Multiple CC chemokines in channel catfish and blue catfish as revealed by analysis of expressed sequence tags

Chemokines represent a superfamily of chemotactic cytokines involved in recruitment, activation and adhesion of a variety of leukocyte types to inflammatory foci, as well as in the organization and maintenance of lymphoid organ architecture and in normal developmental processes. Nearly all chemokines have been identified in human and mouse, but only a handful of fish chemokines have been identified. Here we describe 14 distinct chemokines from channel catfish and blue catfish identified by analysis of 30,000 expressed sequence tags. Based on sequence analysis, sequence similarity, and the arrangement of the conserved cysteine residues, all 14 chemokines were identified as members of the CC subfamily. Phylogenetic analysis did not reveal clear evidence of orthology of the catfish and human or mouse chemokines. Similarity analysis indicated that nine of the 14 CC chemokines were identified for the first time in fish. The availability of this pool of catfish CC chemokines should facilitate rapid identification and phylogenetic analysis of CC chemokines from other fish and related species.     

Generation and analysis of expressed sequence tags for microsatellite marker development in Calamus simplicifolius C. F. Wei

Rattans serve as an important source of raw non-wood materials for furniture and handicraft industries worldwide. However, their genomic sequence information in public databases is very limited. In this study, a set of 2,528 good-quality expressed sequence tags (ESTs) were generated from a full-length cDNA library constructed previously with root, stem and male inflorescence tissues of Calamus simplicifolius C. F. Wei, a rattan species native to Hainan Island, China. The ESTs were assembled into 1,588 unigenes, including 1,221 singletons and 367 contigs. BlastX searches against the GenBank non-redundant protein database revealed that 1,248 (78.6 %) unigenes had at least one significant match (E ≤ 10^?5). The gene ontology functional classification assigned 991, 669 and 977 of the unigenes to the cellular component, molecular function and biological process categories, respectively. A total of 71 simple sequence repeat (SSR) loci were developed among these ESTs, including 65 polymorphic across 19 rattan species representing three genera. High levels of cross-species/genus transferability were observed for the EST-SSRs. For the polymorphic EST-SSR markers, the number of alleles per locus and polymorphic information content ranged from 2 to 25 (mean 11.1) and from 0.135 to 0.949 (mean 0.695), respectively. The EST sequences and the EST-SSR primers have been deposited in GenBank databases of EST (IDs JK838364–40891) and Probe (IDs Pr16718978–9048, to be assigned).     

Prospects and pitfalls in whole genome association studies

Recent large-scale studies of common genetic variation throughout the human genome are making it feasible to conduct whole genome studies of genotype-phenotype associations. Such studies have the potential to uncover novel contributors to common complex traits and thus lead to insights into the aetiology of multifactorial phenotypes. Despite this promise, it is important to recognize that the availability of genetic markers and the ability to assay them at realistic cost does not guarantee success of this approach. There are a number of practical issues that require close attention, some forms of allelic architecture are not readily amenable to the association approach with even the most rigorous design, and doubtless new hurdles will emerge as the studies begin. Here we discuss the promise and current challenges of the whole genome approach, and raise some issues to consider in interpreting the results of the first whole genome studies.     

Simple sequence repeat marker development from bacterial artificial chromosome end sequences and expressed sequence tags of flax (Linum usitatissimum L.)

Flax is an important oilseed crop in North America and is mostly grown as a fibre crop in Europe. As a self-pollinated diploid with a small estimated genome size of ~370 Mb, flax is well suited for fast progress in genomics. In the last few years, important genetic resources have been developed for this crop. Here, we describe the assessment and comparative analyses of 1,506 putative simple sequence repeats (SSRs) of which, 1,164 were derived from BAC-end sequences (BESs) and 342 from expressed sequence tags (ESTs). The SSRs were assessed on a panel of 16 flax accessions with 673 (58 %) and 145 (42 %) primer pairs being polymorphic in the BESs and ESTs, respectively. With 818 novel polymorphic SSR primer pairs reported in this study, the repertoire of available SSRs in flax has more than doubled from the combined total of 508 of all previous reports. Among nucleotide motifs, trinucleotides were the most abundant irrespective of the class, but dinucleotides were the most polymorphic. SSR length was also positively correlated with polymorphism. Two dinucleotide (AT/TA and AG/GA) and two trinucleotide (AAT/ATA/TAA and GAA/AGA/AAG) motifs and their iterations, different from those reported in many other crops, accounted for more than half of all the SSRs and were also more polymorphic (63.4 %) than the rest of the markers (42.7 %). This improved resource promises to be useful in genetic, quantitative trait loci (QTL) and association mapping as well as for anchoring the physical/genetic map with the whole genome shotgun reference sequence of flax.     

Annotated expressed sequence tags for studies of the regulation of reproductive modes in aphids

The damaging effect of aphids to crops is largely determined by the spectacular rate of increase of populational expansion due to their parthenogenetic generations. Despite this, the molecular processes triggering the transition between the parthenogenetic and sexual phases between their annual life cycle have received little attention. Here, we describe a collection of genes from the cereal aphid Rhopalosiphum padi expressed during the switch from parthenogenetic to sexual reproduction. After cDNA cloning and sequencing, 726 expressed sequence tags (EST) were annotated. The R. padi EST collection contained a substantial number (139) of bacterial endosymbiont sequences. The majority of R. padi cDNAs encoded either unknown proteins (56%) or housekeeping polypeptides (38%). The large proportion of sequences without similarities in the databases is related to both their small size and their high GC content, corresponding probably to the presence of 5'-unstranslated regions. Fifteen genes involved in developmental and differentiation events were identified by similarity to known genes. Some of these may be useful candidates for markers of the early steps of sexual differentiation.     

Development and characterization of expressed sequence tags for the turkey (Meleagris gallopavo) genome and comparative sequence analysis with other birds

Twenty-one randomly selected clones from a turkey (Meleagris gallopavo) pituitary complementary DNA (cDNA) library were sequenced to develop expressed sequence tags (ESTs) for this economically important avian species whose genome is among the least understood. Primers specific for the ESTs were used to produce amplicons from the genomic DNA of turkey, chicken (Gallus gallus), guinea fowl (Numidia meleagris), pigeon (Columba domestica), and quail (Corturnix japonica). The amplicons were sequenced and analyzed for sequence variation within- and similarity among-species and with GenBank database sequences. The proportion of shared bases between the turkey sequence and the consensus sequence from each of the other species ranged from 72% to 93% between turkey and pigeon and quail and between turkey and chicken, respectively. The total number of single nucleotide polymorphisms (SNPs) observed ranged from 3 in quail to 18 in chicken out of 4898 and 5265 bases analyzed, respectively. The most frequent nucleotide variation observed was a C-->T transition. Linkage analysis of one such SNP in the backcross progeny of the East Lansing reference DNA panel, localized TUS0005, the chicken sequence derived from primers specific for turkey TUT2E EST, to chromosome 4. The ESTs reported, as well as the SNPs may provide a useful resource for ongoing efforts to develop high utility genome maps for the turkey and chicken. The primers described can also be used as a tool in future investigations directed at further understanding the biology of the guinea fowl, pigeon and quail and their relatedness to the turkey.     

Analysis of expressed sequence tags from grapevine flower and fruit and development of simple sequence repeat markers

A total of 6,230 EST sequences were produced from 7,561 clones in a cDNA library generated from grapevine (Vitis vinifera cv. ‘Summer Black’) flower and fruit tissues in this study. After cluster and assembly analysis of the datasets, 3,582 unigenes (GenBank accession numbers GW836604–GW840185) were established, among which 381 were new grapevine EST sequences. Out of the 381 new ESTs, 289 could be mapped on the 19 grapevine chromosomes. 913 unique ESTs with known or putative functions were assigned to 11 putative cellular roles. 540 potentially workable grapevine EST-SSRs were developed from 3,582 unigenes and about 42.6% of these unigenes were identified as true-to-type SSR loci and could amplify polymorphic bands from 22 individual plants of V. vinifera L, indicating that grapevine EST datasets are a valuable source for the development of functional simple sequence repeat (SSR) markers.     

The first set of expressed sequence tags (EST) from the medicinal mushroom Agaricus subrufescens delivers resource for gene discovery and marker development

Agaricus subrufescens is one of the most important culinary-medicinal cultivable mushrooms with potentially high-added-value products and extended agronomical valorization. The development of A. subrufescens-related technologies is hampered by, among others, the lack of suitable molecular tools. Thus, this mushroom is considered as a genomic orphan species with a very limited number of available molecular markers or sequences. To fill this gap, this study reports the generation and analysis of the first set of expressed sequence tags (EST) for A. subrufescens. cDNA fragments obtained from young sporophores (SP) and vegetative mycelium in liquid culture (CL) were sequenced using 454 pyrosequencing technology. After assembly process, 4,989 and 5,125 sequences were obtained in SP and CL libraries, respectively. About 87 % of the EST had significant similarity with Agaricus bisporus-predicted proteins, and 79 % correspond to known proteins. Functional categorization according to Gene Ontology could be assigned to 49 % of the sequences. Some gene families potentially involved in bioactive compound biosynthesis could be identified. A total of 232 simple sequence repeats (SSRs) were identified, and a set of 40 EST-SSR polymorphic markers were successfully developed. This EST dataset provides a new resource for gene discovery and molecular marker development. It constitutes a solid basis for further genetic and genomic studies in A. subrufescens.     

A high-density intervarietal map of the wheat genome enriched with markers derived from expressed sequence tags

Bread wheat (Triticum aestivum L.) is a hexaploid species with a large and complex genome. A reference genetic marker map, namely the International Triticeae Mapping Initiative (ITMI) map, has been constructed with the recombinant inbred line population derived from a cross involving a synthetic line. But it is not sufficient for a full understanding of the wheat genome under artificial selection without comparing it with intervarietal maps. Using an intervarietal mapping population derived by crossing Nanda2419 and Wangshuibai, we constructed a high-density genetic map of wheat. The total map length was 4,223.1 cM, comprising 887 loci, 345 of which were detected by markers derived from expressed sequence tags (ESTs). Two-thirds of the high marker density blocks were present in interstitial and telomeric regions. The map covered, mostly with the EST-derived markers, approximately 158 cM of telomeric regions absent in the ITMI map. The regions of low marker density were largely conserved among cultivars and between homoeologous subgenomes. The loci showing skewed segregation displayed a clustered distribution along chromosomes and some of the segregation distortion regions (SDR) are conserved in different mapping populations. This map enriched with EST-derived markers is important for structure and function analysis of wheat genome as well as in wheat gene mapping, cloning, and breeding programs.     

Mapping of 1400 expressed sequence tags in the bovine genome using a somatic cell hybrid panel

A bovine/hamster hybrid cell panel consisting of 30 independent hybrids was developed to locate genes. Polymerase chain reaction analysis of 279 microsatellites on the cattle linkage map in this panel revealed the presence of all chromosomes in either entire or fragmented form. Among primer pairs prepared from bovine 3'-expressed sequence tags (ESTs), 1400 ESTs were assigned to specific chromosomes, of which 1303 were newly assigned in this study, and mapped 854 (61%) to 1 of 192 chromosomal segments using this panel. The regional mapping of new genes to cattle chromosomes can be rapidly achieved using this panel.     

Mining for SSRs and FDMs from expressed sequence tags of Camellia sinensis

Simple Sequence Repeats (SSRs) developed from Expressed Sequence Tags (ESTs), known as EST-SSRs are most widely used and potentially valuable source of gene based markers for their high levels of crosstaxon portability, rapid and less expensive development. The EST sequence information in the publicly available databases is increasing in a faster rate. The emerging computational approach provides a better alternative process of development of SSR markers from the ESTs than the conventional methods. In the present study, 12,851 EST sequences of Camellia sinensis, downloaded from National Center for Biotechnology Information (NCBI) were mined for the development of Microsatellites. 6148 (4779 singletons and 1369 contigs) non redundant EST sequences were found after preprocessing and assembly of these sequences using various computational tools. Out of total 3822.68 kb sequence examined, 1636 (26.61%) EST sequences containing 2371 SSRs were detected with a density of 1 SSR/1.61 kb leading to development of 245 primer pairs. These mined EST-SSR markers will help further in the study of variability, mapping, evolutionary relationship in Camellia sinensis. In addition, these developed SSRs can also be applied for various studies across species.     

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.     

Generation of a total of 6483 expressed sequence tags from 60 day-old bovine whole fetus and fetal placenta

Expressed sequence tags (ESTs) generated based on characterization of clones isolated randomly from cDNA libraries are used to study gene expression profiles in specific tissues and to provide useful information for characterizing tissue physiology. In this study, two directionally cloned cDNA libraries were constructed from 60 day-old bovine whole fetus and fetal placenta. We have characterized 5357 and 1126 clones, and then identified 3464 and 795 unique sequences for the fetus and placenta cDNA libraries: 1851 and 504 showed homology to already identified genes, and 1613 and 291 showed no significant matches to any of the sequences in DNA databases, respectively. Further, we found 94 unique sequences overlapping in both the fetus and the placenta, leading to a catalog of 4165 genes expressed in 60 day-old fetus and placenta. The catalog is used to examine expression profile of genes in 60 day-old bovine fetus and placenta.     

Generation and analysis of expressed sequence tags from Botrytis cinerea

Botrytis cinerea is a filamentous plant pathogen of a wide range of plant species, and its infection may cause enormous damage both during plant growth and in the post-harvest phase. We have constructed a cDNA library from an isolate of B. cinerea and have sequenced 11,482 expressed sequence tags that were assembled into 1,003 contigs sequences and 3,032 singletons. Approximately 81% of the unigenes showed significant similarity to genes coding for proteins with known functions: more than 50% of the sequences code for genes involved in cellular metabolism, 12% for transport of metabolites, and approximately 10% for cellular organization. Other functional categories include responses to biotic and abiotic stimuli, cell communication, cell homeostasis, and cell development. We carried out pair-wise comparisons with fungal databases to determine the B. cinerea unisequence set with relevant similarity to genes in other fungal pathogenic counterparts. Among the 4,035 non-redundant B. cinerea unigenes, 1,338 (23%) have significant homology with Fusarium verticillioides unigenes. Similar values were obtained for Saccharomyces cerevisiae and Aspergillus nidulans (22% and 24%, respectively). The lower percentages of homology were with Magnaporthe grisae and Neurospora crassa (13% and 19%, respectively). Several genes involved in putative and known fungal virulence and general pathogenicity were identified. The results provide important information for future research on this fungal pathogen.