Pool Assay Research Articles

High-throughput analysis of adaptation using barcoded strains of Saccharomyces cerevisiae.

BackgroundExperimental evolution of microbes can be used to empirically address a wide range of questions about evolution and is increasingly employed to study complex phenomena ranging from genetic evolution to evolutionary rescue. Regardless of experimental aims, fitness assays are a central component of this type of research, and low-throughput often limits the scope and complexity of experimental evolution studies. We created an experimental evolution system in Saccharomyces cerevisiae that utilizes genetic barcoding to overcome this challenge.ResultsWe first confirm that barcode insertions do not alter fitness and that barcode sequencing can be used to efficiently detect fitness differences via pooled competition-based fitness assays. Next, we examine the effects of ploidy, chemical stress, and population bottleneck size on the evolutionary dynamics and fitness gains (adaptation) in a total of 76 experimentally evolving, asexual populations by conducting 1,216 fitness assays and analyzing 532 longitudinal-evolutionary samples collected from the evolving populations. In our analysis of these data we describe the strengths of this experimental evolution system and explore sources of error in our measurements of fitness and evolutionary dynamics.ConclusionsOur experimental treatments generated distinct fitness effects and evolutionary dynamics, respectively quantified via multiplexed fitness assays and barcode lineage tracking. These findings demonstrate the utility of this new resource for designing and improving high-throughput studies of experimental evolution. The approach described here provides a framework for future studies employing experimental designs that require high-throughput multiplexed fitness measurements.

Ginkgetin derived from Ginkgo biloba leaves enhances the therapeutic effect of cisplatin via ferroptosis-mediated disruption of the Nrf2/HO-1 axis in EGFR wild-type non-small-cell lung cancer

BackgroundCisplatin (DDP) is the first-in-class drug for advanced and non-targetable non-small-cell lung cancer (NSCLC). A recent study indicated that DDP could slightly induce non-apoptotic cell death ferroptosis, and the cytotoxicity was promoted by ferroptosis inducer. The agents enhancing the ferroptosis may therefore increase the anticancer effect of DDP. Several lines of evidence supporting the use of phytochemicals in NSCLC therapy. Ginkgetin, a bioflavonoid derived from Ginkgo biloba leaves, showed anticancer effects on NSCLC by triggering autophagy. Ferroptosis can be triggered by autophagy, which regulates redox homeostasis. Thus, we aimed to elucidate the possible role of ferroptosis involved in the synergistic effect of ginkgetin and DDP in cancer therapy. MethodsThe promotion of DDP-induced anticancer effects by ginkgetin was examined via a cytotoxicity assay and western blot. Ferroptosis triggered by ginkgetin in DDP-treated NSCLC was observed via a lipid peroxidation assay, a labile iron pool assay, western blot, and qPCR. With ferroptosis blocking, the contribution of ferroptosis to ginkgetin + DDP-induced cytotoxicity, the Nrf2/HO-1 axis, and apoptosis were determined via a luciferase assay, immunostaining, chromatin immunoprecipitation (CHIP), and flow cytometry. The role of ferroptosis in ginkgetin + DDP-treated NSCLC cells was illustrated by the application of ferroptosis inhibitors, which was further demonstrated in a xenograft nude mouse model. ResultsGinkgetin synergized with DDP to increase cytotoxicity in NSCLC cells, which was concomitant with increased labile iron pool and lipid peroxidation. Both these processes were key characteristics of ferroptosis. The induction of ferroptosis mediated by ginkgetin was further confirmed by the decreased expression of SLC7A11 and GPX4, and a decreased GSH/GSSG ratio. Simultaneously, ginkgetin disrupted redox hemostasis in DDP-treated cells, as demonstrated by the enhanced ROS formation and inactivation of the Nrf2/HO-1 axis. Ginkgetin also enhanced DDP-induced mitochondrial membrane potential (MMP) loss and apoptosis in cultured NSCLC cells. Furthermore, blocking ferroptosis reversed the ginkgetin-induced inactivation of Nrf2/HO-1 as well as the elevation of ROS formation, MMP loss, and apoptosis in DDP-treated NSCLC cells. ConclusionThis study is the first to report that ginkgetin derived from Ginkgo biloba leaves promotes DDP-induced anticancer effects, which can be due to the induction of ferroptosis.

"Sample pooling of RNA extracts to speed up SARS-CoV-2 diagnosis using CDC FDA EUA RT-qPCR kit"

BackgroundThe CDC protocol for SARS-CoV2 RT-PCR diagnosis (2019-nCoV CDC kit) is considered a gold standard worldwide; based on three different FAM probes (N1 and N2 for viral detection; RP for RNA extraction quality control), three reactions per sample are needed for SARS-CoV-2 diagnosis. ResultsWe herein describe a sample pooling protocol: pooling 3 RNA extractions into a single PCR reaction; we tested this protocol with 114 specimens grouped in 38 pools and found no significant differences for N1 and N2 Ct values between pool and single sample PCR reaction. ConclusionThis pool of three protocol has a sensitivity of 100 % compared to the standard single sample protocol. For a typical 96-well plate, this pool assay allows 96 samples processing, speeding up diagnosis and reducing cost while maintaining clinical performance, particularly useful for SARS-CoV-2 diagnosis at developing countries.

Serum galactomannan level for the diagnosis of invasive pulmonary aspergillosis in hospitalized patients with chronic obstructive pulmonary disease (COPD): Meta-analysis

Background: The serum galactomannan (GM) assay is used to diagnose invasive pulmonary aspergillosis (IPA). We conducted a meta-analysis to assess the overall accuracy of the serum GM test for detecting IPA in critically ill COPD patients. Methods: The web of science, Pubmed, Embase, China National Knowledge Infrastructure, WanFang, Weipu (VIP) Science China databases were searched for all the eligible studies, including randomized controlled trials and prospective studies up to February 2019. We selected and assessed articles that reported diagnostic data related to serum GM for diagnosis of invasive fungal disease in hospitalized COPD patients. Additionally, pooled sensitivity (SEN), specificity (SPE), true positive (TP), false positive (FP), true negative (TN), false negative (FN) were estimated for summarizing overall test performance. Results: Of 1104 articles screened, 14 studies were involved in the meta-analysis with 1127 patients in the COPD serum sample. The pooled serum GM assay results with cutoff value of 0.5 were as follows: DOR: 15 (95% CI, 6–38), sensitivity: 0.57 (95% CI, 0.37–0.75), specificity: 0.92 (95%,CI, 0.85–0.96), and the SROC was 0.89 (95% CI, 0.86–0.91). When the cutoff value was 1, the results were as follows: DOR: 33 (95% CI, 15–70), sensitivity: 0.57 (95% CI, 0.39–0.73), specificity: 0.96 (95% CI, 0.93–0.98), and SROC was 0.95 (95% CI, 0.92–0.96). Conclusion: Serum GM can be used to assist in diagnosis of IPA in critically ill COPD patients. When cutoff value was set as 1, serum GM had better diagnostic value compared to those with cutoff of 0.5.

Quantitative trait locus for resistance to the aphid Rhopalosiphum padi L. in barley (Hordeum vulgare L.) is not linked with a genomic region for gramine concentration

Gramine is an indole alkaloid found in certain grass species. Results of previous studies have diverged as to whether this compound might cause resistance to aphids or not. In a breeding program aiming to introduce resistance to the cereal pest Rhopalosiphum padi L. in barley, a cultivar has been crossed with the progenitor of cultivated barley, Hordeum vulgare ssp. spontaneum, and an F1-derived population of doubled haploid (DH) lines was screened both for seedling gramine concentration and resistance to R. padi. The resistance was measured as individual aphid growth in the laboratory. The present study aims to determine if there is a genetic relationship between aphid growth retardation and gramine concentration. To do so, the lines were genotyped with a 384 SNP oligonucleotide pool assay and QTL analyses were performed for both traits. A previously identified aphid resistance locus on the distal part of chromosome 2HS was thereby confirmed, with resistance inherited from H. v. ssp. spontaneum, whereas one or more QTL for gramine concentration were potentially indicated on chromosome 3H, thus corroborating that the two traits are not linked genetically.

Pathway sensor-based functional genomics screening identifies modulators of neuronal activity

Neuronal signal transduction shapes brain function and malfunction may cause mental disorders. Despite the existence of functional genomics screens for proliferation and toxicity, neuronal signalling has been difficult to address so far. To overcome this limitation, we developed a pooled screening assay which combines barcoded activity reporters with pooled genetic perturbation in a dual-expression adeno-associated virus (AAV) library. With this approach, termed pathScreener, we comprehensively dissect signalling pathways in postmitotic neurons. This overcomes several limitations of lentiviral-based screens. By applying first a barcoded and multiplexed reporter assay, termed cisProfiler, we identified the synaptic-activity responsive element (SARE) as top performance sensor of neuronal activity. Next, we targeted more than 4,400 genes and screened for modulatory effects on SARE activity in primary cortical neurons. We identified with high replicability many known genes involved in glutamatergic synapse-to-nucleus signalling of which a subset was validated in orthogonal assays. Several others have not yet been associated with the regulation of neuronal activity such as the hedgehog signalling members Ptch2 and Ift57. This assay thus enhances the toolbox for analysing regulatory processes during neuronal signalling and may help identifying novel targets for brain disorders.

Systematic Complex Haploinsufficiency-Based Genetic Analysis of Candida albicans Transcription Factors: Tools and Applications to Virulence-Associated Phenotypes.

Genetic interaction analysis is a powerful approach to the study of complex biological processes that are dependent on multiple genes. Because of the largely diploid nature of the human fungal pathogen Candida albicans, genetic interaction analysis has been limited to a small number of large-scale screens and a handful for gene-by-gene studies. Complex haploinsufficiency, which occurs when a strain containing two heterozygous mutations at distinct loci shows a phenotype that is distinct from either of the corresponding single heterozygous mutants, is an expedient approach to genetic interactions analysis in diploid organisms. Here, we describe the construction of a barcoded-library of 133 heterozygous TF deletion mutants and deletion cassettes for designed to facilitate complex haploinsufficiency-based genetic interaction studies of the TF networks in C. albicans. We have characterized the phenotypes of these heterozygous mutants under a broad range of in vitro conditions using both agar-plate and pooled signature tag-based assays. Consistent with previous studies, haploinsufficiency is relative uncommon. In contrast, a set of 12 TFs enriched in mutants with a role in adhesion were found to have altered competitive fitness at early time points in a murine model of disseminated candidiasis. Finally, we characterized the genetic interactions of a set of biofilm related TFs in the first two steps of biofilm formation, adherence and filamentation of adherent cells. The genetic interaction networks at each stage of biofilm formation are significantly different indicating that the network is not static but dynamic.

Main and epistatic QTL analyses for Sclerotinia Head Rot resistance in sunflower.

Sclerotinia Head Rot (SHR), a disease caused by Sclerotinia sclerotiorum, is one of the most limiting factors in sunflower production. In this study, we identified genomic loci associated with resistance to SHR to support the development of assisted breeding strategies. We genotyped 114 Recombinant Inbred Lines (RILs) along with their parental lines (PAC2 –partially resistant–and RHA266 –susceptible–) by using a 384 single nucleotide polymorphism (SNP) Illumina Oligo Pool Assay to saturate a sunflower genetic map. Subsequently, we tested these lines for SHR resistance using assisted inoculations with S. sclerotiorum ascospores. We also conducted a randomized complete-block assays with three replicates to visually score disease incidence (DI), disease severity (DS), disease intensity (DInt) and incubation period (IP) through four field trials (2010–2014). We finally assessed main effect quantitative trait loci (M-QTLs) and epistatic QTLs (E-QTLs) by composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively. As a result of this study, the improved map incorporates 61 new SNPs over candidate genes. We detected a broad range of narrow sense heritability (h2) values (1.86–59.9%) as well as 36 M-QTLs and 13 E-QTLs along 14 linkage groups (LGs). On LG1, LG10, and LG15, we repeatedly detected QTLs across field trials; which emphasizes their putative effectiveness against SHR. In all selected variables, most of the identified QTLs showed high determination coefficients, associated with moderate to high heritability values. Using markers shared with previous Sclerotinia resistance studies, we compared the QTL locations in LG1, LG2, LG8, LG10, LG11, LG15 and LG16. This study constitutes the largest report of QTLs for SHR resistance in sunflower. Further studies focusing on the regions in LG1, LG10, and LG15 harboring the detected QTLs are necessary to identify causal alleles and contribute to unraveling the complex genetic basis governing the resistance.

Association of candidate gene polymorphisms with milk technological traits, yield, composition, and somatic cell score in Italian Holstein-Friesian sires

Advances in DNA-based marker technology have enabled the identification of genomic regions underlying complex phenotypic traits in livestock species. The incorporation of detected quantitative trait loci into genetic evaluation provides great potential to enhance selection accuracies, hence expediting the genetic improvement of economically important traits. The objective of the present study was to investigate 96 single nucleotide polymorphisms (SNP) located in 53 candidate genes previously reported to have effects on milk production and quality traits in a population of highly selected Holstein-Friesian bulls. A total of 423 semen samples were used to genotype the bulls through a custom oligo pool assay. Forty-five SNP in 32 genes were found to be associated with at least 1 of the tested traits. Most significant and favorable SNP trait associations were observed for polymorphisms located in CCL3 and AGPAT6 genes for fat yield (0.037 and 0.033 kg/d, respectively), DGKG gene for milk yield (0.698 kg/d), PPARGC1A, CSN1S1, and AGPAT6 genes for fat percentage (0.127, 0.113, and 0.093%, respectively), GHR gene for protein (0.064%) and casein percentage (0.053%), and TLR4 gene for fat (0.090%), protein (0.066%), and casein percentage (0.050%). Somatic cell score was favorably affected by GHR (-0.095) and POU1F1 (-0.137), and interesting SNP-trait associations were observed for polymorphisms located in CSN2, POU1F1, and AGPAT6 genes for rennet coagulation time (-0.592, -0.558, and -0.462 min, respectively), and GHR and CSN2 genes for curd firmness 30 min after rennet addition (1.264 and 1.183 mm, respectively). In addition to the influence of individual SNP, the effects of composite genotypes constructed by grouping SNP according to their individual effects on traits considered in the analysis were also examined. Favorable and significant effects on milk traits were observed for 2 composite genotypes, one including 10 SNP and the other 4 SNP. The former was associated with an increase of milk (0.075 kg/d), fat (0.097 kg/d), protein (0.083 kg/d), and casein yields (0.065 kg/d), and the latter was associated with an increase of fat (0.244%), protein (0.071%), and casein percentage (0.047%). Although further research is required to validate the identified SNP loci in other populations and breeds, our results can be considered as a preliminary foundation for further replication studies on gene-assisted selection programs.

Effect of Collection Methods on Salivary Cortisol

Salivary cortisol is a useful measure of hypothalmic-pituitary axis function, but the effects of collection method, sample volume, and environmental conditions on hormone recovery are poorly described. These potential sources of pre-analytical variation were assessed using spiked pooled saliva and assay by liquid chromatography and tandem mass spectrometry (LC-MS/MS). When saliva was collected onto cotton swabs, hormone recovery was determined primarily by volume recovery, which was in turn determined by sample volume. To ensure adequate cortisol recovery, ≥2 ml of saliva must be absorbed onto swabs. This may be difficult for many children and some adults to achieve. Salivary cortisol is remarkably stable to a range of environmental conditions, including repeated freeze-thaw cycles, storage at room temperature and exposure to sunlight. This study shows that it is preferable to obtain saliva by passive drool without swabs if adequate sample volume (≥2 ml) cannot be achieved, and that saliva samples can be returned by standard post without affecting hormone recovery.

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes-such as transcriptional profiles-at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations andtheir genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.

A statistical approach to determining responses to individual peptides from pooled-peptide ELISpot data

To investigate in detail the effect of infection or vaccination on the human immune system, ELISpot assays are used to simultaneously test the immune response to a large number of peptides of interest. Scientists commonly use “peptide pools”, where, instead of an individual peptide, a test well contains a group of peptides. Since the response from a well may be due to any or many of the peptides in the pool, pooled assays usually need to be followed by confirmatory assays of a number of individual peptides. We present a statistical method that enables estimation of individual peptide responses from pool responses using the Expectation Maximization (EM) algorithm for “incomplete data”. We demonstrate the accuracy and precision of these estimates in simulation studies of ELISpot plates with 90 pools of 6 or 7 peptides arranged in three dimensions and three Mock wells for the estimation of background. In analysis of real pooled data from 6 subjects in a HIV-1 vaccine trial, where 199 peptides were arranged in 80 pools if size 9 or 10, our estimates were in very good agreement with the results from individual-peptide confirmatory assays. Compared to the classical approach, we could identify almost all the same peptides with high or moderate response, with less than half the number of confirmatory tests. Our method facilitates efficient use of the information available in pooled ELISpot data to avoid or reduce the need for confirmatory testing. We provide an easy-to-use free online application for implementing the method, where on uploading two spreadsheets with the pool design and pool responses, the user obtains the estimates of the individual peptide responses.

Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism.

Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. Here, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the test species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.

Genetic dissection of chlorate respiration in Pseudomonas stutzeri PDA reveals syntrophic (per)chlorate reduction.

Genes important for growth of Pseudomonas stutzeri PDA on chlorate were identified using a randomly DNA bar-coded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB and clrC were essential for chlorate reduction, while clrD, clrE and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness defects in Bar-seq experiments. We hypothesized this was enabled through chlorite dismutation by the community, as most strains in the Bar-seq library contained an intact cld. In support of this, Δcld grew with wild-type PDA or ΔclrA, and purified Cld also restored growth to the Δcld mutant. Expanding on this, wild-type PDA and a Δcld mutant of the perchlorate reducer Azospira suillum PS grew on perchlorate in co-culture, but not individually. These results demonstrate that co-occurrence of cld and a chloroxyanion reductase within a single organism is not necessary and raises the possibility of syntrophic (per)chlorate respiration in the environment.

Abstract PR13: Systems and structural biology approaches to elucidate new effectors in KRAS mutant tumors

Abstract Activated KRAS interacts directly and indirectly with numerous downstream effector pathways that are still elusive or poorly characterized. Furthermore, it has become clear over the past few years that the KRAS regulatory gene network (interactome) is highly cell-context dependent and thus tumor specific. We sought to computationally elucidate novel KRAS effectors, modulators, and interactors in Lung Adenocarcinomas (LUAD) and Colon Adenocarcinomas (COAD) by integrating computational and biochemical reverse engineering methods. Specifically, results from ARACNe and MINDy based analysis of KRAS networks were combined with Protein-protein interaction (PPI) predictions based on the PrePPI algorithm. These algorithms detect, respectively, transcriptional, post-translational, and protein-protein interactions respectively. The MARINa/VIPER algorithm was used to analyze the networks resulting from these analyses to identify key modulators and effectors of KRAS activity, including modulators harboring genetic alterations that phenocopy aberrant KRAS activity. In addition, we collaborated with the Jackson (Stanford) and Vidal (Harvard) labs to include high-throughput tandem affinity mass spectrometry data and yeast 2 hybrid screens in these analyses. Validation of computationally inferred effectors, using pooled shRNA silencing assays in primary lung cancer cells isolated from a KrasLSL/p53f/f mouse model of LUAD validated novel genes as likely KRASmut effectors and putative synthetic lethal genes. At least two hairpins against RNF43, E4F1, HMCN1, MYO9A, RHOT2, SETD1B and MAP3K2 scored in this assay. These results suggest that KRAS network analyses using computational methods can identify novel members of the KRAS interactome. Citation Format: Joshua Broyde, David Simpson, David A. Wah, Federico M. Giorgi, Donald Petrey, Mariano J. Alvarez, Antonina Silkov, Alexander Lachmann, David E. Hill, Marc Vidal, Peter Jackson, Barry Honig, Alejandro Sweet-Cordero, Andrea Califano. Systems and structural biology approaches to elucidate new effectors in KRAS mutant tumors. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR13.

A single-islet microplate assay to measure mouse and human islet insulin secretion

One complication to comparing β-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest.

Fruit Quality Traits Have Played Critical Roles in Domestication of the Apple

With its long history of cultivation, the domesticated apple, Malus × domestica Borkh., is an excellent model for studying domestication in long‐lived perennial plants. As apples have been transported from their center of origin in the Tian Shan region in central Asia and moved along the famous Silk Road trading path, they have undergone selection for distinct phenotypic traits. In this study, a high‐throughput single nucleotide polymorphism (SNP) genotyping assay is used to investigate relationships of Malus species, draw inferences on the domestication history, identify traits critical for domestication, and assess potential genetic loci under selection. A total of 160 Malus accessions, including wild species, old and new apple cultivars, and advanced selections, were genotyped. Of 1536 SNPs from GoldenGate oligonucleotide pool assays (OPAs), 901 SNPs fulfilled filtering criteria. A principal component analysis (PCA) revealed that most M. × domestica accessions grouped together. A total of 67 loci, including 13 genomic SNPs and 54 genic SNPs, have been identified as potential targets for selection during evolution of the apple genome from its wild progenitor M. sieversii. Genes introgressed from local wild species into M. × domestica are associated with adaptation to local environments, while genes for fruit quality traits are derived from M. sieversii.

Association Mapping of Agronomic QTLs in U.S. Spring Barley Breeding Germplasm

The use of genome‐wide association studies (GWAS) to detect quantitative trait loci (QTL) controlling complex traits has become a popular approach for studying key traits in crop plants. The goal of this study was to identify the genomic regions of barley (Hordeum vulgare L.) that impact five agronomic and one quality trait in U.S. elite barley breeding lines, as well as to identify markers tightly linked with these loci for further use in barley improvement. Advanced recombinant inbred lines submitted to the U.S. Barley Coordinated Agricultural Project (CAP) were genotyped using a platform of 3072 single nucleotide polymorphism (SNP) markers from the barley oligonucleotide pool assays (BOPAs) 1 and 2. In each of 4 yr, approximately 770 lines were evaluated in a replicated, randomized complete block design under both irrigated and dryland conditions. This gave an overall population size of >3000 lines, which we analyzed in a hierarchical fashion, including analyzing the lines in aggregate using a mixed model to account for population structure and relatedness among the lines. We identified 41 significant marker–trait associations, of which 31 had been previously reported as QTL using biparental mapping techniques; 10 novel marker‐trait associations were identified. The results of this work show that genes with major effects are still segregating in U.S. barley germplasm and demonstrate the utility of GWAS in barley breeding populations.

The associations between immunity-related genes and breast cancer prognosis in Korean women.

We investigated the role of common genetic variation in immune-related genes on breast cancer disease-free survival (DFS) in Korean women. 107 breast cancer patients of the Seoul Breast Cancer Study (SEBCS) were selected for this study. A total of 2,432 tag single nucleotide polymorphisms (SNPs) in 283 immune-related genes were genotyped with the GoldenGate Oligonucleotide pool assay (OPA). A multivariate Cox-proportional hazard model and polygenic risk score model were used to estimate the effects of SNPs on breast cancer prognosis. Harrell’s C index was calculated to estimate the predictive accuracy of polygenic risk score model. Subsequently, an extended gene set enrichment analysis (GSEA-SNP) was conducted to approximate the biological pathway. In addition, to confirm our results with current evidence, previous studies were systematically reviewed. Sixty-two SNPs were statistically significant at p-value less than 0.05. The most significant SNPs were rs1952438 in SOCS4 gene (hazard ratio (HR) = 11.99, 95% CI = 3.62–39.72, P = 4.84E-05), rs2289278 in TSLP gene (HR = 4.25, 95% CI = 2.10–8.62, P = 5.99E-05) and rs2074724 in HGF gene (HR = 4.63, 95% CI = 2.18–9.87, P = 7.04E-05). In the polygenic risk score model, the HR of women in the 3rd tertile was 6.78 (95% CI = 1.48–31.06) compared to patients in the 1st tertile of polygenic risk score. Harrell’s C index was 0.813 with total patients and 0.924 in 4-fold cross validation. In the pathway analysis, 18 pathways were significantly associated with breast cancer prognosis (P<0.1). The IL-6R, IL-8, IL-10RB, IL-12A, and IL-12B was associated with the prognosis of cancer in data of both our study and a previous study. Therefore, our results suggest that genetic polymorphisms in immune-related genes have relevance to breast cancer prognosis among Korean women.

First insights into the transcriptome and development of new genomic tools of a widespread circum‐Mediterranean tree species, Pinus halepensis Mill

Aleppo pine (Pinus halepensis Mill.) is a relevant conifer species for studying adaptive responses to drought and fire regimes in the Mediterranean region. In this study, we performed Illumina next-generation sequencing of two phenotypically divergent Aleppo pine accessions with the aims of (i) characterizing the transcriptome through Illumina RNA-Seq on trees phenotypically divergent for adaptive traits linked to fire adaptation and drought, (ii) performing a functional annotation of the assembled transcriptome, (iii) identifying genes with accelerated evolutionary rates, (iv) studying the expression levels of the annotated genes and (v) developing gene-based markers for population genomic and association genetic studies. The assembled transcriptome consisted of 48,629 contigs and covered about 54.6 Mbp. The comparison of Aleppo pine transcripts to Picea sitchensis protein-coding sequences resulted in the detection of 34,014 SNPs across species, with a Ka /Ks average value of 0.216, suggesting that the majority of the assembled genes are under negative selection. Several genes were differentially expressed across the two pine accessions with contrasted phenotypes, including a glutathione-s-transferase, a cellulose synthase and a cobra-like protein. A large number of new markers (3334 amplifiable SSRs and 28,236 SNPs) have been identified which should facilitate future population genomics and association genetics in this species. A 384-SNP Oligo Pool Assay for genotyping with the Illumina VeraCode technology has been designed which showed an high overall SNP conversion rate (76.6%). Our results showed that Illumina next-generation sequencing is a valuable technology to obtain an extensive overview on whole transcriptomes of nonmodel species with large genomes.