Expressed Sequence Tag Database Research Articles

Proteomic Analysis of the Acid‐Soluble Nacre Matrix of the BivalveUnio pictorum: Detection of Novel Carbonic Anhydrase and Putative Protease Inhibitor Proteins

The matrix extracted from mollusc shell nacre is a mixture of proteins and glycoproteins that is thought to play a major role in controlling biomineral synthesis and in increasing its mechanical properties. We investigated the nacreous shell of the freshwater mussel Unio pictorum, to which we applied a proteomics approach adapted to mollusc shell proteins. On one hand, the acid-soluble nacre matrix was fractionated by SDS-PAGE and the five main protein bands (P95, P50, P29, P16, and P12) were digested with trypsin and analyzed by nanoLC-MS/MS followed by de novo sequencing. On the other hand, the acid-soluble nacre matrix was analyzed in a similar manner, without any preliminary fractionation. In total, we obtained about 140 peptides, of between 9 and 21 residues, as well as several shorter peptides. Interestingly, it appears that the different protein bands share several identical peptides; this has implications for the underlying genetic machinery that synthesizes nacre proteins. Homology searches against sequences in the Swiss-Prot protein database and the 800,000 mollusc expressed sequence tag database were performed, but surprisingly, only a few obvious homologies were established. Among the peptides that match with known sequences, some from P50 and P16/P12 proteins align with carbonic anhydrase (CA) and with the protease inhibitor, respectively. The evolutionary implications of our findings are discussed.

Analysis of ESTs generated from inner bark tissue of an Acacia auriculiformis x Acacia mangium hybrid

The majority of plant expressed sequence tags (ESTs) available in the public databases are from non-woody species such as Arabidopsis, maize, soybean, and rice, with the exception of the model tree species, Populus. In this study, we report the first EST database constructed from the commercially important tree species Acacia. ESTs were generated from pooled RNA extracted from the mature, intermediate, and young inner bark tissues of an Acacia auriculiformis x Acacia mangium hybrid. Finally, 3,182 high-quality ESTs were analyzed representing a total of 1,982 unique transcripts (663 contigs and 1,319 singletons) for the inner bark cDNA library. A total of 1,053 unigenes (1,750 ESTs) showed similarities with protein sequences in public database where 867 were significant matches (E value ≤ e−10). Further analysis performed on unigenes with significant matches and known functions identified 231 genes including genes involved in cellulose, lignin, and hemicellulose biosynthesis. For cDNA library validation, quantitative real-time RT-PCR was performed to study the expression levels of the cellulose and lignin genes identified from this EST database in the phloem tissue of A. mangium, A. auriculiformis, and A. auriculiformis x A. mangium hybrid. All the seven candidate genes were expressed in all the individuals studied confirming the dependability of the EST database. The cDNA library and the EST database constructed are valuable resources for forest tree research aiming towards understanding the genetic control of wood formation and in the future endeavors to modify wood and fiber profile through marker-assisted breeding programs.

Sugar transporter genes of the brown planthopper, Nilaparvata lugens: A facilitated glucose/fructose transporter

The brown planthopper (BPH), Nilaparvata lugens, attacks rice plants and feeds on their phloem sap, which contains large amounts of sugars. The main sugar component of phloem sap is sucrose, a disaccharide composed of glucose and fructose. Sugars appear to be incorporated into the planthopper body by sugar transporters in the midgut. A total of 93 expressed sequence tags (ESTs) for putative sugar transporters were obtained from a BPH EST database, and 18 putative sugar transporter genes ( Nlst1– 18) were identified. The most abundantly expressed of these genes was Nlst1. This gene has previously been identified in the BPH as the glucose transporter gene NlHT1, which belongs to the major facilitator superfamily. Nlst1, 4, 6, 9, 12, 16, and 18 were highly expressed in the midgut, and Nlst2, 7, 8, 10, 15, 17, and 18 were highly expressed during the embryonic stages. Functional analyses were performed using Xenopus oocytes expressing NlST1 or 6. This showed that NlST6 is a facilitative glucose/fructose transporter that mediates sugar uptake from rice phloem sap in the BPH midgut in a manner similar to NlST1.

Comparative analysis of expressed sequence tags from three castes and two life stages of the termite Reticulitermes flavipes

BackgroundTermites (Isoptera) are eusocial insects whose colonies consist of morphologically and behaviorally specialized castes of sterile workers and soldiers, and reproductive alates. Previous studies on eusocial insects have indicated that caste differentiation and behavior are underlain by differential gene expression. Although much is known about gene expression in the honey bee, Apis mellifera, termites remain relatively understudied in this regard. Therefore, our objective was to assemble an expressed sequence tag (EST) data base for the eastern subterranean termite, Reticulitermes flavipes, for future gene expression studies.ResultsSoldier, worker, and alate caste and two larval cDNA libraries were constructed, and approximately 15,000 randomly chosen clones were sequenced to compile an EST data base. Putative gene functions were assigned based on a BLASTX Swissprot search. Categorical in silico expression patterns for each library were compared using the R-statistic. A significant proportion of the ESTs of each caste and life stages had no significant similarity to those in existing data bases. All cDNA libraries, including those of non-reproductive worker and soldier castes, contained sequences with putative reproductive functions. Genes that showed a potential expression bias among castes included a putative antibacterial humoral response and translation elongation protein in soldiers and a chemosensory protein in alates.ConclusionsWe have expanded upon the available sequences for R. flavipes and utilized an in silico method to compare gene expression in different castes of an eusocial insect. The in silico analysis allowed us to identify several genes which may be differentially expressed and involved in caste differences. These include a gene overrepresented in the alate cDNA library with a predicted function of neurotransmitter secretion or cholesterol absorption and a gene predicted to be involved in protein biosynthesis and ligase activity that was overrepresented in the late larval stage cDNA library. The EST data base and analyses reported here will be a valuable resource for future studies on the genomics of R. flavipes and other termites.

Taxonomic distribution and origins of the extended LHC (light-harvesting complex) antenna protein superfamily

BackgroundThe extended light-harvesting complex (LHC) protein superfamily is a centerpiece of eukaryotic photosynthesis, comprising the LHC family and several families involved in photoprotection, like the LHC-like and the photosystem II subunit S (PSBS). The evolution of this complex superfamily has long remained elusive, partially due to previously missing families.ResultsIn this study we present a meticulous search for LHC-like sequences in public genome and expressed sequence tag databases covering twelve representative photosynthetic eukaryotes from the three primary lineages of plants (Plantae): glaucophytes, red algae and green plants (Viridiplantae). By introducing a coherent classification of the different protein families based on both, hidden Markov model analyses and structural predictions, numerous new LHC-like sequences were identified and several new families were described, including the red lineage chlorophyll a/b-binding-like protein (RedCAP) family from red algae and diatoms. The test of alternative topologies of sequences of the highly conserved chlorophyll-binding core structure of LHC and PSBS proteins significantly supports the independent origins of LHC and PSBS families via two unrelated internal gene duplication events. This result was confirmed by the application of cluster likelihood mapping.ConclusionsThe independent evolution of LHC and PSBS families is supported by strong phylogenetic evidence. In addition, a possible origin of LHC and PSBS families from different homologous members of the stress-enhanced protein subfamily, a diverse and anciently paralogous group of two-helix proteins, seems likely. The new hypothesis for the evolution of the extended LHC protein superfamily proposed here is in agreement with the character evolution analysis that incorporates the distribution of families and subfamilies across taxonomic lineages. Intriguingly, stress-enhanced proteins, which are universally found in the genomes of green plants, red algae, glaucophytes and in diatoms with complex plastids, could represent an important and previously missing link in the evolution of the extended LHC protein superfamily.

In silico identification of MicroRNAs and their targets in diatoms

As one of the most successful groups of microalgae in the contemporary ocean, diatoms are of ecological, biotechnological and evolutionary significance. Recent research has revealed for the first time the presence of functional silencing machinery in diatoms. Nevertheless, no microRNAs (miRNAs) participating in their gene regulation have been reported. Based on the principle of sequence conservation, previous known plant miRNAs were blasted against the expressed sequence tag databases of the marine diatoms Phaeodactylum tricornutum andThalassiosira pseudonana and according to a series of filtering criteria, 6 conserved miRNAs were identified and 5 potential target genes of them were subsequently predicted. Alignmental and phylogenetic analyses showed that the miRNA precursor sequences were unexpectedly poorly conserved and distantly related to other family members. Above all, the findings from this study will contribute not only to further research of miRNAs features and regulatory mechanisms in diatoms, but also to their evolutionary research by virtue of new molecular tools. Key words: Diatom, microRNA, evolution, bioinformatics.

High-frequency Oligonucleotides in Watermelon Expressed Sequenced Tag-unigenes Are Useful in Producing Polymorphic Polymerase Chain Reaction Markers among Watermelon Genotypes

In this study, we report a simple procedure for developing and using new types of polymerase chain reaction (PCR) primers, named “high-frequency oligonucleotides–targeting active genes” (HFO-TAG). The HFO-TAG primers were constructed by first using a “practical extraction and report language” script to identify oligonucleotides (8, 9, and 10 bases) that exist in high frequency in 4700 expressed sequence tag (EST)-unigenes of watermelon (Citrullus lanatus) fruit. This computer-based screening yielded 3162 oligonucleotides that exist 32 to 335 times in the 4700 EST-unigenes. Of these, 192 HFO-TAG primers (found 51 to 269 times in the 4700 EST-unigenes) were used to amplify genomic DNA of four closely related watermelon cultivars (Allsweet, Crimson Sweet, Charleston Gray, and Dixielee). The average number of DNA fragments produced by a single HFO-TAG primer among these four watermelon cultivars was considerably higher (an average of 5.74 bands per primer) than the number of fragments produced by intersimple sequence repeat (ISSR) or randomly amplified polymorphic DNA (RAPD) primers (an average of 2.32 or 4.15 bands per primer, respectively). The HFO-TAG primers produced a higher number of polymorphic fragments (an average of 1.77 polymorphic fragments per primer) compared with the ISSR and RAPD primers (an average of 0.89 and 0.47 polymorphic fragments per primer, respectively). Amplification of genomic DNA from 12 watermelon cultivars and two U.S. Plant Introductions with the HFO-TAG primers produced a significantly higher number of fragments than RAPD primers. Also, in PCR experiments examining the ability of primers to amplify fragments from a watermelon cDNA library, the HFO-TAG primers produced considerably more fragments (an average of 6.44 fragments per primer) compared with ISSR and RAPD primers (an average of 3.59 and 2.49 fragments per primer, respectively). These results indicate that the HFO-TAG primers should be more effective than ISSR or RAPD primers in targeting active gene loci. The extensive EST database available for a large number of plant and animal species should be a useful source for developing HFO-TAG primers that can be used in genetic mapping and phylogenic studies of important crop plants and animal species.

SSR mining in oil palm EST database: application in oil palm germplasm diversity studies

This study reports on the detection of additional expressed sequence tags (EST) derived simple sequence repeat (SSR) markers for the oil palm. A large collection of 19243 Elaeis guineensis ESTs were assembled to give 10258 unique sequences, of which 629 ESTs were found to contain 722 SSRs with a variety of motifs. Dinucleotide repeats formed the largest group (45.6%) consisting of 66.9% AG/CT, 21.9% AT/AT, 10.9% AC/GT and 0.3% CG/CG motifs. This was followed by trinucleotide repeats, which is the second most abundant repeat types (34.5%) consisting of AAG/CTT (23.3%), AGG/CCT (13.7%), CCG/CGG (11.2%), AAT/ATT (10.8%), AGC/GCT (10.0%), ACT/AGT (8.8%), ACG/CGT (7.6%), ACC/GGT (7.2%), AAC/GTT (3.6%) and AGT/ACT (3.6%) motifs. Primer pairs were designed for 405 unique EST-SSRs and 15 of these were used to genotype 105 E. guineensis and 30 E. oleifera accessions. Fourteen SSRs were polymorphic in at least one germplasm revealing a total of 101 alleles. The high percentage (78.0%) of alleles found to be specific for either E. guineensis or E. oleifera has increased the power for discriminating the two species. The estimates of genetic differentiation detected by EST-SSRs were compared to those reported previously. The transferability across palm taxa to two Cocos nucifera and six exotic palms is also presented. The polymerase chain reaction (PCR) products of three primer-pairs detected in E. guineensis, E. oleifera, C. nucifera and Jessinia bataua were cloned and sequenced. Sequence alignments showed mutations within the SSR site and the flanking regions. Phenetic analysis based on the sequence data revealed that C. nucifera is closer to oil palm compared to J. bataua; consistent with the taxanomic classification.

Quantitative gene expression profiles in real time from expressed sequence tag databases.

An accumulation of expressed sequence tag (EST) data in the public domain and the availability of bioinformatic programs have made EST gene expression profiling a common practice. However, the utility and validity of using EST databases (e.g., dbEST) has been criticized, particularly for quantitative assessment of gene expression. Problems with EST sequencing errors, library construction, EST annotation, and multiple paralogs make generation of specific and sensitive qualitative arid quantitative expression profiles a concern. In addition, most EST-derived expression data exists in previously assembled databases. The Virtual Northern Blot (VNB) (http: //tlab.bu.edu/vnb.html) allows generation, evaluation, and optimization of expression profiles in real time, which is especially important for alternatively spliced, novel, or poorly characterized genes. Representative gene families with variable nucleotide sequence identity, tissue specificity, and levels of expression (bcl-xl, aldoA, and cyp2d9) are used to assess the quality of VNB's output. The profiles generated by VNB are more sensitive and specific than those constructed with ESTs listed in preindexed databases at UCSC and NCBI. Moreover, quantitative expression profiles produced by VNB are comparable to quantization obtained from Northern blots and qPCR. The VNB pipeline generates real-time gene expression profiles for single-gene queries that are both qualitatively and quantitatively reliable.

EST-Database Search of Plant Defensins – An Example Using Sugarcane, a Large and Complex Genome

EST (Expressed Sequence Tags) databases are increasing in number and size, especially regarding cultivated plants. Sugarcane is one of the most important tropical and subtropical crops, presenting a complex polyploid genome of hybrid origin, bearing a challenge for the understanding of genetic processes in higher plants. In the present work a general search was carried out on the largest Sugarcane EST Database (SUCEST) that includes 237,954 ESTs aiming to identify defensin antimicrobial peptides - a class of small, basic, cysteine-rich peptides distributed throughout the kingdoms. Using a computational approach 17 new defensin isoforms could be identified. Main steps for the search, characterization and evaluation of the defensin expression profile are presented. Prevalent expression tissues were leaf roll, lateral bark, root apex, base of inflorescence, developing seed, and calli. Bioinformatics and phylogenetic analysis of the primary structure of the sugarcane defensin candidates as well as the 3D structures obtained by comparative modeling support their role as antimicrobials.

The Genetics, Transcriptional Profiles, and Catalytic Properties of UDP-α-d-Xylose 4-Epimerases from Barley

Abstract Cell walls in the grasses contain relatively high levels of heteroxylans and, in particular, arabinoxylans. Enzymes and corresponding genes that are involved in the provision of sugar nucleotide substrates represent potential control points for arabinoxylan biosynthesis. Following expressed sequence tag database analyses, three genes encoding barley (Hordeum vulgare) UDP-d-xylose 4-epimerases (UXE; EC 5.1.3.5), designated HvUXE1, HvUXE2, and HvUXE3, were cloned and their positions on genetic maps defined. To confirm the identity of the genes, a cDNA construct encoding HvUXE1 was expressed in Pichia pastoris. The purified, recombinant HvUXE1 catalyzed the freely reversible interconversion of UDP-α-d-xylopyranose and UDP-β -l-arabinopyranose, with K m values of 1.8 and 1.4 mm, respectively. At equilibrium, the ratio of substrate to product was approximately 1:1. Each molecule of heterologously expressed HvUXE1 enzyme contained about one molecule of noncovalently bound NAD+. Molecular modeling provided a structural rationale for the substrate specificity of the UDP-d-xylose 4-epimerase and, in particular, explained its tight specificity for UDP-xylose compared with other sugar nucleotide epimerases. Quantitative transcript analyses performed for each of the three genes in a range of organs showed, inter alia, that in developing barley endosperm HvUXE1 and HvUXE3 mRNA levels peaked at a time when UDP-α-d-xylopyranose synthase (UXS) transcripts also reached a maximum and when arabinoxylan biosynthesis was initiated. Furthermore, the data revealed that the transcription of HvUXE and HvUXS gene family members is coordinated with the incorporation of pentose sugars onto cell walls in barley leaves, roots, and developing endosperm.

EST analysis reveals putative genes involved in glycyrrhizin biosynthesis

BackgroundGlycyrrhiza uralensis is one of the most popular medicinal plants in the world and is also widely used in the flavoring of food and tobacco. Due to limited genomic and transcriptomic data, the biosynthetic pathway of glycyrrhizin, the major bioactive compound in G. uralensis, is currently unclear. Identification of candidate genes involved in the glycyrrhizin biosynthetic pathway will significantly contribute to the understanding of the biosynthetic and medicinal chemistry of this compound.ResultsWe used the 454 GS FLX platform and Titanium regents to produce a substantial expressed sequence tag (EST) dataset from the vegetative organs of G. uralensis. A total of 59,219 ESTs with an average read length of 409 bp were generated. 454 ESTs were combined with the 50,666 G. uralensis ESTs in GenBank. The combined ESTs were assembled into 27,229 unique sequences (11,694 contigs and 15,535 singletons). A total of 20,437 unique gene elements representing approximately 10,000 independent transcripts were annotated using BLAST searches (e-value ≤ 1e-5) against the SwissProt, KEGG, TAIR, Nr and Nt databases. The assembled sequences were annotated with gene names and Gene Ontology (GO) terms. With respect to the genes related to glycyrrhizin metabolism, genes encoding 16 enzymes of the 18 total steps of the glycyrrhizin skeleton synthesis pathway were found. To identify novel genes that encode cytochrome P450 enzymes and glycosyltransferases, which are related to glycyrrhizin metabolism, a total of 125 and 172 unigenes were found to be homologous to cytochrome P450s and glycosyltransferases, respectively. The cytochrome P450 candidate genes were classified into 32 CYP families, while the glycosyltransferase candidate genes were classified into 45 categories by GO analysis. Finally, 3 cytochrome P450 enzymes and 6 glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis through an organ-specific expression pattern analysis based on real-time PCR.ConclusionsUsing the 454 GS FLX platform and Titanium reagents, our study provides a high-quality EST database for G. uralensis. Based on the EST analysis, novel candidate genes related to the secondary metabolite pathway of glycyrrhizin, including novel genes encoding cytochrome P450s and glycosyltransferases, were found. With the assistance of organ-specific expression pattern analysis, 3 unigenes encoding cytochrome P450s and 6 unigenes encoding glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis.

Abstract 4767: Neo-peptides from gene fusions as cancer vaccine antigens

Abstract Background: Of all possible sources of antigens for cancer vaccines, frameshift (FS) peptides are attractive in that they may represent neo-antigens. FS peptides may be induced by insertions/deletions or by chimeric transcripts that result from mis-splicing, chromosomal translocations or trans-splicing of mRNA. Aim: In order to discover FS neo-antigens, we have taken a bioinformatical approach at identifying tumor specific, neo-peptides that are generated when chimeric transcripts are translated. Specifically we have focused our attention on identifying chimeric transcripts that create a frameshift when the exons of two different genes are joined. As a result of this gene fusion, approximately 50% of the time a frameshift occurs thus creating a neo-peptide. Method: In order to quickly identify these types of transcripts that are specific to cancer and not present in normal tissue, we have developed an algorithm that can be used to screen publically available databases and high-throughput sequencing data. As proof of principle we have applied this algorithm to the NCBI Expressed Sequence Tag database that contains cDNA sequencing information for normal and cancer tissues. Results: Based on this approach, we have identified and validated 35 chimeric transcripts that are present in breast cancer cell lines and in primary breast tumors. In addition, 9 chimeric transcripts are also present in seven different pancreatic cancer cell lines. Of the 35 chimeric transcripts, 11 chimeric transcripts produce frameshift peptides that are at least nine amino acids or longer and are predicted by SYFPEITHI and Rankpep to be immunogenic and capable of being presented in the most frequent human class I MHC alleles. Moreover, our analyses also identified chimeric transcripts that have already been confirmed by 454 sequencing for breast and prostate cancer. Discussion: Defining the chimeric transcriptome in both healthy and cancer patients could be critical for the development of new therapeutic agents. Of more interest to us, is their potential contribution to a prophylactic cancer vaccine. (This work is supported by a DoD Innovator Award and a Keck Foundation grant to SAJ) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4767.

Survey of transcripts expressed by the invasive juvenile stage of the liver fluke Fasciola hepatica

BackgroundThe common liver fluke Fasciola hepatica is the agent of a zoonosis with significant economic consequences in livestock production worldwide, and increasing relevance to human health in developing countries. Although flukicidal drugs are available, re-infection and emerging resistance are demanding new efficient and inexpensive control strategies. Understanding the molecular mechanisms underlying the host-parasite interaction provide relevant clues in this search, while enlightening the physiological adaptations to parasitism. Genomics and transcriptomics are still in their infancy in F. hepatica, with very scarce information available from the invasive newly excysted juveniles (NEJ). Here we provide an initial glimpse to the transcriptomics of the NEJ, the first stage to interact with the mammalian host.ResultsWe catalogued more than 500 clusters generated from the analysis of F. hepatica juvenile expressed sequence tags (EST), several of them not detected in the adult stage. A set of putative F. hepatica specific transcripts, and a group of sequences conserved exclusively in flatworms were identified. These novel sequences along with a set of parasite transcripts absent in the host genomes are putative new targets for future anti-parasitic drugs or vaccine development.Comparisons of the F. hepatica sequences with other metazoans genomes or EST databases were consistent with the basal positioning of flatworms in the bilaterian phylogeny. Notably, GC content, codon usage and amino acid frequencies are remarkably different in Schistosomes to F. hepatica and other trematodes.Functional annotation of predicted proteins showed a general representation of diverse biological functions. Besides proteases and antioxidant enzymes expected to participate in the early interaction with the host, various proteins involved in gene expression, protein synthesis, cell signaling and mitochondrial enzymes were identified. Differential expression of secreted protease gene family members between juvenile and adult stages may respond to different needs during host colonization.ConclusionThe knowledge of the genes expressed by the invasive stage of Fasciola hepatica is a starting point to unravel key aspects of this parasite's biology. The integration of the emerging transcriptomics, and proteomics data and the advent of functional genomics tools in this organism are positioning F. hepatica as an interesting model for trematode biology.

Alkylresorcinol Synthases Expressed in Sorghum bicolor Root Hairs Play an Essential Role in the Biosynthesis of the Allelopathic Benzoquinone Sorgoleone

Sorghum bicolor is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone, which likely accounts for many of the allelopathic properties of Sorghum spp. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells and involves the production of an alkylresorcinolic intermediate (5-[(Z,Z)-8',11',14'-pentadecatrienyl]resorcinol) derived from an unusual 16:3Delta(9,12,15) fatty acyl-CoA starter unit. This led to the suggestion of the involvement of one or more alkylresorcinol synthases (ARSs), type III polyketide synthases (PKSs) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. In an effort to characterize the enzymes responsible for the biosynthesis of the pentadecyl resorcinol intermediate, a previously described expressed sequence tag database prepared from isolated S. bicolor (genotype BTx623) root hairs was first mined for all PKS-like sequences. Quantitative real-time RT-PCR analyses revealed that three of these sequences were preferentially expressed in root hairs, two of which (designated ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units in recombinant enzyme studies. Furthermore, RNA interference experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 are likely to participate in the biosynthesis of sorgoleone in planta. The sequences of ARS1 and ARS2 were also used to identify several rice (Oryza sativa) genes encoding ARSs, which are likely involved in the production of defense-related alkylresorcinols.

Development of Type I Genetic Markers from Expressed Sequence Tags in Highly Polymorphic Species

Expressed sequence tag (EST) databases provide a primary source of nuclear DNA sequences for genetic marker development in non-model organisms. To date, the process has been relatively inefficient for several reasons: 1) priming site polymorphism in the template leads to inferior or erratic amplification; 2) introns in the target amplicon are too large and/or numerous to allow effective amplification under standard screening conditions; and 3) at least occasionally, a PCR primer straddles an exon-intron junction and is unable to bind to genomic DNA template. The first is only a minor issue for species or strains with low heterozygosity but becomes a significant problem for species with high genomic variation, such as marine organisms with extremely large effective population sizes. Problems arising from unanticipated introns are unavoidable but are most pronounced in intron-rich species, such as vertebrates and lophotrochozoans. We present an approach to marker development in the Pacific oyster Crassostrea gigas, a highly polymorphic and intron-rich species, which minimizes these problems, and should be applicable to other non-model species for which EST databases are available. Placement of PCR primers in the 3' end of coding sequence and 3' UTR improved PCR success rate from 51% to 97%. Almost all (37 of 39) markers developed for the Pacific oyster were polymorphic in a small test panel of wild and domesticated oysters.

Development of EST-derived simple sequence repeat markers for wheat leaf rust fungus, Puccinia triticina Eriks

Gene-associated simple sequence repeat (SSR) markers were developed for Puccinia triticina through the data mining of existing EST libraries. Analysis of 7134 expressed sequence tags (ESTs) from cDNA libraries of P. triticina detected 204 EST-SSRs with a minimum of 12 repeating nucleotides. The majority of EST-SSRs contained short di- or tri-nucleotide repeats. These EST-SSRs were evaluated on 35 P. triticina isolates collected in Canada and 21 EST-SSRs were polymorphic and informative in determining intraspecific genetic diversity. A comparison of virulence and EST-SSR genotypes showed a strong correlation between virulence to Lr2a, Lr2c and Lr17a and EST-SSRs genotypes. The differentiation of the P. triticina population based on EST-SSR genotypes was comparable to that obtained with genomic SSRs, despite differences between two types of SSR markers. Eight of the 21 EST-SSRs produced the cross amplification in Puccinia coronata and Puccinia graminis, suggesting that EST-SSRs are more applicable than genomic SSRs for interspecific analysis. In summary, our study suggests that the data mining of EST databases is a feasible way to generate informative molecular markers for genetic studies of P. triticina.

Development of Juglans Regia SSR Markers by Data Mining of the EST Database

Walnut (Juglans regia), an economically important woody plant, is widely cultivated in temperate regions for its timber and nutritional fruits. Despite abundant studies in germplasm, systemic molecular evaluations of walnut are sparsely reported mainly due to the limited molecular markers available. Expressed sequence tags (EST) provide a valuable resource for developing simple sequence repeat (SSR) markers. In this study, a total of 5,025 walnut ESTs (covering 16.41 Mb) were retrieved from the National Center for Biotechnology Information database. The SSR motifs were then analyzed by the SSRHunter software. In total, 398 SSRs were obtained with an average frequency of 1/4.08 kb. Dinucleotide (di-) repeat motifs accounted for 69.85% of all SSRs, followed by trinucleotide (tri-) with a frequency of 27.64%, while low frequency (2.51%) of tetranucleotide (tetra-) to hexanucleotide (hexa-) was observed. Meanwhile, GCA and TC motifs were prevalent among di- and tri- loci, respectively. Subsequently, a total of 123 primer pairs were designed from the non-redundant SSR-containing unigenes with the selection threshold of SSR length set to 10 bp or more. To examine the efficiency of candidate markers, seven DNA pools were collected from geographically different accessions. Results demonstrated that 41 SSR primer sets could generate high polymorphic amplification products (33.3%), and these polymorphic loci were mainly located in the 3′-untranslated region. Annotation analysis revealed that only two of these 41 loci were located inside open reading frames of characterized proteins (E ≤ 1E−30).

Construction of a genetic linkage map using simple sequence repeat markers from expressed sequence tags for cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta) is an economically important crop that is grown in tropical and sub-tropical regions. Use of molecular technology for genetic improvement of cassava has been limited by the lack of a large set of DNA markers and a genetic map. Therefore, the aims here were to develop additional simple sequence repeat (SSR) markers from the public expressed sequence tags (ESTs), and to construct a genetic linkage map. In this study, we designed 425 EST-SSR markers from sequences obtained from the cassava EST database in GenBank, and integrated them with 667 SSR markers from a microsatellite-enriched genomic sequence received from the International Center for Tropical Agriculture (CIAT). Of these, 107 EST-SSR and 500 genomic SSR primer pairs showed polymorphic patterns when screened in two cassava varieties, Hauy Bong 60 and Hanatee, which were used as female and male parental lines, respectively. Within the 107 and 500 primer pairs, 81 and 226 EST-SSR and SSR primer pairs were successfully genotyped with 100 samples of F1 progeny, respectively. The results showed 20 linkage groups consisting of 211 markers—56 EST-SSR and 155 SSR markers—spanning 1,178 cM, with an average distance between markers of 5.6 cM and about 11 markers per linkage group. These novel EST-SSR markers provided genic PCR-based co-dominant markers that were useful, reliable and economical. The EST-SSRs were used together with SSR markers to construct the cassava genetic linkage map which will be useful for the identification of quantitative trait loci controlling the traits of interest in cassava breeding programs.

An efficient method for developing SNP markers based on EST data combined with high resolution melting (HRM) analysis

BackgroundIn order to identify single nucleotide polymorphisms (SNPs) efficiently from a species with a large genome, SNPs were identified from an expressed sequence tag (EST) database combined with High Resolution Melting (HRM) analysis.FindingsA total of 574 sequence tagged sites (STSs) were generated from Cryptomeria japonica and HRM analysis was used to screen for polymorphisms in these STS markers. STSs were designed in two ways: 1) putative SNP sites were identified by comparing ESTs from specific contigs, then 226 primer pairs designed for the purpose to amplify these SNPs; 2) 348 primer pairs were randomly designed using reads from the 3' end of cDNA. HRM analysis revealed that 325 markers among eight individuals were polymorphic, and that STSs, including putative SNP sites, exhibited higher levels of polymorphism.ConclusionOur results indicate that the combination of SNP screening from an EST database combined with HRM analysis is a highly efficient way to develop SNP markers for expressed genes. This method will contribute to both genetic mapping and the identification of SNPs in non-model organisms.