The olfactory gene families include odorant binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs). To investigate the molecular function of olfactory perception in Macrobrachium rosenbergii, we integrated the full-length transcripts and whole-genome sequences to identify the olfactory gene families. In this study, a total of 38,955 full-length transcripts with an N50 length of 3383 bp were obtained through PacBio SMRT sequencing. Through the annotation of full-length transcripts and whole-genome sequences, several olfactory gene families were identified, including 18 MrORs, 16 MrIRs, 151 MrIGluRs (ionotropic glutamate receptors), 2 MrVIGluRs (variant ionotropic glutamate receptors) and 3 MrCRs (chemosensory receptors). Notably, the CRs were first identified in prawns and shrimps. Additionally, the olfactory gene families in M. nipponense were identified, comprising 4 MnORs, 21 MnIRs, 79 MnIGluRs, 5 MnVIGluRs, 1 MnGR and 1 MnOBP, using the available whole-genome sequences. Meanwhile, the external morphology of the chemical sensory organs of M. rosenbergii was explored, and the presence of plumose setae (PS), hard thorn setae (HTS), bamboo shoot setae (BSS), soft thorn setae (STS) and aesthetascs (AE) on the antennules, HTS and BSS on the second antennae, and PS on the pereiopods were observed by scanning electron microscope. This study provides valuable insights for future functional studies into the olfactory perception of crustaceans and establishes a theoretical basis for molecular design breeding in M. rosenbergii.

Alternative splicing is an essential post-transcriptional regulatory mechanism that diversifies gene function by generating multiple protein isoforms from a single gene and act as a crucial role in insect environmental adaptation. Olfaction, a key sense for insect adaptation, relies heavily on the antennae, which are the primary olfactory organs expressing most of the olfactory genes. Despite the extensive annotation of olfactory genes within insect antennal tissues facilitated by high-throughput sequencing technology advancements, systematic analyses of alternative splicing are still relatively less. In this study, we focused on the oriental fruit fly (Bactrocera dorsalis), a significant pest of fruit crops. We performed a detailed analysis of alternative splicing in its antennae by utilizing the full-length transcriptome of its antennal tissue and the insect\'s genome. The results revealed 8600 non-redundant full-length transcripts identified in the oriental fruit fly antennal full-length transcriptome, spanning 4,145 gene loci. Over 40% of these loci exhibited multiple isoforms. Among these, 161 genes showed sex-biased isoform switching, involving seven different types of alternative splicing. Notably, events involving alternative transcription start sites (ATSS) and alternative transcription termination sites (ATTS) were the most common. Of all the genes undergoing ATSS and ATTS alternative splicing between male and female, 32 genes were alternatively spliced in protein coding regions, potentially affecting protein function. These genes were categorized based on the length of the sex-biased isoforms, with the highest difference in isoform fraction (dIF) associated with the ATSS type, including genes such as BdorABCA13, BdorCAT2, and BdorTSN3. Additionally, transcription factor binding sites for doublesex were identified upstream of both BdorABCA13 and BdorCAT2. Besides being expressed in the antennal tissues, BdorABCA13 and BdorCAT2 are also expressed in the mouthparts, legs, and genitalia of both female and male adults, suggesting their functional diversity. This study reveals alternative splicing events in the antennae of Bactrophora dorsalis from two aspects: odorant receptor genes and other types of genes expressed in the antennae. This study not only provides a research foundation for understanding the regulation of gene function by alternative splicing in the oriental fruit fly but also offers new insights for utilizing olfaction-based behavioral manipulation techniques to manage this pest.

Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer\'s disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.

Dysregulation of alternative splicing has been repeatedly associated with neurodevelopmental disorders, but the extent of cell-type-specific splicing in human neural development remains largely uncharted. Here, single-cell long-read sequencing in induced pluripotent stem cell (iPSC)-derived cerebral organoids identifies over 31,000 uncatalogued isoforms and 4,531 cell-type-specific splicing events. Long reads uncover coordinated splicing and cell-type-specific intron retention events, which are challenging to study with short reads. Retained neuronal introns are enriched in RNA splicing regulators, showing shorter lengths, higher GC contents, and weaker 5\' splice sites. We use this dataset to explore the biological processes underlying neurological disorders, focusing on autism. In comparison with prior transcriptomic data, we find that the splicing program in autistic brains is closer to the progenitor state than differentiated neurons. Furthermore, cell-type-specific exons harbor significantly more de novo mutations in autism probands than in siblings. Overall, these results highlight the importance of cell-type-specific splicing in autism and neuronal gene regulation.

Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae) is an important forest pest in China that mainly infests timber and economic forests. This pest primarily causes plant tissue to necrotize, rot, and eventually die by feeding on the woody parts of tree trunks. To gain a deeper understanding of the genetic mechanism of B. horsfieldi, this study employed single-molecule real-time sequencing (SMRT) and Illumina RNA-seq technologies to conduct full-length transcriptome sequencing of the insect. Total RNA extracted from male and female adults was mixed and subjected to SMRT sequencing, generating a complete transcriptome. Transcriptome analysis, prediction of long non-coding RNA (lncRNA), coding sequences (CDs), analysis of simple sequence repeats (SSR), prediction of transcription factors, and functional annotation of transcripts were performed in this study. The collective 20,356,793 subreads (38.26 G, clean reads) were generated, including 432,091 circular consensus sequences and 395,851 full-length non-chimera reads. The full-length non-chimera reads (FLNC) were clustered and redundancies were removed, resulting in 39,912 consensus reads. SSR and ANGEL software v3.0 were used for predicting SSR and CDs. In addition, four tools were used for annotating 6058 lncRNAs, identifying 636 transcription factors. Furthermore, a total of 84,650 transcripts were functionally annotated in seven different databases. This is the first time that the full-length transcriptome of B. horsfieldi has been obtained using SMRT sequencing. This provides an important foundation for investigating the gene regulation underlying the interaction between B. horsfieldi and its host plants through gene editing in the future and provides a scientific basis for the prevention and control of B. horsfieldi.

Background: Alfalfa (Medicago sativa L.), serves as a legume with high drought tolerance, is a major forage crop with a high biomass of production. However, the molecular mechanism of Alfalfa in response to drought stress are still unclear. Results: We constructed the first full-length transcriptome for Alfalfa root. 21.53Gb clean data were obtained by further data filtering, in which incorporate 566,076 reads of Insert (ROI), and 409,291 full length reads non-Chimeric (FLNC) sequences. Combined with second-generation sequencing (SGS), there were 2615, 6011, and 4617 differentially expressed genes (DEGs) in three comparisons. KEGG pathway analysis showed enrichment of ribosome, glutathione metabolism, and biosynthesis of amino acids are among the DEGs. The majority of transcription factors (TFs) from DEGs were AP2/ERF-ERF (37), C2H2 (32), and bHLH (22) bZIP (22), followed by C3H (19), MYB (18), WRKY (18), GRAS (16), and NAC (15). 32 C2H2 genes were differentially expressed in three groups. In addition, TFs annotated as C3H (19), MYB (18), GRAS (16), and NAC (15) also changed significantly in expression in the three comparisons. We found 24 genes participate in the abscisic acid (ABA) and auxin hormone signaling pathway in response to drought stress, and monitored the expression patterns of these related genes. Conclusion: The present study enhanced our understanding of the genetic diversity and complexity, and provides greater insight into the fundamental transcriptome reprogramming of Alfalfa under drought.

In mammals, testis and epididymis are critical components of the male reproductive system for androgen production, spermatogenesis, sperm transportation, as well as sperm maturation. Here, we report single-molecule real-time sequencing data from the testis and epididymis of the Banna mini-pig inbred line (BMI), a promising laboratory animal for medical research. We obtained high-quality full-length transcriptomes and identified 9879 isoforms and 8761 isoforms in the BMI testis and epididymis, respectively. Most of the isoforms we identified have novel exon structures that will greatly improve the annotation of testis- and epididymis-expressed genes in pigs. We also found that 3055 genes (over 50%) were shared between BMI testis and epididymis, indicating widespread expression profiles of genes related to reproduction. We characterized extensive alternative splicing events in BMI testis and epididymis and showed that 96 testis-expressed genes and 79 epididymis-expressed genes have more than six isoforms, revealing the complexity of alternative splicing. We accurately defined the transcribed isoforms in BMI testis and epididymis by combining Pacific Biotechnology Isoform-sequencing (PacBio Iso-Seq) and Illumina RNA Sequencing (RNA-seq) techniques. The refined annotation of some key genes governing male reproduction will facilitate further understanding of the molecular mechanisms underlying BMI male sterility. In addition, the high-confident identification of 548 and 669 long noncoding RNAs (lncRNAs) in these two tissues has established a candidate gene set for future functional investigations. Overall, our study provides new insights into the role of the testis and epididymis during BMI reproduction, paving the path for further studies on BMI male infertility.

Characterization of the subcellular distribution of RNA is essential for understanding the molecular basis of biological processes. Here, the subcellular nanopore direct RNA-sequencing (DRS) of four lung cancer cell lines (A549, H1975, H358, and HCC4006) is performed, coupled with a computational pipeline, Low-abundance Aware Full-length Isoform clusTEr (LAFITE), to comprehensively analyze the full-length cytoplasmic and nuclear transcriptome. Using additional DRS and orthogonal data sets, it is shown that LAFITE outperforms current methods for detecting full-length transcripts, particularly for low-abundance isoforms that are usually overlooked due to poor read coverage. Experimental validation of six novel isoforms exclusively identified by LAFITE further confirms the reliability of this pipeline. By applying LAFITE to subcellular DRS data, the complexity of the nuclear transcriptome is revealed in terms of isoform diversity, 3\'-UTR usage, m6A modification patterns, and intron retention. Overall, LAFITE provides enhanced full-length isoform identification and enables a high-resolution view of the RNA landscape at the isoform level.

Plant responses to single or combined abiotic stresses between aboveground and underground parts are complex and require crosstalk signaling pathways. In this study, we explored the transcriptome data of common vetch (Vicia sativa L.) subjected to cold and drought stress between leaves and roots via meta-analysis to identify the hub abiotic stress-responsive genes. A total of 4,836 and 3,103 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Transcriptome analysis results showed that the set of stress-responsive DEGs to concurrent stress is distinct from single stress, indicating a specialized and unique response to combined stresses in common vetch. Gene Ontology (GO) enrichment analyses identified that \"Photosystem II,\" \"Defence response,\" and \"Sucrose synthase/metabolic activity\" were the most significantly enriched categories in leaves, roots, and both tissues, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results indicated that \"ABC transporters\" are the most enriched pathway and that all of the genes were upregulated in roots. Furthermore, 29 co-induced DEGs were identified as hub genes based on the consensus expression profile module of single and co-occurrence stress analysis. In transgenic yeast, the overexpression of three cross-stress tolerance candidate genes increased yeast tolerance to cold-drought combined stress. The elucidation of the combined stress-responsive network in common vetch to better parse the complex regulation of abiotic responses in plants facilitates more adequate legume forage breeding for combined stress tolerance.

Owing to its strong environmental suitability to adverse abiotic stress conditions, common vetch (Vicia sativa) is grown worldwide for both forage and green manure purposes and is an important protein source for human consumption and livestock feed. The germination of common vetch seeds and growth of seedlings are severely affected by salinity stress, and the response of common vetch to salinity stress at the molecular level is still poorly understood. In this study, we report the first comparative transcriptomic analysis of the leaves and roots of common vetch under salinity stress. A total of 6361 differentially expressed genes were identified in leaves and roots. In the roots, the stress response was dominated by genes involved in peroxidase activity. However, the genes in leaves focused mainly on Ca2+ transport. Overexpression of six salinity-inducible transcription factors in yeast further confirmed their biological functions in the salinity stress response. Our study provides the most comprehensive transcriptomic analysis of common vetch leaf and root responses to salinity stress. Our findings broaden the knowledge of the common and distinct intrinsic molecular mechanisms within the leaves and roots of common vetch and could help to develop common vetch cultivars with high salinity tolerance.