De Novo Transcriptome Assembly Research Articles

Differential gene expression in leaves and roots of Hydrangea serrata treated with aluminium chloride.

Hydrangea serrata, also knowen as the Japanese tea hortensia, is known for its sweet taste and health properties of bevarages produced from this plant. The H. serrata 3,4-dihydroisocoumarins, hydrangenol and phyllodulcin harbour a variety of biological activities and pharmacological properties. Therefore, a detailed understanding of dihydroisocoumarin biosynthesis in H. serrata is of major interest. Their biosynthesis is assumed to be enhanced by elicitors and mediated by polyketide synthases like in cases of phenylpropanoid derived phytoalexins. A de-novo transcriptome assembly of leaves and roots from the aluminium chloride treatment group versus the control group alongside with annotation was generated. Secondary plant metabolites were analysed by LC-MS. It revealed that a terpene synthase and a triterpenoid synthase gene as well as lignin biosynthesis encoding genes were upregulated in roots. Many genes for transporters, glycosyl, and other transferases as well as glycosylases were found to be differentially expressed in both organs. As no differentially expressed polyketide synthase gene homolog was found, the relative leaf and root 3,4-dihydroisocoumarin content was analysed by LC-MS measurement. Although Hydrangea species are known for their aluminium detoxification using phenylpropanoid-derived compounds, the levels of 3,4- dihydroisocoumarins were not enhanced. In this metabolite analysis, an organ- specific accumulation profile of hydrangenol, phyllodulcin, hydrangeic acid and their mono- and di-glycosides was figured out.

Assessment and Partial Characterization of Candidate Genes in Dihydrochalcone and Arbutin Biosynthesis in an Apple-Pear Hybrid by De Novo Transcriptome Assembly.

Apples (Malus × domestica Borkh.) and pears (Pyrus communis L.) are valuable crops closely related within the Rosaceae family with reported nutraceutical properties derived from secondary metabolites including phloridzin and arbutin, which are distinctive phenolic metabolites characterizing apples and pears, respectively. Here, we generated a de novo transcriptome assembly of an intergeneric hybrid between apple and pear, accumulating intermediate levels of phloridzin and arbutin. Combining RNA-seq, in silico functional annotation prediction, targeted gene expression analysis, and expression-metabolite correlations, we identified candidate genes for functional characterization, resulting in the identification of active arbutin synthases in the hybrid and parental genotypes. Despite exhibiting an active arbutin synthase in vitro, the natural lack of arbutin in apples is reasoned by the absence of the substrate and broad substrate specificity. Altogether, our study serves as the basis for future assessment of potential physiological roles of identified genes by genome editing of hybrids and pears.

Immunomodulatory functions of the gut protist Pentatrichomonas hominis in health and disease

Abstract Protists have been identified as part of the mammalian gut microbiome and are known to impact gut immunity and complex intestinal diseases such as inflammatory bowel disease (IBD). In humans, studies mainly describe host interactions with disease-causing species, while little is known about how commensal protists impact the immune system. Protists from the Parabasalid family, including Pentatrichomonas hominis (P. hominis), have been identified in about 30% of healthy individuals; however, the role of these non-pathogenic commensal Parabasalids remains unstudied. Our data shows that P. hominis promotes Treg differentiation in vitro. Interestingly, this ability was found to be contact-independent, as culture supernatant was sufficient to induce Tregs. Moreover, by doing de-novo genome and transcriptome assembly and high-resolution mass spectrometry we have identified immunomodulatory candidate pathways and metabolites that might be mediating this effect. Re-establishment of tolerance is a major therapeutic strategy for treating intestinal diseases. Hence, we were interested in studying P. hominis impact on the gut immune landscape. To that aim, we successfully and stably colonized mice with P. hominis in gnotobiotic housing conditions and under antibiotic regimen and we are testing P. hominis role under steady state and IBD mouse models. Altogether, our research will address a present gap in knowledge, potentiating the study of protists in the microbiome and their association with intestinal disorders.

De Novo Transcriptome Assembly and Comparative Analysis of Differentially Expressed Genes Involved in Cold Acclimation and Freezing Tolerance of the Arctic Moss Aulacomnium turgidum (Wahlenb.) Schwaegr.

Cold acclimation refers to a phenomenon in which plants become more tolerant to freezing after exposure to non-lethal low temperatures. Aulacomnium turgidum (Wahlenb.) Schwaegr is a moss found in the Arctic that can be used to study the freezing tolerance of bryophytes. To improve our understanding of the cold acclimation effect on the freezing tolerance of A. turgidum, we compared the electrolyte leakage of protonema grown at 25 °C (non-acclimation; NA) and at 4 °C (cold acclimation; CA). Freezing damage was significantly lower in CA plants frozen at -12 °C (CA-12) than in NA plants frozen at -12 °C (NA-12). During recovery at 25 °C, CA-12 demonstrated a more rapid and greater level of the maximum photochemical efficiency of photosystem II than NA-12, indicating a greater recovery capacity for CA-12 compared to NA-12. For the comparative analysis of the transcriptome between NA-12 and CA-12, six cDNA libraries were constructed in triplicate, and RNA-seq reads were assembled into 45,796 unigenes. The differential gene expression analysis showed that a significant number of AP2 transcription factor genes and pentatricopeptide repeat protein-coding genes related to abiotic stress and the sugar metabolism pathway were upregulated in CA-12. Furthermore, starch and maltose concentrations increased in CA-12, suggesting that cold acclimation increases freezing tolerance and protects photosynthetic efficiency through the accumulation of starch and maltose in A. turgidum. A de novo assembled transcriptome can be used to explore genetic sources in non-model organisms.

De-novo transcriptome assembly and analysis of lettuce plants grown under red, blue or white light

Lettuce (Lactuca sativa) is grown in various parts of the world for use as a leafy vegetable. Although the use of light-emitting diode (LED) in controlled plant production systems has been successfully used to enhance nutritional quality and plant growth efficiently, the molecular basis of lettuce’s response to varying light spectra is not studied. Using next-generation sequencing, we have analyzed the transcriptomes of leaf lettuce (Lactuca sativa var. ‘New Red Fire’) grown hydroponically in a modular agricultural production system under three different types of LED lighting: red, blue, and white light. Illumina HiSeq sequencing platform was used to generate paired-end sequence reads (58 Gb raw and 54 Gb clean data) of the transcriptome of lettuce leaves exposed to varying light spectra. The de novo assembled final transcriptome contained 74,096 transcripts. Around 53% and 39% of the assembled transcripts matched to the UniProt and RefSeq RNA sequences, respectively. The validation of the differentially expressed transcripts using RT-qPCR showed complete agreement with RNA-Seq data for 27 transcripts. A comparison of the blue versus red light treatments showed the highest number of significantly differentially expressed transcripts. Among the transcripts significantly up-regulated in blue-light-exposed leaves compared to white-light-exposed leaves, ~ 26% were involved in the ‘response to stress’. Among the transcripts significantly upregulated under red light compared to white light, ~ 6% were associated with ‘nucleosome assembly’ and other processes, such as ‘oxidation–reduction process’ and ‘response to water deprivation’ were significantly enriched. Thus, the result from the current study provides deeper insights into differential gene expression patterns and associated functional aspects under varying light qualities.

RNA-Sequencing of Heterorhabditis nematodes to identify factors involved in symbiosis with Photorhabdus bacteria

BackgroundNematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria.ResultsA de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified.ConclusionsThe symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.

De Novo transcriptome assembly and differential expression analysis of catharanthus roseus in response to salicylic acid

The anti-cancer vinblastine and vincristine alkaloids can only be naturally found in periwinkle (Catharanthus roseus). Both of these alkaloids' accumulations are known to be influenced by salicylic acid (SA). The transcriptome data to reveal the induction effect (s) of SA, however, seem restricted at this time. In this study, the de novo approach of transcriptome assembly was performed on the RNA-Sequencing (RNA-Seq) data in C. roseus. The outcome demonstrated that SA treatment boosted the expression of all the genes in the Terpenoid Indole Alkaloids (TIAs) pathway that produces the vinblastine and vincristine alkaloids. These outcomes supported the time-course measurements of vincristine alkaloid, the end product of the TIAs pathway, and demonstrated that SA spray had a positive impact on transcription and alkaloid synthesis. Additionally, the abundance of transcription factor families including bHLH, C3H, C2H2, MYB, MYB-related, AP2/ ERF, NAC, bZIP, and WRKY suggests a role for a variety of transcription families in response to the SA stimuli. Di-nucleotide and tri-nucleotide SSRs were the most prevalent SSR markers in microsatellite analyses, making up 39% and 34% of all SSR markers, respectively, out of the 77,192 total SSRs discovered.

Development of de-novo transcriptome assembly and SSRs in allohexaploid Brassica with functional annotations and identification of heat-shock proteins for thermotolerance.

Crop Brassicas contain monogenomic and digenomic species, with no evidence of a trigenomic Brassica in nature. Through somatic fusion (Sinapis alba + B. juncea), a novel allohexaploid trigenomic Brassica (H1 = AABBSS; 2n = 60) was produced and used for transcriptome analysis to uncover genes for thermotolerance, annotations, and microsatellite markers for future molecular breeding. Illumina Novaseq 6000 generated a total of 76,055,546 paired-end raw reads, which were used for de-novo assembly, resulting in the development of 486,066 transcripts. A total of 133,167 coding sequences (CDSs) were predicted from transcripts with a mean length of 507.12 bp and 46.15% GC content. The BLASTX search of CDSs against public protein databases showed a maximum of 126,131 (94.72%) and a minimum of 29,810 (22.39%) positive hits. Furthermore, 953,773 gene ontology (GO) terms were found in 77,613 (58.28%) CDSs, which were divided into biological processes (49.06%), cellular components (31.67%), and molecular functions (19.27%). CDSs were assigned to 144 pathways by a pathway study using the KEGG database and 1,551 pathways by a similar analysis using the Reactome database. Further investigation led to the discovery of genes encoding over 2,000 heat shock proteins (HSPs). The discovery of a large number of HSPs in allohexaploid Brassica validated our earlier findings for heat tolerance at seed maturity. A total of 15,736 SSRs have been found in 13,595 CDSs, with an average of one SSR per 4.29 kb length and an SSR frequency of 11.82%. The first transcriptome assembly of a meiotically stable allohexaploid Brassica has been given in this article, along with functional annotations and the presence of SSRs, which could aid future genetic and genomic studies.

Delineating genome-wide alternative splicing landscapes and their functional significance in orchids

Orchids are one of the most attractive groups of flowers with exceptional morphological diversity and high commercial value. Orchid plants are evolved to thrive in diverse habitats under different climatic conditions. Alternative splicing (AS) is an important post-transcriptional regulatory mechanism to modulate transcript diversity for necessary physiological functions in plants. Present study deals with the genome-wide AS landscapes and their functional relevance in six orchid species, information on which was inadequate so far. A total of 180 transcriptome datasets representing different tissues and treatments were chosen from the orchid taxa with well-annotated, publicly accessible genome asssemblies. Cleaned RNA-seq reads were aligned against corresponding reference genomes followed by de-novo transcriptome assembly and detection of novel AS events with significant read support. Alternative acceptor sites (A3S) and intron retentions (IR) were the most prevalent type of AS events. Under cold acclimation, exon skipping events were observed to be elevated in D. officinale leaves. Incidences of intron retentions were elevated in response to pathogen stress, that further amplified offering jasmonate mediated immunity in D. catenatum. GO enrichment of AS-affected genes suggested that most of them are functionally involved in cellular metabolism and RNA processing. A major fraction of genes could be detected that are not differentially expressed in general, however, specific splice variants of them are significantly up/down-regulated with the biotic or abiotic stress mediated responses. Functional information on the AS-affected genes and expressional dynamics of the resultant isoforms would serve as potential genomic resources for orchid research.

Assembly-free rapid differential gene expression analysis in non-model organisms using DNA-protein alignment

BackgroundRNA-seq is being increasingly adopted for gene expression studies in a panoply of non-model organisms, with applications spanning the fields of agriculture, aquaculture, ecology, and environment. For organisms that lack a well-annotated reference genome or transcriptome, a conventional RNA-seq data analysis workflow requires constructing a de-novo transcriptome assembly and annotating it against a high-confidence protein database. The assembly serves as a reference for read mapping, and the annotation is necessary for functional analysis of genes found to be differentially expressed. However, assembly is computationally expensive. It is also prone to errors that impact expression analysis, especially since sequencing depth is typically much lower for expression studies than for transcript discovery.ResultsWe propose a shortcut, in which we obtain counts for differential expression analysis by directly aligning RNA-seq reads to the high-confidence proteome that would have been otherwise used for annotation. By avoiding assembly, we drastically cut down computational costs – the running time on a typical dataset improves from the order of tens of hours to under half an hour, and the memory requirement is reduced from the order of tens of Gbytes to tens of Mbytes. We show through experiments on simulated and real data that our pipeline not only reduces computational costs, but has higher sensitivity and precision than a typical assembly-based pipeline. A Snakemake implementation of our workflow is available at: https://bitbucket.org/project_samar/samar.ConclusionsThe flip side of RNA-seq becoming accessible to even modestly resourced labs has been that the time, labor, and infrastructure cost of bioinformatics analysis has become a bottleneck. Assembly is one such resource-hungry process, and we show here that it can be avoided for quick and easy, yet more sensitive and precise, differential gene expression analysis in non-model organisms.

Proteomic characterization of pilot scale hot-water extracts from the industrial carrageenan red seaweed Eucheuma denticulatum

Seaweeds have a long history as a resource for polysaccharides/hydrocolloids extraction for use in the food industry due to their functionality as stabilizing agents. In addition to the carbohydrate content, seaweeds also contains a significant amount of protein, which may find application in food and feed. Here, we present a novel combination of transcriptomics, proteomics, and bioinformatics to determine the protein composition in two pilot-scale extracts from Eucheuma denticilatum (Spinosum) obtained via hot-water extraction. Although the quality of extracted protein appeared quite poor based on SDS-PAGE analysis, extracts were characterized by qualitative and quantitative proteomics using LC-MS/MS and a de-novo transcriptome assembly for construction of a suitable protein database. A novel concept of length-normalization for relative quantification of sub-optimal protein extracts with partial, non-specific digestion is introduced and validated against conventional methods for relative quantification of proteins. Despite a limited number of protein identifications due to poor protein quality, our data suggest that the majority of quantified protein in the extracts (>75%) is constituted by merely three previously uncharacterized proteins. Putative subcellular localization for the quantified proteins was determined by bioinformatic prediction using several predictors, and by correlating with the expected copy number from the transcriptome analysis, we find that the extracts appear highly enriched in extracellular proteins. This implies that the extraction method used predominantly extracts extracellular proteins, and thus appear ineffective for cellular disruption and subsequent release of intracellular proteins. Nevertheless, the highly abundant proteins may be potential substrates for targeted hydrolysis and release of bioactive peptides. Ultimately, this study highlight the potential of quantitative proteomics for characterization of alternative protein sources intended for use in foods and evaluating protein extraction process efficiency through novel combinations with bioinformatic analysis.

Developmental changes and metabolic reprogramming during establishment of infection and progression of Trypanosoma brucei brucei through its insect host.

Trypanosoma brucei ssp., unicellular parasites causing human and animal trypanosomiasis, are transmitted between mammals by tsetse flies. Periodic changes in variant surface glycoproteins (VSG), which form the parasite coat in the mammal, allow them to evade the host immune response. Different isolates of T. brucei show heterogeneity in their repertoires of VSG genes and have single nucleotide polymorphisms and indels that can impact on genome editing. T. brucei brucei EATRO1125 (AnTaR1 serodeme) is an isolate that is used increasingly often because it is pleomorphic in mammals and fly transmissible, two characteristics that have been lost by the most commonly used laboratory stocks. We present a genome assembly of EATRO1125, including contigs for the intermediate chromosomes and minichromosomes that serve as repositories of VSG genes. In addition, de novo transcriptome assemblies were performed using Illumina sequences from tsetse-derived trypanosomes. Reads of 150 bases enabled closely related members of multigene families to be discriminated. This revealed that the transcriptome of midgut-derived parasites is dynamic, starting with the expression of high affinity hexose transporters and glycolytic enzymes and then switching to proline uptake and catabolism. These changes resemble the transition from early to late procyclic forms in culture. Further metabolic reprogramming, including upregulation of tricarboxylic acid cycle enzymes, occurs in the proventriculus. Many transcripts upregulated in the salivary glands encode surface proteins, among them 7 metacyclic VSGs, multiple BARPs and GCS1/HAP2, a marker for gametes. A novel family of transmembrane proteins, containing polythreonine stretches that are predicted to be O-glycosylation sites, was also identified. Finally, RNA-Seq data were used to create an optimised annotation file with 5' and 3' untranslated regions accurately mapped for 9302 genes. We anticipate that this will be of use in identifying transcripts obtained by single cell sequencing technologies.

Single-cell Long Non-coding RNA Landscape of T Cells in Human Cancer Immunity

The development of new biomarkers or therapeutic targets for cancer immunotherapies requires deep understanding of T cells. To date, the complete landscape and systematic characterization of long noncoding RNAs (lncRNAs) in T cells in cancer immunity are lacking. Here, by systematically analyzing full-length single-cell RNA sequencing (scRNA-seq) data of more than 20,000 libraries of T cells across three cancer types, we provided the first comprehensive catalog and the functional repertoires of lncRNAs in human T cells. Specifically, we developed a custom pipeline for de novotranscriptome assembly and obtained a novel lncRNA catalog containing 9433 genes. This increased the number of current human lncRNA catalog by 16% and nearly doubled the number of lncRNAs expressed in T cells. We found that a portion of expressed genes in single T cells were lncRNAs which had been overlooked by the majority of previous studies. Based on metacell maps constructed by the MetaCell algorithm that partitions scRNA-seq datasets into disjointed and homogenous groups of cells (metacells), 154 signature lncRNA genes were identified. They were associated with effector, exhausted, and regulatory T cell states. Moreover, 84 of them were functionally annotated based on the co-expression networks, indicating that lncRNAs might broadly participate in the regulation of T cell functions. Our findings provide a new point of view and resource for investigating the mechanisms of T cell regulation in cancer immunity as well as for novel cancer-immune biomarker development and cancer immunotherapies

Proteotranscriptomics assisted gene annotation and spatial proteomics of Bombyx mori BmN4 cell line

BackgroundThe process of identifying all coding regions in a genome is crucial for any study at the level of molecular biology, ranging from single-gene cloning to genome-wide measurements using RNA-seq or mass spectrometry. While satisfactory annotation has been made feasible for well-studied model organisms through great efforts of big consortia, for most systems this kind of data is either absent or not adequately precise.ResultsCombining in-depth transcriptome sequencing and high resolution mass spectrometry, we here use proteotranscriptomics to improve gene annotation of protein-coding genes in the Bombyx mori cell line BmN4 which is an increasingly used tool for the analysis of piRNA biogenesis and function. Using this approach we provide the exact coding sequence and evidence for more than 6200 genes on the protein level. Furthermore using spatial proteomics, we establish the subcellular localization of thousands of these proteins. We show that our approach outperforms current Bombyx mori annotation attempts in terms of accuracy and coverage.ConclusionsWe show that proteotranscriptomics is an efficient, cost-effective and accurate approach to improve previous annotations or generate new gene models. As this technique is based on de-novo transcriptome assembly, it provides the possibility to study any species also in the absence of genome sequence information for which proteogenomics would be impossible.

TRANSCRIPTOME ANALASYS OF ADULT GULL-TAPEWORM DIBOTHRIOCHEPHALLUS DENDRITICUS (CESTODA)

In certain regions of Siberia and Baikal area, the main causative agent of diphyllobothriasis of human and animals is gull-tipeworm Dibothriochephallus dendriticus. In last decade, there was heightened interest to the study of molecules secreted by helminth which can modulate the immune response of their mammal hosts, particularly human. Bioinformatic analysis of transcriptomes gives opportunity to obtain data on proteins synthetized in certain time point and find among them ones the most significant for immunological functions. The aim of our study was to carry out transcriptome analysis of adult D. dendriticus. Adult D. dendriticus were pulled from the gut of herring gull. Total RNA was extracted with TRIzol reagent. Bar-coding transcriptome libraries were created. Sequencing obtained libraries was carried out using high-performance sequencer Illumina NextSeq550. De-novo assembly of transcriptome was performed. Annotation of obtained transcripts was done with Blast2Go software. Annotation allowed to describe their classification in terms of biological processes, molecular functions, and cellular components. At the next step, we plan to use D. dendriticus, assembled and annotated with transcriptome of plerocercoids, to search genes, potentially coding proteins that have an immunomodulatory effect on their hosts and also for identification of the proteins in secretome of parasites.

De Novo Transcriptome Assembly and Annotation of Liver and Brain Tissues of Common Brushtail Possums (Trichosurus vulpecula) in New Zealand: Transcriptome Diversity after Decades of Population Control.

The common brushtail possum (Trichosurus vulpecula), introduced from Australia in the mid-nineteenth century, is an invasive species in New Zealand where it is widespread and forms the largest self-sustained reservoir of bovine tuberculosis (Mycobacterium bovis) among wild populations. Conservation and agricultural authorities regularly apply a series of population control measures to suppress brushtail possum populations. The evolutionary consequence of more than half a century of intensive population control operations on the species’ genomic diversity and population structure is hindered by a paucity of available genomic resources. This study is the first to characterise the functional content and diversity of brushtail possum liver and brain cerebral cortex transcriptomes. Raw sequences from hepatic cells and cerebral cortex were assembled into 58,001 and 64,735 transcripts respectively. Functional annotation and polymorphism assignment of the assembled transcripts demonstrated a considerable level of variation in the core metabolic pathways that represent potential targets for selection pressure exerted by chemical toxicants. This study suggests that the brushtail possum population in New Zealand harbours considerable variation in metabolic pathways that could potentially promote the development of tolerance against chemical toxicants.

De novo sequencing and transcriptome analysis of Indian Bael (Aegle marmelos L.)

Aegle marmelos (L.) also known as Bael is a medicinally important tree known for its antidiabetic properties. It is considered as a sacred plant but lacks sufficient genomic and transcriptomic resources. The present study investigated functional profile of A. marmelos via RNA sequencing technology. The denovo transcriptome sequencing and assembly of 4.7 million high quality reads yielded 27,393 unigenes, which were annotated against the databases such as NCBI (National Centre for Biotechnology Information), NR (Non redundant) database, KEGG, Pfam (Protein Family), CDD (Conserved Domains database) and Uniprot. The annotation of assembled transcripts showed a total of 1291 transcripts mapped to 81 different transcription factor families, whereas 55 transcripts were assigned to CYP450 belonging to 21 different classes. KEGG mapping showed transcripts involved in the biosynthesis of terpenoids, flavonoids, including transcripts for synthesis of important compounds like Vitamin E, 4-coumarate and Vitamin K epoxide. 1211 simple sequence repeats (SSRs) were identified which may aid in molecular breeding studies. The study provides an insight into the expression profile of Bael and an opportunity for future genomic and metabolic studies in A.marmelos.

De novo leaf transcriptome assembly of Bougainvillea spectabilis for the identification of genes involves in the secondary metabolite pathways.

Bougainvillea spectabilis is known as a vital medicinal, ornamental as well as an essential oil producing plant. It is also a rich source of important secondary metabolites with several therapeutic properties. Various studies on its pharmacological and toxicological aspects have been published but there is no genomic or transcriptomic resource available in the public databases. To address this important issue, the de-novo transcriptome assembly of B. spectabilis leaf tissue has been done for the identification of genes involved in various important secondary metabolites, Single nucleotide polymorphism (SNPs) and Simple sequence repeats (SSRs). The transcriptome sequencing of B. spectabilis leaf tissue generated 79,811,024 raw reads with GC value 42.77%. The transcriptomic assembly was performed by Trinity software which generated 100,374 transcripts and 99,793 unigenes with minimum and maximum length of 201bp and 13,237bp and N50 value of 1470 and 1472 respectively. Annotation of these unigenes was performed using seven databases including NR, PFAM, GO and KEGG. Approximately, 44,302 unigenes were annotated in GO database. The KEGG pathway analysis revealed 23,102 unigenes in which 19,054 genes were assigned to five groups in KEGG and 130 biochemical pathways. The highest group among the five groups was Metabolism with 9230 unigenes. Moreover, about 63,226 SNPs and 30,333 SSRs in the leaf transcriptome of B. spectabilis were identified. To the best of my understanding it will be the first comprehensive transcriptome analysis of B. spectabilis from family Nyctaginaceae which will help as a reference line for further genomic and transcriptomic studies.

De Novo Transcriptome Assembly of Eucalyptus nitens and the Expression of R2R3-MYB Genes in Response to Cold Acclimation in Eucalyptus Spp.

Most of Eucalyptus species used in commercial forestry are frost sensitive. One of the few species with a higher frost tolerance is Eucalyptus nitens which can tolerate temperatures of − 15 °C, in contrast to Eucalyptus globulus, a frost-sensitive species, but an important species for cellulose production. Considering the excellent wood traits of E. globulus, relevant to the forestry industry, and the frost tolerance of E. nitens breeding programs has led to the generation of interspecific hybrids (Eucalyptus nitens × Eucalyptus globulus). MYB transcription factors (TFs) have been reported as key elements in the response to cold and freezing temperatures, especially the R2R3-MYB-type proteins involved in the biosynthesis of flavonoids, such as AtMYB12 and AtMYB111. In order to unravel the relation of MYB TFs and the cold acclimation (CA) process, a de novo transcriptome assembly from RNA-seq data was performed from non-acclimated, cold acclimated, and deacclimated plants of E. nitens. Twenty MYBs were identified by in silico analysis and ten of them were R2R3-type. MYB64 and MYB68, two R2R3-MYBs, showed the higher transcript abundance in response to CA. MYB64 and MYB68 were validated by qRT-PCR in samples from cold acclimated plants of a cold-tolerant species E. nitens, a cold-sensitive species, E. globulus, and a hybrid of E.nitens × E.globulus, characterized as a cold-tolerant genotype. MYB64, homologous to AtMYB12, showed a higher expression in the cold-tolerant species revealing a possible mechanism mediated by MYB TFs in response to CA in tolerant species of Eucalyptus spp.

Novel DNA Barcode Sequence Discovery from Transcriptome of <i>Acheta domesticus</i>: A Partial Mitochondrial DNA

The potential of mitochondrial DNA (mtDNA) genes are well-known for species identification and to establish a phylogenetic relationship. The De-novo transcriptome assembly of Acheta domesticus commonly known as house cricket, is provides important segments of DNA fragments from mitochondrial DNA due to higher abundance of its mRNA. When the reference sequence with gene annotation is absent for assembling and aligning desire gene sequences, like in the present case, the most similar sequence is obtained from online insect mitochondrial genome database to find mitochondrial DNA conserved domains of interested gene from high throughput RNA sequencing (RNA-seq) data. The RNA-seq data of Acheta domesticus transcriptome is used to retrieve single nucleotide fragment out of 50,046 assembled contigs to discover three important genes from mtDNA of the house cricket. Present study provides effective workflow to identify genes like cytochrome c oxidase subunit II (COX2), NADH dehydrogenase subunit 2 (ND2), cytochrome c oxidase subunit I (COX1) from mtDNA in large sequence archive of RNA-seq data. These three novel barcode sequences will be useful for genetic identification and evolution investigation of Acheta domesticus. The partial mtDNA sequence with these genes will be important for mitochondrial genome construction.