Putative SNP discovery in interspecific hybrids of catfish by comparative EST analysis

In this study, we identified putative SNP markers within genes by comparative analysis of expressed sequence tags (ESTs). Comparison of 849 ESTs from blue catfish (Ictalurus furcatus) with >11,000 ESTs from channel catfish (I. punctatus) deposited in GenBank resulted in the identification of 1020 putative SNPs within 161 genes, of which 145 were nuclear genes of known function. The observed frequency of SNPs within ESTs of the two closely related catfish species was 1.32 SNP per 100 bp. The majority of identified SNPs differed between the two species and, therefore, these SNPs are useful for mapping genes in channel catfish x blue catfish interspecific resource families. The SNPs that differed within species were also observed; these can be applied to genome scans in channel catfish resource families.     

Bioinformatic Mining of Type I Microsatellites from Expressed Sequence Tags of Channel Catfish (<Emphasis Type="Italic">Ictalurus punctatus</Emphasis>)

Gene-derived markers are pivotal to the analysis of genome structure, organization, and evolution and necessary for comparative genomics. However, gene-derived markers are relatively difficult to develop. This project utilized the genomic resources of channel catfish expressed sequence tags (ESTs) to identify simple sequence repeats (SSRs), or microsatellites. It took the advantage of ESTs for the establishment of gene identities, and of microsatellites for the acquisition of high polymorphism. When microsatellites are tagged to genes, the microsatellites can then be used as gene markers. A bioinformatic analysis of 43,033 ESTs identified 4855 ESTs containing microsatellites. Cluster analysis indicated that 1312 of these ESTs fell into 569 contigs, and the remaining 3534 ESTs were singletons. A total of 4103 unique microsatellite-containing genes were identified. The dinucleotide CA/TG and GA/TC pairs were the most abundant microsatellites. AT-rich microsatellite types were predominant among trinucleotide and tetranucleotide microsatellites, consistent with our earlier estimation that the catfish genome is highly AT-rich. Our preliminary results indicated that the majority of the identified microsatellites were polymorphic and, therefore, useful for genetic linkage mapping of catfish. Mapping of these gene-derived markers is under way, which will set the foundation for comparative genome analysis in catfish.     

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.     

High-resolution mapping of the barley leaf rust resistance gene Rph5 using barley expressed sequence tags (ESTs) and synteny with rice

The rapidly growing expressed sequence tag (EST) resources of species representing the Poacea family and availability of comprehensive sequence information for the rice (Oryza sativa) genome create an excellent opportunity for comparative genome analysis. Extensive synteny between rice chromosome 1 and barley (Hordeum vulgare L.) chromosome 3 has proven extremely useful for saturation mapping of chromosomal regions containing target genes of large-genome barley with conserved orthologous genes from the syntenic regions of the rice genome. Rph5 is a gene conferring resistance to the barley leaf rust pathogen Puccinia hordei. It was mapped to chromosome 3HS, which is syntenic with rice chromosome 1S. The objective of this study was to increase marker density within the sub-centimorgan region around Rph5, using sequence-tagged site (STS) markers that were developed based on barley ESTs syntenic to the phage (P1)-derived artificial chromosome (PAC) clones comprising the distal region of rice chromosome 1S. Five rice PAC clones were used as queries in a blastn search to screen 375,187 barley ESTs. Ninety-four non-redundant EST sequences were identified from the EST database and used as templates to design 174 pairs of primer combinations. As a result, 9 barley EST-based STS markers were incorporated into the ‘Bowman’ × ‘Magnif 102’ high-resolution map of the Rph5 region. More importantly, six markers, including five EST-derived STS sequences, were found to co-segregate with Rph5. The results of this study demonstrate the usefulness of rice genomic resources for efficient deployment of barley ESTs for marker saturation of targeted barley genomic regions.     

Isolation, in silico characterization and chromosomal localization of a group of cDNAs from ciliated epithelial cells after in vitro ciliogenesis

Background

Immotile cilia syndrome (ICS) or primary ciliary dyskinesia (PCD) is an autosomal recessive disorder in humans in which the beating of cilia and sperm flagella is impaired. Ciliated epithelial cell linings are present in many tissues. To understand ciliary assembly and motility, it is important to isolate those genes involved in the process.

Results

Total RNA was isolated from cultured ciliated nasal epithelial cells after in vitro ciliogenesis and expressed sequenced tags (ESTs) were generated. The functions and locations of 63 of these ESTs were derived by BLAST from two public databases. These ESTs are grouped into various classes. One group has high homology not only with the mitochondrial genome but also with one or more chromosomal DNAs, suggesting that very similar genes, or genes with very similar domains, are expressed from both mitochondrial and nuclear DNA. A second class comprises genes with complete homology with part of a known gene, suggesting that they are the same genes. A third group has partial homology with domains of known genes. A fourth group, constituting 33% of the ESTs characterized, has no significant homology with any gene or EST in the database.

Conclusions

We have shown that sufficient information about the location of ESTs could be derived electronically from the recently completed human genome sequences. This strategy of EST localization should be significantly useful for mapping and identification of new genes in the forthcoming human genome sequences with the vast number of ESTs in the dbEST database.     

Identification and characterization of SSRs from soybean (Glycine max) ESTs

Microsatellites, or simple sequence repeats (SSRs), are very useful molecular markers for a number of plant species. We used a new publicly available module (TROLL) to extract microsatellites from the public database of soybean expressed sequence tag (EST) sequences. A total of 12,833 sequences containing di- to penta-type SSRs were identified from 200,516 non-redundant soybean ESTs. On average, one SSR was found per 7.25?kb of EST sequences, with the tri-nucleotide motifs being the most abundant. Primer sequences flanking the SSR motifs were successfully designed for 9,638 soybean ESTs using the software primer3.0 and only 59 pairs of them were found in earlier studies. We synthesized 124 pairs of the primers to determine the polymorphism and heterozygosity among eight genotypes of soybean cultivars, which represented a wide range of the cultivated soybean cultivars. PCR amplification products with anticipated SSRs were obtained with 81 pairs of primers; 36 PCR products appeared to be homozygous and the remaining 45 PCR products appeared to be heterozygous and displayed polymorphism among the eight cultivars. We further analysed the EST sequences containing 45 polymorphic EST-SSR markers using the programs BLASTN and BLASTX. Sequence alignment showed that 29 ESTs have homologous sequences and 15 ESTs could be classified into a Uni-gene cluster with comparatively convincing protein products. Among these 15 ESTs belonging to a Uni-gene cluster, 9 SSRs were located in 3'-UTR, 4 SSRs were located in the intron region and 2 SSRs were located in the CDS region. None of these SSRs was located in the 5'-UTR. These novel SSRs identified in the ESTs of soybean provide useful information for gene mapping and cloning in future studies.     

Chromosomal localization of ESTs obtained from human fetal liver via BAC-mediated FISH mapping.

A total of 55 expressed sequence tags (ESTs) randomly chosen from our collection of fetal liver ESTs were mapped to chromosomes by fluorescence in situ hybridization (FISH) mapping techniques. To generate FISH mapping probes, the genomic DNAs for each EST were selected by screening an arrayed human bacterial artificial chromosome (BAC) library. In total, 73 BACs were used for mapping of the 55 ESTs. Among them, 70 BACs representing 52 ESTs unequivocally mapped to single chromosomal regions. The remaining 3 BACs representing 3 ESTs were localized to multiple regions, suggesting that BACs may have very low chimerism. Our mapping results were compared with EST mapping databases deposited in NCBI. Thirty-six of 55 ESTs corresponded to previously mapped positions of ESTs, 2 ESTs mapped to different positions from previously determined ones, and it was found that 17 ESTs have been mapped on new locations from this study. These mapping data may be used for completing the framework of the human physical map, and also for providing a good starting point for searching disease-related genes.     

Development of polymorphic expressed sequence tag-derived microsatellites for the extension of the genetic linkage map of the black tiger shrimp (Penaeus monodon)

In this study, microsatellite markers were developed for the genetic linkage mapping and breeding program of the black tiger shrimp Penaeus monodon. A total of 997 unique microsatellite-containing expressed sequence tags (ESTs) were identified from 10 100 EST sequences in the P. monodon EST database. AT-rich microsatellite types were predominant in the EST sequences. Homology searching by the blastn and blastx programs revealed that these 997 ESTs represented 8.6% known gene products, 27.8% hypothetical proteins and 63.6% unknown gene products. Characterization of 50 markers on a panel of 35-48 unrelated shrimp indicated an average number of alleles of 12.6 and an average polymorphic information content of 0.723. These EST microsatellite markers along with 208 other markers (185 amplified fragment length polymorphisms, one exon-primed intron-crossing, six single strand conformation polymorphisms, one single nucleotide polymorphism, 13 non-EST-associated microsatellites and two EST-associated microsatellites) were analysed across the international P. monodon mapping family. A total of 144 new markers were added to the P. monodon maps, including 36 of the microsatellite-containing ESTs. The current P. monodon male and female linkage maps have 47 and 36 linkage groups respectively with coverage across half the P. monodon genome.     

Isolation and Enrichment of Abundant Microsatellites from a Channel Catfish (Ictalurus punctatus) Brain cDNA Library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid, single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.     

Characterization and radiation hybrid mapping of expressed sequence tags from the canine brain

Abstract Maps of the canine genome are now developing rapidly. Most of the markers on the current integrated canine radiation hybrid/genetic linkage/cytogenetic map are highly polymorphic microsatellite (type II) markers that are very useful for mapping disease loci. However, there is still an urgent need for the mapping of gene-based (type I) markers that are required for comparative mapping, as well as identifying candidate genes for disease loci that have been genetically mapped. We constructed an adult brain cDNA library as a resource to increase the number of gene-based markers on the canine genome map. Eighty-one percent of the 2700 sequenced expressed sequence tags (ESTs) represented unique sequences. The canine brain ESTs were compared with sequences in public databases to identify putative canine orthologs of human genes. One hundred nine of the canine ESTs were mapped on the latest canine radiation hybrid (RH) panel to determine the location of the respective canine gene. The addition of these new gene-based markers revealed three conserved segments (CS) between human and canine genomes previously detected by fluorescence in situ hybridization (FISH), but not by RH mapping. In addition, five new CS between dog and human were identified that had not been detected previously by RH mapping or FISH. This work has increased the number of gene-based markers on the canine RH map by approximately 30% and indicates the benefit to be gained by increasing the gene content of the current canine comparative map.     

Development of EST derived SSRs and SNPs as a genomic resource in Indian catfish, <Emphasis Type="Italic">Clarias batrachus</Emphasis>

Clarias batrachus, an Indian catfish species, is endemic to the Indian subcontinent and potential cultivable species. The genomic resources in C. batrachus in the form of ESTs containing microsatellite repeats (EST-SSR) and single nucleotide polymorphisms (SNPs) that are associated with the expressed genes from spleen were mined. From a total of 1,937 ESTs generated, 1,698 unique sequences were obtained, out of which 221 EST-SSRs were identified and 54% could be functionally annotated by similarity searches. A total of 23 contigs containing 3 or more ESTs were found to contain 31 SNP loci, out of which 8 ESTs showed similarity to genes of known function and 1 for hypothetical protein. Nine ESTs with SSRs and/or SNPs identified in this study were reported to be associated with diseases in human and animals. These identified loci can be developed into markers in C. batrachus, which can be useful in linkage mapping, comparative genomics studies and for its genetic improvement programmes.     

Translational machinery of channel catfish: II. Complementary DNA and expression of the complete set of 47 60S ribosomal proteins

Ribosomal protein genes have become widely used as markers for phylogenetic studies and comparative genomics, but they have not been available in fish. We have cloned and sequenced a complete set of all 47 60S ribosomal protein cDNAs from channel catfish (Ictalurus punctatus), of which 43 included the complete protein encoding regions. Most ribosomal protein mRNAs in channel catfish are highly similar to their mammalian counterparts. However, L4, L14, and L29 are significantly shorter in channel catfish than in mammals due to deletions in the 3' end of the gene. Two distantly related L5 cDNAs, L5a and L5b, were found in channel catfish. L5a is more similar to L5 in other vertebrates, while L5b showed significant levels of divergence, suggesting independent evolution of the two L5-encoding genes. The 47 ribosomal protein genes are generally highly expressed and together account for 11-14% of overall gene expression, depending on the tissues. Expression levels were highly variable both within a single tissue among different ribosomal protein genes, and among tissues with regard to a single ribosomal protein gene. Strong tissue preference expression was also observed for some ribosomal proteins. This set of ribosomal protein gene sequences represents one of the most complete sets from any single organism and will aid in fish phylogenetic and comparative genomic studies.     

Isolation and enrichment of abundant microsatellites from a channel catfish (Ictalurus punctatus) brain cDNA library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid; single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.