Genome sequence and genetic linkage analysis of Shiitake mushroom _Lentinula edodes_

_Lentinula edodes_ (Shiitake/Xianggu) is an important cultivated mushroom. Understanding the genomics and functional genomics of _L. edodes_ allows us to improve its cultivation and quality. Genome sequence is a key to develop molecular genetic markers for breeding and genetic manipulation. We sequenced the genome of _L. edodes_ monokaryon L54A using Roche 454 and ABI SOLiD genome sequencing. Sequencing reads of about 1400Mb were de novo assembled into a 40.2 Mb genome sequence. We compiled the genome sequence into a searchable database with which we have been annotating the genes and analyzing the metabolic pathways. In addition, we have been using many molecular techniques to analyze genes differentially expressed during development. Gene ortholog groups of _L. edodes_ genome sequence compared across genomes of several fungi including mushrooms identified gene families unique to mushroom-forming fungi. We used a mapping population of haploid basidiospores of dikaryon L54 for genetic linkage analysis. High-quality variations such as single nucleotide polymorphisms, insertions, and deletions of the mapping population formed a high-density genetic linkage map. We compared the linkage map to the _L. edodes_ L54A genome sequence and located selected quantitative trait loci. The Shiitake community will benefit from these resources for genetic studies and breeding.
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A cost-effective and universal strategy for complete prokaryotic genomic sequencing proposed by computer simulation

Background: Pyrosequencing techniques allow scientists to perform prokaryotic genome sequencing to achieve the draft genomic sequences within a few days. However, the assemblies with shotgun sequencing are usually composed of hundreds of contigs. A further multiplex PCR procedure is needed to fill all the gaps and link contigs into complete chromosomal sequence, which is the basis for prokaryotic comparative genomic studies. In this article, we study various pyrosequencing strategies by simulated assembling from 100 prokaryotic genomes. Findings. Simulation study shows that a single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) can produce: 1) ∼90% of 100 assemblies with 99.99%; 4) average false gene duplication rate is < 0.7%; 5) average false gene loss rate is < 0.4%. Conclusions: A single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) is a cost-effective way for prokaryotic whole genome sequencing. This strategy provides solution to produce high quality draft assemblies for most of prokaryotic organisms within days. Due to the small number of assembled scaffolds, the following multiplex PCR procedure (for gap filling) would be easy. As a result, large scale prokaryotic whole genome sequencing projects may be finished within weeks. © 2012 Jiang et al; BioMed Central Ltd.published_or_final_versio

5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea

Abstract: Background: The transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development. Results: We evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom. Conclusions: We implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea

A Novel <i>Lentinula edodes</i> Laccase and Its Comparative Enzymology Suggest Guaiacol-Based Laccase Engineering for Bioremediation

<div><p>Laccases are versatile biocatalysts for the bioremediation of various xenobiotics, including dyes and polyaromatic hydrocarbons. However, current sources of new enzymes, simple heterologous expression hosts and enzymatic information (such as the appropriateness of common screening substrates on laccase engineering) remain scarce to support efficient engineering of laccase for better “green” applications. To address the issue, this study began with cloning the laccase family of <i>Lentinula edodes</i>. Three laccases <i>perfectio sensu stricto</i> (Lcc4A, Lcc5, and Lcc7) were then expressed from <i>Pichia pastoris</i>, characterized and compared with the previously reported Lcc1A and Lcc1B in terms of kinetics, stability, and degradation of dyes and polyaromatic hydrocarbons. Lcc7 represented a novel laccase, and it exhibited both the highest catalytic efficiency (assayed with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS]) and thermostability. However, its performance on “green” applications surprisingly did not match the activity on the common screening substrates, namely, ABTS and 2,6-dimethoxyphenol. On the other hand, correlation analyses revealed that guaiacol is much better associated with the decolorization of multiple structurally different dyes than are the two common screening substrates. Comparison of the oxidation chemistry of guaiacol and phenolic dyes, such as azo dyes, further showed that they both involve generation of phenoxyl radicals in laccase-catalyzed oxidation. In summary, this study concluded a robust expression platform of <i>L. edodes</i> laccases, novel laccases, and an indicative screening substrate, guaiacol, which are all essential fundamentals for appropriately driving the engineering of laccases towards more efficient “green” applications.</p></div

Specific dye decolorization rate of (A) the recombinant laccases, (B) their laccase-HBT system, and (C) laccase-TEMPO system.

<p>The reaction was performed in 1×McIlvanie buffer (pH 4) at 30°C by using 5 µg of enzyme with or without 1 mM HBT or TEMPO. The specific decolorization rate was defined as the amount of dye decolorized in one hour by one mg of protein under the assay condition. Detailed decolorization profiles are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066426#pone.0066426.s007" target="_blank">Fig. S7</a>. Results shown are the average of three independent experiments ± S.D.</p

Biodegradation of (A) ANT and (B) BaA by the recombinant laccases and their LMSs.

<p>The reaction was performed in 1×McIlvanie buffer (pH 4) at 30°C by using 5 µg of enzyme with or without 1 mM HBT or TEMPO. Residual amount of PAHs was resolved by high-performance liquid chromatography (Waters) equipped with a C-18 reverse-phase column (4.6 mm×250 mm, Grace) at ambient temperature and quantified by a photodiode array detector at 252 nm. Results shown are the average of three independent experiments ± S.D.</p

A heat map displaying the association between activity on benchmark substrates, dyes, and PAHs.

<p>Clustering was performed by MeV v4.8 based on the Pearson’s correlation, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066426#pone-0066426-t004" target="_blank">Table 4</a>. +H: with 1 mM HBT; +T: with 1 mM TEMPO.</p