Whole-genome association mapping in elite inbred crop varieties

We have previously shown that linkage disequilibrium (LD) in the elite cultivated barley (Hordeum vulgare) gene pool extends, on average, for <1-5 cM. Based on this information, we have developed a platform for whole genome association studies that comprises a collection of elite lines that we have characterized at 3060 genome-wide single nucleotide polymorphism (SNP) marker loci. Interrogating this data set shows that significant population substructure is present within the elite gene pool and that diversity and LD vary considerably across each of the seven barley chromosomes. However, we also show that a subpopulation comprised of only the two-rowed spring germplasm is less structured and well suited to whole genome association studies without the need for extensive statistical intervention to account for structure. At the current marker density, the two-rowed spring population is suited for fine mapping simple traits that are located outside of the genetic centromeres with a resolution that is sufficient for candidate gene identification by exploiting conservation of synteny with fully sequenced model genomes and the emerging barley physical map.     

Next-generation RAD sequencing identifies thousands of SNPs for assessing hybridization between rainbow and westslope cutthroat trout

The increased numbers of genetic markers produced by genomic techniques have the potential to both identify hybrid individuals and localize chromosomal regions responding to selection and contributing to introgression. We used restriction-site-associated DNA sequencing to identify a dense set of candidate SNP loci with fixed allelic differences between introduced rainbow trout (Oncorhynchus mykiss) and native westslope cutthroat trout (Oncorhynchus clarkii lewisi). We distinguished candidate SNPs from homeologs (paralogs resulting from whole-genome duplication) by detecting excessively high observed heterozygosity and deviations from Hardy-Weinberg proportions. We identified 2923 candidate species-specific SNPs from a single Illumina sequencing lane containing 24 barcode-labelled individuals. Published sequence data and ongoing genome sequencing of rainbow trout will allow physical mapping of SNP loci for genome-wide scans and will also provide flanking sequence for design of qPCR-based TaqMan(?) assays for high-throughput, low-cost hybrid identification using a subset of 50-100 loci. This study demonstrates that it is now feasible to identify thousands of informative SNPs in nonmodel species quickly and at reasonable cost, even if no prior genomic information is available.     

Patterns of polymorphism and linkage disequilibrium in cultivated barley

We carried out a genome-wide analysis of polymorphism (4,596 SNP loci across 190 elite cultivated accessions) chosen to represent the available genetic variation in current elite North West European and North American barley germplasm. Population sub-structure, patterns of diversity and linkage disequilibrium varied considerably across the seven barley chromosomes. Gene-rich and rarely recombining haplotype blocks that may represent up to 60% of the physical length of barley chromosomes extended across the ‘genetic centromeres’. By positioning 2,132 bi-parentally mapped SNP markers with minimum allele frequencies higher than 0.10 by association mapping, 87.3% were located to within 5 cM of their original genetic map position. We show that at this current marker density genetically diverse populations of relatively small size are sufficient to fine map simple traits, providing they are not strongly stratified within the sample, fall outside the genetic centromeres and population sub-structure is effectively controlled in the analysis. Our results have important implications for association mapping, positional cloning, physical mapping and practical plant breeding in barley and other major world cereals including wheat and rye that exhibit comparable genome and genetic features.     

Barley Cbf3 gene identification,expression pattern,and map location

Although cold and drought adaptation in cereals and other plants involve the induction of a large number of genes, inheritance studies in Triticeae (wheat [Triticum aestivum], barley [Hordeum vulgare], and rye [Secale cereale]) have revealed only a few major loci for frost or drought tolerance that are consistent across multiple genetic backgrounds and environments. One might imagine that these loci could encode highly conserved regulatory factors that have global effects on gene expression; therefore, genes encoding central regulators identified in other plants might be orthologs of these Triticeae stress tolerance genes. The CBF/DREB1 regulators, identified originally in Arabidopsis as key components of cold and drought regulation, merit this consideration. We constructed barley cDNA libraries, screened these libraries and a barley bacterial artificial chromosome library using rice (Oryza sativa) and barley Cbf probes, found orthologs of Arabidopsis CBF/DREB1 genes, and examined the expression and genetic map location of the barley Cbf3 gene, HvCbf3. HvCbf3 was induced by a chilling treatment. HvCbf3 is located on barley chromosome 5H between markers WG364b and saflp58 on the barley cv Dicktoo x barley cv Morex genetic linkage map. This position is some 40 to 50 cM proximal to the winter hardiness quantitative trait locus that includes the Vrn-1H gene, but may coincide with the wheat 5A Rcg1 locus, which governs the threshold temperature at which cor genes are induced. From this, it remains possible that HvCbf3 is the basis of a minor quantitative trait locus in some genetic backgrounds, though that possibility remains to be thoroughly explored.     

Germ-line DNA copy number variation frequencies in a large North American population

Genomic copy number variation (CNV) is a recently identified form of global genetic variation in the human genome. The Affymetrix GeneChip 100 and 500 K SNP genotyping platforms were used to perform a large-scale population-based study of CNV frequency. We constructed a genomic map of 578 CNV regions, covering approximately 220 Mb (7.3%) of the human genome, identifying 183 previously unknown intervals. Copy number changes were observed to occur infrequently (<1%) in the majority (>93%) of these genomic regions, but encompass hundreds of genes and disease loci. This North American population-based map will be a useful resource for future genetic studies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

SNP Haplotype Mapping in a Small ALS Family

The identification of genes for monogenic disorders has proven to be highly effective for understanding disease mechanisms, pathways and gene function in humans. Nevertheless, while thousands of Mendelian disorders have not yet been mapped there has been a trend away from studying single-gene disorders. In part, this is due to the fact that many of the remaining single-gene families are not large enough to map the disease locus to a single site in the genome. New tools and approaches are needed to allow researchers to effectively tap into this genetic gold-mine. Towards this goal, we have used haploid cell lines to experimentally validate the use of high-density single nucleotide polymorphism (SNP) arrays to define genome-wide haplotypes and candidate regions, using a small amyotrophic lateral sclerosis (ALS) family as a prototype. Specifically, we used haploid-cell lines to determine if high-density SNP arrays accurately predict haplotypes across entire chromosomes and show that haplotype information significantly enhances the genetic information in small families. Panels of haploid-cell lines were generated and a 5 centimorgan (cM) short tandem repeat polymorphism (STRP) genome scan was performed. Experimentally derived haplotypes for entire chromosomes were used to directly identify regions of the genome identical-by-descent in 5 affected individuals. Comparisons between experimentally determined and in silico haplotypes predicted from SNP arrays demonstrate that SNP analysis of diploid DNA accurately predicted chromosomal haplotypes. These methods precisely identified 12 candidate intervals, which are shared by all 5 affected individuals. Our study illustrates how genetic information can be maximized using readily available tools as a first step in mapping single-gene disorders in small families.     

Discovery, characterization and validation of single nucleotide polymorphisms within 206 bovine genes that may be considered as candidate genes for beef production and quality

A large number of putative single nucleotide polymorphisms (SNPs) have been identified from the bovine genome-sequencing project. However, few of these have been validated and many will turn out to be sequencing artefacts or have low minor allele frequencies. In addition, there is little information available on SNPs within coding regions, which are likely to be responsible for phenotypic variation. Therefore, additional SNP discovery is necessary to identify and validate polymorphisms both in specific genes and genome-wide. Sequence-tagged sites within 286 genes were resequenced from a panel of animals representing a wide range of European cattle breeds. For 80 genes, no polymorphisms were identified, and 672 putative SNPs were identified within 206 genes. Fifteen European cattle breeds (436 individuals plus available parents) were genotyped with these putative SNPs, and 389 SNPs were confirmed to have minor allele frequencies above 10%. The genes containing SNPs were localized on chromosomes by radiation hybrid mapping and on the bovine genome sequence by Blast . Flanking microsatellite loci were identified, to facilitate the alignment of the genes containing the SNPs in relation to mapped quantitative trait loci. Of the 672 putative SNPs discovered in this work, only 11 were found among the validated SNPs and 100 were found among the approximately 2.3 million putative SNPs currently in dbSNP. The genes studied in this work could be considered as candidates for traits associated with beef production and the SNPs reported will help to assess the role of the genes in the genetic control of muscle development and meat quality. The allele frequency data presented allows the general utility of the SNPs to be assessed.     

Sequence differentiation in regions identified by a genome scan for local adaptation

Genome scans using large numbers of randomly selected markers have revealed a small proportion of loci that deviate from neutral expectations and so may mark genomic regions that contribute to local adaptation. Measurements of sequence differentiation and identification of genes in these regions is important but difficult, especially in organisms with limited genetic information available. We have followed up a genome scan in the marine gastropod, Littorina saxatilis, by searching a bacterial artificial chromosome library with differentiated and undifferentiated markers, sequencing four bacterial artificial chromosomes and then analysing sequence variation in population samples for fragments at, and close to the original marker polymorphisms. We show that sequence differentiation follows the patterns expected from the original marker frequencies, that differentiated markers identify independent and highly localized sites and that these sites fall outside coding regions. Two differentiated loci are characterized by insertions of putative transposable elements that appear to have increased in frequency recently and which might influence expression of downstream genes. These results provide strong candidate loci for the study of local adaptation in Littorina. They demonstrate an approach that can be applied to follow up genome scans in other taxa and they show that the genome scan approach can lead rapidly to candidate genes in nonmodel organisms.     

Identifying the Genetic Variation of Gene Expression Using Gene Sets: Application of Novel Gene Set eQTL Approach to PharmGKB and KEGG

Genetic variation underlying the regulation of mRNA gene expression in humans may provide key insights into the molecular mechanisms of human traits and complex diseases. Current statistical methods to map genetic variation associated with mRNA gene expression have typically applied standard linkage and/or association methods; however, when genome-wide SNP and mRNA expression data are available performing all pair wise comparisons is computationally burdensome and may not provide optimal power to detect associations. Consideration of different approaches to account for the high dimensionality and multiple testing issues may provide increased efficiency and statistical power. Here we present a novel approach to model and test the association between genetic variation and mRNA gene expression levels in the context of gene sets (GSs) and pathways, referred to as gene set - expression quantitative trait loci analysis (GS-eQTL). The method uses GSs to initially group SNPs and mRNA expression, followed by the application of principal components analysis (PCA) to collapse the variation and reduce the dimensionality within the GSs. We applied GS-eQTL to assess the association between SNP and mRNA expression level data collected from a cell-based model system using PharmGKB and KEGG defined GSs. We observed a large number of significant GS-eQTL associations, in which the most significant associations arose between genetic variation and mRNA expression from the same GS. However, a number of associations involving genetic variation and mRNA expression from different GSs were also identified. Our proposed GS-eQTL method effectively addresses the multiple testing limitations in eQTL studies and provides biological context for SNP-expression associations.     

Alignment of a <Emphasis Type="Italic">Salix</Emphasis> linkage map to the <Emphasis Type="Italic">Populus</Emphasis> genomic sequence reveals macrosynteny between willow and poplar genomes

Poplars (genus Populus) and willows (genus Salix) are members of the Salicaceae, a family of catkin-bearing trees, shrubs and sub-shrubs. Poplar is considered the model system for biological studies in trees and considerable genetic and genomic resources have become available in recent years. The transfer of information to research studies in willow, for which fewer resources are currently available, would be highly beneficial. However, the extent of conservation between poplar and willow genomes has not yet been extensively studied. To address this, we have constructed a linkage map of willow based on a large mapping population derived from a cross between two Salix viminalis × (S. viminalis × S. schwerinii) hybrid sibs, and aligned this to the publicly available poplar genome sequence. A set of genome-wide, expressed poplar sequences was selected and used to design primer sets that efficiently amplified homeologous regions in willow. Direct sequencing of the willow products confirmed homology with the poplar target in the majority of instances and allowed identification of single nucleotide polymorphisms (SNPs) that were used to map these loci. In total, 202 amplified fragment length polymorphisms (AFLPs), 75 microsatellites and 79 SNPs were used to construct a willow consensus map that spanned 1,856.7 cM with an average interval between markers of 6.3 cM. Poplar sequences homologous to those of the mapped willow microsatellite loci were identified and used in addition to the SNP markers to putatively align all but two minor linkage groups to the poplar genome sequence. A high degree of macrosynteny was revealed.     

Chromosomal assignments for porcine genes encoding enzymes in hepatic metabolic pathways

Increasing the number of mapped genes will facilitate (1) the identification of potential candidate genes for a trait of interest within quantitative trait loci regions and (2) comparative mapping. The metabolic activities of the liver are essential for providing fuel to peripheral organs, for regulation of amino acid, carbohydrate and lipid metabolism and for homoeostasis of vitamins, minerals and electrolytes. We aimed to identify and map genes coding for enzymes active in the liver by somatic cell genetics in order to contribute to the improvement of the porcine gene map. We mapped 28 genes of hepatic metabolic pathways including six genes whose locations could be confirmed and 22 new assignments. Localization information in human was available for all but one gene. In total 24 genes were assigned to in the expected chromosomal regions on the basis of the currently available information on the comparative human and pig map while for four genes our results suggest a new correspondence or extended regions of conservation between porcine and human chromosomes.     

SNPsFinder--a web-based application for genome-wide discovery of single nucleotide polymorphisms in microbial genomes

SUMMARY: Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variations in closely related microbial species, strains or isolates. Some SNPs confer selective advantages for microbial pathogens during infection and many others are powerful genetic markers for distinguishing closely related strains or isolates that could not be distinguished otherwise. To facilitate SNP discovery in microbial genomes, we have developed a web-based application, SNPsFinder, for genome-wide identification of SNPs. SNPsFinder takes multiple genome sequences as input to identify SNPs within homologous regions. It can also take contig sequences and sequence quality scores from ongoing sequencing projects for SNP prediction. SNPsFinder will use genome sequence annotation if available and map the predicted SNP regions to known genes or regions to assist further evaluation of the predicted SNPs for their functional significance. SNPsFinder can generate PCR primers for all predicted SNP regions according to user's input parameters to facilitate experimental validation. The results from SNPsFinder analysis are accessible through the World Wide Web. AVAILABILITY: The SNPsFinder program is available at http://snpsfinder.lanl.gov/. SUPPLEMENTARY INFORMATION: The user's manual is available at http://snpsfinder.lanl.gov/UsersManual/     

In silico whole-genome EST analysis reveals 2322 novel microsatellites for the silver-lipped pearl oyster, Pinctada maxima

Molecular stock improvement techniques such as marker assisted selection have great potential in accelerating selective breeding programmes for animal production industries. However, the discovery and application of trait/marker associations usually requires a large number of genome-wide polymorphic loci. Here, we present 2322 unique microsatellites for the silver-lipped pearl oyster, Pinctada maxima, a species of aquaculture importance throughout the Indo-Australian Archipelago for production of the highly valued South Sea pearl. More than 1.2 million Roche 454 expressed sequence tag (EST) reads were screened for microsatellite repeat motifs. A total of 12,604 sequences contained either a di, tri, tetra, penta or hexa microsatellite repeat motif (n ≥ 6), with 6435 of these sequences having sufficient flanking regions for primer development. All identified microsatellites with designed primers were condensed into 2322 unique clusters (i.e., unique loci) of which 360 were shown to be polymorphic based on multiple sequence reads with different repeat motifs. Genotyping of five microsatellite loci demonstrated that in silico evaluation of polymorphism levels was a very useful method for identification of polymorphic loci, with the variation uncovered being a lower bound. Gene Ontology annotations of sequences containing microsatellites suggest that most are derived from a diverse array of unique genes. This EST derived microsatellite database will be a valuable resource for future studies in genetic map construction, diversity analysis, quantitative trait loci analysis, association mapping and marker assisted selection, not only for P. maxima, but also closely related species within the genus Pinctada.     

SNP-guided identification of monoallelic DNA-methylation events from enrichment-based sequencing data

Monoallelic gene expression is typically initiated early in the development of an organism. Dysregulation of monoallelic gene expression has already been linked to several non-Mendelian inherited genetic disorders. In humans, DNA-methylation is deemed to be an important regulator of monoallelic gene expression, but only few examples are known. One important reason is that current, cost-affordable truly genome-wide methods to assess DNA-methylation are based on sequencing post-enrichment. Here, we present a new methodology based on classical population genetic theory, i.e. the Hardy–Weinberg theorem, that combines methylomic data from MethylCap-seq with associated SNP profiles to identify monoallelically methylated loci. Applied on 334 MethylCap-seq samples of very diverse origin, this resulted in the identification of 80 genomic regions featured by monoallelic DNA-methylation. Of these 80 loci, 49 are located in genic regions of which 25 have already been linked to imprinting. Further analysis revealed statistically significant enrichment of these loci in promoter regions, further establishing the relevance and usefulness of the method. Additional validation was done using both 14 whole-genome bisulfite sequencing data sets and 16 mRNA-seq data sets. Importantly, the developed approach can be easily applied to other enrichment-based sequencing technologies, like the ChIP-seq-based identification of monoallelic histone modifications.