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Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery.

ABSTRACT: BACKGROUND: Massively parallel sequencing of cDNA is now an efficient route for generating enormous sequence collections that represent expressed genes. This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. The large and complex genomes of pines (Pinus spp.) have hindered the development of genomic resources, despite the ecological and economical importance of the group. While most genomic studies have focused on a single species (P. taeda), genomic level resources for other pines are insufficiently developed to facilitate ecological genomic research. Lodgepole pine (P. contorta) is an ecologically important foundation species of montane forest ecosystems and exhibits substantial adaptive variation across its range in western North America. Here we describe a sequencing study of expressed genes from P. contorta, including their assembly and annotation, and their potential for molecular marker development to support population and association genetic studies. RESULTS: We obtained 586,732 sequencing reads from a 454 GS XLR70 Titanium pyrosequencer (mean length: 306 base pairs). A combination of reference-based and de novo assemblies yielded 63,657 contigs, with 239,793 reads remaining as singletons. Based on sequence similarity with known proteins, these sequences represent approximately 17,000 unique genes, many of which are well covered by contig sequences. This sequence collection also included a surprisingly large number of retrotransposon sequences, suggesting that they are highly transcriptionally active in the tissues we sampled. We located and characterized thousands of simple sequence repeats and single nucleotide polymorphisms as potential molecular markers in our assembled and annotated sequences. High quality PCR primers were designed for a substantial number of the SSR loci, and a large number of these were amplified successfully in initial screening. CONCLUSIONS: This sequence collection represents a major genomic resource for P. contorta, and the large number of genetic markers characterized should contribute to future research in this and other pines. Our results illustrate the utility of next generation sequencing as a basis for marker development and population genomics in non-model species.

SUBMITTER: Parchman TL

PROVIDER: S-EPMC2851599 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery.

Parchman Thomas L TL Geist Katherine S KS Grahnen Johan A JA Benkman Craig W CW Buerkle C Alex CA

BMC genomics 20100316

<h4>Background</h4>Massively parallel sequencing of cDNA is now an efficient route for generating enormous sequence collections that represent expressed genes. This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. The large and complex genomes of pines (Pinus spp.) have hindered the development of genomic resources, despite the ecological an ...[more]

PMID: 20233449

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Project description:In this study, we reported the characterization of the first transcriptome of the mud crab (Scylla paramamosain). Pooled cDNAs of four tissue types from twelve wild individuals were sequenced using the Roche 454 FLX platform. Analysis performed included de novo assembly of transcriptome sequences, functional annotation, and molecular marker discovery. A total of 1,314,101 high quality reads with an average length of 411 bp were generated by 454 sequencing on a mixed cDNA library. De novo assembly of these 1,314,101 reads produced 76,778 contigs (consisting of 818,154 reads) with 5.4-fold average sequencing coverage. The remaining 495,947 reads were singletons. A total of 78,268 unigenes were identified based on sequence similarity with known proteins (E?0.00001) in UniProt and non-redundant protein databases. Meanwhile, 44,433 sequences were identified (E?0.00001) using a BLASTN search against the NCBI nucleotide database. Gene Ontology (GO) analysis indicated that biosynthetic process, cell part, and ion binding were the most abundant terms in biological process, cellular component, and molecular function categories, respectively. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis revealed that 4,878 unigenes distributed in 281 different pathways. In addition, 19,011 microsatellites and 37,063 potential single nucleotide polymorphisms were detected from the transcriptome of S. paramamosain. Finally, thirty polymorphic microsatellite markers were developed and used to assess genetic diversity of a wild population of S. paramamosain. So far, existing sequence resources for S. paramamosain are extremely limited. The present study provides a characterization of transcriptome from multiple tissues and individuals, as well as an assessment of genetic diversity of a wild population. These sequence resources will facilitate the investigation of population genetic diversity, the development of genetic maps, and the conduct of molecular marker-assisted breeding in S. paramamosain and related crab species.

Project description:Sainfoin (Onobrychis viciifolia) is a highly nutritious tannin-containing forage legume. In the diet of ruminants sainfoin can have anti-parasitic effects and reduce methane emissions under in vitro conditions. Many of these benefits have been attributed to condensed tannins or proanthocyanidins in sainfoin. A combination of increased use of industrially produced nitrogen fertilizer, issues with establishment and productivity in the first year and more reliable alternatives, such as red clover led to a decline in the use of sainfoin since the middle of the last century. In recent years there has been a resurgence of interest in sainfoin due to its potential beneficial nutraceutical and environmental attributes. However, genomic resources are scarce, thus hampering progress in genetic analysis and improvement. To address this we have used next generation RNA sequencing technology to obtain the first transcriptome of sainfoin. We used the library to identify gene-based simple sequence repeats (SSRs) and potential single nucleotide polymorphisms (SNPs).One genotype from each of five sainfoin accessions was sequenced. Paired-end (PE) sequences were generated from cDNA libraries of RNA extracted from 7 day old seedlings. A combined assembly of 92,772 transcripts was produced de novo using the Trinity programme. About 18,000 transcripts were annotated with at least one GO (gene ontology) term. A total of 63 transcripts were annotated as involved in the tannin biosynthesis pathway. We identified 3786 potential SSRs. SNPs were identified by mapping the reads of the individual assemblies against the combined assembly. After stringent filtering a total of 77,000 putative SNPs were identified. A phylogenetic analysis of single copy number genes showed that sainfoin was most closely related to red clover and Medicago truncatula, while Lotus japonicus, bean and soybean are more distant relatives.This work describes the first transcriptome assembly in sainfoin. The 92 K transcripts provide a rich source of SNP and SSR polymorphisms for future use in genetic studies of this crop. Annotation of genes involved in the condensed tannin biosynthesis pathway has provided the basis for further studies of the genetic control of this important trait in sainfoin.

Project description:BackgroundAs a major stored-product pest insect, Liposcelis entomophila has developed high levels of resistance to various insecticides in grain storage systems. However, the molecular mechanisms underlying resistance and environmental stress have not been characterized. To date, there is a lack of genomic information for this species. Therefore, studies aimed at profiling the L. entomophila transcriptome would provide a better understanding of the biological functions at the molecular levels.Methodology/principal findingsWe applied Illumina sequencing technology to sequence the transcriptome of L. entomophila. A total of 54,406,328 clean reads were obtained and that de novo assembled into 54,220 unigenes, with an average length of 571 bp. Through a similarity search, 33,404 (61.61%) unigenes were matched to known proteins in the NCBI non-redundant (Nr) protein database. These unigenes were further functionally annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A large number of genes potentially involved in insecticide resistance were manually curated, including 68 putative cytochrome P450 genes, 37 putative glutathione S-transferase (GST) genes, 19 putative carboxyl/cholinesterase (CCE) genes, and other 126 transcripts to contain target site sequences or encoding detoxification genes representing eight types of resistance enzymes. Furthermore, to gain insight into the molecular basis of the L. entomophila toward thermal stresses, 25 heat shock protein (Hsp) genes were identified. In addition, 1,100 SSRs and 57,757 SNPs were detected and 231 pairs of SSR primes were designed for investigating the genetic diversity in future.Conclusions/significanceWe developed a comprehensive transcriptomic database for L. entomophila. These sequences and putative molecular markers would further promote our understanding of the molecular mechanisms underlying insecticide resistance or environmental stress, and will facilitate studies on population genetics for psocids, as well as providing useful information for functional genomic research in the future.

Dataset Information

Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery.

Publications

Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery.

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