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Deciphering the molecular basis of complex traits requires understanding how natural genetic variation interacts with underlying biological pathways. In this study, we explored how natural genetic variation influences traits in maize affected by a semi-dominant maize dwarfing allele,Dwarf13–1 (D13-1)which encodes a defective ionotropic glutamate receptor (GLR). This allowed us to investigate natural genetic variation in the genome affecting GLR signaling in maize. We implemented an F1 association mapping (FOAM) approach, where heterozygous mutants carrying the semi-dominantD13-1allele were crossed with a maize association panel. The resulting F1 families segregated 1:1 for mutant and wild-type phenotypes allowing comparisons between the congenic F1 hybrid siblings to identify and map natural alleles that interact with theD13-1mutant allele. FOAM mapping detected two loci that modify the expression of theD13-1/+ mutant phenotype. The phenotypic impacts of both loci were epistatically controlled byD13-1, and only affected the phenotypes of mutant F1 hybrids. One,tropotriskaideka1(tod1), encoded a maize homolog of the GLR-interactingcornichongene and modifiedD13-1/+mutant severity. A second, encoded by thed13locus itself, affected the severity of theD13-1/+phenotype via variation in the wild-type allele in the heterozygous mutants. By integrating gene expression analyses, these epistatic interactions, and SNP linkage information we identified multiple, unlinked, alleles affecting expression of the wild-type D13 transcript that modify mutant trait expression. Greater expression of the wild-type D13 allele increased plant height and suppressedD13-1/+ mutant severity, consistent with a multi-subunit complex GLR structure and complex-poisoning mode-of-action for the semi-dominantD13-1allele. This approach identifies natural alleles affecting the GLR pathway in maize and establishes GLRs and their interactors as dose-dependent regulators of plant architecture. Our pathway-focused framework and epistasis testing of natural variants provides greater confidence in identifying genes contributing to complex traits. 

Abstract Tetrapyrroles are macrocyclic compounds present in and required for all life on Earth. Mutants with defects in tetrapyrrole pathway enzymes can be used to uncover natural variation in this pathway and study pathway regulation. We report the effects of the Oil yellow1 mutation, Oy1-N1989, a semi-dominant allele of subunit I in the Mg-chelatase enzyme with reduced chlorophyll biosynthesis in maize (Zea mays), on global gene expression and chlorophyll content. In Oy1-N1989/+ mutants, coordinate feedback regulation of the tetrapyrrole pathway was observed as transcriptional feedback regulation of genes encoding steps in the tetrapyrrole pathway. Natural variation in the wild-type allele at oy1 modulated the severity of the impact of Oy1-N1989/+ on gene expression. Previously identified cis-acting expression variation at oy1 in wild-type plants affected similar transcriptional co-regulation of genes encoding steps in the tetrapyrrole pathway as observed in the RNA-seq of Oy1-N1989/+ mutants. This demonstrated that the coordinate regulation of the pathway also occurs during physiologically relevant variation in OY1 abundance. Cis variants at 7 tetrapyrrole pathway genes were linked to variation in chlorophyll accumulation in Oy1-N1989/+ mutant or wild-type plants. Analysis of trans-acting transcriptional variation by eGWAS detected multiple transcriptional hotspots, which affected the expression of a subset of tetrapyrrole pathway genes, indicating that these genes are repeated targets of transcriptional regulation. The hotspots were encoded at locations with no known regulators of the tetrapyrrole pathway, indicating as yet undiscovered molecular mechanisms of feedback regulation operating in natural populations. The trans-regulatory hotspots coordinately regulate this pathway and may work to limit the accumulation of phototoxic intermediates. 

Most ferns, unlike all seed plants, are homosporous and produce sexually undifferentiated spores. Sex ratio in many homosporous species is environmentally established by the secretion of antheridiogen from female/hermaphrodite gametophytes. Nearby undetermined gametophytes perceive antheridiogen, which induces male development. In the fern Ceratopteris richardii (Ceratopteris), hermaphroditic (her) mutants develop as hermaphrodites even in the presence of antheridiogen. Modern sequencing and genomic tools make the molecular identification of mutants in the 11-Gbp genome of this fern possible. We mapped 2 linked mutants, her7-14 and her7-19, to the same 16-Mbp interval on chromosome 29 of the Ceratopteris genome. An ortholog of the receptor kinase gene BRASSINOSTEROID INSENSITIVE 1 (BRI1) within this interval encoded a deletion mutation in her7-14 and a missense mutation in her7-19. Three other linked her mutants encoded missense mutations in the same gene, which we name HER7. Consistent with a function as a receptor kinase, HER7-GFP fusion protein localized to the plasma membrane and cytoplasm. Analysis of gene expression showed that brassinosteroid biosynthesis was upregulated in hermaphrodites compared with male gametophytes. Our work demonstrates that HER7 is required for sex determination in Ceratopteris and opens avenues for studying the evolution of antheridiogen systems. 

Creating mutations in maize has provided key foundational information for our mechanistic understanding of genetics, evolution, and even the role of chromosomes as units of inheritance. Chemical mutagenesis is used in biological research to create novel genetic variation. Ethyl methanesulfonate (EMS) is an alkylating agent and a highly potent and frequently used mutagen. EMS mutagenesis can be used to identify genes based on phenotypes induced by mutagenesis (forward genetics) and to validate the functions of genes by independently creating multiple mutant alleles in known genes (reverse genetics). Due to our ability to collect huge quantities of maize pollen and to easily apply pollen to the silks of maize ears to conduct pollination and achieve hundreds of fertilization events, pollen EMS mutagenesis is uniquely facile in maize. While pollen EMS mutagenesis is commonly performed, treatment of maize seeds with EMS is also highly effective, and can be used for certain research objectives that are difficult to achieve with pollen mutagenesis, such as recovering mutant sectors. The alkylation of guanine residues by EMS primarily results in G > A or C > T transitions in the DNA, making the molecular profiling of mutations caused by EMS easy, with an extremely low false positive rate. EMS is hydrophilic, has a moderate half-life in water, and is sensitive to light and high temperatures. With appropriate precautions in research settings, EMS can be relatively safe to handle. Here, we provide an introduction to chemical mutagenesis via EMS, including some history on its use in maize and the considerations for the effective and safe design of mutagenesis experiments with EMS in maize. 

Seed mutagenesis using alkylating chemical agents such as ethyl methanesulfonate (EMS) can generate somatic and germinal mutations in many plant species. In monoecious plants like maize, the sperm- and egg-producing reproductive germlines are derived from distinct cell lineages in the embryo. This separation results in independent mutations inherited via the egg and sperm lineages and prevents the recovery of recessive mutant phenotypes in diploid progeny after the first round of self-pollination. Thus, two generations of self-pollination are required to screen for recessive mutations when conducting seed mutagenesis. The additional time and manual self-pollination make this approach laborious. However, a high mutation rate and the ability to screen for somatic sectors in heterozygous mutant plants and other defined genetic backgrounds make seed mutagenesis an effective but underutilized mutagenesis tool for maize research. This protocol provides the directions and optimization steps to perform effective seed mutagenesis in maize. A high frequency of somatic mutations from seed mutagenesis can be achieved, but comes at the expense of poor and disordered growth, failure to form reproductive structures, and low or no seed production at high EMS concentrations or long contact times. In experiments where germinal mutations are a goal, an optimum dose of EMS is required in the first generation. Maize genetic backgrounds vary in their sensitivity to EMS, requiring some pilot testing in new genetic backgrounds. Researchers using this protocol can carry out seed mutagenesis safely and effectively to develop libraries of mutants or alleles for various experiments. 

In maize, abundant pollen production and easy controlled pollination permit the direct mutagenesis of pollen to produce populations of independent mutant lines. Pollen can be treated with alkylating agents, such as ethyl methanesulfonate (EMS), to induce point mutations. The ease of applying and decontaminating this mutagen after the mutagenesis application and the advantages provided by the mutation spectra for subsequent bioinformatic analysis make EMS an attractive mutagen. We provide a maize pollen mutagenesis protocol with a list of critical supplies, a step-by-step procedure, and troubleshooting tips. Pollen is freshly collected and suspended in an emulsion of EMS and paraffin oil. The slurry of pollen, oil, and EMS is then directly placed on prepared maize silks to perform pollinations. Mutations result during embryogenesis due to replication-dependent mispairing at alkylated residues contributed by sperm nuclei. Thus, each seed bears an independent set of mutations. These progenies can be analyzed directly, as is the case in targeted mutagenesis experiments or the exploration of dominant genetic variation. Alternatively, the progenies of self-pollinated plants can be screened in the next generation to discover novel recessive mutations. In addition to the dose of EMS and contact time, the genetic background of maize can significantly influence outcomes, and some optimization of dose and contact time may be required for a genetic background and specific use case. Although we outline good practices for safe handling of EMS and waste, researchers should consult their local safety officers to ensure safe handling, decontamination, and disposal of EMS, which is toxic. 

Abstract Convenient and economical genotyping methods and simplified bioinformatic workflows are critical for genetic studies and breeding. The declining cost of sequencing library construction, sample multiplexing, and the advent of skim sequencing has reduced costs and enabled large-scale genetic and genomic experiments. Here, we present a simple skim sequencing and bioinformatics pipeline sufficient for various genotyping applications. Our low-depth skim sequencing method costs 21 USD per sample and provides an average of 144k reads. Our approach uses a double-stranded DNA sample to prepare libraries for genome sequencing. We demonstrate various uses for this strategy in maize, a complex and large (2.5 Gbp) genome. DNA from multiple pedigreed populations, including advanced backcrossed progenies, bi-parental populations, near-isogenic lines, and recombinant inbred lines, were used to map loci, detect donor introgressions, and determine introgression haplotypes. Read counts at known polymorphic positions detected donor genotypes even when derived from parents of unknown origin and could localize mutations of phenotypic impact via bulked segregant analysis. Remarkably, the small amount of sequencing data produced were sufficient to identify the haplotypes of introgressions of unknown origin from by comparison to known genotypes. Correct haplotype identification enabled more accurate allele frequencies to be calculated when mapping loci. This is of exceptional value in maize, where a rich collection of mutants from the 20^thcentury are of unknown pedigree. One sentence summaryEconomical and efficient whole genome ultra-low pass sequencing of DNA samples for numerous genetic and genomic applications. 

Abstract Brassinosteroids (BR) and gibberellins (GA) regulate plant height and leaf angle in maize (Zea mays). Mutants with defects in BR or GA biosynthesis or signaling identify components of these pathways and enhance our knowledge about plant growth and development. In this study, we characterized three recessive mutant alleles of GRAS transcription factor 42 (gras42) in maize, a GRAS transcription factor gene orthologous to the DWARF AND LOW TILLERING (DLT) gene of rice (Oryza sativa). These maize mutants exhibited semi-dwarf stature, shorter and wider leaves, and more upright leaf angle. Transcriptome analysis revealed a role for GRAS42 as a determinant of BR signaling. Analysis of the expression consequences from loss of GRAS42 in the gras42-mu1021149 mutant indicated a weak loss of BR signaling in the mutant, consistent with its previously demonstrated role in BR signaling in rice. Loss of BR signaling was also evident by the enhancement of weak BR biosynthetic mutant alleles in double mutants of nana plant1-1 and gras42-mu1021149. The gras42-mu1021149 mutant had little effect on GA-regulated gene expression, suggesting that GRAS42 is not a regulator of core GA signaling genes in maize. Single-cell expression data identified gras42 expressed among cells in the G2/M phase of the cell cycle consistent with its previously demonstrated role in cell cycle gene expression in Arabidopsis (Arabidopsis thaliana). Cis-acting natural variation controlling GRAS42 transcript accumulation was identified by expression genome-wide association study (eGWAS) in maize. Our results demonstrate a conserved role for GRAS42/SCARECROW-LIKE 28 (SCL28)/DLT in BR signaling, clarify the role of this gene in GA signaling, and suggest mechanisms of tillering and leaf angle control by BR. 

Identifying the genetic basis of local adaptation and fitness trade-offs across environments is a central goal of evolutionary biology. Cold acclimation is an adaptive plastic response for surviving seasonal freezing, and costs of acclimation may be a general mechanism for fitness trade-offs across environments in temperate zone species. Starting with locally adapted ecotypes ofArabidopsis thalianafrom Italy and Sweden, we examined the fitness consequences of a naturally occurring functional polymorphism inCBF2. This gene encodes a transcription factor that is a major regulator of cold-acclimated freezing tolerance and resides within a locus responsible for a genetic trade-off for long-term mean fitness. We estimated the consequences of alternate genotypes ofCBF2on 5-y mean fitness and fitness components at the native field sites by comparing near-isogenic lines with alternate genotypes ofCBF2to their genetic background ecotypes. The effects ofCBF2were validated at the nucleotide level using gene-edited lines in the native genetic backgrounds grown in simulated parental environments. The foreignCBF2genotype in the local genetic background reduced long-term mean fitness in Sweden by more than 10%, primarily via effects on survival. In Italy, fitness was reduced by more than 20%, primarily via effects on fecundity. At both sites, the effects were temporally variable and much stronger in some years. The gene-edited lines confirmed thatCBF2encodes the causal variant underlying this genetic trade-off. Additionally, we demonstrated a substantial fitness cost of cold acclimation, which has broad implications for potential maladaptive responses to climate change.