The Chemosensory Transcriptome of a Diving Beetle

Insects astoundingly dominate Earth’s land ecosystems and have a huge impact on human life. Almost every aspect of their life relies upon their highly efficient and adaptable chemosensory system. In the air, most chemical signals that are detected at long range are hydrophobic molecules, which insects detect using proteins encoded by multigenic families that emerged following land colonization by insect ancestors, namely the odorant-binding proteins (OBPs) and the odorant receptors (ORs). However, land-to-freshwater transitions occurred in many lineages within the insect tree of life. Whether chemosensory gene repertoires of aquatic insects remained essentially unchanged or underwent more or less drastic modifications to cope with physico-chemical constraints associated with life underwater remains virtually unknown. To address this issue, we sequenced and analyzed the transcriptome of chemosensory organs of the diving beetle Rhantus suturalis (Coleoptera, Dytiscidae). A reference transcriptome was assembled de novo using reads from five RNA-seq libraries (male and female antennae, male and female palps, and wing muscle). It contained 47,570 non-redundant unigenes encoding proteins of more than 50 amino acids. Within this reference transcriptome, we annotated sequences coding 53 OBPs, 48 ORs, 73 gustatory receptors (GRs), and 53 ionotropic receptors (IRs). Phylogenetic analyses notably revealed a large OBP gene expansion (35 paralogs in R. suturalis) as well as a more modest OR gene expansion (9 paralogs in R. suturalis) that may be specific to diving beetles. Interestingly, these duplicated genes tend to be expressed in palps rather than in antennae, suggesting a possible adaptation with respect to the land-to-water transition. This work provides a strong basis for further evolutionary and functional studies that will elucidate how insect chemosensory systems adapted to life underwater.

BaRTv2: A highly resolved barley reference transcriptome for accurate transcript-specific RNA-seq quantification

Accurate characterization of splice junctions as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data and enable detailed investigations of transcriptional and post-transcriptional regulation. Using novel computational methods and a combination of PacBio Iso-seq and Illumina short read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset (RTD) from the European 2-row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the splice junctions and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared to an earlier version, BaRTv1.0. The accuracy of transcript level quantification, splice junctions and transcript start and end sites has been validated extensively using parallel technologies and analysis, including high resolution RT PCR and 5 prime RACE. BaRTv2.18 contains 39,434 genes and 148,260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high-quality resource for advanced transcriptomic analyses, including both transcriptional and post-transcriptional regulation, with exceptional resolution and precision.

Reference Transcriptome Data in Silkworm Bombyx mori

Herein, we performed RNA-seq analysis of ten major tissues/subparts of silkworm larvae. The sequences were mapped onto the reference genome assembly and the reference transcriptome data were successfully constructed. The reference data provided a nearly complete sequence for sericin-1, a major silk gene with a complex structure. We also markedly improved the gene model for other genes. The transcriptomic expression was investigated in each tissue and a number of transcripts were identified that were exclusively expressed in tissues such as the testis. Transcripts strongly expressed in the midgut formed tight genomic clusters, suggesting that they originated from tandem gene duplication. Transcriptional factor genes expressed in specific tissues or the silk gland subparts were also identified. We successfully constructed reference transcriptome data in the silkworm and found that a number of transcripts showed unique expression profiles. These results will facilitate basic studies on the silkworm and accelerate its applications, which will contribute to further advances in lepidopteran and entomological research as well as the practical use of these insects.

Novel Variance-Component TWAS method for studying complex human diseases with applications to Alzheimer’s dementia

Transcriptome-wide association studies (TWAS) have been widely used to integrate transcriptomic and genetic data to study complex human diseases. Within a test dataset lacking transcriptomic data, traditional two-stage TWAS methods first impute gene expression by creating a weighted sum that aggregates SNPs with their corresponding cis-eQTL effects on reference transcriptome. Traditional TWAS methods then employ a linear regression model to assess the association between imputed gene expression and test phenotype, thereby assuming the effect of a cis-eQTL SNP on test phenotype is a linear function of the eQTL’s estimated effect on reference transcriptome. To increase TWAS robustness to this assumption, we propose a novel Variance-Component TWAS procedure (VC-TWAS) that assumes the effects of cis-eQTL SNPs on phenotype are random (with variance proportional to corresponding reference cis-eQTL effects) rather than fixed. VC-TWAS is applicable to both continuous and dichotomous phenotypes, as well as individual-level and summary-level GWAS data. Using simulated data, we show VC-TWAS is more powerful than traditional TWAS methods based on a two-stage Burden test, especially when eQTL genetic effects on test phenotype are no longer a linear function of their eQTL genetic effects on reference transcriptome. We further applied VC-TWAS to both individual-level (N = ~3.4K) and summary-level (N = ~54K) GWAS data to study Alzheimer’s dementia (AD). With the individual-level data, we detected 13 significant risk genes including 6 known GWAS risk genes such as TOMM40 that were missed by traditional TWAS methods. With the summary-level data, we detected 57 significant risk genes considering only cis-SNPs and 71 significant genes considering both cis- and trans- SNPs, which also validated our findings with the individual-level GWAS data. Our VC-TWAS method is implemented in the TIGAR tool for public use.

Construction of a reference transcriptome for the analysis of male sterility in sugi (Cryptomeria japonica D. Don) focusing on MALE STERILITY 1 (MS1)

Sugi (Cryptomeria japonica D. Don) is an important conifer used for afforestation in Japan. As the genome of this species is 11 Gbps, it is too large to assemble within a short timeframe. Transcriptomics is one approach that can address this deficiency. Here we designed a workflow consisting of three stages to de novo assemble transcriptome using Oases and Trinity. The three transcriptomic stage used were independent assembly, automatic and semi-manual integration, and refinement by filtering out potential contamination. We identified a set of 49,795 cDNA and an equal number of translated proteins. According to the benchmark set by BUSCO, 87.01% of cDNAs identified were complete genes, and 78.47% were complete and single-copy genes. Compared to other full-length cDNA resources collected by Sanger and PacBio sequencers, the extent of the coverage in our dataset was the highest, indicating that these data can be safely used for further studies. When two tissue-specific libraries were compared, there were significant expression differences between male strobili and leaf and bark sets. Moreover, subtle expression difference between male-fertile and sterile libraries were detected. Orthologous genes from other model plants and conifer species were identified. We demonstrated that our transcriptome assembly output (CJ3006NRE) can serve as a reference transcriptome for future functional genomics and evolutionary biology studies.

Whole Community Metatranscriptomes and Lipidomes Reveal Diverse Responses Among Antarctic Phytoplankton to Changing Ice Conditions

The transition from winter to spring represents a major shift in the basal energy source for the Antarctic marine ecosystem from lipids and other sources of stored energy to sunlight. Because sea ice imposes a strong control on the transmission of sunlight into the water column during the polar spring, we hypothesized that the timing of the sea ice retreat influences the timing of the transition from stored energy to photosynthesis. To test the influence of sea ice on water column microbial energy utilization we took advantage of unique sea ice conditions in Arthur Harbor, an embayment near Palmer Station on the western Antarctic Peninsula, during the 2015 spring–summer seasonal transition. Over a 5-week period we sampled water from below land-fast sea ice, in the marginal ice zone at nearby Palmer Station B, and conducted an ice removal experiment with incubations of water collected below the land-fast ice. Whole-community metatranscriptomes were paired with lipidomics to better understand how lipid production and utilization was influenced by light conditions. We identified several different phytoplankton taxa that responded similarly to light by the number of genes up-regulated, and in the transcriptional complexity of this response. We applied a principal components analysis to these data to reduce their dimensionality, revealing that each of these taxa exhibited a strikingly different pattern of gene up-regulation. By correlating the changes in lipid concentration to the first principal component of log fold-change for each taxa we could make predictions about which taxa were associated with different changes in the community lipidome. We found that genes coding for the catabolism of triacylglycerol storage lipids were expressed early on in phytoplankton associated with a Fragilariopsis kerguelensis reference transcriptome. Phytoplankton associated with a Corethron pennatum reference transcriptome occupied an adjacent niche, responding favorably to higher light conditions than F. kerguelensis. Other diatom and dinoflagellate taxa had distinct transcriptional profiles and correlations to lipids, suggesting diverse ecological strategies during the polar winter–spring transition.

Pitchers of Nepenthes khasiana express several digestive-enzyme encoding genes, harbor mostly fungi and probably evolved through changes in the expression of leaf polarity genes

Background A structural phenomenon seen in certain lineages of angiosperms that has captivated many scholars including Charles Darwin is the evolution of plant carnivory. Evidently, these structural features collectively termed carnivorous syndrome, evolved to aid nutritional acquisition from attracted, captured and digested prey. We now understand why plant carnivory evolved but how carnivorous plants acquired these attributes remains a mystery. In an attempt to understand the evolution of Nepenthes pitcher and to shed more light on its role in prey digestion, we analyzed the transcriptome data of the highly specialized Nepenthes khasiana leaf comprising the leaf base lamina, tendril and the different parts/zones of the pitcher tube viz. digestive zone, waxy zone and lid. Results In total, we generated around 262 million high-quality Illumina reads. Reads were pooled, normalized and de novo assembled to generate a reference transcriptome of about 412,224 transcripts. We then estimated transcript abundance along the N. khasiana leaf by mapping individual reads from each part/zone to the reference transcriptome. Correlation-based hierarchical clustering analysis of 27,208 commonly expressed genes indicated functional relationship and similar cellular processes underlying the development of the leaf base and the pitcher, thereby implying that the Nepenthes pitcher is indeed a modified leaf. From a list of 2386 differentially expressed genes (DEGs), we identified transcripts encoding key enzymes involved in prey digestion and protection against pathogen attack, some of which are expressed at high levels in the digestive zone. Interestingly, many of these enzyme-encoding genes are also expressed in the unopened N. khasiana pitcher. Transcripts showing homology to both bacteria and fungi were also detected; and in the digestive zone, fungi are more predominant as compared to bacteria. Taking cues from histology and scanning electron microscopy (SEM) photomicrographs, we found altered expressions of key regulatory genes involved in leaf development. Of particular interest, the expression of class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) and ARGONAUTE (AGO) genes were upregulated in the tendril. Conclusions Our findings suggest that N. khasiana pitchers employ a wide range of enzymes for prey digestion and plant defense, harbor microbes and probably evolved through altered expression of leaf polarity genes.

De novo assembly of the Brown trout (Salmo trutta m. fario) brain and muscle transcriptome: transcript annotation, tissue differential expression profile and SNP discovery

Objectives The Brown trout is a salmonid species with a high commercial value in Europe. Life history and spawning behaviour include resident (Salmo trutta m. fario) and migratory (Salmo trutta m. trutta) ecotypes. The main objective is to apply RNA-seq technology in order to obtain a reference transcriptome of two key tissues, brain and muscle, of the riverine trout Salmo trutta m. fario. Having a reference transcriptome of the resident form will complement genomic resources of salmonid species. Data description We generate two cDNA libraries from pooled RNA samples, isolated from muscle and brain tissues of adult individuals of Salmo trutta m. fario, which were sequenced by Illumina technology. Raw reads were subjected to de-novo transcriptome assembly using Trinity, and coding regions were predicted by TransDecoder. A final set of 35,049 non-redundant ORF unigenes were annotated. Tissue differential expression analysis was evaluated by Cuffdiff. A False Discovery Rate (FDR) ≤ 0.01 was considered for significant differential expression, allowing to identify key differentially expressed unigenes. Finally, we have identified SNP variants that will be useful tools for population genomic studies.