Vol. 62, No 4/2015 683–689 http://dx.doi.org/10.18388/abp.2015_1102 Regular paper Determination of biodiversity of Coprinus comatus using genotyping and metabolic profiling tools* Anna Pawlik1, Anna Malinowska1, Marek Siwulski2, Magdalena Frąc3, Jerzy Rogalski1 and Grzegorz Janusz1* 1Department of Biochemistry, Maria Curie-Skłodowska University, Lublin, Poland; 2Department of Vegetable Crops, Poznań University of Life Sciences, Poznań, Poland; 3Department of Plant and Soil System, Laboratory of Molecular and Environmental Microbiology, Institute of Agro- physics PAS, Lublin, Poland Coprinus comatus strains (CCMs) originating from Po- Up to date, only a few of laboratory-based techniques land were identified using ITS region sequencing. Based have been used to study the genetic diversity in Coprinus, on the sequences obtained, the genetic relationship be- such as random amplified polymorphism DNA (RAPD) tween the CCM strains was determined and a clear sepa- (Muraguchi et al., 2003; Jang et al., 2009), internal tran- ration of all strains into two main clusters was obtained. scribed spacers (ITS) 25S ribosomal DNA sequenc- The Coprinus strains were also genetically characterized ing technique (Ko et al., 2001; Keirle et al., 2004), large for the first time by the AFLP technique. The analysis subunit rDNA sequencing technique (Hopple & Vilgalys, showed that the CCMs separated into four main clusters 1999), RFLP markers (Muraguchi et al., 2003), sequence and a high complication of a UPGMA-based dendrogram related amplified polymorphism (SRAP) (Cai et al., 2010), was achieved. C. comatus strains included in the analy- and functional characterization of specific gene families sis displayed an AFLP profile similarity level in the range (Agger et al., 2009). from 44 to 66%. The highest similarity coefficient, 0.490, Although the Agaricales include many edible and me- was found between CCM12 and CCM13, and the lowest dicinal species (Stamets, 2000), these mushrooms are of- (0.202) between the CCM2 and CCM5 isolates. Biolog FF ten poorly characterized or intractable to genetic analysis MicroPlates were applied to obtain data on utilization (Muraguchi et al., 2003), and there are many gaps to be of 95 carbon sources and mycelial growth. The analysis filled in the current knowledge on their taxonomy and allowed comparison of the functional diversity of the biology. The traditional generic concept for Coprinus Pers. CCM strains and revealed a broad variability within the has existed for over 200 years. Members of the genus analyzed Coprinus species based on substrate utilization were recognized by a suite of morphological characters. profiles. Significant differences (2–48) have been shown Phylogenetic analyses of the DNA sequences are begin- in the substrate richness values. The Biolog experiments ning to demonstrate relationships among fungi that have proved to be a good profiling technology for studying not been obtainable previously through morphological the diversity in shaggy manes due to metabolic differ- characterization alone (Keirle et al., 2004). The develop- ences and demonstrated that all the strains might be ment of tools aimed at clear-cut and safe identification considered individually. It is evident that the strain met- and assessment of the genetic variability of fungal strains abolic grouping does not correlate with the grouping is thus a fundamental goal of molecular genetics research based on the ITS sequences and AFLP profiles, however, (Urbanelli et al., 2007). However, the use of genetic tech- some similarities may be observed. niques alone in fungal diversity studies has sometimes failed (Hoyos-Carvajal et al., 2009). Additionally, phylog- Key words: AFLP, ITS, Biolog, fungal diversity, Coprinus comatus, shag- enies based only on a selected molecular method do not gy mane mushroom necessarily have the same topology as trees made from Received: 15 July, 2015; revised: 17 September, 2015; accepted: morphological or biochemical data (Kubicek et al., 2003; 02 October, 2015; available on-line: 26 October, 2015 Tripathi et al., 2011). Recently, metabolic profiling tech- nologies have been applied to investigate the taxonomy and metabolic relationships within fungi (Tripathi et al., INTRODUCTION 2011; Janusz et al., 2015; Pawlik et al., 2015). Bearing this in mind, it seems highly reasonable and fully justified to Coprinus comatus, the shaggy mane mushroom, is com- use a comprehensive approach, taking into account mo- monly seen on newly disturbed grounds, grassy places, lecular, morphological, physiological, and metabolic data and road sides. Moreover, it has been cultivated as a de- in the research concerning identification and differentia- licious and highly nutritious edible species in recent years tion of fungal species. in China (Sabo et al., 2010; Stojkovic et al., 2013). Beside Therefore, the aim of the present work was to deter- its culinary value, C. comatus is regarded as a medicinal mine the intraspecific diversity of Coprinus comatus based mushroom and in recent years numerous publications *e-mail: [email protected] have been produced indicating that it may possess an- *The results concerning Coprinus comatus genetic identification tioxidant, antitumor, antidiabetic, immuno-modulating, (ITS sequencing) were presented in the form of a poster at the 47th National Scientific Conference “Microorganisms-plants the hypolipidemic, and antibacterial properties (Han et al., environment under changing climate conditions” (2013 Puławy, 2006; Li, Lu, et al., 2010; Sabo et al., 2010; Ren et al., Poland). The results on C. comatus Biolog metabolic profiling were 2012; Zhao et al., 2014). In addition, its ability to kill presented in the form of a poster at the 6th International Weigl nematodes is intensively studied (Luo et al., 2004; Luo Conference on Microbiology, Gdańsk, Poland (8–10 July, 2015). et al., 2007). Abbreviations: CCMs,Coprinus comatus strains 684 A. Pawlik and others 2015 on a complex approach using genetic and biochemical Table 1. List of Coprinus comatus strains (CCMs) used in this profiling tools. In addition, we investigated the useful- study ness of these methods for identification and establishing the genomic and metabolic relationships between C. co- a,b GenBank matus strains. Strain number Geographical origin Accession 1. CCM1 Poznań (Poland) JQ901432 MATERIALS AND METHODS 2. CCM2 Poznań (Poland) JQ901433 Fungal strains and cultivation. Coprinus comatus 3. CCM3 Wojnowice (Poland) JQ901434 strains (CCMs, Table 1) were obtained from the De- 4. CCM4 Przyprostynia (Poland) JQ901435 partment of Vegetable Crops, University of Life Sci- ences, Poznan, Poland (ULSP) and deposited at the 5. CCM5 Wojnowo (Poland) JQ901436 Fungal Culture Collection (FCL) of the Department of 6. CCM6 Wilkanowo (Poland) JQ901437 Biochemistry, Maria Curie-Sklodowska University, Lub- 7. CCM7 Jarnatów (Poland) JQ901438 lin, Poland. Pure cultures of C. camatus isolates were ob- tained by excising pieces of trama from carpophores and 8. CCM8 Borne Sulinowo (Poland) JQ901439 transferring them onto Malt Extract Agar medium (Dif- 9. CCM9 Swochowo (Poland) JQ901440 co, BD, USA). Next, the incubation was carried out for 14 days at 25°C and the mycelium was transferred onto 10. CCM10 Smołdzino (Poland) JQ901441 PDA medium (HiMedia, India). The stock culture of 11. CCM11 Skierniewice (Poland) JQ901442 the fungal strains was maintained on Malt Extract Agar 12. CCM12 Kobiór (Poland) JQ901443 slants. The slants were inoculated with mycelia and incu- bated at 24°C for 8 days, and then used for seed culture 13. CCM13 Tychy (Poland) JQ901444 inoculation. The mycelia of C. comatus strains were trans- 14. CCM14 Dębska Kuźnia (Poland) JQ901445 ferred into a 40 ml liquid Lindeberg-Holm (LH) medium aFCL, Fungal Collection of Lublin, Department of Biochemistry, Maria (1952) in 100 ml Erlenmayer flask by punching out ca. 5 Curie-Sklodowska University, Lublin, Poland; bULSP, Department of 2 mm of the slants with a sterilized cutter. The seeds were Vegetable Crops, University of Life Sciences, Poznan, Poland cultivated for 14 days at 24°C. Next, broth cultures were harvested by centrifugation (10 000 × g; 10 min) and used for DNA isolation. base searches were performed with the BLAST program DNA extraction. The mycelia from liquid cultures at the National Centre for Biotechnology Information were used for DNA extraction according to the modi- (Bethesda, MD, USA). The multiple DNA sequence fied protocol developed by Borges et al. (1990). To ex- alignments were performed with the Clustal-W algorithm tract DNA, 20 mg of fresh mycelium was transferred to (Thompson et al., 1994). The neighbour-joining (NJ) al- a 1.2 ml Lysing Matrix A tube (MP Biomedicals, USA) gorithm was employed to construct phylogenetic tree for supplemented with 1 ml of spermidine–SDS buffer C. comatus strains as implemented in MEGA v.6.0 soft- (4 mM spermidine, 10 mM EDTA, 0.1 M NaCl, 0.5% ware. Fewer than 5% alignment gaps, missing data, and SDS, 10 mM β–mercaptoethanol, 40 mM Tris–HCl, pH ambiguous bases were allowed at any position. The to- 8.0) and homogenized 3 × 40 seconds using a benchtop pology of the tree was evaluated by bootstrap analysis of homogenizer FastPrep-24 Instrument (MP Biomedicals, the sequence data based on 1 000 random resamplings. USA). The mixture was then centrifuged, transferred Phylogenetic tree visualization was performed using the (0.6 ml) to sterile 1.5 ml Eppendorf tubes, and imme- TreeView applet (Page, 1996). diately extracted two times with 1 volume of phenol. AFLP fingerprinting. The AFLP (Amplified Frag- Subsequently, the aqueous phase was extracted with 1 ment Length Polymorphism) analysis was performed as volume of chloroform–isoamyl alcohol (24:1) and cen- described by Vos et al. (1995) with some modifications trifuged (10 000 × g, 10 min, 4°C). Next, a 0.1 volume of (Pawlik et al., 2012). Adapters and primers were synthe- 3 M sodium acetate (pH 5.5) was added to the aqueous sized by GensetOligos, France, and IBB PAN, Poland. phase. DNA was then precipitated by the addition of All reagents and chemicals were of molecular biology 2 volumes of ice-cold 96% ethanol and recovered by grade.