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Drought is one of the main environmental factors affecting plant survival and growth. Atraphaxis bracteata is a common desert plant mainly utilized in afforestation and desertification control. This study analyzed the morphological, physiological and molecular regulatory characteristics of different organs of A. bracteata under drought stress. The results showed that with the intensification of drought stress, the height, root length and leaf area of A. bracteata seedlings decreased, while the content of osmotic substances and antioxidant enzyme activity increased. Furthermore, a total of 63 907 non-redundant transcript sequences, of which 55 574 transcripts were annotated, 248 178 FLNC sequences, 107 112 high-quality consistent sequences and 291 314 CCSs were obtained from Iso-Seq. Meanwhile, a total of 2 039 AS events, 22 919 SSR, 40 404 CDS and 5 902 lncRNA were also obtained. The RNA-Seq analysis results revealed that a total of 2 821, 3 907 and 5 532 DETs were identified from roots, stems and leaves, respectively, and which had significantly enrichment in "circadian rhythm-plant" and "starch and sucrose metabolism" pathway. These results would be great significance for further research on the stress resistance of A. bracteata and these DETs function. [ABSTRACT FROM AUTHOR]

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 highthroughput 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Paeonia lactiflora Pall., a Paeoniaceae family member, is a herbaceous perennial flower of significant horticultural importance. It holds prominence in traditional Chinese medicine due to its therapeutic applications in pain relief, inflammation reduction, and immune disorder treatment. Paeoniflorin, the active monoterpene compound in P. lactiflora, exhibits diverse biological activities, such as anti-inflammatory and immune-regulatory effects. Although the pharmacological effects of paeoniflorin have been extensively studied, its biosynthetic pathway remains elusive. Here, we combined PacBio Iso-Seq and RNA-seq techniques to investigate three distinct tissues from two different P. lactiflora ecotypes with varying paeoniflorin contents. The primary objective was to identify candidate genes involved in the biosynthesis of paeoniflorin and related monoterpenes. Differentially expressed unigenes encoding cytochrome P450s, UDP-glucoronosyl and UDP-glucosyl transferases, and transcription factors, which might be associated with monoterpene diversity in P. lactiflora, were identified. In addition, we analyzed genetic differentiation between the two P. lactiflora ecotypes. Notably, gene expression in the terpene pathway within root and rhizome tissues was downregulated compared to leaf tissues. This intriguing observation led us to speculate that paeoniflorin synthesis primarily occurs in the leaves and is subsequently transported to the roots. However, further experimentation is required to provide substantial evidence and verify this hypothesis. [ABSTRACT FROM AUTHOR]

Simple Summary: Batocera horsfieldi (Hope) is an important wood-boring pest in China. This pest primarily infests tree trunks by feeding on the woody tissue, creating a network of interconnected tunnels within the trunk. These tunnels become blocked with insect feces and wood debris, causing damage, decay, and even the death of the host plant's tissues. In this study, single-molecule real-time sequencing (SMRT) and Illumina RNA-seq technologies were employed to conduct full-length transcriptome sequencing of male and female adults of B. horsfieldi. A total of 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. Clustering and redundancy removal of the full-length non-chimera reads resulted in 39,912 consensus reads. Additionally, functional annotation was performed on a total of 84,650 transcripts in seven different databases. This study provides an important foundation for future exploration of gene regulation in the interaction between B. horsfieldi and host plants using RNA interference (RNAi), and it offers a scientific basis for the prevention and control of B. horsfieldi. 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. [ABSTRACT FROM AUTHOR]

Abstract Background High-throughput next-generation sequencing technologies offer a powerful approach to characterizing the transcriptomes of plants. Long read sequencing has been shown to support the discovery of novel isoforms of transcripts. This approach enables the generation of full-length sequences revealing splice variants that may be important in regulating gene action. Investigation of the diversity of transcripts in the rice transcriptome including splice variants was conducted using PacBio long-read sequence data to improve the annotation of the rice genome. Results A cDNA library was prepared from RNA extracted from leaves, roots, seeds, inflorescences, and panicles of O. sativa ssp. japonica var Nipponbare and sequenced on a PacBio Sequel platform. This produced 346,190 non-redundant full-length non-chimeric reads (FLNC) resulting in 33,504 high-quality transcripts. Half of the transcripts were multi-exonic and entirely matched with the reference transcripts. However, 14,874 novel isoforms were also identified resulting predominantly from intron retention and at least one novel splice site. Intron retention was the prevalent alternative splicing event and exon skipping was the least observed. Of 73,659 splice junctions, 12,755 (17%) represented novel splice junctions with canonical and non-canonical intron boundaries. The complexity of the transcriptome was examined in detail for 19 starch synthesis-related genes, defining 276 spliced isoforms of which 94 splice variants were novel. Conclusion The data reveal the great complexity of the rice transcriptome. The novel transcripts provide new insights that may be a key input in future research to improve the annotation of the rice genome.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The European starling, Sturnus vulgaris, is an ecologically significant, globally invasive avian species that is also suffering from a major decline in its native range. Here, we present the genome assembly and long‐read transcriptome of an Australian‐sourced European starling (S. vulgaris vAU), and a second, North American, short‐read genome assembly (S. vulgaris vNA), as complementary reference genomes for population genetic and evolutionary characterization. S. vulgaris vAU combined 10× genomics linked‐reads, low‐coverage Nanopore sequencing, and PacBio Iso‐Seq full‐length transcript scaffolding to generate a 1050 Mb assembly on 6222 scaffolds (7.6 Mb scaffold N50, 94.6% busco completeness). Further scaffolding against the high‐quality zebra finch (Taeniopygia guttata) genome assigned 98.6% of the assembly to 32 putative nuclear chromosome scaffolds. Species‐specific transcript mapping and gene annotation revealed good gene‐level assembly and high functional completeness. Using S. vulgaris vAU, we demonstrate how the multifunctional use of PacBio Iso‐Seq transcript data and complementary homology‐based annotation of sequential assembly steps (assessed using a new tool, saaga) can be used to assess, inform, and validate assembly workflow decisions. We also highlight some counterintuitive behaviour in traditional busco metrics, and present buscomp, a complementary tool for assembly comparison designed to be robust to differences in assembly size and base‐calling quality. This work expands our knowledge of avian genomes and the available toolkit for assessing and improving genome quality. The new genomic resources presented will facilitate further global genomic and transcriptomic analysis on this ecologically important species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Portunus trituberculatus is an ideal model for elucidating crustacean genetic networks. Here we combined single molecule real-time (SMRT) sequencing and Illumina RNA-seq to characterize the coding genes, non-coding RNAs and pseudogenes and further to improve the genome annotation information of P. trituberculatus. In this study, we assembled 9 694 non-redundancy full-length transcripts, and 658 737 307-bp repetitive sequences were identified in the P. trituberculatus full-length transcriptome. We also predicted the P. trituberculatus genome structure based on full-length transcripts, including 18 602 genes, 28 686 non-coding RNAs, 1 407 pseudogenes, 740 motif, and 26 434 domain. Meanwhile, 14 460, 10 211, 5 412, 7 314, and 14 448 genes had significant matches with sequences in the NR, KOG, GO, KEGG, and TrEMBL database, respectively. Overall, our work firstly provided the long-read transcriptome and we believed that these data are very necessary to improve the annotation information of P. trituberculatus genome structure, and useful information for the future studies on evolution and physiological regulation of P. trituberculatus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: High-throughput next-generation sequencing technologies offer a powerful approach to characterizing the transcriptomes of plants. Long read sequencing has been shown to support the discovery of novel isoforms of transcripts. This approach enables the generation of full-length sequences revealing splice variants that may be important in regulating gene action. Investigation of the diversity of transcripts in the rice transcriptome including splice variants was conducted using PacBio long-read sequence data to improve the annotation of the rice genome. Results: A cDNA library was prepared from RNA extracted from leaves, roots, seeds, inflorescences, and panicles of O. sativa ssp. japonica var Nipponbare and sequenced on a PacBio Sequel platform. This produced 346,190 non-redundant full-length non-chimeric reads (FLNC) resulting in 33,504 high-quality transcripts. Half of the transcripts were multi-exonic and entirely matched with the reference transcripts. However, 14,874 novel isoforms were also identified resulting predominantly from intron retention and at least one novel splice site. Intron retention was the prevalent alternative splicing event and exon skipping was the least observed. Of 73,659 splice junctions, 12,755 (17%) represented novel splice junctions with canonical and non-canonical intron boundaries. The complexity of the transcriptome was examined in detail for 19 starch synthesis-related genes, defining 276 spliced isoforms of which 94 splice variants were novel. Conclusion: The data reveal the great complexity of the rice transcriptome. The novel transcripts provide new insights that may be a key input in future research to improve the annotation of the rice genome. [ABSTRACT FROM AUTHOR]

Key message: Combination analysis of single-molecule long-read and Illumina sequencing provide full-length transcriptome information and shed new light on the anthocyanin accumulation mechanism of Pennisetum setaceum cv. 'Rubrum'. Pennisetum setaceum cv. 'Rubrum' is an ornamental grass with purple leaves widely used in landscaping. However, the current next-generation sequencing (NGS) transcriptome information of this species is not satisfactory due to the difficulties in obtaining full-length transcripts. Furthermore, the molecular mechanisms of anthocyanin accumulation in P. setaceum have not been thoroughly studied. In this study, we used PacBio full-length transcriptome sequencing (SMRT) combined with NGS technology to build and improve the transcriptomic datasets and reveal the molecular mechanism of anthocyanin accumulation in P. setaceum cv. 'Rubrum'. Therefore, 280,413 full-length non-chimeric reads sequences were obtained using the SMRT technology. We obtained 97,450 high-quality non-redundant transcripts and identified 5352 alternative splicing events. In addition, 93,066 open reading frames (ORFs), including 57,457 full ORFs and 2910 long non-coding RNA (lncRNAs) were screened out. Furthermore, 10,795 differentially expressed genes were identified using NGS. We also explored key genes, synthesis pathways, and detected lncRNA involved in anthocyanin accumulation, providing new insights into anthocyanin accumulation in P. setaceum cv. 'Rubrum'. To our best knowledge, we provided the full-length transcriptome information of P. setaceum cv. 'Rubrum' for the first time. The results of this study will provide baseline information for gene function studies and pave the way for future P. setaceum cv. 'Rubrum' breeding projects. [ABSTRACT FROM AUTHOR]

The ridgetail white shrimp Exopalaemon carinicauda has the potential to be used as a useful experimental organism in the field of crustacean research due to its transparent body, large egg, and short reproductive cycle. However, little is known about the regulatory mechanisms of ovarian maturation in E. carinicauda. In this study, we applied PacBio single-molecule long-read sequencing technology to unveil the whole transcriptome landscape of Exopalaemon carinicauda to better understand the molecular mechanisms of ovarian development. A total of 49.05 G polymerase read bases were generated, finally, 15, 151 unigenes were successfully identified with a mean length of 4, 257 bp and an N50 of 4, 884 bp from ovary tissues (five different ovarian developmental stages). A total of 14, 007 unigenes were successfully annotated in at least one public database. In addition, 8, 861 coding sequences, 4, 594 lncRNAs and 895 transcription factors were identified. Furthermore, we performed RNA-Seq analysis combined with Iso-Seq results to investigate ovarian maturation regulation mechanism and validated the results by quantitative real-time PCR. We annotated five vitellogenin isoforms in the full-length transcriptome, meanwhile these five genes and vitellogenin receptor suggested an important role during previtellogenesis of E. carinicauda. Genes were involved in PI3K-Akt signaling pathway, retinol metabolism, cell cycle and hedgehog signaling pathway, which showed obvious associations with ovarian maturation of E. carinicauda. We identified 40 DEGs, such as forkhead box protein L2, which may be related to ovarian development in the comparisons. The expression profiles of genes such as ecdysone receptor, ecdysone-induced protein 74EF, ecdysone inducible gene E75 and post-molt protein suggest that they were significantly associated with reproductive molting in the ovarian mature stage. In conclusion, the present study identified important genes and pathways involved in ovarian maturation, which might be useful for studying the reproductive regulation and mechanisms of ovarian maturation in E. carinicauda.

Abstract Background Common vetch (Vicia sativa L.) is an important self-pollinating annual forage legume and is of interest for drought prone regions as a protein source to feed livestock and human consumption. However, the development and production of common vetch are negatively affected by drought stress. Plants have evolved common or distinct metabolic pathways between the aboveground and underground in response to drought stress. Little is known regarding the coordinated response of aboveground and underground tissues of common vetch to drought stress. Results Our results showed that a total of 30,427 full-length transcripts were identified in 12 samples, with an average length of 2278.89 bp. Global transcriptional profiles of the above 12 samples were then analysed via Illumina-Seq. A total of 3464 and 3062 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Gene Ontology (GO) enrichment analyses identified that the dehydrin genes and Δ1-pyrroline-5-carboxylate synthase were induced for the biosynthesis of proline and water conservation. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results indicated that the DEGs were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and arginine and proline metabolism, and various drought response candidate genes were also identified. Abscisic acid (ABA; the AREB/ABF-SnRK2 pathway) regulates the activity of AMY3 and BAM1 to induce starch degradation in leaves and increase carbon export to roots, which may be associated with the drought stress responses in common vetch. Among the co-induced transcription factors (TFs), AREB/ABF, bHLH, MYB, WRKY, and AP2/ERF had divergent expression patterns and may be key in the crosstalk between leaves and roots during adaption to drought stress. In transgenic yeast, the overexpression of four TFs increased yeast tolerance to osmotic stresses. Conclusion The multipronged approach identified in the leaves and roots broadens our understanding of the coordinated mechanisms of drought response in common vetch, and further provides targets to improve drought resistance through genetic engineering.

The domesticated silkworm, Bombyx mori, is an important model system for the order Lepidoptera. Currently, based on third-generation sequencing, the chromosome-level genome of Bombyx mori has been released. However, its transcripts were mainly assembled by using short reads of second-generation sequencing and expressed sequence tags which cannot explain the transcript profile accurately. Here, we used PacBio Iso-Seq technology to investigate the transcripts from 45 developmental stages of Bombyx mori. We obtained 25,970 non-redundant high-quality consensus isoforms capturing ∼60% of previous reported RNAs, 15,431 (∼47%) novel transcripts, and identified 7,253 long non-coding RNA (lncRNA) with a large proportion of novel lncRNA (∼56%). In addition, we found that transposable elements (TEs) exonization account for 11,671 (∼45%) transcripts including 5,980 protein-coding transcripts (∼32%) and 5,691 lncRNAs (∼79%). Overall, our results expand the silkworm transcripts and have general implications to understand the interaction between TEs and their host genes. These transcripts resource will promote functional studies of genes and lncRNAs as well as TEs in the silkworm.

The domesticated silkworm, Bombyx mori , is an important model system for the order Lepidoptera. Currently, based on third-generation sequencing, the chromosome-level genome of Bombyx mori has been released. However, its transcripts were mainly assembled by using short reads of second-generation sequencing and expressed sequence tags which cannot explain the transcript profile accurately. Here, we used PacBio Iso-Seq technology to investigate the transcripts from 45 developmental stages of Bombyx mori. We obtained 25,970 non-redundant high-quality consensus isoforms capturing ∼60% of previous reported RNAs, 15,431 (∼47%) novel transcripts, and identified 7,253 long non-coding RNA (lncRNA) with a large proportion of novel lncRNA (∼56%). In addition, we found that transposable elements (TEs) exonization account for 11,671 (∼45%) transcripts including 5,980 protein-coding transcripts (∼32%) and 5,691 lncRNAs (∼79%). Overall, our results expand the silkworm transcripts and have general implications to understand the interaction between TEs and their host genes. These transcripts resource will promote functional studies of genes and lncRNAs as well as TEs in the silkworm. [ABSTRACT FROM AUTHOR]

Genus Gnetum, of which the majority species are pantropical liana, have broad industrial uses including for string, nets, and paper production. Although numerous studies have investigated anatomical structures during stem development, the underlying molecular mechanisms that regulate this developmental trajectory in Gnetum species remain poorly understood. A total of 12 full-length transcriptomes were generated from four stem developmental stages of an arborescent representative of this genus, Gnetum luofuense, using Oxford Nanopore Technologies. The results of this analysis reveal a total of 24,151 alternative splicing (AS) and 134,391 alternative polyadenylation events. A remarkably dynamic pattern of AS events, especially in the case of intron retentions, was found across the four developmental stages while no dynamic pattern was found among transcript numbers with varied poly(A) sites. A total of 728 long non-coding RNAs were also detected; the number of cis-regulated target genes dramatically increased while no changes were found among trans-regulated target genes. In addition, a K-means clustering analysis of all full-length transcripts revealed that primary growth is associated with carbohydrate metabolism and fungi defense, while secondary growth is closely linked with photosynthesis, nitrogen transportation, and leaf ontogenesis. The use of weighted gene co-expression network analysis as well as differentially expressed transcripts reveals that bHLH, GRF, and MYB-related transcription factors are involved in primary growth, while AP2/ERF, MYB, NAC, PLAZ, and bZIP participate in G. luofuense stem secondary growth. The results of this study provide further evidence that Nanopore sequencing technology provides a cost-effective method for generating full-length transcriptome data as well as for investigating seed plant organ development.

Genus Gnetum , of which the majority species are pantropical liana, have broad industrial uses including for string, nets, and paper production. Although numerous studies have investigated anatomical structures during stem development, the underlying molecular mechanisms that regulate this developmental trajectory in Gnetum species remain poorly understood. A total of 12 full-length transcriptomes were generated from four stem developmental stages of an arborescent representative of this genus, Gnetum luofuense , using Oxford Nanopore Technologies. The results of this analysis reveal a total of 24,151 alternative splicing (AS) and 134,391 alternative polyadenylation events. A remarkably dynamic pattern of AS events, especially in the case of intron retentions, was found across the four developmental stages while no dynamic pattern was found among transcript numbers with varied poly(A) sites. A total of 728 long non-coding RNAs were also detected; the number of cis -regulated target genes dramatically increased while no changes were found among trans -regulated target genes. In addition, a K-means clustering analysis of all full-length transcripts revealed that primary growth is associated with carbohydrate metabolism and fungi defense, while secondary growth is closely linked with photosynthesis, nitrogen transportation, and leaf ontogenesis. The use of weighted gene co-expression network analysis as well as differentially expressed transcripts reveals that bHLH, GRF, and MYB-related transcription factors are involved in primary growth, while AP2/ERF, MYB, NAC, PLAZ, and bZIP participate in G. luofuense stem secondary growth. The results of this study provide further evidence that Nanopore sequencing technology provides a cost-effective method for generating full-length transcriptome data as well as for investigating seed plant organ development. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that circular RNA (circRNA) plays an important role in tumorigenesis by regulating gene expression at the transcriptional and post-transcriptional levels. Alternative splicing events permit multiple transcript isoforms of circRNA to be produced; however, changes in the expression of circRNA full-length transcripts in cervical cancer remain unclear. Here, we systematically explored the dysregulation circRNA full-length transcripts and constructed an improved circRNA-miRNA-mRNA regulatory network to provide potential biomarkers and possible treatment targets in cervical cancer. We identified 9359 circular full-length transcripts from RNase R-treated RNA-seq data in cervical cancer, of which 353 circular full-length transcripts were significantly differentially expressed (DE) between the tumor and normal group. A total of 881 DE mRNA transcript isoforms were also identified from total RNA-seq data in cervical cancer, of which 421 (47.8%) transcript isoforms were up-regulated, and 460 (52.2%) transcript isoforms were down-regulated in tumor samples. Two circRNA-miRNA-mRNA competitively regulated networks, including 33 circRNA transcripts, 2 miRNAs, and 189 mRNA transcripts were constructed. Three genes (COPE, RAB3B, and TFPI) in the network were significantly associated with overall survival (P < 0.05), which indicated that these genes could act as prognostic biomarkers for patients with cervical cancer. Our study revealed genome-wide differential expression of full-length circRNA transcripts and constructed a more accurate circRNA-miRNA-mRNA network at the full-length transcript expression level in cervical cancer. CircRNA may thus be involved in the development of cervical cancer by regulating the expression of COPE, RAB3B, and TFPI. However, the specific regulatory mechanism in cervical cancer requires further study.

Abstract Background Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa’s productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress. Results In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24 h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of 2551 bp. Global transcriptional changes in the same 33 stressed samples were then analyzed via BGISEQ-500 RNA-Seq. Totals of 8861 NaCl-regulated and 8016 mannitol-regulated differentially expressed genes (DEGs) were identified. Metabolic analyses revealed that these DEGs overlapped or diverged in the cascades of molecular networks involved in signal perception, signal transduction, transcriptional regulation, and antioxidative defense. Notably, several well characterized signalling pathways, such as CDPK, MAPK, CIPK, and PYL-PP2C-SnRK2, were shown to be involved in osmotic stress, while the SOS core pathway was activated by ionic stress. Moreover, the physiological shifts of catalase and peroxidase activity, glutathione and proline content were in accordance with dynamic transcript profiles of the relevant genes, indicating that antioxidative defense system plays critical roles in response to salinity stress. Conclusions Overall, our study provides evidence that the response to salinity stress in alfalfa includes both osmotic and ionic components. The key osmotic and ionic stress-related genes are candidates for future studies as potential targets to improve resistance to salinity stress via genetic engineering.

Keywords: African swine fever virus (ASFV); long-read sequencing; short-read sequencing; transcriptomics; direct RNA sequencing; full-length transcripts EN African swine fever virus (ASFV) long-read sequencing short-read sequencing transcriptomics direct RNA sequencing full-length transcripts 1 7 7 07/31/20 20200728 NES 200728 Introduction Methodological breakthroughs in sequencing technologies have revolutionized transcriptome profiling in recent years. RNA Purification Isolation of Total RNA A NucleoSpin® RNA (Macherey-Nagel) kit was used for RNA purification following the manufacturer's instructions. Technical Validation The total RNA, polyA(+) RNA, and rRNA-depleted samples; generated cDNAs; and final sequencing libraries were quantified by a Qubit 4 Fluorometer using Qubit RNA Broad-Range, High Sensitivity RNA, and High Sensitivity dsDNA Assay Kits. Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules. [Extracted from the article]

Background: Common vetch (Vicia sativa L.) is an important self-pollinating annual forage legume and is of interest for drought prone regions as a protein source to feed livestock and human consumption. However, the development and production of common vetch are negatively affected by drought stress. Plants have evolved common or distinct metabolic pathways between the aboveground and underground in response to drought stress. Little is known regarding the coordinated response of aboveground and underground tissues of common vetch to drought stress. Results: Our results showed that a total of 30,427 full-length transcripts were identified in 12 samples, with an average length of 2278.89 bp. Global transcriptional profiles of the above 12 samples were then analysed via Illumina-Seq. A total of 3464 and 3062 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Gene Ontology (GO) enrichment analyses identified that the dehydrin genes and Δ1-pyrroline-5-carboxylate synthase were induced for the biosynthesis of proline and water conservation. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results indicated that the DEGs were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and arginine and proline metabolism, and various drought response candidate genes were also identified. Abscisic acid (ABA; the AREB/ABF-SnRK2 pathway) regulates the activity of AMY3 and BAM1 to induce starch degradation in leaves and increase carbon export to roots, which may be associated with the drought stress responses in common vetch. Among the co-induced transcription factors (TFs), AREB/ABF, bHLH, MYB, WRKY, and AP2/ERF had divergent expression patterns and may be key in the crosstalk between leaves and roots during adaption to drought stress. In transgenic yeast, the overexpression of four TFs increased yeast tolerance to osmotic stresses. Conclusion: The multipronged approach identified in the leaves and roots broadens our understanding of the coordinated mechanisms of drought response in common vetch, and further provides targets to improve drought resistance through genetic engineering. [ABSTRACT FROM AUTHOR]

Transcriptome complexity plays crucial roles in regulating the biological functions of eukaryotes. Except for functional genes, alternative splicing and fusion transcripts produce a vast expansion of transcriptome diversity. In this study, we applied PacBio single-molecule long-read sequencing technology to unveil the whole transcriptome landscape of Lateolabrax maculatus. We obtained 28,809 high-quality non-redundant transcripts, including 18,280 novel isoforms covering 8,961 annotated gene loci within the current reference genome and 3,172 novel isoforms. A total of 10,249 AS events were detected, and intron retention was the predominant AS event. In addition, 1,359 alternative polyadenylation events, 3,112 lncRNAs, 29,609 SSRs, 365 fusion transcripts, and 1,194 transcription factors were identified in this study. Furthermore, we performed RNA-Seq analysis combined with Iso-Seq results to investigate salinity regulation mechanism at the transcripts level. A total of 518 transcripts were differentially expressed, which were further divided into 8 functional groups. Notably, transcripts from the same genes exhibited similar or opposite expression patterns. Our study provides a comprehensive view of the transcriptome complexity in L. maculatus, which significantly improves current gene models. Moreover, the diversity of the expression patterns of transcripts may enhance the understanding of salinity regulatory mechanism in L. maculatus and other euryhaline teleosts.

Transcriptome complexity plays crucial roles in regulating the biological functions of eukaryotes. Except for functional genes, alternative splicing and fusion transcripts produce a vast expansion of transcriptome diversity. In this study, we applied PacBio single-molecule long-read sequencing technology to unveil the whole transcriptome landscape of Lateolabrax maculatus. We obtained 28,809 high-quality non-redundant transcripts, including 18,280 novel isoforms covering 8,961 annotated gene loci within the current reference genome and 3,172 novel isoforms. A total of 10,249 AS events were detected, and intron retention was the predominant AS event. In addition, 1,359 alternative polyadenylation events, 3,112 lncRNAs, 29,609 SSRs, 365 fusion transcripts, and 1,194 transcription factors were identified in this study. Furthermore, we performed RNA-Seq analysis combined with Iso-Seq results to investigate salinity regulation mechanism at the transcripts level. A total of 518 transcripts were differentially expressed, which were further divided into 8 functional groups. Notably, transcripts from the same genes exhibited similar or opposite expression patterns. Our study provides a comprehensive view of the transcriptome complexity in L. maculatus , which significantly improves current gene models. Moreover, the diversity of the expression patterns of transcripts may enhance the understanding of salinity regulatory mechanism in L. maculatus and other euryhaline teleosts. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Over 31,000 uncatalogued isoforms are identified in human cerebral organoids • 4,531 cell-type-specific splicing events are identified in human cerebral organoids • Alternative exons are enriched for autism-associated exons and de novo mutations • 142 autism genes display cell-type-specific isoforms Yang et al. perform single-cell long-read sequencing in the human cerebral organoids, identifying thousands of uncatalogued isoforms, differentially spliced exons, and retained introns. Their results reveal the enrichement of autism-associated exons and de novo mutations in cell-type-specific exons. [ABSTRACT FROM AUTHOR]

Background Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs. Methods We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors. Results A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants. Discussion The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

Background: Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa's productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress. Results: In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24 h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of 2551 bp. Global transcriptional changes in the same 33 stressed samples were then analyzed via BGISEQ-500 RNA-Seq. Totals of 8861 NaCl-regulated and 8016 mannitol-regulated differentially expressed genes (DEGs) were identified. Metabolic analyses revealed that these DEGs overlapped or diverged in the cascades of molecular networks involved in signal perception, signal transduction, transcriptional regulation, and antioxidative defense. Notably, several well characterized signalling pathways, such as CDPK, MAPK, CIPK, and PYL-PP2C-SnRK2, were shown to be involved in osmotic stress, while the SOS core pathway was activated by ionic stress. Moreover, the physiological shifts of catalase and peroxidase activity, glutathione and proline content were in accordance with dynamic transcript profiles of the relevant genes, indicating that antioxidative defense system plays critical roles in response to salinity stress. Conclusions: Overall, our study provides evidence that the response to salinity stress in alfalfa includes both osmotic and ionic components. The key osmotic and ionic stress-related genes are candidates for future studies as potential targets to improve resistance to salinity stress via genetic engineering. [ABSTRACT FROM AUTHOR]

Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza. [ABSTRACT FROM AUTHOR]

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

• Oxford Nanopore full-length transcriptomes technology was applied. • Cold environment stimulation induces honeybees to use different AS and APA mechanism. • Hippo, FoxO and MARK signal pathway help honeybees countering the stress of cold. Apis cerana in Changbai Mountain is an ecological type of Apis cerana , which is an excellent breeding material with cold-resistant developed by long-term natural selection under the ecological conditions. However, the physiological and molecular mechanisms of Changbai Mountain population under cold stress are still unclear. In this study, the Nanopore sequencing was carried out for the transcriptome of Apis cerana in Changbai Mountain in the coldest period of overwintering, which will provide a reference to the cold-resistant mechanism. We determined 5,941 complete ORF sequences, 1,193 lncRNAs, 619 TFs, 10,866 SSRs and functional annotations of 11,599 new transcripts. Our results showed that the myosin family and the C2H2 zinc finger protein transcription factor family possibly have significant impacts on the response mechanism of cold stress during overwintering. In addition, the cold environment alters genes expression profiles in honeybees via different AS and APA mechanisms. These altered genes in Hippo, Foxo, and MARK pathways help them counter the stress of cold in overwinter period. Our results might provide clues about the response of eastern honeybees to extreme cold, and reflect the possible genetic basis of physiological changes. [ABSTRACT FROM AUTHOR]

Psammochloa villosa is a perennial herbaceous plant that is dominant within arid regions of the Inner Mongolian Plateau and the Qinghai-Tibet Plateau in China, where it is an endemic species and exhibits strong drought tolerance and wind resistance. To study drought tolerance in P. villosa and determine its molecular basis, we simulated high and moderate drought stress in a controlled environment and then analyzed transcriptome sequences by combining long-read sequences from a representative, wild-grown individual with short reads from the treatment groups. We obtained 184,076 high-quality isoforms as a reference and 168,650 genes (91.6%), which we were able to annotate according to public databases. Ultimately, we obtained 119,005 unigenes representing the transcriptome of P. villosa under drought stress and, among these, we identified 3089 differentially expressed genes and 1484 transcription factors. Physiologically, P. villosa that was exposed to high and moderate drought stress had reduced germination rates and shorter buds but generated more chlorophyll, which is atypical under drought stress and possibly reflects an adaptation of these plants to their arid environment. We inferred that significantly upregulated genes were annotated as 'Chlorophyll a - b binding protein' and 'Light-harvesting chlorophyll-protein' among drought and control groups. Broadly, our analyses revealed that drought stress triggered many genome-level responses, especially related to mitigation of radical oxygen species (ROS), which increase in concentration under drought stress. In particular, in the high drought stress group compared with the control, GO enrichment analysis revealed a significant enrichment of upregulated genes (n = 10) involved in mitigation of oxidative stress. Similarly, using KEGG we found significant enrichment of genes in the phenylpropanoid biosynthesis pathway (11 genes), which yields phenols that scavenge ROS. We also inferred that many genes involved in metabolism of arginine and proline, which may serve as both scavengers of ROS and osmoprotectants that interact with stress response genes based on our protein–protein interaction network analysis. We verified the relative expression levels of eight genes associated with mitigation of ROS, DNA repair, and transmembrane transporter activity using qRT-PCR, and the results were consistent with our inferences from transcriptomes. This study provides insights into the genomic and physiological basis of drought tolerance in P. villosa and represents a resource for development of the species as a forage crop via molecular breeding within arid lands. [ABSTRACT FROM AUTHOR]

Circular RNA (circRNA) is a special type of non-coding RNA that participates in diverse biological processes in both animals and plants. Five years ago, we developed a comprehensive plant circRNA database (PlantcircBase), which has attracted much attention from the plant circRNA community. Here, we report an updated PlantcircBase (v.7.0), which contains 171,118 circRNAs from 21 plant species. Over 31,000 of the circRNAs have full-length sequences constructed based on analysis of 749 bulk RNA sequencing (RNA-seq) datasets downloaded from the public domain and Nanopore long-read sequencing results of rice RNAs newly generated in this study. A plant multiple conservation score (PMCS), based on the conservation of both sequence and expression profiles, was calculated for each circRNA to quantify and compare the conservation of all circRNAs. A new parameter, plant circRNA confidence level (PCCL), is introduced to measure the identity reliability of each circRNA based on experimental validation results and the number of references that support the circRNA. All this information and other details of circRNAs can be browsed, searched, and downloaded from PlantcircBase 7.0, which also provides online bioinformatics tools for visualization and sequence alignment. PlantcircBase 7.0 is publicly and freely accessible at http://ibi.zju.edu.cn/plantcircbase/., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Iris lactea var. chinensis (I. lactea var. chinensis) is a perennial herb halophyte with salt and drought tolerance. In this study, full-length transcripts of I. lactea var. chinensis were sequenced using the PacBio RSII sequencing platform. Moreover, the transcriptome was investigated under NaCl or polyethylene glycol (PEG) stress. Approximately 30.89 G subreads were generated and 31,195 unigenes were obtained by clustering the same isoforms by the PacBio RSII platform. A total of 15,466 differentially expressed genes (DEGs) were obtained under the two stresses using the Illumina platform. Among them, 9266 and 8390 DEGs were obtained under high concentrations of NaCl and PEG, respectively. In total, 3897 DEGs with the same expression pattern under the two stresses were obtained. The transcriptome expression profiles of I. lactea var. chinensis under NaCl or PEG stress obtained in this study may provide a resource for the same and different response mechanisms against different types of abiotic stress. Furthermore, the stress-related genes found in this study can provide data for future molecular breeding. [ABSTRACT FROM AUTHOR]

• The first full-length reference transcriptome was constructed in common vetch. • The diversity of cold response mechanisms were identified between leaf and root. • Co-induced and tissue-specifically expressed genes were discovered. • Overexpression of six upregulated genes increased yeast cold tolerance. Due to the stronger ability to grow in extreme abiotic stress conditions, common vetch (Vicia sativa L.) has been generally cultivated worldwide and commonly utilized in methane biofuel production and health-promoting foods, and considered as an important protein source to feed livestock and human consumption. Nevertheless, the comprehensive molecular mechanisms on how the aboveground and underground tissues of common vetch response to various abiotic stresses, especially in cold stress, remain largely elusive and unknown. In this study, a total of 30,525 non-redundant full-length transcripts were identified in common vetch, with an average length of 2,291.56 bp and N50 of 3,130 bp. Among these transcripts, 7,689 and 3,415 differentially expressed genes (DEGs) were identified in leaves and roots, respectively, and 1,560 DEGs were induced in both tissues. The Gene Ontology (GO) enrichment results displayed that the DEGs were significantly enriched in the photosynthetic related categories in leaves. Correspondingly, the "response to cold" category was significantly enriched in roots, and most of them were upregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that Ca2+, redox, hormonal signalling, circadian clock, and photosynthesis-antenna proteins might play crucial roles through CBF dependent or independent transcriptional pathways to respond cold stress in common vetch. Besides, a total of 36 DEGs involved in ICE-CBF-COR signalling cascade and cold stress were identified. Furthermore, three cold response-related DEGs and three upregulated transcription factors were overexpressed in yeast and indicated their biological functions to confer the tolerance to cold. This work will advance the understanding of common and distinct intrinsic molecular mechanisms beneath the cold response in common vetch leaf and root tissues. The further investigation of the promising potential candidate genes identified in this study will serve as a valuable resource for next step functional genomics research and raise the possibility to improve the cold tolerance through a transgenic approach in common vetch. [ABSTRACT FROM AUTHOR]

Faithful analysis of transcripts represents a critical component of the gene discovery, genomic characterization and annotation process in species without a reference genome. The present study generated 28,001 full-length transcripts of Misgurnus anguillicaudatus using the Pacific Bioscience (PacBio) single-molecule real-time (SMRT) sequencing system. A total of 77,346 consensus isoforms were identified, and 18,991 complete open reading frames (ORFs) detected. In total, 1345 lncRNAs were identified with high-confidence, with additional identification of a number of well-known transcription factors. The present study may facilitate additional exploration of the genomic signatures of M. anguillicaudatus. [ABSTRACT FROM AUTHOR]