Natural Genetic Variation Research Articles

Characterization of sequence determinants of enhancer function using natural genetic variation.

Sequence variation in enhancers that control cell-type-specific gene transcription contributes significantly to phenotypic variation within human populations. However, it remains difficult to predict precisely the effect of any given sequence variant on enhancer function due to the complexity of DNA sequence motifs that determine transcription factor (TF) binding to enhancers in their native genomic context. Using F1-hybrid cells derived from crosses between distantly related inbred strains of mice, we identified thousands of enhancers with allele-specific TF binding and/or activity. We find that genetic variants located within the central region of enhancers are most likely to alter TF binding and enhancer activity. We observe that the AP-1 family of TFs (Fos/Jun) are frequently required for binding of TEAD TFs and for enhancer function. However, many sequence variants outside of core motifs for AP-1 and TEAD also impact enhancer function, including sequences flanking core TF motifs and AP-1 half sites. Taken together, these data represent one of the most comprehensive assessments of allele-specific TF binding and enhancer function to date and reveal how sequence changes at enhancers alter their function across evolutionary timescales.

Antinutritional factors, nutritional improvement, and future food use of common beans: A perspective.

Common bean seeds are an excellent source of protein as well as of carbohydrates, minerals, vitamins, and bioactive compounds reducing, when in the diet, the risks of diseases. The presence of bioactive compounds with antinutritional properties (e.g., phytic acid, lectins, raffinosaccharides, protease inhibitors) limits, however, the bean’s nutritional value and its wider use in food preparations. In the last decades, concerted efforts have been, therefore, made to develop new common bean genotypes with reduced antinutritional compounds by exploiting the natural genetic variability of common bean and also applying induced mutagenesis. However, possible negative, or positive, pleiotropic effects due to these modifications, in terms of plant performance in response to stresses or in the resulting technological properties of the developed mutant genotypes, have yet not been thoroughly investigated. The purpose of the perspective paper is to first highlight the current advances, which have been already made in mutant bean characterization. A view will be further provided on future research directions to specifically explore further advantages and disadvantages of these bean mutants, their potential use in innovative foods and representing a valuable genetic reservoir of combinations to assess the true functional role of specific seed bioactive components directly in the food matrix.

Quantification of noradrenergic-, dopaminergic-, and tectal-neurons during aging in the short-lived killifish Nothobranchius furzeri.

Parkinson's disease (PD) is characterized by phosphorylation and aggregation of the protein α‐Synuclein and ensuing neuronal death progressing from the noradrenergic locus coeruleus to midbrain dopaminergic neurons. In 2019, Matsui and colleagues reported a spontaneous age‐dependent degeneration of dopaminergic neurons and an even greater neurodegeneration of the noradrenergic neurons in the short‐lived killifish Nothobranchius furzeri. Given the great possible relevance of a spontaneous model for PD, we assessed neurodegeneration of noradrenergic and dopaminergic neurons in two further laboratory strains of N. furzeri. We implemented, for the first time in N. furzeri, a whole‐brain clarification technique and proceeded to entire 3D nuclei reconstruction to quantify total cell numbers in two different stains of N. furzeri. In both strains, we observed that age‐dependent neurodegeneration is limited to the locus coeruleus and does not involve the posterior tuberculum. We also applied 3D counting to the optic tectum, an area of active adult neurogenesis, and detected an increase of neurons with age. Our results confirm age‐dependent neurodegeneration of noradrenergic neurons, a condition reminiscent of the presymptomatic stage of PD indicating that N. furzeri could be used in the future to identify modifying factors for age‐dependent neurodegeneration and open the intriguing possibility that natural genetic variation may influence the susceptibility of dopaminergic neurons.

Sample-multiplexing approaches for single-cell sequencing.

Single-cell sequencing is widely used in biological and medical studies. However, its application with multiple samples is hindered by inefficient sample processing, high experimental costs, ambiguous identification of true single cells, and technical batch effects. Here, we introduce sample-multiplexing approaches for single-cell sequencing in transcriptomics, epigenomics, genomics, and multiomics. In single-cell transcriptomics, sample multiplexing uses variants of native or artificial features as sample markers, enabling sample pooling and decoding. Such features include: (1) natural genetic variation, (2) nucleotide-barcode anchoring on cellular or nuclear membranes, (3) nucleotide-barcode internalization to the cytoplasm or nucleus, (4) vector-based barcode expression in cells, and (5) nucleotide-barcode incorporation during library construction. Other single-cell omics methods are based on similar concepts, particularly single-cell combinatorial indexing. These methods overcome current challenges, while enabling super-loading of single cells. Finally, selection guidelines are presented that can accelerate technological application.

A theory of resistance to multiplexed gene drive demonstrates the significant role of weakly deleterious natural genetic variation

Evolution of resistance is a major barrier to successful deployment of gene-drive systems to suppress natural populations, which could greatly reduce the burden of many vector-borne diseases. Multiplexed guide RNAs (gRNAs) that require resistance mutations in all target cut sites are a promising antiresistance strategy since, in principle, resistance would only arise in unrealistically large populations. Using stochastic simulations that accurately model evolution at very large population sizes, we explore the probability of resistance due to three important mechanisms: 1) nonhomologous end-joining mutations, 2) single-nucleotide mutants arising de novo, or 3) single-nucleotide polymorphisms preexisting as standing variation. Our results explore the relative importance of these mechanisms and highlight a complexity of the mutation-selection-drift balance between haplotypes with complete resistance and those with an incomplete number of resistant alleles. We find that this leads to a phenomenon where weakly deleterious naturally occurring variants greatly amplify the probability of multisite resistance compared to de novo mutation. This key result provides design criterion for antiresistance multiplexed systems, which, in general, will need a larger number of gRNAs compared to de novo expectations. This theory may have wider application to the evolution of resistance or evolutionary rescue when multiple changes are required before selection can act.

Association analyses of host genetics, root-colonizing microbes, and plant phenotypes under different nitrogen conditions in maize.

The root-associated microbiome (rhizobiome) affects plant health, stress tolerance, and nutrient use efficiency. However, it remains unclear to what extent the composition of the rhizobiome is governed by intraspecific variation in host plant genetics in the field and the degree to which host plant selection can reshape the composition of the rhizobiome. Here, we quantify the rhizosphere microbial communities associated with a replicated diversity panel of 230 maize (Zea mays L.) genotypes grown in agronomically relevant conditions under high N (+N) and low N (-N) treatments. We analyze the maize rhizobiome in terms of 150 abundant and consistently reproducible microbial groups and we show that the abundance of many root-associated microbes is explainable by natural genetic variation in the host plant, with a greater proportion of microbial variance attributable to plant genetic variation in -N conditions. Population genetic approaches identify signatures of purifying selection in the maize genome associated with the abundance of several groups of microbes in the maize rhizobiome. Genome-wide association study was conducted using the abundance of microbial groups as rhizobiome traits, and n=622 plant loci were identified that are linked to the abundance of n=104 microbial groups in the maize rhizosphere. In 62/104 cases, which is more than expected by chance, the abundance of these same microbial groups was correlated with variation in plant vigor indicators derived from high throughput phenotyping of the same field experiment. We provide comprehensive datasets about the three-way interaction of host genetics, microbe abundance, and plant performance under two N treatments to facilitate targeted experiments toward harnessing the full potential of root-associated microbial symbionts in maize production.

Improving bread wheat yield through modulating an unselected AP2/ERF gene.

Crop breeding heavily relies on natural genetic variation. However, additional new variations are desired to meet the increasing human demand. Inflorescence architecture determines grain number per spike, a major determinant of bread wheat (Triticum aestivum L.) yield. Here, using Brachypodium distachyon as a wheat proxy, we identified DUO-B1, encoding an APETALA2/ethylene response factor (AP2/ERF) transcription factor, regulating spike inflorescence architecture in bread wheat. Mutations of DUO-B1 lead to mild supernumerary spikelets, increased grain number per spike and, importantly, increased yield under field conditions without affecting other major agronomic traits. DUO-B1 suppresses cell division and promotes the expression of BHt/WFZP, whose mutations could lead to branched 'miracle-wheat'. Pan-genome analysis indicated that DUO-B1 has not been utilized in breeding, and holds promise to increase wheat yield further.

Wheat Biofortification: Utilizing Natural Genetic Diversity, Genome-Wide Association Mapping, Genomic Selection, and Genome Editing Technologies.

Alleviating micronutrients associated problems in children below five years and women of childbearing age, remains a significant challenge, especially in resource-poor nations. One of the most important staple food crops, wheat attracts the highest global research priority for micronutrient (Fe, Zn, Se, and Ca) biofortification. Wild relatives and cultivated species of wheat possess significant natural genetic variability for these micronutrients, which has successfully been utilized for breeding micronutrient dense wheat varieties. This has enabled the release of 40 biofortified wheat cultivars for commercial cultivation in different countries, including India, Bangladesh, Pakistan, Bolivia, Mexico and Nepal. In this review, we have systematically analyzed the current understanding of availability and utilization of natural genetic variations for grain micronutrients among cultivated and wild relatives, QTLs/genes and different genomic regions regulating the accumulation of micronutrients, and the status of micronutrient biofortified wheat varieties released for commercial cultivation across the globe. In addition, we have also discussed the potential implications of emerging technologies such as genome editing to improve the micronutrient content and their bioavailability in wheat.

A natural uORF variant confers phosphorus acquisition diversity in soybean

Phosphorus (P) is an essential element for all organisms. Because P fertilizers are a non-renewable resource and high fixation in soils, sustainable agriculture requires researchers to improve crop P acquisition efficiency. Here, we report a strong association signal at a locus of CPU1 (component of phosphorus uptake 1), from a genome-wide association study of P acquisition efficiency in a soybean core collection grown in the field. A SEC12-like gene, GmPHF1, is identified as the causal gene for CPU1. GmPHF1 facilitates the ER (endoplasmic reticulum) exit of the phosphate transporter, GmPT4, to the plasma membrane of root epidermal cells. A common SNP in an upstream open reading frame (uORF) of GmPHF1, which alters the abundance of GmPHF1 in a tissue-specific manner, contributes to P acquisition diversity in soybean. A natural genetic variation conditions diversity in soybean P acquisition, which can be used to develop P-efficient soybean genotypes.

Effects of sublethal single, simultaneous and sequential abiotic stresses on phenotypic traits of Arabidopsis thaliana.

Plant responses to abiotic stresses are complex and dynamic, and involve changes in different traits, either as the direct consequence of the stress, or as an active acclimatory response. Abiotic stresses frequently occur simultaneously or in succession, rather than in isolation. Despite this, most studies have focused on a single stress and single or few plant traits. To address this gap, our study comprehensively and categorically quantified the individual and combined effects of three major abiotic stresses associated with climate change (flooding, progressive drought and high temperature) on 12 phenotypic traits related to morphology, development, growth and fitness, at different developmental stages in four Arabidopsis thaliana accessions. Combined sublethal stresses were applied either simultaneously (high temperature and drought) or sequentially (flooding followed by drought). In total, we analysed the phenotypic responses of 1782 individuals across these stresses and different developmental stages. Overall, abiotic stresses and their combinations resulted in distinct patterns of effects across the traits analysed, with both quantitative and qualitative differences across accessions. Stress combinations had additive effects on some traits, whereas clear positive and negative interactions were observed for other traits: 9 out of 12 traits for high temperature and drought, 6 out of 12 traits for post-submergence and drought showed significant interactions. In many cases where the stresses interacted, the strength of interactions varied across accessions. Hence, our results indicated a general pattern of response in most phenotypic traits to the different stresses and stress combinations, but it also indicated a natural genetic variation in the strength of these responses. This includes novel results regarding the lack of a response to drought after submergence and a decoupling between leaf number and flowering time after submergence. Overall, our study provides a rich characterization of trait responses of Arabidopsis plants to sublethal abiotic stresses at the phenotypic level and can serve as starting point for further in-depth physiological research and plant modelling efforts.

Naturally segregating genetic variation in circadian period exhibits a regional elevational and climatic cline.

Circadian clocks confer adaptation to predictable 24-h fluctuations in the exogenous environment, but it has yet to be determined what ecological factors maintain natural genetic variation in endogenous circadian period outside of the hypothesized optimum of 24 h. We estimated quantitative genetic variation in circadian period in leaf movement in 30 natural populations of the Arabidopsis relative Boechera stricta sampled within only 1° of latitude but across an elevation gradient spanning 2460-3300 m in the Rocky Mountains. Measuring ~3800 plants from 473 maternal families (7-20 per population), we found that genetic variation was of similar magnitude among versus within populations, with population means varying between 21.9 and24.9 h and maternal family means within populations varying by up to ~6 h. After statistically accounting for spatial autocorrelation at a habitat extreme, we found that elevation explained a significant proportion of genetic variation in the circadian period, such that higher-elevation populations had shorter mean period lengths and reduced intrapopulation ranges. Environmental data indicate that these spatial trends could be related to steep regional climatic gradients in temperature, precipitation, and their intra-annual variability. Our findings suggest that spatially fine-grained environmental heterogeneity contributes to naturally occurring genetic variation in circadian traits in wild populations.

Single Nucleotide Polymorphisms (SNP) and SNP-SNP Interactions of the Surfactant Protein Genes Are Associated With Idiopathic Pulmonary Fibrosis in a Mexican Study Group; Comparison With Hypersensitivity Pneumonitis.

Surfactant proteins (SPs) are important for normal lung function and innate immunity of the lungs and their genes have been identified with significant genetic variability. Changes in quantity or quality of SPs due to genetic mutations or natural genetic variability may alter their functions and contribute to the host susceptibility for particular diseases. Alternatively, SP single nucleotide polymorphisms (SNPs) can serve as markers to identify disease risk or response to therapies, as shown for other genes in a number of other studies. In the current study, we evaluated associations of SFTP SNPs with idiopathic pulmonary fibrosis (IPF) by studying novel computational models where the epistatic effects (dominant, additive, recessive) of SNP-SNP interactions could be evaluated, and then compared the results with a previously published hypersensitivity pneumonitis (HP) study where the same novel models were used. Mexican Hispanic patients (IPF=84 & HP=75) and 194 healthy control individuals were evaluated. The goal was to identify SP SNPs and SNP-SNP interactions that associate with IPF as well as SNPs and interactions that may be unique to each of these interstitial diseases or common between them. We observed: 1) in terms of IPF, i) three single SFTPA1 SNPs to associate with decreased IPF risk, ii) three SFTPA1 haplotypes to associate with increased IPF risk, and iii) a number of three-SNP interactions to associate with IPF susceptibility. 2) Comparison of IPF and HP, i) three SFTPA1 and one SFTPB SNP associated with decreased risk in IPF but increased risk in HP, and one SFTPA1 SNP associated with decreased risk in both IPF and HP, ii) a number of three-SNP interactions with the same or different effect pattern associated with IPF and/or HP susceptibility, iii) one of the three-SNP interactions that involved SNPs of SFTPA1, SFTPA2, and SFTPD, with the same effect pattern, was associated with a disease-specific outcome, a decreased and increased risk in HP and IPF, respectively. This is the first study that compares the SP gene variants in these two phenotypically similar diseases. Our findings indicate that SNPs of all SFTPs may play an important role in the genetic susceptibility to IPF and HP. Importantly, IPF and HP share some SP genetic variants, suggesting common pathophysiological mechanisms and pathways regarding surfactant biogenesis, but also some differences, highlighting the diverse underlying pathogenic mechanisms between an inflammatory-driven fibrosis (HP) and an epithelial-driven fibrosis (IPF). Alternatively, the significant SNPs identified here, along with SNPs of other genes, could serve as markers to distinguish these two devastating diseases.

Trade-offs in defence to pathogen species revealed in expanding nematode populations.

Many host organisms live in polymicrobial environments and must respond to a diversity of pathogens. The degree to which host defences towards one pathogen species affect susceptibility to others is unclear. We used a panel of Caenorhabditis elegans nematode isolates to test for natural genetic variation in fitness costs of immune upregulation and pathogen damage, as well as for trade-offs in defence against two pathogen species, Staphylococcus aureus and Pseudomonas aeruginosa. We examined the fitness impacts of transient pathogen exposure (pathogen damage and immune upregulation) or exposure to heat-killed culture (immune upregulation only) by measuring host population sizes, which allowed us to simultaneously capture changes in reproductive output, developmental time and survival. We found significant decreases in population sizes for hosts exposed to live versus heat-killed S. aureus and found increased reproductive output after live P. aeruginosa exposure, compared with the corresponding heat-killed challenge. Nematode isolates with relatively higher population sizes after live P. aeruginosa infection produced fewer offspring after live S. aureus challenge. These findings reveal that wild C. elegans genotypes display a trade-off in defences against two distinct pathogen species that are evident in subsequent generations.

Natural variation further increases resilience of sorghum bred for chronically drought-prone environments.

Drought stress is one of the major constraints for crop production in the Sahel region of Africa. Here, we explore the potential to use natural genetic variation to build on the inherent drought tolerance of an elite sorghum cultivar, Teshale, that has been bred for Ethiopian conditions including chronic drought. We evaluated a backcross nested-association mapping population using 12 diverse founder lines crossed with Teshale under three drought-prone environments in Ethiopia. All 12 populations averaged higher head exsertion and lower leaf senescence than the recurrent parent in the two most stressful environments, reflecting new drought resilience mechanisms from the donors. A total of 154 quantitative trait loci (QTLs) were detected for eight drought-responsive traits, and their validity was supported by the fact that 113 (73.4%) overlapped with QTLs previously detected for the same traits, concentrated in regions previously associated with 'stay-green' traits. Allele effects showed that some favourable alleles are already present in the Ethiopian cultivar; however, the exotic donors offer rich scope for increasing drought resilience. Using model-selected SNPs associated with the eight traits identified in this study and three in a companion study, phenotypic prediction accuracies for grain yield were equivalent to genome-wide SNPs and were significantly better than random SNPs, indicating that the selected traits are predictive of sorghum grain yield.

Tissue Culture and Somatic Embryogenesis in Warm-Season Grasses-Current Status and Its Applications: A Review.

Warm-season grasses are C4 plants and have a high capacity for biomass productivity. These grasses are utilized in many agricultural production systems with their greatest value as feeds for livestock, bioethanol, and turf. However, many important warm-season perennial grasses multiply either by vegetative propagation or form their seeds by an asexual mode of reproduction called apomixis. Therefore, the improvement of these grasses by conventional breeding is difficult and is dependent on the availability of natural genetic variation and its manipulation through breeding and selection. Recent studies have indicated that plant tissue culture system through somatic embryogenesis complements and could further develop conventional breeding programs by micropropagation, somaclonal variation, somatic hybridization, genetic transformation, and genome editing. This review summarizes the tissue culture and somatic embryogenesis in warm-season grasses and focus on current status and above applications including the author’s progress.

Modulation of the Drosophila transcriptome by developmental exposure to alcohol.

BackgroundPrenatal exposure to ethanol can cause fetal alcohol spectrum disorder (FASD), a prevalent, preventable pediatric disorder. Identifying genetic risk alleles for FASD is challenging since time, dose, and frequency of exposure are often unknown, and manifestations of FASD are diverse and evident long after exposure. Drosophila melanogaster is an excellent model to study the genetic basis of the effects of developmental alcohol exposure since many individuals of the same genotype can be reared under controlled environmental conditions.ResultsWe used 96 sequenced, wild-derived inbred lines from the Drosophila melanogaster Genetic Reference Panel (DGRP) to profile genome-wide transcript abundances in young adult flies that developed on ethanol-supplemented medium or standard culture medium. We found substantial genetic variation in gene expression in response to ethanol with extensive sexual dimorphism. We constructed sex-specific genetic networks associated with alcohol-dependent modulation of gene expression that include protein-coding genes, Novel Transcribed Regions (NTRs, postulated to encode long non-coding RNAs) and female-specific coordinated regulation of snoRNAs that regulate pseudouridylation of ribosomal RNA. We reared DGRP lines which showed extreme upregulation or downregulation of snoRNA expression during developmental alcohol exposure on standard or ethanol supplemented medium and demonstrated that developmental exposure to ethanol has genotype-specific effects on adult locomotor activity and sleep.ConclusionsThere is significant and sex-specific natural genetic variation in the transcriptional response to developmental exposure to ethanol in Drosophila that comprises networks of genes affecting nervous system development and ethanol metabolism as well as networks of regulatory non-coding RNAs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-022-08559-9.

Experimental demonstration and pan-structurome prediction of climate-associated riboSNitches in Arabidopsis

BackgroundGenome-wide association studies (GWAS) aim to correlate phenotypic changes with genotypic variation. Upon transcription, single nucleotide variants (SNVs) may alter mRNA structure, with potential impacts on transcript stability, macromolecular interactions, and translation. However, plant genomes have not been assessed for the presence of these structure-altering polymorphisms or “riboSNitches.”ResultsWe experimentally demonstrate the presence of riboSNitches in transcripts of two Arabidopsis genes, ZINC RIBBON 3 (ZR3) and COTTON GOLGI-RELATED 3 (CGR3), which are associated with continentality and temperature variation in the natural environment. These riboSNitches are also associated with differences in the abundance of their respective transcripts, implying a role in regulating the gene's expression in adaptation to local climate conditions. We then computationally predict riboSNitches transcriptome-wide in mRNAs of 879 naturally inbred Arabidopsis accessions. We characterize correlations between SNPs/riboSNitches in these accessions and 434 climate descriptors of their local environments, suggesting a role of these variants in local adaptation. We integrate this information in CLIMtools V2.0 and provide a new web resource, T-CLIM, that reveals associations between transcript abundance variation and local environmental variation.ConclusionWe functionally validate two plant riboSNitches and, for the first time, demonstrate riboSNitch conditionality dependent on temperature, coining the term “conditional riboSNitch.” We provide the first pan-genome-wide prediction of riboSNitches in plants. We expand our previous CLIMtools web resource with riboSNitch information and with 1868 additional Arabidopsis genomes and 269 additional climate conditions, which will greatly facilitate in silico studies of natural genetic variation, its phenotypic consequences, and its role in local adaptation.

A natural genetic variation screen identifies insulin signaling, neuronal communication, and innate immunity as modifiers of hyperglycemia in the absence of Sirt1.

Variation in the onset, progression, and severity of symptoms associated with metabolic disorders such as diabetes impairs the diagnosis and treatment of at-risk patients. Diabetes symptoms, and patient variation in these symptoms, are attributed to a combination of genetic and environmental factors, but identifying the genes and pathways that modify diabetes in humans has proven difficult. A greater understanding of genetic modifiers and the ways in which they interact with metabolic pathways could improve the ability to predict a patient’s risk for severe symptoms, as well as enhance the development of individualized therapeutic approaches. In this study, we use the Drosophila Genetic Reference Panel to identify genetic variation influencing hyperglycemia associated with loss of Sirt1 function. Through analysis of individual candidate functions, physical interaction networks, and gene set enrichment analysis, we identify not only modifiers involved in canonical glucose metabolism and insulin signaling, but also genes important for neuronal signaling and the innate immune response. Furthermore, reducing the expression of several of these candidates suppressed hyperglycemia, making them potential candidate therapeutic targets. These analyses showcase the diverse processes contributing to glucose homeostasis and open up several avenues of future investigation.

The induction of natural competence adapts staphylococcal metabolism to infection

A central question concerning natural competence is why orthologs of competence genes are conserved in non-competent bacterial species, suggesting they have a role other than in transformation. Here we show that competence induction in the human pathogen Staphylococcus aureus occurs in response to ROS and host defenses that compromise bacterial respiration during infection. Bacteria cope with reduced respiration by obtaining energy through fermentation instead. Since fermentation is energetically less efficient than respiration, the energy supply must be assured by increasing the glycolytic flux. The induction of natural competence increases the rate of glycolysis in bacteria that are unable to respire via upregulation of DNA- and glucose-uptake systems. A competent-defective mutant showed no such increase in glycolysis, which negatively affects its survival in both mouse and Galleria infection models. Natural competence foster genetic variability and provides S. aureus with additional nutritional and metabolic possibilities, allowing it to proliferate during infection.

Towards improved dynamic photosynthesis in C3 crops by utilizing natural genetic variation.

Under field environments, fluctuating light conditions induce dynamic photosynthesis, which affects carbon gain by crop plants. Elucidating the natural genetic variations among untapped germplasm resources and their underlying mechanisms can provide an effective strategy to improve dynamic photosynthesis and, ultimately, improve crop yields through molecular breeding approaches. In this review, we first overview two processes affecting dynamic photosynthesis, namely (i) biochemical processes associated with CO2 fixation and photoprotection and (ii) gas diffusion processes from the atmosphere to the chloroplast stroma. Next, we review the intra- and interspecific variations in dynamic photosynthesis in relation to each of these two processes. It is suggested that plant adaptations to different hydrological environments underlie natural genetic variation explained by gas diffusion through stomata. This emphasizes the importance of the coordination of photosynthetic and stomatal dynamics to optimize the balance between carbon gain and water use efficiency under field environments. Finally, we discuss future challenges in improving dynamic photosynthesis by utilizing natural genetic variation. The forward genetic approach supported by high-throughput phenotyping should be introduced to evaluate the effects of genetic and environmental factors and their interactions on the natural variation in dynamic photosynthesis.