NCBI Non-redundant Protein Research Articles

De novo transcriptome analysis of Tibetan medicinal plant Dysphania schraderiana.

Dysphania schraderiana is widely distributed in Lhasa (Tibet, China) and used as a traditional medicine. However, the lack of genetic information hinders the understanding of its physiological processes, such as the biosynthesis of secondary metabolites. Herein, we used Illumina Hiseq4000 platform to sequence the transcriptome of flower and leaf tissues from D. schraderiana for the first time. Totally, 40,142 unigenes were assembled from approximately 5.2 million clean reads. All unigenes underwent gene prediction and were subsequently annotated in a NR (NCBI non-redundant protein) database, COG (Clusters of Orthologous Groups of proteins) database, and KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Among the 40,142 unigenes, 2,579 genes were identified as differentially expressed between flowers and leaves, and used in further enrichment analysis. Also, 2,156 unigenes were annotated as transcription factors. Furthermore, our transcriptome analysis resulted in the identification of candidate unigenes annotated to enzymes involved in terpenoid biosynthesis. Taken together, this work has laid the foundation for the investigation of secondary metabolite biosynthesis and other physiological processes of D. schraderiana.

De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax.

The velvet belly lanternshark (Etmopterus spinax) is a small deep-sea shark commonly found in the Eastern Atlantic and the Mediterranean Sea. This bioluminescent species is able to emit a blue-green ventral glow used in counter-illumination camouflage, mainly. In this study, paired-end Illumina HiSeqTM technology has been employed to generate transcriptome data from eye and ventral skin tissues of the lanternshark. About 64 and 49 million Illumina reads were generated from skin and eye tissues respectively. The assembly allowed us to predict 119,749 total unigenes including 94,569 for the skin transcriptome and 94,365 for the eye transcriptome while 74,753 were commonly found in both transcriptomes. A taxonomy filtering was applied to extract a reference transcriptome containing 104,390 unigenes among which 38,836 showed significant similarities to known sequences in NCBI non-redundant protein sequences database. Around 58% of the annotated unigenes match with predicted genes from the Elephant shark (Callorhinchus milii) genome. The transcriptome completeness has been evaluated by successfully capturing around 98% of orthologous genes of the « Core eukaryotic gene dataset » within the E. spinax reference transcriptome. We identified potential “light-interacting toolkit” genes including multiple genes related to ocular and extraocular light perception processes such as opsins, phototransduction actors or crystallins. Comparative gene expression analysis reveals eye-specific expression of opsins, ciliary phototransduction actors, crystallins and vertebrate retinoid pathway actors. In particular, mRNAs from a single rhodopsin gene and its potentially associated peropsin were detected in the eye transcriptome, only, confirming a monochromatic vision of the lanternshark. Encephalopsin mRNAs were mainly detected in the ventral skin transcriptome. In parallel, immunolocalization of the encephalopsin within the ventral skin of the shark suggests a functional relation with the photophores, i.e. epidermal light-producing organs. We hypothesize that extraocular photoreception might be involved in the bioluminescence control possibly acting on the shutter opening and/or the photocyte activity itself. The newly generated reference transcriptome provides a valuable resource for further understanding of the shark biology.

Transcriptome analysis of the brain of the Chinese mitten crab, Eriocheir sinensis, for neuropeptide abundance profiles during ovarian development

Neuropeptides, important messenger molecules, regulate various physiological processes, such as growth, development, and reproduction. In the present study, cDNA libraries from brains of E. sinensis were constructed and sequenced using the Illumina technique for transcript analysis and neuropeptides discovery. There were 233,887 transcripts assembled for 194,286 unigenes. According to the annotations of NCBI non-redundant protein (NR) database, 2487 (11.31%) unigenes were annotated successfully. In total, 1273 transcripts were assigned to the “signal transduction mechanisms” category using KOG analysis. The results of KEGG indicate signal transduction and translation pathways were the dominant and enriched signal pathways. Additionally, results indicated C2H2 was the main transcription factor (TF) family. Analysis of the assembled transcripts indicated there were 22 neuropeptide transcripts, such as allatostatin, crustacean female sex hormone, crustacean hyperglycemic hormone, diuretic hormone 31, and eclosion hormone. The detection of these neuropeptides provide for a basic understanding for future study of functions in development, reproduction, and sexual maturation in crustaceans.

The Population Genetics of Alternaria tenuissima in Four Regions of China as Determined by Microsatellite Markers Obtained by Transcriptome Sequencing.

A total of 32,284 unigenes were obtained from the transcriptome of Alternaria tenuissima, a pathogenic fungus causing foliar disease in tomato, using next-generation sequencing (NGS) technology. In total, 24,670 unigenes were annotated using five databases, including NCBI non-redundant protein, Swiss-Prot, euKaryotic Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and the Gene Ontology. A total of 1,140 simple sequence repeats were also identified for use as molecular markers. Sixteen of the simple sequence repeat loci were selected to study the population structure of A. tenuissima. A population genetic analysis of 191 A. tenuissima isolates, sampled from four geographic regions in China, indicated that A. tenuissima had a high level of genetic diversity, and that the selected simple sequence repeat markers could reliably capture the genetic variation. The null hypothesis of random mating was rejected for all four geographic regions in China. Isolation by distance was observed for the entire data set, but not within clusters, which is indicative of barriers to gene flow among geographic regions. The analyses of Bayesian and principal coordinates, however, did not separate four geographic regions into four separate genetic clusters. The different levels of historical migration rates suggest that isolation by distance did not represent a major biological obstacle to the spread of A. tenuissima. The potential epidemic spread of A. tenuissima in China may occur through the transport of plant products or other factors. The presented results provide a basis for a comprehensive understanding of the population genetics of A. tenuissima in China.

Transcriptome characterization and SSR discovery in Squaliobarbus curriculus

Squaliobarbus curriculus is a eurythermic fish found mainly in East Asia. The natural germplasm resources of this species, an organism of potential economic aquacultural importance, have recently been threatened by overfishing and habitat degradation. The objective of this study was to obtain functional genomic resources and candidate microsatellite markers for future genetic observation and germplasm protection of S. curriculus. Illumina paired-end sequencing generated over 22.20 million clean reads, which were assembled into 204 027 transcripts with an average length of 1 457 bp. A total of 112 570 protein-coding sequences (55.17%) were predicted, 111 934 of which had significant matches in the NCBI nonredundant protein database. Overall, 60 450, 34 830, and 77 755 transcripts were assigned to Gene Ontology, Clusters of Orthologous Groups, and Eukaryotic Orthologous Groups categories, respectively. In addition, 44 781 transcripts (21.95%) were mapped to 230 KEGG pathways. A total of 51 426 candidate simple sequence repeat (SSR) markers were identified and used to design 36 149 PCR primer pairs. Fifty of these primer pairs were randomly selected for PCR validation in 32 S. curriculus specimens, which resulted in 25 pairs yielding the expected products. Further analysis revealed that 40% (10) of the 25 amplified loci were polymorphic. The transcriptomic profile and SSR repertoire obtained in this study should facilitate population genetic studies and germplasm resource protection of S. curriculus.

De novo transcriptomic analysis of the alimentary tract of the tephritid gall fly, Procecidochares utilis.

The tephritid gall fly, Procecidochares utilis, is an important obligate parasitic insect of the malignant weed Eupatorium adenophorum which biosynthesizes toxic secondary metabolites. Insect alimentary tracts secrete several enzymes that are used for detoxification, including cytochrome P450s, glutathione S-transferases, and carboxylesterases. To explore the adaptation of P. utilis to its toxic host plant, E. adenophorum at molecular level, we sequenced the transcriptome of the alimentary tract of P. utilis using Illumina sequencing. Sequencing and de novo assembly yielded 62,443 high-quality contigs with an average length of 604 bp that were further assembled into 45,985 unigenes with an average length of 674 bp and an N50 of 983 bp. Among the unigenes, 30,430 (66.17%) were annotated by alignment against the NCBI non-redundant protein (Nr) database, while 16,700 (36.32%), 16,267 (35.37%), and 11,530 (25.07%) were assigned functions using the Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases, respectively. Using the comprehensive transcriptome data set, we manually identified several important gene families likely to be involved in the detoxification of toxic compounds including 21 unigenes within the glutathione S-transferase (GST) family, 22 unigenes within the cytochrome P450 (P450) family, and 16 unigenes within the carboxylesterase (CarE) family. Quantitative PCR was used to verify eight, six, and two genes of GSTs, P450s, and CarEs, respectively, in different P. utilis tissues and at different developmental stages. The detoxification enzyme genes were mainly expressed in the foregut and midgut. Moreover, the unigenes were higher expressed in the larvae, pupae, and 3-day adults, while they were expressed at lower levels in eggs. These transcriptomic data provide a valuable molecular resource for better understanding the function of the P. utilis alimentary canal. These identified genes could be pinpoints to address the molecular mechanisms of P. utilis interacting with toxic plant host.

Gonadal transcriptome sequencing of the critically endangered Acipenser dabryanus to discover candidate sex-related genes.

BackgroundAcipenser dabryanus, an endemic Chinese species, has been listed as a first-class protected animal in China. Sturgeons are among the oldest and most primitive group of existing fish in the world and occupy a special place in the evolutionary history of fish. Thus, a study of the reproduction and sex differentiation of sturgeon will be of great value for fish as well as the whole vertebrate group.MethodsIn this study, we conducted comparative analysis of the testes and ovaries transcriptomes of A. dabryanus to screen for sex-differentiation and sexual development-related genes.ResultsThe transcriptome sequencing of six cDNA libraries generated 265 million clean reads, encompassing 79 Gb of sequences. The N50 and mean length of the identified 91,375 unigenes were 1,718 and 989 bp, respectively. A total of 6,306, 9,961, 13,170, 15,484, and 23,588 unigenes were annotated in the clusters of orthologous groups, gene ontology categories, Kyoto Encyclopedia of Genes and Genomes Pathway, euKaryotic orthologous groups, and NCBI non-redundant protein databases, respectively. A total of 5,396 differentially expressed genes were found between the two sexes, with 1,938 predicted to be up-regulated in ovaries and 3,458 in testes. A total of 73 candidate genes known to be involved in sex differentiation and sexual development were searched in the transcriptome of A. dabryanus of which 52 showed significant similarity. We highlighted six genes that are differentially expressed between the two sexes and may play important roles in sex differentiation and gonad maintenance. In addition, 24,271 simple sequence repeats (SSRs) and 550,519 single-nucleotide polymorphisms (SNPs) were detected.DiscussionThis work represents the first transcriptome study comparing the ovary and testis in A. dabryanus. The putative differentially expressed genes between the gonads provide an important source of information for further study of the sex-differentiation related genes and the sex-differentiation mechanism in sturgeons. The SSRs or SNPs identified in this study will be helpful in the discovery of sex-related markers in A. dabryanus.

Transcriptomic Analysis of the Candidate Genes Related to Aroma Formation in Osmanthus fragrans.

Osmanthus fragrans, or “RiXiangGui”, is an ornamental, woody, evergreen plant that is cultivated widely because it blooms recurrently and emits a strong fragrance. Recently, the germplasm resources, classification, and aroma compositions of O. fragrans have been investigated. However, the molecular mechanisms of the floral scent formation and regulation have remained largely unknown. To obtain a global perspective on the molecular mechanism of the aroma formation during blooming, nine RNA Sequencing (RNA-Seq) libraries were constructed from three flowering stages: The initial, full, and final flowering stage. In short, a total of 523,961,310 high-quality clean reads were assembled into 136,611unigenes, with an average sequence length of 792 bp. About 47.43% of the unigenes (64,795) could be annotated in the NCBI non-redundant protein database. A number of candidate genes were identified in the terpenoid metabolic pathways and 1327 transcription factors (TFs), which showed differential expression patterns among the floral scent formation stages, were also identified, especially OfMYB1, OfMYB6, OfWRKY1, and OfWRKY3, which could play critical roles in the floral scent formation. These results indicated that the floral scent formation of O. fragrans was a very complex process which involved a large number of TFs. This study provides reliable resources for further studies of the O.fragrans floral scent formation.

De novo assembly and comparative transcriptome analysis of Monilinia fructicola, Monilinia laxa and Monilinia fructigena, the causal agents of brown rot on stone fruits

BackgroundBrown rots are important fungal diseases of stone and pome fruits. They are caused by several Monilinia species but M. fructicola, M. laxa and M. fructigena are the most common all over the world. Although they have been intensively studied, the availability of genomic and transcriptomic data in public databases is still scant. We sequenced, assembled and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per each species for comparative transcriptome analyses.ResultsIllumina sequencing was used to generate about 70 million of paired-end reads per species, that were de novo assembled in 33,861 contigs for M. fructicola, 31,103 for M. laxa and 28,890 for M. fructigena. Approximately, 50% of the assembled contigs had significant hits when blasted against the NCBI non-redundant protein database and top-hits results were represented by Botrytis cinerea, Sclerotinia sclerotiorum and Sclerotinia borealis proteins. More than 90% of the obtained sequences were complete, the percentage of duplications was always less than 14% and fragmented and missing transcripts less than 5%. Orthologous transcripts were identified by tBLASTn analysis using the B. cinerea proteome as reference. Comparative transcriptome analyses revealed 65 transcripts over-expressed (FC ≥ 8 and FDR ≤ 0.05) or unique in M. fructicola, 30 in M. laxa and 31 in M. fructigena. Transcripts were involved in processes affecting fungal development, diversity and host-pathogen interactions, such as plant cell wall-degrading and detoxifying enzymes, zinc finger transcription factors, MFS transporters, cell surface proteins, key enzymes in biosynthesis and metabolism of antibiotics and toxins, and transposable elements.ConclusionsThis is the first large-scale reconstruction and annotation of the complete transcriptomes of M. fructicola, M. laxa and M. fructigena and the first comparative transcriptome analysis among the three pathogens revealing differentially expressed genes with potential important roles in metabolic and physiological processes related to fungal morphogenesis and development, diversity and pathogenesis which need further investigations. We believe that the data obtained represent a cornerstone for research aimed at improving knowledge on the population biology, physiology and plant-pathogen interactions of these important phytopathogenic fungi.

Transcriptomic analysis of adaptive mechanisms in response to sudden salinity drop in the mud crab, Scylla paramamosain

BackgroundScylla paramamosain (Crustacea: Decapoda: Portunidae: Syclla De Hann) is a commercially important mud crab distributed along the coast of southern China and other Indo-Pacific countries (Lin Z, Hao M, Zhu D, et al, Comp Biochem Physiol B Biochem Mol Biol 208-209:29–37, 2017; Walton ME, Vay LL, Lebata JH, et al, Estuar Coast Shelf Sci 66(3–4):493–500, 2006; Wang Z, Sun B, Zhu F, Fish Shellfish Immunol 67:612–9, 2017). While S. paramamosain is a euryhaline species, a sudden drop in salinity induces a negative impact on growth, molting, and reproduction, and may even cause death. The mechanism of osmotic regulation of marine crustaceans has been recently under investigation. However, the mechanism of adapting to a sudden drop in salinity has not been reported.MethodsIn this study, transcriptomics analysis was conducted on the gills of S. paramamosain to test its adaptive capabilities over 120 h with a sudden drop in salinity from 23 ‰ to 3 ‰.ResultsAt the level of transcription, 135 DEGs (108 up-regulated and 27 down-regulated) annotated by NCBI non-redundant (nr) protein database were screened. GO analysis showed that the catalytic activity category showed the most participating genes in the 24 s-tier GO terms, indicating that intracellular metabolic activities in S. paramamosain were enhanced. Of the 164 mapped KEGG pathways, seven of the top 20 pathways were closely related to regulation of the Na+ / K+ -ATPase. Seven additional amino acid metabolism-related pathways were also found, along with other important signaling pathways.ConclusionIon transport and amino acid metabolism were key factors in regulating the salinity adaptation of S. paramamosain in addition to several important signaling pathways.

De novo transcriptomic analysis of leaf and fruit tissue of Cornus officinalis using Illumina platform.

Cornus officinalis is one of the most widely used medicinal plants in China and other East Asian countries to cure diseases such as liver, kidney, cardiovascular diseases and frequent urination for thousands of years. It is a Level 3 protected species, and is one of the 42 national key protected wild species of animals and plants in China. However, the genetics and molecular biology of C. officinalis are poorly understood, which has hindered research on the molecular mechanism of its metabolism and utilization. Hence, enriching its genomic data and information is very important. In recent years, the fast-growing technology of next generation sequencing has provided an effective path to gain genomic information from nonmodel species. This study is the first to explore the leaf and fruit tissue transcriptome of C. officinalis using the Illumina HiSeq 4000 platform. A total of 57,954,134 and 60,971,652 clean reads from leaf and fruit were acquired, respectively (GenBank number SRP115440). The pooled reads from all two libraries were assembled into 56,392 unigenes with an average length 856 bp. Among these, 41,146 unigenes matched with sequences in the NCBI nonredundant protein database. The Gene Ontology database assigned 24,336 unigenes with biological process (83.26%), cellular components (53.58%), and molecular function (83.93%). In addition, 10,808 unigenes were assigned a KOG functional classification by the KOG database. Searching against the KEGG pathway database indicated that 18,435 unigenes were mapped to 371 KEGG pathways. Moreover, the edgeR database identified 4,585 significant differentially expressed genes (DEGs), of which 1,392 were up-regulated and 3,193 were down-regulated in fruit tissue compared with leaf tissue. Finally, we explored 581 transcription factors with 50 transcription factor gene families. Most DEGs and transcription factors were related to terpene biosynthesis and secondary metabolic regulation. This study not only represented the first de novo transcriptomic analysis of C. officinalis but also provided fundamental information on its genes and biosynthetic pathway. These findings will help us explore the molecular metabolism mechanism of terpene biosynthesis in C. officinalis.

De novo transcriptome analysis of Rhododendron molle G. Don flowers by Illumina sequencing

Rhododendron molle G. Don occupies an important phylogenetic node in the genus rhododendron with unique yellow flower and medicinal functions. However, only limited genetic resources and their genome information are available for the generation of rhododendron flowers. The next generation sequencing technologies enables generation of genomic resources in a short time and at a minimal cost, and therefore provide a turning point for rhododendron research. Our goal is to use the genetic information to facilitate the relevant research on flowering and flower color formation in R. molle. In total, 66,026 unigenes were identified, among which 31,298 were annotated in the NCBI non-redundant protein database and 22,410 were annotated in the Swiss-Prot database. Of these annotated unigenes, 9490 and 18,680 unigenes were assigned to clusters of orthologous groups and gene ontology categories, respectively. A total of 7177 genes were mapped to 118 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. In addition, 8266 simple sequence repeats (SSRs) were detected, and these SSRs will undoubtedly benefit rhododendron breeding work. Metabolic pathway analysis revealed that 32 unigenes were predicted to be involved in carotenoid biosynthesis. Our transcriptome revealed 32 engines that encode key enzymes in the carotenoid biosynthesis pathway, including PSY, PDS, LCYB, LCYE, etc. The content of β-carotene was much higher than the other carotenoids throughout the flower development. It was consistent with the key genes expression level in the carotenoid biosynthesis pathway by the Illumina expression profile analysis and the qRT-PCR analysis. Our study identified genes associated with carotenoid biosynthesis in R. molle and provides a valuable resource for understanding the flowering and flower color formation mechanisms in R. molle.

Illumina-based de novo transcriptome sequencing and analysis of Chinese forest musk deer.

The Chinese forest musk deer (Moschus berezovskii Flerov) is an endangered artiodactyl mammal. The musk secreted by sexually mature males is highly valued for alleged pharmaceutical properties and perfume manufacturing. However, the genomic and transcriptomic resources of musk deer remain deficiently represented and poorly understood. Next-generation sequencing technique is an efficient method for generating an enormous amount of sequence data that can represent a large number of genes and their expression levels. In the present study, we used Illumina HiSeq technology to perform de novo assembly of heart and musk gland transcriptomes from the Chinese forest musk deer. A total of 239,383 transcripts and 176,450 unigenes were obtained, of which 37,329 unigenes were matched to known sequences in the NCBI nonredundant protein (Nr) database; 31,039 unigenes were assigned to 61 GO terms, and 11,782 to 332 KEGG pathways. Additionally, 592 and 2282 differentially expressed genes were found to be specifically expressed in the heart and musk gland, respectively. The abundant transcriptomic data generated in the present report will provide a comprehensive sequence resource for Chinese forest musk deer as well as lay down a foundation which will help in accelerating genetic and functional genomics research in this species.

HT-SuperSAGE of the gut tissue of a Vip3Aa-resistant Heliothis virescens (Lepidoptera: Noctuidae) strain provides insights into the basis of resistance.

Multitoxin Bt‐crops expressing insecticidal toxins with different modes of action, for example, Cry and Vip, are expected to improve resistance management in target pests. While Cry1A resistance has been relatively well characterized in some insect species, this is not the case for Vip3A, for which no mechanism of resistance has yet been identified. Here we applied HT‐SuperSAGE to analyze the transcriptome of the gut tissue of tobacco budworm Heliothis virescens (F.) laboratory‐selected for Vip3Aa resistance. From a total of 1 324 252 sequence reads, 5 895 126‐bp tags were obtained representing 17 751 nonsingleton unique transcripts (UniTags) from genetically similar Vip3Aa‐resistant (Vip–Sel) and susceptible control (Vip–Unsel) strains. Differential expression was significant (≥2.5 fold or ≤0.4; P < 0.05) for 1989 sequences (11.2% of total UniTags), where 420 represented overexpressed (OE) and 1569 underexpressed (UE) genes in Vip–Sel. BLASTN searches mapped 419 UniTags to H. virescens sequence contigs, of which, 416 (106 OE and 310 UE) were unambiguously annotated to proteins in NCBI nonredundant protein databases. Gene Ontology distributed 345 of annotated UniTags in 14 functional categories with metabolism (including serine‐type hydrolases) and translation/ribosome biogenesis being the most prevalent. A UniTag homologous to a particular member of the REsponse to PAThogen (REPAT) family was found among most overexpressed, while UniTags related to the putative Vip3Aa‐binding ribosomal protein S2 (RpS2) were underexpressed. qRT‐PCR of a subset of UniTags validated the HT‐SuperSAGE data. This study is the first providing lepidopteran gut transcriptome associated with Vip3Aa resistance and a foundation for future attempts to elucidate the resistance mechanism.

Transcriptome profiling and digital gene expression analysis of sweet potato for the identification of putative genes involved in the defense response against Fusarium oxysporum f. sp. batatas.

Sweet potato production is constrained by Fusarium wilt, which is caused by Fusarium oxysporum f. sp. batatas (Fob). The identification of genes related to disease resistance and the underlying mechanisms will contribute to improving disease resistance via sweet potato breeding programs. In the present study, we performed de novo transcriptome assembly and digital gene expression (DGE) profiling of sweet potato challenged with Fob using Illumina HiSeq technology. In total, 89,944,188 clean reads were generated from 12 samples and assembled into 101,988 unigenes with an average length of 666 bp; of these unigenes, 62,605 (61.38%) were functionally annotated in the NCBI non-redundant protein database by BLASTX with a cutoff E-value of 10−5. Clusters of Orthologous Groups (COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were examined to explore the unigenes’ functions. We constructed four DGE libraries for the sweet potato cultivars JinShan57 (JS57, highly resistant) and XinZhongHua (XZH, highly susceptible), which were challenged with pathogenic Fob. Genes that were differentially expressed in the four libraries were identified by comparing the transcriptomes. Various genes that were differentially expressed during defense, including chitin elicitor receptor kinase 1 (CERK), mitogen-activated protein kinase (MAPK), WRKY, NAC, MYB, and ethylene-responsive transcription factor (ERF), as well as resistance genes, pathogenesis-related genes, and genes involved in salicylic acid (SA) and jasmonic acid (JA) signaling pathways, were identified. These data represent a sequence resource for genetic and genomic studies of sweet potato that will enhance the understanding of the mechanism of disease resistance.

ESnail: A transcriptome‐based molecular resource of the central nervous system for terrestrial gastropods

To expand on emerging terrestrial gastropod molecular resources, we have undertaken transcriptome-based sequencing of the central nervous system (CNS) from six ecologically invasive terrestrial gastropods. Focusing on snail species Cochlicella acuta and Helix aspersa and reticulated slugs Deroceras invadens, Deroceras reticulatum, Lehmannia nyctelia and Milax gagates, we obtained a total of 367,869,636 high-quality reads and compared them with existing CNS transcript resources for the invasive Mediterranean snail, Theba pisana. In total, we obtained 419,289 unique transcripts (unigenes) from 1,410,569 assembled contigs, with blast search analysis of multiple protein databases leading to the annotation of 124,268 unigenes, of which 92,544 mapped to ncbi nonredundant protein databases. We found that these transcriptomes have representatives in most biological functions, based on comparison of gene ontology, kegg pathway and protein family contents, demonstrating a high range of transcripts responsible for regulating metabolic activities and molecular functions occurring within the CNS. To provide an accessible genetic resource, we also demonstrate the presence of 66,687 microsatellites and 304,693 single-nucleotide variants, which can be used for the design of potentially thousands of unique primers for functional screening. An online "eSnail" database with a user-friendly web interface was implemented to query all the information obtained herein (http://soft.bioinfo-minzhao.org/esnail). We demonstrate the usefulness of the database through the mining of molluscan neuropeptides. As the most comprehensive CNS transcriptome resource for terrestrial gastropods, eSnail may serve as a useful gateway for researchers to explore gastropod CNS function for multiple purposes, including for the development of biocontrol approaches.

De novo transcriptomic analysis during Lentinula edodes fruiting body growth

The fruiting body of Lentinula edodes is a popular edible mushroom, and extracts from the mycelium and the fruiting body of this species have diverse therapeutic potential. To gain insights into the molecular mechanisms underlying the fruiting body growth of L. edodes from the early bud stage (EBS), through the intermediate developing stage (IDS), to the fully developed stage (FDS), we performed de novo transcriptomic analysis using high-throughput Illumina RNA-sequencing. First, we generated three cDNA libraries representative of the three respective stages. We then obtained 38,933,148, 44,594,472, and 37,905,646 high-quality reads from the respective libraries and assembled the reads into 25,104 transcriptional contigs, containing 15,199 unigenes. We found that only 9331 of the unigenes had been annotated in the NCBI non-redundant protein database, and we functionally annotated 4758 of them through Gene Ontology (GO) analysis and 2921 of them through Clusters of Orthologous Groups of proteins (COGs) analysis. We also assigned 3995 unigenes to metabolic pathways by using the Kyoto Encyclopedia of Genes and Genomes (KEGG). We further identified 399 differentially expressed genes (DEGs) between EBS and IDS, 1428 between IDS and FDS, and 1830 between EBS and FDS, uncovering 769 DEGs in multiple metabolic and signaling pathways. Interestingly, there were a limited number of DEGs whose expression was dramatically associated with FDS. Finally, genes, whose expression was either highly up-regulated in FDS or remained at a high level during fruiting body growth, were annotated specifically in the pathways of purine metabolism, unsaturated fatty acid metabolism and meiosis, suggesting that these key molecular events were actively occurring in the fruiting body. Our work is the first high-throughput transcriptome study on the growth of L. edodes fruiting bodies, and the results uncovered candidate genes for future gene identification and utilization of this commercially and medically important mushroom.

De novo transcriptome sequencing of Isaria cateniannulata and comparative analysis of gene expression in response to heat and cold stresses.

Isaria cateniannulata is a very important and virulent entomopathogenic fungus that infects many insect pest species. Although I. cateniannulata is commonly exposed to extreme environmental temperature conditions, little is known about its molecular response mechanism to temperature stress. Here, we sequenced and de novo assembled the transcriptome of I. cateniannulata in response to high and low temperature stresses using Illumina RNA-Seq technology. Our assembly encompassed 17,514 unigenes (mean length = 1,197 bp), in which 11,445 unigenes (65.34%) showed significant similarities to known sequences in NCBI non-redundant protein sequences (Nr) database. Using digital gene expression analysis, 4,483 differentially expressed genes (DEGs) were identified after heat treatment, including 2,905 up-regulated genes and 1,578 down-regulated genes. Under cold stress, 1,927 DEGs were identified, including 1,245 up-regulated genes and 682 down-regulated genes. The expression patterns of 18 randomly selected candidate DEGs resulting from quantitative real-time PCR (qRT-PCR) were consistent with their transcriptome analysis results. Although DEGs were involved in many pathways, we focused on the genes that were involved in endocytosis: In heat stress, the pathway of clathrin-dependent endocytosis (CDE) was active; however at low temperature stresses, the pathway of clathrin-independent endocytosis (CIE) was active. Besides, four categories of DEGs acting as temperature sensors were observed, including cell-wall-major-components-metabolism-related (CWMCMR) genes, heat shock protein (Hsp) genes, intracellular-compatible-solutes-metabolism-related (ICSMR) genes and glutathione S-transferase (GST). These results enhance our understanding of the molecular mechanisms of I. cateniannulata in response to temperature stresses and provide a valuable resource for the future investigations.

Characterization of the peripheral blood transcriptome and adaptive evolution of the MHC I and TLR gene families in the wolf (Canis lupus)

BackgroundThe wolf (Canis lupus) is one of the most widely distributed terrestrial mammals, because it is well adapted to various ecological niches and their corresponding pathogen environments. Immunological competence is a crucial factor involved in adapting to a changing environment and fighting pathogen infection in animals. In this study, the peripheral blood transcriptome of wolves was generated via RNA-seq to advance understanding of the wolf immunome, with a special focus on the major histocompatibility complex class I (MHC I) and toll-like receptor (TLR) gene families, which are involved in pathogen recognition and defense.ResultsThe blood transcriptomic libraries of eight wolves originating from Tibet and Inner Mongolia were sequenced, and approximately 383 million reads were generated. Using a genome-guided assembly strategy, we obtained 123,851 unigenes, with a mean length of 845 bp and an N50 length of 1121 bp. On the basis of BLAST searches against the NCBI non-redundant protein database (Nr), a total of 36,192 (29.22%) unigenes were annotated. For functional classification, 24,663 unigenes were assigned to 13,016 Gene Ontology (GO) terms belonging to 51 sub-categories of the three main GO categories. Additionally, 7682 unigenes were classified into 6 Kyoto Encyclopedia of Genes and Genomes (KEGG) categories, in which the most represented functional sub-categories were signal transduction and the immune system, and 16,238 unigenes were functionally classified into 25 Eukaryotic Orthologous Groups (KOG) categories. We observed an overall higher ω (dN/dS) value at antigen-binding sites (ABSs) than at non-ABS regions as well as clear evidence of intergenic/intragenic recombination events at wolf MHC I loci. Additionally, our analysis revealed that carnivorous TLRs were dominated by purifying selection, with mean ω values at each TLR locus ranging from 0.173 to 0.527. However, we also found significant instances of positive selection that acted on several codons in pathogen recognition domains and were linked to species-specific differences in pathogen recognition.ConclusionsThis study represents the first attempt to characterize the blood transcriptome of the wolf and to highlight the value of investigating the immune system. Balancing selection and recombination have contributed to the historical evolution of wolf MHC I genes. Moreover, TLRs in carnivores have undergone adaptive evolution against the background of purifying selection, and a high level of adaptive evolution was detected in the wolf TLR system.

The developmental transcriptome of the bamboo snout beetle Cyrtotrachelus buqueti and insights into candidate pheromone-binding proteins.

Cyrtotrachelus buqueti is an extremely harmful bamboo borer, and the larvae of this pest attack clumping bamboo shoots. Pheromone-binding proteins (PBPs) play an important role in identifying insect sex pheromones, but the C. buqueti genome is not readily available for PBP analysis. Developmental transcriptomes of eggs, larvae from the first instar to the prepupal stage, pupae, and adults (females and males) from emergence to mating were built by RNA sequencing (RNA-Seq) in the present study to establish a sequence background of C. buqueti to help understand PBPs. Approximately 164.8 million clean reads were obtained and annotated into 108,854 transcripts. These were assembled into 24,338, 21,597, 24,798, 21,886, 24,642, and 83,115 unigenes for eggs, larvae, pupae, females, males, and the combined datasets, respectively. Unigenes were annotated against NCBI non-redundant protein sequences, NCBI non-redundant nucleotide sequences, Gene Ontology (GO), Protein family, Clusters of Orthologous Groups of Proteins/ Clusters of Eukaryotic Orthologous Groups (KOG), Swiss-Prot, and KEGG Orthology databases. A total of 17,213 unigenes were annotated into 55 sub-categories belonging to three main GO categories; 10,672 unigenes were classified into 26 functional categories by KOG classification, and 8,063 unigenes were classified into five functional KEGG categories. RSEM software for RNA sequencing showed that 4,816, 3,176, 3,661, 2,898, 4,316, 8,019, 7,273, 5,922, 5,844, and 4,570 genes were differentially expressed between larvae and males, larvae and eggs, larvae and pupae, larvae and females, males and females, males and eggs, males and pupae, females and eggs, females and pupae, and eggs and pupae, respectively. Of these, three were confirmed to be significantly differentially expressed between larvae, females, and males. Furthermore, PBP Cbuq7577_g1 was highly expressed in the antenna of males. A comprehensive sequence resource of a desirable quality was constructed from developmental transcriptomes of C. buqueti eggs, larvae, pupae, and adults. This work enriches the genomic data of C. buqueti, and facilitates our understanding of its metamorphosis, development, and response to environmental change. The identified candidate PBP Cbuq7577_g1 might play a crucial role in identifying sex pheromones, and could be used as a targeted gene to control C. buqueti numbers by disrupting sex pheromone communication.