EST-derived Single Nucleotide Polymorphism Research Articles

Construction of a high density linkage map and its application in the identification of QTLs for soluble sugar and organic acid components in apple

Soluble sugars and organic acids have a strong impact on the overall organoleptic quality of fruits. In this study, we report the identification of quantitative trait loci (QTLs) for individual sugars and organic acids in apple. A high density linkage map of apple was constructed using the 1536 EST-derived SNP GoldenGate genotyping platform. The linkage map consists of 601 molecular markers, including 540 single nucleotide polymorphisms (SNPs) and 61 simple sequence repeats (SSRs), spanning 1368.4 cM with an average of 2.28 cM per marker. The contents of soluble sugars, including sucrose, glucose, fructose, sorbitol, and organic acids, including malic acid and citric acid, were used as the phenotypic data in QTL analysis. Two QTLs for malic acid content were detected on linkage groups (LGs) 8 and 16, while no QTL was found for citric acid content. Four QTLs for the glucose, sucrose, fructose, and sorbitol content were found to be clustered in one region on LG 3. Moreover, an additional QTL for glucose content was detected on the LG 4. Our study not only expands our understanding of the genetic basis for fruit organoleptic quality but it also provides molecular markers that will aid in marker-assisted selection for fruit quality in apple breeding programs.

Large-scale SNP discovery through RNA sequencing and SNP genotyping by targeted enrichment sequencing in cassava (Manihot esculenta Crantz).

Cassava (Manihot esculenta Crantz) is one of the most important crop species being the main source of dietary energy in several countries. Marker-assisted selection has become an essential tool in plant breeding. Single nucleotide polymorphism (SNP) discovery via transcriptome sequencing is an attractive strategy for genome complexity reduction in organisms with large genomes. We sequenced the transcriptome of 16 cassava accessions using the Illumina HiSeq platform and identified 675,559 EST-derived SNP markers. A subset of those markers was subsequently genotyped by capture-based targeted enrichment sequencing in 100 F1 progeny segregating for starch viscosity phenotypes. A total of 2,110 non-redundant SNP markers were used to construct a genetic map. This map encompasses 1,785 cM and consists of 19 linkage groups. A major quantitative trait locus (QTL) controlling starch pasting properties was identified and shown to coincide with the QTL previously reported for this trait. With a high-density SNP-based linkage map presented here, we also uncovered a novel QTL associated with starch pasting time on LG 10.

Transferability of Cupped Oyster EST (Expressed Sequence Tag)-Derived SNP (Single Nucleotide Polymorphism) Markers to Related Crassostrea and Ostrea Species

Single nucleotide polymorphisms (SNPs) are widely acknowledged as the marker of choice for many genetic and genomic applications because they show co-dominant inheritance, are highly abundant across genomes and are suitable for high-throughput genotyping. Here we evaluated the applicability of SNP markers developed from Crassostrea gigas and C. virginica expressed sequence tags (ESTs) in closely related Crassostrea and Ostrea species. A total of 213 putative interspecific level SNPs were identified from re-sequencing data in six amplicons, yielding on average of one interspecific level SNP per seven bp. High polymorphism levels were observed and the high success rate of transferability show that genic EST-derived SNP markers provide an efficient method for rapid marker development and SNP discovery in closely related oyster species. The six EST-SNP markers identified here will provide useful molecular tools for addressing questions in molecular ecology and evolution studies including for stock analysis (pedigree monitoring) in related oyster taxa.

Association of eight EST-derived SNPs with carcass and meat quality traits in pigs.

The identification of genetic markers associated with important economic traits is fundamental to improving the productivity and quality of livestock. In this investigation, we searched for 177 expressed sequence tags (ESTs) putatively involved in meat quality from the available pig EST database, and detected eight single nucleotide polymorphisms (SNPs) in eight ESTs. We investigated the associations of these SNPs with 18 carcass and meat quality traits in a Landrace × Lantang F2 resource population (n = 257). Association analysis revealed that seven SNPs (except E42) were associated with some of the carcass- and meat quality-related traits. Particularly, significant associations of three SNPs (E53, E82, and E36) with backfat thickness traits were observed. Further, the genetic effects of E53 on four live backfat thickness traits were validated in an independent population (n = 221). More investigations about E53 sequence characteristics were performed, i.e., radiation hybrid (RH) mapping, 3'-RACE, and screening analysis of the positive BAC clones. Our research identified the genetic effects of eight EST-derived SNPs on carcass and meat quality traits, and suggested that E53 may be a useful marker for live backfat thickness traits in pig breeding programs.

Comparative assessment of genomic SSR, EST–SSR and EST–SNP markers for evaluation of the genetic diversity of wild and cultured Pacific oyster, Crassostrea gigas Thunberg

Abstract A set of eight genomic simple sequence repeat (SSR), 10 expressed sequences tag (EST)-derived SSR and 10 EST-derived single nucleotide polymorphism (SNP) markers were analyzed on two wild and three cultured Pacific oyster (Crassostrea gigas Thunberg) populations to access their applicability for population genetic studies. Genomic SSR markers were found to be the most polymorphic, with an average PIC value of 0.870, higher than EST–SSRs (0.721) and SNPs (0.346). The Nei's gene diversity (HE) per locus for the genomic SSRs ranged from 0.821 to 0.936 (mean HE = 0.882), while that of the EST–SSRs ranged from 0.624 to 0.825 (mean HE = 0.755) and that of the EST–SNPs ranged from 0.372 to 0.499 (mean HE = 0.448). No significant differences in the number of alleles, HE and PIC values were identified between the wild and cultured populations in terms of all three types of markers. The effective marker index (EMI) was the highest (0.653) for the genomic SSR markers and the lowest (0.346) for the SNP markers, while the EST–SSR markers had an intermediate EMI (0.541). Comparable population pairwise estimates of FST were obtained for all marker types, but while the absolute values for the genomic and EST–SSRs were similar (FST = 0.041 and 0.032, respectively), the differentiation level was higher for EST–SNPs in pairwise and global comparisons (global EST–SNPs FST = 0.077). The dendrogram of the combined dataset, based on the shared-allele distance among populations, classified the wild and cultured population into two major clusters. Our results suggest 1) that genomic SSRs and EST–SSRs are more suitable for routine genetic diversity analysis than are EST–SNPs, and 2) that EST–SSR markers represent a useful addition to the tools available for population genetic analysis, particularly for aquaculture species with existing EST databases.

RadishBase: A Database for Genomics and Genetics of Radish

Radish is an economically important vegetable crop. During the past several years, large-scale genomics and genetics resources have been accumulated for this species. To store, query, analyze and integrate these radish resources efficiently, we have developed RadishBase (http://bioinfo.bti.cornell.edu/radish), a genomics and genetics database of radish. Currently the database contains radish mitochondrial genome sequences, expressed sequence tag (EST) and unigene sequences and annotations, biochemical pathways, EST-derived single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers, and genetic maps. RadishBase is designed to enable users easily to retrieve and visualize biologically important information through a set of efficient query interfaces and analysis tools, including the BLAST search and unigene annotation query interfaces, and tools to classify unigenes functionally, to identify enriched gene ontology (GO) terms and to visualize genetic maps. A database containing radish pathways predicted from unigene sequences is also included in RadishBase. The tools and interfaces in RadishBase allow efficient mining of recently released and continually expanding large-scale radish genomics and genetics data sets, including the radish genome sequences and RNA-seq data sets.

Development of 101 novel EST-derived single nucleotide polymorphism markers for Zhikong scallop (Chlamys farreri)

Zhikong scallop (Chlamys farreri) is an important maricultured species in China. Many researches on this species, such as population genetics and QTL fine-mapping, need a large number of molecular markers. In this study, based on the expressed sequence tags (EST), a total of 300 putative single nucleotide polymorphisms (SNPs) were selected and validated using high resolution melting (HRM) technology with unlabeled probe. Of them, 101 (33.7%) were found to be polymorphic in 48 individuals from 4 populations. Further evaluation with 48 individuals from Qingdao population showed that all the polymorphic loci had two alleles with the minor allele frequency ranged from 0.046 to 0.500. The observed and expected heterozygosities ranged from 0.000 to 0.925 and from 0.089 to 0.505, respectively. Fifteen loci deviated significantly from Hardy-Weinberg equilibrium and significant linkage disequilibrate was detected in one pair of markers. BLASTx gave significant hits for 72 of the 101 polymorphic SNP-containing ESTs. Thirty four polymorphic SNP loci were predicted to be non-synonymous substitutions as they caused either the change of codons (33 SNPs) or pretermination of translation (1 SNP). The markers developed can be used for the population studies and genetic improvement on Zhikong scallop.

Characterization of novel EST-derived SNP markers using 454 pyrosequencing in Sinonovacula constricta

The razor clam (Sinonovacula constricta) is an important aquaculture clam. But no single nucleotide polymorphisms (SNP) markers were characterized from expressed sequence tags (ESTs) in the species previously. Here, we designed 18 primers for 25 SNP loci generated from the transtriptome of S. constricta by 454 pyrosequencing. All the SNP loci were characterized on 24 individuals. The observed (Ho) and expected (He) heterozygosity ranged from 0.042 to 0.833 (mean 0.325) and from 0.082 to 0.515 (mean 0.360), respectively. Nine loci showed significant departure from Hardy–Weinberg equilibrium (p < 0.05). No linkage disequilibrium was detected between loci pairs. These new EST-SNP markers are useful for conservation genetics and genetic breeding of S. constricta.

EST-derived SNP discovery and selective pressure analysis in Pacific white shrimp (Litopenaeus vannamei)

Pacific white shrimp has become a major aquaculture and fishery species worldwide. Although a large scale EST resource has been publicly available since 2008, the data have not yet been widely used for SNP discovery or transcriptome-wide assessment of selective pressure. In this study, a set of 155 411 expressed sequence tags (ESTs) from the NCBI database were computationally analyzed and 17 225 single nucleotide polymorphisms (SNPs) were predicted, including 9 546 transitions, 5 124 transversions and 2 481 indels. Among the 7 298 SNP substitutions located in functionally annotated contigs, 58.4% (4 262) are non-synonymous SNPs capable of introducing amino acid mutations. Two hundred and fifty nonsynonymous SNPs in genes associated with economic traits have been identified as candidates for markers in selective breeding. Diversity estimates among the synonymous nucleotides were on average 3.49 times greater than those in non-synonymous, suggesting negative selection. Distribution of non-synonymous to synonymous substitutions (Ka/Ks) ratio ranges from 0 to 4.01, (average 0.42, median 0.26), suggesting that the majority of the affected genes are under purifying selection. Enrichment analysis identified multiple gene ontology categories under positive or negative selection. Categories involved in innate immune response and male gamete generation are rich in positively selected genes, which is similar to reports in Drosophila and primates. This work is the first transcriptome-wide assessment of selective pressure in a Penaeid shrimp species. The functionally annotated SNPs provide a valuable resource of potential molecular markers for selective breeding.

Characterization of 38 EST-derived SNP markers in Zhikong scallop (Chlamys farreri) and their cross-species utility in Yesso scallop (Patinopecten yessoensis)

Scallops are bivalve molluscs of worldwide commercial significance. The advent of next-generation sequencing technologies provides an excellent opportunity to rapidly and cost-effectively discover a large number of single nucleotide polymorphisms (SNPs) in scallops. Here we described an approach that could be potentially used to screen interspecific transferable SNPs from transcriptomic data. Following this approach, 38 polymorphic SNP markers were developed in Zhikong scallop, of which 8 were also polymorphic in Yesso scallop, resulting in a transferability rate of 21 %, which is 4.6 times higher than that from randomly selected SNP markers. To our knowledge, this is the first report on the survey of SNP transferability in scallops. SNP markers developed in this study would be useful in genetic studies as well as evolutionary studies in the two scallop species.

The Cassava Genome: Current Progress, Future Directions

The starchy swollen roots of cassava provide an essential food source for nearly a billion people, as well as possibilities for bioenergy, yet improvements to nutritional content and resistance to threatening diseases are currently impeded. A 454-based whole genome shotgun sequence has been assembled, which covers 69% of the predicted genome size and 96% of protein-coding gene space, with genome finishing underway. The predicted 30,666 genes and 3,485 alternate splice forms are supported by 1.4 M expressed sequence tags (ESTs). Maps based on simple sequence repeat (SSR)-, and EST-derived single nucleotide polymorphisms (SNPs) already exist. Thanks to the genome sequence, a high-density linkage map is currently being developed from a cross between two diverse cassava cultivars: one susceptible to cassava brown streak disease; the other resistant. An efficient genotyping-by-sequencing (GBS) approach is being developed to catalog SNPs both within the mapping population and among diverse African farmer-preferred varieties of cassava. These resources will accelerate marker-assisted breeding programs, allowing improvements in disease-resistance and nutrition, and will help us understand the genetic basis for disease resistance.

Development and characterisation of an expressed sequence tags (EST)-derived single nucleotide polymorphisms (SNPs) resource in rainbow trout

BackgroundThere is considerable interest in developing high-throughput genotyping with single nucleotide polymorphisms (SNPs) for the identification of genes affecting important ecological or economical traits. SNPs are evenly distributed throughout the genome and are likely to be functionally relevant. In rainbow trout, in silico screening of EST databases represents an attractive approach for de novo SNP identification. Nevertheless, EST sequencing errors and assembly of EST paralogous sequences can lead to the identification of false positive SNPs which renders the reliability of EST-derived SNPs relatively low. Further validation of EST-derived SNPs is therefore required. The objective of this work was to assess the quality of and to validate a large number of rainbow trout EST-derived SNPs.ResultsA panel of 1,152 EST-derived SNPs was selected from the INRA Sigenae SNP database and was genotyped in standard and double haploid individuals from several populations using the Illumina GoldenGate BeadXpress assay. High-quality genotyping data were obtained for 958 SNPs representing a genotyping success rate of 83.2 %, out of which, 350 SNPs (36.5 %) were polymorphic in at least one population and were designated as true SNPs. They also proved to be a potential tool to investigate genetic diversity of the species, as the set of SNP successfully sorted individuals into three main groups using STRUCTURE software. Functional annotations revealed 28 non-synonymous SNPs, out of which four substitutions were predicted to affect protein functions. A subset of 223 true SNPs were polymorphic in the two INRA mapping reference families and were integrated into the INRA microsatellite-based linkage map.ConclusionsOur results represent the first study of EST-derived SNPs validation in rainbow trout, a species whose genome sequences is not yet available. We designed several specific filters in order to improve the genotyping yield. Nevertheless, our selection criteria should be further improved in order to reduce the observed high rate of false positive SNPs which results from the occurrence of whole genome duplications.

Cowpea-Soybean Synteny Clarified through an Improved Genetic Map

Linkage mapping is relevant to modern plant biology and provides a framework for downstream analyses including quantitative trait loci identification, map-based cloning, assessment of diversity, association mapping, and molecular breeding. Here, we report a consensus genetic map of cowpea [Vigna unguiculata (L.) Walp.] and synteny to other legumes based on expressed sequence tag (EST)-derived single nucleotide polymorphisms (SNPs). In total, 1293 individuals representing 13 mapping populations were genotyped using an Illumina 1536 GoldenGate Assay. A consensus map containing 1107 EST-derived SNP markers (856 bins) on 11 linkage groups (680 cM) was constructed from 13 population-specific maps. This effort combined six new population-specific maps and seven revised population-specific maps to construct an improved consensus map with 33% more bins, 19% more markers, and improved marker order when compared to the previous cowpea SNP consensus map. Comparative and whole genome visualizations are presented as a framework for discussing map quality and synteny with soybean [Glycine max (L.) Merr.].

The First Set of EST-derived Single Nucleotide Polymorphism Markers for Japanese Scallop, Patinopecten yessoensis

The First Set of EST-derived Single Nucleotide Polymorphism Markers for Japanese Scallop, Patinopecten yessoensis

Identification of single nucleotide polymorphism in ginger using expressed sequence tags.

Ginger (Zingiber officinale Rosc) (Family: Zingiberaceae) is a herbaceous perennial, the rhizomes of which are used as a spice. Ginger is a plant which is well known for its medicinal applications. Recently EST-derived SNPs are a free by-product of the currently expanding EST (Expressed Sequence Tag) databases. The development of high-throughput methods for the detection of SNPs (Single Nucleotide Polymorphism) and small indels (insertion/deletion) has led to a revolution in their use as molecular markers. Available (38139) Ginger EST sequences were mined from dbEST of NCBI. CAP3 program was used to assemble EST sequences into contigs. Candidate SNPs and Indel polymorphisms were detected using the perl script AutoSNP version 1.0 which has used 31905 ESTs for detecting SNPs and Indel sites. We found 64026 SNP sites and 7034 indel polymorphisms with frequency of 0.84 SNPs / 100 bp. Among the three tissues from which the EST libraries had been generated, Rhizomes had high frequency of 1.08 SNPs/indels per 100 bp whereas the leaves had lowest frequency of 0.63 per 100 bp and root is showing relative frequency 0.82/100bp. Transitions and transversion ratio is 0.90. In overall detected SNP, transversion is high when compare to transition. These detected SNPs can be used as markers for genetic studies. The results of the present study hosted in our webserver www.spices.res.in/spicesnip.

Bioinformatics Analysis of Distribution of Microsatellite Markers (SSRs) / Single Nucleotide Polymorphism (SNPs) in Expressed Transcripts of Prosopis Juliflora: Frequency and Distribution

The present paper aims to identify the distribution of Microsatellite markers (SSRs)/Single Nucleotide Polymorphism (SNPs) as resource tools for the analysis of interspecies hypervariability in Prosopis spp. Microsatellites are ubiquitously repeated extension of 1-5 bp motif extended throughout the genome. The dbEST division of Genbank contains 1467 ESTs of Prosopis juliflora which have been utilized for the development of genic microsatellite markers (SSRs). Locally installed assembling program was used for the cluster analysis of the EST. Analysis was performed on a Linux Cluster system. The analysis of the putative unigenes has been shown to have most abundant motif of A/T followed by dinucleotide AG/CT and trinucleotide repeat AAG/CTT. EST-derived SNPs are becoming the resources for the development of SNP markers. The relative rates of development of the high throughput computational methods for the detection of SNPs (Single Nucleotide Polymorphism) and small indels (insertion / deletion) has gained wide applications in the field of the molecular markers.

A linkage map of the Atlantic salmon (Salmo salar) based on EST-derived SNP markers

BackgroundThe Atlantic salmon is a species of commercial and ecological significance. Like other salmonids, the species displays residual tetrasomy and a large difference in recombination rate between sexes. Linkage maps with full genome coverage, containing both type I and type II markers, are needed for progress in genomics. Furthermore, it is important to estimate levels of linkage disequilibrium (LD) in the species. In this study, we developed several hundred single nucleotide polymorphism (SNP) markers for the Atlantic salmon, and constructed male and female linkage maps containing SNP and microsatellite markers. We also investigated further the distribution of male and female recombination events across the genome, and estimated levels of LD between pairs of markers.ResultsThe male map had 29 linkage groups and was 390 cM long. The female map had 30 linkage groups as was 1983 cM long. In total, the maps contained 138 microsatellite markers and 304 SNPs located within genes, most of which were successfully annotated. The ratio of male to female recombination events was either close to zero or very large, indicating that there is little overlap between regions in which male and female crossovers occur. The female map is likely to have close to full genome coverage, while the majority of male linkage groups probably lack markers in telomeric regions where male recombination events occur. Levels of r2 increased with decreasing inter-marker distance in a bimodal fashion; increasing slowly from ~60 cM, and more rapidly more from ~12 cM. Long-ranging LD may be consequence of recent admixture in the population, the population being a 'synthetic' breeding population with contributions from several distinct rivers. Levels of r2 dropped to half its maximum value (above baseline) within 15 cM, and were higher than 0.2 above baseline for unlinked markers ('useful LD') at inter-marker distances less than 5 cM.ConclusionThe linkage map presented here is an important resource for genetic, comparative, and physical mapping of the Atlantic salmon. The female map is likely to have a map coverage that is not far from complete, whereas the male map length is likely to be significantly shorter than the true map, due to suboptimal marker coverage in the apparently small physical regions where male crossovers occur. 'Useful LD' was found at inter-marker distances less than 5 cM.

Mining for single nucleotide polymorphisms and insertions / deletions in expressed sequence tag libraries of oil palm

The oil palm is a tropical oil bearing tree. Recently EST-derived SNPs and SSRs are a free by-product of the currently expanding EST (Expressed Sequence Tag) data bases. The development of high-throughput methods for the detection of SNPs (Single Nucleotide Polymorphism) and small indels (insertion / deletion) has led to a revolution in their use as molecular markers. Available (5452) Oil palm EST sequences were mined from dbEST of NCBI. CAP3 program was used to assemble EST sequences into contigs. Candidate SNPs and Indel polymorphisms were detected using the perl script auto_snip version 1.0 which has used 576 ESTs for detecting SNPs and Indel sites. We found 1180 SNP sites and 137 indel polymorphisms with frequency 1.36 SNPs / 100 bp. Among the six tissues from which the EST libraries had been generated, mesocarp had high frequency of 2.91 SNPs and indels per 100 bp whereas the zygotic embryos had lowest frequency of 0.15 per 100 bp. We also used the Shannon index to analyze the proportion of ten possible types of SNP/indels. ESTs from tissues of normal apex showed highest values of Shannon index (0.60) whereas abnormal apex had least value (0.02). The present report deals the use of Shannon index for comparing SNP/ indel frequencies mined from ESTlibraries and also confirm that the frequency of SNP occurrence in oil palm to use them as markers for genetic studies.

EST-derived single nucleotide polymorphism markers for assembling genetic and physical maps of the barley genome

In a panel of seven genotypes, 437 expressed sequence tag (EST)-derived DNA fragments were sequenced. Single nucleotide polymorphisms (SNPs) that were polymorphic between the parents of three mapping populations were mapped by heteroduplex analysis and a genome-wide consensus map comprising 216 EST-derived SNPs and 4 InDel (insertion/deletion) markers was constructed. The average frequency of SNPs amounted to 1/130 bp and 1/107.8 bp for a set of randomly selected and a set of mapped ESTs, respectively. The calculated nucleotide diversities (pi) ranged from 0 to 40.0 x 10(-3) (average 3.1 x 10(-3)) and 0.52 x 10(-3) to 39.51 x 10(-3) (average 4.37 x 10(-3)) for random and mapped ESTs, respectively. The polymorphism information content value for mapped SNPs ranged from 0.24 to 0.50 with an average of 0.34. As expected, combination of SNPs present in an amplicon (haplotype) exhibited a higher information content ranging from 0.24 to 0.85 with an average of 0.50. Cleaved amplified polymorphic sequence assays (including InDels) were designed for a total of 87 (39.5%) SNP markers. The high abundance of SNPs in the barley genome provides avenues for the systematic development of saturated genetic maps and their integration with physical maps.

Comparative assessment of EST-SSR, EST-SNP and AFLP markers for evaluation of genetic diversity and conservation of genetic resources using wild, cultivated and elite barleys

A set of 16 expressed sequence tag (EST)-derived simple sequence repeat (SSR) and 15 EST-derived single nucleotide polymorphism (SNP) markers together with 4 amplified fragment length polymorphism (AFLP) primer combinations were analyzed on 43 wild ( Hordeum vulgare ssp. s pontaneum – HS), 35 cultivated ( H. vulgare ssp. vulgare – HV) and 12 elite ( H. vulgare ssp. vulgare – from EU) barley lines. SSR markers were found most polymorphic with an average PIC value of 0.593 and eight alleles per marker, while AFLP markers showed the highest effective multiplex ratio (26.4) and marker index (5.042). The effective marker index (EMI) was recorded highest (0.468) for AFLP markers and lowest (0.341) for the SNP markers while the SSR markers had an intermediate EMI (0.442). Cluster analysis on combined set of SSR, SNP and AFLP genotyping data classified wild, cultivated and elite barley lines in three distinct groups. The present study suggests the SNP markers as the best class of markers for characterizing and conserving the genebank materials and the AFLP and SSR markers more suitable for diversity analysis and fingerprinting.