Tentative Consensus Sequences Research Articles

An expressed sequence tag analysis of the intertidal brown seaweeds Fucus serratus (L.) and F. vesiculosus (L.) (Heterokontophyta, Phaeophyceae) in response to abiotic stressors.

In order to aid gene discovery and uncover genes responding to abiotic stressors in stress-tolerant brown algae of the genus Fucus, expressed sequence tags (ESTs) were studied in two species, Fucus serratus and Fucus vesiculosus. Clustering of over 12,000 ESTs from three libraries for heat shock/recovery and desiccation/rehydration resulted in identification of 2,503, 1,290, and 2,409 unigenes from heat-shocked F. serratus, desiccated F. serratus, and desiccated F. vesiculosus, respectively. Low overall annotation rates (18-31%) were strongly associated with the presence of long 3' untranslated regions in Fucus transcripts, as shown by analyses of predicted protein-coding sequence in annotated and nonannotated tentative consensus sequences. Posttranslational modification genes were overrepresented in the heat shock/recovery library, including many chaperones, the most abundant of which were a family of small heat shock protein transcripts, Hsp90 and Hsp70 members. Transcripts of LI818-like light-harvesting genes implicated in photoprotection were also expressed during heat shock in high light. The expression of several heat-shock-responsive genes was confirmed by quantitative reverse transcription polymerase chain reaction. However, candidate genes were notably absent from both desiccation/rehydration libraries, while the responses of the two species to desiccation were divergent, perhaps reflecting the species-specific physiological differences in stress tolerance previously established. Desiccation-tolerant F. vesiculosus overexpressed at least 17 ribosomal protein genes and two ubiquitin-ribosomal protein fusion genes, suggesting that ribosome function and/or biogenesis are important during cycles of rapid desiccation and rehydration in the intertidal zone and possibly indicate parallels with other poikilohydric organisms such as desiccation-tolerant bryophytes.

Analysis of Expressed Sequence Tags in Porcine Uterus Tissue

Two non-normalized cDNA libraries of uteri from Danish Landrace and Chinese Erhualian pigs were constructed, and 13,756 expressed sequence tags (ESTs) were randomly sequenced. The ESTs were clustered by Phrap software, and 6,139 distinct tentative consensus sequences were produced, including 2,730 contigs and 3,409 singlets. Using Blast tools, these 6,139 candidate genes were compared to the nr and nt databases; 5,210 of them were assigned putative functions, whereas 929 potentially represent new genes. Highly expressed genes appear to be associated with basic energy metabolism, transferase activity, localization, cellular physiological process, protein binding, and nucleic acid binding. Antileukoproteinase was the most highly expressed gene, corresponding to endometrial differentiation and conceptus or fetal development.

Analysis of Gene Expression Profiles in Leaf Tissues of Cultivated Peanuts and Development of EST-SSR Markers and Gene Discovery.

Peanut is vulnerable to a range of foliar diseases such as spotted wilt caused by Tomato spotted wilt virus (TSWV), early (Cercospora arachidicola) and late (Cercosporidium personatum) leaf spots, southern stem rot (Sclerotium rolfsii), and sclerotinia blight (Sclerotinia minor). In this study, we report the generation of 17,376 peanut expressed sequence tags (ESTs) from leaf tissues of a peanut cultivar (Tifrunner, resistant to TSWV and leaf spots) and a breeding line (GT-C20, susceptible to TSWV and leaf spots). After trimming vector and discarding low quality sequences, a total of 14,432 high-quality ESTs were selected for further analysis and deposition to GenBank. Sequence clustering resulted in 6,888 unique ESTs composed of 1,703 tentative consensus (TCs) sequences and 5185 singletons. A large number of ESTs (5717) representing genes of unknown functions were also identified. Among the unique sequences, there were 856 EST-SSRs identified. A total of 290 new EST-based SSR markers were developed and examined for amplification and polymorphism in cultivated peanut and wild species. Resequencing information of selected amplified alleles revealed that allelic diversity could be attributed mainly to differences in repeat type and length in the SSR regions. In addition, a few additional INDEL mutations and substitutions were observed in the regions flanking the microsatellite regions. In addition, some defense-related transcripts were also identified, such as putative oxalate oxidase (EU024476) and NBS-LRR domains. EST data in this study have provided a new source of information for gene discovery and development of SSR markers in cultivated peanut. A total of 16931 ESTs have been deposited to the NCBI GenBank database with accession numbers ES751523 to ES768453.

Identification of differentially expressed genes in a porcine in vivo model of adipogenesis using suppression subtractive hybridization

Although they provide valuable information, in vitro models of adipocyte development often require high doses of hormones and growth factors, which may influence gene expression and adipocyte differentiation patterns. To overcome these problems, a novel in vivo model of adipose tissue development was used to characterize genes involved in adipogenesis. The suppression subtractive hybridization technique was used to identify genes showing differential expression between the adipose tissue of a day 90 gestating sow, which is enriched in adipocytes, and day 90 fetal adipose tissue, which is enriched in preadipocytes. A total of 149 expressed sequence tags corresponding to identified genes and tentative consensus sequences emerged. Thirty-seven clones matched expressed sequence tags or genomic DNA sequences and six novel sequences were also identified. Adipogenesis-related genes were identified, many of which have never been reported to be expressed in mammalian adipose tissue, and may play a role in regulation of adipose tissue differentiation. Validation of differentially expressed genes was confirmed for perilipin, monocyte to macrophage differentiation-associated, myocilin, paraoxonase 3, stearoyl-CoA desaturase, angiotensinogen and adiponectin genes using real-time RT-PCR.

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening.

BackgroundKiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs).ResultsThe ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified.ConclusionThis large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Expressed cDNAs from Embryonic and Larval Stages of the Horn Fly (Diptera: Muscidae)

We used an expressed sequence tag approach to initiate a study of the genome of the horn fly, Hematobia irritans (L.) (Diptera: Muscidae). Two normalized cDNA libraries were synthesized from RNA isolated from embryos and first instars from a field population of horn flies. Approximately 10,000 clones were sequenced from both the 5' and 3' directions. Sequence data from each library was assembled into a database of tentative consensus sequences (TCs) and singletons and used to search public protein databases and annotate the sequences. Additionally, the sequences from both the egg and larval libraries were combined into a single database consisting of 16,702 expressed sequence tags (ESTs) assembling into 2886 TCs and 1,522 singleton entries. Several sequences were identified that may have roles in the horn fly's resistance to insecticides. The availability of this database will facilitate the design of microarray and other experiments to study horn fly gene expression on a larger scale than previously possible. This would include studies designed to investigate metabolic-based insecticide resistance, identify novel antigens for vaccine-based control approaches, and discover new proteins to serve as targets for new pesticide development.

Development of a 37 k high‐density oligonucleotide microarray: a new tool for functional genome research in rainbow trout

A rainbow trout high‐density oligonucleotide microarray was constructed using all tentative consensus (TC) sequences that are publicly available from all international rainbow trout Oncorhynchus mykiss genomic research projects through the Rainbow Trout Gene Index database. The new array contains 60‐mer oligonucleotide probes representing 37 394 unique TC sequences and 1417 control spots. The array (4 × 44 format) was manufactured according to the design by Agilent Technologies using the inkjet‐based SurePrint technology (design number 016320). The performance of the new microarray platform was evaluated by analysing gene expression associated with rainbow trout, vitellogenesis‐induced muscle atrophy. This microarray will open new avenues of research that will aid in the development of novel strategies for genetic improvement for economically important traits benefiting the salmonid aquaculture industries.

SNP discovery, linkage analysis and microsynteny in tentative consensus sequences derived from roots cDNA in a supernodulating soybean mutant

A soybean transcript map with genes mainly expressed in roots of a supernodulating mutant was constructed by mapping of single nucleotide polymorphism (SNP) markers into pre-existing SSR-based map, using a recombinant inbred line (RIL) population from Pureunkong and Jinpumkong 2. For SNP discovery, primer pairs were designed from 793 tentative consensus sequences (TCs) derived from the roots of a supernodulating soybean mutant. Among 520 loci showing high-quality sequence, more than 300 SNPs were identified in 116 loci between the parental genotypes. Average SNP frequency between the parental genotypes was one SNP per 1,010 bp. A total of 325 markers, including 98 SNPs, 208 SSRs, 15 AFLPs and four classical markers, were used to construct a genetic linkage map with 20 linkage groups (LGs) and a total length of 2,379 cM. Ten nonsynonymous changes were detected out of these SNP markers, indicating the possible usage of these markers as functional markers. With 40 of the SNP markers, putative synteny between soybean and Medicago truncatula (Mt) was revealed by identification of 19 short colinear blocks between the two genomes. This genetic map will provide the basis for the identification of gene in relation to nodulation and nitrogen fixation in partially sequenced genome like soybean by comparisons of the model genome (Mt). And, the integration of SNP markers and expression data with available genetic and phenotypic information would be helpful for clarifying function of cloned genes.

Expression Pattern Profiling of Testis Specific Tentative Consensus Sequences in Chicken.

Expression Pattern Profiling of Testis Specific Tentative Consensus Sequences in Chicken.

Testis-Specific Novel Transcripts in Chicken: In Situ Localization and Expression Pattern Profiling During Sexual Development1

Tissue-specific novel transcripts expressed during sexual development were examined by RT-PCR, quantitative RT-PCR (qRT-PCR), and in situ hybridization to provide data for chicken genomics. Public databases for transcript data have been constructed with known and unknown sequences of various tissues from different animals. However, the expression patterns and functions of the transcripts are less known. From the The Institute for Genomics Research Gallus gallus library, we examined 291 tentative consensus (TC) sequences that assembled 100% with transcripts by RT-PCR during male and female sexual development from Embryonic Day 6 to 25 wk of age. We found 85 TC sequences that were specific to testicular development; of these, 43 TC sequences were exclusively upregulated in 25-wk-old testis. Another 52 TC sequences were not specific to one tissue, but occurred in the testis and ovary at different developmental ages. Twelve testis-specific TC sequences upregulated in 25-wk-old testis were randomly selected and further examined with qRT-PCR. For precise localization, these 12 testis-specific TC sequences were examined by in situ hybridization with 25-wk-old adult testis. Six TC sequences were strongly expressed in secondary spermatocytes and haploid spermatids until spermatozoa release. Another six TC sequences were differentially expressed in the adluminal compartment of seminiferous tubules. Among the testis-specific TC sequences, TC120901 is a known gene, phospholipase C, zeta (PLCZ1). Our data provide potential insight into gene expression and genomic information on novel transcripts that are important to avian reproduction.

Host Genes Involved in Nodulation Preference in Common Bean (Phaseolus vulgaris)–Rhizobium etli Symbiosis Revealed by Suppressive Subtractive Hybridization

Common bean cultivars are nodulated preferentially by Rhizobium etli lineages from the same center of host diversification. Nodulation was found to be earlier and numerous in bean plants inoculated with the cognate strain. We predicted that analysis of transcripts at early stages of the interaction between host and rhizobium would identify plant genes that are most likely to be involved in this preferential nodulation. Therefore, we applied a suppressive subtractive hybridization approach in which cDNA from a Mesoamerican cultivar inoculated with either the more- or less-efficient strain of R. etli was used as the driver and the tester, respectively. Forty-one independent tentative consensus sequences (TCs) were obtained and classified into different functional categories. Of 11 selected TCs, 9 were confirmed by quantitative reverse-transcriptase polymerase chain reaction. Two genes show high homology to previously characterized plant receptors. Two other upregulated genes encode for Rab11, a member of the small GTP-binding protein family, and HAP5, a subunit of the heterotrimeric CCAAT-transcription factor. Interestingly, one of the TCs encodes for an isoflavone reductase, which may lead to earlier Nod factor production by specific strains of rhizobia. The transcript abundance of selected cDNAs also was found to be higher in mature nodules of the more efficient interaction. Small or no differences were observed when an Andean bean cultivar was inoculated with a cognate strain, suggesting involvement of these genes in the strain-specific response. The potential role of these genes in the early preferential symbiotic interaction is discussed.

MELOGEN: an EST database for melon functional genomics

BackgroundMelon (Cucumis melo L.) is one of the most important fleshy fruits for fresh consumption. Despite this, few genomic resources exist for this species. To facilitate the discovery of genes involved in essential traits, such as fruit development, fruit maturation and disease resistance, and to speed up the process of breeding new and better adapted melon varieties, we have produced a large collection of expressed sequence tags (ESTs) from eight normalized cDNA libraries from different tissues in different physiological conditions.ResultsWe determined over 30,000 ESTs that were clustered into 16,637 non-redundant sequences or unigenes, comprising 6,023 tentative consensus sequences (contigs) and 10,614 unclustered sequences (singletons). Many potential molecular markers were identified in the melon dataset: 1,052 potential simple sequence repeats (SSRs) and 356 single nucleotide polymorphisms (SNPs) were found. Sixty-nine percent of the melon unigenes showed a significant similarity with proteins in databases. Functional classification of the unigenes was carried out following the Gene Ontology scheme. In total, 9,402 unigenes were mapped to one or more ontology. Remarkably, the distributions of melon and Arabidopsis unigenes followed similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome. Bioinformatic analyses primarily focused on potential precursors of melon micro RNAs (miRNAs) in the melon dataset, but many other genes potentially controlling disease resistance and fruit quality traits were also identified. Patterns of transcript accumulation were characterised by Real-Time-qPCR for 20 of these genes.ConclusionThe collection of ESTs characterised here represents a substantial increase on the genetic information available for melon. A database (MELOGEN) which contains all EST sequences, contig images and several tools for analysis and data mining has been created. This set of sequences constitutes also the basis for an oligo-based microarray for melon that is being used in experiments to further analyse the melon transcriptome.

Molecular Cloning and Expression of a cDNA Encoding a Coccidioides posadasii Cu,Zn Superoxide Dismutase Identified by Proteomic Analysis of the Coccidioidal T27K Vaccine

Previous studies have demonstrated that the coccidioidal T27K vaccine preparation is protective in mice against respiratory challenge using Coccidioides posadasii (C. posadasii) arthroconidia. Proteomic methods have been employed to define the molecular components within the vaccine. This method has led to the identification of novel and previously uncharacterized coccidioidal proteins including a Cu,Zn superoxide dismutase. A two-dimensional gel of the T27K vaccine was run and spots were excised for mass spectrometric analysis. One peptide was obtained from the T27K gel that matched a TIGR C. posadasii 2.0 gene index tentative consensus sequence, TC1072, which is similar to fungal Cu,Zn superoxide dismutase. Activity assays performed with native PAGE gels of the T27K vaccine showed that the vaccine contains superoxide dismutase. The cDNA encoding the enzyme has been cloned and sequenced and expressed as a recombinant protein.

Molecular Cloning and Expression of a cDNA Encoding a Coccidioides posadasii 1,2‐α‐Mannosidase Identified in the Coccidioidal T27K Vaccine by Immunoproteomic Methods

The coccidioidal T27K vaccine is protective in mice against respiratory challenge with Coccidioides posadasii (C. posadasii) arthroconidia. The vaccine is a subcellular multicomponent preparation that has not been fully characterized. To identify potential protective antigens in the heterogeneous mixture, the vaccine has been separated by two-dimensional gel electrophoresis and then analyzed for seroreactive proteins using immunoblot analysis with pooled sera from patients with coccidioidomycosis. Two seroreactive spots of identical apparent molecular weight were identified and sequenced using tandem mass spectrometry. Three peptides were generated, two of which matched a tentative consensus sequence in the TIGR C. posadasii 2.0 gene index database that is similar to fungal 1,2-alpha-mannosidases. The 5' and 3' ends of the mannosidase cDNA were mapped using rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR), and a full-length cDNA was then obtained using reverse-transcription (RT) PCR. The cDNA was cloned and sequenced and expressed as a recombinant protein. The predicted protein consists of 519 amino acids, has a theoretical molecular weight and pI of 56,918 Da and 4.84, respectively, and is very similar (>60%) to other fungal 1,2-alpha-mannosidases. Class I 1,2-alpha-mannosidase enzyme activity was also detected in the T27K vaccine using the substrate, Man-alpha-1,2-Man-alpha-OCH(3) in a spectrophotometric assay.

닭에서 생식세포 발달에 관여하는 유전자 검색

본 연구는 한우에서 이유시 체중과 도체형질들 간의 상관관계를 구명하고자 실시하였다. 조사된 도체 형질은 배최장근 단면적, 등지방두께, 21단계로 구분하여 평가한 근내지방도(근내지방도 I)와 7단계로 구분하여 평가한 근내지방도(근내지방도 II), 그리고 7단계로 구분하여 평가한 육색 등이었다. 유전모수의 추정은 DFREML 방법을 적용하여 실시하였는데 이유시 체중에 대한 통계모형은 동기우 그룹효과(년도－계절－성) 외에 이유시 송아지 일령 및 어미소 일령의 1차식 효과와 2차식 효과를 고정효과로 포함하였고 개체효과를 임의효과로 포함하였다. 도체형질에 대한 통계 모형은 동기우 그룹효과(년도－계절－성) 외에 도축시 일령의 일차식 효과를 고정효과에 포함하였고 개체효과를 임의효과로 포함하였다. 조사된 형질별 유전력 추정치는 이유시 체중이 0.25, 배최장근 단면적이 0.20, 등지방두께가 0.20, 근내지방도Ⅰ이 0.32, 근내지방도 Ⅱ가 0.32 그리고 육색이 0.22였다. 이유시 체중과 배최장근 단면적, 등지방두께, 근내지방도 I, 근내지방도 II 및 육색 간의 유전(표현형) 상관계수는 각각 0.75(0.16), 0.18(0.05), －0.41(－0.09), －0.40(0.11) 및 －0.07(0.05)였다. 본 연구 결과는 이유시 체중이 무거운 방향으로 단형질 선발을 진행할 경우 등심단면적이 넓어지고, 등지방두께가 두꺼워지며 근내지방도가 감소하는 도체를 생산하는 후손집단이 형성될 가능성이 있음을 시사하고 있다. We examined the expression patterns of the chicken TCs(tentative consensus sequences) originated from GermOnline genes in various chicken tissues, applying information from GermOnline to chicken organisms. 42 TCs among 84 chicken homologous TCs from the pool of 84 genes related to germ cell lineage in mouse(10), rat(71) and human(3) had high homology based on a BLAST search. Of these, Hmgcs2 and Sycp3 was shown to be expressed in a testis- specific manner and a reproductive organ(testis and ovary)-specific manner, respectively, by RT- PCR analysis. Crmp4, Cyct, Ldhc, Epha7, Pcsk4 and Dnmt3a are expressed in brain, testis, and ovary. The characterization of chicken genes originated from GermOnline in this research may give an enormously useful source of information related to germ cell development.

Genome-wide Detection and Analysis of Alternative Splicing for Nucleotide Binding Site-Leucine-Rich Repeats Sequences in Rice

Alternative splicing is a major contributor to genomic complexity and proteome diversity, yet the analysis of alternative splicing for the sequence containing nucleotide binding site and leucine-rich repeats (NBS-LRR) domain has not been explored in rice ( Oryza sativa L.). Hidden Markov model (HMM) searches were performed for NBS-LRR domain. 875 NBS-LRR-encoding sequences were obtained from the Institute for Genomic Research (TIGR). All of them were used to blast Knowledge-based Oryza Molecular Biological Encyclopaedia (KOME), TIGR rice gene index (TGI), and Universal Protein Resource (UniProt) to obtain homologous full-length cDNAs (FL-cDNAs), tentative consensus sequences, and protein sequences. Alternative splicing events were detected from genomic alignment of FL-cDNAs, tentative consensus sequences, and protein sequences, which provide valuable information on splice variants of genes. These sequences were aligned to the corresponding BAC sequences using the Spidey and Sim4 programs and each of the proteins was aligned by tBLASTn. Of the 875 NBS-LRR sequences, 119 (13.6%) sequences had alternative splicing where multiple FL-cDNAs, TGI sequences and proteins corresponded to the same gene. 71 intron retention events, 20 exon skipping events, 16 alternative termination events, 25 alternative initiation events, 12 alternative 5′ splicing events, and 16 alternative 3′ splicing events were identified. Most of these alternative splices were supported by two or more transcripts. The data sets are available at http://www.bioinfor.org Furthermore, the bioinformatics analysis of splice boundaries showed that exon skipping and intron retention did not exhibit strong consensus. This implies a different regulation mechanism that guides the expression of splice isoforms. This article also presents the analysis of the effects of intron retention on proteins. The C-terminal regions of alternative proteins turned out to be more variable than the N-terminal regions. Finally, tissue distribution and protein localization of alternative splicing were explored. The largest categories of tissue distributions for alternative splicing were shoot and callus. More than one-thirds of protein localization for splice forms was plasma membrane and cytoplasm. All the NBS-LRR proteins for splice forms may have important function in disease resistance and activate downstream signaling pathways.

Gene models from ESTs (GeneModelEST): an application on the Solanum lycopersicum genome

BackgroundThe structure annotation of a genome is based either on ab initio methodologies or on similaritiy searches versus molecules that have been already annotated. Ab initio gene predictions in a genome are based on a priori knowledge of species-specific features of genes. The training of ab initio gene finders is based on the definition of a data-set of gene models. To accomplish this task the common approach is to align species-specific full length cDNA and EST sequences along the genomic sequences in order to define exon/intron structure of mRNA coding genes.ResultsGeneModelEST is the software here proposed for defining a data-set of candidate gene models using exclusively evidence derived from cDNA/EST sequences.GeneModelEST requires the genome coordinates of the spliced-alignments of ESTs and of contigs (tentative consensus sequences) generated by an EST clustering/assembling procedure to be formatted in a General Feature Format (GFF) standard file. Moreover, the alignments of the contigs versus a protein database are required as an NCBI BLAST formatted report file.The GeneModelEST analysis aims to i) evaluate each exon as defined from contig spliced alignments onto the genome sequence; ii) classify the contigs according to quality levels in order to select candidate gene models; iii) assign to the candidate gene models preliminary functional annotations.We discuss the application of the proposed methodology to build a data-set of gene models of Solanum lycopersicum, whose genome sequencing is an ongoing effort by the International Tomato Genome Sequencing Consortium.ConclusionThe contig classification procedure used by GeneModelEST supports the detection of candidate gene models, the identification of potential alternative transcripts and it is useful to filter out ambiguous information. An automated procedure, such as the one proposed here, is fundamental to support large scale analysis in order to provide species-specific gene models, that could be useful as a training data-set for ab initio gene finders and/or as a reference gene list for a human curated annotation.

Inferring angiosperm phylogeny from EST data with widespread gene duplication

BackgroundMost studies inferring species phylogenies use sequences from single copy genes or sets of orthologs culled from gene families. For taxa such as plants, with very high levels of gene duplication in their nuclear genomes, this has limited the exploitation of nuclear sequences for phylogenetic studies, such as those available in large EST libraries. One rarely used method of inference, gene tree parsimony, can infer species trees from gene families undergoing duplication and loss, but its performance has not been evaluated at a phylogenomic scale for EST data in plants.ResultsA gene tree parsimony analysis based on EST data was undertaken for six angiosperm model species and Pinus, an outgroup. Although a large fraction of the tentative consensus sequences obtained from the TIGR database of ESTs was assembled into homologous clusters too small to be phylogenetically informative, some 557 clusters contained promising levels of information. Based on maximum likelihood estimates of the gene trees obtained from these clusters, gene tree parsimony correctly inferred the accepted species tree with strong statistical support. A slight variant of this species tree was obtained when maximum parsimony was used to infer the individual gene trees instead.ConclusionDespite the complexity of the EST data and the relatively small fraction eventually used in inferring a species tree, the gene tree parsimony method performed well in the face of very high apparent rates of duplication.

Large-scale identification of transcripts expressed in a symbiotic fungus (Termitomyces) during plant biomass degradation

Fungus-growing termites have a symbiotic relationship with the basidiomycetes of the genus Termitomyces. This symbiotic system is able to degrade dead plant material efficiently. We conducted expressed sequence tag (EST) analysis of a symbiotic Termitomyces fungus degrading plant material in a field nest of the termite Macrotermes gilvus. A subtractive cDNA library was also investigated to facilitate the discovery of genes expressed specifically under the symbiotic conditions. A total of 2,613 ESTs were collected and resulted in 1,582 nonredundant tentative consensus sequences, of which approximately 59% showed significant similarity to known protein sequences. A number of homologous sequences to genes involved in plant cell wall degradation were identified and a majority of them encoded putative pectinolytic enzymes. Real-time quantitative reverse transcriptase polymerase chain reaction analyses confirmed significant upregulation of putative stress response genes under symbiotic conditions. The present ESTs database provides a valuable resource for molecular biological study of plant material degradation in the symbiosis between termites and fungi.

Accumulation of genome‐specific transcripts, transcription factors and phytohormonal regulators during early stages of fiber cell development in allotetraploid cotton

Gene expression during the early stages of fiber cell development and in allopolyploid crops is poorly understood. Here we report computational and expression analyses of 32 789 high-quality ESTs derived from Gossypium hirsutum L. Texas Marker-1 (TM-1) immature ovules (GH_TMO). The ESTs were assembled into 8540 unique sequences including 4036 tentative consensus sequences (TCs) and 4504 singletons, representing approximately 15% of the unique sequences in the cotton EST collection. Compared with approximately 178 000 existing ESTs derived from elongating fibers and non-fiber tissues, GH_TMO ESTs showed a significant increase in the percentage of genes encoding putative transcription factors such as MYB and WRKY and genes encoding predicted proteins involved in auxin, brassinosteroid (BR), gibberellic acid (GA), abscisic acid (ABA) and ethylene signaling pathways. Cotton homologs related to MIXTA, MYB5, GL2 and eight genes in the auxin, BR, GA and ethylene pathways were induced during fiber cell initiation but repressed in the naked seed mutant (N1N1) that is impaired in fiber formation. The data agree with the known roles of MYB and WRKY transcription factors in Arabidopsis leaf trichome development and the well-documented phytohormonal effects on fiber cell development in immature cotton ovules cultured in vitro. Moreover, the phytohormonal pathway-related genes were induced prior to the activation of MYB-like genes, suggesting an important role of phytohormones in cell fate determination. Significantly, AA sub-genome ESTs of all functional classifications including cell-cycle control and transcription factor activity were selectively enriched in G. hirsutum L., an allotetraploid derived from polyploidization between AA and DD genome species, a result consistent with the production of long lint fibers in AA genome species. These results suggest general roles for genome-specific, phytohormonal and transcriptional gene regulation during the early stages of fiber cell development in cotton allopolyploids.