Analysis of genetic diversity among Chinese Cyclocybe chaxingu strains using ISSR and SRAP markers

Mushroom strains

A total of twenty-four strains of C. chaxingu strains were collected from the main producing areas of China (Table 1), which were deposited in the Culture Collection of Jiangxi Agricultural University (JAUCC), were used in this study. A previous study based on ITS, SSU and RPB2 phylogenetic analysis showed that all the test strains belonged to C. chaxingu (Liu, 2020).

DNA extraction

The mycelia grew on PDA at 25 °C for 8 days were used to extract DNA for molecular marker analysis. Genomic DNA was extracted from 100 mg of dry mycelia using a modified cetyltrimethyl ammonium bromide (CTAB) method (Huang, Ge & Sun, 2000; Doyle & Doyle, 1987). The quality and quantity of the genomic DNA were determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and electrophoresis on a 1.0% agarose gel. DNA samples were diluted to 50 ng/µl for PCR amplification.

ISSR and SRAP analysis

A total of 23 primers/primer pairs (Biomed, Beijing, China) that produced clearly distinguishable and reproducible fragments were selected and used in this study for ISSR and SRAP analysis (Table 2). All of the amplification reactions were performed in a PCR Amplifier (Bio-Rad T100TM Thermal Cycler; Bio-Rad, Hercules, CA, USA) in 25-µL reaction mixtures.

For ISSR analysis, the reaction mixtures contained 12.5 µL of 2 × Taq PCR Master Mix (Vazyme, Nanjing, China), 1 µL of primer (10 µM/L), 1 µL of template DNA, and 10.5 µL of ddH₂O. Amplification program was: 4 min of denaturing at 94 °C, 35 cycles of 35 s at 94 °C, 45 s at 46–55 °C (see Table 2 for primer annealing temperature), 2 min at 72 °C and followed by a final extension of 10 min at 72 °C.

For SRAP analysis, the reaction mixtures contained 12.5 µL of 2 × Taq PCR Master Mix (Vazyme, Nanjing, China), 1 µL of each primer (10 µM/L), 1 µL of template DNA, and 9.5 µL of ddH₂O. The amplification included an initial denaturation at 94 °C for 5 min, 5 cycles of 94 °C for 1 min, 35 °C for 1 min and 72 °C for 1 min, followed by 35 cycles of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C, and a final extension of 10 min at 72 °C.

Amplified products were fractionated by electrophoresis in 2% (w/v) agarose/TAE gels, visualized under UV after staining with TS-Gelred (Tsingke Biotechnology Co., Ltd., Beijing, China), and documented using a gel documentation and image analysis system (GenoSens 2000; CLiNX, Shanghai, China).

Data analysis for genetic diversity

Qualitative scoring of bands was done from gel photographs obtained from ISSR and SRAP analysis with “1″for presence and “0″for absence to generate a binary matrix. Only those bands amplified consistently were considered. Smeared and weak bands were excluded from the analysis. For data structures with only 0 and 1, Jaccard similarity coefficient is generally adopted (Ivchenko & Honov, 1998). The genetic distances were estimated based on Jaccard similarity coefficient. A cluster analysis was performed based on the genetic distances using the Unweighted Pair Group Method of Arithmetic Average (UPGMA) by R Statistical Software (R Core Team, 2021) and vegan package (Oksanen et al., 2020). The goodness of fit of the clustering to the data matrix was calculated. And the optimal grouping strategy is to select the number of groups corresponding to the maximum average contour width (Rousseeuw, 1987). Genetic diversity analysis was performed using the POPGENE program (version 1.32) (Yeh et al., 1999). The number of amplified loci (N), the percentage of polymorphic loci (PPL), the number of effective alleles (Ne), the Nei’s gene diversity (H) and the Shannon information index (I) were calculated for each primer and among all primers (Nei, 1973; Lewontin, 1972). In addition, a Principal Coordinate Analysis (PCoA) (Longya, Talumphai & Jantasuriyarat, 2020) was performed using the R Statistical Software to obtain a graphical representation of the relationship between the 24 test genotypes.

Genetic diversity based on ISSR marker

A total of 24 primers were initially screened to produce polymorphic patterns and only 8 of them were selected which gave reproducible and distinct polymorphic amplified products. For representational purposes, the extent of polymorphism revealed by primer P10 is shown in Fig. 1. The data collected from inter-simple sequence repeat (ISSR), detected total of 75 loci in 24 strains, out of which 61 (81.33%) were polymorphic, with an average of 9.38 polymorphic fragments per primer (Table 3). The ISSR primer P1 and P2 gave the highest polymorphism (100%), while the lowest polymorphism (60%) was detected by the P12 primer. The values of Ne, H and I were 1.547, 0.312 and 0.459, respectively.

For the 24 C. chaxingu strains, the genetic distances estimated based on Jaccard coefficient using ISSR data (see Table S1) varied from 0.06 (JAUCC 2192 and JAUCC 2196) to 0.60 (JAUCC 0727 and JAUCC 1927), with an overall mean of 0.40. The co-phenetic correlation for the ISSR dendrogram was estimated at 0.93, corresponding to a good fit. A dendrogram constructed from the Jaccard distances matrix using the UPGMA method was shown in Fig. 2A. All the test strains were grouped into five main clusters by calculating the maximum average contour width value. Cluster I and Cluster III each comprised a single genotype (JAUCC 0727 and JAUCC 1927), while Cluster II, Cluster IV and Cluster V were delineated into two sub-clusters. Within Cluster II, Cluster IV and Cluster V, JAUCC 1925, JAUCC 2119 and JAUCC 1926 appeared to be distinct from the other genotypes, respectively. Interestingly, most strains cultivated in Yunnan Province were clustered together at the distance level of 0.45. Groupings identified by UPGMA analysis were confirmed by PCoA data (Fig. 2B). The two most informative PCoA components accounted for 44.21% of the variation observed.

Genetic diversity based on SRAP marker

Among the 48 SRAP primer pairs tested in the study, 15 primer pairs were further used to characterize C. chaxingu strains. A representative set of amplification profiles obtained with primer combination me3 + em7 is shown in Fig. 3. The present study showed that out of 166 loci, 132 (79.52%) loci were polymorphic showing an average of 11.07 polymorphic loci per primer pairs tested (Table 3). The maximum polymorphic loci were generated by primer pairs em6+me3 (94.45%). Among the primers pairs studied, primers pairs em6+me3 generated the highest 18 loci, while primer combination em6+me2 and em3+me6 generated the lowest eight loci. The values of Ne, H and I were 1.506, 0.289 and 0.425 based on SRAP marker.

With the cluster analysis on SRAP molecular marker, the two most closely related strains were found to be JAUCC 1847 and JAUCC 1851 (genetic distance was 0.03), and the two most distantly related strains were JAUCC 1847 and JAUCC 2924 with lowest similarity index (genetic distance was 0.57). Genetic distance estimated based on Jaccard coefficient obtained by SRAP profile with an average value of 0.39. Cluster analysis of SRAP data (see Table S2) based on the distance matrix generated a dendrogram with four major groups in a maximum average contour width value (Fig. 4A). The co-phenetic correlation for the SRAP dendrogram was estimated at 0.96, which showed a strong goodness of it. Within Cluster I and Cluster III, JAUCC 1918 and JAUCC 0727 were each comprised a single genotype. In the SRAP analysis, strain JAUCC 0727 showed a closer relationship with the other strains than in ISSR analysis. Similarly, there was a tendency to get together within most strains form Yunnan Province (JAUCC 1920, JAUCC 1921, JAUCC 1922, JAUCC 1925, JAUCC 1927), at genetic distance level of 0.38. Principal coordinate analysis (PCoA) data based on the genetic distance matrix are shown in Fig. 4B. These revealed similar groupings to UPGMA, and confirmed the genetic uniqueness of genotypes JAUCC 0727. The two most informative PCoA components accounted for 63.81% of the variations observed.

Genetic diversity based on combined ISSR and SRAP markers

A total of 23 primers/primer pairs were used for the analysis of combined ISSR and SRAP data, genetic distance among all the test strains ranged from 0.06 (JAUCC 2192 and JAUCC 2196) up to 0.54 (JAUCC 1851 and JAUCC 1920). A total of 241 loci in 24 strains, out of which 193 (80.08%) were polymorphic, with an average of 10.48 polymorphic fragments. The co-phenetic correlation for the combined ISSR and SRAP dendrogram was estimated 0.97, corresponding to a very good fit. Dendrogram by using UPGMA and Jaccard coefficient grouped the 24 test strains into five main clusters with a maximum average contour width value (Fig. 5A). Within Cluster IV, JAUCC 0727 appeared as a single genotype once again. Strains cultivated in Yunnan Province clustered together (genetic distance was 0.4) same as using ISSR or SRAP molecular marker alone.

The PCoA based on ISSR and SRAP data revealed that the strains belonging to a particular cluster were grouped together in the PCoA plot (Fig. 5B). Groupings identified by UPGMA analysis and Jaccard coefficient were confirmed by PCoA data which also revealed that the strain JAUCC 0727 was genetically very distinct from the other genotypes. The two most informative PCoA components accounted for 61.01% of the variation observed.