High-quality Expressed Sequence Tags Research Articles

Transcriptome analysis reveals the peptide toxins diversity of Macrothele palpator venom

Spider venom is a large pharmacological repertoire of different bioactive peptide toxins. However, obtaining crude venom from some spiders is challenging. Thus, studying individual toxins through venom purification is a daunting task. In this study, we constructed the cDNA library and transcriptomic sequencing from the Macrothele palpator venom glands. Subsequently, 718 high-quality expressed sequence tags (ESTs) were identified, and grouped into three categories, including 449 toxin-like (62.53 %), 136 cellular component (18.94 %) and 133 non-matched (18.52 %) based on the gene function annotation. Additionally, 112 non-redundant toxin-like peptides were classified into 13 families (families A-M) based on their sequence homology and cysteine framework. Bioinformatics analysis revealed a high sequence similarity between families A-J and the toxins from Macrothele gigas in the NR database. In contrast, families K-M had a generally low sequence homology with known spider peptide toxins and unpredictable biological functions. Taken together, this study adds many new members to the spider toxin superfamily and provides a basis for identifying various potential biological tools in M. palpator venom.

The Molecular Composition of Peptide Toxins in the Venom of Spider Lycosa coelestis as Revealed by cDNA Library and Transcriptomic Sequencing.

In the so-called "struggle for existence" competition, the venomous animals developed a smart and effective strategy, envenomation, for predation and defense. Biochemical analysis revealed that animal venoms are chemical pools of proteinase, peptide toxins, and small organic molecules with various biological activities. Of them, peptide toxins are of great molecular diversity and possess the capacity to modulate the activity of ion channels, the second largest group of drug targets expressed on the cell membrane, which makes them a rich resource for developing peptide drug pioneers. The spider Lycosa coelestis (L. coelestis) commonly found in farmland in China is a dominant natural enemy of agricultural pests; however, its venom composition and activity were never explored. Herein, we conducted cDNA library and transcriptomic sequencing of the venom gland of L. coelestis, which identified 1131 high-quality expressed sequence tags (ESTs), grouped into three categories denoted as toxin-like ESTs (597, 52.79%), cellular component ESTs (357, 31.56%), and non-matched ESTs (177, 15.65%). These toxin-like ESTs encode 98 non-reductant toxins, which are artificially divided into 11 families based on their sequence homology and cysteine frameworks (2-14 cysteines forming 1-7 disulfide bonds to stabilize the toxin structure). Furthermore, RP-HPLC purification combined with off-line MALDI-TOF analysis have detected 147 different peptides physically existing in the venom of L. coelestis. Electrophysiology analysis confirmed that the venom preferably inhibits the voltage-gated calcium channels in rat dorsal root ganglion neurons. Altogether, the present study has added a great lot of new members to the spider toxin superfamily and built the foundation for characterizing novel active peptides in the L. coelestis venom.

Molecular Diversity of Peptide Toxins in the Venom of Spider Heteropoda pingtungensis as Revealed by cDNA Library and Transcriptome Sequencing Analysis.

The venoms of toxic animals are chemical pools composed of various proteins, peptides, and small organic molecules used for predation and defense, in which the peptidic toxins have been intensively pursued mining modulators targeting disease-related ion channels and receptors as valuable drug pioneers. In the present study, we uncovered the molecular diversity of peptide toxins in the venom of the spider Heteropoda pingtungensis (H. pingtungensis) by using a combinatory strategy of venom gland cDNA library and transcriptome sequencing (RNA-seq). An amount of 991 high-quality expressed sequence tags (ESTs) were identified from 1138 generated sequences, which fall into three categories, such as the toxin-like ESTs (531, 53.58%), the cellular component ESTs (255, 25.73%), and the no-match ESTs (205, 20.69%), as determined by gene function annotations. Of them, 190 non-redundant toxin-like peptides were identified and can be artificially grouped into 13 families based on their sequence homology and cysteine frameworks (families A–M). The predicted mature toxins contain 2–10 cysteines, which are predicted to form intramolecular disulfide bonds to stabilize their three-dimensional structures. Bioinformatics analysis showed that toxins from H. pingtungensis venom have high sequences variability and the biological targets for most toxins are unpredictable due to lack of homology to toxins with known functions in the database. Furthermore, RP-HPLC and MALDI-TOF analyses have identified a total of 110 different peptides physically existing in the H. pingtungensis venom, and many RP-HPLC fractions showed potent inhibitory activity on the heterologously expressed NaV1.7 channel. Most importantly, two novel NaV1.7 peptide antagonists, µ-Sparatoxin-Hp1 and µ-Sparatoxin-Hp2, were characterized. In conclusion, the present study has added many new members to the spider toxin superfamily and built the foundation for identifying novel modulators targeting ion channels in the H. pingtungensis venom.

Transcript profiling of chickpea pod wall revealed the expression of floral homeotic gene AGAMOUS-like X2 (CaAGLX2).

The differentially expressed genes in the chickpea pod wall have been identified for the first time using a forward suppression subtractive hybridization (SSH) library. In all, 226 clones of SSH library were sequenced and analyzed. A total of 179 high-quality expressed sequence tags (ESTs) were generated and based on the CAP3 assembly of these ESTs, 126 genes (97 singletons and 29 contigs) were computationally annotated. The mapping of 88.26% ESTs by gene ontology (GO) annotation distributed them into 751 GO terms of three categories, cellular location, molecular function, and biological process. The KEGG pathway analysis revealed 45 ESTs are involved in 49 different biological pathways. Also, 67 ESTs encodes four different classes of enzymes such as oxidoreductases (29), transferase (20), hydrolases (16) and isomerase (2). Six genes were selected and subjected to qPCR analysis, of these, two genes (FHG Floral homeotic AGAMOUS-like isoform X2, MADS1 MADS-box transcription factor) showed significant up-regulation in the pod wall compared to leaves. Surprisingly, one of the MADS1 box gene, FHG (CaAGLX2), responsible for flower development expressed in the pod wall. Therefore, understanding its specific role in the pod wall could be interesting. Thus, the transcript dynamics of the chickpea pod wall revealed differentially expressed genes in the pod wall, which may be participating in the metabolic build-up of both pod wall and seeds.

Generation and characterization of expressed sequence tags (ESTs) from coralloid root cDNA library of Cycas debaoensis

A normalized full-length cDNA library was constructed from the coralloid roots of Cycas debaoensis by the DSN (duplex-specific nuclease) normalization method combined with the SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique. The titer of the original cDNA library was about 1.5 × 106 cfu·mL−1 and the average insertion size was about 1 kb with a high recombination rate (97%). The 5011 high-quality expressed sequence tags (ESTs) were obtained from 5393 randomly picked cDNA clones. Clustering and assembly of ESTs resulted in 2984 unique sequences, consisting of 618 contigs and 2366 singlets. EST sequence annotation revealed that 2333 and 1901 unigenes were functionally annotated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 1495 (50.1%) unigenes were associated with 4082 Gene Ontology (GO) terms. A total of 847 unigenes were grouped into 22 Cluster of Orthologous Groups (COG) functional categories. Based on the EST dataset, 22 ESTs that encoded putative receptor-like protein kinase (RLK) genes were screened. Furthermore, a total of 94 simple sequence repeats (SSRs) were discovered, of which 20 loci were successfully amplified in C. debaoensis. This study is the first EST analysis for the coralloid roots of C. debaoensis and provides a valuable genomic resource for novel gene discovery, gene expression and comparative genomics, conservation and management studies as well as applications in C. debaoensis and related cycad species.

Analysis of putative sclerotia maturation-related gene expression in Rhizoctonia solani AG1-IA

Rhizoctonia solani AG1-IA (R. solani AG1-IA) is a major soil-borne fungal pathogen of maize that causes significant yield losses in all maize-growing regions worldwide. The sclerotium produced by R. solani AG1-IA can overwinter in grass roots or diseased plants and infect crops the following year. The molecular mechanism underlying sclerotium formation in R. solani is poorly understood. In this study, we constructed the cDNA library of the R. solani AG1-IA pathogenic strain WF-9, from which 329 high-quality expressed sequence tags (ESTs) were obtained. Of the 250 clustered unigenes, 12 genes were selected for further expression analysis during the three stages of sclerotial growth (mycelium, initiation of sclerotium, and maturation of sclerotium). The results of expression analysis support the previously suggested roles of chitin synthase D and exo-beta-1,3-glucanase in facilitating sclerotial growth through preservation of water content and energy. In addition, cytochrome P450, NADPH oxidase, catalase (CAT), acyl-CoA oxidase 1 (ACOX1), mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase HOG1 (HOG 1), and the G-protein ? subunit play important roles in balancing reactive oxygen species (ROS) levels during sclerotial development. The findings of this study can help understand the molecular mechanism of sclerotial development further.

Generation, Annotation, and Analysis of a Large-Scale Expressed Sequence Tag Library from Arabidopsis pumila to Explore Salt-Responsive Genes.

Arabidopsis pumila is an ephemeral plant, and a close relative of the model plant Arabidopsis thaliana, but it possesses higher photosynthetic efficiency, higher propagation rate, and higher salinity tolerance compared to those A. thaliana, thus providing a candidate plant system for gene mining for environmental adaption and salt tolerance. However, A. pumila is an under-explored resource for understanding the genetic mechanisms underlying abiotic stress adaptation. To improve our understanding of the molecular and genetic mechanisms of salt stress adaptation, more than 19,900 clones randomly selected from a cDNA library constructed previously from leaf tissue exposed to high-salinity shock were sequenced. A total of 16,014 high-quality expressed sequence tags (ESTs) were generated, which have been deposited in the dbEST GenBank under accession numbers JZ932319 to JZ948332. Clustering and assembly of these ESTs resulted in the identification of 8,835 unique sequences, consisting of 2,469 contigs and 6,366 singletons. The blastx results revealed 8,011 unigenes with significant similarity to known genes, while only 425 unigenes remained uncharacterized. Functional classification demonstrated an abundance of unigenes involved in binding, catalytic, structural or transporter activities, and in pathways of energy, carbohydrate, amino acid, or lipid metabolism. At least seven main classes of genes were related to salt-tolerance among the 8,835 unigenes. Many previously reported salt tolerance genes were also manifested in this library, for example VP1, H+-ATPase, NHX1, SOS2, SOS3, NAC, MYB, ERF, LEA, P5CS1. In addition, 251 transcription factors were identified from the library, classified into 42 families. Lastly, changes in expression of the 12 most abundant unigenes, 12 transcription factor genes, and 19 stress-related genes in the first 24 h of exposure to high-salinity stress conditions were monitored by qRT-PCR. The large-scale EST library obtained in this study provides first-hand information on gene sequences expressed in young leaves of A. pumila exposed to salt shock. The rapid discovery of known or unknown genes related to salinity stress response in A. pumila will facilitate the understanding of complex adaptive mechanisms for ephemerals.

Analysis of Expressed Sequence Tags (EST) in Date Palm.

Expressed sequence tags (EST) were generated from a normalized cDNA library of the date palm Sukkari cv. to understand the high-quality and better field performance of this well-known commercial cultivar. A total of 6943 high-quality ESTs were generated, out of them 6671 are submitted to the GenBank dbEST (LIBEST_028537). The generated ESTs were assembled into 6362 unigenes, consisting of 494 (14.4%) contigs and 5868 (84.53%) singletons. The functional annotation shows that the majority of the ESTs are associated with binding (44%), catalytic (40%), transporter (5%), and structural molecular (5%) activities. The blastx results show that 73% of unigenes are significantly similar to known plant genes and 27% are novel. The latter could be of particular interest in date palm genetic studies. Further analysis shows that some ESTs are categorized as stress/defense- and fruit development-related genes. These newly generated ESTs could significantly enhance date palm EST databases in the public domain and are available to scientists and researchers across the globe. This knowledge will facilitate the discovery of candidate genes that govern important developmental and agronomical traits in date palm. It will provide important resources for developing genetic tools, comparative genomics, and genome evolution among date palm cultivars.

Screening and identification of genes associated with flower senescence in tree peony (Paeonia x suffruticosa Andrews) using suppression subtractive hybridization

ABSTRACTA short blossom period is a major obstacle for the tree peony industry. In order to preferentially isolate genes associated with flower senescence in the tree peony variety ‘Lu He Hong’, forward and reverse suppression subtractive hybridization cDNA libraries were constructed using senescent and blooming petals as testers, respectively. A total of 712 high-quality expressed sequence tags, representing 211 unigenes, were obtained, and 181 unigenes were annotated on the basis of sequence similarity with data in the National Center for Biotechnology Information’s non-redundant database. In addition, 168 unigenes were functionally annotated using Kyoto Encyclopedia of Genes and Genomes databases, and 44 unigenes were classified into 14 functional categories using the Clusters of Orthologous Groups. Most of the unigenes appear to be involved in cellular components, biological processes, and molecular functions, according to Gene Ontology analysis. Analysis of 23 chosen genes using quantitative reverse transcription PCR confirmed that their transcription levels were consistent with the results of the suppression subtractive hybridization libraries.

Hypoxic stress -responsive genes in air breathing catfish, Clarias magur (Hamilton 1822) and their possible physiological adaptive function

The Indian catfish, Clarias magur (previous name C. batrachus) is an air breathing fish, inhabitant of aquatic bodies characterized by low dissolved oxygen levels. It is exposed to hypoxic conditions in its natural habitat. Thus, it can be useful model to study the mechanism of hypoxia stress tolerance. In C. magur, molecular processes facilitating its adaptation to hypoxia stress remain largely unexplored, in part due to unavailability of genomic resources. The suppression subtractive hybridization technique (SSH) was employed to compare the differential expression of transcripts under experimental hypoxic conditions, to that of normoxic conditions. Twelve subtracted cDNA libraries (six each forward and reverse) were constructed from brain, heart, liver, muscle, spleen and head kidney tissues. A total of 2020 clones were screened and sequenced, resulting into 1805 high quality expressed sequence tags (ESTs). Annotation of these differentially expressed ESTs resulted into the identification of genes involved in vast majority of pathways/processes affecting metabolism, cellular processes, signal transduction and/or immune functions. Additionally, 18 potential novel genes expressed in hypoxia stress exposed fish were also identified. The study had catalogued the differentially expressed genes from hypoxia stress induced C. magur, where most of them are reported for the first time in a hypoxia-tolerant fish species. The results not only provided insights for the hypoxia stress altered cellular functions in C. magur, but also generated a valuable functional genomics resource to assist targeted studies on functional genomics and future genome projects.

Identification of Multiple Stress Responsive Genes by Sequencing a Normalized cDNA Library from Sea-Land Cotton (Gossypium barbadense L.)

BackgroundPlants often face multiple stresses including drought, extreme temperature, salinity, nutrition deficiency and biotic stresses during growth and development. All the stresses result in a series of physiological and metabolic reactions and then generate reversible inhibition of metabolism and growth and can cause seriously irreversible damage, even death. At each stage of cotton growth, environmental stress conditions pose devastating threats to plant growth and development, especially yield and quality. Due to the complex stress conditions and unclear molecular mechanisms of stress response, there is an urgent need to explore the mechanisms of cotton response against abiotic stresses.Methodology and Principal FindingsA normalized cDNA library was constructed using Gossypium barbadense Hai-7124 treated with different stress conditions (heat, cold, salt, drought, potassium and phosphorus deficit and Verticillium dahliae infection). Random sequencing of this library generated 6,047 high-quality expressed sequence tags (ESTs). The ESTs were clustered and assembled into 3,135 uniESTs, composed of 2,497 contigs and 638 singletons. The blastx results demonstrated 2,746 unigenes showing significant similarity to known genes, 74 uniESTs displaying significant similarity to genes of predicted proteins, and 315 uniESTs remain uncharacterized. Functional classification unveiled the abundance of uniESTs in binding, catalytic activity, and structural molecule activity. Annotations of the uniESTs by the plant transcription factor database (PlantTFDB) and Plant Stress Protein Database (PSPDB) disclosed that transcription factors and stress-related genes were enriched in the current library. The expression of some transcription factors and specific stress-related genes were verified by RT-PCR under various stress conditions.Conclusions/SignificanceAnnotation results showed that a huge number of genes respond to stress in our study, such as MYB-related, C2H2, FAR1, bHLH, bZIP, MADS, and mTERF. These results will improve our knowledge of stress tolerance in cotton. In addition, they are also helpful in discovering candidate genes related to stress tolerance. The publicly available ESTs from G. barbadense are a valuable genomic resource that will facilitate further molecular study and breeding of stress-tolerant cotton.

Gene expression profiling of the venom gland from the Venezuelan mapanare (Bothrops colombiensis) using expressed sequence tags (ESTs).

BackgroundBothrops colombiensis is a highly dangerous pit viper and responsible for over 70 % of snakebites in Venezuela. Although the composition in B. colombiensis venom has been identified using a proteome analysis, the venom gland transcriptome is currently lacking.ResultsWe constructed a cDNA library from the venom gland of B. colombiensis, and a set of 729 high quality expressed sequence tags (ESTs) was identified. A total number of 344 ESTs (47.2 % of total ESTs) was related to toxins. The most abundant toxin transcripts were metalloproteinases (37.5 %), phospholipases A2s (PLA2, 29.7 %), and serine proteinases (11.9 %). Minor toxin transcripts were linked to waprins (5.5 %), C-type lectins (4.1 %), ATPases (2.9 %), cysteine-rich secretory proteins (CRISP, 2.3 %), snake venom vascular endothelium growth factors (svVEGF, 2.3 %), L-amino acid oxidases (2 %), and other putative toxins (1.7 %). While 160 ESTs (22 % of total ESTs) coded for translation proteins, regulatory proteins, ribosomal proteins, elongation factors, release factors, metabolic proteins, and immune response proteins. Other proteins detected in the transcriptome (87 ESTs, 11.9 % of total ESTs) were undescribed proteins with unknown functions. The remaining 138 (18.9 %) cDNAs had no match with known GenBank accessions.ConclusionThis study represents the analysis of transcript expressions and provides a physical resource of unique genes for further study of gene function and the development of novel molecules for medical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-016-0059-7) contains supplementary material, which is available to authorized users.

Identification of Conserved Orthologous Set Markers in Cultivated Vigna radiata (L.) Wilczek

Identification of Conserved Orthologous Set Markers in Cultivated Vigna radiata (L.) Wilczek With an aim to develop widely applicable conserved gene markers to identify and tag orthologous genes from related Vigna species to reveal phylogenetic relationships and the nature of genes conserved in genus Vigna across the evolution, low copy nuclear Conserved Ortholog Set (COS) genes were tested to explore the interspecific genetic relationship of Vigna radiata (L.) Wilczek with other species such as V. mungo (L.), V. umbellata Thunb., V. angularis Wild and V. unguiculata (L.) Walp. To detect COS regions, computational approach was followed utilizing the available expressed sequence tags (EST) database of Vigna radiata and its related species. The ESTs were processed to eliminate sequence repeats, contaminants and low-complexity sequences. Upon alignment with Soybean genome, only high quality ESTs were grouped into clusters from which consensus sequences representing putative genes are generated for each Vigna species. From 6443 ESTs, 2550 contigs were acquired to develop 230 primer pairs to amplify conserved orthologous sequences across Vigna species. Among 14 primer pairs used for validation with genotypes of V. radiata, 3 gave double bands (paralogs) and 9 produced single bands indicating single copy orthologs. This infers that DNA sequences identified by this comparative genomics approach would be of great use in analyzing genomic information of related, unexplored crop genomes to augment gene discovery and plant breeding in other legumes.

Identification and expression of proteolysis-response genes forMacrophthalmus japonicusexposure to irgarol toxicity

Macrophthalmus japonicus is one of the most abundant macrobenthic animals in estuarine zone, which are transition areas between freshwater and marine environments. The crabs are potential bio-indicators reflecting aquatic sediment toxicity. So far, limited genetic research has focused on this group of animals largely due to the limited genomic information. In this study, we performed de novo transcriptome sequencing to produce the most comprehensive expressed sequence tag resource for M. japonicus , and identified stress response genes in M. japonicus exposed to antifouling biocides irgarol. Using 454 pyrosequencing, a total of 887 690 reads were obtained, which were assembled into 24 217 high-quality expressed sequence tags. These contigs were then clustered into 17 289 isotigs and further grouped into 12 923 isogroups. About 48% of the isogroups showed significant matches to known proteins based on sequence similarity. Moreover, irgarol toxicity induced up-regulation of stress response genes, associated with proteolysis and oxidation–reduction in biological process by Gene ontology analyses. Our data provide the most comprehensive transcriptome resource available for M. japonicus . This resource allowed us to identify genes associated with proteolysis processes, which facilitated the quantitative analysis of differential gene expression respond to irgarol toxicity. These data would provide foundation for future genetic and genomic studies of crustacean species and monitoring to freshwater as well as marine ecosystems.

Gene discovery in the developing xylem tissue of a tropical timber tree species: Neolamarckia cadamba (Roxb.) Bosser (kelampayan)

A complementary DNA (cDNA) library was con- structed from the developing xylem tissues of Neolamarckia cadamba. A total of 10,368 single-pass sequences was gener- ated through high-throughput 5'-expressed sequence tag (EST) sequencing of the cDNA clones, and 6622 high- quality ESTs were obtained after removing the low-quality sequences; this gave approximately 3.17 Mb of data. Clustering of the high-quality ESTs revealed 4728 unigenes, consisting of 2100 consensus and 2628 singletons. A total of 2405 ESTs were successfully annotated with 7753 gene on- tology (GO) terms that distributed among three main GO cat- egories, which were biological processes (2333), molecular function (3056) and cellular component (2364). Simple se- quence repeat (SSR) mining revealed that the frequency of SSR in the N. cadamba EST database (NcbdEST) was 3.3 %, with the GCT/AGC motif being the most abundant repeat motif. The most abundant transcript with known func- tion found in this database was 60S ribosomal protein follow- ed by 40S ribosomal protein. Some of the important genes involved in xylogenesis and lignin biosynthesis were found in NcdbEST; these include tubulin genes, cellulose synthase (CesA), xyloglucan endotransglycosylase (XET), arabinogalactan, cinnamate 4-hydroxylase (C4H), caffeoyl- coenzyme A O-methyltransferase (CCoAOMT) and peroxi- dase. The data obtained from this study will provide a power- ful means for identifying mechanisms controlling wood for- mationpathwaysofkelampayanandsupplymanynew cloned genes for future endeavours to modify wood and fibre properties.

Molecular Cloning and Differential Expression of Cytosolic Class I Small Hsp Gene Family in Pennisetum glaucum (L.).

Small heat shock protein (Hsp) family genes have been reported in several plant species that function as molecular chaperones to protect proteins from being denatured in extreme conditions. As a first step towards the isolation and characterization of genes that contribute to combating abiotic stresses particularly heat stress, construction and screening of the subtracted complementary DNA (cDNA) library is reported here. In this study, a subtractive heat stress cDNA library was constructed that was used to isolate members of small Hsps (sHsps) using PgsHsp17.9A gene as a probe. As a result, a total of 150 cDNA clones were isolated from the subtracted cDNA library screening, leading to 121 high-quality expressed sequence tags (ESTs), with an average size of 450 bp, comprising of 15 contigs, and majority of these isolated sHsp genes belong to cytosolic class I (CI) family. In silico sequence analysis of CI-sHsp family genes revealed that the length of sHsp proteins varied from 151 to 159 amino acids and showed large variation in isoelectric point value (5.03 to 10.05) and a narrow range of molecular weight (16.09 to 17.94 kDa). The real-time PCR results demonstrated that CI-sHsp genes are differentially expressed in Pennisetum leaves under different abiotic stress conditions particularly at high temperature. The results presented in this study provide basic information on PgCI-sHsp family genes and form the foundation for future functional studies of these genes.

Analysis of expressed sequence tags (ESTs) from a normalized cDNA library and isolation of EST simple sequence repeats from the invasive cotton mealybug Phenacoccus solenopsis.

The cotton mealybug, Phenacoccus solenopsis Tinsley, is a serious and invasive pest. At present, genetic resources for studying P. solenopsis are limited, and this negatively affects genetic research on the organism and, consequently, translational work to improve management of this pest. In the present study, expressed sequence tags (ESTs) were analyzed from a normalized complementary DNA library of P. solenopsis. In addition, EST-derived microsatellite loci (also known as simple sequence repeats or SSRs) were isolated and characterized. A total of 1107 high-quality ESTs were acquired from the library. Clustering and assembly analysis resulted in 785 unigenes, which were classified functionally into 23 categories according to the Gene Ontology database. Seven EST-based SSR markers were developed in this study and are expected to be useful in characterizing how this invasive species was introduced, as well as providing insights into its genetic microevolution.

An expressed sequence tags analysis for leaves of Chinese milk vetch (Astragalus sinicus)

Chinese milk vetch (Astragalus sinicus) is one of the most important green manure crops in temperate zone countries. To identify the genes involved in plant growth, a cDNA library was constructed from the mature leaves of Chinese milk vetch, and expressed sequence tag analysis was performed. Sequencing of the cDNA clones generated 1,027 high-quality expressed sequence tags clustered total 693 unigenes which included 110 Contigs and 583 Singlets. A total of 198 unigenes were most similar to Medicago truncatula genes. Genes related to photosynthesis were highly expressed in our library. The most abundant gene which had 42 clones was annotated as Ribulose bisphosphate carboxylase small chain with the function of carbon utilization by fixation of carbon dioxide. The results suggested that this high-quality library could be a good resource for understanding molecular events of Chinese milk vetch leaf growth, and the full-length clones could be used for crop improvement studies in the future.

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.

MOROKOSHI: transcriptome database in Sorghum bicolor.

In transcriptome analysis, accurate annotation of each transcriptional unit and its expression profile is essential. A full-length cDNA (FL-cDNA) collection facilitates the refinement of transcriptional annotation, and accurate transcription start sites help to unravel transcriptional regulation. We constructed a normalized FL-cDNA library from eight growth stages of aerial tissues in Sorghum bicolor and isolated 37,607 clones. These clones were Sanger sequenced from the 5′ and/or 3′ ends and in total 38,981 high-quality expressed sequence tags (ESTs) were obtained. About one-third of the transcripts of known genes were captured as FL-cDNA clone resources. In addition to these, we also annotated 272 novel genes, 323 antisense transcripts and 1,672 candidate isoforms. These clones are available from the RIKEN Bioresource Center. After obtaining accurate annotation of transcriptional units, we performed expression profile analysis. We carried out spikelet-, seed- and stem-specific RNA sequencing (RNA-Seq) analysis and confirmed the expression of 70.6% of the newly identified genes. We also downloaded 23 sorghum RNA-Seq samples that are publicly available and these are shown on a genome browser together with our original FL-cDNA and RNA-Seq data. Using our original and publicly available data, we made an expression profile of each gene and identified the top 20 genes with the most similar expression. In addition, we visualized their relationships in gene co-expression networks. Users can access and compare various transcriptome data from S, bicolor at http://sorghum.riken.jp.