Environment-specific selection alters flowering-time plasticity and results in pervasive pleiotropic responses in maize.

Crop genetic diversity for climate adaptations is globally partitioned. We performed experimental evolution in maize to understand the response to selection and how plant germplasm can be moved across geographical zones. Initialized with a common population of tropical origin, artificial selection on flowering time was performed for two generations at eight field sites spanning 25° latitude, a 2800 km transect. We then jointly tested all selection lineages across the original sites of selection, for the target trait and 23 other traits. Modeling intergenerational shifts in a physiological reaction norm revealed separate components for flowering-time plasticity. Generalized and local modes of selection altered the plasticity of each lineage, leading to a latitudinal pattern in the responses to selection that were strongly driven by photoperiod. This transformation led to widespread changes in developmental, architectural, and yield traits, expressed collectively in an environment-dependent manner. Furthermore, selection for flowering time alone alleviated a maladaptive syndrome and improved yields for tropical maize in the temperate zone. Our findings show how phenotypic selection can rapidly shift the flowering phenology and plasticity of maize. They also demonstrate that selecting crops to local conditions can accelerate adaptation to climate change.

SPEARS: Standard Performance Evaluation of Ancestral haplotype Reconstruction through Simulation.

MOTIVATION: Ancestral haplotype maps provide useful information about genomic variation and insights into biological processes. Reconstructing the descendent haplotype structure of homologous chromosomes, particularly for large numbers of individuals, can help with characterizing the recombination landscape, elucidating genotype-to-phenotype relationships, improving genomic predictions and more. Inferring haplotype maps from sparse genotype data is an efficient approach to whole-genome haplotyping, but this is a non-trivial problem. A standardized approach is needed to validate whether haplotype reconstruction software, conceived population designs and existing data for a given population provides accurate haplotype information for further inference. RESULTS: We introduce SPEARS, a pipeline for the simulation-based appraisal of genome-wide haplotype maps constructed from sparse genotype data. Using a specified pedigree, the pipeline generates virtual genotypes (known data) with genotyping errors and missing data structure. It then proceeds to mimic analysis in practice, capturing sources of error due to genotyping, imputation and haplotype inference. Standard metrics allow researchers to assess different population designs and which features of haplotype structure or regions of the genome are sufficiently accurate for analysis. Haplotype maps for 1000 outcross progeny from a multi-parent population of maize are used to demonstrate SPEARS. AVAILABILITYAND IMPLEMENTATION: SPEARS, the protocol and suite of scripts, are publicly available under an MIT license at GitHub (https://github.com/maizeatlas/spears). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries.

BACKGROUND: Targeted resequencing with high-throughput sequencing (HTS) platforms can be used to efficiently interrogate the genomes of large numbers of individuals. A critical issue for research and applications using HTS data, especially from long-read platforms, is error in base calling arising from technological limits and bioinformatic algorithms. We found that the community standard long amplicon analysis (LAA) module from Pacific Biosciences is prone to substantial bioinformatic errors that raise concerns about findings based on this pipeline, prompting the need for a new method. RESULTS: A single molecule real-time (SMRT) sequencing-error correction and assembly pipeline, C3S-LAA, was developed for libraries of pooled amplicons. By uniquely leveraging the structure of SMRT sequence data (comprised of multiple low quality subreads from which higher quality circular consensus sequences are formed) to cluster raw reads, C3S-LAA produced accurate consensus sequences and assemblies of overlapping amplicons from single sample and multiplexed libraries. In contrast, despite read depths in excess of 100X per amplicon, the standard long amplicon analysis module from Pacific Biosciences generated unexpected numbers of amplicon sequences with substantial inaccuracies in the consensus sequences. A bootstrap analysis showed that the C3S-LAA pipeline per se was effective at removing bioinformatic sources of error, but in rare cases a read depth of nearly 400X was not sufficient to overcome minor but systematic errors inherent to amplification or sequencing. CONCLUSIONS: C3S-LAA uses a divide and conquer processing algorithm for SMRT amplicon-sequence data that generates accurate consensus sequences and local sequence assemblies. Solving the confounding bioinformatic source of error in LAA allowed for the identification of limited instances of errors due to DNA amplification or sequencing of homopolymeric nucleotide tracts. For research and development in genomics, C3S-LAA allows meaningful conclusions and biological inferences to be made from accurately polished sequence output.

The Genetics of Leaf Flecking in Maize and Its Relationship to Plant Defense and Disease Resistance.

Physiological leaf spotting, or flecking, is a mild-lesion phenotype observed on the leaves of several commonly used maize (Zea mays) inbred lines and has been anecdotally linked to enhanced broad-spectrum disease resistance. Flecking was assessed in the maize nested association mapping (NAM) population, comprising 4,998 recombinant inbred lines from 25 biparental families, and in an association population, comprising 279 diverse maize inbreds. Joint family linkage analysis was conducted with 7,386 markers in the NAM population. Genome-wide association tests were performed with 26.5 million single-nucleotide polymorphisms (SNPs) in the NAM population and with 246,497 SNPs in the association population, resulting in the identification of 18 and three loci associated with variation in flecking, respectively. Many of the candidate genes colocalizing with associated SNPs are similar to genes that function in plant defense response via cell wall modification, salicylic acid- and jasmonic acid-dependent pathways, redox homeostasis, stress response, and vesicle trafficking/remodeling. Significant positive correlations were found between increased flecking, stronger defense response, increased disease resistance, and increased pest resistance. A nonlinear relationship with total kernel weight also was observed whereby lines with relatively high levels of flecking had, on average, lower total kernel weight. We present evidence suggesting that mild flecking could be used as a selection criterion for breeding programs trying to incorporate broad-spectrum disease resistance.

An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy.

We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process.

Limits on the reproducibility of marker associations with southern leaf blight resistance in the maize nested association mapping population.

BACKGROUND: A previous study reported a comprehensive quantitative trait locus (QTL) and genome wide association study (GWAS) of southern leaf blight (SLB) resistance in the maize Nested Association Mapping (NAM) panel. Since that time, the genomic resources available for such analyses have improved substantially. An updated NAM genetic linkage map has a nearly six-fold greater marker density than the previous map and the combined SNPs and read-depth variants (RDVs) from maize HapMaps 1 and 2 provided 28.5 M genomic variants for association analysis, 17 fold more than HapMap 1. In addition, phenotypic values of the NAM RILs were re-estimated to account for environment-specific flowering time covariates and a small proportion of lines were dropped due to genotypic data quality problems. Comparisons of original and updated QTL and GWAS results confound the effects of linkage map density, GWAS marker density, population sample size, and phenotype estimates. Therefore, we evaluated the effects of changing each of these parameters individually and in combination to determine their relative impact on marker-trait associations in original and updated analyses. RESULTS: Of the four parameters varied, map density caused the largest changes in QTL and GWAS results. The updated QTL model had better cross-validation prediction accuracy than the previous model. Whereas joint linkage QTL positions were relatively stable to input changes, the residual values derived from those QTL models (used as inputs to GWAS) were more sensitive, resulting in substantial differences between GWAS results. The updated NAM GWAS identified several candidate genes consistent with previous QTL fine-mapping results. CONCLUSIONS: The highly polygenic nature of resistance to SLB complicates the identification of causal genes. Joint linkage QTL are relatively stable to perturbations of data inputs, but their resolution is generally on the order of tens or more Mbp. GWAS associations have higher resolution, but lower power due to stringent thresholds designed to minimize false positive associations, resulting in variability of detection across studies. The updated higher density linkage map improves QTL estimation and, along with a much denser SNP HapMap, greatly increases the likelihood of detecting SNPs in linkage with causal variants. We recommend use of the updated genetic resources and results but emphasize the limited repeatability of small-effect associations.

Multivariate analysis of maize disease resistances suggests a pleiotropic genetic basis and implicates a GST gene.

Plants are attacked by pathogens representing diverse taxonomic groups, such that genes providing multiple disease resistance (MDR) are expected to be under positive selection pressure. To address the hypothesis that naturally occurring allelic variation conditions MDR, we extended the framework of structured association mapping to allow for the analysis of correlated complex traits and the identification of pleiotropic genes. The multivariate analytical approach used here is directly applicable to any species and set of traits exhibiting correlation. From our analysis of a diverse panel of maize inbred lines, we discovered high positive genetic correlations between resistances to three globally threatening fungal diseases. The maize panel studied exhibits rapidly decaying linkage disequilibrium that generally occurs within 1 or 2 kb, which is less than the average length of a maize gene. The positive correlations therefore suggested that functional allelic variation at specific genes for MDR exists in maize. Using a multivariate test statistic, a glutathione S-transferase (GST) gene was found to be associated with modest levels of resistance to all three diseases. Resequencing analysis pinpointed the association to a histidine (basic amino acid) for aspartic acid (acidic amino acid) substitution in the encoded protein domain that defines GST substrate specificity and biochemical activity. The known functions of GSTs suggested that variability in detoxification pathways underlie natural variation in maize MDR.

Enhancing adaptation of tropical maize to temperate environments using genomic selection.

Tropical maize can be used to diversify the genetic base of temperate germplasm and help create climate-adapted cultivars. However, tropical maize is unadapted to temperate environments, in which sensitivities to long photoperiods and cooler temperatures result in severely delayed flowering times, developmental defects, and little to no yield. Overcoming this maladaptive syndrome can require a decade of phenotypic selection in a targeted, temperate environment. To accelerate the incorporation of tropical diversity in temperate breeding pools, we tested if an additional generation of genomic selection can be used in an off-season nursery where phenotypic selection is not very effective. Prediction models were trained using flowering time recorded on random individuals in separate lineages of a heterogenous population grown at two northern U.S. latitudes. Direct phenotypic selection and genomic prediction model training was performed within each target environment and lineage, followed by genomic prediction of random intermated progenies in the off-season nursery. Performance of genomic prediction models was evaluated on self-fertilized progenies of prediction candidates grown in both target locations in the following summer season. Prediction abilities ranged from 0.30 to 0.40 among populations and evaluation environments. Prediction models with varying marker effect distributions or spatial field effects had similar accuracies. Our results suggest that genomic selection in a single off-season generation could increase genetic gains for flowering time by more than 50% compared to direct selection in summer seasons only, reducing the time required to change the population mean to an acceptably adapted flowering time by about one-third to one-half.

Characterization of integration sites and transfer DNA structures in Agrobacterium-mediated transgenic events of maize inbred B104.

In maize, the community-standard transformant line B104 is a useful model for dissecting features of transfer DNA (T-DNA) integration due to its compatibility with Agrobacterium-mediated transformation and the availability of its genome sequence. Knowledge of transgene integration sites permits the analysis of the genomic environment that governs the strength of gene expression and phenotypic effects due to the disruption of an endogenous gene or regulatory element. In this study, we optimized a fusion primer and nested integrated PCR (FPNI-PCR) technique for T-DNA detection in maize to characterize the integration sites of 89 T-DNA insertions in 81 transformant lines. T-DNA insertions preferentially occurred in gene-rich regions and regions distant from centromeres. Integration junctions with and without microhomologous sequences as well as junctions with de novo sequences were detected. Sequence analysis of integration junctions indicated that T-DNA was incorporated via the error-prone repair pathways of nonhomologous (predominantly) and microhomology-mediated (minor) end-joining. This report provides a quantitative assessment of Agrobacterium-mediated T-DNA integration in maize with respect to insertion site features, the genomic distribution of T-DNA incorporation, and the mechanisms of integration. It also demonstrates the utility of the FPNI-PCR technique, which can be adapted to any species of interest.

2018-2019 field seasons of the Maize Genomes to Fields (G2F) G x E project.

OBJECTIVES: This report provides information about the public release of the 2018-2019 Maize G X E project of the Genomes to Fields (G2F) Initiative datasets. G2F is an umbrella initiative that evaluates maize hybrids and inbred lines across multiple environments and makes available phenotypic, genotypic, environmental, and metadata information. The initiative understands the necessity to characterize and deploy public sources of genetic diversity to face the challenges for more sustainable agriculture in the context of variable environmental conditions. DATA DESCRIPTION: Datasets include phenotypic, climatic, and soil measurements, metadata information, and inbred genotypic information for each combination of location and year. Collaborators in the G2F initiative collected data for each location and year; members of the group responsible for coordination and data processing combined all the collected information and removed obvious erroneous data. The collaborators received the data before the DOI release to verify and declare that the data generated in their own locations was accurate. ReadMe and description files are available for each dataset. Previous years of evaluation are already publicly available, with common hybrids present to connect across all locations and years evaluated since this project's inception.

Multivariate mixed linear model analysis of longitudinal data: an information-rich statistical technique for analyzing plant disease resistance.

ABSTRACT The mixed linear model (MLM) is an advanced statistical technique applicable to many fields of science. The multivariate MLM can be used to model longitudinal data, such as repeated ratings of disease resistance taken across time. In this study, using an example data set from a multi-environment trial of northern leaf blight disease on 290 maize lines with diverse levels of resistance, multivariate MLM analysis was performed and its utility was examined. In the population and environments tested, genotypic effects were highly correlated across disease ratings and followed an autoregressive pattern of correlation decay. Because longitudinal data are often converted to the univariate measure of area under the disease progress curve (AUDPC), comparisons between univariate MLM analysis of AUDPC and multivariate MLM analysis of longitudinal data were made. Univariate analysis had the advantage of simplicity and reduced computational demand, whereas multivariate analysis enabled a comprehensive perspective on disease development, providing the opportunity for unique insights into disease resistance. To aid in the application of multivariate MLM analysis of longitudinal data on disease resistance, annotated program syntax for model fitting is provided for the software ASReml.

Physiological adaptive traits are a potential allele reservoir for maize genetic progress under challenging conditions.

Combined phenomic and genomic approaches are required to evaluate the margin of progress of breeding strategies. Here, we analyze 65 years of genetic progress in maize yield, which was similar (101 kg ha-1 year-1) across most frequent environmental scenarios in the European growing area. Yield gains were linked to physiologically simple traits (plant phenology and architecture) which indirectly affected reproductive development and light interception in all studied environments, marked by significant genomic signatures of selection. Conversely, studied physiological processes involved in stress adaptation remained phenotypically unchanged (e.g. stomatal conductance and growth sensitivity to drought) and showed no signatures of selection. By selecting for yield, breeders indirectly selected traits with stable effects on yield, but not physiological traits whose effects on yield can be positive or negative depending on environmental conditions. Because yield stability under climate change is desirable, novel breeding strategies may be needed for exploiting alleles governing physiological adaptive traits.

Shades of gray: the world of quantitative disease resistance.

A thorough understanding of quantitative disease resistance (QDR) would contribute to the design and deployment of durably resistant crop cultivars. However, the molecular mechanisms that control QDR remain poorly understood, largely due to the incomplete and inconsistent nature of the resistance phenotype, which is usually conditioned by many loci of small effect. Here, we discuss recent advances in research on QDR. Based on inferences from analyses of the defense response and from the few isolated QDR genes, we suggest several plausible hypotheses for a range of mechanisms underlying QDR. We propose that a new generation of genetic resources, complemented by careful phenotypic analysis, will produce a deeper understanding of plant defense and more effective utilization of natural resistance alleles.

Genome assembly of a Mesoamerican derived variety of lima bean: a foundational cultivar in the Mid-Atlantic USA.

Lima bean, Phaseolus lunatus, is closely related to common bean and is high in fiber and protein, with a low glycemic index. Lima bean is widely grown in the state of Delaware, where late summer and early fall weather are conducive to pod production. The same weather conditions also promote diseases such as pod rot and downy mildew, the latter of which has caused previous epidemics. A better understanding of the genes underlying resistance to this and other pathogens is needed to keep this industry thriving in the region. Our current study sought to sequence, assemble, and annotate a commercially available cultivar called Bridgeton, which could then serve as a reference genome, a basis of comparison to other Phaseolus taxa, and a resource for the identification of potential resistance genes. Combined efforts of sequencing, linkage, and comparative analysis resulted in a 623 Mb annotated assembly for lima bean, as well as a better understanding of an evolutionarily dynamic resistance locus in legumes.

The importance of dominance and genotype-by-environment interactions on grain yield variation in a large-scale public cooperative maize experiment.

High-dimensional and high-throughput genomic, field performance, and environmental data are becoming increasingly available to crop breeding programs, and their integration can facilitate genomic prediction within and across environments and provide insights into the genetic architecture of complex traits and the nature of genotype-by-environment interactions. To partition trait variation into additive and dominance (main effect) genetic and corresponding genetic-by-environment variances, and to identify specific environmental factors that influence genotype-by-environment interactions, we curated and analyzed genotypic and phenotypic data on 1918 maize (Zea mays L.) hybrids and environmental data from 65 testing environments. For grain yield, dominance variance was similar in magnitude to additive variance, and genetic-by-environment variances were more important than genetic main effect variances. Models involving both additive and dominance relationships best fit the data and modeling unique genetic covariances among all environments provided the best characterization of the genotype-by-environment interaction patterns. Similarity of relative hybrid performance among environments was modeled as a function of underlying weather variables, permitting identification of weather covariates driving correlations of genetic effects across environments. The resulting models can be used for genomic prediction of mean hybrid performance across populations of environments tested or for environment-specific predictions. These results can also guide efforts to incorporate high-throughput environmental data into genomic prediction models and predict values in new environments characterized with the same environmental characteristics.

Utility of Climatic Information via Combining Ability Models to Improve Genomic Prediction for Yield Within the Genomes to Fields Maize Project.

Genomic prediction provides an efficient alternative to conventional phenotypic selection for developing improved cultivars with desirable characteristics. New and improved methods to genomic prediction are continually being developed that attempt to deal with the integration of data types beyond genomic information. Modern automated weather systems offer the opportunity to capture continuous data on a range of environmental parameters at specific field locations. In principle, this information could characterize training and target environments and enhance predictive ability by incorporating weather characteristics as part of the genotype-by-environment (G×E) interaction component in prediction models. We assessed the usefulness of including weather data variables in genomic prediction models using a naïve environmental kinship model across 30 environments comprising the Genomes to Fields (G2F) initiative in 2014 and 2015. Specifically four different prediction scenarios were evaluated (i) tested genotypes in observed environments; (ii) untested genotypes in observed environments; (iii) tested genotypes in unobserved environments; and (iv) untested genotypes in unobserved environments. A set of 1,481 unique hybrids were evaluated for grain yield. Evaluations were conducted using five different models including main effect of environments; general combining ability (GCA) effects of the maternal and paternal parents modeled using the genomic relationship matrix; specific combining ability (SCA) effects between maternal and paternal parents; interactions between genetic (GCA and SCA) effects and environmental effects; and finally interactions between the genetics effects and environmental covariates. Incorporation of the genotype-by-environment interaction term improved predictive ability across all scenarios. However, predictive ability was not improved through inclusion of naive environmental covariates in G×E models. More research should be conducted to link the observed weather conditions with important physiological aspects in plant development to improve predictive ability through the inclusion of weather data.

Selection mapping of loci for quantitative disease resistance in a diverse maize population.

The selection response of a complex maize population improved primarily for quantitative disease resistance to northern leaf blight (NLB) and secondarily for common rust resistance and agronomic phenotypes was investigated at the molecular genetic level. A tiered marker analysis with 151 simple sequence repeat (SSR) markers in 90 individuals of the population indicated that on average six alleles per locus were available for selection. An improved test statistic for selection mapping was developed, in which quantitative trait loci (QTL) are identified through the analysis of allele-frequency shifts at mapped multiallelic loci over generations of selection. After correcting for the multiple tests performed, 25 SSR loci showed evidence of selection. Many of the putatively selected loci were unlinked and dispersed across the genome, which was consistent with the diffuse distribution of previously published QTL for NLB resistance. Compelling evidence for selection was found on maize chromosome 8, where several putatively selected loci colocalized with published NLB QTL and a race-specific resistance gene. Analysis of F2 populations derived from the selection mapping population suggested that multiple linked loci in this chromosomal segment were, in part, responsible for the selection response for quantitative resistance to NLB.

Validation and Characterization of Maize Multiple Disease Resistance QTL.

Southern Leaf Blight, Northern Leaf Blight, and Gray Leaf Spot, caused by ascomycete fungi, are among the most important foliar diseases of maize worldwide. Previously, disease resistance quantitative trait loci (QTL) for all three diseases were identified in a connected set of chromosome segment substitution line (CSSL) populations designed for the identification of disease resistance QTL. Some QTL for different diseases co-localized, indicating the presence of multiple disease resistance (MDR) QTL. The goal of this study was to perform an independent test of several of the MDR QTL identified to confirm their existence and derive a more precise estimate of allele additive and dominance effects. Twelve F2:3 family populations were produced, in which selected QTL were segregating in an otherwise uniform genetic background. The populations were assessed for each of the three diseases in replicated trials and genotyped with markers previously associated with disease resistance. Pairwise phenotypic correlations across all the populations for resistance to the three diseases ranged from 0.2 to 0.3 and were all significant at the alpha level of 0.01. Of the 44 QTL tested, 16 were validated (identified at the same genomic location for the same disease or diseases) and several novel QTL/disease associations were found. Two MDR QTL were associated with resistance to all three diseases. This study identifies several potentially important MDR QTL and demonstrates the importance of independently evaluating QTL effects following their initial identification.

Optimal Designs for Genomic Selection in Hybrid Crops.

Improved capacity of genomics and biotechnology has greatly enhanced genetic studies in different areas. Genomic selection exploits the genotype-to-phenotype relationship at the whole-genome level and is being implemented in many crops. Here we show that design-thinking and data-mining techniques can be leveraged to optimize genomic prediction of hybrid performance. We phenotyped a set of 276 maize hybrids generated by crossing founder inbreds of nested association mapping populations for flowering time, ear height, and grain yield. With 10 296 310 SNPs available from the parental inbreds, we explored the patterns of genomic relationships and phenotypic variation to establish training samples based on clustering, graphic network analysis, and genetic mating scheme. Our analysis showed that training set designs outperformed random sampling and earlier methods that either minimize the mean of prediction error variance or maximize the mean of generalized coefficient of determination. Additional analyses of 2556 wheat hybrids from an early-stage hybrid breeding system and 1439 rice hybrids from an established hybrid breeding system validated the approaches. Together, we demonstrated that effective genomic prediction models can be established with a training set 2%-13% of the size of the whole set, enabling an efficient exploration of enormous inference space of genetic combinations.

The Genomic Basis for Short-Term Evolution of Environmental Adaptation in Maize.

Understanding the evolutionary capacity of populations to adapt to novel environments is one of the major pursuits in genetics. Moreover, for plant breeding, maladaptation is the foremost barrier to capitalizing on intraspecific variation in order to develop new breeds for future climate scenarios in agriculture. Using a unique study design, we simultaneously dissected the population and quantitative genomic basis of short-term evolution in a tropical landrace of maize that was translocated to a temperate environment and phenotypically selected for adaptation in flowering time phenology. Underlying 10 generations of directional selection, which resulted in a 26-day mean decrease in female-flowering time, [Formula: see text] of the heritable variation mapped to [Formula: see text] of the genome, where, overall, alleles shifted in frequency beyond the boundaries of genetic drift in the expected direction given their flowering time effects. However, clustering these non-neutral alleles based on their profiles of frequency change revealed transient shifts underpinning a transition in genotype-phenotype relationships across generations. This was distinguished by initial reductions in the frequencies of few relatively large positive effect alleles and subsequent enrichment of many rare negative effect alleles, some of which appear to represent allelic series. With these genomic shifts, the population reached an adapted state while retaining [Formula: see text] of the standing molecular marker variation in the founding population. Robust selection and association mapping tests highlighted several key genes driving the phenotypic response to selection. Our results reveal the evolutionary dynamics of a finite polygenic architecture conditioning a capacity for rapid environmental adaptation in maize.