Altered GC- and AT-biased genotypes ofOphiocordyceps sinensisin the stromal fertile portions and ascospores of naturalCordyceps sinensis

Abstract

Objective: To examine multiple genotypes of Ophiocordyceps sinensis in the stromal fertile portion (SFP) densely covered with numerous ascocarps and ascospores of natural Cordyceps sinensis. Methods: Mature C. sinensis specimens were harvested and continuously cultivated in our laboratory (altitude 2,200 m). The SFPs (with ascocarps) and fully and semi-ejected ascospores were collected for histological and molecular examinations. Biochip-based single nucleotide polymorphism (SNP) MALDI-TOF mass spectrometry (MS) was used to genotype multiple O. sinensis mutants in the SFPs and ascospores. Results: Microscopic analysis demonstrated distinct morphologies of the SFPs (with ascocarps) before and after ascospore ejection and the SFP of developmental failure, which, along with the fully and semi-ejected ascospores, were subjected to SNP MS genotyping analysis. Mass spectra showed the coexistence of GC- and AT-biased genotypes of O. sinensis that were genetically and phylogenetically distinct in the SFPs before and after ejection and of developmental failure and in fully and semi-ejected ascospores. The intensity ratios of MS peaks were dynamically altered in the SFPs and the fully and semi-ejected ascospores. Mass spectra also showed transversion mutation alleles of unknown sequences with altered intensities in the SFPs and ascospores. Genotype #5 of AT-biased Cluster-A maintained a high intensity in all SFPs and ascospores. An MS peak with a high intensity containing AT-biased Genotypes #6 and #15 in pre-ejection SFPs was significantly attenuated after ascospore ejection. The abundance of Genotypes #5‒6 and #16 of AT-biased Cluster-A was differentially altered in the fully and semi-ejected ascospores that were collected from the same C. sinensis specimens. Conclusion: Multiple O. sinensis genotypes coexisted in different combinations with altered abundances in the SFPs prior to and after ejection, the SFP of developmental failure and in the two types of ascospores of C. sinensis, demonstrating their genomic independence. Fungal metagenomic members present in different combinations and with dynamic alterations play symbiotic roles in different compartments of natural C. sinensis.

Differential coexistence of multiple genotypes of Ophiocordyceps sinensis in the stromata, ascocarps and ascospores of natural Cordyceps sinensis

“…The fully ejected ascospores of C. sinensis were diluted in normal saline, placed on a glass slide, and air-dried for histological examination under an optical microscope (Model BX51, OLYMPUS, Japan) without staining. The ascospores were fixed on a glass slide with 4% paraformaldehyde for 1 h, incubated in 0.01% calmodulin for 5 min for the visualization of septa, washed 3 times with PBS, and observed under a fluorescence microscope with UV epi-illumination (Model XZ51, OLYMPUS, Japan) [69].…”

“…The sequences of Genotypes #2-17 of O. sinensis, however, were not present in the genome assemblies (ANOV00000000, JAAVMX000000000, LKHE00000000, LWBQ00000000 and NGJJ00000000) of 5 Genotype #1 H. sinensis strains, Co18, IOZ07, 1229, ZJB12195 and CC1406-203, and instead belonged to the genomes of independent O. sinensis fungi [7][8][9][10][11][12][27][28][30][31][51][52][53][54][55][56]69].…”

“…In fact, Genotypes #6 and #16 were detected in both types of ascospores using the biochip-based MassARRAY SNP mass spectrometry technique [69].…”

“…These studies did not report the maturation stages of natural and cultivated C. sinensis specimens that were used as the study materials. However, other studies using genotype-specific primers, amplicon cloning and biochip-based SNP mass spectrometry genotyping techniques reported the detection of Genotype #4 of O. sinensis in natural C. sinensis coexisting with Genotype #1 H. sinensis with or without Genotypes #5-6[16][17][18]24,[27][28]61,69].Genotype #4 of AT-biased Cluster-B was found to predominate in the stroma of immature C. sinensis and to decline during C. sinensis maturation (cf. Fig7)[16][17][27][28][30][31]69].…”

“…However, other studies using genotype-specific primers, amplicon cloning and biochip-based SNP mass spectrometry genotyping techniques reported the detection of Genotype #4 of O. sinensis in natural C. sinensis coexisting with Genotype #1 H. sinensis with or without Genotypes #5-6[16][17][18]24,[27][28]61,69].Genotype #4 of AT-biased Cluster-B was found to predominate in the stroma of immature C. sinensis and to decline during C. sinensis maturation (cf. Fig7)[16][17][27][28][30][31]69]. Genotype #4 and the newly discovered Genotype #15 of AT-biased Cluster-B are present in the SFP (with ascocarps) but not in ascospores of natural C. sinensis (cf.…”