MATERIALS AND METHODS

Study area: The sample was collected in the natural land in Sovenga Hills (GPS coordinates: S 23°53'41.763''E, 29°44'21.292''), in Limpopo Province, South Africa, under the pine tree. This study was carried out in December, 2020.

Molecular analysis: DNA extraction was done using the Chelex method¹³. A piece of A. bisporus was picked up with a sterilized needle and then transferred to a 1.5 mL

Eppendorf tube containing 30 μL double distilled water. The mushroom pieces in the tube were crushed with the fine needle and vortexed. Fifty microliters of 5% Chelex^® 50 and 5 μL of proteinase K were added to the tube containing crushed mushroom. These separate microcentrifuge tubes with the mushroom lysate were kept at 56°C for 2 hrs and then incubated at 95°C for 10 min to deactivate the proteinase K and finally spin for 3 min at 16000 rpm¹⁴. The supernatant was then extracted from each of the tubes and stored at -20°C. Following this step, the forward and reverse primers, 18s (5'-TTGATTACGTCCCTGCCCTTT–3') and 26s (5'-TTTCACTCGCCGTTACTAAGG–3'), was used to amplify the ITS rDNA. PCR was conducted with 5 μL of the DNA template, 12.5 μL of 2X PCR Master Mix Red (Promega, USA), 1 μL of each primer (10 pmol μL^–1) and nuclease-free water for a final volume of 25 μL. The amplification was processed using a Thermo Fisher Scientific Thermal Cycler (USA), with the following program: Initial denaturation for 3 min at 94°C, 40 cycles of denaturation for 45 sec at 94°C, 53°C annealing temperatures for ITS rDNA, extension for 45 sec to 1 min at 72°C and finally an extension step of 5 min at 72°C followed by a temperature on hold at 4°C. After DNA amplification, 5 μL of PCR product was loaded on a 1.5% agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid and one mM EDTA) to observe the DNA. The bands were stained with RedGel and pictured under a UV illuminator. The PCR product was stored at -20°C. Finally, the PCR product was purified for sequencing by Inqaba Biotech Company (South Africa, Pretoria). The phylogenetic analysis was done using maximum likelihood implemented in Mega-X software¹⁵. The 18S rDNA sequence for A. bisporus from South Africa was deposited in the GenBank under the accession number MZ087879.

RESULTS AND DISCUSSION

Molecular characterization: An 860 bp for 18S rDNA was obtained from a strain of A. bisporus from South Africa. The sequence was analyzed and compared with other 18S rDNA sequences of A. bisporus available from GenBank. The NBLAST for 18S rDNA revealed a 99% similarity with the American strain of A. bisporus (acc. No. AY787216) and Chinese strain (acc. No. FJ869172). The genetic distance estimation ranged from 0.011-0.633. The pairwise genetic distance for the strains of A. bisporus revealed the highest distance (0.633) between the South African and a Chinese (acc. No. JX268572) strain of A. bisporus in Table 1. In contrast, the lowest genetic distance (0.011) was observed between the South African and the Chinese (acc. No. FJ869172) and American (acc. No. AY787216) strain of A. bisporus (Table 1).

Fig. 1:

Maximum likelihood tree for 18S rDNA of the button strain of A. bisporus recovered from South Africa under the pine tree in Sovenga Hills

In addition, the South African A. bisporus, showed a low genetic distance (0.011) with the Taiwanese strains (acc. No. AJ012402, AJ012403, AJ012404) of A. bisporus.

Phylogeny of A. bisporus, as derived from the corresponding tree in Fig.1, reveals a clear relationship with A. bisporus strains. Additionally, the A. bisporus strains were placed separately from the outgroup A. heterocystis (acc. No. KU366695). The phylogenetic analysis revealed that the South African strains placed close to the Taiwanese (acc. No. AJ012402, AJ012403, AJ012404) and American (acc. No. AY787216) strains of the same species with 100 bootstrap value. Besides, the Egyptian strains (acc. No. MH751906) were grouped separately from other A. bisporus. This is in agreement with the previous record by Wang et al.¹ using IGS rDNA.

The 18S rDNA tree placed the present South African A. bisporus close to the same strain from China (acc. No. FJ869172), USA (acc. No. AY787216) and Taiwan (acc. No. AJ012404) with 100 bootstrap values (Fig.1).

The small subunit ribosomal DNA (SSU) is useful for the examination of close relationships in fungus¹⁶. Recently, domestic breeding has been analyzed for its divergences in the nucleotide sequences of IGS1¹⁷. Their result indicated that A. bisporus is a monophyletic group, which is in agreement with the present study. Kwon et al.¹⁸ showed that the IGS1 rDNA region in A. bisporus is not inheritable with entirely homologous sequences. It seems that it is a quickly shifting region in the process of breeding. Nevertheless, using IGS regions has been suggested for discriminatory analysis among intra-specific individuals of mushroom species¹⁹. DNA barcoding is an approach to rapidly identify species using short and standard genetic markers⁸. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus²⁰, but its utility for barcoding in mushrooms has not been established. Determining and realizing the best DNA barcode markers for identifying mushrooms is essential⁴. Dentinger et al.²¹ compared COI and ITS as DNA barcode markers for mushrooms their result indicated a similar performance of COI and ITS as a barcode. The 18S rDNA phylogenetic tree indicated that genetic variation exists among the sequenced mushrooms from South Africa and those deposited in the gene bank (https://www.ncbi.nlm.nih.gov/). However, 18S rDNA is a useful marker for the phylogenetic and molecular identification of the natural button mushroom. The result indicated that the chelex was also a suitable method for edible mushroom DNA extraction. This is the first report of A. bisporus using 18S rDNA from South Africa.

CONCLUSION

In molecular studies, we need to know the different methods for extracting DNA. Also, increase the credibility of work by using different primers. This study, used 18S rDNA for A. bisporus in South Africa for the first time. Current results suggest that 18S rDNA could be a helpful marker for the natural strain of the South African A. bisporus. On the other hand, the result indicated that the Chelex method was a good option for extracting DNA in A. bisporus which is cheap and affordable.

SIGNIFICANCE STATEMENT

This study was discovered for identification of the A. bisporus, using 18S rDNA was useful that can be beneficial for molecular analysis of this mushroom. Furthermore, this study will help the researchers uncover the critical areas of molecular research that many researchers could not explore. Thus, a new theory on identifying button mushrooms may be arrived at.

ACKNOWLEDGMENT

I acknowledge the Aquaculture and Genetic Lab of the Faculty of Health for using the laboratory for processing the specimen.