EST‐derived polymorphic microsatellites from cultivated strawberry (Fragaria×ananassa) are useful for diversity studies and varietal identification among Fragaria species

Microsatellite or simple sequence repeat markers derived from expressed sequence tags (ESTs) provide genetic markers within potentially functional genes, which could be very useful for breeding programs. To date, the development of microsatellite markers in the genus Fragaria has focused mainly on Fragaria vesca. However, most of the interests of breeding programs relate to specific characteristics of cultivated strawberry. Here, we describe a set of 10 EST‐derived microsatellites from Fragaria × ananassa. These markers showed high levels of polymorphism within strawberry cultivars and among different Fragaria species, indicating their potential for genetic studies not only on strawberry but also in other species within the genus.     

Chromosome‐specific physical localisation of expressed sequence tag loci in Corchorus olitorius L.

Jute (Corchorus spp.), as a natural fibre‐producing species, ranks next only to cotton. Inadequate understanding of its genetic architecture is a major lacuna for genetic improvement of this crop in terms of yield and quality. Establishment of a physical map provides a genomic tool that helps in positional cloning of valuable genes. In this report, an attempt was initiated to study association and localisation of single copy expressed sequence tag (EST) loci in the genome of Corchorus olitorius. The chromosome‐specific association of EST was determined based on the appearance of an extra signal for a single copy cDNA probe in mitotic interphase nuclei of specific trisomic(s) for fluorescence in situ hybridisation, and validated using a cDNA fragment of the 26S rRNA gene (600 bp) as molecular probe. The probe exhibited three signals in meiotic interphase nuclei of trisomic 5, instead of two as observed in diploids and other trisomics, indicating its association with chromosome 5. Subsequent hybridisation of the same probe on the pachytene chromosomes of diploids confirmed that 26S rRNA occupies the terminal end of the short arm of chromosome 5 in C. olitorius. Subsequently, chromosome‐specific association of 63 single copy EST and their physical localisation were determined on chromosomes 2, 4, 5 and 7. The study describes chromosome‐specific physical localisation of genes in jute. The approach used here could be a step towards construction of genome‐wide physical maps for any recalcitrant plant species like jute.     

phorest: a web‐based tool for comparative analyses of expressed sequence tag data

Comparative analysis of expressed sequence tags is becoming an important tool in molecular ecology for comparing gene expression in organisms grown in certain environments. Additionally, expressed sequence tag database information can be used for the construction of DNA microarrays and for the detection of single nucleotide polymorphisms. For such applications, we present phorest , a web‐based tool for managing, analysing and comparing various collections of expressed sequence tags. It is written in PHP (PHP: Hypertext Preprocessor) and runs on UNIX, Microsoft Windows and Macintosh (Mac OS X) platforms.     

Genomewide gene‐associated microsatellite markers for the model invasive ascidian,Ciona intestinalis species complex

The vase tunicate, Ciona intestinalis species complex, has become a good model for ecological and evolutionary studies, especially those focusing on microevolution associated with rapidly changing environments. However, genomewide genetic markers are still lacking. Here, we characterized a large set of genomewide gene‐associated microsatellite markers for C. intestinalis spA (=C. robusta). Bioinformatic analysis identified 4654 microsatellites from expressed sequence tags (ESTs), 2126 of which successfully assigned to chromosomes were selected for further analysis. Based on the distribution evenness on chromosomes, function annotation and suitability for primer design, we chose 545 candidate microsatellites for further characterization. After amplification validation and variation assessment, 218 loci were polymorphic in at least one of the two populations collected from the coast of Arenys de Mar, Spain (N = 24–48), and Cape Town, South Africa (N = 24–33). The number of alleles, observed heterozygosity and expected heterozygosity ranged from 2 to 11, 0 to 0.833 and 0.021 to 0.818, and from 2 to 10, 0 to 0.879 and 0.031 to 0.845 for the Spanish and African populations, respectively. When all microsatellites were tested for cross‐species utility, only 60 loci (25.8%) could be successfully amplified and all loci were polymorphic in C. intestinalis spB. A high level of genomewide polymorphism is likely responsible for the low transferability. The large set of microsatellite markers characterized here is expected to provide a useful genomewide resource for ecological and evolutionary studies using C. intestinalis as a model.     

Analysis of expressed sequence tags from Chaetomium cupreum grown under conditions associated with mycoparasitism

Aims:  To elucidate the molecular mechanisms associated with mycoparasitism from Chaetomium cupreum , an effective biocontrol agent with ability against plant pathogenic fungi.
Methods and Results:  One cDNA library was constructed from conditions predicted to resemble mycoparasitic process. A total of 1876 ESTs were generated and assembled into 1035 unigenes. B last X search revealed that 585 unigenes had similarities with sequences available from public databases. Based on the ESTs abundance, MFS monosaccharide transporter was found as the gene expressed at the highest level. A KEGG analysis allowed mapping of 60 metabolic pathways well represented by the glycolysis/gluconeogenesis, d -arginine and ornithine metabolism, and tryptophan metabolism. The genes related to mycoparasitism were detected.
Conclusions:  The results revealed that the cell walls of the fungal pathogen can simulate some aspects of the mycoparasitic interaction between C. cupreum and its targets.
Significance and Impact of the Study:  This is the first report to study genes expression under conditions associated with the mycoparasitic process. The findings contribute to elucidate the molecular mechanisms involved in mycoparasitism and will help to advance our efforts in developing novel strategies for biocontrol of plant fungal diseases.     

Identification and characterization of expressed sequence tags‐derived simple sequence repeats markers from robusta coffee variety ‘CxR’ (an interspecific hybrid of Coffea canephora × Coffea congensis)

SSR (simple sequence repeats) markers derived from ESTs (expressed sequence tags), commonly called EST‐SSRs or genic SSRs provide useful genetic markers for crop improvement. These are easy and economical to develop as by‐products of large‐scale EST resources that have become available as part of the functional genomic studies in many plant species. Here, we describe for the first time, nine genic‐SSRs of coffee that are developed from the microsatellite containing ESTs from a cDNA library of moisture‐stressed leaves of coffee variety, ‘CxR’ (a commercial interspecific hybrid between Coffea congensis and Coffea canephora). The markers show considerable allelic diversity with PIC values up to 0.70 and 0.75 for Coffea arabica and Coffea canephora, respectively, and robust cross‐species amplification in 16 other related taxa of coffee. The validation studies thus demonstrate the potential utility of the EST‐SSRs for genetic analysis of coffee germplasm.     

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.     

Development of 25 gene‐associated microsatellite markers of Atlantic cod (Gadus morhua L.)

Microsatellites were identified by screening 2294 GenBank entries available for Atlantic cod (Gadus morhua L.), mainly representing expressed sequence tags and cDNA sequences. Ninety‐two novel microsatellite loci (tetra‐, tri‐ and dinucleotides) were characterized on 96 individuals. This strategy yielded 25 gene‐associated polymorphic microsatellite markers (11 tri‐ and 14 dinucleotides) with two to 20 alleles and an average heterozygosity of 0.48 in the population studied (range 0.02–0.89). One marker exhibited significant homozygote excess, and one of the primer pairs amplified two linked markers. The gene identity was determined at nine of the loci, confirming the associated microsatellites as type I markers.     

The identification of nuclear and mitochondrial genes by sequencing randomly chosen clones from a marsupial mammary gland cDNA library

To increase the number of genes that can be mapped to the genome of the tammar wallaby (Macropus eugenii), we sequenced 100 randomly chosen clones from a mammary gland cDNA library. Provisional identifications were made of seven nuclear genes and one mitochondrial gene encoding two caseins, -galactosidase, acetyl-coenzyme A synthetase, lipoprotein lipase, inorganic pyrophosphatase, an ATP-dependent RNA helicase, and cytochromec oxidase I. Highly conserved genes, such as that encoding acetyl-coenzyme A synthetase, were easily identified even from cross-kingdom matches. Genes which are highly divergent, however, such as those encoding themature casein peptides, could not be aligned with homologues in the databases. Even in an organ where there is high mRNA species redundancy, the sequence characterization of expressed sequence tags provides a rapid means of gene identification for mapping purposes.     

Characterization of EST derived SSRs from the bay scallop,Argopecten irradians

Interest in bay scallop conservation has resulted in organized stock enhancement efforts and increased attention to fisheries management issues. Genetic markers can facilitate the monitoring of enhancement efforts, characterization of wild populations, and optimize hatchery practices. We have identified eight polymorphic simple sequence repeat markers including one dinucleotide, six trinucleotide and one compound dinucleotide repeats, in expressed sequence tags generated from multiple bay scallop cDNA libraries. The numbers of alleles range from two to five. The expected and observed heterozygosities range from 0.093 to 0.720 and 0.095 to 0.600, respectively.     

Gene expression profiling in porcine mammary gland during lactation and identification of breed-and developmental-stage-specific genes

A total of 28941 ESTs were sequenced from five 5′-directed non-normalized cDNA libraries, which were assembled into 2212 contigs and 5642 singlets using CAP3. These sequences were annotated and clustered into 6857 unique genes, 2072 of which having no functional annotations were considered as novel genes. These genes were further classified into Gene Ontology categories. By comparing the expression profiles, we identified some breed-and developmental-stage-specific gene groups. These genes may be relative to reproductive performance or play important roles in milk synthesis, secretion and mammary involution. The unknown EST sequences and expression profiles at different developmental stages and breeds are very important resources for further research.     

Gene expression profiling in porcine mammary gland during lactation and identification of breed-and developmental-stage-specific genes

The mammary gland provides an excellent system to study questions pertaining to organogenesis, cell differentiation and oncogenesis. Intensive efforts have been made to understand the development of the mammary gland, particularly in terms of lactogenesis…