Expressed Sequence Tag Libraries Research Articles

Identification and validation of differentially expressed genes from pig skeletal muscle

Pig is an important animal for meat production; this is generally associated with characteristics determined prenatally during myogenesis. Expressed sequence tags (EST) can provide direct information on the transcriptome and indirect information on the relation between the genome and phenotype, giving information about differentially expressed genes (DEG). In this work, the identification and annotation of DEG from EST libraries of three pig breeds (Duroc, Large White and Local Breed Piau) were performed followed by real-time PCR analyses during pre- and postnatal stages (21, 40, 70 and 90days of pregnancy and 107, 121 and 171days postnatal) from commercial breed animals for analysis of genes expression levels. Therefore, 34 genes differentially expressed were identified, of which 21 grouped in a network related with muscle development. From this, the expression profile of 13 genes was measured, to confirm their relationship with myogenesis like ANKRD2, MYBPC1, NEB and MYL2. These genes showed a prenatal high expression in this study. Besides, novels candidates for muscle development (TP53 and DCTN1) were listed. These findings can contribute to better explaining gene function mechanism and are helpful in uncovering the pathways that mediate pre- and postnatal skeletal muscle development in vertebrates.

Identification, Cloning, and Functional Characterization of the IL-1 Receptor Antagonist in the Chicken Reveal Important Differences between the Chicken and Mammals

The human IL-1 family contains 11 genes encoded at three separate loci. Nine, including IL-1R antagonist (IL-1RN), are present at a single locus on chromosome 2, whereas IL-18 and IL-33 lie on chromosomes 11 and 9, respectively. There are currently only two known orthologs in the chicken, IL-1β and IL-18, which are encoded on chromosomes 22 and 24, respectively. Two novel chicken IL-1 family sequences were identified from expressed sequence tag libraries, representing secretory and intracellular (icIL-1RN) structural variants of the IL-1RN gene, as seen in mammals. Two further putative splice variants (SVs) of both chicken IL-1RN (chIL-1RN) structural variants were also isolated. Alternative splicing of human icIL-1RN gives three different transcripts; there are no known SVs for human secretory IL-1RN. The chicken icIL-1RN SVs differ from those found in human icIL-1RN in terms of the rearrangements involved. In mammals, IL-1RN inhibits IL-1 activity by physically occupying the IL-1 type I receptor. Both full-length structural variants of chIL-1RN exhibited biological activity similar to their mammalian orthologs in a macrophage cell line bioassay. The four SVs, however, were not biologically active. The chicken IL-1 family is more fragmented in the genome than those of mammals, particularly in that the large multigene locus seen in mammals is absent. This suggests differential evolution of the family since the divergence of birds and mammals from a common ancestor, and makes determination of the full repertoire of chicken IL-1 family members more challenging.

Unique nucleocytoplasmic dsDNA and +ssRNA viruses are associated with the dinoflagellate endosymbionts of corals

The residence of dinoflagellate algae (genus: Symbiodinium) within scleractinian corals is critical to the construction and persistence of tropical reefs. In recent decades, however, acute and chronic environmental stressors have frequently destabilized this symbiosis, ultimately leading to coral mortality and reef decline. Viral infection has been suggested as a trigger of coral-Symbiodinium dissociation; knowledge of the diversity and hosts of coral-associated viruses is critical to evaluating this hypothesis. Here, we present the first genomic evidence of viruses associated with Symbiodinium, based on the presence of transcribed +ss (single-stranded) RNA and ds (double-stranded) DNA virus-like genes in complementary DNA viromes of the coral Montastraea cavernosa and expressed sequence tag (EST) libraries generated from Symbiodinium cultures. The M. cavernosa viromes contained divergent viral sequences similar to the major capsid protein of the dinoflagellate-infecting +ssRNA Heterocapsa circularisquama virus, suggesting a highly novel dinornavirus could infect Symbiodinium. Further, similarities to dsDNA viruses dominated (∼69%) eukaryotic viral similarities in the M. cavernosa viromes. Transcripts highly similar to eukaryotic algae-infecting phycodnaviruses were identified in the viromes, and homologs to these sequences were found in two independently generated Symbiodinium EST libraries. Phylogenetic reconstructions substantiate that these transcripts are undescribed and distinct members of the nucleocytoplasmic large DNA virus (NCLDVs) group. Based on a preponderance of evidence, we infer that the novel NCLDVs and RNA virus described here are associated with the algal endosymbionts of corals. If such viruses disrupt Symbiodinium, they are likely to impact the flexibility and/or stability of coral-algal symbioses, and thus long-term reef health and resilience.

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.

Validation and comparison of microsatellite markers derived from Senegalese sole (Solea senegalensis, Kaup) genomic and expressed sequence tags libraries

In this work, we tested 100 potential new microsatellites (SSRs) equally derived from expressed sequence tag (EST) and enriched genomic-DNA libraries from Senegalese sole (Solea senegalensis, Kaup), a valuable cultured flatfish species. A final set of 69 new polymorphic microsatellites were validated after a population analysis, 37 of which corresponded to the first EST library constructed for Senegalese sole (EST-SSR). Although differences were not significant, EST sequences provided a higher proportion of quality markers (74%) than anonymous ones (64%). Most of the rejected anonymous SSRs (17 loci) were discarded because they did not generate PCR products; only one was monomorphic. On the contrary, all EST-SSRs gave PCR products, although monomorphism was more frequent (26%). Altogether, the number of alleles per locus was fairly similar in both SSR types, ranging from 2 to 19. The observed and expected heterozygosities varied from 0.105 to 1 and from 0.108 to 0.937, respectively. The main difference between the two sets was the percentage of annotated loci, being higher in EST-SSRs, as expected. Within the EST-SSRs, 46% of them showed flanking regions that significantly matched with EST sequences from other three flatfish species; however, the microsatellite itself was present only on half of these cases. These two new SSR sets constitute a suitable tool for fingerprinting, gene flow, genetic diversity, genome mapping studies and molecular-assisted breeding in this species.

Expression sequence tag library derived from peripheral blood mononuclear cells of the chlorocebus sabaeus

BackgroundAfrican Green Monkeys (AGM) are amongst the most frequently used nonhuman primate models in clinical and biomedical research, nevertheless only few genomic resources exist for this species. Such information would be essential for the development of dedicated new generation technologies in fundamental and pre-clinical research using this model, and would deliver new insights into primate evolution.ResultsWe have exhaustively sequenced an Expression Sequence Tag (EST) library made from a pool of Peripheral Blood Mononuclear Cells from sixteen Chlorocebus sabaeus monkeys. Twelve of them were infected with the Simian Immunodeficiency Virus. The mononuclear cells were or not stimulated in vitro with Concanavalin A, with lipopolysacharrides, or through mixed lymphocyte reaction in order to generate a representative and broad library of expressed sequences in immune cells. We report here 37,787 sequences, which were assembled into 14,410 contigs representing an estimated 12% of the C. sabaeus transcriptome. Using data from primate genome databases, 9,029 assembled sequences from C. sabaeus could be annotated. Sequences have been systematically aligned with ten cDNA references of primate species including Homo sapiens, Pan troglodytes, and Macaca mulatta to identify ortholog transcripts. For 506 transcripts, sequences were quasi-complete. In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes.ConclusionsThe EST library we provide here will prove useful in gene annotation efforts for future sequencing of the African Green Monkey genomes. Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

Molecular Cloning and Characterization of First Organic Matrix Protein from Sclerites of Red Coral, Corallium rubrum

We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.

A High-Throughput Virus-Induced Gene-Silencing Vector for Screening Transcription Factors in Virus-Induced Plant Defense Response in Orchid

The large number of species and worldwide spread of species of Orchidaceae indicates their successful adaptation to environmental stresses. Thus, orchids provide rich resources to study how plants have evolved to cope with stresses. This report describes our improvement of our previously reported orchid virus-induced gene silencing vector, pCymMV-pro60, with a modified Gateway cloning system which requires only one recombination and can be inoculated by agroinfiltration. We cloned 1,700 DNA fragments, including 187 predicted transcription factors derived from an established expression sequence tag library of orchid, into pCymMV-Gateway. Phalaenopsis aphrodite was inoculated with these vectors that contained DNA fragments of the 187 predicted transcription factors. The viral vector initially triggered the expression of the salicylic acid (SA)-related plant defense responses and later induced silencing of the endogenous target transcription factor genes. By monitoring the expression of the SA-related plant defense marker PhaPR1 (homolog of PR1), we identified a gene, PhaTF15, involved in the expression of PhaPR1. Knockdown of PhaTF15 by virus-induced gene silencing and by transient delivery of double-stranded RNA (dsRNA) reduced expression of the orchid homolog of the conserved positive defense regulator NPR1, PhaNPR1. Cymbidium mosaic virus also accumulated to high levels with knockdown of PhaTF15 by transient delivery of dsRNA. We demonstrated efficient cloning and screening strategies for high-throughput analysis of orchid and identify a gene, PhaTF15, involved in regulation of SA-related plant defense.

Transcriptome changes after genome‐wide admixture in invasive sculpins (Cottus)

Models on hybrid speciation assume that hybridization generates increased phenotypic variance that is utilized to invade new adaptive peaks. We test to what extent this prediction can be traced using gene expression data in the fish species Cottus perifretum and Cottus rhenanus as well as a natural hybrid lineage referred to as invasive sculpins. In addition, interspecies crosses were used to explore evolutionary trajectories from initial stages to the hybrid lineage. EST (expressed sequence tag) libraries were sequenced to design an oligonucleotide microarray that was calibrated for probe-specific differences in binding behaviour. Levels of gene expression divergence between species correlate with genetic divergence at neutral markers and, accordingly, invasive sculpins were intermediate between the parental species overall. However, the hybrid lineage is distinguished through unique patterns of gene expression that are enriched for biological functions which represent candidates for the fitness properties of invasive sculpins. We compare F(2) crosses with natural invasive sculpins to show that the variance in gene expression decreases in invasives. Moreover, few of the transgressive patterns of gene expression that distinguish invasives can be directly observed in F(2) crosses. This suggests that the invasive transcriptome was subject to secondary changes after admixture. The result is in line with an evolutionary process that reduces maladaptive variance and optimizes the phenotype of an emerging hybrid lineage.

Transcriptome analysis of the entomopathogenic fungus Beauveria bassiana grown on cuticular extracts of the coffee berry borer (Hypothenemus hampei)

The coffee berry borer (CBB; Hypothenemus hampei) is a major pest of coffee responsible for significant crop losses worldwide. The entomopathogenic fungus Beauveria bassiana represents a natural means of controlling this insect pest; however, little is known concerning the molecular determinants that contribute to the virulence of the fungus towards the CBB. In order to examine genes involved in insect virulence, two expressed sequence tag (EST) libraries, representing germinating conidia and growing hyphae/mycelia of B. bassiana cells grown on cuticular extracts of the CBB were constructed and analysed. In total, 4186 cDNA transcripts were obtained, which included 2141 from the cuticle-germinated conidia and 2045 from the cuticle-grown mycelium libraries, respectively. The average sequence length obtained was 470 bp and transcript assembly resulted in a set of 1271 and 1305 unique gene sequences for the conidial and mycelia libraries, respectively. Around 50 % of the sequences in each library could be annotated by gene ontology terms. An analysis of the two generated libraries as well as a previously reported EST library of B. bassiana grown on chitin was performed. Between the cuticle-germinated conidia and the cuticle-grown mycelia libraries, 322 unique gene sequences were shared, of which 90 % could be annotated, leaving 949 unique cuticle-germinated conidial genes and 983 unique growing hyphae/mycelia genes of which around 65 % were annotated. ESTs shared between the libraries indicated a basic response pattern for B. bassiana against H. hampei, which included genes implicated in pathogenicity. The expression profiles of four genes were evaluated with a cyclophilin, an alkaline-like serine protease and a mitogen-activated protein kinase (MAPK), showing elevated expression during initial phases of infection, i.e. conidia germinating on insect extracts. These data provide clues and gene candidates for further exploration concerning the biology and molecular mechanisms of entomopathogenicity by this fungus.

Glandular trichomes: what comes after expressed sequence tags?

Glandular trichomes cover the surface of many plant species. They exhibit tremendous diversity, be it in their shape or the compounds they secrete. This diversity is expressed between species but also within species or even individual plants. The industrial uses of some trichome secretions and their potential as a defense barrier, for example against arthropod pests, has spurred research into the biosynthesis pathways that lead to these specialized metabolites. Because complete biosynthesis pathways take place in the secretory cells, the establishment of trichome-specific expressed sequence tag libraries has greatly accelerated their elucidation. Glandular trichomes also have an important metabolic capacity and may be considered as true cell factories. To fully exploit the potential of glandular trichomes as breeding or engineering objects, several research areas will have to be further investigated, such as development, patterning, metabolic fluxes and transcription regulation. The purpose of this review is to provide an update on the methods and technologies which have been used to investigate glandular trichomes and to propose new avenues of research to deepen our understanding of these specialized structures.

Development and application of single nucleotide polymorphism markers in the polyploid Brassica napus by 454 sequencing of expressed sequence tags

With 4 figures and 4 tablesAbstractSingle nucleotide polymorphisms (SNPs) are currently the important classes of genetic markers for major crop species. It has the abundant polymorphism, however, is not widely applicable for genetic linkage mapping. In this study, methods for developing SNP markers in polyploid rapeseed by incorporation of expressed sequence tag (EST) redundancy, quality index and lines information between parents in the absence of reference genomic sequence data are demonstrated. For the development of SNP markers, ESTs were generated from two rapeseed parents ZY036 and 51070. Putative SNPs were computationally identified using 201 190 high‐quality 454 ESTs from the two parents. A total of 655 putative SNPs were detected, among which transition changes accounted for 56% and transversion changes accounted for 44%. Of 50 randomly selected, putative SNPs 42 could be successfully validated, and six of these were successfully mapped in an F2 population from the two parents that were used for generating the EST libraries. These results demonstrate the usefulness of our approach for SNP marker development.

Characterization of the early response of the orchid, Phalaenopsis amabilis, to Erwinia chrysanthemi infection using expression profiling

Erwinia chrysanthemi is a devastating bacterial pathogen in Phalaenopsis amabilis and causes soft-rotting disease by secretion of cell wall-degrading enzymes. However, the molecular mechanisms underlying the interaction of P. amabilis with E. chrysanthemi remain elusive. In this study, early molecular events of the plant in response to the pathogen attack were investigated. The alteration in reactive oxygen species accumulation and peroxidase activity occurred at the site of infection. Subsequently, a systematic sequencing of expressed sequence tags (ESTs) using suppression subtractive hybridization (SSH) was performed to obtain the first global picture of the assembly of genes involved in the pathogenesis. The majority of the SSH clones showed a high identity with genes coding for proteins that have known roles in redox homeostasis, responses to pathogens and metabolism. A notable number of the SSH clones were those encoding WRKY, MYB and basic leucine zipper transcription factors, indicating the stimulation of intracellular signal transduction. An orchid gene encoding trans-2-enoyl-CoA reductase (ECR) was the most abundant transcripts in the EST library. ECR is an enzyme catalyzing the very long chain fatty acids (VLCFAs) biosynthesis, and the full-length cDNA of the ECR gene (PaECR1) was obtained. Functional analysis of PaECR1 was conducted by virus-induced gene silencing to knock down the gene expression in P. amabilis. The PaECR1-silenced plants were more susceptible to E. chrysanthemi infection, implying potential roles for VLCFAs in the pathogenesis. In summary, the pathogen-responsive gene expression profiles facilitated a more comprehensive view of the molecular events that underlie this economically important plant-pathogen interaction.

ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca — Cephalopoda)

New molecular resources regarding the so-called “non-standard models” in biology extend the present knowledge and are essential for molecular evolution and diversity studies (especially during the development) and evolutionary inferences about these zoological groups, or more practically for their fruitful management. Sepia officinalis, an economically important cephalopod species, is emerging as a new lophotrochozoan developmental model. We developed a large set of expressed sequence tags (ESTs) from embryonic stages of S. officinalis, yielding 19,780 non-redundant sequences (NRS). Around 75% of these sequences have no homologs in existing available databases. This set is the first developmental ESTs library in cephalopods. By exploring these NRS for tubulin, a generic protein family, and reflectin, a cephalopod specific protein family, we point out for both families a striking molecular diversity in S. officinalis.

Genomics of Compositae weeds: EST libraries, microarrays, and evidence of introgression

Weeds cause considerable environmental and economic damage. However, genomic characterization of weeds has lagged behind that of model plants and crop species. Here we describe the development of genomic tools and resources for 11 weeds from the Compositae family that will serve as a basis for subsequent population and comparative genomic analyses. Because hybridization has been suggested as a stimulus for the evolution of invasiveness, we also analyze these genomic data for evidence of hybridization. We generated 22 expressed sequence tag (EST) libraries for the 11 targeted weeds using Sanger, 454, and Illumina sequencing, compared the coverage and quality of sequence assemblies, and developed NimbleGen microarrays for expression analyses in five taxa. When possible, we also compared the distributions of Ks values between orthologs of congeneric taxa to detect and quantify hybridization and introgression. Gene discovery was enhanced by sequencing from multiple tissues, normalization of cDNA libraries, and especially greater sequencing depth. However, assemblies from short sequence reads sometimes failed to resolve close paralogs. Substantial introgression was detected in Centaurea and Helianthus, but not in Ambrosia and Lactuca. Transcriptome sequencing using next-generation platforms has greatly reduced the cost of genomic studies of nonmodel organisms, and the ESTs and microarrays reported here will accelerate evolutionary and molecular investigations of Compositae weeds. Our study also shows how ortholog comparisons can be used to approximately estimate the genome-wide extent of introgression and to identify genes that have been exchanged between hybridizing taxa.

Carbohydrate‐active enzymes revealed in Coptotermes formosanus (Isoptera: Rhinotermitidae) transcriptome

Coptotermes formosanus is one of the most destructive wood-feeding termites. To understand the molecular mechanisms that regulate the development of the termite, a normalized C. formosanus cDNA library was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. The sequencing of this library generated 131 636 expressed sequence tags (ESTs) and 25 939 assembled unigenes. The carbohydrate-active enzymes (CAZymes) revealed in this library were analysed in the present report. A total of 509 putative CAZymes were identified. Diverse cellulolytic enzymes were uncovered from both the host termite and from symbionts harboured by the termite, which were possibly the result of the high efficiency of cellulose utilization. CAZymes associated with trehalose biosynthetic and metabolic pathways were also identified, which are potential regulators of the physiological activities of trehalose, an important insect blood sugar. Representative CAZyme coding genes in glycoside hydrolase family 1 (GH1) were quantitatively analysed. The results showed that the five GH1 β-glucosidase genes were expressed differentially among different castes and one of them was female alate-specific. Overall, the normalized EST library provides a comprehensive genetic resource of C. formosanus and will serve a diverse range of research areas. The CAZymes represent one of the repositories of enzymes useful for physiological studies and applications in sugar-based biofuel production.

Progress in Blueberry Research in New Zealand

Blueberries were first introduced to New Zealand in the 1950s from North America and by 1970 around 30 cultivars had been imported. Selection for new material better suited to the New Zealand climate then began using F1 seedlings and open pollinated crosses. The Plant & Food Research Ltd blueberry breeding program (led by Dr. Jessica Scalzo) is currently run at three sites: at Hamilton and Motueka on the North and South Island, respectively, of New Zealand, and a smaller program at Dierking's Nursery in Germany. Plant & Food Research Ltd's breeding targets are to improve yield, ripening season, and disease resistance, as well as fruit quality traits, such as fruit size, firmness, flavor, color, and grittiness. Areas of research include molecular genetics, functional genomics, biochemical composition, and health, and in 2008, we joined the Speciality Crop Research Initiative project to generate genomic tools for blueberry improvement, led by Dr. Lisa Rowland. We present a summary of the microsatellite and single nucleotide polymorphism-based markers we have developed as part of the Speciality Crop Research Initiative program, as well as an update on other blueberry research undertaken at Plant & Food Research Ltd. This will include anthocyanin composition studies, research into polyphenolics and skeletal muscle damage, as well as work on our expressed sequence tag library and candidate genes within the anthocyanin pathway. We will also discuss the impact of this research on the future of blueberry breeding at Plant & Food Research Ltd.

Application of real-time PCR-based SNP detection for mapping of <i>Net2</i>, a causal D-genome gene for hybrid necrosis in interspecific crosses between tetraploid wheat and <i>Aegilops tauschii</i>

Available information on genetically assigned molecular markers is not sufficient for efficient construction of a high-density linkage map in wheat. Here, we report on application of high resolution melting (HRM) analysis using a real-time PCR apparatus to develop single nucleotide polymorphism (SNP) markers linked to a hybrid necrosis gene, Net2, located on wheat chromosome 2D. Based on genomic information on barley chromosome 2H and wheat expressed sequence tag libraries, we selected wheat cDNA sequences presumed to be located near the Net2 chromosomal region, and then found SNPs between the parental Ae. tauschii accessions of the synthetic wheat mapping population. HRM analysis of the PCR products from F(2) individuals' DNA enabled us to assign 44.4% of the SNP-representing cDNAs to chromosome 2D despite the presence of the A and B genomes. In addition, the designed SNP markers were assigned to chromosome 2D of Ae. tauschii. The order of the assigned SNP markers in synthetic hexaploid wheat was confirmed by comparison with the markers in barley and Ae. tauschii. Thus, the SNP-genotyping method based on HRM analysis is a useful tool for development of molecular markers at target loci in wheat.

Generating Genomic Tools for Blueberry Improvement

Because of their recognized health benefits, there has been increased demand and consumption of blueberries in recent years. Great strides have been made in blueberry cultivar development since its domestication using traditional breeding approaches. However, genomic tools that could be used to hasten improvement are lacking. The aim of our Specialty Crop Research Initiative project, funded at the end of 2008, is to develop genomic tools for molecular breeding and assessing genetic diversity of blueberry. Marker-assisted breeding would be particularly useful for combining traits for climatic adaptation with those for improved fruit and nutritional quality in highbush blueberry (Vaccinium corymbosum). Genomic resources being developed include expressed sequence tag libraries, expressed sequence tag-based molecular markers, and genetic linkage maps. Transcriptome sequences have been generated from fruit at different stages of development, flower buds at different stages of cold acclimation, and leaves by 454 sequencing. About 600,000 sequences have been assembled into approximately 15,000 contigs. Markers derived from expressed sequence tags (simple sequence repeats and expressed sequence tag-polymerase chain reaction markers) are being used to identify quantitative trait loci associated with cold hardiness, chilling requirement, and fruit quality traits, in studies of genetic diversity, spatial genetic structure, and gene flow in the wild lowbush blueberry (V. angustifolium), and to construct a phylogenetic tree of Vaccinium species in the section Cyanococcus. Availability of these genomic tools will allow future advances, such as the development of a blueberry microarray to facilitate studying gene expression and the use of marker-assisted breeding.

Development of ESTs and data mining of pineapple EST-SSRs

Improving the quality of the non-climacteric fruit, pineapple, is possible with information on the expression of genes that occur during the process of fruit ripening. This can be made known though the generation of partial mRNA transcript sequences known as expressed sequence tags (ESTs). ESTs are useful not only for gene discovery but also function as a resource for the identification of molecular markers, such as simple sequence repeats (SSRs). This paper reports on firstly, the construction of a normalized library of the mature green pineapple fruit and secondly, the mining of EST-SSRs markers using the newly obtained pineapple ESTs as well as publically available pineapple ESTs deposited in GenBank. Sequencing of the clones from the EST library resulted in 282 good sequences. Assembly of sequences generated 168 unique transcripts (UTs) consisting of 34 contigs and 134 singletons with an average length of ≈500bp. Annotation of the UTs categorized the known proteins transcripts into the three ontologies as: molecular function (34.88%), biological process (38.43%), and cellular component (26.69%). Approximately 7% (416) of the pineapple ESTs contained SSRs with an abundance of trinucleotide SSRs (48.3%) being identified. This was followed by dinucleotide and tetranucleotide SSRs with frequency of 46 and 57%, respectively. From these EST-containing SSRs, 355 (85.3%) matched to known proteins while 133 contained flanking regions for primer design. Both the ESTs were sequenced and the mined EST-SSRs will be useful in the understanding of non-climacteric ripening and the screening of biomarkers linked to fruit quality traits.