High-density Single Nucleotide Polymorphisms Map Research Articles

Quantitative trait locus mapping for important yield traits of a sorghum-sudangrass hybrid using a high-density single nucleotide polymorphism map.

The sorghum-sudangrass hybrid is a vital gramineous herbage.The F2 population was obtained to clarify genetic regularities among the traits of sorghum-sudangrass hybrids by bagging and selfing in the F1 generation using 'scattered ear sorghum' and 'red hull sudangrass.' This hybrid combines the characteristics of the strong resistance of parents, high yield, and good palatability and has clear heterosis. A thorough understanding of the genetic mechanisms of yield traits in sorghum-sudangrass hybrids is essential in improving their yield. Therefore, we conducted quantitative trait locus (QTL) mapping for plant height, stem diameter, tiller number, leaf number, leaf length, leaf width, and fresh weight of each plant in three different environments, using a high-density genetic linkage map based on single nucleotide polymorphism markers previously constructed by our team. A total of 55 QTLs were detected, uniformly distributed over the 10 linkage groups (LGs), with logarithm of odds values ranging between 2.5 and 7.1, which could explain the 4.9-52.44% phenotypic variation. Furthermore, 17 yield-related relatively high-frequency QTL (RHF-QTL) loci were repeatedly detected in at least two environments, with an explanatory phenotypic variation of 4.9-30.97%. No RHF-QTLs were associated with the tiller number. The genes within the confidence interval of RHF-QTL were annotated, and seven candidate genes related to yield traits were screened. Three QTL sites overlapping or adjacent to previous studies were detected by comparative analysis. We also found that QTL was enriched and that qLL-10-1 and qFW-10-4 were located at the same location of 25.81 cM on LG10. The results of this study provide a foundation for QTL fine mapping, candidate gene cloning, and molecular marker-assisted breeding of sorghum-sudangrass hybrids.

Quantitative trait locus mapping for plant height and branch number in an upland cotton recombinant inbred line with an SNP-based high-density genetic map

Cotton is one of the most important cash crops around the world, providing natural fiber for the textile industry. Agronomic traits play an important role in the mechanized harvesting of cotton. Plant height (PH) and branch number (BN) are two important traits that could affect plant architecture and ultimately, economic yield of cotton. The quantitative trait locus (QTL) for PH and BN across seven environments was identified with a high-density single nucleotide polymorphism map constructed using recombinant inbred lines from upland cotton 0–153 and sGK9708. A total of 68 QTLs for PH (nine stable) and 64 QTLs for BN (eight stable) were identified. Among these stable QTLs, three (two for PH and one for BN) have been identified in previous studies. Four hundred genes for PH and 624 genes for BN were located on the confidence intervals of these stable QTLs. Among them, 134 for PH and 224 for BN were expressed in at least one tissue of root, stem and leaf. Based on the annotation information, expression pattern and the function validated on the other species, ten genes (six for PH and four for BN) could be considered as potential candidate genes. These results could contribute to understanding the mechanism of PH and branch formation, and to improving the method of cotton breeding for molecular marker-assisted selection.

High-density SNP mapping reveals closely linked QTL for resistance to Stagonospora nodorum blotch (SNB) in flag leaf and glume of hexaploid wheat.

The genetic control of adult plant resistance to Stagonospora nodorum blotch (SNB) is complex, consisting of genes with minor effects interacting in an additive manner. Earlier studies detected quantitative trait loci (QTL) for flag leaf resistance in successive years on chromosomes 1B, 2A, 2D, and 5B using SSR- and DArT-based genetic maps of progeny from the crosses EGA Blanco/Millewa, 6HRWSN125/WAWHT2074, and P92201D5/P91193D1. Similarly, QTL for glume resistance detected in successive years and multiple environments were identified on chromosomes 2D and 4B from genetic maps of P92201D5/P91193D1 and 6HRWSN125/WAWHT2074, respectively. The SSR- and DArT-based genetic maps had an average distance of 6.5, 7.8, and 9.7 cM between marker loci for populations EGA/Millewa, P92201D5/P91193D1, and 6HRWSN125/WAWHT2074, respectively. This study used single nucleotide polymorphism (SNP) markers from the iSelect Infinium 90K genotyping array to fine-map genomic regions harbouring QTL for flag leaf and glume SNB resistance, reducing the average distance between markers to 2.9, 3.3, and 3.4 cM for populations P92201D5/P91193D1, EGA/Millewa, and 6HRWSN125/WAWHT2074, respectively. Increasing the marker density of the genetic maps with SNPs did not identify any new QTL for SNB resistance but discriminated previously identified co-located QTL into separate but closely linked QTL.

QTL Analysis of Spike Morphological Traits and Plant Height in Winter Wheat (Triticum aestivum L.) Using a High-Density SNP and SSR-Based Linkage Map.

Wheat yield can be enhanced by modifying the spike morphology and the plant height. In this study, a population of 191 F9 recombinant inbred lines (RILs) was developed from a cross between two winter cultivars Yumai 8679 and Jing 411. A dense genetic linkage map with 10,816 markers was constructed by incorporating single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) marker information. Five spike morphological traits and plant height were evaluated under nine environments for the RILs and parental lines, and the number of detected environmentally stable QTLs were 18 and three, respectively. The 1RS/1BL (rye) translocation increased both spike length and spikelet number with constant spikelet compactness. The QPht.cau-2D.1 was identical to gene Rht8, which decreased spike length without modifying spikelet number. Notably, four novel QTLs locating on chromosomes 1AS (QSc.cau-1A.1), 2DS (QSc.cau-2D.1), and 7BS (QSl.cau-7B.1 and QSl.cau-7B.2) were firstly identified in this study, which provide further insights into the genetic factors that shaped the spike morphology in wheat. Moreover, SNP markers tightly linked to previously reported QTLs will eventually facilitate future studies including their positional cloning or marker-assisted selection.

Fine mapping of a major locus controlling plant height using a high-density single-nucleotide polymorphism map in Brassica napus.

A saturated map was constructed using SNP markers to fine-map a Brassica napus dominant locus for dwarf mutant onto a 152-kb interval of chromosome A09 containing 14 genes. Major dwarf loci in crops may play important roles in crop improvement and developmental genetics. The present study investigated and fine-mapped a Brassica napus dwarf-dominant locus BnDWF1. Plants carrying the BnDWF1 locus in populations derived from 'zhongshuang11' and Bndwf1 have deep-green leaves and dwarf architecture that differ sharply from tall plants with normal green leaves. BnDWF1, as a major locus controlling plant height, showed a very high heritability (0.91-0.95). To map this locus, a high-density single-nucleotide polymorphism map was constructed, and the BnDWF1 locus was mapped at an interval between single-nucleotide polymorphism markers, M19704 and M19695, on linkage group A09 of B. napus, with five co-segregating single-nucleotide polymorphism markers. Furthermore, fine mapping narrowed the interval harboring BnDWF1 to 152kb in length in B. napus. This interval contains 14 annotated or predicted genes, seven of which are candidates responsible for the dwarf trait. This study provides an effective foundation for the study of plant height regulation and plant type breeding in B. napus.

High-Density SNP Map Construction and QTL Identification for the Apetalous Character in Brassica napus L.

The apetalous genotype is a morphological ideotype for increasing seed yield and should be of considerable agricultural use; however, only a few studies have focused on the genetic control of this trait in Brassica napus. In the present study, a recombinant inbred line, the AH population, containing 189 individuals was derived from a cross between an apetalous line ‘APL01’ and a normally petalled variety ‘Holly’. The Brassica 60 K Infinium BeadChip Array harboring 52,157 single nucleotide polymorphism (SNP) markers was used to genotype the AH individuals. A high-density genetic linkage map was constructed based on 2,755 bins involving 11,458 SNPs and 57 simple sequence repeats, and was used to identify loci associated with petalous degree (PDgr). The linkage map covered 2,027.53 cM, with an average marker interval of 0.72 cM. The AH map had good collinearity with the B. napus reference genome, indicating its high quality and accuracy. After phenotypic analyses across five different experiments, a total of 19 identified quantitative trait loci (QTLs) distributed across chromosomes A3, A5, A6, A9 and C8 were obtained, and these QTLs were further integrated into nine consensus QTLs by a meta-analysis. Interestingly, the major QTL qPD.C8-2 was consistently detected in all five experiments, and qPD.A9-2 and qPD.C8-3 were stably expressed in four experiments. Comparative mapping between the AH map and the B. napus reference genome suggested that there were 328 genes underlying the confidence intervals of the three steady QTLs. Based on the Gene Ontology assignments of 52 genes to the regulation of floral development in published studies, 146 genes were considered as potential candidate genes for PDgr. The current study carried out a QTL analysis for PDgr using a high-density SNP map in B. napus, providing novel targets for improving seed yield. These results advanced our understanding of the genetic control of PDgr regulation in B. napus.

A High-Density SNP and SSR Consensus Map Reveals Segregation Distortion Regions in Wheat.

Segregation distortion is a widespread phenomenon in plant and animal genomes and significantly affects linkage map construction and identification of quantitative trait loci (QTLs). To study segregation distortion in wheat, a high-density consensus map was constructed using single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers by merging two genetic maps developed from two recombinant-inbred line (RIL) populations, Ning7840 × Clark and Heyne × Lakin. Chromosome regions with obvious segregation distortion were identified in the map. A total of 3541 SNPs and 145 SSRs were mapped, and the map covered 3258.7 cM in genetic distance with an average interval of 0.88 cM. The number of markers that showed distorted segregation was 490 (18.5%) in the Ning7840 × Clark population and 225 (10.4%) in the Heyne × Lakin population. Most of the distorted markers (630) were mapped in the consensus map, which accounted for 17.1% of mapped markers. The majority of the distorted markers clustered in the segregation distortion regions (SDRs) on chromosomes 1B, 2A, 2B, 3A, 3B, 4B, 5A, 5B, 5D, 6B, 7A, and 7D. All of the markers in a given SDR skewed toward one of the parents, suggesting that gametophytic competition during zygote formation was most likely one of the causes for segregation distortion in the populations.

Identification of quantitative trait loci for phosphorus use efficiency traits in rice using a high density SNP map.

BackgroundSoil phosphorus (P) deficiency is one of the major limiting factors to crop production. The development of crop varieties with improved P use efficiency (PUE) is an important strategy for sustainable agriculture. The objectives of this research were to identify quantitative trait loci (QTLs) linked to PUE traits using a high-density single nucleotide polymorphism (SNP) map and to estimate the epistatic interactions and environmental effects in rice (Oryza sativa L.).ResultsWe conducted a two-year field experiment under low and normal P conditions using a recombinant inbred population of rice derived from Zhenshan 97 and Minghui 63 (indica). We investigated three yield traits, biomass (BIOM), harvest index (HI), and grain yield (Yield), and eight PUE traits: total P uptake (PUP), P harvest index (PHI), grain P use efficiency (gPUE) based on P accumulation in grains, straw P use efficiency (strPUE) based on P accumulation in straw, P use efficiency for biomass (PUEb) and for grain yield (PUEg) based on P accumulation in the whole plant, P translocation (PT), and P translocation efficiency (PTE). Of the 36 QTLs and 24 epistatic interactions identified, 26 QTLs and 12 interactions were detected for PUE traits. The environment affected seven QTLs and three epistatic interactions. Four QTLs (qPHI1 and qPHI2 for PHI, qPUEg2 for PUEg, and qPTE8 for PTE) with strong effects were environmentally independent. By comparing our results with similar QTLs in previous studies, three QTLs for PUE traits (qPUP1 and qPUP10 for PUP, and qPHI6 for PHI) were found across various genetic backgrounds. Seven regions were shared by QTLs for yield and PUE traits.ConclusionMost QTLs linked to PUE traits were different from those linked to yield traits, suggesting different genetic controls underlying these two traits. Those chromosomal regions with large effects that are not affected by different environments are promising for improving P use efficiency. The seven regions shared by QTLs linked to yield and PUE traits imply the possibility of the simultaneous improvement of yield and PUE traits.

An SNP-based saturated genetic map and QTL analysis of fruit-related traits in cucumber using specific-length amplified fragment (SLAF) sequencing.

BackgroundCucumber, Cucumis sativus L., is an economically important vegetable crop which is processed or consumed fresh worldwide. However, the narrow genetic base in cucumber makes it difficult for constructing high-density genetic maps. The development of massively parallel genotyping methods and next-generation sequencing (NGS) technologies provides an excellent opportunity for developing single nucleotide polymorphisms (SNPs) for linkage map construction and QTL analysis of horticultural traits. Specific-length amplified fragment sequencing (SLAF-seq) is a recent marker development technology that allows large-scale SNP discovery and genotyping at a reasonable cost. In this study, we constructed a high-density SNP map for cucumber using SLAF-seq and detected fruit-related QTLs.ResultsAn F2 population of 148 individuals was developed from an intra-varietal cross between CC3 and NC76. Genomic DNAs extracted from two parents and 148 F2 individuals were subjected to high-throughput sequencing and SLAF library construction. A total of 10.76 Gb raw data and 75,024,043 pair-end reads were generated to develop 52,684 high-quality SLAFs, out of which 5,044 were polymorphic. 4,817 SLAFs were encoded and grouped into different segregation patterns. A high-resolution genetic map containing 1,800 SNPs was constructed for cucumber spanning 890.79 cM. The average distance between adjacent markers was 0.50 cM. 183 scaffolds were anchored to the SNP-based genetic map covering 46% (168.9 Mb) of the cucumber genome (367 Mb). Nine QTLs for fruit length and weight were detected, a QTL designated fl3.2 explained 44.60% of the phenotypic variance. Alignment of the SNP markers to draft genome scaffolds revealed two mis-assembled scaffolds that were validated by fluorescence in situ hybridization (FISH).ConclusionsWe report herein the development of evenly dispersed SNPs across cucumber genome, and for the first time an SNP-based saturated linkage map. This 1,800-locus map would likely facilitate genetic mapping of complex QTL loci controlling fruit yield, and the orientation of draft genome scaffolds.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1158) contains supplementary material, which is available to authorized users.

Analysis of agronomic and domestication traits in a durum × cultivated emmer wheat population using a high-density single nucleotide polymorphism-based linkage map.

Development of a high-density SNP map and evaluation of QTL shed light on domestication events in tetraploid wheat and the potential utility of cultivated emmer wheat for durum wheat improvement. Cultivated emmer wheat (Triticumturgidum ssp. dicoccum) is tetraploid and considered as one of the eight founder crops that spawned the Agricultural Revolution about 10,000years ago. Cultivated emmer has non-free-threshing seed and a somewhat fragile rachis, but mutations in genes governing these and other agronomic traits occurred that led to the formation of today's fully domesticated durum wheat (T. turgidum ssp. durum). Here, we evaluated a population of recombinant inbred lines (RILs) derived from a cross between a cultivated emmer accession and a durum wheat variety. A high-density single nucleotide polymorphism (SNP)-based genetic linkage map consisting of 2,593 markers was developed for the identification of quantitative trait loci. The major domestication gene Q had profound effects on spike length and compactness, rachis fragility, and threshability as expected. The cultivated emmer parent contributed increased spikelets per spike, and the durum parent contributed higher kernel weight, which led to the identification of some RILs that had significantly higher grain weight per spike than either parent. Threshability was governed not only by the Q locus, but other loci as well including Tg-B1 on chromosome 2B and a putative Tg-A1 locus on chromosome 2A indicating that mutations in the Tg loci occurred during the transition of cultivated emmer to the fully domesticated tetraploid. These results not only shed light on the events that shaped wheat domestication, but also demonstrate that cultivated emmer is a useful source of genetic variation for the enhancement of durum varieties.

Gene-based SNP discovery and genetic mapping in pea.

Key message Gene-based SNPs were identified and mapped in pea using five recombinant inbred line populations segregating for traits of agronomic importance. Pea (Pisum sativum L.) is one of the world’s oldest domesticated crops and has been a model system in plant biology and genetics since the work of Gregor Mendel. Pea is the second most widely grown pulse crop in the world following common bean. The importance of pea as a food crop is growing due to its combination of moderate protein concentration, slowly digestible starch, high dietary fiber concentration, and its richness in micronutrients; however, pea has lagged behind other major crops in harnessing recent advances in molecular biology, genomics and bioinformatics, partly due to its large genome size with a large proportion of repetitive sequence, and to the relatively limited investment in research in this crop globally. The objective of this research was the development of a genome-wide transcriptome-based pea single-nucleotide polymorphism (SNP) marker platform using next-generation sequencing technology. A total of 1,536 polymorphic SNP loci selected from over 20,000 non-redundant SNPs identified using deep transcriptome sequencing of eight diverse Pisum accessions were used for genotyping in five RIL populations using an Illumina GoldenGate assay. The first high-density pea SNP map defining all seven linkage groups was generated by integrating with previously published anchor markers. Syntenic relationships of this map with the model legume Medicago truncatula and lentil (Lens culinaris Medik.) maps were established. The genic SNP map establishes a foundation for future molecular breeding efforts by enabling both the identification and tracking of introgression of genomic regions harbouring QTLs related to agronomic and seed quality traits.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-014-2375-y) contains supplementary material, which is available to authorized users.

A High-Density SNP Map for Accurate Mapping of Seed Fibre QTL in Brassica napus L

A high density genetic linkage map for the complex allotetraploid crop species Brassica napus (oilseed rape) was constructed in a late-generation recombinant inbred line (RIL) population, using genome-wide single nucleotide polymorphism (SNP) markers assayed by the Brassica 60 K Infinium BeadChip Array. The linkage map contains 9164 SNP markers covering 1832.9 cM. 1232 bins account for 7648 of the markers. A subset of 2795 SNP markers, with an average distance of 0.66 cM between adjacent markers, was applied for QTL mapping of seed colour and the cell wall fiber components acid detergent lignin (ADL), cellulose and hemicellulose. After phenotypic analyses across four different environments a total of 11 QTL were detected for seed colour and fiber traits. The high-density map considerably improved QTL resolution compared to the previous low-density maps. A previously identified major QTL with very high effects on seed colour and ADL was pinpointed to a narrow genome interval on chromosome A09, while a minor QTL explaining 8.1% to 14.1% of variation for ADL was detected on chromosome C05. Five and three QTL accounting for 4.7% to 21.9% and 7.3% to 16.9% of the phenotypic variation for cellulose and hemicellulose, respectively, were also detected. To our knowledge this is the first description of QTL for seed cellulose and hemicellulose in B. napus, representing interesting new targets for improving oil content. The high density SNP genetic map enables navigation from interesting B. napus QTL to Brassica genome sequences, giving useful new information for understanding the genetics of key seed quality traits in rapeseed.

High density SNP mapping and QTL analysis for fruit quality characteristics in peach (Prunus persica L.)

Single nucleotide polymorphisms (SNPs) were used to construct an integrated SNP linkage map of peach (Prunus persica (L.) Batsch). A set of 1,536 SNPs were evaluated with the GoldenGate® Genotyping assay in two mapping populations, Pop-DF, and Pop-DG. After genotyping and filtering, a final set of 1,400 high quality SNPs in Pop-DF and 962 in Pop-DG with full map coverage were selected and used to construct two linkage maps with JoinMap®4.0. The Pop-DF map covered 422 cM of the peach genome and included 1,037 SNP markers, and Pop-DG map covered 369 cM and included 738 SNPs. A consensus map was constructed with 588 SNP markers placed in eight linkage groups (n = 8 for peach), with map coverage of 454 cM and an average distance of 0.81 cM/marker site. Placements of SNPs on the “peach v1.0” physical map were compared to placement on the linkage maps and several differences were observed. Using the SNP linkage map of Pop-DG and phenotypic data collected for three harvest seasons, a QTL analysis for fruit quality traits and chilling injury symptoms was carried out with the mapped SNPs. Significant QTL effects were detected for mealiness (M) and flesh bleeding (FBL) QTLs on linkage group 4 and flesh browning (FBr) on linkage group 5. This study represents one of the first examples of QTL detection for quality traits and chilling injury symptoms using a high-density SNP map in a single peach F1 family.

TMEM106B is associated with frontotemporal lobar degeneration in a clinically diagnosed patient cohort

In a genome-wide association study of frontotemporal lobar degeneration with pathological inclusions of TAR DNA-binding protein, significant association was obtained with three single nucleotide polymorphisms at 7p21.3, in a region encompassing the gene TMEM106B. This study also suggested a potential modifying effect of TMEM106B on disease since the association was strongest in progranulin mutation carriers. Further, the risk effect seemed to correlate with increased TMEM106B expression in patients. In the present study, we sought to replicate these three findings using an independent Flanders–Belgian cohort of primarily clinically diagnosed patients with frontotemporal lobar degeneration (n = 288). We were able to confirm the association with TMEM106B with a P-value of 0.008 for rs1990622, the top marker from the genome-wide association study [odds ratio 0.75 (95% confidence interval 0.61–0.93)]. Further, high-density single nucleotide polymorphism mapping suggested that the association was solely driven by the gene TMEM106B. Homozygous carriers of the TMEM106B protective alleles had a 50% reduced risk of developing frontotemporal lobar degeneration. However, we were unable to detect a modifying effect of the TMEM106B single nucleotide polymorphisms on onset age in progranulin mutation carriers belonging to an extended, clinical and pathological well-documented founder family segregating a progranulin null mutation. Also, we could not observe significant differences in messenger RNA expression between patients and control individuals in lymphoblast cell lines and in brain frontal cortex. In conclusion, we replicated the genetic TMEM106B association in a primarily clinically diagnosed cohort of patients with frontotemporal lobar degeneration from Flanders–Belgium. Additional studies are needed to unravel the molecular role of TMEM106B in disease onset and pathogenesis.

Extent and consistency of linkage disequilibrium and identification of DNA markers for production and egg quality traits in commercial layer chicken populations

BackgroundThe genome sequence and a high-density SNP map are now available for the chicken and can be used to identify genetic markers for use in marker-assisted selection (MAS). Effective MAS requires high linkage disequilibrium (LD) between markers and quantitative trait loci (QTL), and sustained marker-QTL LD over generations. This study used data from a 3,000 SNP panel to assess the level and consistency of LD between single nucleotide polymorphisms (SNPs) over consecutive years in two egg-layer chicken lines, and analyzed one line by two methods (SNP-wise association and genome-wise Bayesian analysis) to identify markers associated with egg-quality and egg-production phenotypes.ResultsThe LD between markers pairs was high at short distances (r2 > 0.2 at < 2 Mb) and remained high after one generation (correlations of 0.80 to 0.92 at < 5 Mb) in both lines. Single- and 3-SNP regression analyses using a mixed model with SNP as fixed effect resulted in 159 and 76 significant tests (P < 0.01), respectively, across 12 traits. A Bayesian analysis called BayesB, that fits all SNPs simultaneously as random effects and uses model averaging procedures, identified 33 SNPs that were included in the model >20% of the time (φ > 0.2) and an additional ten 3-SNP windows that had a sum of φ greater than 0.35. Generally, SNPs included in the Bayesian model also had a small P-value in the 1-SNP analyses.ConclusionHigh LD correlations between markers at short distances across two generations indicate that such markers will retain high LD with linked QTL and be effective for MAS. The different association analysis methods used provided consistent results. Multiple single SNPs and 3-SNP windows were significantly associated with egg-related traits, providing genomic positions of QTL that can be useful for both MAS and to identify causal mutations.

High-density SNP genotyping to define β-globin locus haplotypes

Five major β-globin locus haplotypes have been established in individuals with sickle cell disease (SCD) from the Benin, Bantu, Senegal, Cameroon, and Arab-Indian populations. Historically, β-haplotypes were established using restriction fragment length polymorphism (RFLP) analysis across the β-locus, which consists of five functional β-like globin genes located on chromosome 11. Previous attempts to correlate these haplotypes as robust predictors of clinical phenotypes observed in SCD have not been successful. We speculate that the coverage and distribution of the RFLP sites located proximal to or within the globin genes are not sufficiently dense to accurately reflect the complexity of this region. To test our hypothesis, we performed RFLP analysis and high-density single nucleotide polymorphism (SNP) genotyping across the β-locus using DNA samples from healthy African Americans with either normal hemoglobin A (HbAA) or individuals with homozygous SS (HbSS) disease. Using the genotyping data from 88 SNPs and Haploview analysis, we generated a greater number of haplotypes than that observed with RFLP analysis alone. Furthermore, a unique pattern of long-range linkage disequilibrium between the locus control region and the β-like globin genes was observed in the HbSS group. Interestingly, we observed multiple SNPs within the HindIII restriction site located in the Gγ-globin intervening sequence II which produced the same RFLP pattern. These findings illustrated the inability of RFLP analysis to decipher the complexity of sequence variations that impacts genomic structure in this region. Our data suggest that high-density SNP mapping may be required to accurately define β-haplotypes that correlate with the different clinical phenotypes observed in SCD.

High-resolution, high-throughput SNP mapping in Drosophila melanogaster

Single nucleotide polymorphisms (SNPs) are useful markers for genetic mapping experiments in model organisms. Here we report the establishment of a high-density SNP map and high-throughput genotyping assays for Drosophila melanogaster. Our map comprises 27,367 SNPs in common laboratory Drosophila stocks. These SNPs were clustered within 2,238 amplifiable markers at an average density of 1 marker every 50.3 kb, or 6.3 genes. We have also constructed a set of 62 Drosophila stocks, each of which facilitates the generation of recombinants within a defined genetic interval of 1-2 Mb. For flexible, high-throughput SNP genotyping, we used fluorescent tag-array mini-sequencing (TAMS) assays. We designed and validated TAMS assays for 293 SNPs at an average resolution of 391.3 kb, and demonstrated the utility of these tools by rapidly mapping 14 mutations that disrupt embryonic muscle patterning. These resources enable high-resolution high-throughput genetic mapping in Drosophila.

A SNP in the ACT gene associated with astrocytosis and rapid cognitive decline in AD

There is biochemical and animal model evidence supporting a pathological role of the ACT gene in AD. However, direct genetic evidence remains controversial and has been mostly limited to individual single nucleotide polymorphism (SNP) analysis. To resolve this apparent conflict we have used a high-density ACT SNP map, constructed haplotypes and explored correlations with phenotype. SNPs were identified by sequencing and used to construct haplotypes in 668 AD patients and 419 controls and a case–control association study was performed. Five SNPs, comprising five common haplotypes, represented 93% of ACT gene variation. Although no single SNP or haplotype was associated with AD status, a SNP in intron 2 was associated with later onset and more rapid cognitive decline ( P = 0.04). This SNP was both individually associated with severe astrocytosis ( P = 0.004) in AD patients and when combined with the signal sequence SNP ( P = 0.002). This suggests that astrocytosis may have a protective function for a limited period in some patients. These SNP associations either support a direct role for the ACT gene, in AD pathology or alternatively reflect linkage with polymorphisms in other genes nearby.

Integrating QTL and high-density SNP analyses in mice to identify Insig2 as a susceptibility gene for plasma cholesterol levels

The use of inbred strains of mice to dissect the genetic complexity of common diseases offers a viable alternative to human studies, given the control over experimental parameters that can be exercised. Central to efforts to map susceptibility loci for common diseases in mice is a comprehensive map of DNA variation among the common inbred strains of mice. Here we present one of the most comprehensive high-density, single nucleotide polymorphism (SNP) maps of mice constructed to date. This map consists of 10,350 SNPs genotyped in 62 strains of inbred mice. We demonstrate the utility of these data via a novel integrative genomics approach to mapping susceptibility loci for complex traits. By integrating in silico quantitative trait locus (QTL) mapping with progressive QTL mapping strategies in segregating mouse populations that leverage large-scale mapping of the genetic determinants of gene expression traits, we not only facilitate identification of candidate quantitative trait genes, but also protect against spurious associations that can arise in genetic association studies due to allelic association among unlinked markers. Application of this approach to our high-density SNP map and two previously described F2 crosses between strains C57BL/6J (B6) and DBA/2J and between B6 ApoE −/− and C3H/HeJ ApoE −/− results in the identification of Insig2 as a strong candidate susceptibility gene for total plasma cholesterol levels.

Identification of 156 novel SNPs in 29 genes encoding G-protein coupled receptors

We have been performing extensive screening on single nucleotide polymorphisms (SNPs) in and around genes encoding drug metabolizing enzymes, transporters, and receptors and have constructed the high-density SNP maps of such gene regions. In addition to genetic information reported earlier, we identified a total of 390 genetic variations, 358 SNPs and 32 genetic variations of other types, detected in 29 genes encoding G-protein coupled receptors in Japanese populations. Following a comparison of our data with SNPs in the dbSNP database in the US National Center for Biotechnology Information, 156 SNPs from these gene loci are considered to be novel. The fine-scale SNP maps constructed in this study should serve an important resource for studies of linkage-disequilibrium mapping for complex genetic diseases and drug-response phenotypes.