDNA were amplified using the following The obtained sequence data were deposited in GenBank primer pairs: V9G-F, 5’-TTACGTCCCTGCCCTTTGTA-3’ with accession numbers. 577 DEMİREL / Turk J Bot 3. Results and discussion compared by BLAST, reference sequences with poor