Jason G. Wallace

Entering the second century of maize quantitative genetics.

Maize is the most widely grown cereal in the world. In addition to its role in global agriculture, it has also long served as a model organism for genetic research. Maize stands at a genetic crossroads, as it has access to all the tools available for plant genetics but exhibits a genetic architecture more similar to other outcrossing organisms than to self-pollinating crops and model plants. In this review, we summarize recent advances in maize genetics, including the development of powerful populations for genetic mapping and genome-wide association studies (GWAS), and the insights these studies yield on the mechanisms underlying complex maize traits. Most maize traits are controlled by a large number of genes, and linkage analysis of several traits implicates a ‘common gene, rare allele’ model of genetic variation where some genes have many individually rare alleles contributing. Most natural alleles exhibit small effect sizes with little-to-no detectable pleiotropy or epistasis. Additionally, many of these genes are locked away in low-recombination regions that encourage the formation of multi-gene blocks that may underlie maize’s strong heterotic effect. Domestication left strong marks on the maize genome, and some of the differences in trait architectures may be due to different selective pressures over time. Overall, maize’s advantages as a model system make it highly desirable for studying the genetics of outcrossing species, and results from it can provide insight into other such species, including humans.

Association mapping across numerous traits reveals patterns of functional variation in maize.

Phenotypic variation in natural populations results from a combination of genetic effects, environmental effects, and gene-by-environment interactions. Despite the vast amount of genomic data becoming available, many pressing questions remain about the nature of genetic mutations that underlie functional variation. We present the results of combining genome-wide association analysis of 41 different phenotypes in ∼5,000 inbred maize lines to analyze patterns of high-resolution genetic association among of 28.9 million single-nucleotide polymorphisms (SNPs) and ∼800,000 copy-number variants (CNVs). We show that genic and intergenic regions have opposite patterns of enrichment, minor allele frequencies, and effect sizes, implying tradeoffs among the probability that a given polymorphism will have an effect, the detectable size of that effect, and its frequency in the population. We also find that genes tagged by GWAS are enriched for regulatory functions and are ∼50% more likely to have a paralog than expected by chance, indicating that gene regulation and gene duplication are strong drivers of phenotypic variation. These results will likely apply to many other organisms, especially ones with large and complex genomes like maize.

Genome-Wide Association of Carbon and Nitrogen Metabolism in the Maize Nested Association Mapping Population

Genetic variants of maize identify genes and regions that control core carbon and nitrogen metabolism. Carbon (C) and nitrogen (N) metabolism are critical to plant growth and development and are at the basis of crop yield and adaptation. We performed high-throughput metabolite analyses on over 12,000 samples from the nested association mapping population to identify genetic variation in C and N metabolism in maize (Zea mays ssp. mays). All samples were grown in the same field and used to identify natural variation controlling the levels of 12 key C and N metabolites, namely chlorophyll a, chlorophyll b, fructose, fumarate, glucose, glutamate, malate, nitrate, starch, sucrose, total amino acids, and total protein, along with the first two principal components derived from them. Our genome-wide association results frequently identified hits with single-gene resolution. In addition to expected genes such as invertases, natural variation was identified in key C4 metabolism genes, including carbonic anhydrases and a malate transporter. Unlike several prior maize studies, extensive pleiotropy was found for C and N metabolites. This integration of field-derived metabolite data with powerful mapping and genomics resources allows for the dissection of key metabolic pathways, providing avenues for future genetic improvement.

Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments

Pearl millet [Cenchrus americanus (L.) Morrone] is a staple food for more than 90 million farmers in arid and semi-arid regions of sub-Saharan Africa, India and South Asia. We report the ∼1.79 Gb draft whole genome sequence of reference genotype Tift 23D2B1-P1-P5, which contains an estimated 38,579 genes. We highlight the substantial enrichment for wax biosynthesis genes, which may contribute to heat and drought tolerance in this crop. We resequenced and analyzed 994 pearl millet lines, enabling insights into population structure, genetic diversity and domestication. We use these resequencing data to establish marker trait associations for genomic selection, to define heterotic pools, and to predict hybrid performance. We believe that these resources should empower researchers and breeders to improve this important staple crop.

Population Genetics and Structure of a Global Foxtail Millet Germplasm Collection

Foxtail millet [Setaria italica (L.) P. Beauv.] is one among the most ancient crops of dryland agriculture. It is the second most important crop among millets grown for grains or forage. Foxtail millet germplasm resources provide reservoirs of novel alleles and genes for crop improvement that have remained mostly unexplored. We genotyped a set of 190 foxtail millet germplasm accessions (including 155 accessions of the foxtail millet core collection) using genotyping‐by‐sequencing (GBS) for rapid single nucleotide polymorphisms (SNP) characterization to study population genetics and structure, which enable allele mining through association mapping approaches. After filtering a total 350,000 raw SNPs identified across 190 germplasm accessions for minor allele frequency (MAF), coverage for samples and coverage for sites, we retained 181 accessions with 17,714 high‐quality SNPs with ≥5% MAF. Genetic structure analyses revealed that foxtail millet germplasm accessions are structured along both on the basis of races and geographic origin, and the maximum proportion of variation was due to among individuals within populations. Accessions of race indica were less diverse and are highly differentiated from those of maxima and moharia. Genome‐wide linkage disequilibrium (LD) analysis showed on an average LD extends up to ∼150 kbp and varied with individual chromosomes. The utility of the data for performing genome‐wide association studies (GWASs) was tested with plant pigmentation and days to flowering and identified significant marker–trait associations. This SNP data provides a foundation for exploration of foxtail millet diversity and for mining novel alleles and mapping genes for economically important traits.

Association of OLE RNA with bacterial membranes via an RNA-protein interaction

Ornate, large, extremophilic (OLE) RNAs are large, non‐coding transcripts characterized by their ornate secondary structure and presence predominantly in Gram‐positive, extremophilic bacteria. A gene for an OLE‐associated protein (OAP) is almost always located immediately downstream of the OLE gene. OAP has no extensive homology to other proteins and is predicted to form multiple transmembrane domains. We show that this protein forms a ribonucleoprotein complex with OLE RNA using at least 2:1 protein : RNA stoichiometry. A series of truncated OLE RNA constructs was used to establish that most of the RNA can be deleted without eliminating protein binding. Two primary binding sites are present within the RNA, although additional binding determinants exist and extensive structural stabilization is induced by OAP. RNA fluorescence in situ hybridization (FISH) was used in Escherichia coli to demonstrate that ribonucleoprotein complex formation localizes the RNA near cell membranes of this heterologous system. Therefore, the majority of the complex structure formed by OLE RNA may perform a biochemical function that requires membrane localization.

The Genetic Makeup of a Global Barnyard Millet Germplasm Collection

Barnyard millet (Echinochloa spp.) is an important crop for many smallholder farmers in southern and eastern Asia. It is valued for its drought tolerance, rapid maturation, and superior nutritional qualities. Despite these characteristics there are almost no genetic or genomic resources for this crop in either cultivated species [E. colona (L.) Link and E. crus‐galli (L.) P. Beauv.]. Recently, a core collection of 89 barnyard millet accessions was developed at the genebank at the International Crops Research Institute for the Semi‐Arid Tropics (ICRISAT). To enhance the use of this germplasm and genomic research in barnyard millet improvement, we report the genetic characterization of this core collection using whole‐genome genotyping‐by‐sequencing. We identified several thousand single‐nucleotide polymorphisms segregating in the core collection, and we use them to show patterns of population structure and phylogenetic relationships among the accessions. We determine that there are probably four population clusters within the E. colona accessions and three such clusters within E. crus‐galli. These clusters match phylogenetic relationships but by and large do not correspond to classification into individual races or clusters based on morphology. Geospatial data available for a subset of samples indicates that the clusters probably originate from geographic divisions. In all, these data will be useful to breeders working to improve this crop for smallholder farmers. This work also serves as a case study of how modern genomics can rapidly characterize crops, including ones with little to no prior genetic data.

Fast-Flowering Mini-Maize: Seed to Seed in 60 Days.

Two lines of Zea mays were developed as a short-generation model for maize. The Fast-Flowering Mini-Maize (FFMM) lines A and B are robust inbred lines with a significantly shorter generation time, much smaller stature, and better greenhouse adaptation than traditional maize varieties. Five generations a year are typical. FFMM is the result of a modified double-cross hybrid between four fast-flowering lines: Neuffer’s Early ACR (full color), Alexander’s Early Early Synthetic, Tom Thumb Popcorn, and Gaspe Flint, followed by selection for early flowering and desirable morphology throughout an 11-generation selfing regime. Lines A and B were derived from different progeny of the initial hybrid, and crosses between Mini-Maize A and B exhibit heterosis. The ancestry of each genomic region of Mini-Maize A and B was inferred from the four founder populations using genotyping by sequencing. Other genetic and genomic tools for these lines include karyotypes for both lines A and B, kernel genetic markers y1 (white endosperm) and R1-scm2 (purple endosperm and embryo) introgressed into Mini-Maize A, and ∼24× whole-genome resequencing data for Mini-Maize A.

OLE RNA protects extremophilic bacteria from alcohol toxicity

OLE (Ornate, Large, Extremophilic) RNAs represent a recently discovered non-coding RNA class found in extremophilic anaerobic bacteria, including certain human pathogens. OLE RNAs exhibit several unusual characteristics that indicate a potentially novel function, including exceptionally high expression and localization to cell membranes via interaction with a protein partner called OLE-associated protein (OAP). In the current study, new genetic and phenotypic characteristics of OLE RNA from Bacillus halodurans C-125 were established. OLE RNA is transcribed at high levels from its own promoter under normal growth conditions and the transcript is exceptionally stable compared to most other RNAs. Expression is increased by ∼7-fold when cells are exposed to near lethal concentrations of short-chain alcohols such as ethanol or methanol. Strains wherein the genes for OLE and/or OAP are deleted are more susceptible to growth inhibition by alcohol and also become more sensitive to cold. Normal growth characteristics can be restored by expressing the genes for OLE and OAP from plasmids or from elsewhere on the chromosome. Our findings confirm a functional link between OLE and OAP and reveal the importance of a large non-coding RNA in the response to alcohol-induced stress.

Maize YABBY Genes drooping leaf1 and drooping leaf2 Regulate Plant Architecture

Maize drooping leaf genes are necessary for proper leaf development and reside within quantitative trait locus regions for agronomically important leaf architecture traits. Leaf architecture directly influences canopy structure, consequentially affecting yield. We discovered a maize (Zea mays) mutant with aberrant leaf architecture, which we named drooping leaf1 (drl1). Pleiotropic mutations in drl1 affect leaf length and width, leaf angle, and internode length and diameter. These phenotypes are enhanced by natural variation at the drl2 enhancer locus, including reduced expression of the drl2-Mo17 allele in the Mo17 inbred. A second drl2 allele, produced by transposon mutagenesis, interacted synergistically with drl1 mutants and reduced drl2 transcript levels. The drl genes are required for proper leaf patterning, development and cell proliferation of leaf support tissues, and for restricting auricle expansion at the midrib. The paralogous loci encode maize CRABS CLAW co-orthologs in the YABBY family of transcriptional regulators. The drl genes are coexpressed in incipient and emergent leaf primordia at the shoot apex, but not in the vegetative meristem or stem. Genome-wide association studies using maize NAM-RIL (nested association mapping-recombinant inbred line) populations indicated that the drl loci reside within quantitative trait locus regions for leaf angle, leaf width, and internode length and identified rare single nucleotide polymorphisms with large phenotypic effects for the latter two traits. This study demonstrates that drl genes control the development of key agronomic traits in maize.