Trichome hairs affect diverse agronomic characters such as seed weight and yield, prevent insect damage and reduce loss of water but their molecular control has not been extensively studied in soybean. Several detailed models for trichome development have been proposed for Arabidopsis thaliana, but their applicability to important crops such as cotton and soybean is not fully known.
Two high throughput transcript sequencing methods, Digital Gene Expression (DGE) Tag Profiling and RNA-Seq, were used to compare the transcriptional profiles in wild-type (cv. Clark standard, CS) and a mutant (cv. Clark glabrous, i.e., trichomeless or hairless, CG) soybean isoline that carries the dominant P1 allele. DGE data and RNA-Seq data were mapped to the cDNAs (Glyma models) predicted from the reference soybean genome, Williams 82. Extending the model length by 250 bp at both ends resulted in significantly more matches of authentic DGE tags indicating that many of the predicted gene models are prematurely truncated at the 5' and 3' UTRs. The genome-wide comparative study of the transcript profiles of the wild-type versus mutant line revealed a number of differentially expressed genes. One highly-expressed gene, Glyma04g35130, in wild-type soybean was of interest as it has high homology to the cotton gene GhRDL1 gene that has been identified as being involved in cotton fiber initiation and is a member of the BURP protein family. Sequence comparison of Glyma04g35130 among Williams 82 with our sequences derived from CS and CG isolines revealed various SNPs and indels including addition of one nucleotide C in the CG and insertion of ~60 bp in the third exon of CS that causes a frameshift mutation and premature truncation of peptides in both lines as compared to Williams 82.
Although not a candidate for the P1 locus, a BURP family member (Glyma04g35130) from soybean has been shown to be abundantly expressed in the CS line and very weakly expressed in the glabrous CG line. RNA-Seq and DGE data are compared and provide experimental data on the expression of predicted soybean gene models as well as an overview of the genes expressed in young shoot tips of two closely related isolines.
Systematic research on fish immunogenetics is indispensable in understanding the origin and evolution of immune systems. This has long been a challenging task because of the limited number of deep sequencing technologies and genome backgrounds of non-model fish available. The newly developed Solexa/Illumina RNA-seq and Digital gene expression (DGE) are high-throughput sequencing approaches and are powerful tools for genomic studies at the transcriptome level. This study reports the transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus using RNA-seq and DGE in an attempt to gain insights into the immunogenetics of marine fish.
RNA-seq analysis generated 169,950 non-redundant consensus sequences, among which 48,987 functional transcripts with complete or various length encoding regions were identified. More than 52% of these transcripts are possibly involved in approximately 219 known metabolic or signalling pathways, while 2,673 transcripts were associated with immune-relevant genes. In addition, approximately 8% of the transcripts appeared to be fish-specific genes that have never been described before. DGE analysis revealed that the host transcriptome profile of Vibrio harveyi-challenged L. japonicus is considerably altered, as indicated by the significant up- or down-regulation of 1,224 strong infection-responsive transcripts. Results indicated an overall conservation of the components and transcriptome alterations underlying innate and adaptive immunity in fish and other vertebrate models. Analysis suggested the acquisition of numerous fish-specific immune system components during early vertebrate evolution.
This study provided a global survey of host defence gene activities against bacterial challenge in a non-model marine fish. Results can contribute to the in-depth study of candidate genes in marine fish immunity, and help improve current understanding of host-pathogen interactions and evolutionary history of immunogenetics from fish to mammals.
We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing.
The key of RNAi approach success for potential insect pest control is mainly dependent on careful target selection and a convenient delivery system. We adopted second-generation sequencing technology to screen RNAi targets. Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) technologies were used to screen optimal RNAi targets from Ostrinia furnalalis. Total 14690 stage specific genes were obtained which can be considered as potential targets, and 47 were confirmed by qRT-PCR. Ten larval stage specific expression genes were selected for RNAi test. When 50 ng/µl dsRNAs of the genes DS10 and DS28 were directly sprayed on the newly hatched larvae which placed on the filter paper, the larval mortalities were around 40∼50%, while the dsRNAs of ten genes were sprayed on the larvae along with artificial diet, the mortalities reached 73% to 100% at 5 d after treatment. The qRT-PCR analysis verified the correlation between larval mortality and the down-regulation of the target gene expression. Topically applied fluorescent dsRNA confirmed that dsRNA did penetrate the body wall and circulate in the body cavity. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost less and an easy way to select the candidate target genes for RNAi. More importantly, it demonstrated that dsRNAs are able to penetrate the integument and cause larval developmental stunt and/or death in a lepidopteron insect. This finding largely broadens the target selection for RNAi from just gut-specific genes to the targets in whole insects and may lead to new strategies for designing RNAi-based technology against insect damage.
Tea is the most popular non-alcoholic health beverage in the world. The tea plant (Camellia sinensis (L.) O. Kuntze) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in tea plants. To elucidate the molecular mechanisms of cold acclimation, we employed RNA-Seq and digital gene expression (DGE) technologies to the study of genome-wide expression profiles during cold acclimation in tea plants.
Using the Illumina sequencing platform, we obtained approximately 57.35 million RNA-Seq reads. These reads were assembled into 216,831 transcripts, with an average length of 356 bp and an N50 of 529 bp. In total, 1,770 differentially expressed transcripts were identified, of which 1,168 were up-regulated and 602 down-regulated. These include a group of cold sensor or signal transduction genes, cold-responsive transcription factor genes, plasma membrane stabilization related genes, osmosensing-responsive genes, and detoxification enzyme genes. DGE and quantitative RT-PCR analysis further confirmed the results from RNA-Seq analysis. Pathway analysis indicated that the “carbohydrate metabolism pathway” and the “calcium signaling pathway” might play a vital role in tea plants’ responses to cold stress.
Our study presents a global survey of transcriptome profiles of tea plants in response to low, non-freezing temperatures and yields insights into the molecular mechanisms of tea plants during the cold acclimation process. It could also serve as a valuable resource for relevant research on cold-tolerance and help to explore the cold-related genes in improving the understanding of low-temperature tolerance and plant-environment interactions.
Camellia Sinensis; Cold Acclimation; RNA-Seq; DGE; Genome-wide Expression Profiles; Tea Plants
Detailed characterization of the gene expression patterns in spermatogonia and primary spermatocytes is critical to understand the processes which occur prior to meiosis during normal spermatogenesis. The genome-wide expression profiles of mouse type B spermatogonia and primary spermatocytes were investigated using the Solexa/Illumina digital gene expression (DGE) system, a tag based high-throughput transcriptome sequencing method, and the developmental processes which occur during early spermatogenesis were systematically analyzed. Gene expression patterns vary significantly between mouse type B spermatogonia and primary spermatocytes. The functional analysis revealed that genes related to junction assembly, regulation of the actin cytoskeleton and pluripotency were most significantly differently expressed. Pathway analysis indicated that the Wnt non-canonical signaling pathway played a central role and interacted with the actin filament organization pathway during the development of spermatogonia. This study provides a foundation for further analysis of the gene expression patterns and signaling pathways which regulate the molecular mechanisms of early spermatogenesis.
Japanese scallop has been cultured on a large-scale in China for many years. However, serious marine pollution in recent years has resulted in considerable loss to this industry. Moreover, due to the lack of genomic resources, limited research has been carried out on this species. To facilitate the understanding at molecular level immune and stress response mechanism, an extensive transcriptomic profiling and digital gene expression (DGE) database of Japanese scallop upon cadmium exposure was carried out using the Illumina sequencing platform.
RNA-seq produced about 112 million sequencing reads from the tissues of adult Japanese scallops. These reads were assembled into 194,839 non-redundant sequences with open reading frame (ORF), of which 14,240 putative amino acid sequences were assigned biological function annotation and were annotated with gene ontology and eukaryotic orthologous group terms. In addition, we identified 720 genes involved in response to stimulus and 302 genes involved in immune-response pathways. Furthermore, we investigated the transcriptomic changes in the gill and digestive gland of Japanese scallops following cadmium exposure using a tag-based DGE system. A total of 7,556 and 3,002 differentially expressed genes were detected, respectively, and functionally annotated with KEGG pathway annotations.
This study provides a comprehensive transcripts sequence resource for the Japanese scallop and presents a survey of gene expression in response to heavy metal exposure in a non-model marine invertebrate via the Illumina sequencing platform. These results may contribute to the in-depth elucidation of the molecular mechanisms involved in bivalve responses to marine pollutants.
Epidermal Growth Factor (EGF) is a key regulatory growth factor activating many processes relevant to normal development and disease, affecting cell proliferation and survival. Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.
By applying a procedure for cross-platform data meta-analysis based on RankProd and GlobalAncova tests, we establish a well validated gene set with transcript levels altered after EGF treatment. We use this robust gene list to build higher order networks of gene interaction by interconnecting associated networks, supporting and extending the important role of the EGF signaling pathway in cancer. In addition, we find an entirely new set of genes previously unrelated to the currently accepted EGF associated cellular functions.
We propose that the use of global genomic cross-validation derived from high content technologies (microarrays or deep sequencing) can be used to generate more reliable datasets. This approach should help to improve the confidence of downstream in silico functional inference analyses based on high content data.
Despite striking differences in cognition and behavior between humans and our closest primate relatives, several studies have found little evidence for adaptive change in protein-coding regions of genes expressed primarily in the brain. Instead, changes in gene expression may underlie many cognitive and behavioral differences. Here, we used digital gene expression: tag profiling (here called Tag-Seq, also called DGE:tag profiling) to assess changes in global transcript abundance in the frontal cortex of the brains of 3 humans, 3 chimpanzees, and 3 rhesus macaques. A substantial fraction of transcripts we identified as differentially transcribed among species were not assayed in previous studies based on microarrays. Differentially expressed tags within coding regions are enriched for gene functions involved in synaptic transmission, transport, oxidative phosphorylation, and lipid metabolism. Importantly, because Tag-Seq technology provides strand-specific information about all polyadenlyated transcripts, we were able to assay expression in noncoding intragenic regions, including both sense and antisense noncoding transcripts (relative to nearby genes). We find that many noncoding transcripts are conserved in both location and expression level between species, suggesting a possible functional role. Lastly, we examined the overlap between differential gene expression and signatures of positive selection within putative promoter regions, a sign that these differences represent adaptations during human evolution. Comparative approaches may provide important insights into genes responsible for differences in cognitive functions between humans and nonhuman primates, as well as highlighting new candidate genes for studies investigating neurological disorders.
gene expression; transcriptional evolution; Tag-Seq; noncoding RNA
Digital gene expression (DGE) profiling has become an established tool to study RNA expression. Here, we provide an in-depth analysis of small RNA DGE profiles from two different rat strains (BN-Lx and SHR) from six different rat tissues (spleen, liver, brain, testis, heart, kidney). We describe the expression patterns of known and novel micro (mi)RNAs and piwi-interacting (pi)RNAs.
We confirmed the expression of 588 known miRNAs (54 in antisense orientation) and identified 56 miRNAs homologous to known human or mouse miRNAs, as well as 45 new rat miRNAs. Furthermore, we confirmed specific A to I editing in brain for mir-376a/b/c and identified mir-377 as a novel editing target. In accordance with earlier findings, we observed a highly tissue-specific expression pattern for all tissues analyzed. The brain was found to express the highest number of tissue-specific miRNAs, followed by testis. Notably, our experiments also revealed robust strain-specific differential miRNA expression in the liver that is caused by genetic variation between the strains. Finally, we identified two types of germline-specific piRNAs in testis, mapping either to transposons or in strand-specific clusters.
Taken together, the small RNA compendium described here advances the annotation of small RNAs in the rat genome. Strain and tissue-specific expression patterns furthermore provide a strong basis for studying the role of small RNAs in regulatory networks as well as biological process like physiology and neurobiology that are extensively studied in this model system.
Phytoplasmas are plant pathogenic bacteria that have no cell wall and are responsible for major crop losses throughout the world. Phytoplasma-infected plants show a variety of symptoms and the mechanisms they use to physiologically alter the host plants are of considerable interest, but poorly understood. In this study we undertook a detailed analysis of Paulownia infected by Paulownia witches’-broom (PaWB) Phytoplasma using high-throughput mRNA sequencing (RNA-Seq) and digital gene expression (DGE). RNA-Seq analysis identified 74,831 unigenes, which were subsequently used as reference sequences for DGE analysis of diseased and healthy Paulownia in field grown and tissue cultured plants. Our study revealed that dramatic changes occurred in the gene expression profile of Paulownia after PaWB Phytoplasma infection. Genes encoding key enzymes in cytokinin biosynthesis, such as isopentenyl diphosphate isomerase and isopentenyltransferase, were significantly induced in the infected Paulownia. Genes involved in cell wall biosynthesis and degradation were largely up-regulated and genes related to photosynthesis were down-regulated after PaWB Phytoplasma infection. Our systematic analysis provides comprehensive transcriptomic data about plants infected by Phytoplasma. This information will help further our understanding of the detailed interaction mechanisms between plants and Phytoplasma.
The brown planthopper (BPH) Nilaparvata lugens (Stål) is one of the most serious insect pests of rice in Asia. However, little is known about the mechanisms responsible for the development, wing dimorphism and sex difference in this species. Genomic information for BPH is currently unavailable, and, therefore, transcriptome and expression profiling data for this species are needed as an important resource to better understand the biological mechanisms of BPH.
In this study, we performed de novo transcriptome assembly and gene expression analysis using short-read sequencing technology (Illumina) combined with a tag-based digital gene expression (DGE) system. The transcriptome analysis assembles the gene information for different developmental stages, sexes and wing forms of BPH. In addition, we constructed six DGE libraries: eggs, second instar nymphs, fifth instar nymphs, brachypterous female adults, macropterous female adults and macropterous male adults. Illumina sequencing revealed 85,526 unigenes, including 13,102 clusters and 72,424 singletons. Transcriptome sequences larger than 350 bp were subjected to Gene Orthology (GO) and KEGG Orthology (KO) annotations. To analyze the DGE profiling, we mainly compared the gene expression variations between eggs and second instar nymphs; second and fifth instar nymphs; fifth instar nymphs and three types of adults; brachypterous and macropterous female adults as well as macropterous female and male adults. Thousands of genes showed significantly different expression levels based on the various comparisons. And we randomly selected some genes to confirm their altered expression levels by quantitative real-time PCR (qRT-PCR).
The obtained BPH transcriptome and DGE profiling data provide comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the molecular mechanisms from various physiological aspects including development, wing dimorphism and sex difference in BPH.
We determined the genome-wide digital gene expression (DGE) profiles of primary acute lymphoblastic leukemia (ALL) cells from 21 patients taking advantage of ‘second-generation' sequencing technology. Patients included in this study represent four cytogenetically distinct subtypes of B-cell precursor (BCP) ALL and T-cell lineage ALL (T-ALL). The robustness of DGE combined with supervised classification by nearest shrunken centroids (NSC) was validated experimentally and by comparison with published expression data for large sets of ALL samples. Genes that were differentially expressed between BCP ALL subtypes were enriched to distinct signaling pathways with dic(9;20) enriched to TP53 signaling, t(9;22) to interferon signaling, as well as high hyperdiploidy and t(12;21) to apoptosis signaling. We also observed antisense tags expressed from the non-coding strand of ∼50% of annotated genes, many of which were expressed in a subtype-specific pattern. Antisense tags from 17 gene regions unambiguously discriminated between the BCP ALL and T-ALL subtypes, and antisense tags from 76 gene regions discriminated between the 4 BCP subtypes. We observed a significant overlap of gene regions with alternative polyadenylation and antisense transcription (P<1 × 10−15). Our study using DGE profiling provided new insights into the RNA expression patterns in ALL cells.
pediatric acute lymphoblastic leukemia; ALL; digital gene expression; second-generation sequencing; antisense transcript; alternative polyadenylation
Using RNA-seq digital difference expression profiling methods, we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). A reference transcriptome of hemocyte-expressed transcripts was assembled from 202 million 42-base tags by combining the sequence data of all samples, and the assembled sequences were then subject to BLASTx analysis to determine gene identities. We used the fully sequenced HzSNPV reference genome to align 477,264 Illumina sequence tags from infected hemocytes in order to document expression of HzSNPV genes at early points during infection. A comparison of expression profiles of control insects to those lethally infected with HzSNPV revealed differential expression of key cellular stress response genes and genes involved in lipid metabolism. Transcriptional regulation of specific insect hormones in baculovirus-infected insects was also altered. A number of transcripts bearing homology to retroviral elements that were detected add to a growing body of evidence for extensive invasion of errantiviruses into the insect genome. Using this method, we completed the first and most comprehensive gene expression survey of both baculoviral infection and host immune defense in lepidopteran larvae.
baculovirus; lepidopteran; transcription; RNA-seq
The diamondback moth Plutella xyllostella has developed a high level of resistance to the latest insecticide chlorantraniliprole. A better understanding of P. xylostella’s resistance mechanism to chlorantraniliprole is needed to develop effective approaches for insecticide resistance management.
To provide a comprehensive insight into the resistance mechanisms of P. xylostella to chlorantraniliprole, transcriptome assembly and tag-based digital gene expression (DGE) system were performed using Illumina HiSeq™ 2000. The transcriptome analysis of the susceptible strain (SS) provided 45,231 unigenes (with the size ranging from 200 bp to 13,799 bp), which would be efficient for analyzing the differences in different chlorantraniliprole-resistant P. xylostella stains. DGE analysis indicated that a total of 1215 genes (189 up-regulated and 1026 down-regulated) were gradient differentially expressed among the susceptible strain (SS) and different chlorantraniliprole-resistant P. xylostella strains, including low-level resistance (GXA), moderate resistance (LZA) and high resistance strains (HZA). A detailed analysis of gradient differentially expressed genes elucidated the existence of a phase-dependent divergence of biological investment at the molecular level. The genes related to insecticide resistance, such as P450, GST, the ryanodine receptor, and connectin, had different expression profiles in the different chlorantraniliprole-resistant DGE libraries, suggesting that the genes related to insecticide resistance are involved in P. xylostella resistance development against chlorantraniliprole. To confirm the results from the DGE, the expressional profiles of 4 genes related to insecticide resistance were further validated by qRT-PCR analysis.
The obtained transcriptome information provides large gene resources available for further studying the resistance development of P. xylostella to pesticides. The DGE data provide comprehensive insights into the gene expression profiles of the different chlorantraniliprole-resistant stains. These genes are specifically related to insecticide resistance, with different expressional profiles facilitating the study of the role of each gene in chlorantraniliprole resistance development.
The Eastern hive honey bee, Apis cerana cerana is a native and widely bred honey bee species in China. Molecular biology research about this honey bee species is scarce, and genomic information for A. c. cerana is not currently available. Transcriptome and expression profiling data for this species are therefore important resources needed to better understand the biological mechanisms of A. c. cerana. In this study, we obtained the transcriptome information of A. c. cerana by RNA-sequencing and compared gene expression differences between queens and workers of A. c. cerana by digital gene expression (DGE) analysis.
Using high-throughput Illumina RNA sequencing we obtained 51,581,510 clean reads corresponding to 4.64 Gb total nucleotides from a single run. These reads were assembled into 46,999 unigenes with a mean length of 676 bp. Based on a sequence similarity search against the five public databases (NR, Swissport, GO, COG, KEGG) with a cut-off E-value of 10−5 using BLASTX, a total of 24,630 unigenes were annotated with gene descriptions, gene ontology terms, or metabolic pathways. Using these transcriptome data as references we analyzed the gene expression differences between the queens and workers of A. c. cerana using a tag-based digital gene expression method. We obtained 5.96 and 5.66 million clean tags from the queen and worker samples, respectively. A total of 414 genes were differentially expressed between them, with 189 up-regulated and 225 down-regulated in queens.
Our transcriptome data provide a comprehensive sequence resource for future A. c. cerana study, establishing an important public information platform for functional genomic studies in A. c. cerana. Furthermore, the DGE data provide comprehensive gene expression information for the queens and workers, which will facilitate our understanding of the molecular mechanisms of the different physiological aspects of the two castes.
Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we report on the first genome-wide host transcriptional responses to classical North American type PRRSV (N-PRRSV) strain CH 1a infection using Solexa/Illumina's digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results suggest that N-PRRSV appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processes. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS.
Bud dormancy is a critical developmental process that allows perennial plants to survive unfavorable environmental conditions. Pear is one of the most important deciduous fruit trees in the world, but the mechanisms regulating bud dormancy in this species are unknown. Because genomic information for pear is currently unavailable, transcriptome and digital gene expression data for this species would be valuable resources to better understand the molecular and biological mechanisms regulating its bud dormancy.
We performed de novo transcriptome assembly and digital gene expression (DGE) profiling analyses of ‘Suli’ pear (Pyrus pyrifolia white pear group) using the Illumina RNA-seq system. RNA-Seq generated approximately 100 M high-quality reads that were assembled into 69,393 unigenes (mean length = 853 bp), including 14,531 clusters and 34,194 singletons. A total of 51,448 (74.1%) unigenes were annotated using public protein databases with a cut-off E-value above 10-5. We mainly compared gene expression levels at four time-points during bud dormancy. Between Nov. 15 and Dec. 15, Dec. 15 and Jan. 15, and Jan. 15 and Feb. 15, 1,978, 1,024, and 3,468 genes were differentially expressed, respectively. Hierarchical clustering analysis arranged 190 significantly differentially-expressed genes into seven groups. Seven genes were randomly selected to confirm their expression levels using quantitative real-time PCR.
The new transcriptomes offer comprehensive sequence and DGE profiling data for a dynamic view of transcriptomic variation during bud dormancy in pear. These data provided a basis for future studies of metabolism during bud dormancy in non-model but economically-important perennial species.
‘Suli’ pear (Pyrus pyrifolia white pear group); Transcriptome; Bud dormancy; RNA-Seq
As the only truly flying mammals, bats use their unique wing - consisting of four elongated digits (digits II-V) connected by membranes - to power their flight. In addition to the elongated digits II-V, the forelimb contains one shorter digit (digit I) that is morphologically similar to the hindlimb digits. Here, we capitalized on the morphological variation among the bat forelimb digits to investigate the molecular mechanisms underlying digit elongation and wing formation. Using next generation sequencing technology, we performed digital gene expression tag profiling (DGE-tag profiling) of developing digits in a pooled sample of two Myotis ricketti and validated our sequencing results using real-time quantitative PCR (RT-qPCR) of gene expression in the developing digits of two Hipposideros armiger.
Among hundreds of genes exhibiting significant differences in expression between the short and long digits, we highlight 14 genes most related to digit elongation. These genes include two Tbx genes (Tbx3 and Tbx15), five BMP pathway genes (Bmp3, RGMB, Smad1, Smad4 and Nog), four Homeobox genes (Hoxd8, Hoxd9, Hoxa1 and Satb1), and three other genes (Twist1, Tmeff2 and Enpp2) related to digit malformations or cell proliferation. In addition, our results suggest that Tbx4 and Pitx2 contribute to the morphological similarity and five genes (Acta1, Tnnc2, Atp2a1, Hrc and Myoz1) contribute to the functional similarity between the thumb and hindlimb digits.
Results of this study not only implicate many developmental genes as robust candidates underlying digit elongation and wing formation in bats, but also provide a better understanding of the genes involved in autopodial development in general.
Generating reliable expression profiles from minute cell quantities is critical for scientific discovery and potential clinical applications. Here we present low-quantity digital gene expression (LQ-DGE), an amplification-free approach involving capture of poly(A)+ RNAs from cellular lysates onto poly(dT)-coated sequencing surfaces, followed by on-surface reverse transcription and sequencing. We applied LQ-DGE to profile malignant and nonmalignant mouse and human cells, demonstrating its quantitative power and potential applicability to archival specimens.
Previous transcriptome profiling studies have investigated the molecular mechanisms of pollen and anther development, and identified many genes involved in these processes. However, only 51 anther ESTs of Upland cotton (Gossypium hirsutum) were found in NCBI and there have been no reports of transcriptome profiling analyzing anther development in Upland cotton, a major fiber crop in the word.
Ninety-eight hundred and ninety-six high quality ESTs were sequenced from their 3′-ends and assembled into 6,643 unigenes from a normalized, full-length anther cDNA library of Upland cotton. Combined with previous sequenced anther-related ESTs, 12,244 unigenes were generated as the reference genes for digital gene expression (DGE) analysis. The DGE was conducted on anthers that were isolated at tetrad pollen (TTP), uninucleate pollen (UNP), binucleate pollen (BNP) and mature pollen (MTP) periods along with four other tissues, i.e., roots (RO), stems (ST), leaves (LV) and embryos (EB). Through transcriptome profiling analysis, we identified 1,165 genes that were enriched at certain anther development periods, and many of them were involved in starch and sucrose metabolism, pentose and glucuronate interconversion, flavonoid biosynthesis, and ascorbate and aldarate metabolism.
We first generated a normalized, full-length cDNA library from anthers and performed transcriptome profiling analysis of anther development in Upland cotton. From these results, 10,178 anther expressed genes were identified, among which 1,165 genes were stage-enriched in anthers. And many of these stage-enriched genes were involved in some important processes regulating anther development.
Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR). In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG), and abscisic acid (ABA). Three analytical software packages (geNorm, Bestkeeper, and NormFinder) were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC) and alpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.
Corky split vein caused by boron (B) deficiency in ‘Newhall’ Navel Orange was studied in the present research. The boron-deficient citrus exhibited a symptom of corky split vein in mature leaves. Morphologic and anatomical surveys at four representative phases of corky split veins showed that the symptom was the result of vascular hypertrophy. Digital gene expression (DGE) analysis was performed based on the Illumina HiSeq™ 2000 platform, which was applied to analyze the gene expression profilings of corky split veins at four morphologic phases. Over 5.3 million clean reads per library were successfully mapped to the reference database and more than 22897 mapped genes per library were simultaneously obtained. Analysis of the differentially expressed genes (DEGs) revealed that the expressions of genes associated with cytokinin signal transduction, cell division, vascular development, lignin biosynthesis and photosynthesis in corky split veins were all affected. The expressions of WOL and ARR12 involved in the cytokinin signal transduction pathway were up-regulated at 1st phase of corky split vein development. Furthermore, the expressions of some cell cycle genes, CYCs and CDKB, and vascular development genes, WOX4 and VND7, were up-regulated at the following 2nd and 3rd phases. These findings indicated that the cytokinin signal transduction pathway may play a role in initiating symptom observed in our study.
Stem cells self-renew and repeatedly produce differentiated cells during development and growth. The differentiated cells can be converted into stem cells in some metazoans and land plants with appropriate treatments. After leaves of the moss Physcomitrella patens are excised, leaf cells reenter the cell cycle and commence tip growth, which is characteristic of stem cells called chloronema apical cells. To understand the underlying molecular mechanisms, a digital gene expression profiling method using mRNA 5′-end tags (5′-DGE) was established. The 5′-DGE method produced reproducible data with a dynamic range of four orders that correlated well with qRT-PCR measurements. After the excision of leaves, the expression levels of 11% of the transcripts changed significantly within 6 h. Genes involved in stress responses and proteolysis were induced and those involved in metabolism, including photosynthesis, were reduced. The later processes of reprogramming involved photosynthesis recovery and higher macromolecule biosynthesis, including of RNA and proteins. Auxin and cytokinin signaling pathways, which are activated during stem cell formation via callus in flowering plants, are also activated during reprogramming in P. patens, although no exogenous phytohormone is applied in the moss system, suggesting that an intrinsic phytohormone regulatory system may be used in the moss.
Chrysomya megacephala (Fabricius), a prevalent necrophagous blowfly that is easily mass reared, is noted for being a mechanical vector of pathogenic microorganisms, a pollinator of numerous crops, and a resource insect in forensic investigation in the postmortem interval. In the present study, in order to comprehensively understand the physiological and biochemical functions of C. megacephala, we performed RNA-sequencing and digital gene expression (DGE) profiling using Solexa/Illumina sequencing technology.
A total of 39,098,662 clean reads were assembled into 27,588 unigenes with a mean length of 768 nt. All unigenes were searched against the Nt database, Nr database, Swiss-Prot, Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genome (KEGG) with the BLASTn or BLASTx algorithm (E-value<0.00001) for annotations. In total, 7,081 unigenes and 14,099 unigenes were functionally classified into 25 COG categories and 240 KEGG pathways, respectively. Furthermore, 20,216 unigenes were grouped into 48 sub-categories belonging to 3 main Gene Ontology (GO) categories (ontologies). Using the transcriptome data as references, we analyzed the differential gene expressions between a soybean oil-fed group (SOF) and a lard oil-fed group (LOF), compared to the negative control group (NC), using the DGE approach. We finally obtained 1,566 differentially expressed genes in SOF/NC, and 1,099 genes in LOF/NC. For further analysis, GO and KEGG functional enrichment were performed on all differentially expressed genes, and a group of differentially expressed candidate genes related to lipometabolism were identified.
This study provides a global survey of C. megacephala and provides the basis for further research on the functional genomics of this insect.