Public Expressed Sequence Tag Research Articles

ESAP plus: a web-based server for EST-SSR marker development.

BackgroundSimple sequence repeats (SSRs) have become widely used as molecular markers in plant genetic studies due to their abundance, high allelic variation at each locus and simplicity to analyze using conventional PCR amplification. To study plants with unknown genome sequence, SSR markers from Expressed Sequence Tags (ESTs), which can be obtained from the plant mRNA (converted to cDNA), must be utilized. With the advent of high-throughput sequencing technology, huge EST sequence data have been generated and are now accessible from many public databases. However, SSR marker identification from a large in-house or public EST collection requires a computational pipeline that makes use of several standard bioinformatic tools to design high quality EST-SSR primers. Some of these computational tools are not users friendly and must be tightly integrated with reference genomic databases.ResultsA web-based bioinformatic pipeline, called EST Analysis Pipeline Plus (ESAP Plus), was constructed for assisting researchers to develop SSR markers from a large EST collection. ESAP Plus incorporates several bioinformatic scripts and some useful standard software tools necessary for the four main procedures of EST-SSR marker development, namely 1) pre-processing, 2) clustering and assembly, 3) SSR mining and 4) SSR primer design. The proposed pipeline also provides two alternative steps for reducing EST redundancy and identifying SSR loci. Using public sugarcane ESTs, ESAP Plus automatically executed the aforementioned computational pipeline via a simple web user interface, which was implemented using standard PHP, HTML, CSS and Java scripts. With ESAP Plus, users can upload raw EST data and choose various filtering options and parameters to analyze each of the four main procedures through this web interface. All input EST data and their predicted SSR results will be stored in the ESAP Plus MySQL database. Users will be notified via e-mail when the automatic process is completed and they can download all the results through the web interface.ConclusionsESAP Plus is a comprehensive and convenient web-based bioinformatic tool for SSR marker development. ESAP Plus offers all necessary EST-SSR development processes with various adjustable options that users can easily use to identify SSR markers from a large EST collection. With familiar web interface, users can upload the raw EST using the data submission page and visualize/download the corresponding EST-SSR information from within ESAP Plus. ESAP Plus can handle considerably large EST datasets. This EST-SSR discovery tool can be accessed directly from: http://gbp.kku.ac.th/esap_plus/.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3328-4) contains supplementary material, which is available to authorized users.

Mining microsatellite markers from public expressed sequence tags databases for the study of threatened plants.

BackgroundSimple Sequence Repeats (SSRs) are widely used in population genetic studies but their classical development is costly and time-consuming. The ever-increasing available DNA datasets generated by high-throughput techniques offer an inexpensive alternative for SSRs discovery. Expressed Sequence Tags (ESTs) have been widely used as SSR source for plants of economic relevance but their application to non-model species is still modest.MethodsHere, we explored the use of publicly available ESTs (GenBank at the National Center for Biotechnology Information-NCBI) for SSRs development in non-model plants, focusing on genera listed by the International Union for the Conservation of Nature (IUCN). We also search two model genera with fully annotated genomes for EST-SSRs, Arabidopsis and Oryza, and used them as controls for genome distribution analyses. Overall, we downloaded 16 031 555 sequences for 258 plant genera which were mined for SSRsand their primers with the help of QDD1. Genome distribution analyses in Oryza and Arabidopsis were done by blasting the sequences with SSR against the Oryza sativa and Arabidopsis thaliana reference genomes implemented in the Basal Local Alignment Tool (BLAST) of the NCBI website. Finally, we performed an empirical test to determine the performance of our EST-SSRs in a few individuals from four species of two eudicot genera, Trifolium and Centaurea.ResultsWe explored a total of 14 498 726 EST sequences from the dbEST database (NCBI) in 257 plant genera from the IUCN Red List. We identify a very large number (17 102) of ready-to-test EST-SSRs in most plant genera (193) at no cost. Overall, dinucleotide and trinucleotide repeats were the prevalent types but the abundance of the various types of repeat differed between taxonomic groups. Control genomes revealed that trinucleotide repeats were mostly located in coding regions while dinucleotide repeats were largely associated with untranslated regions. Our results from the empirical test revealed considerable amplification success and transferability between congenerics.ConclusionsThe present work represents the first large-scale study developing SSRs by utilizing publicly accessible EST databases in threatened plants. Here we provide a very large number of ready-to-test EST-SSR (17 102) for 193 genera. The cross-species transferability suggests that the number of possible target species would be large. Since trinucleotide repeats are abundant and mainly linked to exons they might be useful in evolutionary and conservation studies. Altogether, our study highly supports the use of EST databases as an extremely affordable and fast alternative for SSR developing in threatened plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2031-1) contains supplementary material, which is available to authorized users.

A Long-Read Transcriptome Assembly of Cotton (Gossypium hirsutum L.) and Intraspecific Single Nucleotide Polymorphism Discovery.

Upland cotton (Gossypium hirsutum L.) has a narrow germplasm base, which constrains marker development and hampers intraspecific breeding. A pressing need exists for high-throughput single nucleotide polymorphism (SNP) markers that can be readily applied to germplasm in breeding and breeding-related research programs. Despite progress made in developing new sequencing technologies during the past decade, the cost of sequencing remains substantial when one is dealing with numerous samples and large genomes. Several strategies have been proposed to lower the cost of sequencing for multiple genotypes of large-genome species like cotton, such as transcriptome sequencing and reduced-representation DNA sequencing. This paper reports the development of a transcriptome assembly of the inbred line Texas Marker-1 (TM-1), a genetic standard for cotton, its usefulness as a reference for RNA sequencing (RNA-seq)-based SNP identification, and the availability of transcriptome sequences of four other cotton cultivars. An assembly of TM-1 was made using Roche 454 transcriptome reads combined with an assembly of all available public expressed sequence tag (EST) sequences of TM-1. The TM-1 assembly consists of 72,450 contigs with a total of 70 million bp. Functional predictions of the transcripts were estimated by alignment to selected protein databases. Transcriptome sequences of the five lines, including TM-1, were obtained using an Illumina Genome Analyzer-II, and the short reads were mapped to the TM-1 assembly to discover SNPs among the five lines. We identified >14,000 unfiltered allelic SNPs, of which ∼3,700 SNPs were retained for assay development after applying several rigorous filters. This paper reports availability of the reference transcriptome assembly and shows its utility in developing intraspecific SNP markers in upland cotton.

Identification of expressed resistance gene analogs (RGA) and development of RGA-SSR markers in tobacco

Tobacco is an important cash crop and an ideal experimental system for studies of plant-pathogen interactions. Identification of tobacco resistance (R) genes and resistance gene analogs (RGAs) is propitious to elucidate the underlying resistant mechanisms. In recent years, the public tobacco EST (expressed sequence tags) data set, which provides a rich source for identifying expressed RGAs, has enlarged substantially. In this study, 149606 Uni-ESTs were assembled from 412325 tobacco ESTs available in GenBank, scanned with 112 published plant R-genes protein sequences, and 1113 Nicotiana (tobacco) RGAs (NtRGAs) were identified. The majority of them comprised the common R-genes domains, such as NBS-LRR, LRR-PK, LRR, PK and Mlo, while we were unable to identify 109 RGAs using published domains of R-genes. Upon sequence alignment, 1079 NtRGAs were allocated on 712 loci within the Nicotiana benthamiana genome. A total of 78 simple sequence repeats (SSRs) were identified from 72 NtRGAs, and out of 64 newly designed primer pairs, 54 primer pairs generated clear bands upon PCR amplification using tobacco genomic DNA. Only nine primer pairs displayed polymorphism in 24 varieties of tobacco, with 2-4 alleles per locus (2.56 alleles on average), while 41 primer pairs were able to detect polymorphisms in six wild species of genus Nicotiana, with 2-4 alleles per locus (2.61 alleles on average).

Identification and cross-species amplification of microsatellite markers derived from expressed sequence data of rose species

Genic SSR markers derived from public expressed sequence tags (ESTs) data are valuable and cost effective marker resources for genome mapping and diversity studies. Owing to their derivation from the transcribed regions which often have putative functions, these markers can be easily associated with desired trait. In the present study, 19 novel SSR markers were identified from 450 non redundant unigenes derived from 3,726 public ESTs of two rose species. Among SSRs, tri-repeats (61.3 %) were most abundant followed by di-repeat (29 %). Newly identified EST-SSR markers recorded significant homology with the known/putative proteins of Arabidopsis thaliana. The cross transferability to 12 rose species ranged from 63.2 to 100 %. Novel SSR loci found to be moderately to highly polymorphic with locus wise average number of alleles and polymorphism information content (PIC) were 4.1 and 0.33, respectively. Cloning and sequencing of EST-SSR size variant amplicons of marker locus Rches12 revealed that the variation in the number of SSR repeat-units was the main source of fragment polymorphism. The high polymorphic potential coupled with high cross-transferability rate demonstrates wider applicability of novel SSR markers in genetic diversity, genome mapping and evolutionary studies in various rose species.

CancerEST: a web-based tool for automatic meta-analysis of public EST data.

The identification of cancer-restricted biomarkers is fundamental to the development of novel cancer therapies and diagnostic tools. The construction of comprehensive profiles to define tissue- and cancer-specific gene expression has been central to this. To this end, the exploitation of the current wealth of ‘omic’-scale databases can be facilitated by automated approaches, allowing researchers to directly address specific biological questions. Here we present CancerEST, a user-friendly and intuitive web-based tool for the automated identification of candidate cancer markers/targets, for examining tissue specificity as well as for integrated expression profiling. CancerEST operates by means of constructing and meta-analyzing expressed sequence tag (EST) profiles of user-supplied gene sets across an EST database supporting 36 tissue types. Using a validation data set from the literature, we show the functionality and utility of CancerEST.Database URL: http://www.cancerest.org.uk

Development and utilization of genomic and genic microsatellite markers in Assam tea (Camellia assamica ssp. assamica) and related Camellia species

AbstractMicrosatellite or simple sequence repeat (SSR) markers are valuable tools for many purposes, such as phylogenetic, fingerprinting and molecular breeding studies. However, such marker resources are unavailable in Assam tea (Camellia assamica ssp. assamica; Masters). With an objective to enrich the repertoire of microsatellite markers in traditional tea, 185 novel microsatellite (150 genomic and 35 genic) markers were identified from (GA)n‐enriched genomic libraries and public expressed sequence data in Assam tea. High‐quality 0.412‐Mb non‐redundant (NR) genomic data set derived from nucleotide sequencing of 1297 (GA)n‐enriched genomic positive clones and 2723 unigenes (1.33 Mb) predicted from 10 803 random public expressed sequence tags (ESTs) in C. assamica ssp. assamica were utilized for identification of genomic and genic microsatellite markers, respectively. The average number of alleles and polymorphic information content (PIC) recorded for the newly developed SSR markers were 6.17 and 0.398, respectively. The average observed (Ho) and expected (He) heterozygosity varied from 0.626 to 0.697, respectively. These markers were found to be highly transferable (74.5–100%) to cultivated (C. sinensis, C. assamica ssp. lasiocalyx) and five wild Camellia species. Genetic diversity coefficient detected a high level of divergence in 24 cultivated tea accessions (69.3%). Phylogenetic analysis revealed that major groupings were broadly in accordance with taxonomic classification of tea, and all the wild Camellia species remained as an out‐group. The high polymorphic content coupled with high rate of cross‐transferability demonstrates wider applicability of novel microsatellite markers in genotyping, genetic diversity, genome mapping and evolutionary studies in various Camellia species.

De novo assembly and characterization of Camelina sativatranscriptome by paired-end sequencing

BackgroundBiofuels extracted from the seeds of Camelina sativa have recently been used successfully as environmentally friendly jet-fuel to reduce greenhouse gas emissions. Camelina sativa is genetically very close to Arabidopsis thaliana, and both are members of the Brassicaceae. Although public databases are currently available for some members of the Brassicaceae, such as A. thaliana, A. lyrata, Brassica napus, B. juncea and B. rapa, there are no public Expressed Sequence Tags (EST) or genomic data for Camelina sativa. In this study, a high-throughput, large-scale RNA sequencing (RNA-seq) of the Camelina sativa transcriptome was carried out to generate a database that will be useful for further functional analyses.ResultsApproximately 27 million clean “reads” filtered from raw reads by removal of adaptors, ambiguous reads and low-quality reads (2.42 gigabase pairs) were generated by Illumina paired-end RNA-seq technology. All of these clean reads were assembled de novo into 83,493 unigenes and 103,196 transcripts using SOAPdenovo and Trinity, respectively. The average length of the transcripts generated by Trinity was 697 bp (N50 = 976), which was longer than the average length of unigenes (319 bp, N50 = 346 bp). Nonetheless, the assembly generated by SOAPdenovo produced similar number of non-redundant hits (22,435) with that of Trinity (22,433) in BLASTN searches of the Arabidopsis thaliana CDS sequence database (TAIR). Four public databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-prot, NCBI non-redundant protein (NR), and the Cluster of Orthologous Groups (COG), were used for unigene annotation; 67,791 of 83,493 unigenes (81.2%) were finally annotated with gene descriptions or conserved protein domains that were mapped to 25,329 non-redundant protein sequences. We mapped 27,042 of 83,493 unigenes (32.4%) to 119 KEGG metabolic pathways.ConclusionsThis is the first report of a transcriptome database for Camelina sativa, an environmentally important member of the Brassicaceae. We showed that C. savita is closely related to Arabidopsis spp. and more distantly related to Brassica spp. Although the majority of annotated genes had high sequence identity to those of A. thaliana, a substantial proportion of disease-resistance genes (NBS-encoding LRR genes) were instead more closely similar to the genes of other Brassicaceae; these genes included BrCN, BrCNL, BrNL, BrTN, BrTNL in B. rapa. As plant genomes are under long-term selection pressure from environmental stressors, conservation of these disease-resistance genes in C. sativa and B. rapa genomes implies that they are exposed to the threats from closely-related pathogens in their natural habitats.

Mining microsatellite markers from public expressed sequence tag sequences for genetic diversity analysis in pomegranate

Mining microsatellite markers from public expressed sequence tag sequences for genetic diversity analysis in pomegranate

Transcriptome Sequencing and Characterization for the Sea Cucumber Apostichopus japonicus (Selenka, 1867)

BackgroundSea cucumbers are a special group of marine invertebrates. They occupy a taxonomic position that is believed to be important for understanding the origin and evolution of deuterostomes. Some of them such as Apostichopus japonicus represent commercially important aquaculture species in Asian countries. Many efforts have been devoted to increasing the number of expressed sequence tags (ESTs) for A. japonicus, but a comprehensive characterization of its transcriptome remains lacking. Here, we performed the large-scale transcriptome profiling and characterization by pyrosequencing diverse cDNA libraries from A. japonicus.ResultsIn total, 1,061,078 reads were obtained by 454 sequencing of eight cDNA libraries representing different developmental stages and adult tissues in A. japonicus. These reads were assembled into 29,666 isotigs, which were further clustered into 21,071 isogroups. Nearly 40% of the isogroups showed significant matches to known proteins based on sequence similarity. Gene ontology (GO) and KEGG pathway analyses recovered diverse biological functions and processes. Candidate genes that were potentially involved in aestivation were identified. Transcriptome comparison with the sea urchin Strongylocentrotus purpuratus revealed similar patterns of GO term representation. In addition, 4,882 putative orthologous genes were identified, of which 202 were not present in the non-echinoderm organisms. More than 700 simple sequence repeats (SSRs) and 54,000 single nucleotide polymorphisms (SNPs) were detected in the A. japonicus transcriptome.ConclusionPyrosequencing was proven to be efficient in rapidly identifying a large set of genes for the sea cucumber A. japonicus. Through the large-scale transcriptome sequencing as well as public EST data integration, we performed a comprehensive characterization of the A. japonicus transcriptome and identified candidate aestivation-related genes. A large number of potential genetic markers were also identified from the A. japonicus transcriptome. This transcriptome resource would lay an important foundation for future genetic or genomic studies on this species.

Discovery, validation, and in silico functional characterization of EST-SSR markers in Eucalyptus globulus

Eucalyptus globulus is the most commonly planted hardwood species for pulpwood in temperate regions. We aimed to develop and characterize functional molecular markers for population genetic analyses and molecular breeding in this model tree species. Public expressed sequence tag (EST) databases were screened for nonredundant sequences to predict putative gene functions and to discover simple sequence repeats (EST-SSRs), which were then validated in E. globulus and six other Eucalyptus species. A total of 4,924 nonredundant sequences were identified from 12,690 updated E. globulus ESTs. Approximately 19.3% (952) were unigenes and contained 1,140 EST-SSR markers, which were mainly trimeric (58.6%). A set of 979 primers for putative SSR markers was designed after bioinformatic analysis. The predicted functions of these ESTs containing SSR were classified according to their gene ontology (GO) categories (biological process, molecular function, and cellular component). GO categories were assigned to 226 ESTs (30.2%). Most ESTs containing SSR (78.7%) had significant matches (E ≤ 10−5) with the nonredundant protein database using BLASTX. From a set of 56 random primer pairs, 37 could be validated in eight E. globulus genotypes and were also tested for cross-transferability to other six Eucalyptus species (Eucalyptus grandis, Eucalyptus saligna, Eucalyptus dunnii, Eucalyptus viminalis, Eucalyptus camaldulensis, Eucalyptus tereticornis). Seventeen polymorphic EST-SSR markers for E. globulus were evaluated in 60 unrelated trees, being representative of the species’ natural distribution. As a result, six highly informative markers were proposed for genetic diversity analyses, fingerprinting, and comparative population studies, between different species of E. globulus.

Microsatellite marker development in the protozoan parasite Perkinsus olseni

The analysis of an enriched partial genomic library and of public expressed sequence tag (EST) resources allowed the characterization of the first microsatellite loci in the protozoan parasite Perkinsus olseni. Clonal cultures from laboratory isolates derived from infected clams Ruditapes decussatus (from Spain), R. philippinarum (from Spain and Japan), and Austrovenus stutchburyi (from New Zealand) were used for the characterization of 12 microsatellites. Low variation was detected at most loci, with the number of alleles at polymorphic loci ranging from 2 to 7 (average 3.20 +/- 0.51) and gene diversity from 0.11 to 0.79 (average 0.40 +/- 0.07). Preliminary results show that (1) isolates of P. olseni are diploid cells, and (2) multiple infections can occur within a single host. Eight of the loci analyzed successfully cross-amplified in the congeneric species P. mediterraneus. These microsatellite markers will be useful to analyze in detail the population genetic structure of P. olseni, crucial for the efficient management of this parasitic disease.

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.

Putative Vitis vinifera Rop- and Rab-GAP-, GEF-, and GDI-interacting proteins uncovered with novel methods for public genomic and EST database analysis

To understand how grapevine Rop and Rab proteins achieve their functional versatility in signalling, identification of the putative VvRop- and VvRab-interacting proteins was performed using newly designed tools. In this study, sequences encoding eight full-length proteins for VvRop GTPase-activating proteins (GAPs), five for VvRabGAPs, six for VvRop guanine nucleotide exchange factors (GEFs), one for VvRabGEF, five for VvRop GDP dissociation inhibitors (GDIs), and three for VvRabGDIs were identified. These proteins had a CRIB motif or PH domain, a TBC domain, a PRONE domain, a DENN domain, or GDI signatures, respectively. By bootstrap analysis, an unrooted consensus phylogenetic tree was constructed which indicated that VvRopGDIs and VvRopGEFs--but not VvRopGAP--belonged to the same clade, and that VvRabGEF1 protein was more closely related to VvRopGAPs than to the other putative VvRab-interacting proteins. Twenty-two genes out of 28 encoding putative VvRop- and VvRab-interacting proteins could be located on identified grapevine chromosomes. Generally one gene was anchored on one chromosome, but in some cases up to four genes were located on the same chromosome. Expression patterns of the genes encoding putative VvRop- and VvRab-interacting proteins were also examined using a newly developed tool based on public expressed sequence tag (EST) database analysis. Expression patterns were sometimes found to be specific to an organ or a developmental stage. Although some limitations exist, the use of EST database analysis is stressed, in particular in the case of species where expression data are obtained at high costs in terms of time and effort.

Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing

BackgroundIn-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage.ResultsTo explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20–454 pyrosequencing runs of a cDNA library obtained from 2 week-old Palomero Toluqueño maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20–454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20–454 sequences and corresponding levels of gene expression.ConclusionA protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.

Microsatellite Characterization and Marker Development from Public EST and WGS Databases in the Reef-Building Coral Acropora millepora (Cnidaria, Anthozoa, Scleractinia)

Mining for microsatellites (also called simple sequence repeats [SSRs]) in public sequence databases of a common Indo-Pacific coral Acropora millepora identified 191 SSRs from 10 258 expressed sequence tag (EST) and 618 SSRs from 14 625 whole-genome shotgun (WGS) sequences. In contrast to other animals, trinucleotide repeats, rather than dinucleotide repeats, are dominant in the WGS-SSRs, and AAT is the most frequent trinucleotide motif in EST-SSRs. We successfully developed 40 polymorphic markers from EST-SSRs and WGS-SSRs. Both EST- and WGS-SSRs show high levels of polymorphism within corals from the same reef patch. Interestingly, markers WGS079 and WGS227 revealed SSR duplications in a few individuals, suggesting recent duplication events. Genotypic linkage disequilibrium was identified in 5 pairs of SSR markers, which will be invaluable for high-resolution studies of genetic admixture in natural populations of A. millepora. Transferability analysis showed that 25 of these markers can be successfully amplified in one of the most ubiquitous Indo-Pacific corals Acropora hyacinthus. The marker collection reported here is the largest ever developed for any reef-building coral. It holds great potential for addressing coral reef connectivity across the Indo-Pacific with an unprecedented precision, especially taking into account the cross-species transferability of a substantial number of markers.

EST, COSII, and arbitrary gene markers give similar estimates of nucleotide diversity in cultivated tomato (Solanum lycopersicum L.)

Because cultivated tomato (Solanum lycopersicum L.) is low in genetic diversity, public, verified single nucleotide polymorphism (SNP) markers within the species are in demand. To promote marker development we resequenced approximately 23 kb in a diverse set of 31 tomato lines including TA496. Three classes of markers were sampled: (1) 26 expressed-sequence tag (EST), all of which were predicted to be polymorphic based on TA496, (2) 14 conserved ortholog set II (COSII) or unigene, and (3) ten published sequences, composed of nine fruit quality genes and one anonymous RFLP marker. The latter two types contained mostly noncoding DNA. In total, 154 SNPs and 34 indels were observed. The distributions of nucleotide diversity estimates among marker types were not significantly different from each other. Ascertainment bias of SNPs was evaluated for the EST markers. Despite the fact that the EST markers were developed using SNP prediction within a sample consisting of only one TA496 allele and one additional allele, the majority of polymorphisms in the 26 EST markers were represented among the other 30 tomato lines. Fifteen EST markers with published SNPs were more closely examined for bias. Mean SNP diversity observations were not significantly different between the original discovery sample of two lines (53 SNPs) and the 31 line diversity panel (56 SNPs). Furthermore, TA496 shared its haplotype with at least one other line at 11 of the 15 markers. These data demonstrate that public EST databases and noncoding regions are a valuable source of unbiased SNP markers in tomato.

Wounding‐mediated gene expression and accelerated viviparous reproduction of the pea aphid Acyrthosiphon pisum

Most insects mount a potent antimicrobial defence upon contact with microbes or microbe-associated pattern molecules. Using a combined set of methods for analysis of insect innate immunity, we report here that piercing of the pea aphid Acyrthosiphon pisum with a bacteria-contaminated needle elicits lysozyme-like activity in the haemolymph but no detectable activities against live bacteria. Confirming these results, we found no homologues of known antimicrobial peptides in our cDNA library generated by using the suppression subtractive hybridization method or in over 90,000 public expressed sequence tag (EST) sequences, but lysozyme genes have recently been described in A. pisum. Interestingly, we discovered that production of viviparous offspring was significantly accelerated upon wounding. Therefore, we postulate that aphids may increase terminal reproductive investment and limit antibacterial defence in response to a threat to their survival.

Large-scale identification of polymorphic microsatellites using an in silico approach

BackgroundSimple Sequence Repeat (SSR) or microsatellite markers are valuable for genetic research. Experimental methods to develop SSR markers are laborious, time consuming and expensive. In silico approaches have become a practicable and relatively inexpensive alternative during the last decade, although testing putative SSR markers still is time consuming and expensive. In many species only a relatively small percentage of SSR markers turn out to be polymorphic. This is particularly true for markers derived from expressed sequence tags (ESTs). In EST databases a large redundancy of sequences is present, which may contain information on length-polymorphisms in the SSR they contain, and whether they have been derived from heterozygotes or from different genotypes. Up to now, although a number of programs have been developed to identify SSRs in EST sequences, no software can detect putatively polymorphic SSRs.ResultsWe have developed PolySSR, a new pipeline to identify polymorphic SSRs rather than just SSRs. Sequence information is obtained from public EST databases derived from heterozygous individuals and/or at least two different genotypes. The pipeline includes PCR-primer design for the putatively polymorphic SSR markers, taking into account Single Nucleotide Polymorphisms (SNPs) in the flanking regions, thereby improving the success rate of the potential markers. A large number of polymorphic SSRs were identified using publicly available EST sequences of potato, tomato, rice, Arabidopsis, Brassica and chicken.The SSRs obtained were divided into long and short based on the number of times the motif was repeated. Surprisingly, the frequency of polymorphic SSRs was much higher in the short SSRs.ConclusionPolySSR is a very effective tool to identify polymorphic SSRs. Using PolySSR, several hundred putative markers were developed and stored in a searchable database. Validation experiments showed that almost all markers that were indicated as putatively polymorphic by polySSR were indeed polymorphic. This greatly improves the efficiency of marker development, especially in species where there are low levels of polymorphism, like tomato. When combined with the new sequencing technologies PolySSR will have a big impact on the development of polymorphic SSRs in any species.PolySSR and the polymorphic SSR marker database are available from .

SAGE transcript profiling of the juvenile cambial region of Eucalyptus grandis

Despite the importance of Eucalyptus spp. in the pulp and paper industry, functional genomic approaches have only recently been applied to understand wood formation in this genus. We attempted to establish a global view of gene expression in the juvenile cambial region of Eucalyptus grandis Hill ex Maiden. The expression profile was obtained from serial analysis of gene expression (SAGE) library data produced from 3- and 6-year-old trees. Fourteen-base expressed sequence tags (ESTs) were searched against public Eucalyptus ESTs and annotated with GenBank. Altogether 43,304 tags were generated producing 3066 unigenes with three or more copies each, 445 with a putative identity, 215 with unknown function and 2406 without an EST match. The expression profile of the juvenile cambial region revealed the presence of highly frequent transcripts related to general metabolism and energy metabolism, cellular processes, transport, structural components and information pathways. We made a quantitative analysis of a large number of genes involved in the biosynthesis of cellulose, pectin, hemicellulose and lignin. Our findings provide insight into the expression of functionally related genes involved in juvenile wood formation in young fast-growing E. grandis trees.