Non-redundant Research Articles

De novo assembly and analysis of the Heortia vitessoides transcriptome via high-throughput Illumina sequencing

Heortia vitessoides Moore, a defoliating pest in Aquilaria sinensis (Loureiro) Sprenger forests, is responsible for significant damage to the trees. There is a lack of nucleotide and protein sequence information for H. vitessoides. Hence, in this study, the transcriptome of H. vitessoides was sequenced using the Illumina Hiseq 2000 platform to evaluate the expression profiles and predict the functional genes. A total of 60,587,900 clean reads were produced and de novo assembled into 66,673 unigenes of average length 957bp; 28,202 and 27,339 identified unigenes were annotated using the National Center for Biotechnology Information (NCBI) non-redundant (NR) database and Swiss-Prot database, respectively. All assembled unigenes were categorized into 64 biological processes, cellular components, or molecular functions using Gene Ontology (GO). In total, 24,278 unigenes were annotated using a database of eukaryotic orthologous groups (KOG) and assigned to 25 functional categories. Of these, 9172 unigenes had matches in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and were assigned to 344 KEGG pathways. In addition, there were 11,670 simple sequence repeats (SSRs) in 66,673 unigenes and 64 repeated motif types, of which A/T was the most frequent. We also found that 1000 unigenes were related to insecticide resistance. To the best of our knowledge, this is the first comprehensive transcriptome analysis for H. vitessoides, providing valuable genome data sources for the study of the molecular biology of this pest.

Differential gene expression in banana roots in response to Fusarium wilt

Banana wilt disease, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a devastating disease in the banana industry. The molecular mechanism underlying the wilt resistance of AAA-type banana cultivars has not been fully characterized. Our objective was to analyse the genes differentially expressed in roots during the time of flower bud differentiation in plants grown in fields infested with Foc TR4. We compared the Foc TR4-tolerant ‘Formosana’ banana and the susceptible ‘Brazil’ banana. cDNA libraries were constructed from RNA isolated from the roots of both cultivars, and generated approximately 40 million and 35 million high-quality reads, respectively. We mapped 28 353 810 ‘Formosana’ reads and 26 917 421 ‘Brazil’ reads to the banana genome. A search of the NR (non-redundant) database resulted in the annotation of 34 408 genes. Additionally, 107 genes were differentially expressed between the two cultivars. Among the 48 differentially expressed genes with known functions, 41 were expressed more highly in ‘Formosana’ than in ‘Brazil’. These genes were divided into several categories, including specific resistance mechanism-related enzyme genes and phytoalexin synthesis-related genes. The expression levels of eight genes were validated by a quantitative real-time polymerase chain reaction. Our results indicate that ‘Formosana’ may enhance its tolerance to Foc TR4 by increasing the expression of defence-related genes (possibly constitutively). This cultivar also exhibits up-regulated expression of non-specific stress-related genes, which may enhance its overall disease tolerance. This study provides important transcript-level details potentially useful for clarifying the molecular mechanism underlying the disease resistance of Cavendish banana cultivars.

Ovarian transcriptomic analysis of Shan Ma ducks at peak and late stages of egg production.

ObjectiveTo assess the differences in ovarian transcriptomes in Shan Ma ducks between their peak and late stages of egg production, and to obtain new transcriptomic data of these egg-producing ducks.MethodsThe Illumina HiSeq 2000 system was used for high throughput sequencing of ovarian transcriptomes from Shan Ma ducks at their peak or late stages of egg production.ResultsGreater than 93% of the sequencing data had a base quality score (Q score) that was not less than 20 (Q20). From ducks at their peak stage of egg production, 42,782,676 reads were obtained, with 4,307,499,083 bp sequenced. From ducks at their late stage of egg production, 45,316,166 reads were obtained, with 4,562,063,363 bp sequenced. A comparison of the two datasets identified 2,002 differentially expressed genes, with 790 upregulated and 1,212 downregulated. Further analysis showed that 1,645 of the 2,002 differentially expressed genes were annotated in the non-redundant (NR) database, with 646 upregulated and 999 downregulated. Among the differentially expressed genes with annotations in the NR database, 696 genes were functionally annotated in the clusters of orthologous groups of proteins database, involving 25 functional categories. One thousand two hundred four of the differentially expressed genes with annotations in the NR database were functionally annotated in the gene ontology (GO) database, and could be divided into three domains and 56 categories. The three domains were cellular component, molecular function, and biological process. Among the genes identified in the GO database, 451 are involved in development and reproduction. Analysis of the differentially expressed genes with annotations in the NR database against the Kyoto encyclopedia of genes and genomes database revealed that 446 of the genes could be assigned to 175 metabolic pathways, of which the peroxisome proliferator-activated receptor signaling pathway, insulin signaling pathway, fructose and mannose metabolic pathways, gonadotropin releasing hormone signaling pathway and transforming growth factor beta signaling pathway were significantly enriched.ConclusionThe differences in ovarian transcriptomes in Shan Ma ducks between their peak and late stages of egg production were elucidated, which greatly enriched the ovarian transcriptomic information of egg-producing ducks.

Discovery and characterization of miRNAs in mouse thymus responses to ionizing radiation by deep sequencing

To investigate the potential regulatory roles of microRNA (miRNA) in mouse response to ionizing radiation (IR)-induced thymus injury, miRNA expression profiles of mouse thymus with or without IR were analyzed using deep sequencing technology. Potential target candidates of the identified miRNA were predicted using RNAhybrid and miRanda. Differently expressed miRNA targets functional annotation and pathways were noted using Swiss-Prot, Gene Ontology (GO), Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and non-redundant (NR) databases. In this study, there were 112 differently expressed miRNAs identified, including 45 known mature and 67 novel miRNAs, which meanwhile contained 77 up-regulated and 35 down-regulated miRNAs. The results of quantitative RT-polymerase chain reaction (qRT-PCR) verification were in agreement with the sequencing analysis. And the target genes of miRNA were annotated. These results revealed the differences of miRNA expression, further extended the biological knowledge and greatly facilitated future studies on the function of miRNA in IR-induced thymus injury.

De novo assembly of the transcriptome of Aegiceras corniculatum, a mangrove species in the Indo-West Pacific region

Aegiceras corniculatum (L.) Blanco is one of the most salt tolerant mangrove species and can thrive in 3% salinity at the seaward edge of mangrove forests. Here we sequenced the transcriptome of A. corniculatum used Illumina GA platform to develop its genomic resources for ecological and evolutionary studies. We obtained about 50 million high-quality paired-end reads with 75bp in length. Using the short read assembler Velvet, we yielded 49,437 contigs with the average length of 625bp. A total of 32,744 (66.23%) contigs showed significant similarity to the GenBank non-redundant (NR) protein database. 30,911 and 18,004 of these sequences were assigned to Gene Ontology and eukaryotic orthologous groups of proteins (KOG). A total of 4942 transcripts from our assemblies had significant similarity with KEGG Orthologs and were involved in 144 KEGG pathways, while 9899 unigenes had enzyme commission (EC) numbers. In addition, 9792 transcriptome-derived SSRs were identified from 7342 sequences. With our strict criteria, 4165 candidate SNPs were also identified from 2058 contigs. Some of these SNPs were further validated by Sanger sequencing. Genomic resources generated in this study should be valuable in ecological, evolutionary, and functional genomics studies for this mangrove species.

MiRNA expression profile of ionizing radiation‐induced liver injury in mouse using deep sequencing

In order to investigate the potential regulatory roles of microRNAs (miRNAs) in mouse response to ionizing radiation (IR), the small RNA libraries from liver tissues of mice with or without ionizing radiation (IR) were sequenced by high-throughput deep sequencing technology. A total of 270 miRNAs including 212 known and 58 potentially novel miRNAs were identified. Within these miRNAs, there were 48 miRNAs that were differentially expressed, including 27 known and 21 novel miRNAs. The results of quantitative RT-polymerase chain reaction (qRT-PCR) were in consistent with the sequencing analysis. Target gene prediction, function annotation, and pathway of the identified miRNAs were analyzed using RNAhybrid, miRanda software and Swiss-Prot, Gene Ontology (GO), Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes, and Genomes (KEGG) and non-redundant (NR) databases. These results should be useful to investigate the biological function of miRNAs under IR-induced liver injury.

De Novo Transcriptome Analysis for Kentucky Bluegrass Dwarf Mutants Induced by Space Mutation.

Kentucky bluegrass (Poa pratensis L.) is a major cool-season turfgrass requiring frequent mowing. Utilization of cultivars with slow growth is a promising method to decrease mowing frequency. In this study, two dwarf mutant selections of Kentucky bluegrass (A12 and A16) induced by space mutation were analyzed for the differentially expressed genes compared with the wild type (WT) by the high-throughput RNA-Seq technology. 253,909 unigenes were obtained by de novo assembly. 24.20% of the unigenes had a significant level of amino acid sequence identity to Brachypodium distachyon proteins, followed by Hordeum vulgare with 18.72% among the non-redundant (NR) Blastx top hits. Assembled unigenes were associated with 32 pathways using KEGG orthology terms and their respective KEGG maps. Between WT and A16 libraries, 4,203 differentially expressed genes (DEGs) were identified, whereas there were 883 DEGs between WT and A12 libraries. Further investigation revealed that the DEG pathways were mainly involved in terpenoid biosynthesis and plant hormone metabolism, which might account for the differences of plant height and leaf blade color between dwarf mutant and WT plants. Our study presents the first comprehensive transcriptomic data and gene function analysis of Poa pratensis L., providing a valuable resource for future studies in plant dwarfing breeding and comparative genome analysis for Pooideae plants.

Comparative transcriptome analysis reveals a potential photosynthate partitioning mechanism between lipid and starch biosynthetic pathways in green microalgae

Comparative transcriptome analysis provides a tool to top-view regulations of biochemical pathways in the cells and predicts the key enzymes by comparing gene expression changes in different conditions. Transcriptomes of a green microalga Neodesmus sp. UTEX 2219-4 treated under different stresses favorable or unfavorable for lipid biosynthesis were sequenced and compared to elucidate the photosynthate partitioning mechanism between fatty acid and starch biosynthesis pathways. This microalga contains 20,556 isotigs, and 8387 of these have homologs in the Non-Redundant (NR) database. The fatty acid and starch biosynthetic pathways of this microalga were reconstructed. The expression levels of the isotigs related to these pathways and involved in photosynthesis were calculated. The photosynthetic light reaction genes were down-regulated but electron carriers were up-regulated under osmotic and salt stresses, suggesting that ATP production was supported by cyclic electron transport in these conditions. In lipid producing conditions, expression of multiple isotigs in the fatty acid biosynthesis pathway was up-regulated, indicating that overexpression of acetyl-CoA carboxylase alone was not sufficient to enhance lipid biosynthesis. Results from this study suggested that triose phosphate isomerase is the key enzyme regulating photosynthate partitioning between fatty acid and starch biosyntheses in green microalgae.

De novo assembly and characterization of farmed blue fox (Alopex lagopus) global transcriptome using Illumina paired-end sequencing.

The blue fox (Alopex lagopus), a coat-color variant of the Arctic fox, is a domesticated fur-bearing mammal. In the present study, transcriptome data generated from a pool of nine different tissues were obtained with Illumina HiSeq2500 paired-end sequencing technology. After filtering from raw reads, 32,358,290 clean reads were assembled into 161,269 transcripts and 97,252 unigenes by the Trinity fragment assembly software. Of the assembled unigenes, 37,967 were annotated in the National Center for Biotechnology Information (NCBI) Non-Redundant (NR) protein database and 26,264 in the Swiss-Prot database. Among the annotated unigenes, 24,839 and 24,267 were assigned using the Gene Ontology (GO) and euKaryotic Orthologous Groups (KOG) databases, respectively. Altogether, 17,057 unigenes were mapped onto 227 pathways using the Kyoto Encyclopedia of Genes and Genomes database. In addition, 6394 simple sequence repeats were identified by examining 12,965 unigenes (>1 kb), which could contribute to the development of molecular markers. This study generated transcriptome data for the blue fox that will promote further progress in expression profiling studies, and provide a good annotation basis for genomic studies.

De Novo Assembly of the Donkey White Blood Cell Transcriptome and a Comparative Analysis of Phenotype-Associated Genes between Donkeys and Horses

Prior to the mechanization of agriculture and labor-intensive tasks, humans used donkeys (Equus africanus asinus) for farm work and packing. However, as mechanization increased, donkeys have been increasingly raised for meat, milk, and fur in China. To maintain the development of the donkey industry, breeding programs should focus on traits related to these new uses. Compared to conventional marker-assisted breeding plans, genome- and transcriptome-based selection methods are more efficient and effective. To analyze the coding genes of the donkey genome, we assembled the transcriptome of donkey white blood cells de novo. Using transcriptomic deep-sequencing data, we identified 264,714 distinct donkey unigenes and predicted 38,949 protein fragments. We annotated the donkey unigenes by BLAST searches against the non-redundant (NR) protein database. We also compared the donkey protein sequences with those of the horse (E. caballus) and wild horse (E. przewalskii), and linked the donkey protein fragments with mammalian phenotypes. As the outer ear size of donkeys and horses are obviously different, we compared the outer ear size-associated proteins in donkeys and horses. We identified three ear size-associated proteins, HIC1, PRKRA, and KMT2A, with sequence differences among the donkey, horse, and wild horse loci. Since the donkey genome sequence has not been released, the de novo assembled donkey transcriptome is helpful for preliminary investigations of donkey cultivars and for genetic improvement.

A global transcriptional analysis of Megalobrama amblycephala revealing the molecular determinants of diet-induced hepatic steatosis

Blunt snout bream (Megalobrama amblycephala), a prevalent species in China's intensive polyculture systems, is highly susceptible to hepatic steatosis, resulting in considerable losses to the fish farming industry. Due to a lack of genomic resources, the molecular mechanisms of lipid metabolism in M. amblycephala are poorly understood. Here, a hepatic cDNA library was generated from equal amounts of mRNAs isolated from M. amblycephala fed normal-fat and high-fat diets. Sequencing of this library using the Illumina/Solexa platform produced approximately 51.87 million clean reads, which were assembled into 48,439 unigenes with an average length of 596bp and an N50 value of 800bp. These unigenes were searched against the nucleotide (NT), non-redundant (NR), Swiss-Prot, Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genome (KEGG) databases using the BLASTn or BLASTx algorithms (E-value≤10−5). A total of 8602 unigenes and 22,155 unigenes were functionally classified into 25 COG categories and 259 KEGG pathways, respectively. Furthermore, 22,072 unigenes were grouped into 62 sub-categories belonging to three main Gene Ontology (GO) terms. Using a digital gene expression analysis and the M. amblycephala transcriptome as a reference, 477 genes (134 up-regulated and 343 down-regulated) were identified as differentially expressed in fish fed a high-fat diet versus a normal-fat diet. KEGG and GO functional enrichment analyses of the differentially expressed unigenes were performed and 12 candidate genes related to lipid metabolism were identified. This study provides a global survey of hepatic transcriptome profiles and identifies candidate genes that may be related to lipid metabolism in M. amblycephala. These findings will facilitate further investigations of the mechanisms underlying hepatic steatosis in M. amblycephala.

Subtraction suppressive hybridisation analysis of differentially expressed genes associated with puberty in the goat hypothalamus.

The cost of developing replacement nanny goats could be reduced by decreasing the age at puberty because this way nanny goats could be brought into production at an earlier age. The aim of the present study was to screen genes related to puberty to investigate the molecular mechanisms of puberty. Subtracted cDNA libraries were constructed for hypothalami from juvenile (Group A), pubertal (Group B) and age-matched control pubertal (Group E) Jining grey (JG) and Liaoning cashmere (LC) goats using suppression subtractive hybridisation (SSH). Differentially expressed genes were analysed by bioinformatics methods. There were 203 expressed sequence tags (ESTs) in the subtracted cDNA libraries that were differentially expressed between JG and LC goats at the juvenile stage, 226 that were differentially expressed at puberty and 183 that were differentially expressed in the age-matched control group. The differentially expressed ESTs in each subtracted cDNA library were classified as known gene, known EST and unknown EST according to sequence homology in the GenBank non-redundant (NR) and EST database. According to gene function analysis in the COG (Cluster of Orthologous Groups) database, the known genes were grouped into 10 subdivisions in Group A, into seven subdivisions in Group E and into nine subdivisions in Group B under three categories: cellular processes and signalling, information storage and processing, and metabolism. Pathway analysis in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database of known genes revealed that the three pathways that most differentially expressed genes were involved in were metabolic pathways, Parkinson's disease and oxidative phosphorylation. Protein interaction analysis of the high homology genes revealed the most dominant network to be structure of ribosome/protein translation, oxidative phosphorylation and carbohydrate metabolism. The results reveal that the onset of puberty is a complex event involving multiple genes in multiple biological processes. The differentially expressed genes include genes related to both neuroendocrine and energy metabolism.

Whole transcriptome analysis using next-generation sequencing of sterile-cultured Eisenia andrei for immune system research.

Recently, earthworms have become a useful model for research into the immune system, and it is expected that results obtained using this model will shed light on the sophisticated vertebrate immune system and the evolution of the immune response, and additionally help identify new biomolecules with therapeutic applications. However, for earthworms to be used as a genetic model of the invertebrate immune system, basic molecular and genetic resources, such as an expressed sequence tag (EST) database, must be developed for this organism. Next-generation sequencing technologies have generated EST libraries by RNA-seq in many model species. In this study, we used Illumina RNA-sequence technology to perform a comprehensive transcriptome analysis using an RNA sample pooled from sterile-cultured Eisenia andrei. All clean reads were assembled de novo into 41,423 unigenes using the Trinity program. Using this transcriptome data, we performed BLAST analysis against the GenBank non-redundant (NR) database and obtained a total of 12,285 significant BLAST hits. Furthermore, gene ontology (GO) analysis assigned 78 unigenes to 24 immune class GO terms. In addition, we detected a unigene with high similarity to beta-1,3-glucuronyltransferase 1 (GlcAT-P), which mediates a glucuronyl transfer reaction during the biosynthesis of the carbohydrate epitope HNK-1 (human natural killer-1, also known as CD57), a marker of NK cells. The identified transcripts will be used to facilitate future research into the immune system using E. andrei.

Identifying DNA-binding proteins by combining support vector machine and PSSM distance transformation.

BackgroundDNA-binding proteins play a pivotal role in various intra- and extra-cellular activities ranging from DNA replication to gene expression control. Identification of DNA-binding proteins is one of the major challenges in the field of genome annotation. There have been several computational methods proposed in the literature to deal with the DNA-binding protein identification. However, most of them can't provide an invaluable knowledge base for our understanding of DNA-protein interactions.ResultsWe firstly presented a new protein sequence encoding method called PSSM Distance Transformation, and then constructed a DNA-binding protein identification method (SVM-PSSM-DT) by combining PSSM Distance Transformation with support vector machine (SVM). First, the PSSM profiles are generated by using the PSI-BLAST program to search the non-redundant (NR) database. Next, the PSSM profiles are transformed into uniform numeric representations appropriately by distance transformation scheme. Lastly, the resulting uniform numeric representations are inputted into a SVM classifier for prediction. Thus whether a sequence can bind to DNA or not can be determined. In benchmark test on 525 DNA-binding and 550 non DNA-binding proteins using jackknife validation, the present model achieved an ACC of 79.96%, MCC of 0.622 and AUC of 86.50%. This performance is considerably better than most of the existing state-of-the-art predictive methods. When tested on a recently constructed independent dataset PDB186, SVM-PSSM-DT also achieved the best performance with ACC of 80.00%, MCC of 0.647 and AUC of 87.40%, and outperformed some existing state-of-the-art methods.ConclusionsThe experiment results demonstrate that PSSM Distance Transformation is an available protein sequence encoding method and SVM-PSSM-DT is a useful tool for identifying the DNA-binding proteins. A user-friendly web-server of SVM-PSSM-DT was constructed, which is freely accessible to the public at the web-site on http://bioinformatics.hitsz.edu.cn/PSSM-DT/.

RNA-Seq analysis and transcriptome assembly for blackberry (Rubus sp. Var. Lochness) fruit.

BackgroundThere is an increasing interest in berries, especially blackberries in the diet, because of recent reports of their health benefits due to their high content of flavonoids. A broad range of genomic tools are available for other Rosaceae species but these tools are still lacking in the Rubus genus, thus limiting gene discovery and the breeding of improved varieties.ResultsDe novo RNA-seq of ripe blackberries grown under field conditions was performed using Illumina Hiseq 2000. Almost 9 billion nucleotide bases were sequenced in total. Following assembly, 42,062 consensus sequences were detected. For functional annotation, 33,040 (NR), 32,762 (NT), 21,932 (Swiss-Prot), 20,134 (KEGG), 13,676 (COG), 24,168 (GO) consensus sequences were annotated using different databases; in total 34,552 annotated sequences were identified. For protein prediction analysis, the number of coding DNA sequences (CDS) that mapped to the protein database was 32,540. Non redundant (NR), annotation showed that 25,418 genes (73.5%) has the highest similarity with Fragaria vesca subspecies vesca. Reanalysis was undertaken by aligning the reads with this reference genome for a deeper analysis of the transcriptome. We demonstrated that de novo assembly, using Trinity and later annotation with Blast using different databases, were complementary to alignment to the reference sequence using SOAPaligner/SOAP2. The Fragaria reference genome belongs to a species in the same family as blackberry (Rosaceae) but to a different genus. Since blackberries are tetraploids, the possibility of artefactual gene chimeras resulting from mis-assembly was tested with one of the genes sequenced by RNAseq, Chalcone Synthase (CHS). cDNAs encoding this protein were cloned and sequenced. Primers designed to the assembled sequences accurately distinguished different contigs, at least for chalcone synthase genes.ConclusionsWe prepared and analysed transcriptome data from ripe blackberries, for which prior genomic information was limited. This new sequence information will improve the knowledge of this important and healthy fruit, providing an invaluable new tool for biological research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1198-1) contains supplementary material, which is available to authorized users.

Transcriptome profiling shows gene regulation patterns in a flavonoid pathway in response to exogenous phenylalanine in Boesenbergia rotunda cell culture.

BackgroundPanduratin A extracted from Boesenbergia rotunda is a flavonoid reported to possess a range of medicinal indications which include anti-dengue, anti-HIV, anti-cancer, antioxidant and anti-inflammatory properties. Boesenbergia rotunda is a plant from the Zingiberaceae family commonly used as a food ingredient and traditional medicine in Southeast Asia and China. Reports on the health benefits of secondary metabolites extracted from Boesenbergia rotunda over the last few years has resulted in rising demands for panduratin A. However large scale extraction has been hindered by the naturally low abundance of the compound and limited knowledge of its biosynthetic pathway.ResultsTranscriptome sequencing and digital gene expression (DGE) analysis of native and phenylalanine treated Boesenbergia rotunda cell suspension cultures were carried out to elucidate the key genes differentially expressed in the panduratin A biosynthetic pathway. Based on experiments that show increase in panduratin A production after 14 days post treatment with exogenous phenylalanine, an aromatic amino acid derived from the shikimic acid pathway, total RNA of untreated and 14 days post-phenylalanine treated cell suspension cultures were extracted and sequenced using next generation sequencing technology employing an Illumina-Solexa platform. The transcriptome data generated 101, 043 unigenes with 50, 932 (50.41%) successfully annotated in the public protein databases; including 49.93% (50, 447) in the non-redundant (NR) database, 34.63% (34, 989) in Swiss-Prot, 24,07% (24, 316) in Kyoto Encyclopedia of Genes and Genomes (KEGG) and 16.26% (16, 426) in Clusters of Orthologous Groups (COG). Through DGE analysis, we found that 14, 644 unigenes were up-regulated and 14, 379 unigenes down-regulated in response to exogenous phenylalanine treatment. In the phenylpropanoid pathway leading to the proposed panduratin A production, 2 up-regulated phenylalanine ammonia-lyase (PAL), 3 up-regulated 4-coumaroyl:coenzyme A ligase (4CL) and 1 up-regulated chalcone synthase (CHS) were found.ConclusionsThis is the first report of Boesenbergia rotunda de novo transcriptome data that could serve as a reference for gene or enzyme functional studies in the Zingiberaceae family. Although enzymes that are directly involved in the panduratin A biosynthetic pathway were not completely elucidated, the data provides an overall picture of gene regulation patterns leading to panduratin A production.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-984) contains supplementary material, which is available to authorized users.

Development and utilization of genomic and genic microsatellite markers in Assam tea (Camellia assamica ssp. assamica) and related Camellia species

AbstractMicrosatellite or simple sequence repeat (SSR) markers are valuable tools for many purposes, such as phylogenetic, fingerprinting and molecular breeding studies. However, such marker resources are unavailable in Assam tea (Camellia assamica ssp. assamica; Masters). With an objective to enrich the repertoire of microsatellite markers in traditional tea, 185 novel microsatellite (150 genomic and 35 genic) markers were identified from (GA)n‐enriched genomic libraries and public expressed sequence data in Assam tea. High‐quality 0.412‐Mb non‐redundant (NR) genomic data set derived from nucleotide sequencing of 1297 (GA)n‐enriched genomic positive clones and 2723 unigenes (1.33 Mb) predicted from 10 803 random public expressed sequence tags (ESTs) in C. assamica ssp. assamica were utilized for identification of genomic and genic microsatellite markers, respectively. The average number of alleles and polymorphic information content (PIC) recorded for the newly developed SSR markers were 6.17 and 0.398, respectively. The average observed (Ho) and expected (He) heterozygosity varied from 0.626 to 0.697, respectively. These markers were found to be highly transferable (74.5–100%) to cultivated (C. sinensis, C. assamica ssp. lasiocalyx) and five wild Camellia species. Genetic diversity coefficient detected a high level of divergence in 24 cultivated tea accessions (69.3%). Phylogenetic analysis revealed that major groupings were broadly in accordance with taxonomic classification of tea, and all the wild Camellia species remained as an out‐group. The high polymorphic content coupled with high rate of cross‐transferability demonstrates wider applicability of novel microsatellite markers in genotyping, genetic diversity, genome mapping and evolutionary studies in various Camellia species.

Characterization of the Zhikong Scallop (Chlamys farreri) Mantle Transcriptome and Identification of Biomineralization-Related Genes

Chlamys farreri is a significant species in aquaculture and fishery in East Asia. A deep understanding of its shell formation by studying the transcriptome of the mantle, a key organ in shell formation, could provide important guidance for its culture. Thus, we sequenced and analyzed the mantle transcriptome of C. farreri. The 77,975 unigenes were generated after Illumina sequencing and de novo assembly. The unigenes were annotated using authoritative databases (non-redundant (NR), COG, Gene Ontology (GO), and KEGG) to obtain functional information. BLASTX alignment was performed between unigenes and reported proteins related to biomineralization. The results identified 53 homologous genes representing 17 matrix proteins, most of which are involved in calcite formation, and 171 homologies with 26 proteins related to general processes of biomineralization. The discovery and unusually high expression of MSP-1 suggested its importance in scallops. Homologous unigenes with aragonite-formation-related matrix proteins were much fewer compared with those related to calcite formation. The results implied that, in C. farreri, the number and proportion of matrix proteins related to aragonite formation is much lower than those related to calcite formation, which was consistent with the proportions of aragonite and calcite in C. farreri shells. Thus, the formation of different polymorphs of calcium carbonate (calcite and aragonite) in molluskan shells is regulated by different groups of proteins. Moreover, 17 candidate unigenes, which are probably involved in biomineralization, were predicted by screening for gene products with secreted domains and tandem-arranged repeat units. Our results contribute to the understanding of biomineralization processes and the evolution of shell formation.

The Annotation-enriched non-redundant patent sequence databases

The EMBL-European Bioinformatics Institute (EMBL-EBI) offers public access to patent sequence data, providing a valuable service to the intellectual property and scientific communities. The non-redundant (NR) patent sequence databases comprise two-level nucleotide and protein sequence clusters (NRNL1, NRNL2, NRPL1 and NRPL2) based on sequence identity (level-1) and patent family (level-2). Annotation from the source entries in these databases is merged and enhanced with additional information from the patent literature and biological context. Corrections in patent publication numbers, kind-codes and patent equivalents significantly improve the data quality. Data are available through various user interfaces including web browser, downloads via FTP, SRS, Dbfetch and EBI-Search. Sequence similarity/homology searches against the databases are available using BLAST, FASTA and PSI-Search. In this article, we describe the data collection and annotation and also outline major changes and improvements introduced since 2009. Apart from data growth, these changes include additional annotation for singleton clusters, the identifier versioning for tracking entry change and the entry mappings between the two-level databases.Database URL: http://www.ebi.ac.uk/patentdata/nr/

Identification and analysis of genes differentially expressed in the Spodoptera litura fat body in response to the biocontrol fungus, Nomuraea rileyi

Nomuraea rileyi is an important pathogenic fungus that can successfully control Spodoptera litura. However, little is known on how S. litura responds to N. rileyi infection. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from the S. litura fat body and the up-regulated genes were identified to isolate differentially expressed genes in response to N. rileyi. A total of 345/1175 random clones screened by cDNA array dot blotting were sequenced, resulting in 117 uniquely expressed sequence tags (ESTs). Potential functional genes were identified by BLAST searches and were categorized into seven groups associated with different biological processes based on the literature and gene ontologies. Among 117 genes, 74 had matches in the non-redundant (NR) protein database and were found to be involved in different biological processes, while 43 of the screened genes were classified to the "unknown function" gene group. Notably, only two genes had previously been reported in S. litura and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 115 genes were found for the first time in S. litura. Semi-quantitative RT-PCR analysis of seven randomly selected genes revealed that most were differentially expressed after N. rileyi infection. qRT-PCR analysis confirmed that four genes (Hsp70, Hsp90, gallerimycin, and cysteine proteinase) were significantly up-regulated after N. rileyi infection. Taken together, the present study identified up-regulated S. litura genes in response to N. rileyi infection. Further investigations are needed to unravel the molecular mechanisms of the genes or proteins potentially involved in the S. litura innate immune defense against N. rileyi infection.