Full-length Transcriptome Sequencing Research Articles

Comparative transcriptomic analysis reveals genes related to the rapid accumulation of oleic acid in Camellia chekiangoleosa, an oil tea plant with early maturity and large fruit

Camellia chekiangoleosa has a higher oleic acid content and a shorter reproductive cycle than typical oil tea plants. It was intensively sampled over six C. chekiangoleosa seed development stages. The content of fatty acids determined by GC showed that the accumulation of fatty acids gradually increased from the S1 to S5 stages, and the maximum concentration was reached in S5. Then, fatty acids declined slightly in S6. The main fatty acid component showed the same accumulation trend as the total fatty acids, except linolenic acid, which remained at a low level throughout seed developmental stages. Changes in the expression of fatty acid accumulation-related genes were monitored using second-generation and SMRT full-length transcriptome sequencing. Finally, 18.92 G accurate and reliable data were obtained. Differential expression analysis and weighted coexpression analysis revealed two “gene modules” significantly associated with oleic acid and linoleic acid contents, and the high expression of ENR, KAS I, and KAS II, which accumulate substrates for oleic acid synthesis, was thought to be responsible for the rapid accumulation of fatty acids in the early stage. The rapid increase in fatty acids in the second stage may be closely related to the synergy between the high expression of SAD and low expression of FAD2. In addition, many transcription factors, such as ERF, GRAS, GRF, MADS, MYB and WRKY, may be involved in the fatty acid synthesis. Our data provide a rich resource for further studies on the regulation of fatty acid synthesis in C. chekiangoleosa.

Expression characteristics of long noncoding RNA and messenger RNA in human traumatic brain injury.

The role of long noncoding RNAs has attracted significant attention in diseases. However, their expression characteristics in human traumatic brain injury are unclear. The brain contusion tissues and tissues adjacent to the brain contusion from 6 server traumatic brain injury patients were used to analyze differential expression signatures of long noncoding RNAs and mRNAs via full-length transcriptome sequencing, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis and establishment of a long noncoding RNA/mRNA coexpression network. We identified 1720 long noncoding RNAs and 1632 mRNAs differential expression. Microarray analysis showed that 874 long noncoding RNAs and 1405 mRNAs were upregulated, 846 long noncoding RNAs and 227 mRNAs were downregulated. Subsequently, we used Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses to reveal signaling pathways that were associated with target genes. Then, a long noncoding RNA/mRNA coexpression network was generated, which showed an absolute correlation coefficient value >0.99 for 559 long noncoding RNA-mRNA pairs. Finally, we comprehensive analyzed long noncoding RNA/mRNA coexpression network and Kyoto Encyclopedia of Genes and Genomes pathway and found the top five pairs of long noncoding RNA/mRNA. Accordingly, we identified that long noncoding RNA tubulin beta 6 class V/nuclear factor E2-related factor 2 was most closely related to the pathological process after traumatic brain injury. Our results indicated that the expression profiles of long noncoding RNAs and mRNAs were different after traumatic brain injury, providing new insight regarding long noncoding RNAs in human traumatic brain injury.

Full-Length Transcriptome Sequencing: An Insight Into the Dog Model of Heart Failure.

Heart failure (HF) leads to a progressive increase in morbidity and mortality rates. This study aimed to explore the transcriptional landscape during HF and identify differentially expressed transcripts (DETs) and alternative splicing events associated with HF. We generated a dog model of HF (n = 3) using right ventricular pacemaker implantation. We performed full-length transcriptome sequencing (based on nanopore platform) on the myocardial tissues and analyzed the transcripts using differential expression analysis and functional annotation methods [Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses]. Additionally, we estimated the expression of the selected genes by quantitative real-time PCR (qRT-PCR) and detected the proportion of immune cells using flow cytometry. We found that increased B-type natriuretic peptide reduced ejection fraction, and apparent clinical signs were observed in the dog model of HF. We identified 67,458 transcripts using full-length transcriptome sequencing. A total of 785 DETs were obtained from the HF and control groups. These DETs were mainly enriched in the immune responses, especially Th1, Th2, and Th17 cell differentiation processes. Furthermore, flow cytometry results revealed that the proportion of Th1 and Th17 cells increased in patients with HF compared to controls, while the proportion of Th2 cells decreased. Differentially expressed genes in the HF and control groups associated with Th1, Th2, and Th17 cell differentiation were quantified using qRT-PCR. We also identified variable splicing events of sarcomere genes (e.g., MYBPC3, TNNT2, TTN, FLNC, and TTNI3). In addition, we detected 4,892 transcription factors and 406 lncRNAs associated with HF. Our analysis based on full-length transcript sequencing provided an analysis perspective in a dog model of HF, which is valuable for molecular research in an increasingly relevant large animal model of HF.

A Comparative Transcriptional Landscape of Two Castor Cultivars Obtained by Single-Molecule Sequencing Comparative Analysis.

Background and Objectives: Castor (Ricinus communis L.) is an important non-edible oilseed crop. Lm-type female strains and normal amphiprotic strains are important castor cultivars, and are mainly different in their inflorescence structures and leaf shapes. To better understand the mechanisms underlying these differences at the molecular level, we performed a comparative transcriptional analysis. Materials and Methods: Full-length transcriptome sequencing and short-read RNA sequencing were employed. Results: A total of 76,068 and 44,223 non-redundant transcripts were obtained from high-quality transcripts of Lm-type female strains and normal amphiprotic strains, respectively. In Lm-type female strains and normal amphiprotic strains, 51,613 and 20,152 alternative splicing events were found, respectively. There were 13,239 transcription factors identified from the full-length transcriptomes. Comparative analysis showed a great variety of gene expression of common and unique transcription factors between the two cultivars. Meanwhile, a functional analysis of the isoforms was conducted. The full-length sequences were used as a reference genome, and a short-read RNA sequencing analysis was performed to conduct differential gene analysis. Furthermore, the function of DEGs were performed to annotation analysis. Conclusion: The results revealed considerable differences and expression diversity between the two cultivars, well beyond what was reported in previous studies and likely reflecting the differences in architecture between these two cultivars.

Genome-wide association study and transcriptome of olecranon-type traits in peach (Prunus persica L.) germplasm

BackgroundThe top of the olecranon honey peach (Prunus persica L.) fruit appears similar to an eagle’s beak. In this study, a single olecranon honey peach with a round-type fruit was observed in our fruit orchard. To explore the genetic mechanism of olecranon formation, we performed full-length transcriptome sequencing analysis of olecranon and round peaches as well as a genome-wide association study of the association of olecranon-type trait loci.ResultsThe gene locus was 26,924,482 base pairs in NC_034014.1. Transcriptome sequencing showed that the clean sequencing data of each sample reached 7.10GB, with 14,360 genes and 23,167 transcripts expressed in both the olecranon honey peach and round peach. Among the 11 differentially expressed genes selected as candidate genes, six were highly expressed in olecranon peach and named as LOC18775282, LOC18772209, LOC18773929, LOC18772013, LOC18773401, and ONT.13798.5. Five genes were highly expressed in round peach and named as LOC18773079, LOC18773525, LOC18773067, LOC18775244, and LOC18772236. Notably, ONT.13798.5 was not previously identified. The genes were within 1 Mb up- or down-stream of the main genome-wide association study locus for olecranon-type traits.ConclusionsThis study revealed loci associated with olecranon and provides useful information for analysis and breeding of olecranon honey peach.

Identification of the diterpenoid biosynthesis genes and their expression status in relation to oleoresin yield of masson pine

Conifer oleoresin is a highly precious raw material for industrial sectors and a precursor for biofuels. Diterpenoid, particularly diterpenoid resin acids (DRAs), is one of the most abundant oleoresin components. Masson pine (Pinus massoniana L.) is one of the most important native species for oleoresin yield in China. To better understand the biosynthesis of diterpenoid in high oleoresin-yield masson pine and develop methods for its production in needle and trunk xylem, we used the combination of SMRT full-length transcriptome and Illumina RNA sequencing technologies to establish a transcriptome dataset. Approximately 20,560 and 17,950 full-length non-redundant transcripts were assembled in trunk xylem and needle, respectively. Of those, 29 and 20 DEGs were identified involving in the Terpenoid Backbone Biosynthesis pathway (TBB) (KO 00900) and the Diterpenoid Biosynthesis pathway (DB) (KO 00904) in trunk xylem, as well as 35 and 12 DEGs in the TBB and the DB in the needle, respectively. Compared with the low oleoresin-yield germplasm, 13 up-regulated DEGs with complete open reading frame (ORF) sequence were obtained by Venn analysis in trunk xylem and needle of a high oleoresin-yield clone. Subsequently, the expression patterns of the 16 diterpenoid biosynthesis-related genes (including three types of DXS genes) were validated by quantitative real-time PCR in both 12-year-old and 1-year-old high and low oleoresin-yield masson pine clones. The results showed that the 13 genes in the higher yield oleoresin trees were proved to be higher expression in comparison with the lower ones irrespectively of the tree age; as well as MCS (2-C-methyl-d-erythritol 2, 4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase), and IDI (Isopentenyl-diphosphate Delta-isomerase) of the methylerythritol 4-phosphate (MEP) pathway demonstrated a higher expression compared to that of other TBB genes. Both cytosol-localized MVA and plastid-localized MEP pathways gave significant contributions to diterpenoid accumulation, and the latter played a more dominant role. To clarify the potential relationships between oleoresin yield and the expression patterns of 16 genes from the TBB and DB pathways, the Pearson correlation analysis was performed using three tissues (young needle, old needle, and trunk xylem) from the 60-year-old tree. Remarkably, HMGR (Hydroxy methylglutaryl-CoA reductase) and ent-CDS (Ent-copalyl diphosphate synthase) were positive correlation with oleoresin yield in the new needle, whereas FPPS (Farnesyl pyrophosphate synthase) was a negative correlation with that in the old needle; furthermore, HMGS (Hydroxy methylglutaryl-CoA synthase), IDI and CDS (Copalyl diphosphate synthase) were positively correlated with oleoresin synthesis in the trunk xylem. Moreover, the Pearson correlation analysis was also carried out to determine the potential relationships between the 13 genes of the TBB pathways and ent-CDS, CDS, and LDS ((13E)-labda-7 13-dien-15-ol synthase) from DB pathways within three tissues of the 60-year-old masson pine. Furthermore, the diterpenoid and diterpene resin acids (DRAs) demonstrated highly positive correlations with oleoresin yield, and DRAs were detected as the most abundant diterpenoids in oleoresin. Promisingly, HMGR, MCS, HDS, IDI, ent-CDS, and CDS may be used as the key target genes of the molecular-assisted selection for high-oleoresin-yielding germplasm in this species.

Single molecule, full-length transcript sequencing provides insight into the TPS gene family in Paeonia ostii.

BackgroundThe tree peony (Paeonia section Moutan DC), one of the traditional famous flowers with both ornamental and medicinal value, was widely used in China. Surprisingly little is known about the full-length transcriptome sequencing in tree peony, limiting the research on its gene function and molecular mechanism. The trehalose phosphate phosphatase (TPS) family genes has been found to affect plant growth and development and the function of TPS genes in Paeonia ostii is unknown.MethodsIn our study, we performed single molecule, full-length transcript sequencing in P. ostii. 10 TPS family members were identified from PacBio sequencing for bioinformatics analysis and transcriptional expression analysis.ResultsA total of 230,736 reads of insert (ROI) sequences and 114,215 full-Length non-chimeric reads (FLNC) were obtained for further ORFs and transcription factors prediction, SSR analysis and lncRNA identification. NR, Swissprot, GO, COG, KOG, Pfam and KEGG databases were used to obtain annotation information of transcripts. 10 TPS family members were identified with molecular weights between 48.0 to 108.5 kD and isoelectric point between 5.61 to 6.37. Furthermore, we found that TPS family members contain conserved TPP or TPS domain. Based on phylogenetic tree analysis, PoTPS1 protein was highly similar to AtTPS1 protein in Arabidopsis. Finally, we analyzed the expression levels of all TPS genes in P. ostii and found PoTPS5 expressed at the highest level. In conclusion, this study combined the results of the transcriptome to systematically analyze the 10 TPS family members, and sets a framework for further research of this important gene family in development of tree peony.

Full-length transcriptome analysis reveals the mechanism of acupuncture at PC6 improves cardiac function in myocardial ischemia model

BackgroundThe pathological process of myocardial ischemia (MI) is very complicated. Acupuncture at PC6 has been proved to be effective against MI injury, but the mechanism remains unclear. This study investigated the mechanism that underlies the effect of acupuncture on MI through full-length transcriptome.MethodsAdult male C57/BL6 mice were randomly divided into control, MI, and PC6 groups. Mice in MI and PC6 group generated MI model by ligating the left anterior descending (LAD) coronary artery. The samples were collected 5 days after acupuncture treatment.ResultsThe results showed that treatment by acupuncture improved cardiac function, decreased myocardial infraction area, and reduced the levels of cTnT and cTnI. Based on full-length transcriptome sequencing, 5083 differential expression genes (DEGs) and 324 DEGs were identified in the MI group and PC6 group, respectively. These genes regulated by acupuncture were mainly enriched in the inflammatory response pathway. Alternative splicing (AS) is a post-transcriptional action that contributes to the diversity of protein. In all samples, 8237 AS events associated with 1994 genes were found. Some differential AS-involved genes were enriched in the pathway related to heart disease. We also identified 602 new genes, 4 of which may the novel targets of acupuncture in MI.ConclusionsOur findings suggest that the effect of acupuncture on MI may be based on the multi-level regulation of the transcriptome.

Single-Molecule Real-Time and Illumina-Based RNA Sequencing Data Identified Vernalization-Responsive Candidate Genes in Faba Bean (Vicia faba L.).

Faba bean (Vicia faba L.) is one of the most widely grown cool season legume crops in the world. Winter faba bean normally has a vernalization requirement, which promotes an earlier flowering and pod setting than unvernalized plants. However, the molecular mechanisms of vernalization in faba bean are largely unknown. Discovering vernalization-related candidate genes is of great importance for faba bean breeding. In this study, the whole transcriptome of faba bean buds was profiled by using next-generation sequencing (NGS) and single-molecule, real-time (SMRT) full-length transcriptome sequencing technology. A total of 29,203 high-quality non-redundant transcripts, 21,098 complete coding sequences (CDS), 1,045 long non-coding RNAs (lncRNAs), and 12,939 simple sequence repeats (SSRs) were identified. Furthermore, 4,044 differentially expressed genes (DEGs) were identified through pairwise comparisons. By Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, these differentially expressed transcripts were found to be enriched in binding and transcription factor activity, electron carrier activity, rhythmic process, and receptor activity. Finally, 50 putative vernalization-related genes that played important roles in the vernalization of faba bean were identified; we also found that the levels of vernalization-responsive transcripts showed significantly higher expression levels in cold-treated buds. The expression of VfSOC1, one of the candidate genes, was sensitive to vernalization. Ectopic expression of VfSOC1 in Arabidopsis brought earlier flowering. In conclusion, the abundant vernalization-related transcripts identified in this study will provide a basis for future researches on the vernalization and faba bean breeding and established a reference full-length transcriptome for future studies on faba bean.

The Single-Molecule Long-Read Sequencing of Intestine After Soy Meal-Induced Enteritis in Juvenile Pearl Gentian Grouper, Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂

Intestinal inflammatory disease induced by excessive soy protein substitutions for fish meal (FM) protein is a common phenomenon. The pearl gentian grouper, Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂, a marine fish with important economical and nutritional values, exhibited a similar problem. As far as we know, there are no reports on the full-length transcriptome of the pearl gentian grouper. In the present study, seven isonitrogenous and isolipidic (10% lipid) diets were prepared and fed to fish for 10 weeks. The water volume in each barrel was about 1 m3, using natural light and temperature. The results showed that 40% dietary soy proteins significantly negatively affected the growth performance of the pearl gentian grouper. Compared to the FM control, the content of immunoglobulin M and the enzyme activities of glutathione reductase, glutathione peroxidase, and total superoxide dismutase in the intestine significantly increased; the content of malondialdehyde in the intestine significantly decreased; and the enzyme activities of alanine transaminase and aspartate transaminase in the liver significantly increased. A library composed of seven different treated distal intestine tissues, including the FM control group, 20% soybean meal substitute for FM (SBM20), SBM40, 20% soybean protein concentrate (SPC20), SPC40, 20% fermented soybean meal substitute for FM (FSBM20), and FSBM40, was constructed and sequenced using PacBio single-molecule real-time (SMRT) and the RNA-Seq technology. As a whole, this study obtained 420,006 full-length non-chimeric (FLNC) reads. After error correction, sequence clustering, and redundancy, 82,351 transcripts with high quality were obtained. In addition, a total of 77,815 transcripts were annotated in seven databases (non-redundant protein sequences, non-redundant nucleotide sequences, Protein family, Clusters of Orthologous Groups of proteins, Gene Ontology, Swiss-Prot, and KEGG Orthology). Also, 49,093 long non-coding RNAs (lncRNAs) and 141,702 simple sequence repeats were identified. Based on full-length transcriptome sequencing, the present study found that the Toll-like receptor/nuclear factor kappa-B signaling pathway plays an important role in the development of SBM- and FSBM-induced enteritis. SPC-induced enteritis is mainly accompanied by a general imbalance of the nutrition absorption-related signaling pathways, which only affects a small part of the immune-related signaling pathways. This study supplies new and valuable reference transcripts, which would better facilitate further research on the pearl gentian grouper.

Full-length sequencing of Ginkgo biloba L. reveals the synthesis of terpenoids during seed development

Full-length transcriptome sequencing based on the PacBio sequencing platform could significantly optimize the annotation of gene structures. As an ancient relic gymnosperm in the monotypic order Ginkgoales, Ginkgo biloba L. contains rich terpenoids that are medicinally valuable. The seeds have abundant edible endosperm, which is delicious and of high nutritional value. However, existing molecular studies on the developmental process of ginkgo seeds are relatively weak, and the biosynthesis of terpenoids in seeds has received little attention. Therefore, single-molecule real-time (SMRT) technology and Illumina sequencing were combined to sequence six tissues related to the reproductive growth and development of ginkgo in order to generate a high-quality full-length transcription database. In total, 20.98 Gb of clean reads containing 178,548 full-length non-chimeric (FLNC) sequences were obtained. From these data, 4019 novel genes and 22,845 novel isoforms were predicted, 52.32 % of the novel genes were annotated, and three novel isoforms were annotated in terpene synthesis related pathways. The enrichment analysis of differentially expressed genes (DEGs) showed that, 95 genes were enriched into 21 categories related to seed development, and 47 DEGs were enriched in the skeletal pathway of terpene synthesis. Combined with the real-time quantitative reverse transcription PCR (qRT-PCR), the phosphosynthase family members synthesizing terpene precursors have diverse and complex expression trends during seed development. Our findings confirm the advantages of SMRT, which facilitated the construction a rich transcript data-set for research on the development of ginkgo seeds, enriching the annotation of the ginkgo genome, and enhancing our understanding of gene regulation of terpene biosynthesis in ginkgo seeds.

Investigation of flavonoid expression and metabolite content patterns during seed formation of Artemisia sphaerocephala Krasch.

AbstractFlavonoids are a group of phenolic secondary metabolites in plants that have important physiological, ecological and economic value. In this study, using the desert plant Artemisia sphaerocephala Krasch. as the sample material, the content and components of the total flavonoids in its seeds at seven different developmental stages were determined. In addition, the genes involved in flavonoid metabolism were identified by full-length transcriptome sequencing (third-generation sequencing technology based on PacBio RS II). Their expression levels were analysed by RNA-seq short reading sequencing, to reveal the patterns and regulation mechanisms of flavonoid accumulation during seed development. The key results were as follows: the content of total flavonoids in mature seeds was 15.05 mg g−1, including five subclasses: flavonols, chalcones, flavones, flavanones and proanthocyanidins, among which flavonols accounted for 45.78%. The period of rapid accumulation of flavonoids was 40–70 d following anthesis. The high expression of phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL) and UDP-glucose:flavonoids 3-o-glucosyltransferase (UF3GT) promoted the accumulation of total flavonoids, while the high expression of flavonoids 3′-hydroxylase (F3′H) and flavonols synthase (FLS) made flavanols the main component. Transcription factors such as the MYB-bHLH-WDR (MBW) complex and Selenium-binding protein (SBP) directly regulated the structural genes of flavonoid metabolism, while C2H2-type zinc finger (C2H2), Zinc-finger transcription factor (GATA), Dehydration-responsive element binding (DREB), Global Transcription factor Group E protein (GTE), Trihelix DNA-binding factors (Trihelix) and Phytochrome-interacting factor (PIF) indirectly promoted the synthesis of flavonoids through hormones such as brassinoidsteroids (BRs) and abscisic acid (ABA). These results provided valuable resources for the application of related genes in genetics and breeding.

Exploring Nanotechnological Detection Background of Heat Shock Proteins (HSPs) in Coral Acropora muricata.

Coral reefs are cornerstone of global marine ecosystems, providing shelter for over one third of marine organisms. Currently, along with global warming and increased human activities, large-scale decline of coral reefs has become a severe ecosystem problem, and now quantitative detection of heat shock protein (HSP) gene by nanotechnology has become a research hotspot in this field. However, Acropora muricata is one of the most important dominant reef-building corals in Indo- Pacific region, encounter an urgent obstacle on the HSP detection research by nanoscience and nanotechnology for lack of sequence background. Here, we combined PacBio single molecular real-time (SMRT) and HiSeq X Ten sequencing technologies to perform full-length transcriptome sequencing of heat shock proteins in Acropora muricata, a reef-building coral dominant in many Indo-Pacific reefs, to annotate them. Thirteen functional heat shock proteins (HSPs) were identified using phylogenetic analysis, classified into three subgroups as HSP60, HSP70 and HSP90. HSPs are widely distributed in all animal phyla, having evolved from the last prokaryotic common ancestor. Additionally, phylogenetic and tertiary nanostructure analyses suggested that HSP70 is the most diverse HSP in A. muricata, with extensive sequence and structure differences indicating adaptations to warming water and suggesting its utility in studies of El Niño and other warming events. A greater understanding of the HSP gene family is likely to also be of value in studies of coral nanotechnological detection that can be used to protect reef ecosystems.

Nanopore-based full-length transcriptome sequencing of Muscovy duck (Cairina moschata) ovary

Unlike mammals, studies on mechanisms that regulate waterfowl ovulation have been rarely reported. To advance our understanding of the ovulation differences in Muscovy duck, we utilized the Oxford Nanopore Technologies (ONT) to generate transcriptome data from 3 groups of female duck ovaries with ovulation differences (i.e., preovulation [PO], consecutive ovulation [CO], and inconsecutive ovulation [IO]). In this study, the full-length transcriptome data qualitative analysis showed that a total of 24,504 nonredundant full-length transcripts were generated, 19,060 new transcripts were discovered and 14,848 novel transcripts were successfully annotated. For the quantitative analysis, differentially expressed genes (DEGs) between the 3 groups were identified and functional properties were characterized. CTNNB1, IGF1, FOXO3, HSPA2, PTEN and SMC4 may be potential hub genes that regulate ovulation. Adhesion-related pathway, mTOR pathway, TGF-β signaling pathway and FoxO signaling pathway have been considered as important pathways that affect follicular development and ovulation. These results provide a more complete data source of full-length transcriptome for the further study of gene expression and genetics in Muscovy duck. The hub genes and potential mechanisms that affect the ovulation of Muscovy duck have been screened out to provide a scientific basis for breeding work to improve the reproduction performance of Muscovy duck.

Integration of early disease-resistance phenotyping, histological characterization, and transcriptome sequencing reveals insights into downy mildew resistance in impatiens

Downy mildew (DM), caused by obligate parasitic oomycetes, is a destructive disease for a wide range of crops worldwide. Recent outbreaks of impatiens downy mildew (IDM) in many countries have caused huge economic losses. A system to reveal plant–pathogen interactions in the early stage of infection and quickly assess resistance/susceptibility of plants to DM is desired. In this study, we established an early and rapid system to achieve these goals using impatiens as a model. Thirty-two cultivars of Impatiens walleriana and I. hawkeri were evaluated for their responses to IDM at cotyledon, first/second pair of true leaf, and mature plant stages. All I. walleriana cultivars were highly susceptible to IDM. While all I. hawkeri cultivars were resistant to IDM starting at the first true leaf stage, many (14/16) were susceptible to IDM at the cotyledon stage. Two cultivars showed resistance even at the cotyledon stage. Histological characterization showed that the resistance mechanism of the I. hawkeri cultivars resembles that in grapevine and type II resistance in sunflower. By integrating full-length transcriptome sequencing (Iso-Seq) and RNA-Seq, we constructed the first reference transcriptome for Impatiens comprised of 48,758 sequences with an N50 length of 2060 bp. Comparative transcriptome and qRT-PCR analyses revealed strong candidate genes for IDM resistance, including three resistance genes orthologous to the sunflower gene RGC203, a potential candidate associated with DM resistance. Our approach of integrating early disease-resistance phenotyping, histological characterization, and transcriptome analysis lay a solid foundation to improve DM resistance in impatiens and may provide a model for other crops.

Mechanisms by Which Fermented Soybean Meal and Soybean Meal Induced Enteritis in Marine Fish Juvenile Pearl Gentian Grouper.

Soy meals can cause intestinal inflammation and even injury in animals, especially infants and juvenile individuals. This study investigated the effects of fermented soybean meal (FSBM) on the growth and intestinal homeostasis of juvenile pearl gentian grouper and examined the mechanisms by which FSBM and soybean meal (SBM) induced enteritis in fish, using “3+2” full-length transcriptome sequencing. We randomly assigned 720 female juvenile groupers into three treatment groups: FM control group, 20% FSBM group (FSBM20), and FSBM40 group (n = 4). Three iso-nitrogenous (50% protein) and iso-lipidic (10% lipid) diets were prepared and fed to fish for 10 weeks. The water volume in each barrel was about 1 m3, using natural light and temperature. Results showed that dietary FSBM, at experimental level, significantly affected fish growth and intestinal structure negatively and significantly increased enteritis indices. The degree of intestinal injury and inflammation was determined by the enzyme activities of trypsin and lysozyme, and the contents of IgM, C3, C4, and malondialdehyde, and the expressions of pro-inflammatory genes (IL1β, IL8, IL17, and TNFα) and anti-inflammatory genes (IL4, IL10, and TGFβ1). Full-length transcriptome analysis identified 2,305 and 3,462 differentially expressed genes (DEGs) in SBM40 and FSBM40 groups, respectively. However, only 18.98% (920/5,445) of DEGs had similar expression patterns, indicating that high levels of SBM40 and FSBM40 have different metabolic strategies. KEGG enrichment analysis indicated that among the significant pathways, ~45% were related to immune diseases/systems, infectious diseases, and signal transduction in both SBM and FSBM groups. Based on PacBio SMRT sequencing, nine toll-like receptor (TLR) members, including TLR1, TLR2, TLR3, TLR5, TLR8, TLR9, TLR13, TLR21, and TLR22, were detected in intestinal tissues of pearl gentian grouper. TLR-MyD88-NF-κB signaling pathway played an important role in the development of FSBM- and SBM-induced enteritis in pearl gentian grouper; however, TLR receptors used in SBM and FSBM groups were different. TLR1, TLR8, TLR13, and TLR22 were the main receptors used in FSBM group, while TLR5, TLR8, TLR9, TLR21, and TLR22 were the main receptors used in SBM group. Present study provides valuable theoretical references for further research on soy protein-induced enteritis in fish.

Identification and expression profiling of genes involved in circadian clock regulation in red dragon fruit (Hylocereus polyrhizus) by full-length transcriptome sequencing

ABSTRACT Crassulacean acid metabolism (CAM) plants fix CO2 at night, exhibiting a reversed regulatory pattern of metabolomic pathways compared with most model plants, which have C3 and C4 pathways. In this study, we used a valuable tropic fruit, red dragon fruit (Hylocereus polyrhizus), as model plant to identify and analyze the circadian regulation genes. Due to the absence of red dragon fruit’s whole-genome dataset, we established a full-length transcriptome dataset using single-molecule real-time (SMRT) sequencing method. A 7.66-Gb dataset with 4,552,474 subreads was generated, with an average length of 1,683 bp and an N50 of 2,446 bp. Using this dataset, we identified center oscillator genes: CCA1 (CIRCADIAN CLOCK ASSOCIATED1), ELF3 (EARLY FLOWERING 3), GI (GIGANTEA), LHY (LATE ELONGATED HYPOCOTYL), LNK1 (NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1), and TOC1 (TIMING OF CAB EXPRESSION 1); a gene for the input pathway: CRY1 (CRYPTOCHROME); a gene for the output pathway: CO (CONSTANS); and genes related to the CAM pathway: MDH (MALATE DEHYDROGENASE), ME (MALIC ENZYMES), and PPDK (PYRUVATE PHOSPHATE DIKINASE). We further established the 24-h rhythmic expression pattern of these genes and classified these into three groups: HpCCA1, HpELF3, HpLHY, HpLNK1, and HpGI have expression peaks during the day; HpTOC1, HpCO, and HpCRY1 have highest expression levels at night; The genes involved in the CAM pathways, namely, HpMDH, HpME1, and HpPPDK, have double expression peaks in the day and night. Comparison of these expression patterns between red dragon fruit and model plants could provide clues in understanding the circadian clock regulation and the activity of the CAM pathways in cactus plants.

Full-Length Transcriptome Sequencing Provides Insights into Flavonoid Biosynthesis in Fritillaria hupehensis.

One of the most commonly utilized medicinal plants in China is Fritillaria hupehensis (Hsiao et K.C. Hsia). However, due to a lack of genomic resources, little is known about the biosynthesis of relevant compounds, particularly the flavonoid biosynthesis pathway. A PacBio RS II sequencing generated a total of 342,044 reads from the bulb, leaf, root, and stem, of which 316,438 were full-length (FL) non-redundant reads with an average length of 1365 bp and a N50 of 1888 bp. There were also 38,607 long non-coding RNAs and 7914 simple sequence repeats detected. To improve our understanding of processes implicated in regulating secondary metabolite biosynthesis in F. hupehensis tissues, we evaluated potential metabolic pathways. Overall, this study provides a repertoire of FL transcripts in F. hupehensis for the first time, and it will be a valuable resource for marker-assisted breeding and research into bioactive compounds for medicinal and pharmacological applications.

Full length sequencing reveals novel transcripts of detoxification genes along with related alternative splicing events and lncRNAs in Phyllotreta striolata.

The striped flea beetle, Phyllotreta striolata (Fabricius), damages crops in the Brassicaceae. The genetic data for this pest are insufficient to reveal its insecticide resistance mechanisms or to develop molecular markers for resistance monitoring. We used PacBio Iso-Seq technology to sequence the full-length transcriptome of P. striolata. After isoform sequence clustering and removal of redundant transcripts, a total of 41,293 transcripts were obtained, and 35,640 of these were annotated in the database of gene products. Structure analysis uncovered 4,307 alternative splicing events, and 3,836 sequences were recognized as lncRNAs. Transcripts with the complete coding region of important detoxification enzymes were further classified. There were 57 transcripts of P450s distributed in CYP2, CYP3, CYP4, and Mito CYP clades, 29 transcripts of ESTs from 4 functional groups, 17 transcripts of GSTs classified into 5 families, 51 transcripts of ABCs distributed in 6 families, and 19 transcripts of UGTs. Twenty-five lncRNAs were predicted to be regulators of these detoxification genes. Full-length transcriptome sequencing is an efficient method for molecular study of P. striolata and it is also useful for gene function analysis.

Molecular Basis of the Distinct Metabolic Features in Shoot Tips and Roots of Tea Plants (Camellia sinensis): Characterization of MYB Regulator for Root Theanine Synthesis.

The physiological and metabolic differences between shoot tips and roots of tea plants are significant, and understanding them is required for improvement of tea quality and plant growth. A high-quality full-length transcriptome sequencing on tea plant roots and shoot tips by PacBio SMRT technology was done to gain a further understanding. Approximately 160699 and 166120 full-length transcripts were recovered in roots and shoots, respectively, including 31232 and 41068 novel isoforms and 16960 and 26029 alternative splicing (AS) isoforms. These supported 21699 full-length reads and 31232 and 41068 novel transcripts from root and shoot, respectively, including 1679 long noncoding RNAs (lncRNAs) and 2772 fusion transcripts, which significantly upgrade the Camellia sinensis genome annotation. The respective 6475 and 6981 transcripts in roots and shoots differ in 3'-untranslated regions. Meanwhile, extensive analyses of novel transcripts, ASs, and lncRNAs also revealed a large number of ASs and lincRNAs closely related to the regulation of characteristic secondary metabolites including catechins, theanine, and caffeine. Finally, a root-specific CsMYB6 was characterized to regulate theanine biosynthesis by genetic and molecular analyses. CsMYB6 directly bound to and activate the promoter of theanine synthetase gene (CsTSI). The study lays a foundation for the further investigation of metabolic genomics and regulation in tea plants.