The 20 fastest growing independent mycelia with distinct colony shapes were selected from each parental strain. Mating was conducted by placing mycelial blocks (3 × 3 mm) from opposite strains on the same PDA plate 1 cm apart. Mating was confirmed by the formation of clamp connections under the microscope after incubation at 30 °C for 7 days. RAPD analysis has shown some
strain-specific DNA bands in the gel. To develop the unique DNA bands as the strain-specific DNA markers, the DNA bands were excised and extracted with a DNA gel extraction kit (Solgent Co., Korea). The extracted DNA was cloned into pGEM-T-easy cloning vector (Promega Co., USA). The insert DNA sequence was determined by a commercial DNA sequencing BIRB 796 in vivo service. The determined DNA sequences were deposited into GenBank (NCBI) with the accession numbers given in Table 1. Primer sets of 15 nucleotides in length were designed using the 5′- and 3′-ends of the determined sequences (Table 1). Target-specific
primer sets were employed for the detection selleck of specific strains using the following conditions: 94 °C for 5 min; 30 cycles at 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 2 min; 72 °C for 10 min. RAPD analyses with three random primers were conducted for the verification of nine H. marmoreus strains. The RAPD with primers OPS-1, OPS-10, and OPL-13 yielded 22, 16, and 21 distinct DNA bands, respectively, with the sizes ranging from 0.5 to 3.5 kbp (Fig. 1a). The DNA band pattern was clustered using the UPGMA method. The resulting dendrogram, which was a reflection of genetic background, showed that the H. marmoreus
strains could be clustered into three groups (Fig. 1b). The largest group consisted of Hm0-7, Hm1-1, Hm1-6, Hm2-7, Hm3-6, and Hm3-8. Hm1-1 and Hm1-6 were essentially the same strain. Strains Hm3-6 and Hm3-8 are Korean varieties based on the Japanese strain Hm0-7. Taiwanese Hm2-7 could be derived Megestrol Acetate from Hm0-7. Strains Hm0-4 and Hm2-10 were included in the second cluster. These strains were from a mushroom stock belonging to a commercial farm. Hm3-10 showed the most distinct DNA band pattern and thus formed an independent single-member group in the dendrogram. Hm3-10 was a wild strain collected from a mountain in the middle of Korea. Cultivation characteristics of the strains were investigated in terms of mushroom yield, culture period, and taste of fruiting bodies. The results are summarized in the Table 2. Strains Hm1-1, Hm1-6, and Hm3-6 showed the best results among the strains. The former two strains were identical in RAPD and therefore had the same characteristics. The wild strain Hm3-10, which exhibited a distinct genetic background in the RAPD analysis, showed reasonable cultivation characteristics except for poor fruiting body yield, suggesting a potential to be developed as a commercial strain. The morphology of fully grown Hm3-10 and Hm1-1 are shown in Fig. 1c and d, respectively.