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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 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. 

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. 

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. 

Advances in quantitative genetics have enabled researchers to identify genomic regions associated with changes in phenotype. However, genomic regions can contain hundreds to thousands of genes, and progressing from genomic regions to candidate genes is still challenging. In genome-wide association studies (GWAS) measuring elemental accumulation (ionomic) traits, a mere 5% of loci are associated with a known ionomic gene - indicating that many causal genes are still unknown. To select candidates for the remaining 95% of loci, we developed a method to identify conserved genes underlying GWAS loci in multiple species. For 19 ionomic traits, we identified 14,336 candidates across Arabidopsis, soybean, rice, maize, and sorghum. We calculated the likelihood of candidates with random permutations of the data and determined that most of the top 10% of candidates were orthologous genes linked to GWAS loci across all five species. The candidate list also includes orthologous genes with previously established ionomic functions in Arabidopsis and rice. Our methods highlight the conserved nature of ionomic genetic regulators and enable the identification of previously unknown ionomic genes.