Tentative Consensus Sequences Research Articles

Toward Coalescing Gene Expression and Function with QTLs of Water-Deficit Stress in Cotton.

Cotton exhibits moderately high vegetative tolerance to water-deficit stress but lint production is restricted by the available rainfed and irrigation capacity. We have described the impact of water-deficit stress on the genetic and metabolic control of fiber quality and production. Here we examine the association of tentative consensus sequences (TCs) derived from various cotton tissues under irrigated and water-limited conditions with stress-responsive QTLs. Three thousand sixteen mapped sequence-tagged-sites were used as anchored targets to examine sequence homology with 15,784 TCs to test the hypothesis that putative stress-responsive genes will map within QTLs associated with stress-related phenotypic variation more frequently than with other genomic regions not associated with these QTLs. Approximately 1,906 of 15,784 TCs were mapped to the consensus map. About 35% of the annotated TCs that mapped within QTL regions were genes involved in an abiotic stress response. By comparison, only 14.5% of the annotated TCs mapped outside these QTLs were classified as abiotic stress genes. A simple binomial probability calculation of this degree of bias being observed if QTL and non-QTL regions are equally likely to contain stress genes was P (x ≥ 85) = 7.99 × 10−15. These results suggest that the QTL regions have a higher propensity to contain stress genes.

Identification, molecular characterization, and expression analysis of a DOMON-like type 9 carbohydrate-binding module domain-containing protein of Coccidioides posadasii.

Previously, we investigated the effect of N-acetylglucosamine (GlcNAc) on Coccidioides posadasii chitinolytic enzymes during in vitro spherule-endospore (S/E) phase culture. During those studies, sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of supernatants from S/E phase cultures grown in Converse medium with or without added GlcNAc revealed a ∼ 28-kDa band (CFP28), whose abundance was increased by GlcNAc in parallel with the chitinolytic enzymes. Mass spectrometry (MS) of the CFP28 band revealed peptides that matched an open reading frame found in the tentative consensus sequence, TC20325, retrieved from the Dana Farber Cancer Institute C. posadasii Gene Index Database. The TC20325 cDNA sequence was used to design internal primers based on MS peptides and a full-length cDNA was isolated using a combination of rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. The deduced amino acid sequence of the full-length cDNA consists of 231 amino acid residues with a 19 aa signal peptide. The mature protein has a calculated molecular mass of ∼ 24.5 kDa, a theoretical pI of 6.09, and consists of a single DOMON-like type 9 carbohydrate-binding module (CBM9-like-3) conserved domain. The protein shares the highest sequence similarity (≥57%) to hypothetical proteins from fungi within the Pezizomycotina subphylum of Ascomycota. Antiserum against a recombinant version of CFP28 recognized native CFP28 in S/E phase cells and culture supernatants. CFP28 mRNA and protein expression were detectable in S/E phase in Converse medium, but were increased in the presence of added GlcNAc. Purified native CFP28 reacted with pooled sera from patients with coccidioidomycosis.

Analysis of the Alfalfa Root Transcriptome in Response to Salinity Stress

Salinity is one of the major abiotic factors affecting alfalfa productivity. Identifying genes that control this complex trait will provide critical insights for alfalfa breeding programs. To date, no studies have been published on a deep sequencing-based profiling of the alfalfa transcriptome in response to salinity stress. Observations gathered through research on reference genomes may not always be applicable to alfalfa. In this work, Illumina RNA-sequencing was performed in two alfalfa genotypes contrasting in salt tolerance, in order to estimate a broad spectrum of genes affected by salt stress. A total of 367,619,586 short reads were generated from cDNA libraries originated from roots of both lines. More than 60,000 tentative consensus sequences (TCs) were obtained and, among them, 74.5% had a significant similarity to proteins in the NCBI database. Mining of simple sequence repeats (SSRs) from all TCs revealed 6,496 SSRs belonging to 3,183 annotated unigenes. Bioinformatics analysis showed that the expression of 1,165 genes, including 86 transcription factors (TFs), was significantly altered under salt stress. About 40% of differentially expressed genes were assigned to known gene ontology (GO) categories using Arabidopsis GO. A random check of differentially expressed genes by quantitative real-time PCR confirmed the bioinformatic analysis of the RNA-seq data. A number of salt-responsive genes in both tested genotypes were identified and assigned to functional classes, and gene candidates with roles in the adaptation to salinity were proposed. Alfalfa-specific data on salt-responsive genes obtained in this work will be useful in understanding the molecular mechanisms of salinity tolerance in alfalfa.

Analysis of the barley leaf transcriptome under salinity stress using mRNA-Seq

Salinity is a threat to crops in many parts of the world, and together with drought, it is predicted to be a serious constraint to food security. However, understanding the impact of this stressor on plants is a major challenge due to the involvement of numerous genes and regulatory pathways. While transcriptomic analyses of barley (Hordeum vulgare L.) under salt stress have been reported with microarrays, there are no reports as yet of the use of mRNA-Seq. We demonstrate the utility of mRNA-Seq by analysing cDNA libraries derived from acutely salt-stressed and unstressed leaf material of H. vulgare cv. Hindmarsh. The data yielded >50 million sequence tags which aligned to 26,944 sequences in the Unigene reference database. To gain maximum information, we performed de novo assembly of unaligned reads and discovered >3,800 contigs, termed novel tentative consensus sequences, which are either new, or significant improvements on current databases. Differential gene expression screening found 48 significantly up-regulated and 62 significantly down-regulated transcripts. The work provides comprehensive insights into genome-wide effects of salinity and is a new resource for the study of gene regulation in barley and wheat. Further, the bioinformatics workflow may be applicable to other non-model plants to establish their transcriptomes and identify unique sequences.

SymGRASS: a database of sugarcane orthologous genes involved in arbuscular mycorrhiza and root nodule symbiosis

BackgroundThe rationale for gathering information from plants procuring nitrogen through symbiotic interactions controlled by a common genetic program for a sustainable biofuel production is the high energy demanding application of synthetic nitrogen fertilizers. We curated sequence information publicly available for the biofuel plant sugarcane, performed an analysis of the common SYM pathway known to control symbiosis in other plants, and provide results, sequences and literature links as an online database.MethodsSugarcane sequences and informations were downloaded from the nucEST database, cleaned and trimmed with seqclean, assembled with TGICL plus translating mapping method, and annotated. The annotation is based on BLAST searches against a local formatted plant Uniprot90 generated with CD-HIT for functional assignment, rpsBLAST to CDD database for conserved domain analysis, and BLAST search to sorghum's for Gene Ontology (GO) assignment. Gene expression was normalized according the Unigene standard, presented as ESTs/100 kb. Protein sequences known in the SYM pathway were used as queries to search the SymGRASS sequence database. Additionally, antimicrobial peptides described in the PhytAMP database served as queries to retrieve and generate expression profiles of these defense genes in the libraries compared to the libraries obtained under symbiotic interactions.ResultsWe describe the SymGRASS, a database of sugarcane orthologous genes involved in arbuscular mycorrhiza (AM) and root nodule (RN) symbiosis. The database aggregates knowledge about sequences, tissues, organ, developmental stages and experimental conditions, and provides annotation and level of gene expression for sugarcane transcripts and SYM orthologous genes in sugarcane through a web interface. Several candidate genes were found for all nodes in the pathway, and interestingly a set of symbiosis specific genes was found.ConclusionsThe knowledge integrated in SymGRASS may guide studies on molecular, cellular and physiological mechanisms by which sugarcane controls the establishment and efficiency of endophytic associations. We believe that the candidate sequences for the SYM pathway together with the pool of exclusively expressed tentative consensus (TC) sequences are crucial for the design of molecular studies to unravel the mechanisms controlling the establishment of symbioses in sugarcane, ultimately serving as a basis for the improvement of grass crops.

Expressed sequence tags in cultivated peanut (Arachis hypogaea): discovery of genes in seed development and response to Ralstonia solanacearum challenge

Although an important oil crop, peanut has only 162,030 expressed sequence tags (ESTs) publicly available, 86,943 of which are from cultivated plants. More ESTs from cultivated peanuts are needed for isolation of stress-resistant, tissue-specific and developmentally important genes. Here, we generated 63,234 ESTs from our 5 constructed peanut cDNA libraries of Ralstonia solanacearum challenged roots, R. solanacearum challenged leaves, and unchallenged cultured peanut roots, leaves and developing seeds. Among these ESTs, there were 14,547 unique sequences with 7,961 tentative consensus sequences and 6,586 singletons. Putative functions for 47.8% of the sequences were identified, including transcription factors, tissue-specific genes, genes involved in fatty acid biosynthesis and oil formation regulation, and resistance gene analogue genes. Additionally, differentially expressed genes, including those involved in ethylene and jasmonic acid signal transduction pathways, from both peanut leaves and roots, were identified in R. solanacearum challenged samples. This large expression dataset from different peanut tissues will be a valuable source for marker development and gene expression analysis. It will also be helpful for finding candidate genes for fatty acid synthesis and oil formation regulation as well as for studying mechanisms of interactions between the peanut host and R. solanacearum pathogen.

Comparative transcriptome analysis of Pima and Acala cotton during boll development using 454 pyrosequencing technology

Information on gene expression during cotton fiber development for high fiber quality genotypes including Pima (Gossypium barbadense L.) and Acala cotton (G. hirsutum L.) is lacking, impeding the discovery of novel genes and DNA markers for fiber quality traits. In the present study, massively parallel sequencing technology was used to generate a total of 394,301 expressed sequence tag (EST) sequence reads, totaling over 42 million bp, from immature ovaries at 10 days post-anthesis of Pima Phy 76 and Acala 1517-99. For Pima Phy 76, 1,900 EST contigs were tentatively assembled from 21.8% (50,876) ESTs which resulted in 1,541 tentative consensus sequences (TCs). 1,005 and 1,064 EST contigs were homologous to TCs from the A2 and D5 genomes, respectively, while 738 of the EST contigs were shared by both genomes. For Acala 1517-99, 2,151 EST contigs were tentatively assembled from 24.5% (39,368) ESTs and resulted in 1,787 TCs. 1,220 and 1,206 EST contigs were homologous to known transcripts from A2 and D5 genomes and only 452 of the EST contigs were shared by both genomes. 691 EST contigs are also homologous (E-value ≤ 1 × 10−10) between Pima Phy 76 and Acala 1517-99, of which 38 contigs pairs were highly homologous (E-value ≤ 1 × 10−60) with sequence variations, predominantly single nucleotide polymorphisms (SNPs). A Blast search of a sample of contigs to Upland cotton ESTs in GenBank indicated that more than 75% EST contigs may contain SNPs. This report represents the first research in cotton using 454 pyrosequencing technology to enrich ESTs for high fiber quality cotton.

Upgraded genomic information of <i>Jatropha curcas</i> L.

In order to upgrade the genome sequence information of J. curcas L., we integrated de novo assembly of a total of 537 million paired-end reads generated from the Illumina sequencing platform into the current genome assembly which was obtained by a combination of the conventional Sanger method and the Roche/454 sequencing platform. The total length of the upgraded genome sequences thus obtained was 297,661,187 bp consisting of 39,277 contigs. The average and N50 lengths of the generated contigs were 7,579 bp and 15,950 bp, both of which were increased fourfold from the previous genome assembly. Along with genome sequence upgrading, the currently available transcriptome data were collected from the public databases and assembled into 19,454 tentative consensus sequences. Based on a comparison between these tentative consensus sequences of transcripts and the predictions of computer programs, a total of 30,203 complete and partial structures of protein-encoding genes were deduced. The number of genes with complete structures was increased about threefold from the previous genome annotation. By applying the upgraded genome sequence and predicted protein-coding gene information, the number and features of the tandemly arrayed genes, syntenic relations between Jatropha and other plant genomes, and structural features of transposable elements were investigated. The detailed information on the updated J. curcas genome is available at http://www.kazusa.or.jp/jatropha/.

Neospora caninum: Comparative gene expression profiling of Neospora caninum wild type and a temperature sensitive clone

To understand the genetic basis of virulence, gene expression profiles of a temperature-sensitive clone (NCts-8, relatively avirulent) and its wild type (NC-1) of Neospora caninum were characterized and compared using a high-density microarray with approximately 63,000 distinct oligonucleotides. This microarray consists of 5692 unique N. caninum sequences, including 1980 Tentative Consensus sequences and 3712 singleton ESTs from the TIGR N. caninum Gene Index (NCGI, release 5.0). Each sequence was represented by 11 distinct 60mer oligonucleotides synthesized in situ on the microarray. The results showed that 111 genes were significantly repressed and no up-regulated genes were identified in the NCts-8 clone. The level of 10 randomly selected genes from the repressed genes was confirmed using real-time RT-PCR. Of the 111 repressed genes, 58 were hypothetical protein products and 53 were annotated genes. Over 70% of the repressed genes identified in this study are clustered on five chromosomes (I, VII, VIII, X and XII). These results suggest that the down-regulated genes may be in part responsible for the reduced pathogenesis of NCts-8; further characterization of the regulated genes may aid in understanding of molecular basis of virulence and development of countermeasures against neosporosis.

Genome-wide SNP discovery in tetraploid alfalfa using 454 sequencing and high resolution melting analysis

BackgroundSingle nucleotide polymorphisms (SNPs) are the most common type of sequence variation among plants and are often functionally important. We describe the use of 454 technology and high resolution melting analysis (HRM) for high throughput SNP discovery in tetraploid alfalfa (Medicago sativa L.), a species with high economic value but limited genomic resources.ResultsThe alfalfa genotypes selected from M. sativa subsp. sativa var. 'Chilean' and M. sativa subsp. falcata var. 'Wisfal', which differ in water stress sensitivity, were used to prepare cDNA from tissue of clonally-propagated plants grown under either well-watered or water-stressed conditions, and then pooled for 454 sequencing. Based on 125.2 Mb of raw sequence, a total of 54,216 unique sequences were obtained including 24,144 tentative consensus (TCs) sequences and 30,072 singletons, ranging from 100 bp to 6,662 bp in length, with an average length of 541 bp. We identified 40,661 candidate SNPs distributed throughout the genome. A sample of candidate SNPs were evaluated and validated using high resolution melting (HRM) analysis. A total of 3,491 TCs harboring 20,270 candidate SNPs were located on the M. truncatula (MT 3.5.1) chromosomes. Gene Ontology assignments indicate that sequences obtained cover a broad range of GO categories.ConclusionsWe describe an efficient method to identify thousands of SNPs distributed throughout the alfalfa genome covering a broad range of GO categories. Validated SNPs represent valuable molecular marker resources that can be used to enhance marker density in linkage maps, identify potential factors involved in heterosis and genetic variation, and as tools for association mapping and genomic selection in alfalfa.

Transcriptome profiling and molecular marker discovery in red pepper, Capsicum annuum L. TF68

Transcriptome from high throughput sequencing-by-synthesis is a good resource of molecular markers. In this study, we present utility of massively parallel sequencing by synthesis for profiling the transcriptome of red pepper (Capsicum annuum L. TF68) using 454 GS-FLX pyrosequencing. Through the generation of approximately 30.63 megabases (Mb) of expressed sequence tag (EST) data with the average length of 375 base pairs (bp), 9,818 contigs and 23,712 singletons were obtained by raw reads assembly. Using BLAST alignment against NCBI non-redundant and a UniProt protein database, 30% of the tentative consensus sequences were assigned to specific function annotation, while 24% returned alignments of unknown function, leaving up to 46% with no alignment. Functional classification using FunCat revealed that sequences with putative known function were distributed cross 18 categories. All unigenes have an approximately equal distribution on chromosomes by aligning with tomato (Solanum lycopersicum) pseudomolecules. Furthermore, 1,536 high quality single nucleotide discrepancies were discovered using the Bukang mature fruit cDNA collection (dbEST ID: 23667) as a reference. Moreover, 758 simple sequence repeat (SSR) motif loci were mined from 614 contigs, from which 572 primer sets were designed. The SSR motifs corresponded to di- and tri- nucleotide motifs (27.03 and 61.92%, respectively). These molecular markers may be of great value for application in linkage mapping and association mapping research.

Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

BackgroundAlfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling.ResultsCell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes.ConclusionsOur results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.

Transcriptome analysis and SNP/SSR marker information of red pepper variety YCM334 and Taean

Transcriptome analysis is becoming a powerful tool for the identification and characterization of molecular markers, splicing-specific mRNA mutants, and differentially expressed genes. In this study, we present the first comprehensive characterization of the transcriptomes of the mature fruits of two red pepper ( Capsicum annuum) accessions, YCM334 and Taean, using the 454 Genome Sequencer (GS)-FLX Titanium System. For YCM334, sequencing runs yielded 121,152 reads (average length = 409 nucleotides), which were assembled into 11,770 contiguous sequences (contigs) and 13,827 singletons. Sequencing runs for Taean yielded 154,088 reads (average length = 395 nucleotides) assembled into 14,200 contigs and 15,135 singletons. Using a BLAST search against the NCBI non-redundant (NR) and UniProt databases with an arbitrary expectation value of E-5, over 10,000 unigenes were functionally annotated and classified using the FunCat functional annotation scheme, and were found to distribute in 18 FunCat categories for YCM334 and 17 for Taean. Expression pattern analysis identified 540 Tentative Consensus (TC) sequences that were specifically detected in YCM334. Most of these differential TCs assigned with specific annotations covered 16 FunCat categories, of which 4 were annotated with resistance terms. Polymorphic marker discovery revealed a total of 1265 and 1436 simple sequence repeat (SSR) motifs for YCM334 and Taean, respectively, and a total of 3218 single nucleotide polymorphisms (SNPs) with high confidence. The information generated in this study provides valuable resources for characterization of some important agronomic traits and identification of molecular mechanism such as phytopathogen resistance.

A legume specific protein database (LegProt) improves the number of identified peptides, confidence scores and overall protein identification success rates for legume proteomics

A legume specific protein database (LegProt) has been created containing sequences from seven legume species, i.e., Glycine max, Lotus japonicus, Medicago sativa, Medicago truncatula, Lupinus albus, Phaseolus vulgaris, and Pisum sativum. The database consists of amino acid sequences translated from predicted gene models and 6-frame translations of tentative consensus (TC) sequences assembled from expressed sequence tags (ESTs) and singleton ESTs. This database was queried using mass spectral data for protein identification and identification success rates were compared to the NCBI nr database. Specifically, Mascot MS/MS ion searches of tandem nano-LC Q-TOFMS/MS mass spectral data showed that relative to the NCBI nr protein database, the LegProt database yielded a 54% increase in the average protein score (i.e., from NCBI nr 480 to LegProt 739) and a 50% increase in the average number of matched peptides (i.e., from NCBI nr 8 to LegProt 12). The overall identification success rate also increased from 88% (NCBI nr) to 93% (LegProt). Mascot peptide mass fingerprinting (PMF) searches of the LegProt database using MALDI-TOFMS data yielded a significant increase in the identification success rate from 19% (NCBI nr) to 34% (LegProt) while the average scores and average number of matched peptides showed insignificant changes. The results demonstrate that the LegProt database significantly increases legume protein identification success rates and the confidence levels compared to the commonly used NCBI nr. These improvements are primarily due to the presence of a large number of legume specific TC sequences in the LegProt database that were not found in NCBI nr. The LegProt database is freely available for download ( http://bioinfo.noble.org/manuscript-support/legumedb) and will serve as a valuable resource for legume proteomics.

Characterization of the rainbow trout transcriptome using Sanger and 454-pyrosequencing approaches

BackgroundRainbow trout are important fish for aquaculture and recreational fisheries and serves as a model species for research investigations associated with carcinogenesis, comparative immunology, toxicology and evolutionary biology. However, to date there is no genome reference sequence to facilitate the development of molecular technologies that utilize high-throughput characterizations of gene expression and genetic variation. Alternatively, transcriptome sequencing is a rapid and efficient means for gene discovery and genetic marker development. Although a large number (258,973) of EST sequences are publicly available, the nature of rainbow trout duplicated genome hinders assembly and complicates annotation.ResultsHigh-throughput deep sequencing of the Swanson rainbow trout doubled-haploid transcriptome using 454-pyrosequencing technology yielded ~1.3 million reads with an average length of 344 bp, a total of 447 million bases. De novo assembly of the sequences yielded 151,847 Tentative Consensus (TC) sequences (average length of 662 bp) and 224,391 singletons. A combination assembly of both the 454-pyrosequencing ESTs and the pre-existing sequences resulted in 161,818 TCs (average length of 758 bp) and 261,071 singletons. Gene Ontology analysis of the combination assembly showed high similarities to transcriptomes of other fish species with known genome sequences.ConclusionThe 454 library significantly increased the suite of ESTs available for rainbow trout, allowing improved assembly and annotation of the transcriptome. Furthermore, the 454 sequencing enables functional genome research in rainbow trout, providing a wealth of sequence data to serve as a reference transcriptome for future studies including identification of paralogous sequences and/or allelic variation, digital gene expression and proteomic research.

A Novel Cytoplasmic Isopentenyl Diphosphate Isomerase Gene from Tomato (Solanum lycopersicum): Cloning, Expression, and Color Complementation

Isopentenyl diphosphate isomerase (IPI; EC5.3.3.2) catalyzes isomerization between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), both of which are essential precursors for terpenoid biosynthesis. A novel gene encoding isopentenyl diphosphate isomerase (designated as SlIPI) was isolated from tomato based on tentative consensus (TC183769) and unigene SGN-U569721 sequences. The SlIPI cDNA contained a 708-bp open reading frame (ORF) encoding a 235-amino-acid protein. The deduced SlIPI protein had an isoelectric point of 5.06 and molecular weight of about 27.18 kDa. Amino acid sequence comparison analysis showed 83-95% similarity to IPIs from other plant species. Phylogenetic analysis revealed that SlIPI had the closest relationship to IPI from Nicotiana tabacum. The SlIPI was likely to be localized in cytoplasm; while, SlIPI2 contained a chloroplast transit peptide. A three dimensional structure modeling revealed that the structure of SlIPI was similar to that of SlIPI2. Tissue expression analysis indicated that SlIPI was constitutively expressed, with the highest expression level detected in the root. Heterologous expression of the recombinant SlIPI in engineered Escherichia coli resulted in the production and accumulation of carotenoid in E. coli, thus confirming that the SlIPI was a functional gene.

Genetic diversity and selection of genotypes to enhance Zn and Fe content in common bean.

Common bean (Phaseolus vulgaris L.) is an important source of dietary protein and minerals worldwide. Genes conditioning variability for mineral contents are not clearly understood. Our ultimate goal is to identify genes conditioning genetic variation for Zn and Fe content. To establish mapping populations for this objective, we tested mineral content of 29 common bean genotypes. Chemical analyses revealed significant genetic variability for seed Zn and Fe contents among the genotypes. Genetic diversity was evaluated with 49 primer pairs, of which 23 were simple sequence repeats (SSR), 16 were developed from tentative consensus (TC) sequences, and 10 were generated from common bean NBS-LRR gene sequences. The discriminatory ability of molecular markers for identifying allelic variation among genotypes was estimated by polymorphism information content (PIC) and the genetic diversity was measured from genetic similarities between genotypes. Primers developed from NBS-LRR gene sequences were highly polymorphic in both PIC values and number of alleles (0.82 and 5.3), followed by SSRs (0.56 and 3.0), and markers developed from TC (0.39 and 2.0). genetic similarity values between genotypes ranged from 14.0 (JaloEEP558 and DOR364) to 91.4 (MIB152 and MIB465). Cluster analysis clearly discriminated the genotypes into Mesoamerican and Andean gene pools. Common bean genotypes were selected to include in crossing to enhance seed Zn and Fe content based on genetic diversity and seed mineral contents of the genotypes.

HOP EST RESOURCES FOR MARKER DEVELOPMENT

The first large dataset of hop EST sequences has recently become available to the hop research community. In this work we assembled all single pass hop EST sequences into tentative consensus sequences and used this information for development of molecular markers. A total of 14,944 EST sequences were acquired in a total length of 7,786,981 bp. After vector clipping, polyA clipping and removal of low-quality and short sequences, the remaining 14,731 sequences were clustered using the TGICL tool. Clustering yielded 1,318 contigs and only 76 singletons in a total length of 1,067,770 bp. A total of 13 clusters contained more than 100 EST sequences each. The 20 contigs with the highest number of ESTs clustered together were compared with blastn or blastx algorithms for simple annotation. The most abundant were ribosomal DNA and chloroplast transcripts. All sequences were subsequently screened for the presence of microsatellite motifs (2-10 bp motifs) and 235 contained such motifs. The most common type of repeat was dinucleotide (131), followed by trinucleotide (118). PCR primers were developed for chalcone synthase (CHS) and valerophenone synthase (VPS) genes and the sequences were checked for the presence of single nucleotide polymorphisms (SNPs). Markers were found in both genes and will be further used to map the two genes in the hop linkage map.

ANEXdb: an integrated animal ANnotation and microarray EXpression database.

To determine annotations of the sequence elements on microarrays used for transcriptional profiling experiments in livestock species, currently researchers must either use the sparse direct annotations available for these species or create their own annotations. ANEXdb ( http://www.anexdb.org ) is an open-source web application that supports integrated access of two databases that house microarray expression (ExpressDB) and EST annotation (AnnotDB) data. The expression database currently supports storage and querying of Affymetrix-based expression data as well as retrieval of experiments in a form ready for NCBI-GEO submission; these services are available online. AnnotDB currently houses a novel assembly of approximately 1.6 million unique porcine-expressed sequence reads called the Iowa Porcine Assembly (IPA), which consists of 140,087 consensus sequences, the Iowa Tentative Consensus (ITC) sequences, and 103,888 singletons. The IPA has been annotated via transfer of information from homologs identified through sequence alignment to NCBI RefSeq. These annotated sequences have been mapped to the Affymetrix porcine array elements, providing annotation for 22,569 of the 23,937 (94%) porcine-specific probe sets, of which 19,253 (80%) are linked to an NCBI RefSeq entry. The ITC has also been mined for sequence variation, providing evidence for up to 202,383 SNPs, 62,048 deletions, and 958 insertions in porcine-expressed sequence. These results create a single location to obtain porcine annotation of and sequence variation in differently expressed genes in expression experiments, thus permitting possible identification of causal variants in such genes of interest. The ANEXdb application is open source and available from SourceForge.net.

Phenylalanine ammonia-lyase (PAL) from tobacco (Nicotiana tabacum): characterization of the four tobaccoPALgenes and active heterotetrameric enzymes

PAL (L-phenylalanine ammonia-lyase), the first enzyme of phenylpropanoid biosynthesis, is often encoded by multigene families in plants. A PCR-based approach was used to isolate cDNA clones corresponding to the four PAL genes of tobacco (Nicotiana tabacum). By careful comparison of cDNA and genomic clones, a new PAL gene (PAL4) was defined. PCR amplification of PAL sequences from cDNA led to the generation of chimaeric clones between PAL1 and PAL4, and incorrect annotation of PAL4 ESTs (expressed sequence tags) as PAL1 in the EST database has given rise to a randomly shuffled tentative consensus sequence. The PAL2 previously described in the literature was shown, by domain swapping experiments with PAL1, to possess a single nucleotide substitution leading to an inactive enzyme. The altered amino acid resulting from this substitution maps to the base of the active site pocket in the three-dimensional structure of PAL. The inactive PAL2 allele could not be recovered from 13 different tobacco cultivars examined. PALs 1-4 were co-expressed in multiple plant organs, and were also co-induced following exposure of cell cultures to yeast elicitor or methyl jasmonate. All four tobacco PAL proteins expressed in Escherichia coli displayed normal Michaelis-Menten kinetics, with Km values between 36 and 60 muM. Co-expression of different PAL proteins in E. coli resulted in formation of heterotetramers, which possessed kinetic properties within the same range as those of the individual homotetramers. The potential physiological function of heterotetrameric PAL forms is discussed.