Unigene Sequences Research Articles

Transcriptome sequencing and screening of genes related to the MADS-box gene family in Clematis courtoisii.

The MADS-box gene family controls plant flowering and floral organ development; therefore, it is particularly important in ornamental plants. To investigate the genes associated with the MADS-box family in Clematis courtoisii, we performed full-length transcriptome sequencing on C. courtoisii using the PacBio Sequel third-generation sequencing platform, as no reference genome data was available. A total of 12.38 Gb of data, containing 9,476,585 subreads and 50,439 Unigenes were obtained. According to functional annotation, a total of 37,923 Unigenes (75.18% of the total) were assigned with functional annotations, and 50 Unigenes were identified as MADS-box related genes. Subsequently, we employed hmmerscan to perform protein sequence similarity search for the translated Unigene sequences and successfully identified 19 Unigenes associated with the MADS-box gene family, including MIKC*(1) and MIKCC (18) genes. Furthermore, within the MIKCC group, six subclasses can be further distinguished.

Transcriptome analysis of genes involved in flower and leaf color of Oncidium by RNA-seq

Oncidium, an important tropical orchid, has high ornamental value due to its specific color and occupies a significant market position for the worldwide flower. Transcriptome analysis of flower and leaf color formation provides new sources for producing novel Oncidium hybridum cultivars. We sequenced 12 samples of flowers (yellow and white) and leaves (striped and regular) of O. hybridum and assembled 381,136 and 453,566 unigene sequences from RNA-seq data, respectively. Among unigenes, 662 and 1,324 differentially expressed genes were identified in flower and leaf samples, respectively. Gene ontology and pathway enrichment showed that secondary metabolite biosynthetic pathways were responsible for flower and leaf color formation. It was determined that UGT75C1, E2.4.1.115, CCD7, E2.1.1.76, and CCoAOMT are involved in regulating flower color, and UGT75C1, LHCB, UGT, RP-L18Ae, and ABCB1 play crucial roles in regulating leaf color. Kyoto Encyclopedia of Genes and Genomes analysis revealed that UGT75C1 was significantly enriched in the anthocyanin biosynthetic pathway, showing effects on flower and leaf colors. This study was the first detailed analysis of the molecular mechanisms of O. hybridum flower and leaf colors, and the results advanced the understanding of the genetic basis of flower and leaf colors; they also provided additional support for improving commercial value and producing novel cultivars of O. hybridum.

Identification and functional characterization of a novel aldo-keto reductase from Aloe vera.

The present investigation profoundly asserted the catalytic potential of plant-based aldo-ketoreductase, postulating its role in polyketide biosynthesis and providing new insights for tailored biosynthesis of vital plant polyketides for therapeutics. Plants hold great potential as a future source of innovative biocatalysts, expanding the possibilities within chemical reactions and generating a variety of benefits. The aldo-keto reductase (AKR) superfamily includes a huge collection of NAD(P)H-dependent oxidoreductases that carry out a variety of redox reactions essential for biosynthesis, detoxification, and intermediary metabolism. The present study involved the isolation, cloning, and purification of a novel aldo-ketoreductase (AvAKR) from the leaves of Aloe vera (Aloe barbadensis Miller) by heterologous gene expression in Escherichia coli based on the unigene sequences of putative ketoreductase and cDNA library screening by oligonucleotide hybridization. The in-silico structural analysis, phylogenetic relationship, and molecular modeling were outranged to approach the novelty of the sequence. Additionally, agroinfiltration of the candidate gene tagged with a green fluorescent protein (GFP) was employed for transient expression in the Nicotiana benthamiana to evaluate the sub-cellular localization of the candidate gene. The AvAKR preferred cytoplasmic localization and shared similarities with the known plant AKRs, keeping the majority of the conserved active-site residues in the AKR superfamily enzymes. The enzyme facilitated the NADPH-dependent reduction of various carbonyl substrates, including benzaldehyde and sugars, proclaiming a broad spectrum range. Our study successfully isolated and characterized a novel aldo-ketoreductase (AvAKR) from Aloe vera, highlighting its versatile NADPH-dependent carbonyl reduction proficiency therewith showcasing its potential as a versatile biocatalyst in diverse redox reactions.

De novo transcriptome analysis of Dysoxylum binectariferum to unravel the biosynthesis of pharmaceutically relevant specialized metabolites.

The tropical tree, D. binectariferum, is a prominent source of chromone alkaloid rohitukine, which is used in the semi-syntheses of anticancer molecules such as flavopiridol and P-276-00. The biosynthetic pathway of rohitukine or its derivatives is currently unknown in plants. Here, we explored chromone alkaloid biosynthesis in D. binectariferum through targeted transcriptome sequencing. Illumina sequencing of leaves and roots of a year-old D. binectariferum seedling generated, 42.43 and 38.74 million paired-end short reads, respectively. Quality filtering and de novo assembly of the transcriptome generated 274,970 contigs and 126,788 unigenes with an N50 contig length of 1560 bp. The assembly generated 117,619 translated unigene protein sequences and 51,598 non-redundant sequences. Nearly 80% of these non-redundant sequences were annotated to publicly available protein and nucleotide databases, suggesting the completeness and effectiveness of the transcriptome assembly. Using the assembly, we identified a chalcone synthase (CHS) and three type III polyketide synthases (PKS-III; non-CHS type) that are likely to be involved in the biosynthesis of chromone ring/noreugenin moiety of rohitukine. We also identified key enzymes like lysine decarboxylase in the piperidine pathway that make the piperidine moiety of rohitukine. Besides these, the upstream enzymes in flavonoid biosynthesis like phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H),4-coumarate-CoA ligase (4CL), and chalcone isomerase (CHI) have also been identified. Also, terpene synthases that are likely to be involved in the biosynthesis of various terpenoid scaffolds have been identified. Together, the D. binectariferum transcriptome resource forms a basis for further exploration of biosynthetic pathways of these valuable compounds through functional validation of the candidate genes and metabolic engineering in heterologous hosts. Additionally, the transcriptome dataset generated will serve as an important resource for research on functional genomics and enzyme discovery in D. binectariferum and comparative analysis with other Meliaceae family members.

Transcriptome analysis of immune-related genes of Asian corn borer (Ostrinia furnacalis [Guenée]) after oral bacterial infection.

The Asian corn borer (Ostrinia furnacalis) is an important agricultural pest causing serious damage to economic crops, such as corn and sorghum. The gut is the first line of defense against pathogens that enter through the mouth. Staphylococcus aureus was used to infect the O. furnacalis midgut to understand the midgut immune mechanism against exogenous pathogens to provide new ideas and methods for the prevention and control of O. furnacalis. A sequencing platform was used for genome assembly and gene expression. The unigene sequences were annotated and functionally classified by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Significant differences were found in the induced expression profiles before and after infection. Some differentially expressed genes have important relations with lipid metabolism and immune mechanism, suggesting that they play an important role in the innate immune response of O. furnacalis. Furthermore, quantitative real-time polymerase chain reaction assay was used to identify the key genes involved in the signaling pathway, and the expression patterns of these key genes were confirmed. The results could help study the innate immune system of lepidopteran insects and provide theoretical support for the control of related pests and the protection of beneficial insects.

Transcriptome Profiling of Rhipicephalus annulatus Reveals Differential Gene Expression of Metabolic Detoxifying Enzymes in Response to Acaricide Treatment.

Ticks are hematophagous ectoparasites of economic consequence by virtue of being carriers of infectious diseases that affect livestock and other sectors of the agricultural industry. A widely prevalent tick species, Rhipicephalus (Boophilus) annulatus, has been recognized as a prime vector of tick-borne diseases in South Indian regions. Over time, the use of chemical acaricides for tick control has promoted the evolution of resistance to these widely used compounds through metabolic detoxification. Identifying the genes related to this detoxification is extremely important, as it could help detect valid insecticide targets and develop novel strategies for effective insect control. We performed an RNA-sequencing analysis of acaricide-treated and untreated R. (B.) annulatus and mapped the detoxification genes expressed due to acaricide exposure. Our results provided high-quality RNA-sequenced data of untreated and amitraz-treated R. (B.) annulatus, and then the data were assembled into contigs and clustered into 50,591 and 71,711 uni-gene sequences, respectively. The expression levels of the detoxification genes across different developmental stages of R. (B.) annulatu identified 16,635 transcripts as upregulated and 15,539 transcripts as downregulated. The annotations of the differentially expressed genes (DEGs) revealed the significant expression of 70 detoxification genes in response to the amitraz treatment. The qRT-PCR revealed significant differences in the gene expression levels across different life stages of R. (B.) annulatus.

Revealing the Gene Diversity and Candidate Gene Family for Adaption to Environment Depth in Leucosiid Crabs Comparing the Transcriptome Assembly

The study of gene diversity in interspecies contributes to our understanding of the environmental adaptation, evolutionary history, origin, and stability of biodiversity. Crabs are the main component of the macrobenthos community; therefore, how crabs adapt to different environments can be a guide to understand how to maintain community diversity. Previous studies on environmental adaptation have focused on differences in morphology, organ structure, and function, but there is a lack of research that explores this topic from the perspective of gene diversity in benthonic crabs. In this study, the leucosiid crab was selected for transcriptome assembly and was analyzed as this superfamily is one of the main clades in brachyuran crabs. Their transcriptome data were used to understand the gene diversity, phylogeny, and divergence time estimations. Subsequently, candidate gene families for depth adaptation were found in eight species that live in habitats with different depths. The results indicated that the number of total unigene sequences was between 65,617 (Philyra malefactrix) and 98,279 (Arcania heptacantha) in eight species. The total length of the genes was counted to be between 48,006,211 and 95,471,088 bp. The age of the superfamily Leucosioidea is over 150 Ma, dating back to at least the Jurassic geological period. The divergence of the family Leucosiidae would have occurred in the middle Cretaceous (around 100 Ma). After dividing into groups of three depth types, which carried the gene families, it was found that the three groups shared the solute carrier family, whereas bile acid secretion, organic solute transporter subunit alpha-like, and solute carrier organic anion transporter families only existed in the shallow group. This result shown that the gene function of ion concentration regulation might one of the candidate gene families related to the environmental adaptation of the leucosiid crab. Hence, these gene families will be analyzed in future studies to understand the mechanism of depth adaptation regulation in crabs.

Transcriptome analysis of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) and characterization of genes associated to immunity, growth, and reproduction

BackgroundDung beetles recycle organic matter through the decomposition of feces and support ecological balance. However, these insects are threatened by the indiscriminate use of agrochemicals and habitat destruction. Copris tripartitus Waterhouse (Coleoptera: Scarabaeidae), a dung beetle, is listed as a class-II Korean endangered species. Although the genetic diversity of C. tripartitus populations has been investigated through analysis of mitochondrial genes, genomic resources for this species remain limited. In this study, we analyzed the transcriptome of C. tripartitus to elucidate functions related to growth, immunity and reproduction for the purpose of informed conservation planning.ResultsThe transcriptome of C. tripartitus was generated using next-generation Illumina sequencing and assembled de novo using a Trinity-based platform. In total, 98.59% of the raw sequence reads were processed as clean reads. These reads were assembled into 151,177 contigs, 101,352 transcripts, and 25,106 unigenes. A total of 23,450 unigenes (93.40%) were annotated to at least one database. The largest proportion of unigenes (92.76%) were annotated to the locally curated PANM-DB. A maximum of 5,512 unigenes had homologous sequences in Tribolium castaneum. Gene Ontology (GO) analysis revealed a maximum of 5,174 unigenes in the Molecular function category. Further, in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, a total of 462 enzymes were associated with established biological pathways. Based on sequence homology to known proteins in PANM-DB, representative immunity, growth, and reproduction-related genes were screened. Potential immunity-related genes were categorized into pattern recognition receptors (PRRs), the Toll-like receptor signaling pathway, the MyD88- dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related transcripts. Among PRRs, we conducted detailed in silico characterization of TLR-2, CTL, and PGRP_SC2-like. Repetitive elements such as long terminal repeats, short interspersed nuclear elements, long interspersed nuclear elements and DNA elements were enriched in the unigene sequences. A total of 1,493 SSRs were identified among all unigenes of C. tripartitus.ConclusionsThis study provides a comprehensive resource for analysis of the genomic topography of the beetle C. tripartitus. The data presented here clarify the fitness phenotypes of this species in the wild and provide insight to support informed conservation planning.

Development of SSR markers on the basis of the Panax notoginseng transcriptome for agronomic and biochemical trait association analyses

Panax notoginseng (Burk.) F.H. Chen (Araliaceae) is a precious medicinal plant, but little is known about the mechanism underlying the inheritance of its important agronomic and biochemical traits. The development of effective molecular markers and the characterization of genes associated with agronomic and biochemical traits are critical for P. notoginseng genetic analyses and breeding. In this study, 41 pairs of primers for polymorphic simple sequence repeats (SSRs) were developed according to the P. notoginseng transcriptome. Among them, 18 SSR markers can be used to reveal the rich genetic diversity among four wild Panax stipuleanatus populations. In addition, 20 significant correlations between 15 SSR locus and 14 agronomic and biochemical traits were identified using mixed linear models. It is worth noting that P19-FAM loci is significantly correlated with the content of P. notoginseng saponin R1 in rhizomes and tuberous roots, and the corresponding unigene sequence of P19-FAM loci is a dof zinc finger protein-encoding gene (PnDof1). Moreover, the PnDof1 protein was localized in the plant cell nucleus, and transgenic PnDof1-overexpressing P. notoginseng cells had higher notoginsenoside R1 and ginsenoside (Rg1, Re, Rb1, and Rd) contents than non-transgenic cells. Some P. notoginseng saponin (PNS) biosynthesis-related genes were significantly up-regulated in the PnDof1-overexpressing transgenic cells. The transcriptome SSR markers developed in this study are useful for the genetic analysis of Panax germplasm resources as well as for molecular breeding. The PnDof1 gene may encode a transcription factor that regulates PNS biosynthesis in P. notoginseng.

Development and Application of EST-SSR Markers Related to Lead Stress Responses in Kenaf Based on Transcriptome Sequencing Data

Kenaf is an important bast fiber crop. In order to diversify the available kenaf simple sequence repeat (SSR) molecular markers and generate markers potentially useful for kenaf breeding, we developed expression sequence tag simple sequence repeat (EST-SSR) molecular markers based on lead-stressed kenaf transcriptome sequencing data and spliced unigene sequences. Additionally, the distribution of the SSRs in the transcriptome and the potential functions of the SSR-containing genes were determined. Moreover, SSR markers in the differentially expressed genes (DEGs) of a protein–protein interaction (PPI) network were analyzed to screen for polymorphic markers, which were used to examine the genetic diversity and population structure of kenaf germplasm resources. The genetic diversity and population structure of 138 kenaf germplasm materials revealed that 22 EST-SSR markers could be used to distinguish the kenaf germplasms. The 22 EST-SSR markers enrich the kenaf molecular markers database and provide an important tool for future genetic improvement of kenaf resistance to lead stress.

RNA-Seq and genetic diversity analysis of faba bean (Vicia faba L.) varieties in China.

Faba bean (Vicia faba L) is one of the most important legumes in the world. However, there is relatively little genomic information available for this species owing to its large genome. The lack of data impedes the discovery of molecular markers and subsequent genetic research in faba bean. The objective of this study was to analyze the faba bean transcriptome, and to develop simple sequence repeat (SSR) markers to determine the genetic diversity of 226 faba bean varieties derived from different regions in China. Faba bean varieties with different phenotype were used in transcriptome analysis. The functions of the unigenes were analyzed using various database. SSR markers were developed and the polymorphic markers were selected to conduct genetic diversity analysis. A total of 92.43 Gb of sequencing data was obtained in this study, and 133,487 unigene sequences with a total length of 178,152,541 bp were assembled. A total of 5,200 SSR markers were developed on the basis of RNA-Seq analysis. Then, 200 SSR markers were used to evaluate polymorphisms. In total, 103 (51.5%) SSR markers showed significant and repeatable bands between different faba bean varieties. Clustering analysis revealed that 226 faba bean materials were divided into five groups. Genetic diversity analysis revealed that the relationship between different faba beans in China was related, especially in the same region. These results provided a valuable data resource for annotating genes to different categories and developing SSR markers.

Microsatellite analysis and polymorphic marker development based on the full-length transcriptome of Camellia chekiangoleosa

Camellia chekiangoleosa is a popular variety of Oil-camellia that has high oil production and ornamental value. Microsatellite (SSR) markers are the preferred tool for the molecular marker-assisted breeding of C. chekiangoleosa. By focusing on the problems of the low development efficiency of polymorphic SSR markers and the lack of available functional markers in Oil-camellia, we identified 97,510 SSR loci based on the full-length transcriptome sequence of C. chekiangoleosa. An analysis of SSR characteristics showed that mononucleotide (51.29%) and dinucleotide (34.36%) SSRs were the main repeat types. The main SSR distribution areas based on proportion covered were ordered as follows: 5'UTR > 3'UTR > CDS. By comparing our data with those in databases such as GO and KEGG, we obtained functional annotations of unigene sequences containing SSR sites. The data showed that the amplification efficiency of the SSR primers was 51.72%, and the development efficiency of polymorphic SSR primers was 26.72%. Experiments verified that dinucleotide and pentanucleotide SSRs located in UTR regions could produce more polymorphic markers. An investigation into the genetic diversity of several C. chekiangoleosa populations also suggested that the developed SSR markers had higher levels of polymorphism. This study will provide a reference and high-quality markers for the large-scale development of functional SSR markers and genetic research in Oil-camellia.

Development and Characterization of Expressed Sequence-tagged Simple Sequence Repeat Markers for Denphal-type Dendrobium Cultivars and Transferability to Dendrobium Species

Denphal-type Dendrobium is the most popular orchid for cut flower and potted plant trade. To improve commercial traits, many novel cultivars have been produced through hybridization by commercial breeders. However, the genetic relationship of most cultivars is unclear, thus hindering the progress of Denphal-type Dendrobium breeding programs. Therefore, the development of molecular markers is encouraged to identify different cultivars. In this study, based on the transcriptome database of the Denphal-type Dendrobium ‘Red Bull’, the polymorphisms expressed sequence tag-derived simple sequence repeat (EST-SSR) were developed from 100 pairs EST-SSRs that were randomly selected from the EST-SSR database. The genetic relationship of 42 Denphal-type Dendrobium cultivars was analyzed according to the developed EST-SSRs. Then, the transferability of EST-SSRs was analyzed by performing a relationship analysis of 40 Dendrobium species. The results showed that a total of 5174 potential EST-SSR markers were identified with 4486 unigene sequences, and 5289 primer pairs were successfully designed. Of the selected 100 pairs of EST-SSRs, a total of 86 pairs produced the expected polymerase chain reaction products of the primary screening, 58 pairs produced the expected fragment size, and 20 pairs showed polymorphisms. Furthermore, the dendrogram of 42 cultivars showed that at a genetic distance of 0.15, the cultivars collected were grouped into five clusters of three major clusters and two minor clusters; all these clusters had the same characters of each cluster. The transferability analysis showed that 18 of the 20 EST-SSR markers among the 40 Dendrobium species were polymorphic. Overall, this study developed EST-SSR markers and will be valuable to facilitating genetic diversity in Denphal-type Dendrobium cultivars and Dendrobium species.

Development of a genome-wide marker design workflow for onions and its application in target amplicon sequencing-based genotyping.

Onions are one of the most widely cultivated vegetables worldwide; however, the development and utilization of molecular markers have been limited because of the large genome of this plant. We present a genome-wide marker design workflow for onions and its application in a high-throughput genotyping method based on target amplicon sequencing. The efficiency of the method was evaluated by genotyping of F2 populations. In the marker design workflow, unigene and genomic sequence data sets were constructed, and polymorphisms between parental lines were detected through transcriptome sequence analysis. The positions of polymorphisms detected in the unigenes were mapped onto the genome sequence, and primer sets were designed. In total, 480 markers covering the whole genome were selected. By genotyping an F2 population, 329 polymorphic sites were obtained from the estimated positions or the flanking sequences. However, missing or sparse marker regions were observed in the resulting genetic linkage map. We modified the markers to cover these regions by genotyping the other F2 populations. The grouping and order of markers on the linkages were similar across the genetic maps. Our marker design workflow and target amplicon sequencing are useful for genome-wide genotyping of onions owing to their reliability, cost effectiveness, and flexibility.

Expression Profiles of Microsatellites in Fruit Tissues of Akebia trifoliata and Development of Efficient EST-SSR Markers.

Akebia trifoliata, a member of the family Lardizabalaceae, has high exploitation potential for multiple economic purposes, so genetic improvements to meet requirements for commercial demand are needed. However, this progress is largely impeded by a lack of effective selection markers. In this study, we obtained 271.49 Gb of clean transcriptomic data from 12 samples (three tissues at four developmental stages) of A. trifoliata fruit. We identified 175,604, 194,370, and 207,906 SSRs from the de novo assembled 416,363, 463,756, and 491,680 unigene sequences obtained from the flesh, seed, and rind tissues, respectively. The profile and proportion of SSR motifs expressed in each fruit tissue and developmental stage were remarkably similar, but many trinucleotide repeats had differential expression levels among different tissues or at different developmental stages. In addition, we successfully designed 16,869 functional EST-SSR primers according to the annotated unigenes. Finally, 94 and 72 primer pairs out of 100 randomly selected primer pairs produced clear bands and polymorphic bands, respectively. These results were also used to elucidate the expression profiles of different tissues at various stages. Additionally, we provided a set of effective, polymorphic, and reliable EST-SSR markers sufficient for accelerating the discovery of metabolic and pathway-specific functional genes for genetic improvement and increased commercial productivity.

Transcriptome analysis of the bivalve Placuna placenta mantle reveals potential biomineralization-related genes

The shells of window pane oyster Placuna placenta are very thin and exhibit excellent optical transparency and mechanical robustness. However, little is known about the biomineralization-related proteins of the shells of P. placenta. In this work, we report the comprehensive transcriptome of the mantle tissue of P. placenta for the first time. The unigenes of the mantle tissue of P. placenta were annotated by using the public databases such as nr, GO, KOG, KEGG, and Pfam. 24,343 unigenes were annotated according to Pfam database, accounting for 21.48% of the total unigenes. We find that half of the annotated unigenes of the mantle tissue of P. placenta are consistent to the annotated unigenes from pacific oyster Crassostrea gigas according to nr database. The unigene sequence analysis from the mantle tissue of P. placenta indicates that 465,392 potential single nucleotide polymorphisms (SNPs) and 62,103 potential indel markers were identified from 60,371 unigenes. 178 unigenes of the mantle tissue of P. placenta are found to be homologous to those reported proteins related to the biomineralization process of molluscan shells, while 18 of them are highly expressed unigenes in the mantle tissue. It is proposed that four unigenes with the highest expression levels in the mantle tissue are very often related to the biomineralization process, while another three unigenes are potentially related to the biomineralization process according to the Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) analysis. In summary, the transcriptome analysis of the mantle tissue of P. Placenta shows the potential biomineralization-related proteins and this work may shed light for the shell formation mechanism of bivalves.

Comparative Transcriptomic and Metabolic Analyses Reveal the Molecular Mechanism of Ovule Development in the Orchid, Cymbidium sinense.

Ovule development is pivotal to plant reproduction and seed development. Cymbidium sinense (Orchidaceae) has high ornamental value due to its pleasant aroma and elegant floral morphology. The regulatory mechanism underlying ovule development in orchids, especially C. sinense, is largely unknown and information on the C. sinense genome is very scarce. In this study, a combined analysis was performed on the transcriptome and non-targeted metabolomes of 18 C. sinense ‘Qi Jian Hei Mo’ ovule samples. Transcriptome analysis assembled gene-related information related to six growth stages of C. sinense ovules (S1-S6, equivalent to 30, 35, 42, 46, 53, and 60 days after pollination). Illumina sequencing technology was used to obtain the complete set of transcriptome sequences of the 18 samples. A total of 81,585 unigene sequences were obtained after assembly, 24,860 (30.47%) of which were functionally annotated. Using transcriptome sequencing technology, a total of 9845 differentially expressed unigenes (DEUs) were identified in C. sinense ovules that were assigned to specific metabolic pathways according to the Kyoto Encyclopedia of Genes and Genomes (KEGG). DEUs associated with transcription factors (TFs) and phytohormones were identified and analyzed. The TFs homeobox and MADS-box were associated with C. sinense ovule development. In particular, the phytohormones associated with DEUs such as indole-3-acetic acid (IAA), cytokinin (CK), gibberellin (GA), abscisic acid (ABA), brassinosteroid (BR), and jasmonate (JA), may have important regulatory effects on C. sinense ovule development. Metabolomic analysis showed an inconsistent number of KEGG annotations of differential metabolites across comparisons (S2_vs_S4, S2_vs_S5, and S4_vs_S5 contained 23, 26, and 3 annotations, respectively) in C. sinense ovules. This study provides a valuable foundation for further understanding the regulation of orchid ovule development and formation, and establishes a theoretical background for future practical applications during orchid cultivation.

Transcriptome and metabolome analyses of the immune response to light stress in the hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀)

Light intensity is an important environmental factor that affects fish growth and health through multiple physiological activities and metabolism and eventually impacts aquaculture harvest. There is a need to evaluate the fish stress response to light intensities, which will benefit aquaculture. Here, hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀) was treated with three light intensities for evaluation of the light stress response, including high light intensity (1 250 lx), low light intensity (10 lx) and moderate light intensity (250 lx). Transcriptome analysis showed that a total of 71 318 unigene sequences were obtained with an N50 of 2 589 bp. Compared to the control group (250 lx), 1 697 differentially expressed genes (DEGs), a considerable quantity, were detected in the 1 250 lx group. Among those genes, 548 were upregulated, and the remaining 149 genes showed decreased expression. Comparatively small numbers of DEGs were detected in the 10 lx group; 54 out of 103 genes exhibited upregulated expression, and 49 genes showed downregulation. For further KEGG analysis, 82 DEGs were enriched in nine common signalling pathways in immunity, of which 73 DEGs were significantly inhibited in the 1 250 lx group. In contrast, only 11 DEGs were enriched in three immunity pathways, with nine DEGs showing a significant increase in the 10 lx group. The metabolome analysis revealed 59 and 44 differential metabolites (DMs) from the 1 250 lx and 10 lx groups, respectively. Of note, those DMs from the 1 250 lx-treated group were tendentiously involved in amino acid metabolism and lipid metabolism pathways, while the purine metabolism, amino acid metabolism and lipid metabolism pathways were mostly found in the 10 lx treatment group. In summary, our data indicated that high light intensity significantly inhibited the immune response in hybrid grouper, while low light intensity presented low stimulation of immune activity. In addition, both high and low light intensity could inhibit protein synthesis and amino acid metabolism. Taken together, hybrid grouper exhibited a much milder stress response to low light intensity than to high light intensity.

De novo Transcriptome Assembly of Senna occidentalis Sheds Light on the Anthraquinone Biosynthesis Pathway.

Senna occidentalis is an annual leguminous herb that is rich in anthraquinones, which have various pharmacological activities. However, little is known about the genetics of S. occidentalis, particularly its anthraquinone biosynthesis pathway. To broaden our understanding of the key genes and regulatory mechanisms involved in the anthraquinone biosynthesis pathway, we used short RNA sequencing (RNA-Seq) and long-read isoform sequencing (Iso-Seq) to perform a spatial and temporal transcriptomic analysis of S. occidentalis. This generated 121,592 RNA-Seq unigenes and 38,440 Iso-Seq unigenes. Comprehensive functional annotation and classification of these datasets using public databases identified unigene sequences related to major secondary metabolite biosynthesis pathways and critical transcription factor families (bHLH, WRKY, MYB, and bZIP). A tissue-specific differential expression analysis of S. occidentalis and measurement of the amount of anthraquinones revealed that anthraquinone accumulation was related to the gene expression levels in the different tissues. In addition, the amounts and types of anthraquinones produced differ between S. occidentalis and S. tora. In conclusion, these results provide a broader understanding of the anthraquinone metabolic pathway in S. occidentalis.

Use of High Resolution Spatiotemporal Gene Expression Data to Uncover Novel Tissue-Specific Promoters in Tomato

Genetic modification can be an effective strategy for improving the agronomic traits of tomato (Solanum lycopersicum) to meet demands for yield, quality, functional components, and stress tolerance. However, limited numbers of available tissue-specific promoters represent a bottleneck for the design and production of transgenic plants. In the current study, a total of 25 unigenes were collected from an RNA-sequence dataset based on their annotation as being exclusively expressed in five type of tissues of tomato pericarp (outer and inner epidermis, collenchyma, parenchyma, and vascular tissues), and every five unigenes, was respectively selected from each tissue based on transcription expression. The 3-kb 5′ upstream region of each unigene was identified from the tomato genome sequence (SL2.50) using annotated unigene sequences, and the promoter sequences were further analyzed. The results showed an enrichment in T/A (T/A > 70%) in the promoter regions. A total of 15 putative tissue-/organ-specific promoters were identified and analyzed by real-time (RT) quantitative (q) PCR analysis, of which six demonstrated stronger activity than widely used tissue-specific tomato promoters. These results demonstrate how high spatiotemporal and high throughput gene expression data can provide a powerful means of identifying spatially targeted promoters in plants.