Fig 1.
Alignment of ITS sequences of GC- and AT-biased genotypes of O. sinensis and primer locations.
GT refers to genotype. Genotypes #1‒2 and #13‒14 are GC-biased O. sinensis genotypes, and Genotypes #4‒6 and #15‒17 are AT-biased O. sinensis genotypes. The GC and AT contents of the 17 O. sinensis genotypes are shown in S1 Table. The underlined sequences in black represent primers for the first-step PCR. Extension primers 067721–477, 067721–531, 067740–324, 067744–324 (RC, reverse complement) and 067740–328 (RC) in brown were used for SNP mass spectrometry genotyping to distinguish between genotypes of O. sinensis. Arrows indicate the directions of the primer extension reactions toward the SNP alleles in brown. “-” refers to an identical base, and spaces refer to unmatched sequence gaps.
Table 1.
“Universal” primer and genotype-specific primers for the first-step PCR amplification of ITS segments.
Fig 2.
A maximum likelihood (ML) phylogenetic tree.
The ML tree was inferred using MEGA software [53], containing 25 ITS sequences (representing 17 genotypes of O. sinensis) and 27 genomic ITS sequences (including multiple repetitive ITS copies) of Genotype #1 H. sinensis Strain 1229, CC1406-203, Co18, IOZ07, and ZJB12195. S2 Table lists the GenBank accession numbers that are hyperlinked to GenBank database, strain or isolate information and ITS1-5.8S-ITS2 sequence range for the 52 ITS sequences that were analyzed phylogenetically in Fig 2. The phylogenetic topology of the ML tree was verified according to the topologies of Bayesian majority-rule consensus phylogenetic trees [7,10, 11, 31].
Table 2.
SNP extension primers for MassARRAY MALDI-TOF MS genotyping.
Fig 3.
Microscopic images of the SFPs (with ascocarps) of natural Cordyceps sinensis.
Panel 3.1 shows a photo of a mature Cordyceps sinensis specimen labeled with stromal fertile portion (SFP). Panel 3.2 shows a confocal image (bar: 1 mm) of a transverse section of the Cordyceps sinensis SFP densely covered with numerous ascocarps prior to ascospore ejection, with a red circle indicating one of the multiple ascocarps densely covering the SFP. Panel 3.3 shows an optical close-up image (40x) of an HE-stained ascocarp containing multiple ascospores of multicellular ascosporic cells. Panel 3.4 shows a confocal image (bar: 100 μm) of 2 ascocarps; the upper one is a maturing ascocarp, and the lower one is a mature ascocarp containing multiple ascospores aggregating toward the opening of the perithecium. Panel 3.5 shows a confocal image close to the opening of the perithecium and a semi-ejected ascospore hanging out of the opening of the perithecium (bar: 20 μm). Panel 3.6 shows an optical image (10x) of HE-stained SFPs after ascospore ejection. Panels 3.7 (10x) and 3.8 (40x) show optical images of HE-stained SFPs of developmental failure. The confocal images shown in Panels 3.2 and 3.4 are large images created by stitching together tile images automatically by the computer and software of the ZEISS LSM 780 confocal microscope, which allow extremely high resolution from designated large fields of view, because of limited scanning areas of the ZEISS LSM 780 confocal microscope.
Fig 4.
MALDI-TOF mass spectra (MS) of extension primer 067721–477 to distinguish between GC- and AT-genotypes in the SFPs (with ascocarps) prior to and after ascospore ejection.
The extension reaction proceeded from extension primer 067721–477 toward the SNP at position 477 in the AB067721 sequence (cf. Fig 1 for the location). In the allelic peaks, “C” denotes extension of the primer with an extended cytosine; “A” indicates an extended adenine, “G” represents an extended guanine, and “T” refers to an extended thymine. Panels 4.1 and 4.2 show the mass spectra for the SFPs prior to and after ascospore ejection, respectively. Panel 4.3 shows the overlapping mass spectra for Panels 4.1 (black tracing) and 4.2 (red tracing) with alignment of the horizontal and vertical axes of the panels.
Table 3.
Mass intensity ratios of the SNP peaks of transition and transversion mutation genotypes in the SFPs prior to and after ascospore ejection and of developmental failure.
Fig 5.
MALDI-TOF mass spectra of extension primer 067744–324 (reverse complement) to distinguish between the AT-biased genotypes in the SFPs (with ascocarps) prior to and after ascospore ejection.
The extension reaction proceeded from extension primer 067744–324 (reverse complement) toward the SNP at position 324 in the AB067740 sequence (cf. Fig 1 for the location). In the allelic peaks, “G” represents the primer with an extended reverse complement cytosine (guanine in the sense chain; cf. Fig 1); “T” represents an extended reverse complement adenine (thymine in the sense chain); “C” denotes an extended reverse complement guanine (cytosine in sense chain); and “A” indicates an extended reverse complement thymine (adenine in the sense chain). Panel 5.1 shows the mass spectrum of the pre-ejection SFP. Panel 5.2 shows the mass spectrum of the post-ejection SFP. Panel 5.3 shows the overlapping mass spectra for Panels 5.1 (tracing in black) and 5.2 (tracing in red) with alignment of the horizontal and vertical axes of the panels.
Table 4.
Mass intensity ratios of the SNP allelic peaks of transition and transversion mutant genotypes in SFPs prior to and after ascospore ejection and of developmental failure.
Fig 6.
MALDI-TOF mass spectra of extension primer 067721–477 to distinguish between GC- and AT-genotypes in fully and semi-ejected ascospores.
The extension reaction proceeded from extension primer 067721–477 toward the SNP at position 477 in the AB067721 sequence (cf. Fig 1 for the location). In the allelic peaks, “C” denotes the primer with an extended cytosine; “A” indicates an extended adenine; “G” represents an extended guanine; and “T” represents an extended thymine. Panel 6.1 shows the mass spectrum of fully ejected ascospores. Panel 6.2 shows the mass spectrum of semi-ejected ascospores.
Table 5.
Mass intensity ratios of the SNP allelic peaks of transition and transversion mutant genotypes in fully and semi-ejected ascospores.
Fig 7.
MALDI-TOF mass spectra of extension primer 067740–324 to distinguish between AT-biased genotypes in the fully and semi-ejected ascospores.
The extension reaction proceeded from extension primer 067740–324 toward the SNP at position 324 in the AB067740 sequence (cf. Fig 1 for the location). In the allelic peaks, “C” denotes the primer with an extended cytosine; “A” indicates an extended adenine; “G” represents an extended guanine; and “T” indicates an extended thymine. Panel 7.1 shows the mass spectrum of fully ejected ascospores. Panel 7.2 shows the mass spectrum of semi-ejected ascospores. Panel 7.3 shows the overlapping mass spectra for Panels 7.1 (black tracing) and 7.2 (red tracing) with alignment of the horizontal and vertical axes of the panels.
Table 6.
Mass intensity ratios for the SNP allelic peaks for multiple transition and transversion mutation genotypes in the fully and semi-ejected ascospores.