A second HD mating type sublocus of Flammulina velutipes is at least di-allelic and active: new primers for identification of HD-a and HD-b subloci

Background Sexual development in Flammulina velutipes is controlled by two different mating type loci (HD and PR). The HD locus contains homeodomain (Hd) genes on two separate HD subloci: HD-a and HD-b. While the functionality of the HD-b sublocus has been largely confirmed, the status and content of the HD-a sublocus has remained unclear. Methods To examine the function of the HD-a sublocus, genome sequences of a series of F. velutipes strains were analyzed and tested through series of amplification by specific primer sets. Furthermore, activity of di-allelic HD-a locus was confirmed by crossing strains with different combinations of HD-a and HD-b subloci. Results Sublocus HD-b contained a large variety of fixed Hd1/Hd2 gene pairs, while the HD-a sublocus either contained a conserved Hd2 gene or, a newly discovered Hd1 gene that was also conserved. Identification of whole HD loci, that is, the contents of HD-a and HD-b subloci in a strain, revealed that strains with similar HD-b subloci could still form normal dikaryons if the two genes at the HD-a sublocus differed. At least di-allelic HD-a sublocus, is thus indicated to be actively involved in mating type compatibility. Conclusions HD-a sublocus is active and di-allelic. Using the new information on the HD subloci, primers sets were developed that specifically amplify HD-a or HD-b subloci in the majority of F. velutipes strains. In this way, unknown HD mating types of F. velutipes can now be quickly identified, and HD mating type compatibility conferred by HD-a or HD-b can be confirmed by PCR

Comparison of the Mitochondrial Genome Sequences of Six Annulohypoxylon stygium Isolates Suggests Short Fragment Insertions as a Potential Factor Leading to Larger Genomic Size

Mitochondrial DNA (mtDNA) is a core non-nuclear genetic material found in all eukaryotic organisms, the size of which varies extensively in the eumycota, even within species. In this study, mitochondrial genomes of six isolates of Annulohypoxylon stygium (Lév.) were assembled from raw reads from PacBio and Illumina sequencing. The diversity of genomic structures, conserved genes, intergenic regions and introns were analyzed and compared. Genome sizes ranged from 132 to 147 kb and contained the same sets of conserved protein-coding, tRNA and rRNA genes and shared the same gene arrangements and orientation. In addition, most intergenic regions were homogeneous and had similar sizes except for the region between cytochrome b (cob) and cytochrome c oxidase I (cox1) genes which ranged from 2,998 to 8,039 bp among the six isolates. Sixty-five intron insertion sites and 99 different introns were detected in these genomes. Each genome contained 45 or more introns, which varied in distribution and content. Introns from homologous insertion sites also showed high diversity in size, type and content. Comparison of introns at the same loci showed some complex introns, such as twintrons and ORF-less introns. There were 44 short fragment insertions detected within introns, intergenic regions, or as introns, some of them located at conserved domain regions of homing endonuclease genes. Insertions of short fragments such as small inverted repeats might affect or hinder the movement of introns, and these allowed for intron accumulation in the mitochondrial genomes analyzed, and enlarged their size. This study showed that the evolution of fungal mitochondrial introns is complex, and the results suggest short fragment insertions as a potential factor leading to larger mitochondrial genomes in A. stygium

Targeted metabolome and transcriptome analyses reveal changes in gibberellin and related cell wall-acting enzyme-encoding genes during stipe elongation in Flammulina filiformis

Flammulina filiformis, a typical agaric fungus, is a widely cultivated and consumed edible mushroom. Elongation of its stipe (as the main edible part) is closely related to its yield and commercial traits; however, the endogenous hormones during stipe elongation and their regulatory mechanisms are not well understood. Gibberellin (GA) plays an important role in the regulation of plant growth, but little has been reported in macro fungi. In this study, we first treated F. filiformis stipes in the young stage with PBZ (an inhibitor of GA) and found that PBZ significantly inhibited elongation of the stipe. Then, we performed GA-targeted metabolome and transcriptome analyses of the stipe at both the young and elongation stages. A total of 13 types of GAs were detected in F. filiformis; the contents of ten of them, namely, GA3, GA4, GA8, GA14, GA19, GA20, GA24, GA34, GA44, and GA53, were significantly decreased, and the contents of three (GA5, GA9, and GA29) were significantly increased during stipe elongation. Transcriptome analysis showed that the genes in the terpenoid backbone biosynthesis pathway showed varying expression patterns: HMGS, HMGR, GPS, and FPPS were significantly upregulated, while CPS/KS had no significant difference in transcript level during stipe elongation. In total, 37 P450 genes were annotated to be involved in GA biosynthesis; eight of them were upregulated, twelve were downregulated, and the rest were not differentially expressed. In addition, four types of differentially expressed genes involved in stipe elongation were identified, including six signal transduction genes, five cell cycle-controlling genes, twelve cell wall-related enzymes and six transcription factors. The results identified the types and content of GAs and the expression patterns of their synthesis pathways during elongation in F. filiformis and revealed the molecular mechanisms by which GAs may affect the synthesis of cell wall components and the cell cycle of the stipe through the downstream action of cell wall-related enzymes, transcription factors, signal transduction and cell cycle control, thus regulating stipe elongation. This study is helpful for understanding the roles of GAs in stipe development in mushrooms and lays the foundation for the rational regulation of stipe length in agaric mushrooms during production

Low temperature, mechanical wound, and exogenous salicylic acid (SA) can stimulate the SA signaling molecule as well as its downstream pathway and the formation of fruiting bodies in Flammulina filiformis

Low temperature (LT) and mechanical wound (MW), as two common physics methods, have been empirically used in production to stimulate the primordia formation of Flammulina filiformis, which is typically produced using the industrial production mode. However, the detailed effect on the fruiting body formation and important endogenous hormones and signaling pathways in this process is poorly understood. In this study, LT, MW, their combination, i.e., MW + LT, and low concentration of SA (0.1 mM SA) treatments were applied to the physiologically mature mycelia of F. filiformis. The results showed that the primordia under the four treatments began to appear on the 5th−6th days compared with the 12th day in the control (no treatment). The MW + LT treatment produced the largest number of primordia (1,859 per bottle), followed by MW (757), SA (141), and LT (22), compared with 47 per bottle in the control. The HPLC results showed that the average contents of endogenous SA were significantly increased by 1.3 to 2.6 times under four treatments. A total of 11 SA signaling genes were identified in the F. filiformis genome, including 4 NPR genes (FfNpr1-4), 5 TGA genes (FfTga1-5), and 2 PR genes (FfPr1-2). FfNpr3 with complete conserved domains (ANK and BTB/POZ) showed significantly upregulated expression under all four above treatments, while FfNpr1/2/4 with one domain showed significantly upregulated response expression under the partial treatment of all four treatments. FfTga1-5 and FfPr1-2 showed 1.6-fold to 8.5-fold significant upregulation with varying degrees in response to four treatments. The results suggested that there was a correlation between “low temperature/mechanical wound—SA signal—fruiting body formation”, and it will help researchers to understand the role of SA hormone and SA signaling pathway genes in the formation of fruiting bodies in fungi

Pricing decision of a dual-channel supply chain with different payment, corporate social responsibility and service level

Strategies such as price, CSR, and service have an important impact on enterprises and supply chains. This paper proposes a two-echelon dual-channel supply chain composed of a manufacturer and a retailer. Considering the product pricing, CSR level, and service level in the supply chain, this paper employs the Stackelberg game to depict supply chain participants’ optimal decisions and analyze the influence of explanatory variables on the optimal decision with retailer’s payment methods. The results state that market share, service level, CSR, and financing interest rate significantly impact the pricing decision of all participants in the supply chain. In addition, strategies of CSR level and service level are also affected by the discount rate of advance payment, financing interest rate, return on investment, and opportunity cost rate. This paper incorporates CSR and service level into the objective function, considers a variety of retailers’ payment methods, enriches the supply chain’s pricing model, and is of great value to scientific decision-making of enterprises and sustainable development of supply chains

Identification of novel and robust internal control genes from Volvariella volvacea that are suitable for RT-qPCR in filamentous fungi

The selection of appropriate internal control genes (ICGs) is a crucial step in the normalization of real-time quantitative PCR (RT-qPCR) data. Housekeeping genes are habitually selected for this purpose, despite accumulating evidence on their instability. We screened for novel, robust ICGs in the mushroom forming fungus Volvariella volvacea. Nine commonly used and five newly selected ICGs were evaluated for expression stability using RT-qPCR data in eight different stages of the life cycle of V. volvacea. Three different algorithms consistently determined that three novel ICGs (SPRYp, Ras and Vps26) exhibited the highest expression stability in V. volvacea. Subsequent analysis of ICGs in twenty-four expression profiles from nine filamentous fungi revealed that Ras was the most stable ICG amongst the Basidiomycetous samples, followed by SPRYp, Vps26 and ACTB. Vps26 was expressed most stably within the analyzed data of Ascomycetes, followed by HH3 and β-TUB. No ICG was universally stable for all fungal species, or for all experimental conditions within a species. Ultimately, the choice of an ICG will depend on a specific set of experiments. This study provides novel, robust ICGs for Basidiomycetes and Ascomycetes. Together with the presented guiding principles, this enables the efficient selection of suitable ICGs for RT-qPCR

The Accordant Trend of Both Parameters (rgs Expression and cAMP Content) Follows the Pattern of Development of Fruiting Body in Volvariella volvacea

The formation of fruiting body in Volvariella volvacea is affected by endogenous genes and environmental factors. However, its regulation at a molecular level is still poorly understood. To study the genes involved in the formation of fruiting body, we cloned a new regulator of the G protein signaling (RGS) encoding gene (rgs) from V. volvacea. Phylogenetic analysis showed that RGS in V. volvacea and other basidiomycete RGS proteins from Schizophyllum commune and Coprinus cinereus belong to the same clade. In addition, we assayed intracellular cAMP content in the three developmental stages (mycelium, fruiting body primordia, and button). We also found that the expression of rgs was highly positively correlated to the content of intracellular cAMP during fruiting body formation. The conserved protein sequences and expression of rgs, together with high concent of cAMP at primordia tissue, suggested that rgs gene and cAMP may play a crucial role in fruiting body formation in V. volvacea.</p

Locally Oriented Scene Complexity Analysis Real-Time Ocean Ship Detection from Optical Remote Sensing Images

Due to strong ocean waves, broken clouds, and extensive cloud cover interferences, ocean ship detection performs poorly when using optical remote sensing images. In addition, it is a challenge to detect small ships on medium resolution optical remote sensing that cover a large area. In this paper, in order to balance the requirements of real-time processing and high accuracy detection, we proposed a novel ship detection framework based on locally oriented scene complexity analysis. First, the proposed method can separate a full image into two types of local scenes (i.e., simple or complex local scenes). Next, simple local scenes would utilize the fast saliency model (FSM) to rapidly complete candidate extraction, and for complex local scenes, the ship feature clustering model (SFCM) will be applied to achieve refined detection against severe background interferences. The FSM considers a fusion enhancement image as an input of the pulse response analysis in the frequency domain to achieve rapid ship detection in simple local scenes. Next, the SFCM builds the descriptive model of the ship feature clustering algorithm to ensure the detection performance on complex local scenes. Extensive experiments on SPOT-5 and GF-2 ocean optical remote sensing images show that the proposed ship detection framework has better performance than the state-of-the-art methods, and it addresses the tricky problem of real-time ocean ship detection under strong waves, broken clouds, extensive cloud cover, and ship fleet interferences. Finally, the proposed ocean ship detection framework is demonstrated on an onboard processing hardware

Optimal codons in Tremella fuciformis end in C/G, a strong difference with known Tremella species

Tremella fuciformis is a popular edible fungus with fruiting bodies that can be produced in large quantities at low costs, while it is easy to transform and cultivate as yeast. This makes it an attractive potential bioreactor. Enhanced heterologous gene expression through codon optimization would be useful, but until now codon usage preferences in T. fuciformis remain unknown. To precisely determine the preferred codon usage of T. fuciformis we sequenced the genome of strain Tr26 resulting in a 24.2 Mb draft genome with 10,040 predicted genes. 3288 of the derived predicted proteins matched the UniProtKB/Swiss-Prot databases with 40 % or more similarity. Corresponding gene models of this subset were subsequently optimized through repetitive comparison of alternative start codons and selection of best length matching gene models. For experimental confirmation of gene models, 96 random clones from an existing T. fuciformis cDNA library were sequenced, generating 80 complete CDSs. Calculated optimal codons for the 3288 predicted and the 80 cloned CDSs were highly similar, indicating sufficient accuracy of predicted gene models for codon usage analysis. T. fuciformis showed a strong preference for C and then G at the third base pair position of used codons, while average GC content of predicted genes was slightly higher than the total genome sequence average. Most optimal codons ended in C or G except for one, and an increased frequency of C ending codons was observed in genes with higher expression levels. Surprisingly, the preferred codon usage in T. fuciformis strongly differed from T. mesenterica and C. neoformans. Instead, optimal codon usage was similar to more distant related species such as Ustilago maydis and Neurospora crassa. Despite much higher overall sequence homology between T. fuciformis and T. mesenterica, only 7 out of 21 optimal codons were equal, whereas T. fuciformis shared up to 20 out of 21 optimal codons with other species. Clearly, codon usage in Tremella can differ largely and should be estimated for individual species. The precise identification of optimal and high expression related codons is therefore an important step in the development of T. fuciformis as a bioreactor system

Three rapid methods for direct PCR of powdery mildew samples for later sequencing

Powdery mildews are an important obligate parasitic fungal group reported in nearly all regions of the world. DNA isolation for molecular identification of powdery mildew samples is now a standard practice and is becoming more affordable but remains a lengthy process. In this study, three direct DNA isolation methods have been developed for rDNA internal transcribed spacer (ITS) amplification of different powdery mildew species. This reduces time taken for traditional DNA extraction from hours to minutes and rapid PCR means large quantities of DNA are available for sequencing in an hour. Methods proposed in this paper were found rapid and efficient for sequencing. ITS sequences obtained, were neat, clear and comparable to sequences obtained by conventional DNA extraction methods. However, ITS remains insufficient for separation of certain closely related species. In conclusion, our methods are simple, cost-effective and produced enough genomic DNA for quick and high quality sequences. Methods should be trialed further for preparation of DNA libraries of further genomic regions for rapid and accurate powdery mildew identification