Set Of Expressed Sequence Tags Research Articles

Advancements of Breeding and Genomics in Wheat (Triticum aestivum L.): Enhancing Yield and Nutritional Value for Sustainable Agriculture

Advancements in wheat breeding and genomics presently explores the genomic interventions driving focusing on quantitative trait loci (QTL) mapping, marker-assisted selection (MAS) and genomic selection (GS). QTL mapping emerges as a pivotal method for pinpointing markers linked with desirable traits, facilitating MAS. Furthermore, genomic selection (GS) holds immense potential for crop improvement. It also delves into the current landscape of MAS and explores various prospects of GS for wheat biofortification. Looking ahead, accelerated mapping studies combined with MAS and GS schemes are poised to further enhance wheat breeding efficiency. Dense molecular maps and a large set of ESTs (Expressed Sequence Tags) have enabled genome-wide identification of gene-rich and gene-poor regions, as well as QTL, including eQTL (Expression quantitative trait loci). Additionally, markers associated with major economic traits have facilitated MAS programs in some countries and enabled map-based cloning of several genes/QTL. Resources for functional genomics, such as TILLING and RNA interference (RNAi), alongside emerging approaches like epigenetics and association mapping, are further enriching wheat genomics research. In this review, we initially present cutting-edge genome-editing technologies in crop plants, with a specific focus on wheat, addressing both functional genomics and genetic enhancement. We subsequently delineate the utilization of additional technologies, including GWAS, high-throughput genotyping and phenotyping, speed breeding, and synthetic biology, within the context of wheat breeding. We assert that integrating genome editing with other molecular breeding strategies will significantly expedite the genetic enhancement of wheat, thus contributing to sustainable global production.

Exploitation of Walnut (Juglans regia L.) Expressed Sequence Tags for Development of SSR Markers After In Silico Analysis

The walnut (Juglans regia) has been extensively characterized for expressed sequence tags (EST) sequences and currently 6169492 ESTs are available in National Center for Biotechnology Information. Although this is a valuable resource for marker development, the redundancy in sequences makes the mining out of unique candidates for designing markers cumbersome. Keeping this in view, the present study was undertaken with the aim to remove the data redundancy in walnut ESTs and then to develop simple sequence repeats (SSRs) markers. The EST sequences were assembled into a non-redundant set of 85 contigs and 1584 singletons (total sequences 1699), indicating 16.55% reduction in data redundancy. These 1699 sequences were then used to mine out SSR motifs. 132 EST-SSRs were detected, with dinucleotide repeats being predominant (70.45%), followed by trinucleotide repeats (27.27%) and very less frequent hexanucleotides (2.27%). These markers were validated by designing primer pairs. 15 of these designed primers were tested on a group of 37 walnut genotypes. Out of which 7 markers gave robust amplification, generating polymorphism. These findings indicate the usefulness of EST-SSRs in genome analysis. This study further emphasizes the importance of assembly of the vast amounts of data submitted in public databases. Our results have generated a set of non redundant walnut ESTs which is of prime importance for development of marker systems without any repetition or overlapping.

Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology

BackgroundChrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits.ResultsTo obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3′-UTR, and 5′-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums.ConclusionWe confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

Identification and characterization of microsatellites in ESTs of Rosa species: Insight in development of SSR markers

The present investigations were undertaken during the year 2012. In this study, analysis of microsatellites identified in expressed sequence tags (ESTs) of Rosa species was performed. Entire set of ESTs for genus Rosa including 1 792 of Rosa chinensis, 5 495 of R.× hybrida and 1 849 of R. lucieae was retrieved from public database and assembled individually. Furthermore, 668 microsatellites (SSR) were identified in the 5 272 assembled ESTs in which di-nucleotide repeats were most frequent. The highest 55.2 percentage of di-nucleotide was identified in R. chinensis followed by R.× hybrida (50.1%) and R. lucieae (44.3%). Repeats with motif sequences AG/CT and AAG/CTT were observed as most frequent motifs. Moreover, SSRs were also identified in non-EST sequences available for the genus Rosa. The entire sequences were assembled together to develop non-redundant data that was subsequently used to design 293 SSR markers. Analysis of microsatellite and resource of SSR markers developed in present study will help for undertaking the molecular research in Rosa species.

Metatranscriptome profiling reveals versatile N-nutrient utilization, CO2 limitation, oxidative stress, and active toxin production in an Alexandrium fundyense bloom

The globally increasing trend of harmful algal blooms (HAB) is often attributed to coastal eutrophication and climate change, but the physiological processes at play during a bloom are poorly understood due to lack of in situ measurements of these processes or their corresponding molecular machinery. Here we employed a dinoflagellate spliced leader-based 454-pyrosequencing technique to generate time-serial expressed sequence tags (EST) throughout a diel cycle during an Alexandrium fundyense bloom in Long Island Sound. Assembly of the reads yielded 87,273 dinoflagellate genes. To facilitate mapping of these data to species and comparing of gene expression dynamics between natural bloom and laboratory culture conditions, similar diel EST sets were sequenced for A. fundyense strain CCMP1719, which achieved 31,451 genes. The assembled metatranscriptome reveals a metabolically active A. fundyense population. Relative to the laboratory culture, the natural bloom expressed more abundantly genes related to nitrogen (N)-scavenging, CO2-concentrating and saxitoxin production. Most strikingly, the data showed a versatility to exploit various sources of N (cyanate, urea, nitrate/nitrite, and ammonium), likely conferring competitive advantages in A. fundyense for bloom formation or maintenance. The dataset also led to the first characterization of Ni-containing superoxide dismutases (NiSODs) in dinoflagellates with mitochondrial targeting signal identified, which along with other types of SOD found indicated diverse SODs being expressed in the A. fundyense bloom. Our results also demonstrate that metatranscriptomics is an effective approach to unraveling physiological processes conducive to HAB outbreaks.

New EST–SSR markers of Coffea arabica: transferability and application to studies of molecular characterization and genetic mapping

Microsatellite markers (SSR) have broad utility in genetic studies due to a high rate of polymorphisms, a codominant nature and multiallelism. EST–SSRs are markers derived from the expressed sequences of a genome and represent transcribed genes. Despite the importance of the genus Coffea, only a small number of EST–SSR markers are currently available. Thus, this study was designed to mine and develop a new set of EST–SSR markers from the Brazilian Coffee Genome Project. We investigated 130,792 expressed sequence tags (ESTs), from which 24,031 DNA sequences with microsatellites were identified. After stability and amplification testing, 101 new EST–SSR markers were developed and analyzed in different coffee species. The average rate of transferability was 88 %, showing that these markers are useful in genetic studies across the genus Coffea. Polymorphism levels and the degree of diversity were consistent with the evolutionary history of the species. All coffee genotypes were discriminated, even the C. arabica genotypes that have known narrow genetic basis. It was also possible to locate 14 EST–SSRs into different linkage groups of the C. arabica genetic map, which demonstrate that these markers can be useful in QTL mapping studies and in molecular-assisted selection.

Generation and Analysis of Expressed Sequence Tags (ESTs) from Halophyte Atriplex canescens to Explore Salt-Responsive Related Genes

Little information is available on gene expression profiling of halophyte A. canescens. To elucidate the molecular mechanism for stress tolerance in A. canescens, a full-length complementary DNA library was generated from A. canescens exposed to 400 mM NaCl, and provided 343 high-quality ESTs. In an evaluation of 343 valid EST sequences in the cDNA library, 197 unigenes were assembled, among which 190 unigenes (83.1% ESTs) were identified according to their significant similarities with proteins of known functions. All the 343 EST sequences have been deposited in the dbEST GenBank under accession numbers JZ535802 to JZ536144. According to Arabidopsis MIPS functional category and GO classifications, we identified 193 unigenes of the 311 annotations EST, representing 72 non-redundant unigenes sharing similarities with genes related to the defense response. The sets of ESTs obtained provide a rich genetic resource and 17 up-regulated genes related to salt stress resistance were identified by qRT-PCR. Six of these genes may contribute crucially to earlier and later stage salt stress resistance. Additionally, among the 343 unigenes sequences, 22 simple sequence repeats (SSRs) were also identified contributing to the study of A. canescens resources.

Comparative metatranscriptomic signatures of wood and paper feeding in the gut of the termite Reticulitermes flavipes (Isoptera: Rhinotermitidae)

Termites are highly eusocial insects that thrive on recalcitrant materials like wood and soil and thus play important roles in global carbon recycling and also in damaging wooden structures. Termites, such as Reticulitermes flavipes (Rhinotermitidae), owe their success to their ability to extract nutrients from lignocellulose (a major component of wood) with the help of gut-dwelling symbionts. With the aim to gain new insights into this enzymatic process we provided R. flavipes with a complex lignocellulose (wood) or pure cellulose (paper) diet and followed the resulting differential gene expression on a custom oligonucleotide-microarray platform. We identified a set of expressed sequence tags (ESTs) with differential abundance between the two diet treatments and demonstrated the source (host/symbiont) of these genes, providing novel information on termite nutritional symbiosis. Our results reveal: (1) the majority of responsive wood- and paper-abundant ESTs are from host and symbionts, respectively; (2) distinct pathways are associated with lignocellulose and cellulose feeding in both host and symbionts; and (3) sets of diet-responsive ESTs encode putative digestive and wood-related detoxification enzymes. Thus, this study illuminates the dynamics of termite nutritional symbiosis and reveals a pool of genes as potential targets for termite control and functional studies of termite-symbiont interactions.

Genetic Differentiation between Quercus frainetto Ten. and Q. pubescens Willd. in Romania

Little is known about genetic differences among Quercus frainetto and Q. pubescens, two species of section Dascia Kotschy (subgenus Lepidobalanus, white oaks) that reach in Romania the margins of their natural distribution range. A set of genomic SSRs (simple sequence repeats) and EST (expressed sequence tags)-SSRs was used to estimate the genetic differentiation among four natural populations of the two species. Q. pubescens had higher values of genetic diversity than Q. frainetto, although the differences were not significant. Two out of seven marker-loci, QrZAG112 and QpZAG110, displayed very high FST values. Averaged across loci, the genetic differentiation was high and significant (FST = 0.067). Genetic distances were much higher among species than among populations within species. A Bayesian analysis indicated that two is the most appropriate number of genetic clusters. Using a blind procedure (i.e. based on multilocus genotypes only) the vast majority of sampled individuals (90%) could be assigned to the cluster corresponding to their phenotypes. When information about sampling localities was introduced in the assignment test, all individual trees were correctly classified. The higher degree of admixture in Q. frainetto as compared to Q. pubescens may be explained by different rates of introgressive hybridization.

Exploiting expressed sequence tag databases for mapping markers associated with fruit development and fruit quality in apple

Apple (Malus × domestica Borkh.) is one of the most important fruit trees grown in Europe and around the world for human consumption, and therefore plant breeders aim at producing new apple varieties with high fruit quality. The availability of molecular markers suitable for marker-assisted selection could greatly increase the efficiency and power of breeding. The recent release of the cv. Golden Delicious genome sequence contributed to an exponential increase in the number of Malus sequences publicly available, while the successful achievement of two apple expressed sequence tag (EST) projects permitted the development of molecular markers from coding sequences. Here we present the setting up and mapping of new EST-based markers specific for fruit development and fruit quality traits. Since a large proportion of the ESTs used in our work were transcriptionally characterized and some of them co-localize within quantitative trait locus regions controlling fruit quality traits, the data reported will be effective in the identification of candidate genes. Due to the high level of polymorphism present in the Malus genome, 70% of the entire set of ESTs analyzed were polymorphic and 80% of them were successfully located using the conventional map-based approach. Fifty new EST-based markers were placed on the apple reference genetic map Fiesta × Discovery, thus enhancing the saturation of some regions, and 17 on Prima × Fiesta. Finally, another 17 markers were located on the Golden Delicious genome sequence using an in-silico approach.

Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.).

BackgroundCommon bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean.ResultsTwo full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes.ConclusionsThe present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean.

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

BackgroundParasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences.ResultsAbout 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein.An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the Chelonus lineage. Venom components specific to C. inanitus included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins.ConclusionsThe use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of C. inanitus appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.

Unraveling new genes associated with seed development and metabolism in Bixa orellana L. by expressed sequence tag (EST) analysis

The tropical tree Bixa orellana L. produces a range of secondary metabolites which biochemical and molecular biosynthesis basis are not well understood. In this work we have characterized a set of ESTs from a non-normalized cDNA library of B. orellana seeds to obtain information about the main developmental and metabolic processes taking place in developing seeds and their associated genes. After sequencing a set of randomly selected clones, most of the sequences were assigned with putative functions based on similarity, GO annotations and protein domains. The most abundant transcripts encoded proteins associated with cell wall (prolyl 4-hydroxylase), fatty acid (acyl carrier protein), and hormone/flavonoid (2OG-Fe oxygenase) synthesis, germination (MADS FLC-like protein) and embryo development (AP2/ERF transcription factor) regulation, photosynthesis (chlorophyll a-b binding protein), cell elongation (MAP65-1a), and stress responses (metallothionein- and thaumatin-like proteins). Enzymes were assigned to 16 different metabolic pathways related to both primary and secondary metabolisms. Characterization of two candidate genes of the bixin biosynthetic pathway, BoCCD and BoOMT, showed that they belong, respectively, to the carotenoid-cleavage dioxygenase 4 (CCD4) and caffeic acid O-methyltransferase (COMT) families, and are up-regulated during seed development. It indicates their involvement in the synthesis of this commercially important carotenoid pigment in seeds of B. orellana. Most of the genes identified here are the first representatives of their gene families in B. orellana.

Generation and analysis of expressed sequence tags from the medicinal plant Salvia miltiorrhiza

Salvia miltiorrhiza Bge. is a well-known traditional Chinese herb. Its roots have been formulated and used clinically for the treatment of various diseases. However, little genetic information has so far been available and this fact has become a major obstacle for molecular studies. To address this lack of genetic information, an Expressed Sequence Tag (EST) library from whole plantlets of S. miltiorrhiza was generated. From the 12959 cDNA clones that were randomly selected and subjected to single-pass sequencing from their 5' ends, 10288 ESTs (with sizes > or = 100 bp) were selected and assembled into 1288 contigs, leaving 2937 singletons, for a total of 4225 unigenes. These were analyzed using BLASTX (against protein databases), RPS-BLAST (against a conserved domain database) as well as the web-based KEGG Automatic Annotation Server for metabolic enzyme assignment. Based on the metabolic enzyme assignment, expression patterns of 14 secondary metabolic enzyme genes in different organs and under different treatments were verified using real-time PCR analysis. Additionally, a total of 122 microsatellites were identified from the ESTs, with 89 having sufficient flanking sequences for primer design. This set of ESTs represents a significant proportion of the S. miltiorrhiza transcriptome, and gives preliminary insights into the gene complement of S. miltiorrhiza. They will prove useful for uncovering secondary metabolic pathways, analyzing cDNA-array based gene expression, genetic manipulation to improve yield of desirable secondary products, and molecular marker identification.

Analysis and functional annotation of expressed sequence tags from the diesel tree (<i>Copaifera officinalis</i>)

Copaiba (Copaifera officinalis) is a tropical plant that is also known as the 'diesel tree', previously noted for production of diesel-like oleoresin. The advancements in molecular tools such as expressed sequence tag (EST) library construction provide a novel opportunity for insight into the physiology of this tree. We generated a small set of ESTs for a young copaiba, sequencing and annotating a total of 613 unigenes. Of these, 84% showed similarity to the National Center for Biotechnology Information (NCBI) database. Annotation showed 70% of unigenes had at least one associated Gene Ontology (GO) term. We found a majority of ESTs to be associated with heat response genes. Based on these data, this EST library of C. officinalis represents a small but important step in helping to understand the general physiology and heat- response expression patterns. Additionally, this small collection of EST offers a modest starting point in helping to understand this enigmatic tropical plant.

Analysis of expressed sequence tags from theHuperzia serrataleaf for gene discovery in the areas of secondary metabolite biosynthesis and development regulation

Huperzia serrata produces various types of lycopodium alkaloids, especially the huperzine A (HupA) that is a promising drug candidate for Alzheimer's disease. Despite the medicinal importance of H. serrata, little genomic or transcriptomic data are available from the public databases. A cDNA library was thus generated from RNA isolated from the leaves of H. serrata. A total of 4012 clones were randomly selected from the library, and 3451 high-quality expressed sequence tags (ESTs) were assembled to yield 1510 unique sequences with an average length of 712 bp. The majority (79.4%) of the unique sequences were assigned to the putative functions based on the BLAST searches against the public databases. The functions of these unique sequences covered a broad set of molecular functions, biological processes and biochemical pathways according to GO and KEGG assignments. The transcripts involved in the secondary metabolite biosynthesis of alkaloids, terpenoids and flavone/flavonoids, such as cytochrome P450, lysine decarboxylase (LDC), flavanone 3-hydroxylase, squalene synthetase and 2-oxoglutarate 3-dioxygenase, were well represented by 34 unique sequences in this EST dataset. The corresponding peptide sequence of the LDC contained the Pfam 03641 domain and was annotated as a putative LDC. The unique sequences encoding transcription factors, phytohormone biosynthetic enzymes and signaling components were also found in this EST collection. In addition, a total of 501 potential SSR-motif microsatellite loci were identified from the 393 H. serrata leaf unique sequences. This set of non-redundant ESTs and the molecular markers obtained in this study will establish valuable resources for a wide range of applications including gene discovery and identification, genetic mapping and analysis of genetic diversity, cultivar identification and marker-assisted selections in this important medicinal plant.

Comparative Analysis of the Global Transcriptome of Anopheles funestus from Mali, West Africa

Background Anopheles funestus is a principal vector of malaria across much of tropical Africa and is considered one of the most efficient of its kind, yet studies of this species have lagged behind those of its broadly sympatric congener, An. gambiae. In aid of future genomic sequencing of An. funestus, we explored the whole body transcriptome, derived from mixed stage progeny of wild-caught females from Mali, West Africa.Principal FindingsHere we report the functional annotation and comparative genomics of 2,005 expressed sequence tags (ESTs) from An. funestus, which were assembled with a previous EST set from adult female salivary glands from the same mosquito. The assembled ESTs provided for a nonredundant catalog of 1,035 transcripts excluding mitochondrial sequences.Conclusions/SignificanceComparison of the An. funestus and An. gambiae transcriptomes using computational and macroarray approaches revealed a high degree of sequence identity despite an estimated 20–80 MY divergence time between lineages. A phylogenetically broader comparative genomic analysis indicated that the most rapidly evolving proteins– those involved in immunity, hematophagy, formation of extracellular structures, and hypothetical conserved proteins– are those that probably play important roles in how mosquitoes adapt to their nutritional and external environments, and therefore could be of greatest interest in disease control.

Mining of expressed sequence tag libraries of cacao for microsatellite markers using five computational tools

Expressed sequence tags (ESTs) provide researchers with a quick and inexpensive route for discovering new genes, data on gene expression and regulation, and also provide genic markers that help in constructing genome maps. Cacao is an important perennial crop of humid tropics. Cacao EST sequences, as available in the public domain, were downloaded and made into contigs. Microsatellites were located in these ESTs and contigs using five softwares (MISA, TRA, TROLL, SSRIT and SSR primer). MISA gave maximum coverage of SSRs in cacao ESTs and contigs, although TRA was able to detect higher order (5-mer) repeats. The frequency of SSRs was one per 26.9 kb in the known set of ESTs. One-third of the repeats in EST-contigs were found to be trimeric. A few rare repeats like 21-mer repeat were also located. A/T repeats were most abundant among the mononucleotide repeats and the AG/GA/TC/CT type was the most frequent among dimerics. Flanking primers were designed using Primer3 program and verified experimentally for PCR amplification. The results of the study are made available freely online database (http://riju.byethost31.com/cocoa/). Seven primer pairs amplified genomic DNA isolated from leaves were used to screen a representative set of 12 accessions of cacao.

Characterization of new polymorphic functional markers for sugarcane

Expressed sequence tags (ESTs) offer the opportunity to exploit single, low-copy, conserved sequence motifs for the development of simple sequence repeats (SSRs). The authors have examined the Sugarcane Expressed Sequence Tag database for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 342 EST-SSRs, which represent a subset of over 2005 SSR-containing sequences that were located in the sugarcane EST database, could be designed from the nonredundant SSR-positive ESTs for possible use as potential genic markers. These EST-SSR markers were used to screen 18 sugarcane (Saccharum spp.) varieties. A high proportion (65.5%) of the above EST-SSRs, which gave amplified fragments of foreseen size, detected polymorphism. The number of alleles ranged from 2 to 24 with an average of 7.55 alleles per locus, while polymorphism information content values ranged from 0.16 to 0.94, with an average of 0.73. The ability of each set of EST-SSR markers to discriminate between varieties was generally higher than the polymorphism information content analysis. When tested for functionality, 82.1% of these 224 EST-SSRs were found to be functional, showing homology to known genes. As the EST-SSRs are within the expressed portion of the genome, they are likely to be associated to a particular gene of interest, improving their utility for genetic mapping; identification of quantitative trait loci, and comparative genomics studies of sugarcane. The development of new EST-SSR markers will have important implications for the genetic analysis and exploitation of the genetic resources of sugarcane and related species and will provide a more direct estimate of functional diversity.

A comprehensive resource of drought- and salinity- responsive ESTs for gene discovery and marker development in chickpea (Cicer arietinum L.).

BackgroundChickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. However, very limited number of molecular markers and candidate genes are available for undertaking molecular breeding in chickpea to tackle these stresses. This study reports generation and analysis of comprehensive resource of drought- and salinity-responsive expressed sequence tags (ESTs) and gene-based markers.ResultsA total of 20,162 (18,435 high quality) drought- and salinity- responsive ESTs were generated from ten different root tissue cDNA libraries of chickpea. Sequence editing, clustering and assembly analysis resulted in 6,404 unigenes (1,590 contigs and 4,814 singletons). Functional annotation of unigenes based on BLASTX analysis showed that 46.3% (2,965) had significant similarity (≤1E-05) to sequences in the non-redundant UniProt database. BLASTN analysis of unique sequences with ESTs of four legume species (Medicago, Lotus, soybean and groundnut) and three model plant species (rice, Arabidopsis and poplar) provided insights on conserved genes across legumes as well as novel transcripts for chickpea. Of 2,965 (46.3%) significant unigenes, only 2,071 (32.3%) unigenes could be functionally categorised according to Gene Ontology (GO) descriptions. A total of 2,029 sequences containing 3,728 simple sequence repeats (SSRs) were identified and 177 new EST-SSR markers were developed. Experimental validation of a set of 77 SSR markers on 24 genotypes revealed 230 alleles with an average of 4.6 alleles per marker and average polymorphism information content (PIC) value of 0.43. Besides SSR markers, 21,405 high confidence single nucleotide polymorphisms (SNPs) in 742 contigs (with ≥ 5 ESTs) were also identified. Recognition sites for restriction enzymes were identified for 7,884 SNPs in 240 contigs. Hierarchical clustering of 105 selected contigs provided clues about stress- responsive candidate genes and their expression profile showed predominance in specific stress-challenged libraries.ConclusionGenerated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding. Mapping of gene-based markers in chickpea will also add more anchoring points to align genomes of chickpea and other legume species.