Collection Of Expressed Sequence Tags Research Articles

Gene Space and Transcriptome Assemblies of Leafy Spurge (Euphorbia esula) Identify Promoter Sequences, Repetitive Elements, High-Quality Markers, and a Full-Length Chloroplast Genome

AbstractLeafy spurge (Euphorbia esulaL.) is an invasive perennial weed infesting range and recreational lands of North America. Previous research and omics projects withE. esulahave helped develop it as a model for studying many aspects of perennial plant development and response to abiotic stress. However, the lack of an assembled genome forE. esulahas limited the power of previous transcriptomics studies to identify functional promoter elements and transcription factor binding sites. An assembled genome forE. esulawould enhance our understanding of signaling processes controlling plant development and responses to environmental stress and provide a better understanding of genetic factors impacting weediness traits, evolution, and herbicide resistance. A comprehensive transcriptome database would also assist in analyzing future RNA-seq studies and is needed to annotate and assess genomic sequence assemblies. Here, we assembled and annotated 56,234 unigenes from an assembly of 589,235 RNA-seq-derived contigs and a previously published Sanger-sequenced expressed sequence tag collection. The resulting data indicate that we now have sequence for >90% of the expressedE. esulaprotein-coding genes. We also assembled the gene space ofE. esulaby using a limited coverage (18X) genomic sequence database. In this study, the programs Velvet and Trinity produced the best gene-space assemblies based on representation of expressed and conserved eukaryotic genes. The results indicate thatE. esulacontains as much as 23% repetitive sequences, of which 11% are unique. Our sequence data were also sufficient for assembling a full chloroplast and partial mitochondrial genome. Further, marker analysis identified more than 150,000 high-quality variants in ourE. esulaL-RNA–scaffolded, whole-genome, Trinity-assembled genome. Based on these results,E. esulaappears to have limited heterozygosity. This study provides a blueprint for low-cost genomic assemblies in weed species and new resources for identifying conserved and novel promoter regions among coordinately expressed genes ofE. esula.

Genomic and Transcriptomic Resources for Marker Development in Synchytrium endobioticum, an Elusive but Severe Potato Pathogen.

Synchytrium endobioticum is an obligate biotrophic fungus that causes wart diseases in potato. Like other species of the class Chytridiomycetes, it does not form mycelia and its zoospores are small, approximately 3 μm in diameter, which complicates the detection of early stages of infection. Furthermore, potato wart disease is difficult to control because belowground organs are infected and resting spores of the fungus are extremely durable. Thus, S. endobioticum is classified as a quarantine organism. More than 40 S. endobioticum pathotypes have been reported, of which pathotypes 1(D1), 2(G1), 6(O1), 8(F1), and 18(T1) are the most important in Germany. No molecular methods for the differentiation of pathotypes are available to date. In this work, we sequenced both genomic DNA and cDNA of the German pathotype 18(T1) from infected potato tissue and generated 5,422 expressed sequence tags (EST) and 423 genomic contigs. Comparative sequencing of 33 genes, single-stranded confirmation polymorphism (SSCP) analysis with polymerase chain reaction fragments of 27 additional genes, as well as the analysis of 41 simple sequence repeat (SSR) loci revealed extremely low levels of variation among five German pathotypes. From these markers, one sequence-characterized amplified region marker and five SSR markers revealed polymorphisms among the German pathotypes and an extended set of 11 additional European isolates. Pathotypes 8(F1) and 18(T1) displayed discrete polymorphisms which allow their differentiation from other pathotypes. Overall, using the information of the six markers, the 16 isolates could be differentiated into three distinct genotype groups. In addition to the presented markers, the new collection of EST from genus Synchytrium might serve in the future for molecular taxonomic studies as well as for analyses of the host-pathogen interactions in this difficult pathosystem. [Formula: see text] Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Preparing and Analyzing Expressed Sequence Tags (ESTs) Library for the Mammary Tissue of Local Turkish Kivircik Sheep

Kivircik sheep is an important local Turkish sheep according to its meat quality and milk productivity. The aim of this study was to analyze gene expression profiles of both prenatal and postnatal stages for the Kivircik sheep. Therefore, two different cDNA libraries, which were taken from the same Kivircik sheep mammary gland tissue at prenatal and postnatal stages, were constructed. Total 3072 colonies which were randomly selected from the two libraries were sequenced for developing a sheep ESTs collection. We used Phred/Phrap computer programs for analysis of the raw EST and readable EST sequences were assembled with the CAP3 software. Putative functions of all unique sequences and statistical analysis were determined by Geneious software. Total 422 ESTs have over 80% similarity to known sequences of other organisms in NCBI classified by Panther database for the Gene Ontology (GO) category. By comparing gene expression profiles, we observed some putative genes that may be relative to reproductive performance or play important roles in milk synthesis and secretion. A total of 2414 ESTs have been deposited to the NCBI GenBank database (GW996847–GW999260). EST data in this study have provided a new source of information to functional genome studies of sheep.

ESAP plus: a web-based server for EST-SSR marker development.

BackgroundSimple sequence repeats (SSRs) have become widely used as molecular markers in plant genetic studies due to their abundance, high allelic variation at each locus and simplicity to analyze using conventional PCR amplification. To study plants with unknown genome sequence, SSR markers from Expressed Sequence Tags (ESTs), which can be obtained from the plant mRNA (converted to cDNA), must be utilized. With the advent of high-throughput sequencing technology, huge EST sequence data have been generated and are now accessible from many public databases. However, SSR marker identification from a large in-house or public EST collection requires a computational pipeline that makes use of several standard bioinformatic tools to design high quality EST-SSR primers. Some of these computational tools are not users friendly and must be tightly integrated with reference genomic databases.ResultsA web-based bioinformatic pipeline, called EST Analysis Pipeline Plus (ESAP Plus), was constructed for assisting researchers to develop SSR markers from a large EST collection. ESAP Plus incorporates several bioinformatic scripts and some useful standard software tools necessary for the four main procedures of EST-SSR marker development, namely 1) pre-processing, 2) clustering and assembly, 3) SSR mining and 4) SSR primer design. The proposed pipeline also provides two alternative steps for reducing EST redundancy and identifying SSR loci. Using public sugarcane ESTs, ESAP Plus automatically executed the aforementioned computational pipeline via a simple web user interface, which was implemented using standard PHP, HTML, CSS and Java scripts. With ESAP Plus, users can upload raw EST data and choose various filtering options and parameters to analyze each of the four main procedures through this web interface. All input EST data and their predicted SSR results will be stored in the ESAP Plus MySQL database. Users will be notified via e-mail when the automatic process is completed and they can download all the results through the web interface.ConclusionsESAP Plus is a comprehensive and convenient web-based bioinformatic tool for SSR marker development. ESAP Plus offers all necessary EST-SSR development processes with various adjustable options that users can easily use to identify SSR markers from a large EST collection. With familiar web interface, users can upload the raw EST using the data submission page and visualize/download the corresponding EST-SSR information from within ESAP Plus. ESAP Plus can handle considerably large EST datasets. This EST-SSR discovery tool can be accessed directly from: http://gbp.kku.ac.th/esap_plus/.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3328-4) contains supplementary material, which is available to authorized users.

Characterization of aromatic aminotransferases from Ephedra sinica Stapf.

Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using L-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.

Characterization of ESTs from black locust for gene discovery and marker development.

Black locust (Robinia pseudoacacia L.) is an ecologically and economically important species. However, it has relatively underdeveloped genomic resources, and this limits gene discovery and marker-assisted selective breeding. In the present study, we obtained large-scale transcriptome data using a next-generation sequencing platform to compensate for the lack of black locust genomic information. Increasing the amount of transcriptome data for black locust will provide a valuable resource for multi-gene phylogenetic analyses and will facilitate research on the mechanisms whereby conserved genes and functions are maintained in the face of species divergence. We sequenced the black locust transcriptome from a cDNA library of multiple tissues and individuals on an Illumina platform, and this produced 108,229,352 clean sequence reads. The high-quality overlapping expressed sequence tags (ESTs) were assembled into 36,533 unigenes, and 4781 simple sequence repeats were characterized. A large collection of high-quality ESTs was obtained, de novo assembled, and characterized. Our results markedly expand the previous transcript catalogues of black locust and can gradually be applied to black locust breeding programs. Furthermore, our data will facilitate future research on the comparative genomics of black locust and related species.

Transcriptome analysis of extant cotton progenitors revealed tetraploidization and identified genome-specific single nucleotide polymorphism in diploid and allotetraploid cotton.

BackgroundThe most widely cultivated cotton (Gossypium hirsutum L., AD-genome) is derived from tetraploidization between A- and D-genome species. G. arboreum L. (A-genome) and G. raimondii Ulbr. (D-genome) are two of closely-related extant progenitors. Gene expression studies in allotetraploid cotton are complicated by the homoeologous loci of A- and D-genome origins. To develop genomic resources for gene expression and cotton breeding, we sequenced and assembled expressed sequence tags (ESTs) derived from G. arboreum and G. raimondii.ResultsRoche/454 FLX sequencing technology was employed to sequence normalized cDNA libraries prepared from leaves, roots, bolls, ovules, and fibers in G. arboreum and G. raimondii, respectively. Sequencing reads from two independent libraries in each species were combined to assemble high-quality EST contigs. The combined sequencing reads included 1,699,776 from A-genome and 1,464,815 from D-genome, which were clustered into 89,588 contigs in the A-genome and 65,542 contigs in the D-genome. These contigs represented ~80% of EST collections in Cotton Gene Index 11 (CGI11, March 2011). Compared to the D-genome transcript database, 27,537 and 10,452 contigs were unique transcripts in A and D genomes, respectively. Further analysis using self-blastn reduced the unigene contig number by 52% in A-genome and 57% in D-genome, suggesting that 50% or more of contigs are paralogs or isoforms within each species. The majority of EST contigs (73–81%) were conserved between A- and D-genomes, whereas 27% and 19% contigs were specific to A- and D-genomes, respectively. Using these ESTs, we generated a total of 75,754 genome-specific single nucleotide polymorphism (SNP) (gSNPs or GNPs) or homoeologous-specific SNPs (hSNPs) of 10,885 contigs or genes between A and D genomes, indicating a possibility of separating allelic expression for those genes in allotetraploid cotton.ConclusionsExpressed genes are highly redundant within each diploid progenitor and between A and D progenitor species, suggesting that diploid progenitors in cotton are likely ancient tetraploids. This large set of A- and D-genome ESTs and GNPs will be valuable resources for genome annotation, gene expression, and crop improvement in allotetraploid cotton.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-493) contains supplementary material, which is available to authorized users.

ClonorESTdb: a comprehensive database for Clonorchis sinensis EST sequences.

BackgroundClonorchiasis, which is primarily caused by liver fluke (Platyhelminthes), is a fatal infectious disease that is mainly associated with bile duct malignancy and the subsequent development of cholangiocarcinoma. Thus, a genomic approach now represents an important step to further our knowledge of biology and the pathology of these parasites. The results of expressed sequence tags (ESTs) sequencing need to be well organized into databases to provide an integrated set of tools and functional information.FindingsHere, the ClonorESTdb database represents a collection of Clonorchis sinensis ESTs that is intended as a resource for parasite functional genomics. A total of 55,736 successful EST sequences, which are cleaned and clustered into non-redundant 13,305 C. sinensis assembled EST sequences (6,497 clusters and 6,808 singletons), were obtained from three in-house prepared cDNA libraries of C. sinensis at different developmental stages. The assembled consensus sequences were annotated using the BLAST algorithm or/and hmm against NCBI NR, UniProt, KEGG and InterProScan. The ClonorESTdb database provides functional annotation, their expression profiles, tandem repeats and putative single nucleotide polymorphisms with utility tools such as local BLAST search and text retrieval.ConclusionsThis resource enables the researcher to identify and compare expression signatures under different biological stages and promotes ongoing parasite drug and vaccine development and biological research.Database URL:http://pathod.cdc.go.kr/clonorestdb/

High-throughput analysis and characterization of Astragalus membranaceus transcriptome using 454 GS FLX.

Astragalus membranaceus (Fisch.) Bge (AR), one of the most important medicinal plants in Asia, was found to exhibit various bioactivities. Due to limited genomic and transcriptomic data, the biosynthetic pathway of the major bioactive compound in AR, is currently unclear. In this study, 454 GS FLX technology was employed to produce a substantial expressed sequence tag (EST) dataset from the AR. In all, 742721 high-quality reads from the AR were produced using Roche GS FLX Titanium. A total of 9893 unique sequences were obtained and annotated by a similarity search against the public databases, and involved in the secondary metabolic pathway, which would facilitate deciphering the molecular mechanism of secondary metabolism in AR. The assembled sequences were annotated with gene names and Gene Ontology (GO) terms. GO revealed the unique sequences that could be assigned to 34 vocabularies. In the KEGG mapping, unique sequences were established as associated with 46 biochemical pathways. These results provided the largest EST collections in AR and will contribute to biosynthetic and biochemical studies that lead to drug improvement. With respect to the genes related to metabolism and biosynthesis pathway were also found. Our work demonstrated the utility of 454 GS FLX as a method for the rapid and cost-effective identification of AR transcriptome, and this EST dataset will be a powerful resource for further studies such as taxonomy, molecular breeding, and secondary metabolism in AR.

Establishment of cDNA Library and EST Analysis from Leaves of Phyllanthus amarus

DNA sequencing of randomly chosen clones from a cDNA library allows thousands of different transcripts to be identified. However, since the likelihood of observing a given transcript is proportional to the expression level of that transcript in the tissue from which the library is derived, often transcripts are represented by several EST sequences. An expressed sequence tags (EST) analysis was undertaken to identify the genes present in the leaves of Phyllanthus amarus, which is a small tropical, glabrous herb with several health benefits. Phyllanthin and hypophyllanthin, major bioactive components, present in highest amounts in the leaves, are of significant therapeutic importance like hepatoprotective, antioxidant, antiviral, hypoglycemic, etc. Taken together, sequencing of cDNA clones generated high-quality ESTs (Accession number: JK492908 to JK492964) with high similarities with genes from Ricinus communis, Onchocerca volvulus, Eucalyptus globules, Gossypium hirsutum, Nicotiana tabacum, Solanum spp. and many more. A BLASTN analysis along with BLASTX analysis of all the unique sequences was performed and was grouped according to the reported activities. Results represented here is the first reference collection of ESTs from this commercially important medicinal herb. This study indicated that the leaf transcriptome contains series of interesting sequences like ALBINO3, ribulose-1, 5 bisphosphate carboxylase/ oxygenase (RUBISCO), chloroplast Research Article International Journal of Biochemistry Research & Review, 4(1): 1-15, 2014 2 photosystem II chlorophyll A/B-binding protein, stress-responsive proteins like methionine sulfoxide reductase type, etc.

A detailed gene expression study of the Miscanthusgenus reveals changes in the transcriptome associated with the rejuvenation of spring rhizomes

BackgroundThe Miscanthus genus of perennial C4 grasses contains promising biofuel crops for temperate climates. However, few genomic resources exist for Miscanthus, which limits understanding of its interesting biology and future genetic improvement. A comprehensive catalog of expressed sequences were generated from a variety of Miscanthus species and tissue types, with an emphasis on characterizing gene expression changes in spring compared to fall rhizomes.ResultsIllumina short read sequencing technology was used to produce transcriptome sequences from different tissues and organs during distinct developmental stages for multiple Miscanthus species, including Miscanthus sinensis, Miscanthus sacchariflorus, and their interspecific hybrid Miscanthus × giganteus. More than fifty billion base-pairs of Miscanthus transcript sequence were produced. Overall, 26,230 Sorghum gene models (i.e., ~ 96% of predicted Sorghum genes) had at least five Miscanthus reads mapped to them, suggesting that a large portion of the Miscanthus transcriptome is represented in this dataset. The Miscanthus × giganteus data was used to identify genes preferentially expressed in a single tissue, such as the spring rhizome, using Sorghum bicolor as a reference. Quantitative real-time PCR was used to verify examples of preferential expression predicted via RNA-Seq. Contiguous consensus transcript sequences were assembled for each species and annotated using InterProScan. Sequences from the assembled transcriptome were used to amplify genomic segments from a doubled haploid Miscanthus sinensis and from Miscanthus × giganteus to further disentangle the allelic and paralogous variations in genes.ConclusionsThis large expressed sequence tag collection creates a valuable resource for the study of Miscanthus biology by providing detailed gene sequence information and tissue preferred expression patterns. We have successfully generated a database of transcriptome assemblies and demonstrated its use in the study of genes of interest. Analysis of gene expression profiles revealed biological pathways that exhibit altered regulation in spring compared to fall rhizomes, which are consistent with their different physiological functions. The expression profiles of the subterranean rhizome provides a better understanding of the biological activities of the underground stem structures that are essentials for perenniality and the storage or remobilization of carbon and nutrient resources.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-864) contains supplementary material, which is available to authorized users.

Transcriptome characterisation of Pinus tabuliformis and evolution of genes in the Pinusphylogeny

BackgroundThe Chinese pine (Pinus tabuliformis) is an indigenous conifer species in northern China but is relatively underdeveloped as a genomic resource; thus, limiting gene discovery and breeding. Large-scale transcriptome data were obtained using a next-generation sequencing platform to compensate for the lack of P. tabuliformis genomic information.ResultsThe increasing amount of transcriptome data on Pinus provides an excellent resource for multi-gene phylogenetic analysis and studies on how conserved genes and functions are maintained in the face of species divergence. The first P. tabuliformis transcriptome from a normalised cDNA library of multiple tissues and individuals was sequenced in a full 454 GS-FLX run, producing 911,302 sequencing reads. The high quality overlapping expressed sequence tags (ESTs) were assembled into 46,584 putative transcripts, and more than 700 SSRs and 92,000 SNPs/InDels were characterised. Comparative analysis of the transcriptome of six conifer species yielded 191 orthologues, from which we inferred a phylogenetic tree, evolutionary patterns and calculated rates of gene diversion. We also identified 938 fast evolving sequences that may be useful for identifying genes that perhaps evolved in response to positive selection and might be responsible for speciation in the Pinus lineage.ConclusionsA large collection of high-quality ESTs was obtained, de novo assembled and characterised, which represents a dramatic expansion of the current transcript catalogues of P. tabuliformis and which will gradually be applied in breeding programs of P. tabuliformis. Furthermore, these data will facilitate future studies of the comparative genomics of P. tabuliformis and other related species.

SNP Discovery with EST and NextGen Sequencing in Switchgrass (Panicum virgatum L.)

Although yield trials for switchgrass (Panicum virgatum L.), a potentially high value biofuel feedstock crop, are currently underway throughout North America, the genetic tools for crop improvement in this species are still in the early stages of development. Identification of high-density molecular markers, such as single nucleotide polymorphisms (SNPs), that are amenable to high-throughput genotyping approaches, is the first step in a quantitative genetics study of this model biofuel crop species. We generated and sequenced expressed sequence tag (EST) libraries from thirteen diverse switchgrass cultivars representing both upland and lowland ecotypes, as well as tetraploid and octoploid genomes. We followed this with reduced genomic library preparation and massively parallel sequencing of the same samples using the Illumina Genome Analyzer technology platform. EST libraries were used to generate unigene clusters and establish a gene-space reference sequence, thus providing a framework for assembly of the short sequence reads. SNPs were identified utilizing these scaffolds. We used a custom software program for alignment and SNP detection and identified over 149,000 SNPs across the 13 short-read sequencing libraries (SRSLs). Approximately 25,000 additional SNPs were identified from the entire EST collection available for the species. This sequencing effort generated data that are suitable for marker development and for estimation of population genetic parameters, such as nucleotide diversity and linkage disequilibrium. Based on these data, we assessed the feasibility of genome wide association mapping and genomic selection applications in switchgrass. Overall, the SNP markers discovered in this study will help facilitate quantitative genetics experiments and greatly enhance breeding efforts that target improvement of key biofuel traits and development of new switchgrass cultivars.

Characterization of maspardin, responsible for human Mast syndrome, in an insect species and analysis of its evolution in metazoans

Mast syndrome is a complicated form of human hereditary spastic paraplegias, caused by a mutation in the gene acid cluster protein 33, which encodes a protein designated as "maspardin." Maspardin presents similarity to the α/β-hydrolase superfamily, but might lack enzymatic activity and rather be involved in protein-protein interactions. Association with the vesicles of the endosomal network also suggested that maspardin may be involved in the sorting and/or trafficking of molecules in the endosomal pathway, a crucial process for maintenance of neuron health. Despite a high conservation in living organisms, studies of maspardin in other animal species than mammals were lacking. In the cotton armyworm Spodoptera littoralis, an insect pest model, analysis of an expressed sequence tag collection from antenna, the olfactory organ, has allowed identifying a maspardin homolog (SlMasp). We have investigated SlMasp tissue distribution and temporal expression by PCR and in situ hybridization techniques. Noteworthy, we found that maspardin was highly expressed in antennae and associated with the structures specialized in odorant detection. We have, in addition, identified maspardin sequences in numerous "nonmammalian" species and described here their phylogenetic analysis in the context of metazoan diversity. We observed a strong conservation of maspardin in metazoans, with surprisingly two independent losses of this gene in two relatively distant ecdysozoan taxa that include major model organisms, i.e., dipterans and nematodes.

Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus

AbstractThe status of genomics and genetics research in the Myrtaceae, a large family of dicotyledonous woody plants, is reviewed withEucalyptusas the focal genus. The family contains over 5,650 species in 130 to 150 genera, predominantly of neo-tropical and Southern Hemisphere distribution. Several genera are well known for their economic importance worldwide. Myrtaceae are typically diploids with small to intermediate genome size. Microsatellites have been developed for several genera while higher throughput marker systems such as diversity arrays technology and single nucleotide polymorphism are available forEucalyptus. Molecular data have been fundamental to current perspectives on the phylogeny, phylogeography and taxonomy of the Myrtaceae, while numerous studies of genetic diversity have been carried out particularly as it relates to endangered, rare, fragmented, overharvested or economically important species. Large expressed sequence tag collections for species ofEucalyptushave recently become public to support the annotation of theEucalyptus grandisgenome. Transcriptomics inEucalyptushas advanced by microarrays and next-generation sequencing focusing on wood development. Linkage maps forEucalyptusdisplay high synteny across species and have been extensively used to map quantitative trait loci for a number of traits including growth, wood quality, disease and insect resistance. Candidate gene-based association genetics have successfully found marker–trait associations for wood and fiber traits. Genomic selection experiments have demonstrated clear potential to improve the efficiency of breeding programs while freeze-tolerant transgenicEucalyptustrials have recently been initiated. The recently releasedE. grandisgenome, sequenced to an average coverage of 8×, will open up exceptional opportunities to advance Myrtaceae genetics and genomics research.

Development of expressed sequence tag and expressed sequence tag–simple sequence repeat marker resources for Musa acuminata

Banana (Musa acuminata) is a crop contributing to global food security. Many varieties lack resistance to biotic stresses, due to sterility and narrow genetic background. The objective of this study was to develop an expressed sequence tag (EST) database of transcripts expressed during compatible and incompatible banana-Mycosphaerella fijiensis (Mf) interactions. Black leaf streak disease (BLSD), caused by Mf, is a destructive disease of banana. Microsatellite markers were developed as a resource for crop improvement. cDNA libraries were constructed from in vitro-infected leaves from BLSD-resistant M. acuminata ssp. burmaniccoides Calcutta 4 (MAC4) and susceptible M. acuminata cv. Cavendish Grande Naine (MACV). Clones were 5'-end Sanger sequenced, ESTs assembled with TGICL and unigenes annotated using BLAST, Blast2GO and InterProScan. Mreps was used to screen for simple sequence repeats (SSRs), with markers evaluated for polymorphism using 20 diploid (AA) M. acuminata accessions contrasting in resistance to Mycosphaerella leaf spot diseases. A total of 9333 high-quality ESTs were obtained for MAC4 and 3964 for MACV, which assembled into 3995 unigenes. Of these, 2592 displayed homology to genes encoding proteins with known or putative function, and 266 to genes encoding proteins with unknown function. Gene ontology (GO) classification identified 543 GO terms, 2300 unigenes were assigned to EuKaryotic orthologous group categories and 312 mapped to Kyoto Encyclopedia of Genes and Genomes pathways. A total of 624 SSR loci were identified, with trinucleotide repeat motifs the most abundant in MAC4 (54.1 %) and MACV (57.6 %). Polymorphism across M. acuminata accessions was observed with 75 markers. Alleles per polymorphic locus ranged from 2 to 8, totalling 289. The polymorphism information content ranged from 0.08 to 0.81. This EST collection offers a resource for studying functional genes, including transcripts expressed in banana-Mf interactions. Markers are applicable for genetic mapping, diversity characterization and marker-assisted breeding.

Cross‐species transferability of SSR loci developed from transciptome sequencing in lodgepole pine

With the advent of next generation sequencing technologies, transcriptome level sequence collections are arising as prominent resources for the discovery of gene-based molecular markers. In a previous study more than 15,000 simple sequence repeats (SSRs) in expressed sequence tag (EST) sequences resulting from 454 pyrosequencing of Pinus contorta cDNA were identified. From these we developed PCR primers for approximately 4000 candidate SSRs. Here, we tested 184 of these SSRs for successful amplification across P. contorta and eight other pine species and examined patterns of polymorphism and allelic variability for a subset of these SSRs. Cross-species transferability was high, with high percentages of loci producing PCR products in all species tested. In addition, 50% of the loci we screened across panels of individuals from three of these species were polymorphic and allelically diverse. We examined levels of diversity in a subset of these SSRs by collecting genotypic data across several populations of Pinus ponderosa in northern Wyoming. Our results indicate the utility of mining pyrosequenced EST collections for gene-based SSRs and provide a source of molecular markers that should bolster evolutionary genetic investigations across the genus Pinus.

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins.

BackgroundBupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway.ResultsOne-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A de novo assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the Bupleurum genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel Bupleurum genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450s and 7 uridine diphosphate GTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450s and three UGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with β-AS in methyl jasmonate-treated adventitious roots and on their similar expression patterns with β-AS in various B. chinense tissues.ConclusionsA collection of high-quality ESTs for B. chinense obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of B. chinense and other Bupleurum species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the P450s and UGTs, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.

ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE GREEN ALGA DUNALIELLA SALINA (CHLOROPHYTA)1

The unicellular green alga Dunaliella salina (Dunal) Teodor. is a novel model photosynthetic eukaryote for studying photosystems, high salinity acclimation, and carotenoid accumulation. In spite of such significance, there have been limited studies on the Dunaliella genome transcriptome and proteome. To further investigate D. salina, a cDNA library was constructed and sequenced. Here, we present the analysis of the 2,282 expressed sequence tags (ESTs) generated together with 3,990 ESTs from dbEST. A total of 4,148 unique sequences (UniSeqs) were identified, of which 56.1% had sequence similarity with Uniprot entries, suggesting that a large number of unique genes may be harbored by Dunaliella. Additionally, protein family domains were identified to further characterize these sequences. Then, we also compared EST sequences with different complete eukaryotic genomes from several animals, plants, and fungi. We observed notable differences between D. salina and other organisms. This EST collection and its annotation provided a significant resource for basic and applied research on D. salina and laid the foundation for a systematic analysis of the transcriptome basis of green algae development and diversification.

Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles

BackgroundCultivated watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] is an important agriculture crop world-wide. The fruit of watermelon undergoes distinct stages of development with dramatic changes in its size, color, sweetness, texture and aroma. In order to better understand the genetic and molecular basis of these changes and significantly expand the watermelon transcript catalog, we have selected four critical stages of watermelon fruit development and used Roche/454 next-generation sequencing technology to generate a large expressed sequence tag (EST) dataset and a comprehensive transcriptome profile for watermelon fruit flesh tissues.ResultsWe performed half Roche/454 GS-FLX run for each of the four watermelon fruit developmental stages (immature white, white-pink flesh, red flesh and over-ripe) and obtained 577,023 high quality ESTs with an average length of 302.8 bp. De novo assembly of these ESTs together with 11,786 watermelon ESTs collected from GenBank produced 75,068 unigenes with a total length of approximately 31.8 Mb. Overall 54.9% of the unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database and around two-thirds of them matched proteins of cucumber, the most closely-related species with a sequenced genome. The unigenes were further assigned with gene ontology (GO) terms and mapped to biochemical pathways. More than 5,000 SSRs were identified from the EST collection. Furthermore we carried out digital gene expression analysis of these ESTs and identified 3,023 genes that were differentially expressed during watermelon fruit development and ripening, which provided novel insights into watermelon fruit biology and a comprehensive resource of candidate genes for future functional analysis. We then generated profiles of several interesting metabolites that are important to fruit quality including pigmentation and sweetness. Integrative analysis of metabolite and digital gene expression profiles helped elucidating molecular mechanisms governing these important quality-related traits during watermelon fruit development.ConclusionWe have generated a large collection of watermelon ESTs, which represents a significant expansion of the current transcript catalog of watermelon and a valuable resource for future studies on the genomics of watermelon and other closely-related species. Digital expression analysis of this EST collection allowed us to identify a large set of genes that were differentially expressed during watermelon fruit development and ripening, which provide a rich source of candidates for future functional analysis and represent a valuable increase in our knowledge base of watermelon fruit biology.