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Sichuan pepper (Zanthoxylum armatum DC) is a popular spice and is often prescribed in traditional Chinese medicine to treat vomiting, diarrhea, ascariasis and eczema, among other conditions. Volatile oils from Z. armatum leaves contain active ingredients, with terpenoids being one of the main components. In the present study, the combination of sequencing data of Z. armatum from PacBio single molecule real time (SMRT) and Illumina RNA sequencing (RNA-Seq) platforms facilitated an understanding of the gene regulatory network of terpenoid biosynthesis in pepper leaves. The leaves of three developmental stages from two Z. armatum cultivars, 'Rongchangwuci' (WC) and 'Zhuye' (ZY), were selected as test materials to construct sequencing libraries. A total of 143,122 predictions of unique coding sequences, 105,465 simple sequence repeats, 20,145 transcription factors and 4719 long non-coding RNAs (lncRNAs) were identified, and 142,829 transcripts were successfully annotated. The occurrence of alternative splicing events was verified by reverse transcription PCR, and quantitative real-time PCR was used to confirm the expression pattern of six randomly selected lncRNAs. A total of 96,931 differentially expressed genes were filtered from different samples. According to functional annotation, a total of 560 candidate genes were involved in terpenoid synthesis, of which 526 were differentially expressed genes (DEGs). To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and transcription factors genes, were analyzed using the weighted gene co-expression network method, and the co-expression network of genes was constructed. Thirty-one terpenoids were identified by gas chromatography-mass spectrometry. The correlation between 18 compounds with significantly different contents and genes with high connectivity in the module was jointly analyzed in both cultivars, yielding 12 candidate DEGs presumably involved in the regulation of terpenoid biosynthesis. Our findings showed that full-length transcriptome SMRT and Illumina RNA-Seq can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Huanglongbing (HLB) is a systemic plant disease caused by ‘Candidatus Liberibacter asiaticus (CLas)’ and transmitted by Diaphorina citri. D. citri acquires the CLas bacteria in the nymph stage and transmits it in the adult stage, indicating that molting from the nymph to adult stages is crucial for HLB transmission. However, the available D. citri reference genomes are incomplete, and gene function studies have been limited to date. In the current research, PacBio single-molecule real-time (SMRT) and Illumina sequencing were performed to investigate the transcriptome of D. citri nymphs and adults. In total, 10,641 full-length, non-redundant transcripts (FLNRTs), 594 alternative splicing (AS) events, 4522 simple sequence repeats (SSRs), 1086 long-coding RNAs (lncRNAs), 281 transcription factors (TFs), and 4459 APA sites were identified. Furthermore, 3746 differentially expressed genes (DEGs) between nymphs and adults were identified, among which 30 DEGs involved in the Hippo signaling pathway were found. Reverse transcription–quantitative PCR (RT-qPCR) further validated the expression levels of 12 DEGs and showed a positive correlation with transcriptome data. Finally, the spatiotemporal expression pattern of genes involved in the Hippo signaling pathway exhibited high expression in the D. citri testis, ovary, and egg. Silencing of the D. citri transcriptional co-activator (DcYki) gene significantly increased D. citri mortality and decreased the cumulative molting. Our results provide useful information and a reliable data resource for gene function research of D. citri.

Potato virus Y (PVY) mainly infects Solanaceous crops, resulting in considerable losses in the yield and quality. Iron (Fe) is involved in various biological processes in plants, but its roles in resistance to PVY infection has not been reported. In this study, foliar application of Fe could effectively inhibit early infection of PVY, and a full-length transcriptome and Illumina RNA sequencing was performed to investigate its modes of action in PVY-infected Nicotiana tabacum. The results showed that 18,074 alternative splicing variants, 3,654 fusion transcripts, 3,086 long non-coding RNAs and 14,403 differentially expressed genes (DEGs) were identified. Specifically, Fe application down-regulated the expression levels of the DEGs related to phospholipid hydrolysis, phospholipid signal, cell wall biosynthesis, transcription factors (TFs) and photosystem I composition, while those involved with photosynthetic electron transport chain (PETC) were up-regulated at 1 day post inoculation (dpi). At 3 dpi, these DEGs related to photosystem II composition, PETC, molecular chaperones, protein degradation and some TFs were up-regulated, while those associated with light-harvesting, phospholipid hydrolysis, cell wall biosynthesis were down-regulated. At 9 dpi, Fe application had little effects on resistance to PVY infection and transcript profiles. Functional analysis of these potentially critical DEGs was thereafter performed using virus-induced gene silencing approaches and the results showed that NbCat-6A positively regulates PVY infection, while the reduced expressions of NbWRKY26, NbnsLTP, NbFAD3 and NbHSP90 significantly promote PVY infection in N. benthamiana. Our results elucidated the regulatory network of Fe-mediated resistance to PVY infection in plants, and the functional candidate genes also provide important theoretical bases to further improve host resistance against PVY infection.

The lack of high-quality juvenile crabs is the greatest impediment to the growth of the mud crab (Scylla paramamosain) industry. To obtain high-quality hybrid offspring, a novel hybrid mud crab (S. serrata ♀ × S. paramamosain ♂) was successfully produced in our previous study. Meanwhile, an interesting phenomenon was discovered, that some first-generation (F1) hybrid offspring’s eyestalks were displaced during the crablet stage I. To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, both single-molecule real-time (SMRT) and Illumina RNA sequencing were implemented. Using a two-step collapsing strategy, three high-quality reconstructed transcriptomes were obtained from purebred mud crabs (S. paramamosain) with normal eyestalks (SPA), hybrid crabs with normal eyestalks (NH), and hybrid crabs with displaced eyestalks (DH). In total, 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in DH. The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant GO terms were related to the cuticle or chitin. Overall, high-quality reconstructed transcriptomes were obtained for the novel interspecific hybrid crab and provided valuable insights into the genetic mechanisms of eyestalk displacement in mud crab (Scylla spp.) crossbreeding.

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Background: Aedes (Stegomyia) albopictus (Skuse) is an invasive and widespread mosquito species that can transmit dengue, chikungunya, yellow fever, and Zika viruses. Its control heavily relies on the use of insecticides. However, the efficacy of the insecticide-based intervention is threatened by the increasing development of resistance to available insecticides. Understanding the current status and potential mechanisms of insecticide resistance is an important prerequisite for devising strategies to maintain the sustainability of vector control programs. In this study, we investigated the current status and probable candidate detoxification genes associated with insecticide resistance in the Asian tiger mosquito in Beijing, the capital city of China. Methods: Bioassays were conducted on three field populations of Ae. albopictus collected from urban communities in Beijing by exposure to diagnostic doses of permethrin, deltamethrin, malathion, and propoxur. Differentially expressed genes (DEGs) associated with insecticide resistance were screened by transcriptomic analysis using Illumina RNA sequencing data (RNA-seq) from 12 independent RNA libraries constructed from female strains of the three field populations and one susceptible strain. Results: The bioassay results indicated that all the three field populations were resistant to propoxur (carbamate), deltamethrin, and permethrin (pyrethroids), but susceptible to malathion (organophosphate). Eighteen (18) cytochrome P450s (P450s), five (5) glutathione S-transferases (GSTs), four (4) carboxy/cholinesterases (CCEs), eight (8) UDP-glycosyltransferases (UGTs), and three (3) ATP-binding cassette transporters (ABCs) were found to be significantly overexpressed in the three field populations relative to the susceptible strain via transcriptomic analysis. Conclusion: This study demonstrates that the Ae. albopictus field populations in Beijing exhibit multiple phenotypic resistance to commonly used pyrethroids and carbamate. The identification of a number of DEGs associated with insecticide resistance indicates that the mechanisms underlying resistance in field populations are complicated, and detoxifying enzymes may play important roles. The multiple resistance status detected in the three field populations suggests that resistance management strategies such as insecticide rotation and non-chemical-based measures should be implemented in order to sustain effective control of the disease vector and vector-borne diseases. [ABSTRACT FROM AUTHOR]

In allotetraploid common carp, protein-coding homoeologs presented divergent expression levels between the two subgenomes. However, whether subgenome dominance occurs in other transcriptional and post-transcriptional events remains unknown. Using Illumina RNA sequencing and PacBio full-length sequencing, we refined the common carp transcriptome annotation and explored differences in four transcriptional and post-transcriptional events between the two subgenomes. The results revealed that the B subgenome presented more alternative splicing events, as did lncRNAs and circRNAs. However, the expression levels, tissue specificity, sequence features, and functions of lncRNAs and circRNAs did not significantly differ between the two subgenomes, suggesting a common regulatory mechanism shared by the two subgenomes. Furthermore, both the number and base substitution frequency of RNA editing events were greater in the B subgenome. Functional analyses of these transcriptional events also revealed subgenome bias. Genes that undergo alternative splicing in the A subgenome participate in more biological processes, and lncRNA targets show a preference between subgenomes. CircRNA host genes in the B subgenome were associated with more biological functions, and RNA editing preferentially occurred in noncoding regions or led to nonsynonymous mutations in the B subgenome. Taken together, the refined transcriptome annotation revealed complicated and imbalanced expression strategies in allotetraploid common carp. Comprehensive transcriptomic analysis revealed distinct patterns in expression, regulation, and function of alternative splicing, lncRNA, circRNA,and RNA editing events between the two subgenomes of allotetraploid common carp. [ABSTRACT FROM AUTHOR]

Introduction: Nitraria tangutorum Bobr., a prominent xerophytic shrub, exhibits remarkable adaptability to harsh environment and plays a significant part in preventing desertification in northwest China owing to its exceptional drought and salinity tolerance. Methods: To investigate the drought-resistant mechanism underlying N. tangutorum, we treated 8-week-old seedlings with polyethylene glycol (PEG)- 6000 (20%, m/m) to induce drought stress. 27 samples from different tissues (leaves, roots and stems) of N. tangutorum at 0, 6 and 24 h after drought stress treatment were sequenced using PacBio single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing to obtain a comprehensive transcriptome. Results: The PacBio SMRT sequencing generated 44,829 non-redundant transcripts and provided valuable reference gene information. In leaves, roots and stems, we identified 1162, 2024 and 232 differentially expressed genes (DEGs), respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that plant hormone signaling and mitogen-activated protein kinase (MAPK) cascade played a pivotal role in transmitting stress signals throughout the whole N. tangutorum plant following drought stress. The interconversion of starch and sucrose, as well as the biosynthesis of amino acid and lignin, may represent adaptive strategies employed by N. tangutorum to effectively cope with drought. Transcription factor analysis showed that AP2/ ERF-ERF, WRKY, bHLH, NAC and MYB families were mainly involved in the regulation of drought response genes. Furthermore, eight physiological indexes, including content of proline, hydrogen peroxide (H2O2), malondialdehyde (MDA), total amino acid and soluble sugar, and activities of three antioxidant enzymes were all investigate after PEG treatment, elucidating the drought tolerance mechanism from physiological perspective. The weighted gene co-expression network analysis (WGCNA) identified several hub genes serve as key regulator in response to drought through hormone participation, ROS cleavage, glycolysis, TF regulation in N. tangutorum. Discussion: These findings enlarge genomic resources and facilitate research in the discovery of novel genes research in N. tangutorum, thereby establishing a foundation for investigating the drought resistance mechanism of xerophyte. [ABSTRACT FROM AUTHOR]

Faba bean is an important pulse. It provides proteins for the human diet and is used in industrial foodstuffs, such as flours. Drought stress severely reduces the yield of faba bean, and this can be efficiently overcome through the identification and application of key genes in response to drought. In this study, PacBio and Illumina RNA sequencing techniques were used to identify the key pathways and candidate genes involved in drought stress response. During seed germination, a total of 17,927 full-length transcripts and 12,760 protein-coding genes were obtained. There were 1676 and 811 differentially expressed genes (DEGs) between the varieties E1 and C105 at 16 h and 64 h under drought stress, respectively. Six and nine KEGG pathways were significantly enriched at 16 h and 64 h under drought stress, which produced 40 and 184 nodes through protein–protein interaction (PPI) analysis, respectively. The DEGs of the PPI nodes were involved in the ABA (abscisic acid) and MAPK (mitogen-activated protein kinase) pathways, N-glycosylation, sulfur metabolism, and sugar metabolism. Furthermore, the ectopic overexpression of a key gene, AAT, encoding aspartate aminotransferase (AAT), in tobacco, enhanced drought tolerance. The activities of AAT and peroxidase (POD), the contents of cysteine and isoleucine, were increased, and the contents of malonaldehyde (MDA) and water loss decreased in the overexpressed plants. This study provides a novel insight into genetic response to drought stress and some candidate genes for drought tolerance genetic improvements in this plant. [ABSTRACT FROM AUTHOR]

Bovicola ovis, commonly known as the sheep-biting louse, is an ectoparasite that adversely affects the sheep industry. Sheep louse infestation lowers the quality of products, including wool and leather, causing a loss of approximately AUD 123M per annum in Australia alone. The lack of a high-quality genome assembly for the sheep-biting louse, as well as any closely related livestock lice, has hindered the development of louse research and management control tools. In this study, we present the assembly of B. ovis with a genome size of ~123 Mbp based on a nanopore long-read sequencing library and Illumina RNA sequencing, complemented with a chromosome-level scaffolding using the Pore-C multiway chromatin contact dataset. Combining multiple alignment and gene prediction tools, a comprehensive annotation on the assembled B. ovis genome was conducted and recalled 11,810 genes as well as other genomic features including orf, ssr, rRNA and tRNA. A manual curation using alignment with the available closely related louse species, Pediculus humanus, increased the number of annotated genes to 16,024. Overall, this study reported critical genetic resources and biological insights for the advancement of sheep louse research and the development of sustainable control strategies in the sheep industry. [ABSTRACT FROM AUTHOR]

Oxidative stress represents a critical facet of the array of abiotic stresses affecting crop growth and yield. In this paper, we investigated the potential differences in the functions of two highly homologous Arabidopsis DSS1 proteins in terms of maintaining genome integrity and response to oxidative stress. In the context of homologous recombination (HR), it was shown that overexpressing AtDSS1(I) using a functional complementation test increases the resistance of the Δdss1 mutant of Ustilago maydis to genotoxic agents. This indicates its conserved role in DNA repair via HR. To investigate the global transcriptome changes occurring in dss1 plant mutant lines, gene expression analysis was conducted using Illumina RNA sequencing technology. Individual RNA libraries were constructed from three total RNA samples isolated from dss1(I), dss1(V), and wild-type (WT) plants under hydrogen peroxide-induced stress. RNA-Seq data analysis and real-time PCR identification revealed major changes in gene expression between mutant lines and WT, while the dss1(I) and dss1(V) mutant lines exhibited analogous transcription profiles. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed significantly enriched metabolic pathways. Notably, genes associated with HR were upregulated in dss1 mutants compared to the WT. Otherwise, genes of the metabolic pathway responsible for the synthesis of secondary metabolites were downregulated in both dss1 mutant lines. These findings highlight the importance of understanding the molecular mechanisms of plant responses to oxidative stress. [ABSTRACT FROM AUTHOR]

Drought stress can significantly affect plant growth, development, and yield. Fewer comparative studies have been conducted between different species of pines, particularly involving Pinus yunnanensis var. pygmaea (P. pygmaea). In this study, the physiological indices, photosynthetic pigment and related antioxidant enzyme changes in needles from P. pygmaea, P. elliottii and P. massoniana under drought at 0, 7, 14, 21, 28 and 35 d, as well as 7 days after rehydration, were measured. The PacBio single-molecule real-time (SMRT) and Illumina RNA sequencing were used to uncover the gene expression differences in P. pygmaea under drought and rehydration conditions. The results showed that the total antioxidant capacity (TAOC) of P. pygmaea was significantly higher than P. massoniana and P. elliottii. TAOC showed a continuous increase trend across all species. Soluble sugar (SS), starch content and non-structural carbohydrate (NSC) of all three pines displayed a "W" pattern, declining initially, increasing, and then decreasing again. P. pygmaea exhibits stronger drought tolerance and greater recovery ability under prolonged drought conditions. Through the PacBio SMRT-seq, a total of 50,979 high-quality transcripts were generated, and 6,521 SSR and 5,561 long non-coding RNAs (LncRNAs) were identified. A total of 2310, 1849, 5271, 5947, 7710, and 6854 differentially expressed genes (DEGs) were identified compared to the control (Pp0D) in six pair-wise comparisons of treatment versus control. bHLH, NAC, ERF, MYB_related, C3H transcription factors (TFs) play an important role in drought tolerance of P. pygmaea. KEGG enrichment analysis and Gene set enrichment analysis (GSEA) analysis showed that P. pygmaea may respond to drought by enhancing metabolic processes such as ABA signaling pathway, alpha-linolenic acid. Weighted gene co-expression network analysis (WGCNA) revealed GST, CAT, LEC14B, SEC23 were associated with antioxidant enzyme activity and TAOC. This study provides a basis for further research on drought tolerance differences among coniferous species. [ABSTRACT FROM AUTHOR]

The African grass or Nile rat (NR) (Arvicanthis niloticus) is a herbivorous diurnal rodent which is used as a biological model for research on type 2 diabetes mellitus (T2DM) and the circadian rhythm. Similar to humans, male NRs develop T2DM with high-carbohydrate diets. The NR thus provides a unique opportunity to identify the nutritional and underlying genetic factors that characterise human T2DM, as well as the effects of potential anti-diabetic phytochemicals such as Water-Soluble Palm Fruit Extract. Whole genome sequencing (WGS) could help identify possible genetic causes why NRs spontaneously develop T2DM in captivity. In this study, we performed WGS on a hepatic deoxyribonucleic acid (DNA) sample isolated from a male NR using PacBio high-fidelity long-read sequencing. The WGS data obtained were then de novo assembled and annotated using PacBio HiFi isoform sequencing (Iso-Seq) data as well as previous Illumina RNA sequencing (RNA-Seq) data. Genes related to insulin and circadian rhythm pathways were present in the NR genome, similar to orthologues in the rat, mouse and human genomes. T2DM development in the NR is thus most likely not attributable to structural differences in these genes when compared to other biological models. Further studies are warranted to gain additional insights on the genetic-environmental factors which underlie the genetic permissiveness of NRs to develop T2DM. [ABSTRACT FROM AUTHOR]

The larval waste, exoskeleton shedding, and leftover feed components of the black soldier fly and its larvae make up the by-product known as frass. In this study, we subjected channel catfish (Ictalurus punctatus) to a 10-week feeding trial to assess how different dietary amounts of frass inclusion would affect both systemic and mucosal tissue gene expression, especially in regard to growth and immunerelated genes. Fish were divided in quadruplicate aquaria, and five experimental diets comprising 0, 50, 100, 200, and 300 g of frass per kilogram of feed were fed twice daily. At the end of the trial, liver, head kidney, gill, and intestine samples were collected for gene expression analyses. First, liver and intestine samples from fish fed with a no frass inclusion diet (control), low-frass (50 g/kg) inclusion diet, or a high-frass (300 g/kg) inclusion diet were subjected to Illumina RNA sequencing to determine global differential gene expression among diet groups. Differentially expressed genes (DEGs) included the upregulation of growth-related genes such as glucose-6-phosphatase and myostatin, as well as innate immune receptors and effector molecules such as toll-like receptor 5, apolipoprotein A1, C-type lectin, and lysozyme. Based on the initial screenings of low/high frass using RNA sequencing, a more thorough evaluation of immune gene expression of all tissues sampled, and all levels of frass inclusion, was further conducted. Using targeted quantitative PCR panels for both innate and adaptive immune genes from channel catfish, differential expression of genes was identified, which included innate receptors (TLR1, TLR5, TLR9, and TLR20A), proinflammatory cytokines (IL-1ß type a, IL-1ß type b, IL-17, IFN-1, and TNFa), chemokines (CFC3 and CFD), and hepcidin in both systemic (liver and head kidney) and mucosal (gill and intestine) tissues. Overall, frass from black soldier fly larvae inclusion in formulated diets was found to alter global gene expression and activate innate and adaptive immunity in channel catfish, which has the potential to support disease resistance in this species in addition to demonstrated growth benefits. [ABSTRACT FROM AUTHOR]

Background/Aims: Phaeodactylum tricornutum, a model organism of diatoms, plays a crucial role in Earth's primary productivity. Investigating its cellular response to grazing pressure is highly significant for the marine ecological environment. Furthermore, the integration of multi-omics approaches has enhanced the understanding of its response mechanism. Methods: To assess the molecular and cellular responses of P. tricornutum to grazer presence, we conducted transcriptomic, proteomic, and metabolomic analyses, combined with phenotypic data from previous studies. Sequencing data were obtained by Illumina RNA sequencing, TMT Labeled Quantitative Proteomics and Non-targeted Metabolomics, and WGCNA analysis and statistical analysis were performed. Results: Among the differentially expressed genes, we observed complex expression patterns of the core genes involved in the phenotypic changes of P. tricornutum under grazing pressure across different strains and multi-omics datasets. These core genes primarily regulate the levels of various proteins and fatty acids, as well as the cellular response to diverse signals. Conclusion: Our research reveals the association of multi-omics in four strains responses to grazing effects in P. tricornutum. Grazing pressure significantly impacted cell growth, fatty acid composition, stress response, and the core genes involved in phenotype transformation. [ABSTRACT FROM AUTHOR]

Ralstonia insidiosa can survive in a wide range of aqueous environments, including food processing areas, and is harmful to humans. It can induce Listeria monocytogenes to form suspended aggregates, resulting from the co-aggregation of two bacteria, which allows for more persistent survival and increases the risk of L. monocytogenes contamination. In our study, different groups of aggregates were analyzed and compared using Illumina RNA sequencing technology. These included R. insidiosa under normal and barren nutrient conditions and in the presence or absence of L. monocytogenes as a way to screen for differentially expressed genes (DEGs) in the process of aggregate formation. In addition, sterile supernatants of R. insidiosa were analyzed under different nutrient conditions using metabolomics to investigate the effect of nutrient-poor conditions on metabolite production by R. insidiosa. We also undertook a combined analysis of transcriptome and metabolome data to further investigate the induction effect of R. insidiosa on L. monocytogenes in a barren environment. The results of the functional annotation analysis on the surface of DEGs and qPCR showed that under nutrient-poor conditions, the acdx, puuE, and acs genes of R. insidiosa were significantly upregulated in biosynthetic processes such as carbon metabolism, metabolic pathways, and biosynthesis of secondary metabolites, with Log2FC reaching 4.39, 3.96, and 3.95 respectively. In contrast, the Log2FC of cydA, cyoB, and rpsJ in oxidative phosphorylation and ribosomal pathways reached 3.74, 3.87, and 4.25, respectively. Thirty-one key components were identified while screening for differential metabolites, which mainly included amino acids and their metabolites, enriched to the pathways of biosynthesis of amino acids, phenylalanine metabolism, and methionine metabolism. Of these, aminomalonic acid and Proximicin B were the special components of R. insidiosa that were metabolized under nutrient-poor conditions. [ABSTRACT FROM AUTHOR]

Bulblet initiation, including adventitious bud initiation and bulblet formation, is a crucial process for lily and other bulbous flowers that are commercially propagated by vegetative means. Here, by a hybrid strategy combining Pacific Biosciences (PacBio) full-length sequencing and Illumina RNA sequencing (RNA-seq), high-quality transcripts of L. brownii (Lb) and its variety, L. brownii var. giganteum (Lbg), during in vitro bulblet initiation were obtained. A total of 53,576 and 65,050 high-quality non-redundant full-length transcripts of Lbg and Lb were generated, respectively. Morphological observation showed that Lbg possessed a stronger capacity to generate bulblets in vitro than Lb, and 1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) significantly increased bulblet regeneration rate in two lilies. Screening of differentially expressed transcripts (DETs) between different stages and Mfuzz analysis showed 0 DAT to 1 DAT was the crucial stage with the most complex transcriptional change, with carbohydrate metabolism pathway was significantly enriched. In addition, 6,218 and 8,965 DETs were screened between the 2,4-D-treated group and the control group in Lbg and Lb, respectively. 2,4-D application had evident effects on the expression of genes involved in auxin signaling pathway, such as TIRs, ARFs, Aux/IAAs, GH3s and SAURs. Then, we compared the expression profiles of crucial genes of carbohydrate metabolism between different stages and different treatments. SUSs, SUTs, TPSs, AGPLs, GBSSs and SSs showed significant responses during bulblet initiation. The expression of CWINs, SUTs and SWEETs were significantly upregulated by 2,4-D in two lilies. In addition, 2,4- D increased the expression of starch degradation genes (AMYs and BAMs) and inhibited starch synthesis genes (AGPLs, GBSSs and SSs). SBEs were significantly upregulated in Lbg but not in Lb. Significant co-expression was showed between genes involved in carbohydrate metabolism and auxin signaling, together with transcription factors such as bHLHs, MYBs, ERFs and C3Hs. This study indicates the coordinate regulation of bulblet initiation by carbohydrate metabolism and auxin signaling, serving as a basis for further studies on the molecular mechanism of bulblet initiation in lily and other bulbous flowers. [ABSTRACT FROM AUTHOR]

Drought stress is one of the most important environmental factors limiting agricultural productivity of wheat (Triticum aestivum L.), particularly under the conditions of global climate change. Although the molecular mechanisms underlying plant drought stress-responses have been recently studied in various wheat cultivars, to date, profiling of the drought response-related transcriptome of Keumkang, a domestic wheat cultivar cultivated mainly in South Korea, has not been conducted. Therefore, based on Illumina RNA sequencing (RNA-seq), we performed a comparative transcriptome analysis in seedlings of cv. Keumkang kept under drought stress up to 24 h. A total of 4700, 10,560, and 9656 up-regulated and 1128, 6363, and 8428 down-regulated transcripts, relative to control levels, were identified in the aboveground parts of wheat seedlings at 3, 6, and 24 h after drought initiation, respectively. The most representative transcripts, including those of abscisic acid (ABA) response genes, ethylene-activated signaling pathways, and water deficit, desiccation, cold, and wounding responsive genes were expressed at higher levels under drought than under normal conditions; furthermore, many drought tolerance-related genes accumulated under the same conditions. Our results contribute to expanding our understanding of the molecular events associated with drought responses and provide a deeper insight into genetic information for further studies on the adaptability of cv. Keumkang to abiotic stress. [ABSTRACT FROM AUTHOR]

Mice have emerged as a widely employed model for investigating various retinal diseases. However, the availability of comprehensive datasets capturing the entire developmental and aging stages of the mouse retina, particularly during the elderly period, encompassing integrated lncRNA and mRNA expression profiles, is limited. In this study, we assembled a total of 18 retina samples from mice across 6 distinct stages of development and aging (5 days, 3 weeks, 6 weeks, 10 weeks, 6 months, and 15 months) to conduct integrated lncRNA and mRNA sequencing analysis. This invaluable dataset offers a comprehensive transcriptomic resource of mRNA and lncRNA expression profiles during the natural progression of retinal development and aging. The discoveries stemming from this investigation will significantly contribute to the elucidation of the underlying molecular mechanisms associated with various retinal diseases, such as congenital retinal dysplasia and retinal degenerative diseases. Design Type(s) organism development and aging design • transcriptional profiling by RNA sequencing design Measurement Type(s) long noncoding RNA and mRNA profiling assay Technology Type(s) illumina RNA sequencing Factor Type(s) life cycle stage Sample Characteristic(s) Mus musculus • retina [ABSTRACT FROM AUTHOR]

Potato virus Y (PVY) infection causes necrosis and curling of leaves, which seriously affect the yield and quality of Solanaceous crops. The roles of nutrient elements in the regulation of plant resistance to virus infection has been widely reported, while the mechanisms are poorly studied. Previous studies in our laboratory have demonstrated that foliar spraying of MgSO4 could induce Nicotiana tabacum resistance to PVY by increasing the activity of defense-related enzymes. Consistent with the results, we found that exogenous magnesium (Mg) had a certain effect on N. tabacum anti-PVY infection. Meanwhile, Illumina RNA sequencing revealed that Mg induced resistance to PVY infection was mainly by regulating carbohydrate metabolism and transportation, nitrogen metabolism, Ca2+ signal transduction and oxidative phosphorylation. Moreover, we used virus-induced gene silencing assays to verify the function of homologs of five N. tabacum genes involved in above pathways in N. benthamiana. The results showed that NbTPS and NbGBE were conducive to PVY infection, while NbPPases and NbNR were related to resistance to PVY infection. These results suggested a novel strategy for resistance to PVY infection and provided a theoretical basis for virus-resistance breeding. [ABSTRACT FROM AUTHOR]

Davidia involucrata Baill. (D. involucrata), a rare and endangered wild plant, is native to China and is globally recognized as an ornamental tree species. However, D. involucrata exhibits inherent biological characteristics that contribute to its low reproductive efficiency. To address this challenge, somatic embryogenesis, a biotechnological method, offers numerous advantages, including enhanced reproductive efficiency, a large reproductive coefficient, and a complete structural composition. Consequently, somatic embryogenesis holds significant value in the propagation and genetic improvement of this particular tree species. In a previous study, we utilized immature zygotic embryos of D. involucrata as explants and induced somatic embryogenesis from embryogenic callus, thereby establishing a rapid propagation and plant regeneration scheme. In this study, we utilized Illumina RNA sequencing to compare the transcriptomes of the embryogenic callus (EC) and non-embryogenic callus (NEC) of D. involucrata. The analysis revealed 131,109 unigenes assembled from EC and NEC, and 12,806 differentially expressed genes (DEGs) were identified. To verify the authenticity of the transcriptome sequencing results, qRT-PCR was performed and 16 DEGs were screened, with the stable reference gene UBQ being selected. Our analysis focused on genes related to plant growth regulators and somatic embryogenesis, such as the Aux, IAA, ARF, GH3, AHP, ARR, CYCD, BBM, WUS, GRF, SERK, and WOX gene families. We found that certain genes in these families were significantly upregulated in EC induction compared to NEC, indicating that they play crucial roles in D. involucrata cell proliferation, differentiation, and cell totipotency. These results offer new insights into the role of these gene families in EC, and may guide efforts to improve the somatic embryo induction, culture conditions, and genetic transformation efficiency of D. involucrata. [ABSTRACT FROM AUTHOR]

Soybean (Glycine max) is an important agricultural crop, but nutrient deficiencies frequently limit soybean production. While research has advanced our understanding of plant responses to long-term nutrient deficiencies, less is known about the signaling pathways and immediate responses to certain nutrient deficiencies, such as Pi and Fe deficiencies. Recent studies have shown that sucrose acts as a long-distance signal that is sent in increased concentrations from the shoot to the root in response to various nutrient deficiencies. Here, we mimicked nutrient deficiency-induced sucrose signaling by adding sucrose directly to the roots. To unravel transcriptomic responses to sucrose acting as a signal, we performed Illumina RNA-sequencing of soybean roots treated with sucrose for 20 min and 40 min, compared to non-sucrose-treated controls. We obtained a total of 260 million paired-end reads, mapping to 61,675 soybean genes, some of which are novel (not yet annotated) transcripts. Of these, 358 genes were upregulated after 20 min, and 2416 were upregulated after 40 min of sucrose exposure. GO (gene ontology) analysis revealed a high proportion of sucrose-induced genes involved in signal transduction, particularly hormone, ROS (reactive oxygen species), and calcium signaling, in addition to regulation of transcription. In addition, GO enrichment analysis indicates that sucrose triggers crosstalk between biotic and abiotic stress responses. [ABSTRACT FROM AUTHOR]

In termites with a true worker caste, the development pattern splits up from early developmental stages: primary reproductives develop through the nymphal line, whereas workers and soldiers follow the apterous developmental line. In some species, such as Cavitermes tuberosus Emerson (Isoptera: Termitidae, Termitinae), secondary reproductives (or neotenics) may also develop through the nymphal line from a transitional stage called aspirant. Aspirants originate mostly from automictic parthenogenetic reproduction. Therefore, C. tuberosus queens originate from sexual (primary queens) or parthenogenetic (neotenic queens) reproduction. A comparison of these two queen castes offers the possibility to better understand core molecular underpinnings of caste development and plasticity in termites. We investigated these molecular mechanisms by using high‐throughput Illumina RNA sequencing of pooled individuals. We first assembled the de novo reference transcriptome of C. tuberosus, and then identified the transcripts consistently co‐expressed across castes, sexes, and two alternative routes to female reproduction. Cavitermes tuberosus final transcriptome had 130 874 transcripts, N50 of 3398, and total length of 213 549 184 bp. We found that female reproductive maturation was characterized by gene expression down‐regulation: primary queens expressed fewer transcripts overall and had the greatest number of down‐regulated transcripts when compared to all other castes. In both secondary and primary queens, biological processes involved in muscle development and contraction, flight, and olfactory learning were enriched in the down‐regulated gene cluster. In contrast, processes related to reproductive development, insulin receptor signaling pathway, isoprenoid biosynthesis, and multiple metabolic processes were enriched among up‐regulated genes in primary queens. Finally, we found that 17% of all transcripts (21852) were differently co‐expressed when females from sexual and parthenogenetic origins were compared, even though the expression profile of core reproductive‐related gene clusters showed a similar trend in all reproductive females despite their origin. Our findings fit the genomic imprinting model predictions of a maternal effect that commonly regulates the expression of core reproductive genes in females from parthenogenetic and non‐parthenogenetic origins, whereas the expression of non‐reproductive genes varies. [ABSTRACT FROM AUTHOR]

The use of omic modalities to dissect the molecular underpinnings of common diseases and traits is becoming increasingly common. But multi-omic traits can be genetically predicted, which enables highly cost-effective and powerful analyses for studies that do not have multi-omics1. Here we examine a large cohort (the INTERVAL study2; n = 50,000 participants) with extensive multi-omic data for plasma proteomics (SomaScan, n = 3,175; Olink, n = 4,822), plasma metabolomics (Metabolon HD4, n = 8,153), serum metabolomics (Nightingale, n = 37,359) and whole-blood Illumina RNA sequencing (n = 4,136), and use machine learning to train genetic scores for 17,227 molecular traits, including 10,521 that reach Bonferroni-adjusted significance. We evaluate the performance of genetic scores through external validation across cohorts of individuals of European, Asian and African American ancestries. In addition, we show the utility of these multi-omic genetic scores by quantifying the genetic control of biological pathways and by generating a synthetic multi-omic dataset of the UK Biobank3 to identify disease associations using a phenome-wide scan. We highlight a series of biological insights with regard to genetic mechanisms in metabolism and canonical pathway associations with disease; for example, JAK–STAT signalling and coronary atherosclerosis. Finally, we develop a portal () to facilitate public access to all genetic scores and validation results, as well as to serve as a platform for future extensions and enhancements of multi-omic genetic scores.A machine learning approach is used to analyse multi-omics (proteomics, metabolomics and transcriptomics) data, producing genetic scores for more than 17,000 biomolecular traits in human blood, and identifying possible associations with disease. [ABSTRACT FROM AUTHOR]

The development of vaccines against sea lice in salmon farming is complex, expensive, and takes several years for commercial availability. Recently, transcriptome studies in sea louse have provided valuable information for identifying relevant molecules with potential use for fish vaccines. However, the bottleneck is the in vivo testing of recombinant protein candidates, the dosage, and the polyvalent formulation strategies. This study explored a cell-based approach to prospect antigens as candidate vaccines against sea lice by comparison with immunized fish. Herein, SHK-1 cells and Atlantic salmon head kidney tissue were exposed to the antigen cathepsin identified from the sea louse Caligus rogercresseyi. The cathepsin protein was cloned and recombinantly expressed in Escherichia coli, and then SHK-1 cell lines were stimulated with 100 ng/mL cathepsin recombinant for 24 h. In addition, Atlantic salmons were vaccinated with 30 ug/mL recombinant protein, and head kidney samples were then collected 30 days post-immunization. SHK-1 cells and salmon head kidney exposed to cathepsin were analyzed by Illumina RNA sequencing. The statistical comparisons showed differences in the transcriptomic profiles between SHK-1 cells and the salmon head kidney. However, 24.15% of the differentially expressed genes were shared. Moreover, putative gene regulation through lncRNAs revealed tissue-specific transcription patterns. The top 50 up and downregulated lncRNAs were highly correlated with genes involved in immune response, iron homeostasis, pro-inflammatory cytokines, and apoptosis. Also, highly enriched pathways related to the immune system and signal transduction were shared between both tissues. These findings highlight a novel approach to evaluating candidate antigens for sea lice vaccine development, improving the antigens screening in the SHK-1 cell line model. [ABSTRACT FROM AUTHOR]

Potato virus Y (PVY) mainly infects Solanaceous crops, resulting in considerable losses in the yield and quality. Iron (Fe) is involved in various biological processes in plants, but its roles in resistance to PVY infection has not been reported. In this study, foliar application of Fe could effectively inhibit early infection of PVY, and a full-length transcriptome and Illumina RNA sequencing was performed to investigate its modes of action in PVY-infected Nicotiana tabacum. The results showed that 18,074 alternative splicing variants, 3,654 fusion transcripts, 3,086 long non-coding RNAs and 14,403 differentially expressed genes (DEGs) were identified. Specifically, Fe application down-regulated the expression levels of the DEGs related to phospholipid hydrolysis, phospholipid signal, cell wall biosynthesis, transcription factors (TFs) and photosystem I composition, while those involved with photosynthetic electron transport chain (PETC) were upregulated at 1 day post inoculation (dpi). At 3 dpi, these DEGs related to photosystem II composition, PETC, molecular chaperones, protein degradation and some TFs were up-regulated, while those associated with light-harvesting, phospholipid hydrolysis, cell wall biosynthesis were down-regulated. At 9 dpi, Fe application had little effects on resistance to PVY infection and transcript profiles. Functional analysis of these potentially critical DEGs was thereafter performed using virus-induced gene silencing approaches and the results showed that NbCat-6A positively regulates PVY infection, while the reduced expressions of NbWRKY26, NbnsLTP, NbFAD3 and NbHSP90 significantly promote PVY infection in N. benthamiana. Our results elucidated the regulatory network of Fe-mediated resistance to PVY infection in plants, and the functional candidate genes also provide important theoretical bases to further improve host resistance against PVY infection. [ABSTRACT FROM AUTHOR]

Pinus elliottii , an important coniferous timber species, has recently become one of the most popular sources of resin in China. Resinosis is a common disease that may negatively affect pine tree growth and production. In this study, we used single-molecule real-time sequencing and Illumina RNA sequencing to generate an accurate transcriptome for P. elliottii. The transcriptome included 90,026 transcripts, 5160 long non-coding RNAs and 7710 transcription factors. We then analyzed RNA-sequencing, small RNA-sequencing and degradome data to identify genes, miRNAs and key miRNA–target pairs involved in response to resinosis in P. elliottii. We identified 1305 genes and 1151 miRNAs exhibiting significant differential expression in response to resinosis. According to the degradome sequencing analysis, 318 differentially expressed transcripts were targets of 14 differentially expressed miRNAs. Our study has provided resources for further functional characterization of genes and miRNAs involved in resinosis in P. elliottii , which should aid the future disease-resistance breeding of this species. [ABSTRACT FROM AUTHOR]

Thuja koraiensis Nakai (Cupressaceae) is an endangered and ecologically important conifer endemic to Korea. Previously, we established a protocol for micropropagation in T. koraiensis, which involved somatic embryogenesis from embryogenic callus of T. koraiensis. However, the molecular mechanisms underlying somatic embryogenesis remain unclear. Herein, we performed transcriptomic analysis to identify somatic embryogenesis-related genes of T. koraiensis via Illumina RNA sequencing. We conducted de novo transcriptome assembly using a Trinity assembler, which produced 274,077 transcript contigs clustered into 205,843 transcripts (unigenes), with an average length of 825 base pairs. Of all the unigenes, 14.69%, 18.62%, and 7.4% had homologs in the Gene Ontology, NCBI Non-redundant Protein, and NCBI Nucleotide databases, respectively. Among these mRNA sequences, expression of putative embryogenesis-associated transcription factors, namely BABYBOOM (BBM), WUSCHEL-RELATED HOMEOBOX (WOX), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK), was analyzed during somatic embryogenesis. RT-PCR analysis revealed that TkBBM, TkWOX, and TkSERK were highly expressed in embryogenic callus and seedling roots, whereas their expression was reduced in seedling leaves. Our findings provide new insights into the roles of BBM, WOX, and SERK in somatic embryogenesis. Our results may serve as a reference for comparative transcriptome analysis in related species and further aid functional genomics studies in conifers. [ABSTRACT FROM AUTHOR]

Naegleria fowleri is a pathogenic free-living amoeba, commonly found around the world in warm, fresh water and soil. N. fowleri trophozoites can infect humans by entering the brain through the nose and causing usually fatal primary amebic meningoencephalitis (PAM). Trophozoites can encyst to survive under unfavorable conditions such as cold temperature, starvation, and desiccation. Recent technological advances in genomics and bioinformatics have provided unique opportunities for the identification and pre-validation of pathogen-related and environmental resistance through improved understanding of the biology of pathogenic N. fowleri trophozoites and cysts at a molecular level. However, genomic and transcriptomic data on differential expression genes (DEGs) between trophozoites and cysts of N. fowleri are very limited. Here, we report transcriptome Illumina RNA sequencing (RNA-seq) for N. fowleri trophozoites and cysts and de novo transcriptome assembly. RNA-seq libraries were generated from RNA extracted from N. fowleri sampled from cysts, and a reference transcriptome was generated through the assembly of trophozoite data. In the database, the assembly procedure resulted in 42,220 contigs with a mean length of 11,254 nucleotides and a C+G content of 37.21%. RNA sequencing showed that 146 genes in cysts of N. fowleri indicated 2-fold upregulation in comparison with trophozoites of N. fowleri, and 163 genes were downregulated; these genes were found to participate in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The KEGG pathway included metabolic (131 sequences) and genetic information processing (66 sequences), cellular processing (43 sequences), environmental information processing (22 sequences), and organismal system (20 sequences) pathways. On the other hand, an analysis of 11,254 sequences via the Gene Ontology database showed that their annotations contained 1069 biological processes including the cellular process (228 sequences) and metabolic process (214 sequences); 923 cellular components including cells (240 sequences) and cell parts (225 sequences); and 415 molecular functions including catalytic activities (195 sequences) and binding processes (186 sequences). Differential expression levels increased in cysts of N. fowleri compared to trophozoites of N. fowleri, which were mainly categorized as serine/threonine protease, kinase, and lipid metabolism-related proteins. These results may provide new insights into pathogen-related genes or environment-resistant genes in the pathogenesis of N. fowleri. [ABSTRACT FROM AUTHOR]

The house sparrow (Passer domesticus) is a valuable avian model for studying evolutionary genetics, development, neurobiology, physiology, behavior, and ecology, both in laboratory and field-based settings. The current annotation of the P. domesticus genome available at the Ensembl Rapid Release site is primarily focused on gene set building and lacks functional information. In this study, we present the first comprehensive functional reannotation of the P. domesticus genome using intestinal Illumina RNA sequencing (RNA-Seq) libraries. Our revised annotation provides an expanded view of the genome, encompassing 38592 transcripts compared to the current 23574 transcripts in Ensembl. We also predicted 14717 protein-coding genes, achieving 96.4% completeness for Passeriformes lineage BUSCOs. A substantial improvement in this reannotation is the accurate delineation of untranslated region (UTR) sequences. We identified 82.7% and 93.8% of the transcripts containing 5′- and 3′-UTRs, respectively. These UTR annotations are crucial for understanding post-transcriptional regulatory processes. Our findings underscore the advantages of incorporating additional specific RNA-Seq data into genome annotation, particularly when leveraging fast and efficient data processing capabilities. This functional reannotation enhances our understanding of the P. domesticus genome, providing valuable resources for future investigations in various research fields. [ABSTRACT FROM AUTHOR]

Bud dormancy is a crucial strategy for perennial plants to withstand adverse winter conditions. However, the regulatory mechanism of bud dormancy in tree peony (Paeonia suffruticosa) remains largely unknown. Here, we observed dramatically reduced and increased accumulation of abscisic acid (ABA) and bioactive gibberellins (GAs) GA1 and GA3, respectively, during bud endodormancy release of tree peony under prolonged chilling treatment. An Illumina RNA sequencing study was performed to identify potential genes involved in the bud endodormancy regulation in tree peony. Correlation matrix, principal component, and interaction network analyses identified a downregulated MYB transcription factor gene, PsMYB306, the expression of which positively correlated with 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (PsNCED3) expression. Protein modeling analysis revealed 4 residues within the R2R3 domain of PsMYB306 to possess DNA binding capability. Transcription of PsMYB306 was increased by ABA treatment. Overexpression of PsMYB306 in petunia (Petunia hybrida) inhibited seed germination and plant growth, concomitant with elevated ABA and decreased GA contents. Silencing of PsMYB306 accelerated cold-triggered tree peony bud burst and influenced the production of ABA and GAs and the expression of their biosynthetic genes. ABA application reduced bud dormancy release and transcription of ENT-KAURENOIC ACID OXIDASE 1 (PsKAO1), GA20-OXIDASE 1 (PsGA20ox1), and GA3-OXIDASE 1 (PsGA3ox1) associated with GA biosynthesis in PsMYB306-silenced buds. In vivo and in vitro binding assays confirmed that PsMYB306 specifically transactivated the promoter of PsNCED3. Silencing of PsNCED3 also promoted bud break and growth. Altogether, our findings suggest that PsMYB306 negatively modulates cold-induced bud endodormancy release by regulating ABA production. A MYB transcription factor inhibits tree peony bud dormancy release by modulating abscisic acid production. [ABSTRACT FROM AUTHOR]

Background Infantile central nervous system infections (CNSIs) can be life-threatening and cause severe sequelae. However, the causative microorganism remains unknown in >40% of patients with aseptic infections. This study aimed to analyze the metagenome for detection of pathogens and the transcriptome for host immune responses during infection in a single cerebrospinal fluid (CSF) sample using 2 different next-generation sequencing (NGS) platforms, Nanopore and Illumina. Methods Twenty-eight CNSIs patients (<12 months) were enrolled, and 49 clinical samples (28 CSF and 21 blood) were collected. The DNA extracted from all 49 samples was sequenced using the Illumina sequencer for the detection of pathogens. Extracted RNA was obtained in sufficient quantities from 23 CSF samples and subjected to sequencing on both Nanopore and Illumina platforms. Human-derived reads subtracted during pathogen detection were used for host transcriptomic analysis from both Nanopore and Illumina sequencing. Results RNA metagenomic sequencing using both sequencing platforms revealed putative viral pathogens in 10 cases. DNA sequencing using the Illumina sequencer detected 2 pathogens. The results of Nanopore and Illumina RNA sequencing were consistent; however, the mapping coverage and depth to the detected pathogen genome of Nanopore RNA sequencing were greater than those of Illumina. Host transcriptomic analysis of Nanopore sequencing revealed highly expressed genes related to the antiviral roles of innate immunity from pathogen-identified cases. Conclusions The use of Nanopore RNA sequencing for metagenomic diagnostics of CSF samples should help to elucidate both pathogens and host immune responses of CNSI and could shed light on the pathogenesis of these infections. [ABSTRACT FROM AUTHOR]

Simple Summary: The oriental fruit moth Grapholita molesta is an important worldwide fruit tree pest. Its hosts mainly include peach, pear, apple, plum and other Rosaceae fruit trees. In order to explore the mechanism of host adaptation, it is necessary to comprehensively study the effects of feeding and the differences in transcriptional levels of G. molesta. In this study, the growth and development parameters, protease activities, midgut transcriptome analysis and key gene validation of G. molesta fed on five hosts and artificial diet were determined. This study indicated that the significant differences in the growth and development parameters and protease activities, transcriptome of KEGG enrichment and WGCNA analysis of G. molesta fed on different hosts. The transcriptional level of the trypsin gene was consistent with that of qRT-PCR. The trypsin gene played an important role in feeding different hosts of G. molesta. We revealed the adaptive mechanism to different hosts of G. molesta in various aspects and laid a solid foundation for further exploring the molecular mechanism of host adaptation. Grapholita molesta is an important fruit tree worldwide pest which feeds on hosts extensively and does serious harm. In this paper, the growth and development parameters and protease activities of G. molesta fed on different hosts were compared. Using Illumina RNA sequencing technology, 18 midgut samples from five different hosts (apple, pear, plum, peach and peach shoots) and artificial diet were sequenced and compared with the reference genome, resulting in 15269 genes and 2785 predicted new genes. From 15 comparative combinations, DEGs were found from 286 to 4187 in each group, with up-regulated genes from 107 to 2395 and down-regulated genes from 83 to 2665. KEGG pathway analysis showed that DEGs were associated with amino acid metabolism, starch and sucrose metabolism, carbohydrate metabolism, and hydrolase activity. A total of 31 co-expression gene modules of different hosts were identified by WGCNA. qRT-PCR showed that the expression pattern of the trypsin gene was consistent with RNA sequencing. In this study, growth and development parameters, protease activity, DEGs, enrichment analysis and qRT-PCR were combined to reveal the adaptation process to different hosts of G. molesta in many aspects. The results of this study provide a basis for further exploration of the molecular mechanism of host adaptation of G. molesta. [ABSTRACT FROM AUTHOR]

Artemisiae argyi Folium is a traditional herbal medicine used for moxibustion heat therapy in China. The volatile oils in A.argyi leaves are closely related to its medicinal value. Records suggest that the levels of these terpenoids components within the leaves vary as a function of harvest time, with June being the optimal time for A. argyi harvesting, owing to the high levels of active ingredients during this month. However, the molecular mechanisms governing terpenoid biosynthesis and the time-dependent changes in this activity remain unclear. In this study, GC–MS analysis revealed that volatile oil levels varied across four different harvest months (April, May, June, and July) in A. argyi leaves, and the primarily terpenoids components (including both monoterpenes and sesquiterpenes) reached peak levels in early June. Through single-molecule real-time (SMRT) sequencing, corrected by Illumina RNA-sequencing (RNA-Seq), 44 full-length transcripts potentially involved in terpenoid biosynthesis were identified in this study. Differentially expressed genes (DEGs) exhibiting time-dependent expression patterns were divided into 12 coexpression clusters. Integrated chemical and transcriptomic analyses revealed distinct time-specific transcriptomic patterns associated with terpenoid biosynthesis. Subsequent hierarchical clustering and correlation analyses ultimately identified six transcripts that were closely linked to the production of these two types of terpenoid within A. argyi leaves, revealing that the structural diversity of terpenoid is related to the generation of the diverse terpene skeletons by prenyltransferase (TPS) family of enzymes. These findings can guide further studies of the molecular mechanisms underlying the quality of A. argyi leaves, aiding in the selection of optimal timing for harvests of A. argyi. [ABSTRACT FROM AUTHOR]

Circular RNA (circRNA) is a noncoding RNA class with important implications for gene expression regulation, mostly by interaction with other RNA species or RNA-binding proteins. While the commonly applied short-read Illumina RNA-sequencing techniques can be used to detect circRNAs, their full sequence is not revealed. However, the complete sequence information is needed to analyze potential interactions and thus the mechanism of action of circRNAs. Here, we present an improved protocol to enrich and sequence full-length circRNAs by using the Oxford Nanopore long-read sequencing platform. The protocol involves an enrichment of lowly abundant circRNAs by exonuclease treatment and negative selection of linear RNAs. Then, a cDNA library is created and amplified by PCR. This protocol provides enough material for several sequencing runs. The library is used as input for ligation-based sequencing together with native barcoding. Stringent quality control of the libraries is ensured by a combination of Qubit, Fragment Analyzer and qRT-PCR. Multiplexing of up to 4 libraries yields in total more than 1–2 Million reads per library, of which 1–2% are circRNA-specific reads with >99% of them full-length. The protocol works well with human cancer cell lines. We further provide suggestions for the bioinformatic analysis of the created data, as well as the limitations of our approach together with recommendations for troubleshooting and interpretation. Taken together, this protocol enables reliable full-length analysis of circRNAs, a noncoding RNA type involved in a growing number of physiologic and pathologic conditions. Metadata Associated content. https://dx.doi.org/10.17504/protocols.io.rm7vzy8r4lx1/v2. [ABSTRACT FROM AUTHOR]

Simple Summary: The eyestalk is a key organ in crustaceans that produces neurohormones and regulates a range of physiological functions. Eyestalk displacement was discovered in some first-generation (F1) offspring of the novel interspecific hybrid crab (Scylla serrata ♀ × S. paramamosain ♂). To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, high-quality transcriptome was reconstructed using single-molecule real-time (SMRT) sequencing. A total of 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in hybrid crabs with displaced eyestalks (DH). The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant gene ontology (GO) terms were related to the cuticle or chitin. Overall, this study highlights the underlying genetic mechanisms of eyestalk displacement and provide useful knowledge for mud crab (Scylla spp.) crossbreeding. The lack of high-quality juvenile crabs is the greatest impediment to the growth of the mud crab (Scylla paramamosain) industry. To obtain high-quality hybrid offspring, a novel hybrid mud crab (S. serrata ♀ × S. paramamosain ♂) was successfully produced in our previous study. Meanwhile, an interesting phenomenon was discovered, that some first-generation (F1) hybrid offspring's eyestalks were displaced during the crablet stage I. To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, both single-molecule real-time (SMRT) and Illumina RNA sequencing were implemented. Using a two-step collapsing strategy, three high-quality reconstructed transcriptomes were obtained from purebred mud crabs (S. paramamosain) with normal eyestalks (SPA), hybrid crabs with normal eyestalks (NH), and hybrid crabs with displaced eyestalks (DH). In total, 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in DH. The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant GO terms were related to the cuticle or chitin. Overall, high-quality reconstructed transcriptomes were obtained for the novel interspecific hybrid crab and provided valuable insights into the genetic mechanisms of eyestalk displacement in mud crab (Scylla spp.) crossbreeding. [ABSTRACT FROM AUTHOR]

Shewanella putrefaciens is a Gram-negative bacterium that can cause seafood spoilage under low-temperature conditions. The bacterium easily forms biofilms to enhance its survival in challenging environments. Our previous research revealed that the biofilm formed by S. putrefaciens WS13 under the low temperature (4 °C) has larger biomass and tighter structure than at an optimum growth temperature (30 °C). In this study, comparative transcriptome analysis was further performed to get insights into the global-level of gene expression in the biofilm formed by S. putrefaciens WS13 under the refrigerating and optimal temperatures using Illumina RNA-Sequencing technique. The results revealed that a total of 761 genes were differentially expressed, of which 497 were significantly up-regulated and 264 were significantly down-regulated (p <0.05). The qRT-PCR results of randomly selected differentially expressed genes (DEGs) confirmed the RNA sequencing results. Comparison of transcriptome data revealed 28 significantly changed metabolic pathways under the cold stress, including the down-regulated chemotaxis, and motility, and up-regulated tryptophan metabolism, histidine biosynthesis, and quorum sensing, which benefited the biofilm formation of S. putrefaciens WS13 under the adverse circumstance. This study provided useful data for better understanding of the biofilm formation of S. putrefaciens , and also laid a theoretical foundation for novel vaccine and drug targets against the severe spoilage bacterium under the cold stress. [ABSTRACT FROM AUTHOR]

Infectious bronchitis virus (IBV) and avian influenza virus (AIV) are two major respiratory infections in chickens. The coinfection of these viruses can cause significant financial losses and severe complications in the poultry industry across the world. To examine transcriptome profile changes during the early stages of infection, differential transcriptional profiles in tracheal tissue of three infected groups (i.e., IBV, AIV, and coinfected) were compared with the control group. Specific-pathogen-free chickens were challenged with Iranian variant-2-like IBV (IS/1494), UT-Barin isolates of H9N2 (A/chicken/Mashhad/UT-Barin/2017), and IBV-AIV coinfection; then, RNA was extracted from tracheal tissue. The Illumina RNA-sequencing (RNA-seq) technique was employed to investigate changes in the Transcriptome. Up- and downregulated differentially expressed genes (DEGs) were detected in the trachea transcriptome of all groups. The Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology databases were examined to identify possible relationships between DEGs. In the experimental groups, upregulated genes were higher compared to downregulated genes. A more severe immune response was observed in the coinfected group; further, cytokine-cytokine receptor interaction, RIG-I-like receptor signaling, Toll-like receptor signaling, NOD-like receptor signaling, Janus kinase/signal transducer, and activator of transcription, and apoptotic pathways were important upregulated genes in this group. The findings of this paper may give a better understanding of transcriptome changes in the trachea during the early stages of infection with these viruses. [ABSTRACT FROM AUTHOR]

Macrobrachium nipponense is an economically important prawn species and common in Chinese inland capture fisheries. During aquaculture, M. nipponense can survive under freshwater and low salinity conditions. The molecular mechanism underlying the response to salinity acclimation remains unclear in this species; thus, in this study, we used the Illumina RNA sequencing platform for transcriptome analyses of the gill and hepatopancreas tissues of M. nipponense exposed to salinity stress [0.4‰ (S0, control group), 6‰ (S6, low salinity group), and 12‰ (S12, high salinity group)]. Differentially expressed genes were identified, and several important salinity adaptation-related terms and signaling pathways were found to be enriched, such as "ion transport," "oxidative phosphorylation," and "glycometabolism." Quantitative real-time PCR demonstrated the participation of 12 key genes in osmotic pressure regulation in M. nipponense under acute salinity stress. Further, the role of carbonic anhydrase in response to salinity acclimation was investigated by subjecting the gill tissues of M. nipponense to in situ hybridization. Collectively, the results reported herein enhance our understanding of the mechanisms via which M. nipponense adapts to changes in salinity. [ABSTRACT FROM AUTHOR]

As high soil salinity threatens the growth and development of plants, understanding the mechanism of plants' salt tolerance is critical. The Chrysanthemum × grandiflora is a newly developed species with a strong salt resistance that possesses multiple genes controlling its quantitative salt resistance. Because of this multigene control, we chose to investigate the plant stress genes overall responses at the transcriptome level. C. grandiflora were treated with a 200 mM NaCl solution for 12 h to study its effect on the roots and leaves via Illumina RNA sequencing. PAL, CYP73A, and 4CL in the phenylpropanoid biosynthesis pathway were upregulated in roots and leaves. In the salicylic acid signal transduction pathway, TGA7 was upregulated in the roots and leaves, while in the jasmonic acid signal transduction pathway, TIFY9 was upregulated in the roots and leaves. In the ion transporter gene, we identified HKT1 that showed identical expression patterns in the roots and leaves. The impact of NaCl imposition for 12 h was largely due to osmotic effect of salinity on C. grandiflora, and most likely the transcript abundance changes in this study were due to the osmotic effect. In order to verify the accuracy of the Illumina sequencing data, we selected 16 DEGs for transcription polymerase chain reaction (qRT-PCR) analysis. qRT-PCR and transcriptome sequencing analysis revealed that the transcriptome sequencing results were reliable. [ABSTRACT FROM AUTHOR]

Bovine rotavirus (BRV) causes massive economic losses in the livestock industry worldwide. Elucidating the pathogenesis of BRV would help in the development of more effective measures to control BRV infection. The MA-104 cell line is sensitive to BRV and is thereby a convenient tool for determining BRV–host interactions. Thus far, the role of the microRNAs (miRNAs) of MA-104 cells during BRV infection is still ambiguous. We performed Illumina RNA sequencing analysis of the miRNA libraries of BRV-infected and mock-infected MA-104 cells at different time points: at 0 h post-infection (hpi) (just after 90 min of adsorption) and at 6, 12, 24, 36, and 48 hpi. The total clean reads obtained from BRV-infected and uninfected cells were 74,701,041 and 74,184,124, respectively. Based on these, 579 were categorized as known miRNAs and 144 as novel miRNAs. One hundred and sixty differentially expressed (DE) miRNAs in BRV-infected cells in comparison with uninfected MA-104 cells were successfully investigated, 95 of which were upregulated and 65 were downregulated. The target messenger RNAs (mRNAs) of the DE miRNAs were examined by bioinformatics analysis. Functional annotation of the target genes with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) suggested that these genes mainly contributed to biological pathways, endocytosis, apoptotic process, trans-Golgi membrane, and lysosome. Pathways such as the mammalian target of rapamycin (mTOR) (mml-miR-486-3p and mml-miR-197-3p), nuclear factor kappa B (NF-κB) (mml-miR-204-3p and novel_366), Rap1 (mml-miR-127-3p), cAMP (mml-miR-106b-3p), mitogen-activated protein kinase (MAPK) (mml-miR-342-5p), T-cell receptor signaling (mml-miR-369-5p), RIG-I-like receptor signaling (mml-miR-504-5p), AMP-activated protein kinase (AMPK) (mml-miR-365-1-5p), and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling (mml-miR-299-3p) were enriched. Moreover, real-time quantitative PCR (qPCR) verified the expression profiles of 23 selected DE miRNAs, which were consistent with the results of deep sequencing, and the 28 corresponding target mRNAs were mainly of regulatory pathways of the cellular machinery and immune importance, according to the bioinformatics analysis. Our study is the first to report a novel approach that uncovers the impact of BRV infection on the miRNA expressions of MA-104 cells, and it offers clues for identifying potential candidates for antiviral or vaccine strategies. [ABSTRACT FROM AUTHOR]

As an important economic shellfish, the pearl oyster, Pinctada fucata, and its larvae are an ideal model for studying molecular mechanisms of larval development in invertebrates. Larval development directly affects the quantity and quality of pearl oysters. MicroRNAs (miRNAs) may play important roles in development, but the effects of miRNA expression on P. fucata early development remain unknown. In this study, miRNA and mRNA transcriptomics of seven different P. fucata developmental stages were analyzed using Illumina RNA sequencing. A total of 329 miRNAs, including 87 known miRNAs and 242 novel miRNAs, and 33,550 unigenes, including 26,333 known genes and 7217 predicted new genes, were identified in these stages. A cluster analysis showed that the difference in the numbers of miRNAs was greatest between fertilized eggs and trochophores. In addition, the integrated mRNA transcriptome was used to predict target genes for differentially expressed miRNAs between adjacent developmental stages, and the target genes were subjected to a gene ontology enrichment analysis. Using the gene ontology annotation, 100 different expressed genes and 95 differentially expressed miRNAs were identified as part of larval development regulation. Real-time PCR was used to identify eight mRNAs and three miRNAs related to larval development. The present findings will be helpful for further clarifying the regulatory mechanisms of miRNA in invertebrate larval development. [ABSTRACT FROM AUTHOR]

In this study, we characterized the gene expression profile in the roots of Populus ussuriensis at 0, 6, 12, 24, 48 and 120 h after the start of polyethylene glycol (PEG)-induced drought stress using PacBio single-molecule real-time sequencing (SMRT-seq) and Illumina RNA sequencing. Compared to the control, 2244 differentially expressed genes (DEGs) were identified, and many of these DEGs were associated with the signal transduction, antioxidant system, ion accumulation and drought-inducing proteins. Changes in certain physiological and biochemical indexes, such as antioxidant activity and the contents of Ca2+, proline, and total soluble sugars, were further confirmed in P. ussuriensis roots. Furthermore, most of the differentially expressed transcription factors were members of the AP2/ERF, C2H2, MYB, NAC, C2C2 and WRKY families. Additionally, based on PacBio SMRT-seq results, 5955 long non-coding RNAs and 700 alternative splicing events were identified. Our results provide a global view of the gene expression profile that contributes to drought resistance in P. ussuriensis and meaningful information for genetic engineering research in the future. [ABSTRACT FROM AUTHOR]