Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing

BACKGROUND Mesenchymal stem cells(MSCs)modulated by various exogenous signals have been applied extensively in regenerative medicine research.Notably,nanosecond pulsed electric fields(nsPEFs),characterized by short duration and high strength,significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways.Consequently,we used transcriptomics to study changes in messenger RNA(mRNA),long noncoding RNA(lncRNA),microRNA(miRNA),and circular RNA expression during nsPEFs application.AIM To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs.METHODS The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing.MSCs were pretreated with 5-pulse nsPEFs(100 ns at 10 kV/cm,1 Hz),followed by total RNA isolation.Each transcript was normalized by fragments per kilobase per million.Fold change and difference significance were applied to screen the differentially expressed genes(DEGs).Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions,complemented by quantitative polymerase chain reaction verification.RESULTS In total,263 DEGs were discovered,with 92 upregulated and 171 downregulated.DEGs were predominantly enriched in epithelial cell proliferation,osteoblast differentiation,mesenchymal cell differentiation,nuclear division,and wound healing.Regarding cellular components,DEGs are primarily involved in condensed chromosome,chromosomal region,actin cytoskeleton,and kinetochore.From aspect of molecular functions,DEGs are mainly involved in glycosaminoglycan binding,integrin binding,nuclear steroid receptor activity,cytoskeletal motor activity,and steroid binding.Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation.CONCLUSION Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs,2 miRNAs,and 65 lncRNAs.Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways,which are involved in vesicular transport,calcium ion transport,cytoskeleton,and cell differentiation.

Transcriptome sequencing reveals novel biomarkers and immune cell infiltration in esophageal tumorigenesis

BACKGROUND Esophageal squamous cell carcinoma(ESCC)is one of the most common malignancies worldwide,and its development comprises a multistep process from intraepithelial neoplasia(IN)to carcinoma(CA).However,the critical regulators and underlying molecular mechanisms remain largely unknown.AIM To explore the genes and infiltrating immune cells in the microenvironment that are associated with the multistage progression of ESCC to facilitate diagnosis and early intervention.METHODS A mouse model mimicking the multistage development of ESCC was established by providing warter containing 4-nitroquinoline 1-oxide(4NQO)to C57BL/6 mice.Moreover,we established a control group without 4NQO treatment of mice.Then,transcriptome sequencing was performed for esophageal tissues from patients with different pathological statuses,including low-grade IN(LGIN),high-grade IN(HGIN),and CA,and controlled normal tissue(NOR)samples.Differentially expressed genes(DEGs)were identified in the LGIN,HGIN,and CA groups,and the biological functions of the DEGs were analyzed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses.The CIBERSORT algorithm was used to detect the pattern of immune cell infilt-ration.Immunohistochemistry(IHC)was also conducted to validate our results.Finally,the Luminex multiplex cytokine analysis was utilized to measure the serum cytokine levels in the mice.RESULTS Compared with those in the NOR group,a total of 681541,and 840 DEGs were obtained in the LGIN,HGIN,and CA groups,respectively.Using the intersection of the three sets of DEGs,we identified 86 genes as key genes involved in the development of ESCC.Enrichment analysis revealed that these genes were enriched mainly in the keratinization,epidermal cell differentiation,and interleukin(IL)-17 signaling pathways.CIBERSORT analysis revealed that,compared with those in the NOR group,M0 and M1 macrophages in the 4NQO group showed stronger infiltration,which was validated by IHC.Serum cytokine analysis revealed that,compared with those in the NOR group,IL-1βand IL-6 were upregulated,while IL-10 was downregulated in the LGIN,HGIN,and CA groups.Moreover,the expression of the representative key genes,such as S100a8 and Krt6b,was verified in external human samples,and the results of immunohistochemical staining were consistent with the findings in mice.CONCLUSION We identified a set of key genes represented by S100a8 and Krt6b and investigated their potential biological functions.In addition,we found that macrophage infiltration and abnormal alterations in the levels of inflam-mation-associated cytokines,such as IL-1β,IL-6,and IL-10,in the peripheral blood may be closely associated with the development of ESCC.

Analyses of Chicken Tenderness Traits Based on Transcriptome Sequencing

The calpain system is ubiquitous in cells, mainly comprising calpains and calpain inhibitors, and is a widespread calcium-dependent cysteine protease in organisms that is involved in many cellular processes such as muscle degradation in vivo and affects the tenderness of meat after animal slaughter. The study found 128 DEGs that probably regulated tenderness traits were selected from 16 significantly enriched GO terms by transcriptome sequencing analysis, and found that the developmental changes in the expression levels of the CAPN1 gene in the pectoral and leg muscles were significantly positively correlated ( P <0.05) with the cumulative growth values of live weight and comb weight. The developmental changes in the expression levels of the CAST gene in the pectoral and leg muscles were not significantly correlated with the cumulative growth values of live weight and comb weight. Our results helped demonstrate the potential molecular mechanisms of tenderness in chickens and provide valuable information for chicken breeding.

Progress and challenges of cerebrovascular endothelial cells research promoted by single-cell transcriptome sequencing technology

Single-cell transcriptome sequencing has been a rapidly developing and powerful biological tool in recent years,and it plays a vital role in describing tissue development,cell heterogeneity,stress response,etc.Cerebrovascular disease is one of the leading causes affecting human health in the world.Thus,it is important to understand the characteristics of cerebrovascular structure,function,and environmental response.Notably,single-cell transcriptome sequencing provides deeper insights into cerebrovascular research in health and disease states.This article will briefly introduce the basic structure and function of cerebrovascular endothelial cells(ECs),summarize the current research and new findings on cerebrovascular ECs at the single-cell transcriptome level,and discuss the challenges in this field.

Use of transcriptome sequencing to explore the effect of CSRP3 on chicken myoblasts

The mechanisms that regulate the specificity and maintenance of chicken muscle fiber types remain largely unknown. In mammals, CSRP3 has been shown to play a vital role in the maintenance of typical muscle structure and function. This study investigated the role that CSRP3 plays in chicken skeletal muscle. First, the antibody against chicken CSRP3 protein was prepared, and the expression levels of the mRNA and protein of the CSRP3 gene in four chicken skeletal muscles with different myofiber compositions were compared. Then the effects of CSRP3 silencing on the expression profile of chicken myoblast transcriptomes were analyzed. The results showed that the expression levels of the mRNA and protein of the CSRP3 gene were both associated with the composition of fiber types in chicken skeletal muscles. A total of 650 genes with at least 1.5-fold differences(Q<0.05) were identified, of which 255 genes were upregulated and 395 genes were downregulated by CSRP3 silencing. Functional enrichment showed that several pathways, including adrenergic signaling in cardiomyocytes, adipocytokine signaling pathway and apelin signaling pathway, were significantly(P<0.05) enriched both in differentially expressed genes and all expressed genes. The co-expressed gene network suggested that CSRP3 silencing caused a compensatory upregulation(Q<0.05) of genes related to the assembly of myofibrils, muscle differentiation, and contraction. Meanwhile, two fast myosin heavy chain genes(MyH1B and MyH1E)were upregulated(Q<0.05) upon CSRP3 silencing. These results suggested that CSRP3 plays a crucial role in chicken myofiber composition, and affects the distribution of chicken myofiber types, probably by regulating the expression of MyH1B and MyH1E.

Transcriptome sequencing analysis of ursolic acid-mediated proliferation suppression on cutaneous T-cell lymphoma cells

Background:Ursolic acid is a triterpenoid compound found in natural plants that exhibits antiproliferative effects in various cancer cells.Our study is the first to demonstrate the strong inhibitory effects of ursolic acid on the proliferation of cutaneous T-cell lymphoma(CTCL)cells.We aimed to further investigate the underlying mechanism of the proliferation inhibition induced by ursolic acid in CTCL cells using transcriptome sequencing.Methods:Cell counting kit-8 assays were used to observe the effects of six traditional medicine monomers on the proliferation of CTCL cells.Transcriptome sequencing was used to identify differentially expressed genes after ursolic acid treatment.Bioinformatics analysis was performed to determine the potential mechanism.Real-time quantitative PCR and western blotting analyses were performed to confirm the sequencing results and verify the possible mechanisms of ursolic acid-mediated proliferation inhibition in CTCL cells.Results:Ursolic acid exhibited the strongest inhibitory effect on the proliferation of CTCL cells among the six traditional medicine monomers.Transcriptome sequencing analysis showed that 2,466 genes were significantly altered.Combined with Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis and protein-protein interaction network analysis,the interaction of various pathways and signaling molecules,such as tumor necrosis factor-α,NLR family pyrin domain containing 1,c-Jun N-terminal kinase,and melanoma differentiation-associated gene 5,accounted for the anti-tumor effects of ursolic acid in CTCL cells.Conclusion:Ursolic acid significantly inhibited the proliferation of CTCL cells,and our study laid a theoretical foundation for the future treatment of CTCL using ursolic acid.

Comparative transcriptome analysis between rhesus macaques(Macaca mulatta) and crab-eating macaques(M. fascicularis)

Understanding gene expression variations between species is pivotal for deciphering the evolutionary diversity in phenotypes. Rhesus macaques(Macaca mulatta, MMU)and crab-eating macaques(M. fascicularis, MFA) serve as crucial nonhuman primate biomedical models with different phenotypes. To date, however, large-scale comparative transcriptome research between these two species has not yet been fully explored. Here, we conducted systematic comparisons utilizing newly sequenced RNA-seq data from84 samples(41 MFA samples and 43 MMU samples)encompassing 14 common tissues. Our findings revealed a small fraction of genes(3.7%) with differential expression between the two species, as well as 36.5% of genes with tissue-specific expression in both macaques. Comparison of gene expression between macaques and humans indicated that 22.6% of orthologous genes displayed differential expression in at least two tissues. Moreover,19.41% of genes that overlapped with macaque-specific structural variants showed differential expression between humans and macaques. Of these, the FAM220A gene exhibited elevated expression in humans compared to macaques due to lineage-specific duplication. In summary,this study presents a large-scale transcriptomic comparison between MMU and MFA and between macaques and humans. The discovery of gene expression variations not only enhances the biomedical utility of macaque models but also contributes to the wider field of primate genomics.

Single‑cell RNA sequencing opens a new era for cotton genomic research and gene functional analysis

Single-cell RNA sequencing(scRNA-seq)is one of the most advanced sequencing technologies for studying transcriptome landscape at the single-cell revolution.It provides numerous advantages over traditional RNA-seq.Since it was first used to profile single-cell transcriptome in plants in 2019,it has been extensively employed to perform different research in plants.Recently,scRNA-seq was also quickly adopted by the cotton research community to solve lots of scientific questions which have been never solved.In this comment,we highlighted the significant progress in employing scRNA-seq to cotton genetic and genomic study and its future potential applications.

Study on compound Duzhong Jiangu granule in the treatment of knee osteoarthritis based on transcriptome analysis strategy

Duzhong Jiangu Granule in the treatment of primary osteoarthritis(POA)model of postmenopausal kidney deficiency type in Hartley female guinea pigs after ovariectomy,and the correlation between gene expression of bone marrow tissue,cartilage tissue,and knee osteoarthritis.Methods:383-months-old Hartley female guinea pigs after one week of adaptive feeding were weighed about 400 g±20 g,numbered,and sorted by ear tag.Six of them were selected as normal groups by looking up random number tables.The remainder were removed from the bilateral ovaries to construct the postmenopausal kidney deficiency model,and castrated guinea pigs were used to construct the postmenopausal kidney deficiency POA model.After the modeling cycle,a guinea pig from the blank group and a guinea pig from the model group were sacrificed and the right knee was observed.The model was established and the experiments continued.There were five guinea pigs in the blank group,and the remaining model guinea pigs were randomly divided into model control group,high-dose group of compound Duzhong Jiangu granules,middle-dose group of compound Duzhong Jiangu granules,low-dose group of compound Duzhong Jiangu granules and design group,with 5 guinea pigs in each group.Blanks and model groups were given a cellulose sodium solution by gavage.The guinea pigs were sacrificed after 30 days of intragastric administration.The left knee cartilage and bone marrow of the blank group,model group,middle dose group,and high dose group of compound Duzhong Jiangu granule were collected and applied to transcriptome sequencing,and the sequencing data were analyzed,including differential gene expression analysis,functional enrichment analysis of database established by Gene Ontology federation(GO)and Kyoto Encyclopedia of Gene and Genome(KEGG)pathway enrichment analysis.The complete specimens of the right knee joint were collected,and the morphological changes of the cartilage of the right knee joint in each group were observed by saffron rapid green staining,and the subchondral bone was quantitatively analyzed by Micro CT so that the expression of TRAF6,MIP-1βand IL-1βprotein in NF-kappa B signaling pathway was detected by Western Blot technique(WB).Results:The results of Safranin Fast Green staining showed that Compound Duzhong Jiangu Granules could effectively reduce the degree of morphological damage of articular cartilage in guinea pigs with the POA model.According to the analysis results of the subchondral bone structure under Micro CT,Compound Duzhong Jiangu Granules can improve the bone condition of the POA model,thus delaying the process of degenerative changes of the knee joint.From the results of transcriptome analysis,Compound Duzhong Jiangu Granules can inhibit the expression of related genes in POA model guinea pigs.According to the results of Wester Bolt verification,Compound Duzhong Jiangu Granules can effectively improve knee osteoarthritis.Conclusions:The effect of Compound Duzhong Jiangu Granules on OA is obvious,and its mechanism may be related to the expression of genes GZMK,Jchain,igkc,IGHV3-74,IGHV3-11,IGHV4-1,CCL5,and IGKV1–39.

Analysis of differentially expressed genes in Verruca vulgaris vs.adjacent normal skin by RNA-sequencing

Introduction:Verruca vulgaris is one of the most common low-risk HPV infections and is characterized by excessive proliferation of keratinocytes.Currently,very little genetic information is available regarding verruca vulgaris in the Chinese population.This study aimed to obtain comprehensive transcript information of verruca vulgaris by RNA sequencing.Methods:High-throughput sequencing was performed on three fresh verruca vulgaris samples and adjacent normal skin on the Illumina sequencing platform.The transcriptomes were analyzed using bioinformatics and the differentially expressed genes(DEGs)were verified by immunohistochemistry.Verruca vulgaris exhibited a unique molecular signature.Results:In total,1,643 DEGs were identified in verruca vulgaris compared to normal skin.The functions of the DEGs were studies by Gene Ontology(GO)enrichment,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,DEGs Reactome analysis,disease annotation function,and STRING protein-protein interaction(PPI)network analysis.The results revealed 595 GO terms associated with the cell cycle,signal transduction,immune system,signaling molecules,and interaction.The Reactome analysis revealed enrichment in reversible hydration of carbon dioxide and BMP signaling,while the disease annotation function revealed that the enriched DEGs are involved in keratosis disorders.The STRING PPI network showed that the edges with the highest density mainly included the 2′-5′oligoadenylate synthase(OAS)family-related proteins.Furthermore,the M-code analysis found ISG15,IRF7,and OASL were scored as significant modules and their high expression compared to the control was verified by immunohistochemistry.Conclusion:These findings contribute to the genetic information of verruca vulgaris in the Chinese population,revealing that interferon-stimulated genes may play essential roles in verruca vulgaris.

Full-length transcriptome sequence and SSR marker development for genetic diversity research in yellowfin seabream Acanthopagrus latus

Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificial breeding and reproduction.We performed full-length transcriptome sequencing and assembly of the genome of yellowfin seabream.A total of 68086 unigenes were obtained,with an N50 of 3391 bp on average length of 2933 bp.A total number of 50593 expressed sequence tags linked to simple sequence repeats(EST-SSR)were identified,among them dinucleotide repeats(40.6%)and AC/GT motifs(38.5%)were the most frequent.Of the 190 EST-SSRs for which PCR primer pairs were designed,150 primer pairs successfully amplified target loci and 15 SSRs showed high polymorphism.The alleles per locus ranged 6-50 on average of 25.3.The expected and observed heterozygosity varied from 0.632 to 0.969 and from 0.519 to 0.953,respectively.The polymorphic index content(PIC)values of each locus ranged 0.587-0.966 on average of 0.851.Among six yellowfin seabream population samples preliminarily tested for genetic diversity and differentiation,the Fangchenggang(FCG)population in Guangxi Province had the highest mean observed heterozygosity(H_(o))value(0.786),whereas the Zhangzhou(ZZ)population in Fujian Province had the lowest(0.678).The pairwise fixation index(Fst)values indicated significant population differentiation among six yellowfin seabream populations.This study provided evidence for the usefulness of the transcriptomic resource information and EST-SSR markers for natural resource conservation,population genetics,and breeding studies of yellowfin seabream in South China.

Successful multidisciplinary clinical approach and molecular characterization by whole transcriptome sequencing of a cardiac myxofibrosarcoma: A case report

BACKGROUND Cardiac tumors are rare and complex entities.Surgery represents the cornerstone of therapy,while the role of adjuvant treatment remains unclear and,in case of relapse or metastatic disease,the prognosis is very poor.Lack of prospective,randomized clinical trials hinders the generation of high level evidence for the optimal diagnostic workup and multimodal treatment of cardiac sarcomas.Herein,we describe the multidisciplinary clinical management and molecular characterization of a rare case of cardiac myxofibrosarcoma in an elderly woman.CASE SUMMARY A 73-year-old woman presented signs and symptoms of acute left-sided heart failure.Imaging examination revealed a large,left atrial mass.With suspicion of a myxoma,she underwent surgery,and symptoms were promptly relieved.Histology showed a cardiac myxofibrosarcoma,a rare histotype of cardiac sarcoma.Eight months later,disease unfortunately relapsed,and after a multidisciplinary discussion,a chemotherapy with doxorubicin and then gemcitabine was started,achieving partial radiologic and complete metabolic response,which was maintained up to 2 years and is still present.This report is focused on the entire clinical path of our patient from diagnosis to follow-up,through surgery and strategies adopted at relapse.Moreover,due to their rarity,very little is known about the molecular landscape of myxofibrosarcomas.Thus,we also performed and described preliminary genome analysis of the tumor tissue to get further insight on mechanisms involved in tumor growth,and to possibly unveil new clinically actionable targets.CONCLUSION We report a case of cardiac myxofibrosarcoma that achieved a very good prognosis due to an integrated surgical,cardiac and oncologic treatment strategy.

Next Generation Transcriptome Sequencing and Quantitative Real-Time PCR Technologies for Characterisation of the Bemisia tabaci Asia 1 mtCOI Phylogenetic Clade

A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemiptera：Aleyrodidae）,the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing（NGS）has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences（301 094 reads）using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes（e.g.,HSP90）but misassemblies in other datasets（e.g.,the vitellogenin gene family）,highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.

Analysis of Saccharina japonica transcriptome using the high-throughput DNA sequencing technique and its vanadium-dependent haloperoxidase gene

Saccharina is one of the most important cold-water living marine brown algal genera. In this study we ana-lyzed the transcriptome of S. japonica, which belongs to the 1 000 Plants （OneKP） Project, by using a next-generation high-throughput DNA sequencing technique. About 5.16 GB of raw data were generated, and 65 536 scaffolds with an average length of 454 bp were assembled with SOAP de novo assembly method. In total, 19 040 unigenes were identified by BLAST;25 734 scaffolds were clustered into 37 Gene ontology functional groups;6 760 scaffolds were classified into 25 COG categories, as well as 2 665 scaffolds that were assigned to 306 KEGG pathways. Majority of the unigenes exhibited more similarities to algae including brown algae and diatom than other cyanobacteria, marine diatom, and plant. Saccharina japonica has the outstanding capability to accumulate halogen such as Br and I via halogenation processes from seawater. We acquired 42 different vanadium-dependent haloperoxidases （vHPO） in S. japonica transcriptome data, including 5 segments of vanadium-dependent iodoperoxidase （vIPO） and 37 segments of vanadium-de-pendent bromoperoxidase （vBPO）. Complicated analyses of identified fulllength S. japonica vBPO1 and S. japonica vBPO2 revealed the importance of vBPO among species of brown algae and the strong relationship between marine algal vBPOs and vIPOs. This study will enhance our understanding of the biological charac-teristics and economic values of S. japonica species.

Analysis of transcriptome sequencing of sciatic nerves in Sprague-Dawley rats of different ages

An aging-induced decrease in Schwann cell viability can affect regeneration following peripheral nerve injury in mammals. It is therefore necessary to investigate possible age-related changes in gene expression that may affect the biological function of peripheral nerves. Ten 1-week-old and ten 12-month-old healthy male Sprague-Dawley rats were divided into young（1 week old） and adult（12 months old） groups according to their ages. mRNA expression in the sciatic nerve was compared between young and adult rats using next-generation sequencing（NGS） and bioinformatics（n = 4/group）. The 18 groups of differentially expressed mRNA（DEmRNAs） were also tested by quantitative reverse transcription polymerase chain reaction（n = 6/group）. Results revealed that（1） compared with young rats, adult rats had 3608 groups of DEmRNAs. Of these, 2684 were groups of upregulated genes, and 924 were groups of downregulated genes. Their functions mainly involved cell viability, proliferation, differentiation, regeneration, and myelination.（2） The gene with the most obvious increase of all DEmRNAs in adult rats was Thrsp（log2 FC = 9.01, P 〈 0.05）, and the gene with the most obvious reduction was Col2 a1（log2 FC = -8.89, P 〈 0.05）.（3） Gene Ontology analysis showed that DEmRNAs were mainly concentrated in oligosaccharide binding, nucleotide-binding oligomerization domain containing one signaling pathway, and peptide-transporting ATPase activity.（4） Analysis using the Kyoto Encyclopedia of Genes and Genomes showed that, with increased age, DEmRNAs were mainly enriched in steroid biosynthesis, Staphylococcus aureus infection, and graft-versus-host disease.（5） Spearman＇s correlation coefficient method for evaluating NGS accuracy showed that the NGS results and quantitative reverse transcription polymerase chain reaction results were positively correlated（rs = 0.74, P 〈 0.05）. These findings confirm a difference in sciatic nerve gene expression between adult and young rats, suggesting that, in peripheral nerves, cells and the microenvironment change with age, thus influencing the function and repair of peripheral nerves.

Uncovering Small RNAs in Penicillium digitatum by Transcriptome Sequencing

Small RNAs in Penicillium digitatum were identified and analyzed via transcriptome sequencing on the BGISEQ-500 platform. A total of 15 predicted miRNAs and 10718 novel siRNAs were found. Their length distribution, sequence, predicted construction, base bias, expression levels and potential targets were determined as well. Through pathway and KEGG enrichment analysis, the miRNA target genes were mostly involved in carbohydrate metabolism, transport and catabolism, translation and amino acid metabolism. The target genes involved in aflatoxin biosynthesis and proteasome had a higher rich factor value. The results will provide a theoretical foundation for understanding the developmental and pathogenic mechanisms of P. digitatum at the transcriptional level.

Sequencing, de novo assembly and characterization of the spotted scat Scatophagus argus(Linnaeus 1766) transcriptome for discovery of reproduction related genes and SSRs

Spotted scat( Scatophagus argus) is an economically important farmed fish, particularly in East and Southeast Asia. Because there has been little research on reproductive development and regulation in this species, the lack of a mature artificial reproduction technology remains a barrier for the sustainable development of the aquaculture industry. More genetic and genomic background knowledge is urgently needed for an in-depth understanding of the molecular mechanism of reproductive process and identification of functional genes related to sexual differentiation, gonad maturation and gametogenesis. For these reasons, we performed transcriptomic analysis on spotted scat using a multiple tissue sample mixing strategy. The Illumina RNA sequencing generated 118 510 486 raw reads. After trimming, de novo assembly was performed and yielded 99 888 unigenes with an average length of 905.75 bp. A total of 45 015 unigenes were successfully annotated to the Nr, Swiss-Prot, KOG and KEGG databases. Additionally, 23 783 and 27 183 annotated unigenes were assigned to 56 Gene Ontology(GO) functional groups and 228 KEGG pathways, respectively. Subsequently, 2 474 transcripts associated with reproduction were selected using GO term and KEGG pathway assignments, and a number of reproduction-related genes involved in sex differentiation, gonad development and gametogenesis were identified. Furthermore, 22 279 simple sequence repeat(SSR) loci were discovered and characterized. The comprehensive transcript dataset described here greatly increases the genetic information available for spotted scat and contributes valuable sequence resources for functional gene mining and analysis. Candidate transcripts involved in reproduction would make good starting points for future studies on reproductive mechanisms, and the putative sex differentiation-related genes will be helpful for sex-determining gene identification and sex-specific marker isolation. Lastly, the SSRs can serve as marker resources for future research into genetics, marker-assisted selection(MAS) and conservation biology.

Transcriptome Sequencing and Analysis of Agaricus blazei Mycelia Under Cadmium Stress

To explore the mechanism of Agaricus blazei Murrill enrichment of the heavy metal cadmium,we employed Illumina high-throughput sequencing to analyze the transcriptomes of A.blazei mycelia treated with and without exogenous cadmium addition,and then the differentially expressed genes(DEGs)of the strains with high and low cadmium enrichment between the control and cadmium treatment were screened out.The results showed that the DEGs were mainly involved in steroid biosynthesis,antibiotic biosynthesis,protein processing in endoplasmic reticulum,glutathione metabolism and other pathways.Carbon metabolism and glutathione metabolism may play an important role in the response of A.blazei mycelium to cadmium stress.

Transcriptome sequencing of essential marine brown and red algal species in China and its significance in algal biology and phylogeny

Most phaeophytes （brown algae） and rhodophytes （red algae） dwell exclusively in marine habitats and play important roles in marine ecology and biodiversity. Many of these brown and red algae are also important resources for industries such as food, medicine and materials due to their unique metabolisms and me-tabolites. However, many fundamental questions surrounding their origins, early diversification, taxonomy, and special metabolisms remain unsolved because of poor molecular bases in brown and red algal study. As part of the 1 000 Plant Project, the marine macroalgal transcriptomes of 19 Phaeophyceae species and 21 Rhodophyta species from China＇s coast were sequenced, covering a total of 2 phyla, 3 classes, 11 orders, and 19 families. An average of 2 Gb per sample and a total 87.3 Gb of RNA-seq raw data were generated. Approxi-mately 15 000 to 25 000 unigenes for each brown algal sample and 5 000 to 10 000 unigenes for each red algal sample were annotated and analyzed. The annotation results showed obvious differences in gene expres-sion and genome characteristics between red algae and brown algae;these differences could even be seen between multicellular and unicellular red algae. The results elucidate some fundamental questions about the phylogenetic taxonomy within phaeophytes and rhodophytes, and also reveal many novel metabolic pathways. These pathways include algal CO2 fixation and particular carbohydrate metabolisms, and related gene/gene family characteristics and evolution in brown and red algae. These findings build on known algal genetic information and significantly improve our understanding of algal biology, biodiversity, evolution, and potential utilization of these marine algae.

Comparative analysis on transcriptome sequencings of six Sargassum species in China

Species of Sargassum are distributed worldwide, and are of great ecological and economic importance in marine ecosystems and bioresources. In this study, transcriptome sequencings of six Sargassum species were performed for the first time using an Illumina platform. For each sample, a total of 2.1-2.5 Gb of nucle-otides are collected and assembled into 69 871-116 790 scaffolds, with an average length of 410-550 bp and N50 length of 756-1 462 bp. A total of 20 512-28 684 unigenes of each sample were annotated and compared well with known gene sequences from nr database. Clusters of Orthologous Groups （COG）, gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） analyses were also performed for further un-derstanding of gene functions and regulation pathways. Gene expression levels were calculated based on RPKM values and compared among these species, especially for those genes related to carbohydrate metab-olism. Cluster analyses indicated that the differences of global gene expression between S. fusiforme, which was nominated as Hizikia fusiformis before, and other five species were not significant. Further phylogenet-ic analysis of 108 orthologous genes confirmed that S. fusiforme had closer relationship with S. hemiphyllum rather than S. horneri. These transcriptome data provided valuable information for better understanding of genome and gene characteristics of Sargassum algae and benefiting comparative and phylogenetic studies of Phaeophyceae species in future studies.