EST Dataset Research Articles

De Novo Transcriptome Profiling for the Generation and Validation of Microsatellite Markers, Transcription Factors, and Database Development for Andrographis paniculata.

Andrographis paniculata belongs to the family Acanthaceae and is known for its medicinal properties owing to the presence of unique constituents belonging to the lactones, diterpenoids, diterpene glycosides, flavonoids, and flavonoid glycosides groups of chemicals. Andrographolide, a major therapeutic constituent of A. paniculata, is extracted primarily from the leaves of this plant and exhibits antimicrobial and anti-inflammatory activities. Using 454 GS-FLX pyrosequencing, we have generated a whole transcriptome profile of entire leaves of A. paniculata. A total of 22,402 high-quality transcripts were generated, with an average transcript length and N50 of 884 bp and 1007 bp, respectively. Functional annotation revealed that 19,264 (86%) of the total transcripts showed significant similarity with the NCBI-Nr database and were successfully annotated. Out of the 19,264 BLAST hits, 17,623 transcripts were assigned GO terms and distributed into three major functional categories: molecular function (44.62%), biological processes (29.19%), and cellular component (26.18%) based on BLAST2GO. Transcription factor analysis showed 6669 transcripts, belonging to 57 different transcription factor families. Fifteen TF genes that belong to the NAC, MYB, and bHLH TF categories were validated by RT PCR amplification. In silico analysis of gene families involved in the synthesis of biochemical compounds having medicinal values, such as cytochrome p450, protein kinases, heat shock proteins, and transporters, was completed and a total of 102 different transcripts encoding enzymes involved in the biosynthesis of terpenoids were predicted. Out of these, 33 transcripts belonged to terpenoid backbone biosynthesis. This study also identified 4254 EST-SSRs from 3661 transcripts, representing 16.34% of the total transcripts. Fifty-three novel EST-SSR markers generated from our EST dataset were used to assess the genetic diversity among eighteen A. paniculata accessions. The genetic diversity analysis revealed two distinct sub-clusters and all accessions based on the genetic similarity index were distinct from each other. A database based on EST transcripts, EST-SSR markers, and transcription factors has been developed using data generated from the present study combined with available transcriptomic resources from a public database using Meta transcriptome analysis to make genomic resources available in one place to the researchers working on this medicinal plant.

Identification of Long Non-coding RNA Transcripts in Glycyrrhiza uralensis.

Background: Chinese liquorice (Glycyrrhiza uralensis), an important medicinal plant, contains various valuable secondary metabolites. Secondary metabolites biosynthesis is very tightly regulated; therefore, elucidation and manipulation of the biosynthetic pathways are of great interest. Recent studies have shown that lncRNAs play important regulatory roles in many biological processes, thus identification and modification of their expression is essential to metabolic pathways for biosynthesis of secondary metabolites.Objectives: In this study we attempted to identify non-coding RNA transcripts (lncRNAs) that may act as important regulators of diverse biological processes, including stress responses and developmental programs in Glycyrrhiza uralensis.Materials and Methods: Identification of potential lncRNAs in Chinese liquorice was performed using a bioinformatics pipeline from the available EST dataset of G. uralensis.Results: Bioinformatics analysis revealed that 1365 identical sequences in the range of 200 to 1286 base pair are putative lncRNAs. Only less than one percent of the predicted lncRNAs display sequence conservation with lncRNAs from other species. Moreover, 13 lncRNAs were detected as the potential precursors of 16 miRNAs. From this analysis, we also detected possible target genes of 16 known miRNA genes. The majority of the predicted miRNA target genes have important role in response to plant disease and a couple of them contribute to signalling and metabolic pathways.Conclusion: This study demonstrates the existence of lncRNAs in G. uralensis which has not been found before and provides valuable resources for further understanding and characterizing of lncRNAs and also a basis for additional investigation to reveal specific roles of lncRNAs in various biological processes and particularly in response to plant diseases.

Exploring the MiRnome of Carica papaya: A cross kingdom approach

Carica papaya is a native fruit with high nutraceutical value that also has potential therapeutic properties. However, the human diet plays an important role in the prevention of human metabolic disorders. Enormous studies have reported that consumption of plant-derived miRNA could modulate human genes and bring about evidence of plant-animal regulation. The present study aims to understand the role of C. papaya miRNA in a cross-kingdom manner using a computational approach. Two unique miRNA families were predicted from publicly available 77,393 EST dataset of C. papaya. The Plant small RNA (psRNA) target server predicted 38 target genes in human that play a significant role in functional enrichment, network analysis and regulating the initial modulators of EGFR (Epidermal Growth Factor) signaling cascade. This potential miRNA revealed their association with different types of cancer such as breast cancer, colorectal cancer, lung cancer involving in cell migration and proliferation. Important top hub protein namely LCP2, CDK6, RUNX1and KCNMA1 targets of cpa-miR6034 and cpa-miR3629b show their interaction with cancer pathways and in few platelet regulation pathways. Hereby we suggest that the intake of C. papaya miRNA may regulate the different types of cancer with cross-kingdom approach and implicit role in the prevention and control of human diseases.

MiRDetect: A combinatorial approach for automated detection of novel miRNA precursors from plant EST data using homology and Random Forest classification

Identification of microRNAs from plants is a crucial step for understanding the mechanisms of pathways and regulation of genes. A number of tools have been developed for the detection of microRNAs from small RNA-seq data. However, there is a lack of pipeline for detection of miRNA from EST dataset even when a huge resource is publicly available and the method is known. Here we present miRDetect, a python implementation to detect novel miRNA precursors from plant EST data using homology and machine learning approach. 10-fold cross validation was applied to choose best classifier based on ROC, accuracy, MCC and F1-scores using 112 features. miRDetect achieved a classification accuracy of 93.35% on a Random Forest classifier and outperformed other precursor detection tools in terms of performance. The miRDetect pipeline aids in identifying novel plant precursors using a mixed approach and will be helpful to researchers with less informatics background.

Transcripts with systematic nucleotide deletion of 1-12 nucleotide in human mitochondrion suggest potential non-canonical transcription.

Raw transcriptomic data contain numerous RNA reads whose homology with template DNA doesn’t match canonical transcription. Transcriptome analyses usually ignore such noncanonical RNA reads. Here, analyses search for noncanonical mitochondrial RNAs systematically deleting 1 to 12 nucleotides after each transcribed nucleotide triplet, producing deletion-RNAs (delRNAs). We detected delRNAs in the human whole cell and purified mitochondrial transcriptomes, and in Genbank's human EST database corresponding to systematic deletions of 1 to 12 nucleotides after each transcribed trinucleotide. DelRNAs detected in both transcriptomes mapped along with 55.63% of the EST delRNAs. A bias exists for delRNAs covering identical mitogenomic regions in both transcriptomic and EST datasets. Among 227 delRNAs detected in these 3 datasets, 81.1% and 8.4% of delRNAs were mapped on mitochondrial coding and hypervariable region 2 of dloop. Del-transcription analyses of GenBank's EST database confirm observations from whole cell and purified mitochondrial transcriptomes, eliminating the possibility that detected delRNAs are false positives matches, cytosolic DNA/RNA nuclear contamination or sequencing artefacts. These detected delRNAs are enriched in frameshift-inducing homopolymers and are poor in frameshift-preventing circular code codons (a set of 20 codons which regulate reading frame detection, over- and underrepresented in coding and other frames of genes, respectively) suggesting a motif-based regulation of non-canonical transcription. These findings show that rare non-canonical transcripts exist. Such non canonical del-transcription does increases mitochondrial coding potential and non-coding regulation of intracellular mechanisms, and could explain the dark DNA conundrum.

In silico Mining of EST-SSRs from the Drought Tolerant ESTs in Safflower

Drought stress is a major abiotic factor causing yield loss in safflower and limited studies have been carried out to dissect the molecular mechanism at EST-SSR level. In this study, a possible relationship between simple sequence repeats (SSRs) distribution and drought stress was studied using three EST libraries of safflower, Ct-D-EST, Ct-NEST and Co-EST. The Ct-D-EST was generated from drought tolerant safflower cultivar A-1; Ct-N-EST and Co-EST were the EST datasets of safflower and its wild progenitor C. oxycanthus, respectively. In total, 156 (45%), 1194 (5%) and 1550 (9%) EST-SSRs were mined from Ct-D-EST, Ct-N-EST and Co-EST libraries, respectively. Comparison of EST-SSRs from Ct-D-EST with that of SSRs from other two libraries showed reasonable differences for each class of repeats. Large variations were observed for dinucleotide repeats in all the libraries. In drought EST-SSRs, only one kind of amino acid was produced by repeat (ATG)14 which encodes met indicating the loci and repeat observed to be 100%. Since three ESTs with SSRs from Ct-D-EST, annotated to putative candidate genes, S-adenosylmethionine synthetase, one-helix protein and myo-inositol 1-phosphate synthetase, did not express with SSRs in the Ct-N-EST, these can be considered as potential candidate genes for drought tolerance. A trinuceotinde SSR, encoding met, was also found in the EST annotated to putative s-adenosylmethionine synthetase, hence, both can be considered as indices for drought tolerance. The genomic resources and the information generated in this study may be useful for the safflower breeders particularly for the development of drought tolerant cultivar.

Construction of a full-length cDNA library from Typha laxmanni Lepech. and T. angustifolia L. from an EST dataset

Construction of a full-length cDNA library from Typha laxmanni Lepech. and T. angustifolia L. from an EST dataset

15 years of GDR: New data and functionality in the Genome Database for Rosaceae.

The Genome Database for Rosaceae (GDR, https://www.rosaceae.org) is an integrated web-based community database resource providing access to publicly available genomics, genetics and breeding data and data-mining tools to facilitate basic, translational and applied research in Rosaceae. The volume of data in GDR has increased greatly over the last 5 years. The GDR now houses multiple versions of whole genome assembly and annotation data from 14 species, made available by recent advances in sequencing technology. Annotated and searchable reference transcriptomes, RefTrans, combining peer-reviewed published RNA-Seq as well as EST datasets, are newly available for major crop species. Significantly more quantitative trait loci, genetic maps and markers are available in MapViewer, a new visualization tool that better integrates with other pages in GDR. Pathways can be accessed through the new GDR Cyc Pathways databases, and synteny among the newest genome assemblies from eight species can be viewed through the new synteny browser, SynView. Collated single-nucleotide polymorphism diversity data and phenotypic data from publicly available breeding datasets are integrated with other relevant data. Also, the new Breeding Information Management System allows breeders to upload, manage and analyze their private breeding data within the secure GDR server with an option to release data publicly.

Development of SSR Markers and Assessment of Genetic Diversity in Medicinal Chrysanthemum morifolium Cultivars.

Chrysanthemum morifolium, is a well-known flowering plant worldwide, and has a high commercial, floricultural, and medicinal value. In this study, simple-sequence repeat (SSR) markers were generated from EST datasets and were applied to assess the genetic diversity among 32 cultivars. A total of 218 in silico SSR loci were identified from 7300 C. morifolium ESTs retrieved from GenBank. Of all SSR loci, 61.47% of them (134) were hexa-nucleotide repeats, followed by tri-nucleotide repeats (17.89%), di-nucleotide repeats (12.39%), tetra-nucleotide repeats (4.13%), and penta-nucleotide repeats (4.13%). In this study, 17 novel EST-SSR markers were verified. Along with 38 SSR markers reported previously, 55 C. morifolium SSR markers were selected for further genetic diversity analysis. PCR amplification of these EST-SSRs produced 1319 fragments, 1306 of which showed polymorphism. The average polymorphism information content of the SSR primer pairs was 0.972 (0.938–0.993), which showed high genetic diversity among C. morifolium cultivars. Based on SSR markers, 32 C. morifolium cultivars were separated into two main groups by partitioning of the clusters using the unweighted pair group method with arithmetic mean dendrogram, which was further supported by a principal coordinate analysis plot. Phylogenetic relationship among C. morifolium cultivars as revealed by SSR markers was highly consistent with the classification of medicinal C. morifolium populations according to their origin and ecological distribution. Our results demonstrated that SSR markers were highly reproducible and informative, and could be used to evaluate genetic diversity and relationships among medicinal C. morifolium cultivars.

Construction of a Full-length cDNA Library from Cardamine manshurica Nakai and Characterization of EST Dataset

Brassicaceae consists of important species that have significant amounts of metabolites, and many studies have been carried out in order to understand the mechanism that improves the content of these metabolites. In Brassicacea, Cardamine manshurica Nakai is one of the important edible plants and is rich in oil, fiber, and various nutrients. In this study, we constructed cDNA library using leaves from 4 week-old plants and analyzed the ESTs of C. manshurica Nakai. One thousand thirty-nine ESTs were discovered which assembled to form 468 unigenes. The latter contained 116 contigs and 352 singletons. Similarity search of these ESTs with BLASTX revealed similarities with Arabidopsis thaliana 285 (31.9%), Arabidopsis lyrata 172 (19.3%), Capsella rubella 162 (18.1%), and Eutrema salsugineum 137 (15.3%). ESTs were functionally categorized into molecular function, biological process, and cellular component, and each category took 10.6%, 58.5%, and 30.9%, respectively. The functional analysis also found that 94.9% of ESTs showed at least one GO ID. Microsatellite analysis of 468 unigene sequences revealed 225 structures of which Di-, Tri-, Tetra-, Penta-repeats were 35.6% (80/225), 63.1% (142/225), 0.9% (2/225), and 0.4% (1/225), respectively. The results from our study can be a valuable resource for Cardamine research.

Development of expressed sequence tag-based microsatellite markers for the critically endangered Isoëtes sinensis (Isoetaceae) based on transcriptome analysis.

Isoëtes sinensis is a critically endangered quillwort. To facilitate studies on the conservation genetics of this species, we developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. A total of 50,063 unigenes were predicted by transcriptome sequencing, 5294 (10.6%) of which significantly matched 3011 Gene Ontology annotations and 2363 were assigned to Kyoto Encyclopedia of Genes and Genomes metabolic pathways. Most of these (2297) were involved in metabolism. A total of 1982 SSR motifs were identified, with trinucleotides being the dominant repeat motif, and 1438 (72.6%) SSR primers were designed. Eighteen randomly selected primer pairs were used to genotype 24 I. sinensis accessions, which confirmed the suitability of these novel markers for molecular studies of I. sinensis. The heterozygosity index value ranged between 0.0799 and 0.9106, while the Shannon-Wiener diversity index value ranged between 0.1732 and 2.5589. The EST-SSRs reported in this study are linked to genic sequences, and are therefore ideal for investigating the evolutionary history of I. sinensis. These markers, together with the large EST dataset generated in this study, will greatly facilitate conservation genetic studies of I. sinensis.

Molecular marker development from transcript sequences and germplasm evaluation for cultivated peanut (Arachis hypogaea L.).

Molecular markers are important tools for genotyping in genetic studies and molecular breeding. The SSR and SNP are two commonly used marker systems developed from genomic or transcript sequences. The objectives of this study were to: (1) assemble and annotate the publicly available ESTs in Arachis and the in-house short reads, (2) develop and validate SSR and SNP markers, and (3) investigate the genetic diversity and population structure of the peanut breeding lines and the U.S. peanut mini core collection using developed SSR markers. An NCBI EST dataset with 252,951 sequences and an in-house 454 RNAseq dataset with 288,701 sequences were assembled separately after trimming. Transcript sequence comparison and phylogenetic analysis suggested that peanut is closer to cowpea and scarlet bean than to soybean, common bean and Medicago. From these two datasets, 6455 novel SSRs and 11,902 SNPs were identified. Of the discovered SSRs, 380 representing various SSR types were selected for PCR validation. The amplification rate was 89.2%. Twenty-two (6.5%) SSRs were polymorphic between at least one pair of four genotypes. Sanger sequencing of PCR products targeting 110 SNPs revealed 13 true SNPs between tetraploid genotypes and 193 homoeologous SNPs within genotypes. Eight out of the 22 polymorphic SSR markers were selected to evaluate the genetic diversity of Florida peanut breeding lines and the U.S. peanut mini core collection. This marker set demonstrated high discrimination power by displaying an average polymorphism information content value of 0.783, a combined probability of identity of 10(-11), and a combined power of exclusion of 0.99991. The structure analysis revealed four sub-populations among the peanut accessions and lines evaluated. The results of this study enriched the peanut genomic resources, provided over 6000 novel SSR markers and the credentials for true peanut SNP marker development, and demonstrated the power of newly developed SSR markers in genotyping peanut germplasm and breeding materials.

Computer aided gene mining for gingerol biosynthesis.

Inspite of the large body of genomic data obtained from the transcriptome of Zingiber officinale, very few studies have focused on the identification and characterization of miRNAs in gingerol biosynthesis. Zingiber officinale transcriptome was analyzed using EST dataset (38169 total) deposited in public domains. In this paper computational functional annotation of the available ESTs and identification of genes which play a significant role in gingerol biosynthesis are described. Zingiber officinale transcriptome was analyzed using EST dataset (38169 total) from ncbi. ESTs were clustered and assembled, resulting in 8624 contigs and 8821 singletons. Assembled dataset was then submitted to the EST functional annotation workflow including blast, gene ontology (go) analysis, and pathway enrichment by kyoto encyclopedia of genes and genomes (kegg) and interproscan. The unigene datasets were further exploited to identify simple sequence repeats that enable linkage mapping. A total of 409 simple sequence repeats were identified from the contigs. Furthermore we examined the existence of novel miRNAs from the ESTs in rhizome, root and leaf tissues. EST analysis revealed the presence of single hypothetical miRNA in rhizome tissue. The hypothetical miRNA is warranted to play an important role in controlling genes involved in gingerol biosynthesis and hence demands experimental validation. The assembly and associated information of transcriptome data provides a comprehensive functional and evolutionary characterization of genomics of Zingiber officinale. As an effort to make the genomic and transcriptomic data widely available to the public domain, the results were integrated into a web-based Ginger EST database which is freely accessible at http://www.kaubic.in/gingerest/.

A novel sucrose hydrolase from the bombycoid silkworms Bombyx mori, Trilocha varians, and Samia cynthia ricini with a substrate specificity for sucrose

Although membrane-associated sucrase activity has been detected in the midgut of various lepidopteran species, it has not yet been identified and characterized at the molecular level. In the present study, we identified a novel sucrose hydrolase (SUH) gene from the following three bombycoid silkworms: Bombyx mori, Trilocha varians, and Samia cynthia ricini and named them BmSuh, TvSuh, and ScSuh, respectively. The EST dataset showed that BmSuh is one of the major glycoside hydrolase genes in the larval midgut of B. mori. These genes were almost exclusively expressed in the larval midgut in all three species, mainly at the feeding stage. SUHs are classified into the glycoside hydrolase family 13 and show significant homology to insect maltases. Enzymatic assays revealed that recombinant SUHs were distinct from conventional maltases and exhibited substrate specificity for sucrose. The recombinant BmSUH was less sensitive to sugar-mimic alkaloids than TvSUH and ScSUH, which may explain the reason why the sucrase activity in the B. mori midgut was less affected by the sugar-mimic alkaloids derived from mulberry.

De novo transcriptome assembly of the wild relative of tea tree (Camellia taliensis) and comparative analysis with tea transcriptome identified putative genes associated with tea quality and stress response.

BackgroundCamellia taliensis is one of the most important wild relatives of cultivated tea tree, C. sinensis. The species extensively occupies mountainous habitats representing a wide-range abiotic tolerance and biotic resistance and thus harbors valuable gene resources that may greatly benefit genetic improvement of cultivated tea tree. However, owning to a large genome size of ~3 Gb and structurally complex genome, there are fairly limited genetic information and particularly few genomic resources publicly available for this species. To better understand the key pathways determining tea flavor and enhance tea tree breeding programs, we performed a high-throughput transcriptome sequencing for C. taliensis.ResultsIn this study, approximate 241.5 million high-quality paired-end reads, accounting for ~24 Gb of sequence data, were generated from tender shoots, young leaves, flower buds and flowers using Illumina HiSeq 2000 platform. De novo assembly with further processing and filtering yielded a set of 67,923 transcripts with an average length of 685 bp and an N50 of 995 bp. Based on sequence similarity searches against public databases, a total of 39,475 transcripts were annotated with gene descriptions, conserved protein domains or gene ontology (GO) terms. Candidate genes for major metabolic pathways involved in tea quality were identified and experimentally validated using RT-qPCR. Further gene expression profiles showed that they are differentially regulated at different developmental stages. To gain insights into the evolution of these genes, we aligned them to the previously cloned orthologous genes in C. sinensis, and found that considerable nucleotide variation within several genes involved in important secondary metabolic biosynthesis pathways, of which flavone synthase II gene (FNSII) is the most variable between these two species. Moreover, comparative analyses revealed that C. taliensis shows a remarkable expansion of LEA genes, compared to C. sinensis, which might contribute to the observed stronger stress resistance of C. taliensis.ConclusionWe reported the first large-coverage transcriptome datasets for C. taliensis using the next-generation sequencing technology. Such comprehensive EST datasets provide an unprecedented opportunity for identifying genes involved in several major metabolic pathways and will accelerate functional genomic studies and genetic improvement efforts of tea trees in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1494-4) contains supplementary material, which is available to authorized users.

Constitutive and stress-inducible overexpression of a native aquaporin gene (MusaPIP2;6) in transgenic banana plants signals its pivotal role in salt tolerance.

High soil salinity constitutes a major abiotic stress and an important limiting factor in cultivation of crop plants worldwide. Here, we report the identification and characterization of a aquaporin gene, MusaPIP2;6 which is involved in salt stress signaling in banana. MusaPIP2;6 was firstly identified based on comparative analysis of stressed and non-stressed banana tissue derived EST data sets and later overexpression in transgenic banana plants was performed to study its tangible functions in banana plants. The overexpression of MusaPIP2;6 in transgenic banana plants using constitutive or inducible promoter led to higher salt tolerance as compared to equivalent untransformed control plants. Cellular localization assay performed using transiently transformed onion peel cells indicated that MusaPIP2;6 protein tagged with green fluorescent protein was translocated to the plasma membrane. MusaPIP2;6-overexpressing banana plants displayed better photosynthetic efficiency and lower membrane damage under salt stress conditions. Our results suggest that MusaPIP2;6 is involved in salt stress signaling and tolerance in banana.

Transcriptome profiles of Penaeus (Marsupenaeus) japonicus animal and vegetal half-embryos: identification of sex determination, germ line, mesoderm, and other developmental genes.

There is virtually no knowledge of the molecular events controlling early embryogenesis in Penaeid shrimp. A combination of controlled spawning environment, shrimp embryo micro-dissection techniques, and next-generation sequencing was used to produce transcriptome EST datasets of Penaeus japonicus animal and vegetal half-embryos. Embryos were collected immediately after spawning, and then blastomeres were separated at the two-cell stage and allowed to develop to late gastrulation, then pooled for RNA isolation and cDNA synthesis. Ion Torrent sequencing of cDNA from approximately 500 pooled animal and vegetal half-embryos from multiple spawnings resulted in 560,516 and 493,703 reads, respectively. Reads from each library were assembled and Gene Ontogeny analysis produced 3479 annotated animal contigs and 4173 annotated vegetal contigs, with 159/139 hits for developmental processes in the animal/vegetal contigs, respectively. Contigs were subject to BLAST for selected developmental toolbox genes. Some of the genes found included the sex determination genes sex-lethal and transformer; the germ line genes argonaute 1, boule, germ cell-less, gustavus, maelstrom, mex-3, par-1, pumilio, SmB, staufen, and tudor; the mesoderm genes brachyury, mef2, snail, and twist; the axis determination/segmentation genes β-catenin, deformed, distal-less, engrailed, giant, hairy, hunchback, kruppel, orthodenticle, patched, tailless, and wingless/wnt-8c; and a number of cell-cycle regulators. Animal and vegetal contigs were computationally subtracted from each other to produce sets unique to either half-embryo library. Genes expressed only in the animal half included bmp1, kruppel, maelstrom, and orthodenticle. Genes expressed only in the vegetal half included boule, brachyury, deformed, dorsal, engrailed, hunchback, spalt, twist, and wingless/wnt-8c.

Transcriptome analysis of the grass carp (Ctenopharyngodon idella) using 454 pyrosequencing methodology for gene and marker discovery.

Total RNA isolated from the brain, muscle, liver, gonad, and intestinal tissues of grass carp was pooled to construct cDNA libraries. Using 454 pyrosequencing, a total of 738,604 high-quality reads were generated from the normalized cDNAs of the pooled individuals. Clustering and assembly of these reads produced a set of 37,086 all-unigene sequences after BLAST. Of these, 24,010 (64.74%) were annotated in the National Center for Biotechnology Information database, and 3715 simple sequence repeats and 2008 single nucleotide polymorphisms were identified in this EST dataset as potential molecular markers. This study provides new data for functional genomic and biological research on grass carp. The markers identified in this study will enrich the currently used molecular markers and facilitate marker-assisted selection in grass carp-breeding programs. These results also demonstrate that transcriptomic analysis based on 454 sequencing is a powerful tool for gene discovery and molecular marker development in non-model species.

Deep sequencing of the Camellia chekiangoleosa transcriptome revealed candidate genes for anthocyanin biosynthesis

Camellia chekiangoleosa is an important species of genus Camellia. It provides high-quality edible oil and has great ornamental value. The flowers are big and red which bloom between February and March. Flower pigmentation is closely related to the accumulation of anthocyanin. Although anthocyanin biosynthesis has been studied extensively in herbaceous plants, little molecular information on the anthocyanin biosynthesis pathway of C. chekiangoleosa is yet known. In the present study, a cDNA library was constructed to obtain detailed and general data from the flowers of C. chekiangoleosa. To explore the transcriptome of C. chekiangoleosa and investigate genes involved in anthocyanin biosynthesis, a 454 GS FLX Titanium platform was used to generate an EST dataset. About 46,279 sequences were obtained, and 24,593 (53.1%) were annotated. Using Blast search against the AGRIS, 1740 unigenes were found homologous to 599 Arabidopsis transcription factor genes. Based on the transcriptome dataset, nine anthocyanin biosynthesis pathway genes (PAL, CHS1, CHS2, CHS3, CHI, F3H, DFR, ANS, and UFGT) were identified and cloned. The spatio-temporal expression patterns of these genes were also analyzed using quantitative real-time polymerase chain reaction. The study results not only enrich the gene resource but also provide valuable information for further studies concerning anthocyanin biosynthesis.

Development of microsatellite markers in sesame (Sesamum indicum L.)

Sesame (Sesamum indicum L.) is an important oilseed crop with high-quality seed oil and many antioxidant properties. Owing to its commercial and medicinal values, there is a renewed interest among agricultural scientists in this ancient crop. Efforts to strengthen the sesame-specific marker base have been initiated in the recent past; however, the available number of microsatellite markers is still not sufficient for the development of high-resolution genetic linkage maps for important agronomic traits and there is a need to increase the number of informative DNA markers in sesame. In the present study, we developed 25 microsatellite markers by employing the selective hybridization strategy and 95 mining expressed sequence tags of the NCBI database. This new set of microsatellite markers was characterized and screened for genetic diversity in an array of 16 sesame germplasms. Of the 120 SSRs, 92 were polymorphic, consisting of 18 SSRs from selective hybridization and 74 from the EST data set. The number of alleles per microsatellite locus ranged from 2 to 5, with an average of 3.11 alleles. The allele size ranged widely (100-510 bp) among the primer pairs. Polymorphic information content estimates ranged from 0.2982 to 0.912. Jaccard's similarity coefficient ranged from 0.21 to 0.82. The potential of the markers was assessed by diversity analysis using the sequential hierarchical agglomerative nonoverlapping clustering technique of the unweighted pair group method of arithmetic means on a set of 16 genotypes of sesame, including 2 wild species. Results supported the hypothesis that S. malabaricum could be the immediate progenitor of the cultivar species and that S. mulayanum is distinct from S. malabaricum and S. indicum, while suggesting hardly any diversity among the cultivars.