Dataset Information

Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii.

ABSTRACT: BACKGROUND: The plant pathogenic basidiomycete Sclerotium rolfsii produces the industrially exploited exopolysaccharide scleroglucan, a polymer that consists of (1 --> 3)-beta-linked glucose with a (1 --> 6)-beta-glycosyl branch on every third unit. Although the physicochemical properties of scleroglucan are well understood, almost nothing is known about the genetics of scleroglucan biosynthesis. Similarly, the biosynthetic pathway of oxalate, the main by-product during scleroglucan production, has not been elucidated yet. In order to provide a basis for genetic and metabolic engineering approaches, we studied scleroglucan and oxalate biosynthesis in S. rolfsii using different transcriptomic approaches. RESULTS: Two S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding approximately 350,000 reads. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Sequence data were used to obtain first insights into the genomics of scleroglucan and oxalate production and to predict putative proteins involved in the synthesis of both metabolites. Using comparative transcriptomics, namely Agilent microarray hybridization and suppression subtractive hybridization, we identified approximately 800 unigenes which are differently expressed under scleroglucan-producing and non-producing conditions. From these, candidate genes were identified which could represent potential leads for targeted modification of the S. rolfsii metabolism for increased scleroglucan yields. CONCLUSIONS: The results presented in this paper provide for the first time genomic and transcriptomic data about S. rolfsii and demonstrate the power and usefulness of combined transcriptome sequencing and comparative microarray analysis. The data obtained allowed us to predict the biosynthetic pathways of scleroglucan and oxalate synthesis and to identify important genes putatively involved in determining scleroglucan yields. Moreover, our data establish the first sequence database for S. rolfsii, which allows research into other biological processes of S. rolfsii, such as host-pathogen interaction.

SUBMITTER: Schmid J

PROVIDER: S-EPMC2887420 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii.

Schmid Jochen J Müller-Hagen Dirk D Bekel Thomas T Funk Laura L Stahl Ulf U Sieber Volker V Meyer Vera V

BMC genomics 20100526

<h4>Background</h4>The plant pathogenic basidiomycete Sclerotium rolfsii produces the industrially exploited exopolysaccharide scleroglucan, a polymer that consists of (1 --> 3)-beta-linked glucose with a (1 --> 6)-beta-glycosyl branch on every third unit. Although the physicochemical properties of scleroglucan are well understood, almost nothing is known about the genetics of scleroglucan biosynthesis. Similarly, the biosynthetic pathway of oxalate, the main by-product during scleroglucan produ ...[more]

PMID: 20504312

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Project description:Two S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding ~350,000 reads. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Sequence data were used to obtain first insights into the genomics of scleroglucan and oxalate production and to predict putative proteins involved in the synthesis of both metabolites. Using comparative transcriptomics, namely Agilent microarray hybridization and suppression subtractive hybridization, we identified ~ 800 unigenes which are differently expressed under scleroglucan-producing and non-producing conditions. Tailor-made microarrays (44K multiplex chip, Agilent) were designed by imaGenes (Berlin, Germany) using an in-house developed method for empirical selection of best performing probes for each gene (Pre Selection Strategy). Briefly, up to ten probes were designed for each of the 454 unigenes (60 bp long oligomers). The 244K Agilent test array was hybridized with pooled Cy3-labeled cRNAs gained form EPSmax13 and EPSmin17 cultures and (in average) two of the best performing oligos were selected for each unigene. For comparative expression profiling, total RNA was isolated from S. rolfsii, cultured for 37 h in EPSmax13 (biological triplicate) and EPSmin17 media (biological triplicate). RNA quality control, synthesis of Cy3-labeled cRNA including cRNA purification and cRNA quality control, microarray hybridization, scanning and data extraction (Agilent´s feature extraction software) were performed by imaGenes GmbH. Expression data were analyzed by imaGenes GmbH using an in-house developed data analysis pipeline. After quantile normalization, genes were defined as differentially expressed if their expression levels varied at least 2 fold in EPSmax13 samples compared to EPSmin17 samples and if the difference was statistically significant (Student’s t-test, P-value cut-off of 0.05). 454 pyrosequencing cDNA study data was uploaded at SRA and assigned accession number SRP002142.

Project description:IntroductionSclerotium rolfsii Sacc. is a globally dispersed pathogenic fungus that causes southern blight disease in many crops and Chinese herbal medicine. The high degree of variation and diversity in the fungi altered population genetic structure. Therefore, the important factors of variation within the pathogen population should be considered during the development of management strategies for the disease.MethodsIn this study, S. rolfsii isolates from 13 hosts in 7 provinces of China were collected and analyzed to identify their morphological features and perform molecular characterization. To develop EST-SSR primers, transcriptome sequencing was performed on isolated CB1, and its SSR loci were comprehensively analyzed. In addition, we analyzed the polymorphisms among different populations based on screened EST-SSR primers.ResultsThe results showed that all of these clean reads with total 36,165,475 assembled bases were clustered into 28,158 unigenes, ranged from 201 bp to 16,402 bp on the length, of which the average length was 1,284 bp. Of these, the SSR sequence appeared at an average interval of 15.43 kB, and the frequency of SSR was 0.0648 SSR/kB. Polymorphism of 9 primers was observed among 22 populations, and was verified by the Shannon's index (average = 1.414) and polymorphic information index (> 0.50). The genetic diversity analysis revealed diversity in all host populations and geographical populations. Further, molecular variance analysis (AMOVA) showed that the differences between groups were mainly related to geographical location. Based on cluster analysis, the 7 populations were roughly divided into 3 groups, and the results were highly consistent with those based on the geographical location, ultimately aligning with the results of STRUCTURE analysis.DiscussionThe findings build on current knowledge of the distribution of S. rolfsii in the southwest area of China, adding value to current knowledge base on the population structure and genetic diversity of S. rolfsii, specifically in the context of Chinese herbal medicine cultivation in China. Overall, our findings may provide valuable information for breeding of crops with enhanced resistance toward S. rolfsii.

Dataset Information

Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii.

Publications

Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii.

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