Discovery of single nucleotide polymorphisms in Lycopersicon esculentum by computer aided analysis of expressed sequence tags

Single nucleotide polymorphisms (SNPs) are useful for characterizing allelic variation, for genome-wide mapping, and as a tool for marker-assisted selection. Discovery of SNPs through de novo sequencing is inefficient within cultivated tomato (Lycopersicon esculentum Mill.) because the polymorphism rate is more than ten-fold lower than the sequencing error rate. The availability of expressed sequence tag (EST) data has made it feasible to discover putative SNPs in silico prior to experimental verification. By exploiting redundancy among EST data available for different varieties among 148,373 tomato ESTs, we have identified candidate SNPs for use within cultivated germplasm pools. 1,245 contigs having three EST sequences of Rio Grande and three EST sequences of TA496 were used for SNP discovery. We detected 1 SNP for every 8,500 bases analyzed, with 101 candidate SNPs in 44 genes identified. Sixty-six SNPs could be recognized by restriction enzymes, and subsequent experimental verification using restriction digestion or CEL I digestion confirmed 83% of the putative polymorphisms tested. SNPs between TA496 and Rio Grande have a high probability (53%) of detecting polymorphisms between other L. esculentum varieties. Twenty-six SNPs in 18 unigenes were mapped to specific chromosomes. Two SNPs, LEOH23 and LEOH37, were shown to be linked to quantitative trait loci contributing to fruit color within elite breeding populations. These results suggest that the growing databases of DNA sequence will yield information that facilitates improvement within the germplasm pools that have contributed to productive modern varieties.     

Single nucleotide polymorphisms in cytochrome P450 genes from barley

Plant cytochrome P450s are known to be essential in a number of economically important pathways of plant metabolism but there are also many P450s of unknown function accumulating in expressed sequence tag (EST) and genomic databases. To detect trait associations that could assist in the assignment of gene function and provide markers for breeders selecting for commercially important traits, detection of polymorphisms in identified P450 genes is desirable. Polymorphisms in EST sequences provide so-called perfect markers for the associated genes. The International Triticeae EST Cooperative data base of 24,344 ESTs was searched for sequences exhibiting homology to P450 genes representing the nine known clans of plant P450s. Seventy five P450 ESTs were identified of which 24 had best matches in Genbank to P450 genes of known function and 51 to P450s of unknown function. Sequence information from PCR products amplified from the genomic template DNA of 11 barley varieties was obtained using primers designed from six barley P450 ESTs and one durum wheat P450 EST. Single nucleotide polymorphisms (SNPs) between barley varieties were identified using five of the seven PCR products. A maximum of five SNPs and three haplotypes among the 11 barley lines were detected in products from any one primer pair. SNPs in three PCR products led to changes between barley varieties in at least one restriction site enabling genotyping and mapping without the expense of a specialist SNP detection system. The overall frequency of SNPs across the 11 barley varieties was 1 every 131 bases.     

An interactive bovine <Emphasis Type="Italic">in silico</Emphasis> SNP database (IBISS)

An interactive bovine in silico SNP (IBISS) database has been created through the clustering and aligning of bovine EST and mRNA sequences. Approximately 324,000 EST and mRNA sequences were clustered to produce 29,965 clusters (producing 48,679 consensus sequences) and 48,565 singletons. A SNP screening regime was placed on variations detected in the multiple sequence alignment files to determine which SNPs are more likely to be real rather than sequencing errors. A small subset of predicted SNPs was validated on a diverse set of bovine DNA samples using PCR amplification and sequencing. Fifty percent of the predicted SNPs in the putative >1 category were polymorphic in the population sampled. The IBISS database represents more than just a SNP database; it is also a genomic database containing uniformly annotated predicted gene mRNA and protein sequences, gene structure, and genomic organization information.     

A set of EST-SNPs for map saturation and cultivar identification in melon

Background

There are few genomic tools available in melon (Cucumis melo L.), a member of the Cucurbitaceae, despite its importance as a crop. Among these tools, genetic maps have been constructed mainly using marker types such as simple sequence repeats (SSR), restriction fragment length polymorphisms (RFLP) and amplified fragment length polymorphisms (AFLP) in different mapping populations. There is a growing need for saturating the genetic map with single nucleotide polymorphisms (SNP), more amenable for high throughput analysis, especially if these markers are located in gene coding regions, to provide functional markers. Expressed sequence tags (ESTs) from melon are available in public databases, and resequencing ESTs or validating SNPs detected in silico are excellent ways to discover SNPs.

Results

EST-based SNPs were discovered after resequencing ESTs between the parental lines of the PI 161375 (SC) × 'Piel de sapo' (PS) genetic map or using in silico SNP information from EST databases. In total 200 EST-based SNPs were mapped in the melon genetic map using a bin-mapping strategy, increasing the map density to 2.35 cM/marker. A subset of 45 SNPs was used to study variation in a panel of 48 melon accessions covering a wide range of the genetic diversity of the species. SNP analysis correctly reflected the genetic relationships compared with other marker systems, being able to distinguish all the accessions and cultivars.

Conclusion

This is the first example of a genetic map in a cucurbit species that includes a major set of SNP markers discovered using ESTs. The PI 161375 × 'Piel de sapo' melon genetic map has around 700 markers, of which more than 500 are gene-based markers (SNP, RFLP and SSR). This genetic map will be a central tool for the construction of the melon physical map, the step prior to sequencing the complete genome. Using the set of SNP markers, it was possible to define the genetic relationships within a collection of forty-eight melon accessions as efficiently as with SSR markers, and these markers may also be useful for cultivar identification in Occidental melon varieties.     

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.     

Genome wide SNP discovery,analysis and evaluation in mallard (<Emphasis Type="Italic">Anas platyrhynchos</Emphasis>)

Background

Next generation sequencing technologies allow to obtain at low cost the genomic sequence information that currently lacks for most economically and ecologically important organisms. For the mallard duck genomic data is limited. The mallard is, besides a species of large agricultural and societal importance, also the focal species when it comes to long distance dispersal of Avian Influenza. For large scale identification of SNPs we performed Illumina sequencing of wild mallard DNA and compared our data with ongoing genome and EST sequencing of domesticated conspecifics. This is the first study of its kind for waterfowl.

Results

More than one billion base pairs of sequence information were generated resulting in a 16× coverage of a reduced representation library of the mallard genome. Sequence reads were aligned to a draft domesticated duck reference genome and allowed for the detection of over 122,000 SNPs within our mallard sequence dataset. In addition, almost 62,000 nucleotide positions on the domesticated duck reference showed a different nucleotide compared to wild mallard. Approximately 20,000 SNPs identified within our data were shared with SNPs identified in the sequenced domestic duck or in EST sequencing projects. The shared SNPs were considered to be highly reliable and were used to benchmark non-shared SNPs for quality. Genotyping of a representative sample of 364 SNPs resulted in a SNP conversion rate of 99.7%. The correlation of the minor allele count and observed minor allele frequency in the SNP discovery pool was 0.72.

Conclusion

We identified almost 150,000 SNPs in wild mallards that will likely yield good results in genotyping. Of these, ~101,000 SNPs were detected within our wild mallard sequences and ~49,000 were detected between wild and domesticated duck data. In the ~101,000 SNPs we found a subset of ~20,000 SNPs shared between wild mallards and the sequenced domesticated duck suggesting a low genetic divergence. Comparison of quality metrics between the total SNP set (122,000 + 62,000 = 184,000 SNPs) and the validated subset shows similar characteristics for both sets. This indicates that we have detected a large amount (~150,000) of accurately inferred mallard SNPs, which will benefit bird evolutionary studies, ecological studies (e.g. disentangling migratory connectivity) and industrial breeding programs.

     

Mining single-nucleotide polymorphisms from hexaploid wheat ESTs.

Single-nucleotide polymorphisms (SNPs) represent a new form of functional marker, particularly when they are derived from expressed sequence tags (ESTs). A bioinformatics strategy was developed to discover SNPs within a large wheat EST database and to demonstrate the utility of SNPs in genetic mapping and genetic diversity applications. A collection of > 90000 wheat ESTs was assembled into contiguous sequences (contigs), and 45 random contigs were then visually inspected to identify primer pairs capable of amplifying specific alleles. We estimate that homoeologue sequence variants occurred 1 in 24 bp and the frequency of SNPs between wheat genotypes was 1 SNP/540 bp (theta = 0.0069). Furthermore, we estimate that one diagnostic SNP test can be developed from every contig with 10-60 EST members. Thus, EST databases are an abundant source of SNP markers. Polymorphism information content for SNPs ranged from 0.04 to 0.50 and ESTs could be mapped into a framework of microsatellite markers using segregating populations. The results showed that SNPs in wheat can be discovered in ESTs, validated, and be applied to conventional genetic studies.     

Association between fatty acid compositions and genotypes of FABP4 and LXR-alpha in Japanese Black cattle

Background

Single nucleotide polymorphisms (SNPs) and small insertions or deletions (indels) are the most common type of polymorphisms and are frequently used for molecular marker development. Such markers have become very popular for all kinds of genetic analysis, including haplotype reconstruction. Haplotypes can be reconstructed for whole chromosomes but also for specific genes, based on the SNPs present. Haplotypes in the latter context represent the different alleles of a gene. The computational approach to SNP mining is becoming increasingly popular because of the continuously increasing number of sequences deposited in databases, which allows a more accurate identification of SNPs. Several software packages have been developed for SNP mining from databases. From these, QualitySNP is the only tool that combines SNP detection with the reconstruction of alleles, which results in a lower number of false positive SNPs and also works much faster than other programs. We have build a web-based SNP discovery and allele detection tool (HaploSNPer) based on QualitySNP.

Results

HaploSNPer is a flexible web-based tool for detecting SNPs and alleles in user-specified input sequences from both diploid and polyploid species. It includes BLAST for finding homologous sequences in public EST databases, CAP3 or PHRAP for aligning them, and QualitySNP for discovering reliable allelic sequences and SNPs. All possible and reliable alleles are detected by a mathematical algorithm using potential SNP information. Reliable SNPs are then identified based on the reconstructed alleles and on sequence redundancy.

Conclusion

Thorough testing of HaploSNPer (and the underlying QualitySNP algorithm) has shown that EST information alone is sufficient for the identification of alleles and that reliable SNPs can be found efficiently. Furthermore, HaploSNPer supplies a user friendly interface for visualization of SNP and alleles. HaploSNPer is available from http://www.bioinformatics.nl/tools/haplosnper/.     

ESTs as a source for sequence polymorphism discovery in sugarcane: example of the <Emphasis Type="Italic">Adh</Emphasis> genes

Expressed sequence tags (ESTs) have proven to be a valuable tool to discover single nucleotide polymorphism (SNP) in human genes but their use for this purpose is still limited in higher plants. Using a database of approximately 250,000 sugarcane ESTs we have recovered 219 sequences encoding alcohol dehydrogenases ( Adh), which tagged 178 distinct cDNAs from 27 libraries, constructed from at least four different cultivars. The partitioning of these ESTs into paralogous genes revealed three Adh genes expressed in sugarcane, one Adh2 and two Adh1. The soundness of the partition was carefully checked by comparison to external data, especially from the closely related sorghum. Analysis of polymorphism in the alignments of EST sequences revealed a total of 37 highly reliable SNPs in the coding and untranslated regions of the three Adh genes. In the coding regions, the mean occurrence of SNPs was one for every 122 base pair. A total of eight insertion-deletions was observed, their occurrence being limited to untranslated regions. These results show that EST data constitute an invaluable source of sequence polymorphism for sugarcane that is worth carefully collecting for the future development of new marker tools.     

QualitySNP: a pipeline for detecting single nucleotide polymorphisms and insertions/deletions in EST data from diploid and polyploid species

Background  

Single nucleotide polymorphisms (SNPs) are important tools in studying complex genetic traits and genome evolution. Computational strategies for SNP discovery make use of the large number of sequences present in public databases (in most cases as expressed sequence tags (ESTs)) and are considered to be faster and more cost-effective than experimental procedures. A major challenge in computational SNP discovery is distinguishing allelic variation from sequence variation between paralogous sequences, in addition to recognizing sequencing errors. For the majority of the public EST sequences, trace or quality files are lacking which makes detection of reliable SNPs even more difficult because it has to rely on sequence comparisons only.     

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.     

High-throughput single nucleotide polymorphism (SNP) identification and mapping in the sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) genome with genotyping by sequencing (GBS) analysis

Sesame (Sesamum indicum L. syn. Sesamum orientale L.) is considered to be the first oil seed crop known to man. Despite its versatile use as an oil seed and a leafy vegetable, sesame is a neglected crop and has not been a subject of molecular genetic research until the last decade. There is thus limited knowledge regarding genome-specific molecular markers that are indispensible for germplasm enhancement, gene identification, and marker-assisted breeding in sesame. In this study, we employed a genotyping by sequencing (GBS) approach to a sesame recombinant inbred line (RIL) population for high-throughput single nucleotide polymorphism (SNP) identification and genotyping. A total of 15,521 SNPs were identified with 14,786 SNPs (95.26 %) located along sesame genome assembly pseudomolecules. By incorporating sesame-specific simple sequence repeat (SSR) markers developed in our previous work, 230.73 megabases (99 %) of sequence from the genome assembly were saturated with markers. This large number of markers will be available for sesame geneticists as a resource for candidate polymorphisms located along the physical chromosomes of sesame. Defining SNP loci in genome assembly sequences provides the flexibility to utilize any genotyping strategy to survey any sesame population. SNPs selected through a high stringency filtering protocol (770 SNPs) for improved map accuracy were used in conjunction with SSR markers (50 SSRs) in linkage analysis, resulting in 13 linkage groups that encompass a total genetic distance of 914 cM with 432 markers (420 SNPs, 12 SSRs). The genetic linkage map constitutes the basis for future work that will involve quantitative trait locus (QTL) analyses of metabolic and agronomic traits in the segregating RIL population.     

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.     

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.     

Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers

A system to use bovine EST data in conjunction with human genomic sequence to improve the bovine linkage map over the entire genome or on specific chromosomes was evaluated. Bovine EST sequence was used to provide primer sequences corresponding to bovine genes, while human genomic sequence directed primer design to flank introns and produce amplicons of appropriate size for efficient direct sequencing. The sequence tagged sites (STS) produced in this way from the four sires of the MARC reference families were examined for single nucleotide polymorphisms (SNPs) that could be used to map the corresponding genes. With this approach, along with a primer/extension mass spectrometry SNP genotyping assay, 100 ESTs were placed on the bovine genetic linkage map. The first 70 were chosen at random from bovine EST–human genomic comparisons. An additional 30 ESTs were successfully mapped to bovine Chromosome 19 (BTA19), and comparison of the resulting BTA19 map to the position of the corresponding human orthologs on the HSA17 draft sequences revealed differences in the spacing and order of genes. Over 80% of successful amplicons contained SNPs, indicating that this is an efficient approach to generating EST-associated genetic markers. We have demonstrated the feasibility of constructing a linkage map based on SNPs associated with ESTs and the plausibility of utilizing EST, comparative mapping information, and human sequence data to target regions of the bovine genome for SNP marker development.     

Expressed sequence tags and their application for plant research

Expressed Sequence Tags (ESTs) are short, usually unedited sequences obtained by single-pass sequencing of cDNA clones from any cDNA library. Analyzing and comparing ESTs can provide information on gene expression, function and evolution. Large-scale EST sequencing has become an attractive alternative to plant genome sequencing. Currently, plant EST collections comprise over 3.8 million sequences from about 200 species. They have proved to be a valuable tool for gene discovery and plant metabolism analysis. Several plant-specific EST databases have been created which provide access to sequence data and bioinformatics-based tools for data mining. Searching EST collections allows pre-selection of genes for preparing cDNA arrays, targeted to bring maximum information on specialized processes, like stress response, symbiotic nitrogen fixation etc. Also, ESt-based molecular markers such as SNP, SSR, and indels are fast developing tools for breeders and researchers.