The key role of myostatin b in somatic growth in fishes derived from distant hybridization.

The basic mechanism of heterosis has not been systematically and completely characterized. In previous studies, we obtained three economically important fishes that exhibit rapid growth, WR (WCC ♀ × RCC ♂), WR-II (WR ♀ × WCC ♂), and WR-III (WR-II ♀ × 4nAU ♂), through distant hybridization. However, the mechanism underlying this rapid growth remains unclear. In this study, we found that WR, WR-II, and WR-III showed muscle hypertrophy and higher muscle protein and fat contents compared with their parent species (RCC and WCC). Candidate genes responsible for this rapid growth were then obtained through an analysis of 12 muscle transcriptomes. Notably, the mRNA level of mstnb (myostatin b), which is a negative regulator of myogenesis, was significantly reduced in WR, WR-II, and WR-III compared with the parent species. To verify the function of mstnb, a mstnb-deficient mutant RCC line was generated using the CRISPR-Cas9 technique. The average body weight of mstnb-deficient RCC at 12 months of age was significantly increased by 29.57% compared with that in wild-type siblings. Moreover, the area and number of muscle fibers were significantly increased in mstnb-deficient RCC, indicating hypertrophy and hyperplasia. Furthermore, the muscle protein and fat contents were significantly increased in mstnb-deficient RCC. The molecular regulatory mechanism of mstnb was then revealed by transcription profiling, which showed that genes related to myogenesis (myod, myog, and myf5), protein synthesis (PI3K-AKT-mTOR), and lipogenesis (pparγ and fabp3) were highly activated in hybrid fishes and mstnb-deficient RCC. This study revealed that low expression or deficiency of mstnb regulates somatic growth by promoting myogenesis, protein synthesis, and lipogenesis in hybrid fishes and mstnb-deficient RCC, which provides evidence for the molecular mechanism of heterosis via distant hybridization.

Evolutionary patterns of DNA base composition at polymorphic sites highlight the role of the environment in shaping barley and rice genomes.

Insights into changes in genome base composition underlying crop domestication can be gained by using comparative genomics. With this approach, previous studies have reported that crop genomes during domestication accumulate more nucleotides adenine (A) and thymine (T) (termed as [AT]-increase) across polymorphic sites. However, the potential influence of the environment or its factors, for example, solar ultraviolet (UV) radiation and temperature, on the [AT]-increase has not been well elucidated. Here, we investigated the [AT]-increase in barley (Hordeum vulgare L.) and rice (Oryza sativa L.) and the association with natural environments, where accessions are distributed. With 12,798,376 and 2,861,535 single-nucleotide polymorphisms from 368 barley and 1375 rice accessions, respectively, we discovered that [AT] increases from wild accessions to improved cultivars, and genomic regions with larger [AT]-increase tend to have higher UV-related motif frequencies, suggesting solar UV radiation as a potential factor in driving genome variation. To link [AT] change with the geographic distribution, we gathered georeferenced accessions and examined their local environments. Interestingly, negative correlations between [AT] and environmental factors were observed (r = -0.39 ∼ -0.75) and modern accessions with higher [AT] values, as compared with wild relatives, are from the environments with lower solar UV radiation or lower temperature. With [AT] and environmental factors as phenotypes, genome-wide association mapping identified three candidate genes that have the potential to contribute to [AT] variation under the effect of environmental conditions. Our findings provide genomic and environmental insights into evolutionary pattern of DNA base composition and underlying mechanisms.

Multifunctional ZnO@DOX/ICG-LMHP Nanoparticles for Synergistic Multimodal Antitumor Activity.

Multifunctional nanoparticles are of significant importance for synergistic multimodal antitumor activity. Herein, zinc oxide (ZnO) was used as pH-sensitive nanoparticles for loading the chemotherapy agent doxorubicin (DOX) and the photosensitizer agent indocyanine green (ICG), and biocompatible low-molecular-weight heparin (LMHP) was used as the gatekeepers for synergistic photothermal therapy/photodynamic therapy/chemotherapy/immunotherapy. ZnO was decomposed into cytotoxic Zn2+ ions, leading to a tumor-specific release of ICG and DOX. ZnO simultaneously produced oxygen (O2) and reactive oxygen species (ROS) for photodynamic therapy (PDT). The released ICG under laser irradiation produced ROS for PDT and raised the tumor temperature for photothermal therapy (PTT). The released DOX directly caused tumor cell death for chemotherapy. Both DOX and ICG also induced immunogenic cell death (ICD) for immunotherapy. The in vivo and in vitro results presented a superior inhibition of tumor progression, metastasis and recurrence. Therefore, this study could provide an efficient approach for designing multifunctional nanoparticles for synergistic multimodal antitumor therapy.

Copper ion affects oxidant decay and combined aspartic acid transformation during chlorination in water pipes: Differentiated action on the yield of trihalomethanes and haloacetonitriles.

The chlorination of extracellular polymeric substances (EPS) secreted by biofilm often induces the formation of high-toxic disinfection byproducts (DBPs) in drinking water distribution systems. The protein components in EPS are the main precursors of DBPs, which mostly exist in the form of combined amino acids. The paper aimed to study the action of a pipe corrosion product (Cu2+) on the formation of DBPs (trihalomethanes, THMs; haloacetonitriles, HANs) with aspartic acid tetrapeptide (TAsp) as a precursor. Cu2+ mainly promoted the reaction of oxidants with TAsp (i.e., TAsp-induced decay) to produce DBPs, rather than self-decay of oxidants to generate BrO3‒ and ClO3‒. Cu2+ increased THMs yield, but decreased HANs yield due to the catalytic hydrolysis. Cu2+ was more prone to promote the reaction of TAsp with HOCl than with HOBr, leading to a DBPs shift from brominated to chlorinated species. The chemical characterizations of Cu2+-TAsp complexations demonstrate that Cu2+ combined with TAsp at the N and O sites in both amine and amide groups, and the intermediate identification suggests that Cu2+ enhanced the stepwise chlorination process by promoting the substitution of chlorine and the breakage of CC bonds. The effect of Cu2+ on THMs yield changed from promoting to inhibiting with the increase of pH, while that on HANs yield was inhibiting regardless of pH variation. Additionally, the impact of Cu2+ on the formation of DBPs was also affected by Cu2+ dose, Cl2/C ratio and Br- concentration. This study helps to understand the formation of EPS-derived DBPs in water pipes, and provides reference for formulating control strategies during biofilm outbreaks.

Environmental context of phenotypic plasticity in flowering time in sorghum and rice.

Phenotypic plasticity is an important topic in biology and evolution. However, how to generate broadly applicable insights from individual studies remains a challenge. Here, with flowering time observed from a large geographical region for sorghum and rice genetic populations, we examine the consistency of parameter estimation for reaction norms of genotypes across different subsets of environments and searched for potential strategies to inform the study design. Both sample size and environmental mean range of the subset affected the consistency. The subset with either a large range of environmental mean or a large sample size resulted in genetic parameters consistent with the overall pattern. Furthermore, high accuracy through genomic prediction was obtained for reaction norm parameters of untested genotypes using models built from tested genotypes under the subsets of environments with either a large range or a large sample size. With 1428 and 1674 simulated settings, our analyses suggested that the distribution of environmental index values of a site should be considered in designing experiments. Overall, we showed that environmental context was critical, and considerations should be given to better cover the intended range of the environmental variable. Our findings have implications for the genetic architecture of complex traits, plant-environment interaction, and climate adaptation.

Genetic mapping of dynamic control of leaf angle across multiple canopy levels in maize.

Optimizing leaf angle and other canopy architecture traits has helped modern maize (Zea mays L.) become adapted to higher planting densities over the last 60 years. Traditional investigations into genetic control of leaf angle have focused on one leaf or the average of multiple leaves; as a result, our understanding of genetic control across multiple canopy levels is still limited. To address this, genetic mapping across four canopy levels was conducted in the present study to investigate the genetic control of leaf angle across the canopy. We developed two populations of doubled haploid lines derived from three inbreds with distinct leaf angle phenotypes. These populations were genotyped with genotyping-by-sequencing and phenotyped for leaf angle at four different canopy levels over multiple years. To understand how leaf angle changes across the canopy, the four measurements were used to derive three additional traits. Composite interval mapping was conducted with the leaf-specific measurements and the derived traits. A set of 59 quantitative trait loci (QTLs) were uncovered for seven traits, and two genomic regions were consistently detected across multiple canopy levels. Additionally, seven genomic regions were found to contain consistent QTLs with either relatively stable or dynamic effects at different canopy levels. Prioritizing the selection of QTLs with dynamic effects across the canopy will aid breeders in selecting maize hybrids with the ideal canopy architecture that continues to maximize yield on a per area basis under increasing planting densities.

Efficient degradation of aqueous dichloromethane by an enhanced microbial electrolysis cell: Degradation kinetics, microbial community and metabolic mechanisms.

Dichloromethane (DCM) has been listed as a toxic and harmful water pollutant, and its removal needs attention. Microbial electrolysis cells (MECs) are viewed as a promising alternative for pollutant removal, which can be strengthened from two aspects: microbial inoculation and acclimation. In this study, the MEC for DCM degradation was inoculated with the active sludge enhanced by Methylobacterium rhodesianum H13 (strain H13) and then acclimated in the form of a microbial fuel cell (MFC). Both the introduction of strain H13 and the initiation in MFC form significantly promoted DCM degradation. The degradation kinetics were fitted by the Haldane model, with Vmax, Kh, Ki and vmax values of 103.2 mg/L/hr, 97.8 mg/L, 268.3 mg/L and 44.7 mg/L/hr/cm2, respectively. The cyclic voltammogram implies that DCM redox reactions became easier with the setup of MEC, and the electrochemical impedance spectrogram shows that the acclimated and enriched microbes reduced the charge transfer resistance from the electrode to the electrolyte. In the biofilm, the dominant genera shifted from Geobacter to Hyphomicrobium in acclimation stages. Moreover, Methylobacterium played an increasingly important role. DCM metabolism mainly occurred through the hydrolytic glutathione S-transferase pathway, given that the gene dcmA was identified rather than the dhlA and P450/MO. The exogenous electrons facilitated the reduction of GSSG, directly or indirectly accelerating the GSH-catalyzed dehalogenation. This study provides support for the construction of an efficient and stable MEC for DCM removal in water environment.

Discovery of Tumor-Targeted 6-Methyl Substituted Pemetrexed and Related Antifolates with Selective Loss of RFC Transport.

Pemetrexed and related 5-substituted pyrrolo[2,3-d]pyrimidine antifolates are substrates for the ubiquitously expressed reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT) and folate receptors (FRs) which are more tumor-selective. A long-standing goal has been to discover tumor-targeted therapeutics that draw from one-carbon metabolic vulnerabilities of cancer cells and are selective for transport by FRs and PCFT over RFC. We discovered that a methyl group at the 6-position of the pyrrole ring in the bicyclic scaffold of 5-substituted 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidine antifolates 1-4 (including pemetrexed) abolished transport by RFC with modest impacts on FRs or PCFT. From molecular modeling, loss of RFC transport involves steric repulsion in the scaffold binding site due to the 6-methyl moiety. 6-Methyl substitution preserved antiproliferative activities toward human tumor cells (KB, IGROV3) with selectivity over IOSE 7576 normal ovary cells and inhibition of de novo purine biosynthesis. Thus, adding a 6-methyl moiety to 5-substituted pyrrolo[2,3-d]pyrimidine antifolates affords tumor transport selectivity while preserving antitumor efficacy.

Enhancement of bio-S0 recovery and revealing the inhibitory effect on microorganisms under high sulfide loading.

Biodesulfurization is a mature technology, but obtaining biosulfur (S0) that can be easily settled naturally is still a challenge. Increasing the sulfide load is one of the known methods to obtain better settling of S0. However, the inhibitory effect of high levels of sulfide on microbes has also not been well studied. We constructed a high loading sulfide (1.55-10.86 kg S/m3/d) biological removal system. 100% sulfide removal and 0.56-2.53 kg S/m3/d S0 (7.0 ± 0.09-16.4 ± 0.25 µm) recovery were achieved at loads of 1.55-7.75 kg S/m3/d. Under the same load, S0 in the reflux sedimentation tank, which produced larger S0 particles (24.2 ± 0.73-53.8 ± 0.70 µm), increased the natural settling capacity and 45% recovery. For high level sulfide inhibitory effect, we used metagenomics and metatranscriptomics analyses. The increased sulfide load significantly inhibited the expression of flavin cytochrome c sulfide dehydrogenase subunit B (fccB) (Decreased from 615 ± 75 to 30 ± 5 TPM). At this time sulfide quinone reductase (SQR) (324 ± 185-1197 ± 51 TPM) was mainly responsible for sulfide oxidation and S0 production. When the sulfide load reached 2800 mg S/L, the SQR (730 ± 100 TPM) was also suppressed. This resulted in the accumulation of sulfide, causing suppression of carbon sequestration genes (Decreased from 3437 ± 842 to 665 ± 175 TPM). Other inhibitory effects included inhibition of microbial respiration, production of reactive oxygen species, and DNA damage. More sulfide-oxidizing bacteria (SOB) and newly identified potential SOB (99.1%) showed some activity (77.6%) upon sulfide accumulation. The main microorganisms in the sulfide accumulation environment were Thiomicrospiracea and Burkholderiaceae, whose sulfide oxidation capacity and respiration were not significantly inhibited. This study provides a new approach to enhance the natural sedimentation of S0 and describes new microbial mechanisms for the inhibitory effects of sulfide.

Zero-valent iron-peroxydisulfate as synergistic co-milling agents for enhanced mechanochemical destruction of 2,4-dichlorophenol: Coupling reduction with oxidation.

Mechanochemical (MC) remediation with zero-valent iron (ZVI) as co-milling agent enables the non-combustion and solvent-free disposal of solid halogenated organic pollutants (HOPs) via solid-phase reaction, but suffers from incomplete dechlorination (especially for less chlorinated chemicals). Herein, a reduction-oxidation coupling strategy using ZVI and peroxydisulfate as synergistic (ZVI-PDS) co-milling agents was investigated, with 2,4-dichlorophenol (2,4-DCP) as probe contaminant. By revisiting the MC destruction process of 2,4-DCP by ZVI, the contribution of both reductive and oxidative routes is confirmed, and the inefficient •OH generation is addressed. With ball-to-material and reagent-to-pollutant mass ratios of 30:1 and 13:1, respectively, ZVI-PDS achieves higher dechlorination ratio (86.8%) for 2,4-DCP within 5 h, outcompeting sole ZVI (40.3%) or PDS (33.9%), due to the accumulation of numerous SO4•-. As suggested by a two-compartment kinetic model, the optimal ZVI/PDS molar ratio of 4:1 is determined, which balances the relative contribution of reductive/oxidative routes and leads to a maximum mineralization efficiency of 77.4%. The analysis on product distribution verifies the generation of dechlorinated, ring-opening and minor coupling products (with low acute toxicity). This work validates the necessity to couple reduction with oxidation in MC destruction for solid HOPs, and may provide information on reagent formulation.

A FadR-Type Regulator Activates the Biodegradation of Polycyclic Aromatic Hydrocarbons by Mediating Quorum Sensing in Croceicoccus naphthovorans Strain PQ-2.

Bacteria employ multiple transcriptional regulators to orchestrate cellular responses to adapt to constantly varying environments. The bacterial biodegradation of polycyclic aromatic hydrocarbons (PAHs) has been extensively described, and yet, the PAH-related transcriptional regulators remain elusive. In this report, we identified an FadR-type transcriptional regulator that controls phenanthrene biodegradation in Croceicoccus naphthovorans strain PQ-2. The expression of fadR in C. naphthovorans PQ-2 was induced by phenanthrene, and its deletion significantly impaired both the biodegradation of phenanthrene and the synthesis of acyl-homoserine lactones (AHLs). In the fadR deletion strain, the biodegradation of phenanthrene could be recovered by supplying either AHLs or fatty acids. Notably, FadR simultaneously activated the fatty acid biosynthesis pathway and repressed the fatty acid degradation pathway. As intracellular AHLs are synthesized with fatty acids as substrates, boosting the fatty acid supply could enhance AHL synthesis. Collectively, these findings demonstrate that FadR in C. naphthovorans PQ-2 positively regulates PAH biodegradation by controlling the formation of AHLs, which is mediated by the metabolism of fatty acids. IMPORTANCE Master transcriptional regulation of carbon catabolites is extremely important for the survival of bacteria that face changes in carbon sources. Polycyclic aromatic hydrocarbons (PAHs) can be utilized as carbon sources by some bacteria. FadR is a well-known transcriptional regulator involved in fatty acid metabolism; however, the connection between FadR regulation and PAH utilization in bacteria remains unknown. This study revealed that a FadR-type regulator in Croceicoccus naphthovorans PQ-2 stimulated PAH biodegradation by controlling the biosynthesis of the acyl-homoserine lactone quorum-sensing signals that belong to fatty acid-derived compounds. These results provide a unique perspective for understanding bacterial adaptation to PAH-containing environments.

Enhancement mechanism of magnetite on the ball-milling destruction of perfluorooctane sulfonate by iron.

Zero-valent iron (Fe) is commonly employed as an additive for the mechanochemical destruction (MCD) of organic pollutants. The poly- and perfluoroalkyl substances (e.g., perfluorooctane sulfonate, PFOS) are a class of toxic environmental pollutants that are difficult to effectively degrade due to their thermodynamic and chemical stability. In this study, magnetite (Fe3O4) was applied to improve the milling performance of Fe to PFOS and its promoting mechanisms were emphatically explored. The desulfurization rate was in ahead of the defluorination rate because the C-S bond is less stable than the C-F bonds in PFOS. Fe3O4 had an excellent reinforcement effect on the milling performance of Fe, which was mainly through accelerating the electron transfer as a conductor, reacting with Fe to produce FeO, and facilitating the formation of HO●. During the MCD of PFOS with Fe/Fe3O4 as an additive, HO● played a dominant role in the defluorination process (accounting for >67%). After the elimination of sulfonate group (-SO3-), the produced radical (C7F15CF2●) continued to react through two main pathways: one was the stepwise defluorination after hydrogenation, and the other one was oxidation reaction after alcoholization to yield the corresponding aldehydes and carboxylic acids. The optimum Fe fraction (MFe) was 30%, and air atmosphere was more effective than oxygen and nitrogen conditions. This study helps to comprehensively understand the role of Fe3O4 in defluorination and fills the gap of Fe/Fe3O4 application in the MCD of PFASs.

Genomic prediction of tocochromanols in exotic-derived maize.

Tocochromanols (vitamin E) are an essential part of the human diet. Plant products, including maize (Zea mays L.) grain, are the major dietary source of tocochromanols; therefore, breeding maize with higher vitamin content (biofortification) could improve human nutrition. Incorporating exotic germplasm in maize breeding for trait improvement including biofortification is a promising approach and an important research topic. However, information about genomic prediction of exotic-derived lines using available training data from adapted germplasm is limited. In this study, genomic prediction was systematically investigated for nine tocochromanol traits within both an adapted (Ames Diversity Panel [AP]) and an exotic-derived (Backcrossed Germplasm Enhancement of Maize [BGEM]) maize population. Although prediction accuracies up to 0.79 were achieved using genomic best linear unbiased prediction (gBLUP) when predicting within each population, genomic prediction of BGEM based on an AP training set resulted in low prediction accuracies. Optimal training population (OTP) design methods fast and unique representative subset selection (FURS), maximization of connectedness and diversity (MaxCD), and partitioning around medoids (PAM) were adapted for inbreds and, along with the methods mean coefficient of determination (CDmean) and mean prediction error variance (PEVmean), often improved prediction accuracies compared with random training sets of the same size. When applied to the combined population, OTP designs enabled successful prediction of the rest of the exotic-derived population. Our findings highlight the importance of leveraging genotype data in training set design to efficiently incorporate new exotic germplasm into a plant breeding program.

Leveraging prior biological knowledge improves prediction of tocochromanols in maize grain.

With an essential role in human health, tocochromanols are mostly obtained by consuming seed oils; however, the vitamin E content of the most abundant tocochromanols in maize (Zea mays L.) grain is low. Several large-effect genes with cis-acting variants affecting messenger RNA (mRNA) expression are mostly responsible for tocochromanol variation in maize grain, with other relevant associated quantitative trait loci (QTL) yet to be fully resolved. Leveraging existing genomic and transcriptomic information for maize inbreds could improve prediction when selecting for higher vitamin E content. Here, we first evaluated a multikernel genomic best linear unbiased prediction (MK-GBLUP) approach for modeling known QTL in the prediction of nine tocochromanol grain phenotypes (12-21 QTL per trait) within and between two panels of 1,462 and 242 maize inbred lines. On average, MK-GBLUP models improved predictive abilities by 7.0-13.6% when compared with GBLUP. In a second approach with a subset of 545 lines from the larger panel, the highest average improvement in predictive ability relative to GBLUP was achieved with a multi-trait GBLUP model (15.4%) that had a tocochromanol phenotype and transcript abundances in developing grain for a few large-effect candidate causal genes (1-3 genes per trait) as multiple response variables. Taken together, our study illustrates the enhancement of prediction models when informed by existing biological knowledge pertaining to QTL and candidate causal genes.

Regulating Acidosis and Relieving Hypoxia by Platelet Membrane-Coated Nanoparticle for Enhancing Tumor Chemotherapy.

Acidosis and hypoxia of tumor remain a great challenge for cancer therapy. Herein, we developed Hb-LOX-DOX-ZIF8@platelet membrane nanoparticles (H-L-D-Z@PM NPs) to address this problem. Lactate oxidase (LOX) could deplete intratumoral lactate adequately and amplify oxidative stress efficiently. In the meantime, hemoglobin (Hb) was intended to deliver oxygen, relieve hypoxia, and boost the catalytic activity of LOX. The coated PM bestowed active tumor-targeting ability and good biocompatibility to these nanoparticles. Moreover, the encapsulation of zeolitic imidazolate framework-8 (ZIF8) offered the acid response capacity to nanoparticles. With the synergism of chemotherapy drug doxorubicin (DOX), these H-L-D-Z@PM NPs appeared to have excellent antitumor competence. Collectively, this study offered a new strategy for enhancing tumor chemotherapy by regulating acidosis and relieving hypoxia.

Combining GWAS and TWAS to identify candidate causal genes for tocochromanol levels in maize grain.

Tocochromanols (tocopherols and tocotrienols, collectively vitamin E) are lipid-soluble antioxidants important for both plant fitness and human health. The main dietary sources of vitamin E are seed oils that often accumulate high levels of tocopherol isoforms with lower vitamin E activity. The tocochromanol biosynthetic pathway is conserved across plant species but an integrated view of the genes and mechanisms underlying natural variation of tocochromanol levels in seed of most cereal crops remains limited. To address this issue, we utilized the high mapping resolution of the maize Ames panel of ∼1,500 inbred lines scored with 12.2 million single-nucleotide polymorphisms to generate metabolomic (mature grain tocochromanols) and transcriptomic (developing grain) data sets for genetic mapping. By combining results from genome- and transcriptome-wide association studies, we identified a total of 13 candidate causal gene loci, including 5 that had not been previously associated with maize grain tocochromanols: 4 biosynthetic genes (arodeH2 paralog, dxs1, vte5, and vte7) and a plastid S-adenosyl methionine transporter (samt1). Expression quantitative trait locus (eQTL) mapping of these 13 gene loci revealed that they are predominantly regulated by cis-eQTL. Through a joint statistical analysis, we implicated cis-acting variants as responsible for colocalized eQTL and GWAS association signals. Our multiomics approach provided increased statistical power and mapping resolution to enable a detailed characterization of the genetic and regulatory architecture underlying tocochromanol accumulation in maize grain and provided insights for ongoing biofortification efforts to breed and/or engineer vitamin E and antioxidant levels in maize and other cereals.

Interpretation of Manhattan Plots and Other Outputs of Genome-Wide Association Studies.

With increasing marker density, estimation of recombination rate between a marker and a causal mutation using linkage analysis becomes less important. Instead, linkage disequilibrium (LD) becomes the major indicator for gene mapping through genome-wide association studies (GWAS). In addition to the linkage between the marker and the causal mutation, many other factors may contribute to the LD, including population structure and cryptic relationships among individuals. As statistical methods and software evolve to improve statistical power and computing speed in GWAS, the corresponding outputs must also evolve to facilitate the interpretation of input data, the analytical process, and final association results. In this chapter, our descriptions focus on (1) considerations in creating a Manhattan plot displaying the strength of LD and locations of markers across a genome; (2) criteria for genome-wide significance threshold and the different appearance of Manhattan plots in single-locus and multiple-locus models; (3) exploration of population structure and kinship among individuals; (4) quantile-quantile (QQ) plot; (5) LD decay across the genome and LD between the associated markers and their neighbors; (6) exploration of individual and marker information on Manhattan and QQ plots via interactive visualization using HTML. The ultimate objective of this chapter is to help users to connect input data to GWAS outputs to balance power and false positives, and connect GWAS outputs to the selection of candidate genes using LD extent.