Unique Loci Research Articles

Epigenetic adaptation to therapy in glioblastoma: popping the hood on therapeutic resistance

Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults, remains one of most intractable diseases. Median survival remains at only 15 months, despite aggressive combination therapy involving surgical resection, radiation, and alkylating chemotherapy. While initially responsive to these therapies, GBM tumors quickly adapt and generate treatment-resistant recurrent growths. We and others have previously shown that the stress induced by standard of care therapies itself activates a remarkable plasticity in GBM cells, converting previously differentiated cells to a therapy-resistant glioma stem cell (GSC) state. The exact processes governing this conversion, however, remain to be fully elucidated. Preliminary investigation revealed that polycomb group protein EZH2, a well-established epigenetic regulator, plays a crucial role in this process. To determine how EZH2 responds to therapy, we performed Genome-wide chromatin immunoprecipitation (ChIP) in parallel with DNA sequencing analyses (ChIP-seq), which identified 1449 distinct regions with elevated EZH2 binding, including critical genes PTPRT, CDK5R2, and Siglec6. Microarray analysis demonstrated subsequent attenuation in their gene expression, leading to heightened activity of STAT3, a critical regulator for promotion of the GSC phenotype. To better understand the epigenetic adaptions occurring during therapeutic stress, we performed ChIP seq analysis for histone 3 lysine 27 acetylation (H3K27ac), a marker for active chromatin. Chemotherapy induces H3K27ac enrichment in 452 unique loci, while radiation caused H3K27ac enrichment at 1029 sites. Comparison of these sites to canonical H3K27me3 sites revealed specific de novo binding in the homeobox transcription factor binding motif (p=0.025). Combination of our epigenomic data from patient-derived xenograft models and GBM patient data with H3K27me3 enrichment profile allowed us to pinpoint several novel homeobox transcription factors potentially linked to GBM plasticity during therapeutic stress. These results provide critical perspective on the global epigenetic changes driving this plasticity occurring during anti-glioma therapy and provide novel avenues for targeting this adaptation therapeutically.

Brain scans from 21,297 individuals reveal the genetic architecture of hippocampal subfield volumes

The hippocampus is a heterogeneous structure, comprising histologically distinguishable subfields. These subfields are differentially involved in memory consolidation, spatial navigation and pattern separation, complex functions often impaired in individuals with brain disorders characterized by reduced hippocampal volume, including Alzheimer’s disease (AD) and schizophrenia. Given the structural and functional heterogeneity of the hippocampal formation, we sought to characterize the subfields’ genetic architecture. T1-weighted brain scans (n = 21,297, 16 cohorts) were processed with the hippocampal subfields algorithm in FreeSurfer v6.0. We ran a genome-wide association analysis on each subfield, co-varying for whole hippocampal volume. We further calculated the single-nucleotide polymorphism (SNP)-based heritability of 12 subfields, as well as their genetic correlation with each other, with other structural brain features and with AD and schizophrenia. All outcome measures were corrected for age, sex and intracranial volume. We found 15 unique genome-wide significant loci across six subfields, of which eight had not been previously linked to the hippocampus. Top SNPs were mapped to genes associated with neuronal differentiation, locomotor behaviour, schizophrenia and AD. The volumes of all the subfields were estimated to be heritable (h2 from 0.14 to 0.27, all p < 1 × 10–16) and clustered together based on their genetic correlations compared with other structural brain features. There was also evidence of genetic overlap of subicular subfield volumes with schizophrenia. We conclude that hippocampal subfields have partly distinct genetic determinants associated with specific biological processes and traits. Taking into account this specificity may increase our understanding of hippocampal neurobiology and associated pathologies.

An efficient analytic approach in genome-wide identification of methylation quantitative trait loci response to fenofibrate treatment

BackgroundThe study of DNA methylation quantitative trait loci (meQTLs) helps dissect regulatory mechanisms underlying genetic associations of human diseases. In this study, we conducted the first genome-wide examination of genetic drivers of methylation variation in response to a triglyceride-lowering treatment with fenofibrate (response-meQTL) by using an efficient analytic approach.MethodsSubjects (n = 429) from the GAW20 real data set with genotype and both pre- (visit 2) and post- (visit 4) fenofibrate treatment methylation measurements were included. Following the quality control steps of removing certain cytosine-phosphate-guanine (CpG) probes, the post−/premethylation changes (post/pre) were log transformed and the association was performed on 208,449 CpG sites. An additive linear mixed-effects model was used to test the association between each CpG probe and single nucleotide polymorphisms (SNPs) around ±1 Mb region, with age, sex, smoke, batch effect, and principal components included as covariates. Bonferroni correction was applied to define the significance threshold (p < 5.6 × 10− 10, given a total of 89,217,303 tests). Finally, we integrated our response-meQTL (re-meQTL) findings with the published genome-wide association study (GWAS) catalog of human diseases/traits.ResultsWe identified 1087 SNPs as cis re-meQTLs associated with 610 CpG probes/sites located in 351 unique gene loci. Among these 1087 cis re-meQTL SNPs, 229 were unique and 6 were co-localized at 8 unique disease/trait loci reported in the GWAS catalog (enrichment p = 1.51 × 10− 23). Specifically, a lipid SNP, rs10903129, located in intron regions of gene TMEM57, was a re-meQTL (p = 3.12 × 10− 36) associated with the CpG probe cg09222892, which is in the upstream region of the gene RHCE, indicating a new target gene for rs10903129. In addition, we found that SNP rs12710728 has a suggestive association with cg17097782 (p = 1.77 × 10− 4), and that this SNP is in high linkage disequilibrium (LD) (R2 > 0.8) with rs7443270, which was previously reported to be associated with fenofibrate response (p = 5.00 × 10− 6).ConclusionsBy using a novel analytic approach, we efficiently identified thousands of cis re-meQTLs that provide a unique resource for further characterizing functional roles and gene targets of the SNPs that are most responsive to fenofibrate treatment. Our efficient analytic approach can be extended to large response quantitative trait locus studies with large sample sizes and multiple time points data.

Interrogation of the Gulf toadfish intestinal proteome response to hypersalinity exposure provides insights into osmoregulatory mechanisms and regulation of carbonate mineral precipitation

Marine bony fish live in a hyperosmotic environment and maintain osmotic homeostasis by drinking seawater, and absorbing salt and water across their gastrointestinal tract. Although the ion and water transport mechanisms in the intestine have been the subject of much study, numerous questions remain unanswered. To address some of these questions, a shotgun proteomics methodology employing isobaric tandem mass tags (TMT) was used to interrogate the anterior intestine, posterior intestine, and intestinal fluid proteomes of Gulf toadfish (Opsanus beta) acclimated to normal (35 ppt) or hypersaline (60 ppt) seawater. Relative protein abundance between tissues was also investigated using label free quantitation. Protein products from nearly 3000 unique toadfish loci were identified and quantified between the tissues, and pathway analysis was performed to gain insight into biological significance. Numerous proteins potentially involved in ion transport, digestion, nutrient absorption, and intestinal CaCO3 precipitation were found to respond to changing salinity, providing additional insight into the molecular mechanisms behind these processes. Intestinal protein heterogeneity was also observed with proteins involved in ion transport responding to hypersalinity exposure primarily in the anterior intestine, and proteins involved in digestion and nutrient absorption showing higher abundance in the anterior intestine, regardless of salinity.

Wheat resistance to Fusarium head blight

Fusarium head blight (FHB), caused primarily by Fusarium graminearum, has rapidly become the most devastating wheat disease worldwide. Host resistance is the most effective and sustainable strategy to combat the disease. To date, more than 50 unique quantitative trait loci (QTLs) have been identified in diverse wheat populations, and Fhb1 consistently shows the highest level of resistance in different genetic backgrounds. In spite of worldwide efforts to deploy Fhb1 through wheat breeding, only a few cultivars currently used in commercial production carry Fhb1. Recently, Fhb1 has been cloned and diagnostic markers have been developed, which will facilitate successful deployment of Fhb1 in wheat breeding. Using marker-assisted backcross, Fhb1 has been successfully transferred into locally adapted cultivars that carry minor QTLs, and selected Fhb1 lines in different backgrounds showed high levels of FHB resistance. This approach may provide a quick solution to improvement of FHB resistance in commercial cultivars. In addition, gene editing that knocks down the susceptible allele of Fhb1 in commercial cultivars may also improve wheat FHB resistance. Most other QTLs identified to date show much smaller effects than Fhb1. These QTLs are distributed in many locally adapted cultivars, and thus are important QTLs for FHB resistance improvement. Currently, diagnostic markers are still not available for most of these minor QTLs; therefore, genomic selection may improve selection accuracy for these QTLs to create highly FHB-resistant cultivars.

The Persistent Homology Mathematical Framework Provides Enhanced Genotype-to-Phenotype Associations for Plant Morphology.

Efforts to understand the genetic and environmental conditioning of plant morphology are hindered by the lack of flexible and effective tools for quantifying morphology. Here, we demonstrate that persistent-homology-based topological methods can improve measurement of variation in leaf shape, serrations, and root architecture. We apply these methods to 2D images of leaves and root systems in field-grown plants of a domesticated introgression line population of tomato (Solanum pennellii). We find that compared with some commonly used conventional traits, (1) persistent-homology-based methods can more comprehensively capture morphological variation; (2) these techniques discriminate between genotypes with a larger normalized effect size and detect a greater number of unique quantitative trait loci (QTLs); (3) multivariate traits, whether statistically derived from univariate or persistent-homology-based traits, improve our ability to understand the genetic basis of phenotype; and (4) persistent-homology-based techniques detect unique QTLs compared to conventional traits or their multivariate derivatives, indicating that previously unmeasured aspects of morphology are now detectable. The QTL results further imply that genetic contributions to morphology can affect both the shoot and root, revealing a pleiotropic basis to natural variation in tomato. Persistent homology is a versatile framework to quantify plant morphology and developmental processes that complements and extends existing methods.

A broad genomic panel of microsatellite loci from Brycon orbignyanus (Characiformes: Bryconidae) an endangered migratory Neotropical fish

A broad panel of tens of thousands of microsatellite loci is unveiled for an endangered piracema (i.e. migratory) South American fish, Brycon orbignyanus. Once one of the main fisheries resources in the Platine Basin, it is now almost extinct in nature and focus of intense aquaculture activity. A total of 178.2 million paired-end reads (90 bases long) were obtained through the use of sequencing-by-synthesis (from a primary genomic library of 500 bp DNA fragments) and is made available through NCBI’s Sequence Read Archive, SRA accession SRX3350440. Short reads were assembled de novo and screening for perfect microsatellite motifs revealed more than 81 thousands unique microsatellite loci, for which primer pairs were proposed. A total of 29 polymorphic microsatellite markers were already previously validated for this panel. A partial genomic assembly is hereby presented and these genomic resources are publicly made available. These data will foster the rapid development of hundreds of new DNA markers for genetic diversity studies, conservation initiatives and management practices for this important and depleted species. The availability of such preliminary genomic data will also be of use in the areas of bioinformatics, ecology, genetics and evolution.

Genetic characterization of Vitis germplasm collected from the southwestern US and Mexico to expedite Pierce's disease-resistance breeding.

Pierce's disease (PD) limits the cultivation of Vitis vinifera grape cultivars in California, across the southern United States and into South America. Resistance has been well characterized in V. arizonica, and one resistance locus has been identified (PdR1). However, resistance is poorly characterized in most other grape species. We tested a wide range of Vitis species from the southwestern United States for resistance to PD and used nuclear and chloroplast markers to phenotypically and genetically select a diverse set of resistant accessions. Chloroplast SSR markers identified 11 maternal lineage lines within the set of 17 (14 new and three previously identified) PD resistant accessions. A total of 19 breeding populations (F1 and pseudo-BC1) were developed with the 14 PD resistant accessions, and a total of 705 seedlings were analyzed for PD resistance. Using a limited mapping approach, 12 SSR markers, linked to the PdR1 locus, were used to genotype the breeding populations and phenotypic data were analyzed. Nine accessions had a major resistance quantitative trait locus (QTL) within the genomic region containing PdR1. The phenotypic data for these three resistant accessions, ANU67, b41-13, and T03-16, did not associate with PdR1 linked markers, indicating that their resistance is located in other regions of the genome. These three accessions were identified as candidates for use in the development of framework maps with larger populations capable of detecting additional and unique loci for PD resistance breeding and the stacking of PD resistance genes.

QTL mapping uncovers a semi-dwarf 1 (sdw1) allele in the barley (Hordeum vulgare) ND23049 line.

In barley, semi-dwarf varieties are attractive for their superior harvest index and lodging resistance, but many semi-dwarf barley genotypes suffer from poor spike emergence. We performed a genetic characterization of a semi-dwarf line (ND23049) that combines short stature, strong stiff culms, and adequate spike emergence. We developed a doubled haploid (DH) population derived by crossing ND23049 and the cultivar CLE253. A subset of 88 DH lines and parents were characterized for plant height in 2013 and 2014 and genotyped. In total, 1984 SNPs (345 unique loci) were used to produce a linkage map of 1127.1 cM. Three QTLs for plant height were detected in this population and coincided with the HvGA20ox2/Sdw1, HvBRI1/Uzu1, and HvPRR95 gene loci. The phenotypic variation explained by each QTL was 75.8%, 7.7%, and 4.1%, respectively, and jointly explained 83.3% (2013) and 87.7% (2014) of plant height. Our results suggest that ND23049 contributed the "short" allele at the HvGA20ox2/sdw1 locus while CLE253 provided "short" alleles at the HvBRI1/uzu1 and HvPRR95 loci. We identified a large deletion (at least 92.7 Kb), including HvGA20ox2 (Sdw1), as the causal mutation in ND23049. A set of tightly flanked SNP markers will help breeders to develop improved semi-dwarf varieties.

Multi-tissue GAL4-mediated gene expression in all Anopheles gambiae life stages using an endogenous polyubiquitin promoter

The ability to manipulate the Anopheles gambiae genome and alter gene expression effectively and reproducibly is a prerequisite for functional genetic analysis and for the development of novel control strategies in this important disease vector. However, in vivo transgenic analysis in mosquitoes is limited by the lack of promoters active ubiquitously. To address this, we used the GAL4/UAS system to investigate the promoter of the An. gambiae Polyubiquitin-c (PUBc) gene and demonstrated its ability to drive expression in mosquito cell culture before incorporation into An. gambiae transgenic driver lines. To generate such lines, piggyBac-mediated insertion was used to identify genomic regions able to sustain widespread expression and to create φC31 docking lines at these permissive sites. Patterns of expression induced by PUBc-GAL4 drivers carrying single intergenic insertions were assessed by crossing with a novel responder UAS-mCD8:mCherry line that was created by φC31-mediated integration. Amongst the drivers created at single, unique chromosomal integration loci, two were isolated that induced differential expression levels in a similar multiple-tissue spatial pattern throughout the mosquito life cycle. This work expands the tools available for An. gambiae functional analysis by providing a novel promoter for investigating phenotypes resulting from widespread multi-tissue expression, as well as identifying and tagging genomic sites that sustain broad transcriptional activity.

Abstract P127: Gene-Lifestyle Interactions Detect Novel Blood Pressure Loci

Introduction: Gene-lifestyle interaction effects (GxE more generally) on blood pressure (BP), a promising approach for novel loci discovery, is being actively pursued in the CHARGE Gene-Lifestyle Interactions Working Group using individual lifestyle domains, such as smoking status. Objectives: To explore the potential utility of a “lifestyle index” (aggregating multiple lifestyle domains) for identifying novel BP loci in a GxE context, we performed genome-wide interaction analysis with two lifestyle index variables. Methods: For each of 6,257 subjects (19 – 80 years, 46.9% male) in the Framingham Heart Study SHARe dataset, lifestyle index was defined as the sum of risk scores for four lifestyle factors: smoking status (0 = Never, 1 = Former, 2 = Current), alcohol intake (0 = Moderate, 1 = Never, 2 = Heavy), physical activity (0 = Active, 1= Inactive), and educational level (0=College degree, 1 = Some college, and 2 = No college). We examined GxE for systolic BP using the quantitative index (ranging from 0 to 7) and a binary form of the index (≥ vs &lt; the median), while adjusting for sex, age, and kinship. Promising loci were identified using a 2 df joint test of main and interaction effects at α = 5 x 10 -6 . Results: Sixteen promising single nucleotide polymorphisms (SNPs) representing 8 loci were found when using the quantitative lifestyle index, and 43 SNPs representing 4 loci when using the binary lifestyle index (Table 1), with only one locus common to both indices. Of the 11 unique loci detected, 3 are within 500 kb of known BP loci and 6 are in or near a gene with a known function. Conclusions: We demonstrate that a composite of multiple lifestyle factors can enhance novel discovery.

A molecular genetic research of the Triticum sinskajae A. Filat. et Kurk. by RAPD analysis and by comparing the nucleotide sequences of the variable intergenic region of the petN-trnC-GCA chloroplast genome and intron of the histone H3.2 gene

Background. Triticum sinskajae A. Filat. et Kurk. was discovered in the early 70th in the last century at the regular reproduction in the Central Asian and Dagestan VIR-stations of T. monococcum samples.&#x0D; Materials and methods. The objects of the study were 4 species of diploid wheat Triticum urartu Thum. ex Gandil. (lines k-62477, k-62465), Triticum monococcum L. (lines k-20970, k-39471), Triticum boeoticum Boiss. (lines k-59161, k-28132, k-40118) and Triticum sinskajae A. Filat. et Kurk. (line k-48993).&#x0D; Results. We found differences between T. sinskajaeand T. monococcum in the variable region of the histone gene H3.2, and the RAPD analysis showed the presence of unique polymorphic loci in T. sinskajae.&#x0D; Conclusion. In gene ral, T. boeoticum, T. monococcum, and T. sinskajae are most likely to be closely related species of diploid wheat, whereas T. urartu is quite significantly different from them.

Global, integrated analysis of methylomes and transcriptomes from laser capture microdissected bronchial and alveolar cells in human lung

ABSTRACTGene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.

Beyond pathways: genetic dissection of tocopherol content in maize kernels by combining linkage and association analyses.

SummaryAlthough tocopherols play an important role in plants and animals, the genetic architecture of tocopherol content in maize kernels has remained largely unknown. In this study, linkage and association analyses were conducted to examine the genetic architecture of tocopherol content in maize kernels. Forty‐one unique quantitative trait loci (QTLs) were identified by linkage mapping in six populations of recombinant inbred lines (RILs). In addition, 32 significant loci were detected via genome‐wide association study (GWAS), 18 of which colocalized with the QTLs identified by linkage mapping. Fine mapping of a major QTL validated the accuracy of GWAS and QTL mapping results and suggested a role for nontocopherol pathway genes in the modulation of natural tocopherol variation. We provided genome‐wide evidence that genes involved in fatty acid metabolism, chlorophyll metabolism and chloroplast function may affect natural variation in tocopherols. These findings were confirmed through mutant analysis of a particular gene from the fatty acid pathway. In addition, the favourable alleles for many of the significant SNPs/QTLs represented rare alleles in natural populations. Together, our results revealed many novel genes that are potentially involved in the variation of tocopherol content in maize kernels. Pyramiding of the favourable alleles of the newly elucidated genes and the well‐known tocopherol pathway genes would greatly improve tocopherol content in maize.

Population genetic structure of the Culex pipiens (Diptera: Culicidae) complex, vectors of West Nile virus, in five habitats

BackgroundThe Culex pipiens complex consists of several morphologically similar, closely related species. In the United States, Cx. pipiens L. is distributed North of 39° latitude, while Cx. quinquefasciatus Say occurs South of 36° latitude; a hybrid zone occurs between these two latitudes including in the Central Valley of California. Members of the Cx. pipiens complex and their hybrids are vectors for West Nile virus (WNv). Hybrid offspring of Cx. pipiens and Cx. quinquefasciatus have been found to have enhanced transmission rates of WNv over those of pure populations of each species. We investigated whether hybrids of Cx. pipiens and Cx. quinquefasciatus occurred more frequently in any of five habitats which were dairies, rural, suburban, and urban areas, and wetlands. In addition, the proportion of alleles unique to Cx. quinquefasciatus and Cx. pipiens found in each habitat-associated population were determined.MethodsAmplified fragment length polymorphism (AFLP) markers were used to compare the population structure of the Cx. pipiens complex from each habitat to geographically distant populations considered pure Cx. pipiens and Cx. quinquefasciatus. Structure analyses were used to assign individuals to either Cx. pipiens, Cx. quinquefasciatus, or hybrids of the Cx. pipiens complex. The ancestry of hybrids (F1, F2, or backcrossed) in relation to the two parent populations was estimated for each Central Valley population. Loci unique to the pure Cx. pipiens population and the pure Cx. quinquefasciatus population were determined. The proportion of loci unique to Cx. pipiens and Cx. quinquefasciatus populations were subsequently determined for each population from the five Merced habitats and from the Oroville California population. The unique loci found in Merced populations and not in Cx. pipiens or Cx. quinquefasciatus were also determined. A principal components analysis was run, as was an analysis to determine loci under putative selection.ResultsThe Structure Harvester analysis found K = 3, and the Culex pipiens complex mosquitoes formed a genetic cluster distinct from Cx. quinquefasciatus and Cx. pipiens. Individuals collected from each habitat were nearly all hybrids. However, Cx. pipiens complex collected near dairies had more individuals categorized as Cx. pipiens than collections from the other habitats. None of the mosquitoes collected in Merced or Oroville were considered pure Cx. quinquefasciatus. Significant genetic divergence was detected among the Cx. pipiens complex from the five habitats in Merced; Cx. pipiens complex mosquitoes from dairies were divergent from the urban and suburban populations. New Hybrids analysis found that individuals from all five Merced habitat-associated populations and the population from Oroville were primarily categorized as hybrids backcrossed to the Cx. pipiens population. Finally, all five habitat-associated populations shared more alleles with Cx. pipiens than with Cx. quinquefasciatus, even though the pure Cx. quinquefasciatus population was more geographically proximate to Merced. Results from the principal component analysis, and the occurrence of several unique loci in Merced populations, suggest that Cx. pipiens molestus may also occur in the habitats sampled.ConclusionsNearly all mosquitoes in the five habitats in Merced in the Central Valley of California area were hybrids of Cx. pipiens and Cx. quinquefasciatus, consisting of hybrids backcrossed to Cx. pipiens. Habitat-associated mosquitoes collected near dairies had more individuals consisting of pure Cx. pipiens, and no mosquitoes from Merced or Oroville CA classified as pure Cx. quinquefasciatus. The genetic distances among Cx. pipiens and Cx. quinquefasciatus, and hybrid populations agree with previous studies using other molecular markers. Cx. pipiens hybrids in Merced shared more alleles with Cx. pipiens than Cx. quinquefasciatus which was unexpected, since Merced is geographically closer to the northern limit of Cx. quinquefasciatus distribution. Culex pipiens molestus may occur in more habitats in the Central Valley than previously suspected, which warrants further investigation. Future studies could investigate the vector competence of hybrids backcrossed to either Cx. pipiens or Cx. quinquefasciatus parent for their ability to transmit West Nile virus.

A Major Locus for Manganese Tolerance Maps on Chromosome A09 in a Doubled Haploid Population of Brassica napus L.

Soil acidity poses a major threat to productivity of several crops; mainly due to the prevalence of toxic levels of Al3+ and Mn2+. Crop productivity could be harnessed on acid soils via the development of plant varieties tolerant to phytotoxic levels of these cations. In this study, we investigated the extent of natural variation for Mn2+ tolerance among ten parental lines of the Australian and International canola mapping populations. Response to Mn2+ toxicity was measured on the bases of cotyledon chlorosis, shoot biomass, and leaf area in nutrient solution under control (9 μM of MnCl2⋅4H2O) and Mn treatment (125 μM of MnCl2⋅4H2O). Among parental lines, we selected Darmor-bzh and Yudal that showed significant and contrasting variation in Mn2+ tolerance to understand genetic control and identify the quantitative trait loci (QTL) underlying Mn2+ tolerance. We evaluated parental lines and their doubled haploid (DH) progenies (196 lines) derived from an F1 cross, Darmor-bzh/Yudal for Mn2+ tolerance. Mn2+-tolerant genotypes had significantly higher shoot biomass and leaf area compared to Mn2+-sensitive genotypes. A genetic linkage map based on 7,805 DArTseq markers corresponding to 2,094 unique loci was constructed and further utilized for QTL identification. A major locus, BnMn2+.A09 was further mapped with a SNP marker, Bn-A09-p29012402 (LOD score of 34.6) accounting for most of the variation in Mn2+ tolerance on chromosome A09. This is the first report on the genomic localization of a Mn2+ tolerance locus in B. napus. Additionally, an ortholog of A. thaliana encoding for cation efflux facilitator transporter was located within 3,991 bp from significant SNP marker associated with BnMn2+.A09. A suite of genome sequence based markers (DArTseq and Illumina Infinium SNPs) flanking the BnMn2+.A09 locus would provide an invaluable tool for various molecular breeding applications to improve canola production and profitability on Mn2+ toxic soils.

Genome-wide association mapping of black point reaction in common wheat (Triticum aestivum L.)

BackgroundBlack point is a serious threat to wheat production and can be managed by host resistance. Marker-assisted selection (MAS) has the potential to accelerate genetic improvement of black point resistance in wheat breeding. We performed a genome-wide association study (GWAS) using the high-density wheat 90 K and 660 K single nucleotide polymorphism (SNP) assays to better understand the genetic basis of black point resistance and identify associated molecular markers.ResultsBlack point reactions were evaluated in 166 elite wheat cultivars in five environments. Twenty-five unique loci were identified on chromosomes 2A, 2B, 3A, 3B (2), 3D, 4B (2), 5A (3), 5B (3), 6A, 6B, 6D, 7A (5), 7B and 7D (2), respectively, explaining phenotypic variation ranging from 7.9 to 18.0%. The highest number of loci was detected in the A genome (11), followed by the B (10) and D (4) genomes. Among these, 13 were identified in two or more environments. Seven loci coincided with known genes or quantitative trait locus (QTL), whereas the other 18 were potentially novel loci. Linear regression showed a clear dependence of black point scores on the number of favorable alleles, suggesting that QTL pyramiding will be an effective approach to increase resistance. In silico analysis of sequences of resistance-associated SNPs identified 6 genes possibly involved in oxidase, signal transduction and stress resistance as candidate genes involved in black point reaction.ConclusionSNP markers significantly associated with black point resistance and accessions with a larger number of resistance alleles can be used to further enhance black point resistance in breeding. This study provides new insights into the genetic architecture of black point reaction.

Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions

A novel genomic and plasmid target-based PCR platform was developed for the detection of Salmonella serovars Heidelberg, Dublin, Hadar, Kentucky, and Enteritidis. Unique genome loci were obtained through extensive genome mining of protein databases and comparative genomic analysis of these serovars. Assays targeting Salmonella serovars Hadar, Heidelberg, Kentucky, and Dublin had 100% specificity and sensitivity, whereas those for Salmonella Enteritidis had 97% specificity and 88% sensitivity. The limits of detection for Salmonella serovars Heidelberg, Kentucky, Hadar, Enteritidis, and Dublin were 12, 9, 40, 13, and 5,280 CFU, respectively. A sensitivity assay was also performed by using milk artificially inoculated with pooled Salmonella serovars, yielding a detection limit of 1 to10 CFU/25 mL of milk samples after enrichment. The minimum DNA detected using the multiplexed TaqMan assay was 75.8 fg (1.53 × 101 genomic equivalents [GE]) for Salmonella Heidelberg, 140.8 fg (2.8 × 101 GE) for Salmonella Enteritidis, and 3.48 pg (6.96 × 102 GE) for Salmonella Dublin. PCR efficiencies were 89.8% for Salmonella Heidelberg, 94.5% for Salmonella Enteritidis, and 75.5% for Salmonella Dublin. Four types of 30 pasteurized milk samples were tested negative by culture techniques and with a genus-specific Salmonella invA gene PCR assay. Among 30 chicken samples similarly tested, 12 (40%) were positive by both culture and the invA PCR. Testing of these 12 samples with the serovar-specific PCR assay detected single and mixed contamination with Salmonella Kentucky, Salmonella Enteritidis, and Salmonella Heidelberg. Five unique primers were designed and tested by multiplex conventional PCR in conjunction with the use of the multiplex TaqMan assay with three of the primers. The diagnostic assays developed in this study could be used as tools for routine detection of these five Salmonella serovars and for epidemiological investigations of foodborne disease outbreaks.

Similar Genetic Architecture with Shared and Unique Quantitative Trait Loci for Bacterial Cold Water Disease Resistance in Two Rainbow Trout Breeding Populations.

Bacterial cold water disease (BCWD) causes significant mortality and economic losses in salmonid aquaculture. In previous studies, we identified moderate-large effect quantitative trait loci (QTL) for BCWD resistance in rainbow trout (Oncorhynchus mykiss). However, the recent availability of a 57 K SNP array and a reference genome assembly have enabled us to conduct genome-wide association studies (GWAS) that overcome several experimental limitations from our previous work. In the current study, we conducted GWAS for BCWD resistance in two rainbow trout breeding populations using two genotyping platforms, the 57 K Affymetrix SNP array and restriction-associated DNA (RAD) sequencing. Overall, we identified 14 moderate-large effect QTL that explained up to 60.8% of the genetic variance in one of the two populations and 27.7% in the other. Four of these QTL were found in both populations explaining a substantial proportion of the variance, although major differences were also detected between the two populations. Our results confirm that BCWD resistance is controlled by the oligogenic inheritance of few moderate-large effect loci and a large-unknown number of loci each having a small effect on BCWD resistance. We detected differences in QTL number and genome location between two GWAS models (weighted single-step GBLUP and Bayes B), which highlights the utility of using different models to uncover QTL. The RAD-SNPs detected a greater number of QTL than the 57 K SNP array in one population, suggesting that the RAD-SNPs may uncover polymorphisms that are more unique and informative for the specific population in which they were discovered.

Genome-Wide Association Meta-Analysis Reveals Novel Juvenile Idiopathic Arthritis Susceptibility Loci.

Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease and has a strong genomic component. To date, JIA genetic association studies have had limited sample sizes, usedheterogeneous patient populations, or included only candidate regions. The aim of this study was to identify new associations between JIA patients with oligoarticular disease and those with IgM rheumatoid factor (RF)-negative polyarticular disease, which are clinically similar and the most prevalent JIA disease subtypes. Three cohorts comprising 2,751 patients with oligoarticular or RF-negative polyarticular JIA were genotyped using the Affymetrix Genome-Wide SNP Array 6.0 or the Illumina HumanCoreExome-12+ Array. Overall, 15,886 local and out-of-study controls, typed on these platforms or the Illumina HumanOmni2.5, were used forassociation analyses. High-quality single-nucleotide polymorphisms (SNPs) were used for imputation to 1000 Genomes prior to SNP association analysis. Meta-analysis showed evidence of association (P < 1 × 10-6 ) at 9 regions: PRR9_LOR (P = 5.12 × 10-8 ), ILDR1_CD86 (P = 6.73 × 10-8 ), WDFY4 (P = 1.79 × 10-7 ), PTH1R (P = 1.87 × 10-7 ), RNF215 (P = 3.09 × 10-7 ), AHI1_LINC00271 (P = 3.48 × 10-7 ), JAK1 (P = 4.18 × 10-7 ), LINC00951 (P = 5.80 × 10-7 ), and HBP1 (P = 7.29 × 10-7 ). Of these, PRR9_LOR, ILDR1_CD86, RNF215, LINC00951, and HBP1 were shown, for the first time, to be autoimmune disease susceptibility loci. Furthermore, associated SNPs included cis expression quantitative trait loci for WDFY4, CCDC12, MTP18, SF3A1, AHI1, COG5, HBP1, and GPR22. This study provides evidence of both unique JIA risk loci and risk loci overlapping between JIA and other autoimmune diseases. These newly associated SNPs are shown to influence gene expression, and their bounding regions tie into molecular pathways of immunologic relevance. Thus, they likely represent regions that contribute to the pathology of oligoarticular JIA and RF-negative polyarticular JIA.