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Global nighttime temperatures are rising at twice the rate of daytime temperatures and pose a challenge for rice (Oryza sativa) production. High nighttime temperature (HNT) stress affects rice yield by reducing grain weight, size, and fertility. Although the genes associated with these yield parameters have been identified and characterized under normal temperatures, the genetic basis of grain weight regulation under HNT stress remains less explored. We examined the natural variation for rice single grain weight (SGW) under HNT stress imposed during grain development. A genome-wide association analysis identified several loci associated with grain weight under HNT stress. A locus, SGW1, specific to HNT conditions resolved to LONELY GUY-Like 1 (LOGL1), which encodes a putative cytokinin-activation enzyme. We demonstrated that LOGL1 contributes to allelic variation at SGW1. Accessions with lower LOGL1 transcript abundance had higher grain weight under HNT. This was supported by the higher grain weight of logl1-mutants relative to the wild type under HNT. Compared to logl1-mutants, LOGL1 over-expressers showed increased sensitivity to HNT. We showed that LOGL1 regulates the thiamin biosynthesis pathway, which is under circadian regulation, which in turn is likely perturbed by HNT stress. These findings provide a genetic source to enhance rice adaptation to warming night temperatures and improve our mechanistic understanding of HNT stress tolerance pathways.
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Grão Comestível , Oryza , Proteínas de Plantas , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/fisiologia , Estudo de Associação Genômica Ampla , Regulação da Expressão Gênica de Plantas , Variação GenéticaRESUMO
Herein, we report that readily accessible azoxy-triazenes can serve as nitrogen atom sources under visible light excitation for the phthalimido-protected aziridination of alkenes. This approach eliminates the need for external oxidants, precious transition metals, and photocatalysts, marking a departure from conventional methods. The versatility of this transformation extends to the selective aziridination of both activated and unactivated multisubstituted alkenes of varying electronic profiles. Notably, this process avoids the formation of competing C-H insertion products. The described protocol is operationally simple, scalable, and adaptable to photoflow conditions. Mechanistic studies support the idea that the photofragmentation of azoxy-triazenes results in the generation of a free singlet nitrene. Furthermore, a mild photoredox-catalyzed N-N cleavage of the protecting group to furnish the free aziridines is reported. Our findings contribute to the advancement of sustainable and practical methodologies for the synthesis of nitrogen-containing compounds, showcasing the potential for broader applications in synthetic chemistry.
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KEY MESSAGE: Estimating genetic gains and formulating a future salinity elite breeding panel for rice pave the way for developing better high-yielding salinity tolerant lines with enhanced genetic gains. Genetic gain is a crucial parameter to check the breeding program's success and help optimize future breeding strategies for enhanced genetic gains. To estimate the genetic gains in IRRI's salinity breeding program and identify the best genotypes based on high breeding values for grain yield (kg/ha), we analyzed the historical data from the trials conducted in the IRRI, Philippines and Bangladesh. A two-stage mixed-model approach accounting for experimental design factors and a relationship matrix was fitted to obtain the breeding values for grain yield and estimate genetic trends. A positive genetic trend of 0.1% per annum with a yield advantage of 1.52 kg/ha was observed in IRRI, Philippines. In Bangladesh, we observed a genetic gain of 0.31% per annum with a yield advantage of 14.02 kg/ha. In the released varieties, we observed a genetic gain of 0.12% per annum with a 2.2 kg/ha/year yield advantage in the IRRI, Philippines. For the Bangladesh dataset, a genetic gain of 0.14% per annum with a yield advantage of 5.9 kg/ha/year was observed in the released varieties. Based on breeding values for grain yield, a core set of the top 145 genotypes with higher breeding values of > 2400 kg/ha in the IRRI, Philippines, and > 3500 kg/ha in Bangladesh with a reliability of > 0.4 were selected to develop the elite breeding panel. Conclusively, a recurrent selection breeding strategy integrated with novel technologies like genomic selection and speed breeding is highly required to achieve higher genetic gains in IRRI's salinity breeding programs.
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Oryza , Oryza/genética , Reprodutibilidade dos Testes , Salinidade , Melhoramento Vegetal , Bangladesh , Grão ComestívelRESUMO
Herein we report the anaerobic cleavage of alkenes into carbonyl compounds using nitroarenes as oxygen transfer reagents under visible light. This approach serves as a safe and practical alternative to mainstream oxidative cleavage protocols, such as ozonolysis and the Lemieux-Johnson reaction. A wide range of alkenes possessing oxidatively sensitive functionalities underwent anaerobic cleavage to generate carbonyl derivatives with high efficiency and regioselectivity. Mechanistic studies support that the transformation occurs via direct photoexcitation of the nitroarene followed by a nonstereospecific radical cycloaddition event with alkenes. This leads to 1,3,2- and 1,4,2-dioxazolidine intermediates that fragment to give the carbonyl products. A combination of radical clock experiments and in situ photoNMR spectroscopy revealed the identities of the key radical species and the putative aryl dioxazolidine intermediates, respectively.
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Alcenos , Oxigênio , Alcenos/química , Anaerobiose , Reação de Cicloadição , LuzRESUMO
A higher minimum (night-time) temperature is considered a greater limiting factor for reduced rice yield than a similar increase in maximum (daytime) temperature. While the physiological impact of high night temperature (HNT) has been studied, the genetic and molecular basis of HNT stress response remains unexplored. We examined the phenotypic variation for mature grain size (length and width) in a diverse set of rice accessions under HNT stress. Genome-wide association analysis identified several HNT-specific loci regulating grain size as well as loci that are common for optimal and HNT stress conditions. A novel locus contributing to grain width under HNT conditions colocalized with Fie1, a component of the FIS-PRC2 complex. Our results suggest that the allelic difference controlling grain width under HNT is a result of differential transcript-level response of Fie1 in grains developing under HNT stress. We present evidence to support the role of Fie1 in grain size regulation by testing overexpression (OE) and knockout mutants under heat stress. The OE mutants were either unaltered or had a positive impact on mature grain size under HNT, while the knockouts exhibited significant grain size reduction under these conditions.
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Oryza , Grão Comestível/genética , Endosperma/genética , Fertilização , Estudo de Associação Genômica Ampla , Oryza/genética , TemperaturaRESUMO
We report the synthesis of a new class of cyclopenta-fused polyaromatic hydrocarbon (CP-PAH) incorporating fused benzodithiophene subunits. These CP-PAHs were prepared utilizing a two-step process involving a palladium catalyzed cyclopentannulation followed by a Scholl cyclodehydrogenation. This work broadens the scope of annulation chemistry by employing 1,2-bis(5-hexylthiophen-3-yl)ethyne and dibromoaryl derivatives based on anthracene, pyrene, and perylene to give 4,4',4'',4'''-(cyclopenta[hi]aceanthrylene-1,2,6,7-tetrayl)tetrakis(2-hexylthiophene), 4,4',4'',4'''-(dicyclopenta[cd,jk]pyrene-1,2,6,7-tetrayl)tetrakis(2-hexylthiophene), and 1,2,7,8-tetrakis(5-hexylthiophen-3-yl)-1,2,7,8-tetrahydrodicyclopenta[cd,lm]perylene. Scholl cyclodehydrogenation of the pendant thiophene units provided access to the π-extended polyaromatic systems 2,5,11,14-tetrahexylrubiceno[5,4-b:6,7-b':12,11-b'':13,14-b''']tetrathiophene, 2,5,11,14-tetrahexyldithieno-[4,5:6,7]indeno[1,2,3-cd]dithieno[4,5:6,7]indeno-[1,2,3-jk]pyrenes, and 2,9,12,19-tetrahexyldithieno[4,5:6,7]indaceno[1,2,3-cd]dithieno[4,5:6,7]indaceno[1,2,3-lm]perylene that possess helicene-like fragments. The anthracene-based CP-PAH was contorted owing to [5]helicene-like arrangements, while the pyrene- and perylene-based systems were essentially planar. The fully conjugated small molecules give low optical gaps (1.7-2.1 eV) with broad light absorption. The HOMO and LUMO energies of the CP-PAHs were found to be in the range of -5.48 to -5.05 eV and -3.48 to -3.14 eV, respectively. Finally, the anthracene-based CP-PAH was found to be a p-type semiconductor when tested in an organic field effect transistor.
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Oxidative cleavage of alkenes leading to valuable carbonyl derivatives is a fundamental transformation in synthetic chemistry. In particular, ozonolysis is the mainstream method for the oxidative cleavage of alkenes that has been widely implemented in the synthesis of natural products and pharmaceutically relevant compounds. However, due to the toxicity and explosive nature of ozone, alternative approaches employing transition metals and enzymes in the presence of oxygen and/or strong oxidants have been developed. These protocols are often conducted under harsh reaction conditions that limit the substrate scope. Photochemical approaches can provide milder and more practical alternatives for this synthetically useful transformation. In this review, we outline recent visible-light-promoted oxidative cleavage reactions that involve photocatalytic activation of oxygen via electron transfer and energy transfer. Also, an emerging field featuring visible-light-promoted oxidative cleavage under anaerobic conditions is discussed. The methods highlighted in this review represent a transformative step toward more sustainable and efficient strategies for the oxidative cleavage of alkenes.
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The composite fermion theory opened a new chapter in understanding many-body correlations through the formation of emergent particles. The formation of two-flux and four-flux composite fermions is well established. While there are limited data linked to the formation of six-flux composite fermions, topological protection associated with them is conspicuously lacking. Here we report evidence for the formation of a quantized and gapped fractional quantum Hall state at the filling factor ν = 9/11, which we associate with the formation of six-flux composite fermions. Our result provides evidence for the most intricate composite fermion with six fluxes and expands the already diverse family of highly correlated topological phases with a new member that cannot be characterized by correlations present in other known members. Our observations pave the way towards the study of higher order correlations in the fractional quantum Hall regime.
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Herein, we report a protocol for the anaerobic oxidation of alcohols, amines, aldehydes, and imines promoted by photoexcited nitroarenes. Mechanistic studies support the idea that photoexcited nitroarenes undergo double hydrogen atom transfer (HAT) steps with alcohols and amines to provide the respective ketone and imine products. In the presence of aldehydes and imines, successive HAT and oxygen atom transfer (OAT) events occur to yield carboxylic acids and amides, respectively. This transformation is amenable to a continuous-photoflow setup, which led to reduced reaction times.
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The productivity of rice is greatly affected by the infection of the plant pathogenic fungus Rhizoctonia solani, which causes a significant grain yield reduction globally. There exist a limited number of rice accessions that are available to develop sheath blight resistance (ShB). Our objective was to identify a good source of the ShB resistance, understand the heritability, and trait interactions, and identify the genomic regions for ShB resistance traits by genome-wide association studies (GWAS). In the present study, a set of 330 traditional landraces and improved rice varieties were evaluated for ShB resistance and created a core panel of 192 accessions used in the GWAS. This panel provides a more considerable amount of genetic variance and found a significant phenotypic variation among the panel of rice accessions for all the agro-morphological and disease-resistance traits over the seasons. The infection rate of ShB and disease reaction were calculated as percent disease index (PDI) and area under the disease progress curve (AUDPC). The correlation analysis showed a significant positive association between PDIs and AUPDC and a negative association between PDI and plant height, flag leaf length, and grain yield. The panel was genotyped with 133 SSR microsatellite markers, resulting in a genome coverage of 314.83 Mb, and the average distance between markers is 2.53 Mb. By employing GLM and MLM (Q + K) models, 30 marker-trait associations (MTAs) were identified with targeted traits over the seasons. Among these QTLs, eight were found to be novel and located on 2, 4, 8, 10, and 12 chromosomes, which explained the phenotypic variation ranging from 5% to 15%. With the GWAS approach, six candidate genes were identified. Os05t0566400, Os08t0155900, and Os09t0567300 were found to be associated with defense mechanisms against ShB. These findings provided insights into the novel donors of IC283139, IC 277248, Sivappuchithirai Kar, and Bowalia. The promising genomic regions on 10 of 12 chromosomes associated with ShB would be useful in developing rice varieties with durable disease resistance.
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BACKGROUND: Developing a systematic phenotypic data analysis pipeline, creating enhanced visualizations, and interpreting the results is crucial to extract meaningful insights from data in making better breeding decisions. Here, we provide an overview of how the Rainfed Rice Breeding (RRB) program at IRRI has leveraged R computational power with open-source resource tools like R Markdown, plotly, LaTeX, and HTML to develop an open-source and end-to-end data analysis workflow and pipeline, and re-designed it to a reproducible document for better interpretations, visualizations and easy sharing with collaborators. RESULTS: We reported the state-of-the-art implementation of the phenotypic data analysis pipeline and workflow embedded into a well-descriptive document. The developed analytical pipeline is open-source, demonstrating how to analyze the phenotypic data in crop breeding programs with step-by-step instructions. The analysis pipeline shows how to pre-process and check the quality of phenotypic data, perform robust data analysis using modern statistical tools and approaches, and convert it into a reproducible document. Explanatory text with R codes, outputs either in text, tables, or graphics, and interpretation of results are integrated into the unified document. The analysis is highly reproducible and can be regenerated at any time. The analytical pipeline source codes and demo data are available at https://github.com/whussain2/Analysis-pipeline . CONCLUSION: The analysis workflow and document presented are not limited to IRRI's RRB program but are applicable to any organization or institute with full-fledged breeding programs. We believe this is a great initiative to modernize the data analysis of IRRI's RRB program. Further, this pipeline can be easily implemented by plant breeders or researchers, helping and guiding them in analyzing the breeding trials data in the best possible way.
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Prediction models based on pedigree and/or molecular marker information are now an inextricable part of the crop breeding programs and have led to increased genetic gains in many crops. Optimization of IRRI's rice drought breeding program is crucial for better implementation of selections based on predictions. Historical datasets with precise and robust pedigree information have been a great resource to help optimize the prediction models in the breeding programs. Here, we leveraged 17 years of historical drought data along with the pedigree information to predict the new lines or environments and dissect the G × E interactions. Seven models ranging from basic to proposed higher advanced models incorporating interactions, and genotypic specific effects were used. These models were tested with three cross-validation schemes (CV1, CV2, and CV0) to assess the predictive ability of tested and untested lines in already observed environments and tested lines in novel or new environments. In general, the highest prediction abilities were obtained when the model accounting interactions between pedigrees (additive) and environment were included. The CV0 scheme (predicting unobserved or novel environments) reveals very low predictive abilities among the three schemes. CV1 and CV2 schemes that borrow information from the target and correlated environments have much higher predictive abilities. Further, predictive ability was lower when predicting lines in non-stress conditions using drought data as training set and/or vice-versa. When predicting the lines using the data sets under the same conditions (stress or non-stress data sets), much better prediction accuracy was obtained. These results provide conclusive evidence that modeling G × E interactions are important in predictions. Thus, considering G × E interactions would help to build enhanced genomic or pedigree-based prediction models in the rice breeding program. Further, it is crucial to borrow the correlated information from other environments to improve prediction accuracy.
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Heat stress occurring during rice (Oryza sativa) grain development reduces grain quality, which often manifests as increased grain chalkiness. Although the impact of heat stress on grain yield is well-studied, the genetic basis of rice grain quality under heat stress is less explored as quantifying grain quality is less tractable than grain yield. To address this, we used an image-based colorimetric assay (Red, R; and Green, G) for genome-wide association analysis to identify genetic loci underlying the phenotypic variation in rice grains exposed to heat stress. We found the R to G pixel ratio (RG) derived from mature grain images to be effective in distinguishing chalky grains from translucent grains derived from control (28/24°C) and heat stressed (36/32°C) plants. Our analysis yielded a novel gene, rice Chalky Grain 5 (OsCG5) that regulates natural variation for grain chalkiness under heat stress. OsCG5 encodes a grain-specific, expressed protein of unknown function. Accessions with lower transcript abundance of OsCG5 exhibit higher chalkiness, which correlates with higher RG values under stress. These findings are supported by increased chalkiness of OsCG5 knock-out (KO) mutants relative to wildtype (WT) under heat stress. Grains from plants overexpressing OsCG5 are less chalky than KOs but comparable to WT under heat stress. Compared to WT and OE, KO mutants exhibit greater heat sensitivity for grain size and weight relative to controls. Collectively, these results show that the natural variation at OsCG5 may contribute towards rice grain quality under heat stress.
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Estimating genetic trends using historical data is an important parameter to check the success of the breeding programs. The estimated genetic trends can act as a guideline to target the appropriate breeding strategies and optimize the breeding program for improved genetic gains. In this study, 17 years of historical data from IRRI's rice drought breeding program was used to estimate the genetic trends and assess the breeding program's success. We also identified top-performing lines based on grain yield breeding values as an elite panel for implementing future population improvement-based breeding schemes. A two-stage approach of pedigree-based mixed model analysis was used to analyze the data and extract the breeding values and estimate the genetic trends for grain yield under non-stress, drought, and in combined data of non-stress and drought. Lower grain yield values were observed in all the drought trials. Heritability for grain yield estimates ranged between 0.20 and 0.94 under the drought trials and 0.43-0.83 under non-stress trials. Under non-stress conditions, the genetic gain of 0.21% (10.22 kg/ha/year) for genotypes and 0.17% (7.90 kg/ha/year) for checks was observed. The genetic trend under drought conditions exhibited a positive trend with the genetic gain of 0.13% (2.29 kg/ha/year) for genotypes and 0.55% (9.52 kg/ha/year) for checks. For combined analysis showed a genetic gain of 0.27% (8.32 kg/ha/year) for genotypes and 0.60% (13.69 kg/ha/year) for checks was observed. For elite panel selection, 200 promising lines were selected based on higher breeding values for grain yield and prediction accuracy of > 0.40. The breeding values of the 200 genotypes formulating the core panel ranged between 2366.17 and 4622.59 (kg/ha). A positive genetic rate was observed under all the three conditions; however, the rate of increase was lower than the required rate of 1.5% genetic gain. We propose a recurrent selection breeding strategy within the elite population with the integration of modern tools and technologies to boost the genetic gains in IRRI's drought breeding program. The elite breeding panel identified in this study forms an easily available and highly enriched genetic resource for future recurrent selection programs to boost the genetic gains.
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Inferring trait networks from a large volume of genetically correlated diverse phenotypes such as yield, architecture, and disease resistance can provide information on the manner in which complex phenotypes are interrelated. However, studies on statistical methods tailored to multidimensional phenotypes are limited, whereas numerous methods are available for evaluating the massive number of genetic markers. Factor analysis operates at the level of latent variables predicted to generate observed responses. The objectives of this study were to illustrate the manner in which data-driven exploratory factor analysis can map observed phenotypes into a smaller number of latent variables and infer a genomic latent factor network using 45 agro-morphological, disease, and grain mineral phenotypes measured in synthetic hexaploid wheat lines (Triticum aestivum L.). In total, eight latent factors including grain yield, architecture, flag leaf-related traits, grain minerals, yellow rust, two types of stem rust, and leaf rust were identified as common sources of the observed phenotypes. The genetic component of the factor scores for each latent variable was fed into a Bayesian network to obtain a trait structure reflecting the genetic interdependency among traits. Three directed paths were consistently identified by two Bayesian network algorithms. Flag leaf-related traits influenced leaf rust, and yellow rust and stem rust influenced grain yield. Additional paths that were identified included flag leaf-related traits to minerals and minerals to architecture. This study shows that data-driven exploratory factor analysis can reveal smaller dimensional common latent phenotypes that are likely to give rise to numerous observed field phenotypes without relying on prior biological knowledge. The inferred genomic latent factor structure from the Bayesian network provides insights for plant breeding to simultaneously improve multiple traits, as an intervention on one trait will affect the values of focal phenotypes in an interrelated complex trait system.
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Rice genetic improvement is a key component of achieving and maintaining food security in Asia and Africa in the face of growing populations and climate change. In this effort, the International Rice Research Institute (IRRI) continues to play a critical role in creating and disseminating rice varieties with higher productivity. Due to increasing demand for rice, especially in Africa, there is a strong need to accelerate the rate of genetic improvement for grain yield. In an effort to identify and characterize the elite breeding pool of IRRI's irrigated rice breeding program, we analyzed 102 historical yield trials conducted in the Philippines during the period 2012-2016 and representing 15,286 breeding lines (including released varieties). A mixed model approach based on the pedigree relationship matrix was used to estimate breeding values for grain yield, which ranged from 2.12 to 6.27 t·ha-1. The rate of genetic gain for grain yield was estimated at 8.75 kg·ha-1 year-1 (0.23%) for crosses made in the period from 1964 to 2014. Reducing the data to only IRRI released varieties, the rate doubled to 17.36 kg·ha-1 year-1 (0.46%). Regressed against breeding cycle the rate of gain for grain yield was 185 kg·ha-1 cycle-1 (4.95%). We selected 72 top performing lines based on breeding values for grain yield to create an elite core panel (ECP) representing the genetic diversity in the breeding program with the highest heritable yield values from which new products can be derived. The ECP closely aligns with the indica 1B sub-group of Oryza sativa that includes most modern varieties for irrigated systems. Agronomic performance of the ECP under multiple environments in Asia and Africa confirmed its high yield potential. We found that the rate of genetic gain for grain yield found in this study was limited primarily by long cycle times and the direct introduction of non-improved material into the elite pool. Consequently, the current breeding scheme for irrigated rice at IRRI is based on rapid recurrent selection among highly elite lines. In this context, the ECP constitutes an important resource for IRRI and NAREs breeders to carefully characterize and manage that elite diversity.
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New conjugated polymers that incorporate dihexylanthradithiophene (DHADT) in the main chain were prepared by Stille, Sonogashira, and Yamamoto cross-coupling polymerization reactions. The polymerization chemistry is enabled by a soluble 5,11-dibromodihexylanthradithiophene monomer that is capable of cross-coupling reactions. Five readily soluble DHADT containing polymers were prepared and characterized experimentally and computationally. These polymers possess HOMO energies of -5.18 eV to -5.43 eV and LUMO energies of -3.0 eV to -2.82 eV. The notable optical features include broad absorption and band gaps ranging from 1.62 eV to 2.15 eV. Polymers were tested in organic field effect transistors and were found to operate in the p-type regime.
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Genome-wide association mapping identifies quantitative trait loci (QTL) that influence the mean differences between the marker genotypes for a given trait. While most loci influence the mean value of a trait, certain loci, known as variance heterogeneity QTL (vQTL) determine the variability of the trait instead of the mean trait value (mQTL). In the present study, we performed a variance heterogeneity genome-wide association study (vGWAS) for grain cadmium (Cd) concentration in bread wheat. We used double generalized linear model and hierarchical generalized linear model to identify vQTL associated with grain Cd. We identified novel vQTL regions on chromosomes 2A and 2B that contribute to the Cd variation and loci that affect both mean and variance heterogeneity (mvQTL) on chromosome 5A. In addition, our results demonstrated the presence of epistatic interactions between vQTL and mvQTL, which could explain variance heterogeneity. Overall, we provide novel insights into the genetic architecture of grain Cd concentration and report the first application of vGWAS in wheat. Moreover, our findings indicated that epistasis is an important mechanism underlying natural variation for grain Cd concentration.
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Pão , Triticum , Cádmio , Estudo de Associação Genômica Ampla , Genômica , Triticum/genéticaRESUMO
Increasing global surface temperatures is posing a major food security challenge. Part of the solution to address this problem is to improve crop heat resilience, especially during grain development, along with agronomic decisions such as shift in planting time and increasing crop diversification. Rice is a major food crop consumed by more than 3 billion people. For rice, thermal sensitivity of reproductive development and grain filling is well-documented, while knowledge concerning the impact of heat stress (HS) on early seed development is limited. Here, we aim to study the phenotypic variation in a set of diverse rice accessions for elucidating the HS response during early seed development. To explore the variation in HS sensitivity, we investigated aus (1), indica (2), temperate japonica (2), and tropical japonica (4) accessions for their HS (39/35°C) response during early seed development that accounts for transition of endosperm from syncytial to cellularization, which broadly corresponds to 24 and 96 hr after fertilization (HAF), respectively, in rice. The two indica and one of the tropical japonica accessions exhibited severe heat sensitivity with increased seed abortion; three tropical japonicas and an aus accession showed moderate heat tolerance, while temperate japonicas exhibited strong heat tolerance. The accessions exhibiting extreme heat sensitivity maintain seed size at the expense of number of fully developed mature seeds, while the accessions showing relative resilience to the transient HS maintained number of fully developed seeds but compromised on seed size, especially seed length. Further, histochemical analysis revealed that all the tested accessions have delayed endosperm cellularization upon exposure to the transient HS by 96 HAF; however, the rate of cellularization was different among the accessions. These findings were further corroborated by upregulation of cellularization-associated marker genes in the developing seeds from the heat-stressed samples.