This study investigates the genetic diversity and evolutionary relationships of different Artemisia argyi germplasm resources to provide a basis for germplasm identification, variety selection, and resource protection. A total of 192 germplasm resources of A. argyi were studied, and EST-based simple sequence repeat(EST-SSR) primers were designed based on transcriptomic data of A. argyi. Polymerase chain reaction(PCR) amplification was performed on these resources, followed by fluorescence capillary electrophoresis to detect genetic diversity and construct DNA fingerprints. From 197 pairs of primers designed, 28 pairs with polymorphic and clear bands were selected. A total of 278 alleles were detected, with an average of 9.900 0 alleles per primer pair and an average effective number of alleles of 1.407 2. The Shannon's diversity index(I) for the A. argyi germplasm resources ranged from 0.148 1 to 0.418 0, with an average of 0.255 7. The polymorphism information content(PIC) ranged from 0.454 5 to 0.878 0, with an average of 0.766 9, showing high polymorphism. Cluster analysis divided the A. argyi germplasm resources into three major groups: Group Ⅰ contained 136 germplasm samples, Group Ⅱ contained 45, and Group Ⅲ contained 11. Principal component analysis also divided the resources into three groups, which was generally consistent with the clustering results. Mantel test results showed that the genetic variation in A. argyi populations was to some extent influenced by geographic distance, but the effect was minimal. Structure analysis showed that 190 germplasm materials had Q≥ 0.6, indicating that these germplasm materials had a relatively homogeneous genetic origin. Furthermore, 8 core primer pairs were selected from the 28 designed primers, which could distinguish various germplasm types. Using these 8 core primers, DNA fingerprints for the 192 A. argyi germplasm resources were successfully constructed. EST-SSR molecular markers can be used to study the genetic diversity and phylogenetic relationships of A. argyi, providing theoretical support for the identification and molecular-assisted breeding of A. argyi germplasm resources.
Artemisia/classification* ; Microsatellite Repeats ; Genetic Variation ; Expressed Sequence Tags ; DNA Fingerprinting ; Phylogeny ; Polymorphism, Genetic ; DNA, Plant/genetics* ; Genetic Markers

To analysis the SSR loci information in the transcriptome of Cordyceps sinensis and develop SSR molecular markers,MISA(MicroSatellite) software was used to analyze the microsatellites information from 16 875 unigene sequences and SSR primer designed by Primer 3. 0. In total,5 899 SSRs were detected in 4 252 unigene with the distribution frequency of 34. 99%,which was represented by 74 repeat motifs and SSR loci occurred per 7 952 bp in length. In the SSRs,the mono-nucleotide was the most abundant repeat motif(42. 5%),followed by tri-nucleotide(34. 48%),C/G and CCG/CGG were the dominant repeat motifs,respectively. The number of repetitions of the six SSR repeat types was concentrated on 5 to 12 times,and the length was mostly less than 24 bp. A total of 12 282 pairs of primers were screened and selected 20 pairs of primers for validity detection randomly,10 pairs of primers amplified the expected specific bands,and primer P1 has significant polymorphism. Moreover,it was found that unigene containing SSR loci is mainly related to genetic and environmental functions after GO and KEGG annotation. In conclusion,these SSR loci in the transcriptome of O. sinensis are high in frequency,rich in primitive types,high in polymorphism,and highly available,which will provides abundant candidate molecular markers for its genetic diversity analysis,resource identification protection,and gene function research.
Cordyceps/genetics* ; Expressed Sequence Tags ; Microsatellite Repeats ; Polymorphism, Genetic ; Transcriptome

PURPOSE: Cockroach feces are known to be rich in IgE-reactive components. Various protease allergens were identified by proteomic analysis of German cockroach fecal extract in a previous study. In this study, we characterized a novel allergen, a chymotrypsin-like serine protease. METHODS: A cDNA sequence homologous to chymotrypsin was obtained by analysis of German cockroach expressed sequence tag (EST) clones. The recombinant chymotrypsins from the German cockroach and house dust mite (Der f 6) were expressed in Escherichia coli using the pEXP5NT/TOPO vector system, and their allergenicity was investigated by ELISA. RESULTS: The deduced amino acid sequence of German cockroach chymotrypsin showed 32.7 to 43.1% identity with mite group 3 (trypsin) and group 6 (chymotrypsin) allergens. Sera from 8 of 28 German cockroach allergy subjects (28.6%) showed IgE binding to the recombinant protein. IgE binding to the recombinant cockroach chymotrypsin was inhibited by house dust mite chymotrypsin Der f 6, while it minimally inhibited the German cockroach whole body extract. CONCLUSIONS: A novel allergen homologous to chymotrypsin was identified from the German cockroach and was cross-reactive with Der f 6.
Allergens ; Amino Acid Sequence ; Blattellidae* ; Chymotrypsin* ; Clone Cells ; Cockroaches ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Expressed Sequence Tags ; Feces ; Hypersensitivity ; Immunoglobulin E ; Mites ; Pyroglyphidae ; Sequence Homology ; Serine Proteases

The thymus is the central lymphoid organ for the development of bone marrow-derived precursor cells into mature T-cells. Understanding the molecular mechanism of thymic involution and regeneration is critical to develop methods to normalize or improve host immunity from the decreased immune function caused by thymic involution. In this study, the regenerating thymus cDNA library was constructed in the rat from a model of thymic involution and regeneration induced by cyclophosphamide. Expressed sequence tags (ESTs) were obtained by partial sequencing of 700 randomly selected insert-containing clones. A total of 630 ESTs were analyzed, of which 486 ESTs (78%) matched to known genes and 125 ESTs (19%) matched to other ESTs (unknown genes). The 19 ESTs (3%) did not match with any known sequences. The ESTs were grouped into six main functional categories: metabolism (44%), signaling components (20%), membrane transport (7%), cytoskeleton (2%), cell division (2%) and defense (2%). As a result of RT-PCR analysis, expression of putative gene 01, putative E2IG2 gene, musculin and osteoactivin significantly increased in rat thymus during regeneration. The putative gene 01 showed complete homology with mitochondrial ribosomal protein S4 by homology search and multiple alignment of amino acid. These results provide the extensive molecular information on thymus regeneration and will be useful source to identify various genes which may play an important role in the thymus regeneration as well as to clone novel genes. Furthermore, the availability of these data will serve as a basis for further research to understand the molecular mechanism of thymus regeneration.
Animals ; Cell Division ; Clone Cells ; Cyclophosphamide* ; Cytoskeleton ; Expressed Sequence Tags* ; Gene Expression ; Gene Library ; Membranes ; Metabolism ; Rats* ; Regeneration* ; Ribosomal Proteins ; T-Lymphocytes ; Thymus Gland*

Long intergenic non-coding RNAs (lincRNAs) have historically been ignored in cancer biology. However, thousands of lincRNAs have been identified in mammals using recently developed genomic tools, including microarray and high-throughput RNA sequencing (RNA-seq). Several of the lincRNAs identified have been well characterized for their functions in carcinogenesis. Here we performed RNA-seq experiments comparing gastric cancer with normal tissues to find differentially expressed transcripts in intergenic regions. By analyzing our own RNA-seq and public microarray data, we identified 31 transcripts, including a known expressed sequence tag, BM742401. BM742401 was downregulated in cancer, and its downregulation was associated with poor survival in gastric cancer patients. Ectopic overexpression of BM742401 inhibited metastasis-related phenotypes and decreased the concentration of extracellular MMP9. These results suggest that BM742401 is a potential lincRNA marker and therapeutic target.
Animals ; DNA, Intergenic/genetics ; Expressed Sequence Tags/*metabolism ; Extracellular Space/metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Genetic Predisposition to Disease ; Humans ; Male ; Matrix Metalloproteinase 9/metabolism ; Mice ; Mice, Inbred C57BL ; Multivariate Analysis ; Neoplasm Metastasis ; Neoplasm Staging ; Phenotype ; Proportional Hazards Models ; RNA, Long Noncoding/*genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Reproducibility of Results ; Stomach Neoplasms/*genetics/*pathology ; Survival Analysis

OBJECTIVETaxus species are highly valued for the production of taxol. Based on high-throughput sequenceing, EST-SSRs were explored and studied in the transcriptome of Taxus cuspidata.

METHODT cuspidata leaf cDNA was extracted and sequenced by 454 GS FLX Titanium. High-quality sequences were assembled using Newbler Assembler Software, which produced unique sequences. SSRs motif was explored using simple sequence repeat identification tool (Perl Script). Primers were designed using PRIMER3.

RESULTA total of 81 148 high-quality reads from the needles of T. cuspidata were produced using the Roche GS FLX Titanium system. A total of 20 557 unique sequences were obtained. There were 753 simple sequence repeat motifs identified. Primers of PCR were obtained for 519 EST-SSRs, randomly selected cloning sequencing revealed that 87.5% of ESTs were the same as the results of Sanger sequencing.

CONCLUSIONThe results provide the first EST-SSRs collection in Taxus and are essential for future efforts of gene discovery, functional genomics, and genome annotation in related species.

DNA Primers ; genetics ; Expressed Sequence Tags ; Gene Expression Profiling ; methods ; Gene Expression Regulation, Plant ; High-Throughput Nucleotide Sequencing ; methods ; Microsatellite Repeats ; genetics ; Taxus ; genetics ; Transcriptome ; genetics

Recently, the technologies of DNA sequence variation and gene expression profiling have been used widely as approaches in the expertise of genome biology and genetics. The application to genome study has been particularly developed with the introduction of the next-generation DNA sequencer (NGS) Roche/454 and Illumina/Solexa systems, along with bioinformation analysis technologies of whole-genome de novo assembly, expression profiling, DNA variation discovery, and genotyping. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing de novo assembly of novel genome sequencing data. It is necessary to have DNA sequence information from a multiplatform NGS with at least 2x and 30x depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly. Massive short-length reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through de novo assembly in any whole-genome sequenced species. The 20x and 50x coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively, is effective to create novel expressed reference sequences. However, only an average 30x coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference expressed sequence tag sequence.
Base Sequence ; Biology ; Chimera ; DNA ; DNA Fingerprinting ; Expressed Sequence Tags ; Gene Expression Profiling ; Genome ; RNA ; RNA, Messenger ; Sequence Analysis, DNA ; Transcriptome

OBJECTIVETo study the distribution frequency and characteristics of nucleotide repeat of 94 923 ESTs for developing microsatellite primers, providing a theoretical basis and technical support for appropriate conservation and application of sweet wormwood (Artemisia annua).

METHODEST-SSR detection was performed using Perl program MISA. Gene Ontology (GO) annotations were formatted for input into the GOSlim program and the output was parsed to count the occurrence of each GO category. Primer 3 software was used to design 18 pairs primers, amplified products were separated on a 6% denaturing polyacrylamide gel using silver staining.

RESULTBy searching with Active Perl, totally 2 110 SSRs were detected, accounting for 8.6%. The frequency of occurrence of dinucleotide and trinucleotide was 28% and 50.4%, respectively. The most common repeat motifs of trinucleotide were ACC/GGT, accounting for 9.8%. Three hundred and twelve SSR-ESTs were annotated using GO terms. The suitable PCR system of 15 pairs primers was established, and revealed microsatellite polymorphism in 36 individuals.

CONCLUSIONThere are a variety of motifs at EST-SSR locus in sweet wormwood, and more effective amplification and polymorphism in 18 pairs detected primers. Therefore, EST resource is an effective and feasible approach to develop SSR markers, and EST-SSRs will a powerful tool for studies of sweet wormwood genetic resources.

Artemisia annua ; genetics ; Expressed Sequence Tags ; Microsatellite Repeats ; Polymorphism, Genetic

Chinese agarwood is formed in the aromatic resinous wood formed in Aquilaria sinensis (Lour.) Gilg (botanical family: Thymelaeaceae). Only when suffering stress of wound, etc, can A. sinensis produce sesquiterpenes etc. compounds of agarwood around wounds. However, little is known about how wound induced the biosynthesis pathway of sesquiterpenes. To reveal the molecular mechanism of wound-induced agarwood formation, RNA sequencing (RNA-seq) technology was used to investigate the profile of gene expression in A. sinensis treated by mechanical wounding and elucidate its functional gene. A total of 40,295 ESTs with an average read length of 305 bp were generated and 22 095 unigenes were formed by initial gene splicing. 61.6% of these unigenes (13 611) were annotated using BLAST searches against the SwissProt, KEGG, Nr and Nt databases. Twenty-six unigenes (encoding 7 enzymes) were found to be involved in sesquiterpene of agarwood biosynthesis by bioinformatic tools of Gene Ontology and KEGG. Novel genes that are potentially involved in sesquiterpenes biosynthesis were identified in A. sinensis, providing data for further sesquiterpenes biosynthesis pathway by molecular methods and the EST data establish a foundation for future studies in the molecular mechanisms of wound-induce agarwood formation in A. sinensis.
Drugs, Chinese Herbal ; chemistry ; metabolism ; Expressed Sequence Tags ; Genes, Plant ; genetics ; Plants, Medicinal ; chemistry ; genetics ; Sequence Analysis, RNA ; Sesquiterpenes ; chemistry ; metabolism ; Stress, Physiological ; genetics ; Thymelaeaceae ; chemistry ; genetics ; Transcriptome ; genetics ; Wood ; genetics ; metabolism

Squalene synthase (SQS) is a key enzyme in plant terpenoid biosynthetic pathway. This study focused on cloning and analysis of Huperzia serrata SQS (HsSQS1) gene. After searching the transcriptome dataset of H serrata, one unique sequence encoding SQS was discovered. The primers were designed according to the transcript sequence of HsSQS1 from the H. serrata transcriptome dataset. The open reading frame of HsSQS1 was cloned using RT-PCR strategy. The bioinformatic analysis of this gene and its corresponding protein were performed. The cDNA (named as HsSQS1) contains a 1263 bp open reading frame and encodes a predicted protein of 420 amino acids. The GenBank accession number for this gene is JQ004938. HsSQS1 contains two transmembrane regions, without signal peptide. The conserved domain of squalene synthase was presented in HsSQS1. HsSQS1 was more abundant in H. serrata root than in leaf and stem. This study cloned and analyzed squalene synthase gene from H. serrata for the first time. The result will provide a foundation for exploring the mechanism ofterpenoid biosynthesis in H. serrata plants.
Amino Acid Sequence ; Biosynthetic Pathways ; Cloning, Molecular ; DNA, Complementary ; genetics ; Expressed Sequence Tags ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; isolation & purification ; metabolism ; Genes, Plant ; genetics ; Huperzia ; enzymology ; genetics ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; Plant Leaves ; enzymology ; Plant Roots ; enzymology ; Plant Stems ; enzymology ; Plants, Medicinal ; enzymology ; genetics ; Triterpenes ; chemistry