Generation and Analysis of ESTs from the Grass Carp,Ctenopharyngodon idellus

Grass carp, Ctenopharyngodon idellus (Valenciennes, 1844), is an economically important species widely cultured in the world, but its genome research resources are largely lacking. The objectives of this study were to construct normalized cDNA libraries for efficient EST analysis, to generate ESTs from these libraries, and to identify EST-related molecular markers such as microsatellites and single nucleotide polymorphisms (SNPs) for genetic analysis of this species. A total of 6,269 ESTs were generated representing 4,815 unique sequences, from which 105 putative microsatellites and 5,228 SNPs were identified. These genome resources provide the material basis for future genetic and functional analyses in this species.     

Generation and analysis of 10 000 ESTs from the half‐smooth tongue sole Cynoglossus semilaevis and identification of microsatellite and SNP markers

Three normalized cDNA libraries were constructed, two of which were constructed from reproductive tissues ovary and testis, and the other one from pooled immune tissues including head kidney, intestine, liver and spleen. A total of 10 542 clones were sequenced generating 10 128 expressed sequence tags (ESTs). Cluster analysis indicated a total of 5808 unique sequences including 1712 contigs and 4096 singletons. A total of 4249 (73%) of the unique ESTs had significant hits to the non‐redundant protein database, 2253 of which were annotated using Gene Ontology (GO) terms. A total of 311 microsatellites (with 246 having sufficient flanking sequences for primer design) and 6294 putative SNPs were identified. These genome resources provide the material basis for future microarray development, marker validation and genetic linkage and QTL analysis.     

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.     

Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags

The construction of high-density linkage maps for use in identifying loci underlying important traits requires the development of large numbers of polymorphic genetic markers spanning the entire genome at regularly spaced intervals. As part of our efforts to develop markers for rainbow trout (Oncorhynchus mykiss), we performed a comparison of allelic variation between microsatellite markers developed from expressed sequence tag (EST) data and anonymous markers identified from repeat-enriched libraries constructed from genomic DNA. A subset of 70 markers (37 from EST databases and 33 from repeat enriched libraries) was characterized with respect to polymorphism information content (PIC), number of alleles, repeat number, locus duplication within the genome and ability to amplify in other salmonid species. Higher PIC was detected in dinucleotide microsatellites derived from ESTs than anonymous markers (72.7% vs. 54.0%). In contrast, dinucleotide repeat numbers were higher for anonymous microsatellites than for EST derived microsatellites (27.4 vs.18.1). A higher rate of cross-species amplification was observed for EST microsatellites. Approximately half of each marker type was duplicated within the genome. Unlike single-copy markers, amplification of duplicated microsatellites in other salmonids was not correlated to phylogenetic distance. Genomic microsatellites proved more useful than EST derived microsatellites in discriminating among the salmonids. In total, 428 microsatellite markers were developed in this study for mapping and population genetic studies in rainbow trout.     

Expressed sequence tag analysis and development of gene associated markers in a near-isogenic plant system of Eragrostis curvula

Eragrostis curvula (Schrad.) Nees is a forage grass native to the semiarid regions of Southern Africa, which reproduces mainly by pseudogamous diplosporous apomixis. A collection of ESTs was generated from four cDNA libraries, three of them obtained from panicles of near-isogenic lines with different ploidy levels and reproductive modes, and one obtained from 12 days-old plant leaves. A total of 12,295 high-quality ESTs were clustered and assembled, rendering 8,864 unigenes, including 1,490 contigs and 7,394 singletons, with a genome coverage of 22%. A total of 7,029 (79.11%) unigenes were functionally categorized by BLASTX analysis against sequences deposited in public databases, but only 37.80% could be classified according to Gene Ontology. Sequence comparison against the cereals genes indexes (GI) revealed 50% significant hits. A total of 254 EST-SSRs were detected from 219 singletons and 35 from contigs. Di- and tri- motifs were similarly represented with percentages of 38.95 and 40.16%, respectively. In addition, 190 SNPs and Indels were detected in 18 contigs generated from 3 to 4 libraries. The ESTs and the molecular markers obtained in this study will provide valuable resources for a wide range of applications including gene identification, genetic mapping, cultivar identification, analysis of genetic diversity, phenotype mapping and marker assisted selection.     

Development of microsatellite markers in potato and their transferability in some members of Solanaceae

We have developed thirty new microsatellite markers in potato by screening genomic libraries and ESTs. Genomic libraries of potato cultivar Kufri Bahar were screened for sequences containing microsatellite motifs GA, GT, ACA, ATC, GAA, TAA and GATA. Using flanking sequences, PCR primers were designed for microsatellites identified from genomic libraries and ESTs. Sixteen new primer pairs from genomic libraries and fourteen from ESTs along with seven previously published primer pairs amplified PCR products in the selected genotypes comprising of 65 Solanum tuberosum lines and 14 other species of the potato gene pool. Neighbor-joining tree based on genetic distance matrix developed using microsatellite markers successfully distinguished all these genotypes in the expected size range. Seventeen microsatellites could also be cross-amplified in at least one of the five members of solanaceae, namely tomato, eggplant, pepper, petunia and tobacco. The new microsatellite markers obtained in this study will be useful in various genetic and taxonomic studies in potato and related genomes.     

Identification and Validation of Single Nucleotide Polymorphisms in Poplar Using Publicly Expressed Sequence Tags

By using assembled expressed sequence tags （ESTs） from 14 different eDNA libraries that contain 84 132 sequences reads, 556 Populus candidate single nucleotide polymorphisms （SNPs） were identified. Because traces were not available from dbEST （http：//www.ncbi.nlm.nih.gov/dbEST/index.html）, stringent filters were used to identify reliable candidate SNPs. Sequences analysis indicated that the main types of substitutions among candidate SNPs were A/G and T/C transitions, which accounted for 22.0% and 30.8%, respectively. One hundred and ten candidate SNPs were tested. As a result, 38 candidate SNPs were confirmed by directed sequencing of PCR products amplified from six different individuals. Thirteen new SNPs in intron regions were found and multiple SNPs were found to be located in both intron and exon regions of four contigs. Heterozygosis was found in all 47 candidate sites and five SNP sites were heterozygous in all six samples. This is the first report of SNP identification in a tree species which reveals that assembled ESTs from multiple libraries of the public database may provide a rich source of comparative sequences for an SNP search in the poplar genome.     

燕麦EST-SSR标记的开发和利用

为拓展分子标记在燕麦种质资源分析与鉴定中的应用,利用公共数据库中的25376条EST(expressed sequence tags)序列,开展了燕麦EST-SSR功能性标记的开发和利用研究。25376条EST序列经拼接去冗余后获得了11618条序列,从中筛选出含有不同重复基元的SSR且重复次数较多、长度较长的556条EST序列进行引物设计,开发了50对燕麦EST-SSR引物,通过筛选得到40对有效的EST-SSR引物。选取其中4对引物对5个燕麦种质资源进行了PCR扩增及产物测序,结果表明扩增条带多态性是由SSR差异造成的。利用40对ESTSSR引物对15个六倍体燕麦种质资源进行遗传多样性分析,共扩增出89个等位基因,平均每对引物产生2.23个等位基因;UPGMA聚类分析表明,15个六倍体燕麦种质资源在Dice系数为0.93处聚为3支,基本上是按照不同种进行聚类的,在相同种中又根据地理来源分别聚集成支。利用40对EST-SSR引物对31个遗传背景不清的燕麦种质资源进行基因组倍性鉴定,发现这些种质中可能存在有四倍体和二倍体的燕麦新资源。本研究开发的燕麦EST-SSR功能性标记将在燕麦遗传多样性分析、遗传图谱构建及燕麦属内种间基因组鉴定等方面发挥重要作用。     

Rapid evolution of mammalian X-linked testis microRNAs

Background

Microsatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish.

Results

The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65°C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA- and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ± 0.54, while the expected heterozygosity was 0.56 ± 0.03. All the isolated microsatellites inherited in a Mendelian pattern.

Conclusion

Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution.     

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.     

A novel genome‐wide microsatellite resource for species of Eucalyptus with linkage‐to‐physical correspondence on the reference genome sequence

Keystone species in their native ranges, eucalypts, are ecologically and genetically very diverse, growing naturally along extensive latitudinal and altitudinal ranges and variable environments. Besides their ecological importance, eucalypts are also the most widely planted trees for sustainable forestry in the world. We report the development of a novel collection of 535 microsatellites for species of Eucalyptus, 494 designed from ESTs and 41 from genomic libraries. A selected subset of 223 was evaluated for individual identification, parentage testing, and ancestral information content in the two most extensively studied species, Eucalyptus grandis and Eucalyptus globulus. Microsatellites showed high transferability and overlapping allele size range, suggesting they have arisen still in their common ancestor and confirming the extensive genome conservation between these two species. A consensus linkage map with 437 microsatellites, the most comprehensive microsatellite‐only genetic map for Eucalyptus, was built by assembling segregation data from three mapping populations and anchored to the Eucalyptus genome. An overall colinearity between recombination‐based and physical positioning of 84% of the mapped microsatellites was observed, with some ordering discrepancies and sporadic locus duplications, consistent with the recently described whole genome duplication events in Eucalyptus. The linkage map covered 95.2% of the 605.8‐Mbp assembled genome sequence, placing one microsatellite every 1.55 Mbp on average, and an overall estimate of physical to recombination distance of 618 kbp/cM. The genetic parameters estimates together with linkage and physical position data for this large set of microsatellites should assist marker choice for genome‐wide population genetics and comparative mapping in Eucalyptus.     

Linkage mapping of gene-associated SNPs to pig chromosome 11

Single nucleotide polymorphisms (SNPs) were discovered in porcine expressed sequence tags (ESTs) orthologous to genes from human chromosome 13 (HSA13) and predicted to be located on pig chromosome 11 (SSC11). The SNPs were identified as sequence variants in clusters of EST sequences from pig cDNA libraries constructed in the Sino-Danish pig genome project. In total, 312 human gene sequences from HSA13 were used for similarity searches in our pig EST database. Pig ESTs showing significant similarity with HSA13 genes were clustered and candidate SNPs were identified. Allele frequencies for 26 SNPs were estimated in a group of 80 unrelated pigs from Danish commercial pig breeds: Duroc, Hampshire, Landrace and Large White. Eighteen of the 26 SNPs genotyped in the PiGMaP Reference Families were mapped by linkage analysis to SSC11. The EST-based SNPs published here are new genetic markers useful for linkage and association studies in commercial and experimental pig populations. This study represents the first gene-associated SNP linkage map of pig chromosome 11 and adds new comparative mapping information between SSC11 and HSA13. Furthermore, our data facilitate future studies aimed at the identification of interesting regions on pig chromosome 11, positional cloning and fine mapping of quantitative trait loci in pig.     

Construction of Genetic Linkage Maps and Comparative Genome Analysis of Catfish Using Gene-Associated Markers

A genetic linkage map of the channel catfish genome (N = 29) was constructed using EST-based microsatellite and single nucleotide polymorphism (SNP) markers in an interspecific reference family. A total of 413 microsatellites and 125 SNP markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 allowed mapping of 331 markers (259 microsatellites and 72 SNPs) to 29 linkage groups. Each linkage group contained 3–18 markers. The largest linkage group contained 18 markers and spanned 131.2 cM, while the smallest linkage group contained 14 markers and spanned only 7.9 cM. The linkage map covered a genetic distance of 1811 cM with an average marker interval of 6.0 cM. Sex-specific maps were also constructed; the recombination rate for females was 1.6 times higher than that for males. Putative conserved syntenies between catfish and zebrafish, medaka, and Tetraodon were established, but the overall levels of genome rearrangements were high among the teleost genomes. This study represents a first-generation linkage map constructed by using EST-derived microsatellites and SNPs, laying a framework for large-scale comparative genome analysis in catfish. The conserved syntenies identified here between the catfish and the three model fish species should facilitate structural genome analysis and evolutionary studies, but more importantly should facilitate functional inference of catfish genes. Given that determination of gene functions is difficult in nonmodel species such as catfish, functional genome analysis will have to rely heavily on the establishment of orthologies from model species.     

Mining ESTs to Determine the Usefulness of SNPs Across Shrimp Species

Expressed sequence tag (EST) libraries from members of the Penaeidae family and brine shrimp (Artemia franciscana) are currently the primary source of sequence data for shrimp species. Penaeid shrimp are the most commonly farmed worldwide, but selection methods for improving shrimp are limited. A better understanding of shrimp genomics is needed for farmers to use genetic markers to select the best breeding animals. The ESTs from Litopenaeus vannamei have been previously mined for single nucleotide polymorphisms (SNPs). This present study took publicly available ESTs from nine shrimp species, excluding L. vannamei, clustered them with CAP3, predicted SNPs within them using SNPidentifier, and then analyzed whether the SNPs were intra- or interspecies. Major goals of the project were to predict SNPs that may distinguish shrimp species, locate SNPs that may segregate in multiple species, and determine the genetic similarities between L. vannamei and the other shrimp species based on their EST sequences. Overall, 4,597 SNPs were predicted from 4,600 contigs with 703 of them being interspecies SNPs, 735 of them possibly predicting species' differences, and 18 of them appearing to segregate in multiple species. While sequences appear relatively well conserved, SNPs do not appear to be well conserved across shrimp species.     

Comparison of single-nucleotide polymorphisms and microsatellites in inference of population structure

Single-nucleotide polymorphisms (SNPs) are a class of attractive genetic markers for population genetic studies and for identifying genetic variations underlying complex traits. However, the usefulness and efficiency of SNPs in comparison to microsatellites in different scientific contexts, e.g., population structure inference or association analysis, still must be systematically evaluated through large empirical studies. In this article, we use the Collaborative Studies on Genetics of Alcoholism (COGA) data from Genetic Analysis Workshop 14 (GAW14) to compare the performance of microsatellites and SNPs in the whole human genome in the context of population structure inference. A total of 328 microsatellites and 15,840 SNPs are used to infer population structure in 236 unrelated individuals. We find that, on average, the informativeness of random microsatellites is four to twelve times that of random SNPs for various population comparisons, which is consistent with previous studies. Our results also indicate that for the combined set of microsatellites and SNPs, SNPs constitute the majority among the most informative markers and the use of these SNPs leads to better inference of population structure than the use of microsatellites. We also find that the inclusion of less informative markers may add noise and worsen the results.     

Using mtDNA Sequences to Estimate SNP Parameters in ESTs

Discovery of single nucleotide polymorphisms (SNPs) requires analysis of redundant sequences such as those available in large public databases. The ability to detect SNPs, especially those of low frequency, is dependent on the depth and scale of the discovery effort. Large numbers of SNPs have been identified by mining large-scale EST surveys and whole genome sequencing projects. These surveys however are subject to ascertainment bias and the inherent errors in large-scale single pass sequencing efforts. For example, the number of steps involved in the construction and sequencing of cDNA libraries make ESTs highly error prone, resulting in an increased frequency of nonvalid SNPs obtained in these surveys. Sequences of mtDNA genes are often incorporated into cDNA libraries as an artifact of the library construction process and are typically either subtracted from cDNA libraries or are considered superfluous when evaluating the information content of EST datasets. Sequences of mtDNA genes provide a unique resource for the analysis of SNP parameters in EST projects. This study uses sequences from four turkey muscle cDNA libraries to demonstrate how mtDNA sequences gleaned from collections of ESTs can be used to estimate SNP parameters and thus help predict the validity of SNPs.     

Using mtDNA sequences to estimate SNP parameters in ESTs

Discovery of single nucleotide polymorphisms (SNPs) requires analysis of redundant sequences such as those available in large public databases. The ability to detect SNPs, especially those of low frequency, is dependent on the depth and scale of the discovery effort. Large numbers of SNPs have been identified by mining large-scale EST surveys and whole genome sequencing projects. These surveys however are subject to ascertainment bias and the inherent errors in large-scale single pass sequencing efforts. For example, the number of steps involved in the construction and sequencing of cDNA libraries make ESTs highly error prone, resulting in an increased frequency of nonvalid SNPs obtained in these surveys. Sequences of mtDNA genes are often incorporated into cDNA libraries as an artifact of the library construction process and are typically either subtracted from cDNA libraries or are considered superfluous when evaluating the information content of EST datasets. Sequences of mtDNA genes provide a unique resource for the analysis of SNP parameters in EST projects. This study uses sequences from four turkey muscle cDNA libraries to demonstrate how mtDNA sequences gleaned from collections of ESTs can be used to estimate SNP parameters and thus help predict the validity of SNPs.