GrainGenes: a data-rich repository for small grains genetics and genomics.

As one of the US Department of Agriculture-Agricultural Research Service flagship databases, GrainGenes (https://wheat.pw.usda.gov) serves the data and community needs of globally distributed small grains researchers for the genetic improvement of the Triticeae family and Avena species that include wheat, barley, rye and oat. GrainGenes accomplishes its mission by continually enriching its cross-linked data content following the findable, accessible, interoperable and reusable principles, enhancing and maintaining an intuitive web interface, creating tools to enable easy data access and establishing data connections within and between GrainGenes and other biological databases to facilitate knowledge discovery. GrainGenes operates within the biological database community, collaborates with curators and genome sequencing groups and contributes to the AgBioData Consortium and the International Wheat Initiative through the Wheat Information System (WheatIS). Interactive and linked content is paramount for successful biological databases and GrainGenes now has 2917 manually curated gene records, including 289 genes and 254 alleles from the Wheat Gene Catalogue (WGC). There are >4.8 million gene models in 51 genome browser assemblies, 6273 quantitative trait loci and >1.4 million genetic loci on 4756 genetic and physical maps contained within 443 mapping sets, complete with standardized metadata. Most notably, 50 new genome browsers that include outputs from the Wheat and Barley PanGenome projects have been created. We provide an example of an expression quantitative trait loci track on the International Wheat Genome Sequencing Consortium Chinese Spring wheat browser to demonstrate how genome browser tracks can be adapted for different data types. To help users benefit more from its data, GrainGenes created four tutorials available on YouTube. GrainGenes is executing its vision of service by continuously responding to the needs of the global small grains community by creating a centralized, long-term, interconnected data repository. Database URL:https://wheat.pw.usda.gov.

The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

Predicting moisture content during maize nixtamalization using machine learning with NIR spectroscopy.

KEY MESSAGE: Moisture content during nixtamalization can be accurately predicted from NIR spectroscopy when coupled with a support vector machine (SVM) model, is strongly modulated by the environment, and has a complex genetic architecture. Lack of high-throughput phenotyping systems for determining moisture content during the maize nixtamalization cooking process has led to difficulty in breeding for this trait. This study provides a high-throughput, quantitative measure of kernel moisture content during nixtamalization based on NIR scanning of uncooked maize kernels. Machine learning was utilized to develop models based on the combination of NIR spectra and moisture content determined from a scaled-down benchtop cook method. A linear support vector machine (SVM) model with a Spearman's rank correlation coefficient of 0.852 between wet laboratory and predicted values was developed from 100 diverse temperate genotypes grown in replicate across two environments. This model was applied to NIR spectra data from 501 diverse temperate genotypes grown in replicate in five environments. Analysis of variance revealed environment explained the highest percent of the variation (51.5%), followed by genotype (15.6%) and genotype-by-environment interaction (11.2%). A genome-wide association study identified 26 significant loci across five environments that explained between 5.04% and 16.01% (average = 10.41%). However, genome-wide markers explained 10.54% to 45.99% (average = 31.68%) of the variation, indicating the genetic architecture of this trait is likely complex and controlled by many loci of small effect. This study provides a high-throughput method to evaluate moisture content during nixtamalization that is feasible at the scale of a breeding program and provides important information about the factors contributing to variation of this trait for breeders and food companies to make future strategies to improve this important processing trait.

Genetic control of kernel compositional variation in a maize diversity panel.

Maize (Zea mays L.) is a multi-purpose row crop grown worldwide, which, over time, has often been bred for increased yield at the detriment of lower composition grain quality. Some knowledge of the genetic factors that affect quality traits has been discovered through the study of classical maize mutants; however, much of the underlying genetic control of these traits and the interaction between these traits remains unknown. To better understand variation that exists for grain compositional traits in maize, we evaluated 501 diverse temperate maize inbred lines in five unique environments and predicted 16 compositional traits (e.g., carbohydrates, protein, and starch) based on the output of near-infrared (NIR) spectroscopy. Phenotypic analysis found substantial variation for compositional traits and the majority of variation was explained by genetic and environmental factors. Correlations and trade-offs among traits in different maize types (e.g., dent, sweetcorn, and popcorn) were explored, and significant differences and meaningful correlations were detected. In total, 22.9-71.0% of the phenotypic variation across these traits could be explained using 2,386,666 single nucleotide polymorphism (SNP) markers generated from whole-genome resequencing data. A genome-wide association study (GWAS) was conducted using these same markers and found 72 statistically significant SNPs for 11 compositional traits. This study provides valuable insights in the phenotypic variation and genetic control underlying compositional traits that can be used in breeding programs for improving maize grain quality.

Natural variation for gene expression responses to abiotic stress in maize.

Plants respond to abiotic stress through a variety of physiological, biochemical, and transcriptional mechanisms. Many genes exhibit altered levels of expression in response to abiotic stress, which requires concerted action of both cis- and trans-regulatory features. In order to study the variability in transcriptome response to abiotic stress, RNA sequencing was performed using 14-day-old maize seedlings of inbreds B73, Mo17, Oh43, PH207 and B37 under control, cold and heat conditions. Large numbers of genes that responded differentially to stress between parental inbred lines were identified. RNA sequencing was also performed on similar tissues of the F1 hybrids produced by crossing B73 and each of the three other inbred lines. By evaluating allele-specific transcript abundance in the F1 hybrids, we were able to measure the abundance of cis- and trans-regulatory variation between genotypes for both steady-state and stress-responsive expression differences. Although examples of trans-regulatory variation were observed, cis-regulatory variation was more common for both steady-state and stress-responsive expression differences. The genes with cis-allelic variation for response to cold or heat stress provided an opportunity to study the basis for regulatory diversity.

Transposable elements contribute to activation of maize genes in response to abiotic stress.

Transposable elements (TEs) account for a large portion of the genome in many eukaryotic species. Despite their reputation as "junk" DNA or genomic parasites deleterious for the host, TEs have complex interactions with host genes and the potential to contribute to regulatory variation in gene expression. It has been hypothesized that TEs and genes they insert near may be transcriptionally activated in response to stress conditions. The maize genome, with many different types of TEs interspersed with genes, provides an ideal system to study the genome-wide influence of TEs on gene regulation. To analyze the magnitude of the TE effect on gene expression response to environmental changes, we profiled gene and TE transcript levels in maize seedlings exposed to a number of abiotic stresses. Many genes exhibit up- or down-regulation in response to these stress conditions. The analysis of TE families inserted within upstream regions of up-regulated genes revealed that between four and nine different TE families are associated with up-regulated gene expression in each of these stress conditions, affecting up to 20% of the genes up-regulated in response to abiotic stress, and as many as 33% of genes that are only expressed in response to stress. Expression of many of these same TE families also responds to the same stress conditions. The analysis of the stress-induced transcripts and proximity of the transposon to the gene suggests that these TEs may provide local enhancer activities that stimulate stress-responsive gene expression. Our data on allelic variation for insertions of several of these TEs show strong correlation between the presence of TE insertions and stress-responsive up-regulation of gene expression. Our findings suggest that TEs provide an important source of allelic regulatory variation in gene response to abiotic stress in maize.

Comprehensive analysis of imprinted genes in maize reveals allelic variation for imprinting and limited conservation with other species.

In plants, a subset of genes exhibit imprinting in endosperm tissue such that expression is primarily from the maternal or paternal allele. Imprinting may arise as a consequence of mechanisms for silencing of transposons during reproduction, and in some cases imprinted expression of particular genes may provide a selective advantage such that it is conserved across species. Separate mechanisms for the origin of imprinted expression patterns and maintenance of these patterns may result in substantial variation in the targets of imprinting in different species. Here we present deep sequencing of RNAs isolated from reciprocal crosses of four diverse maize genotypes, providing a comprehensive analysis that allows evaluation of imprinting at more than 95% of endosperm-expressed genes. We find that over 500 genes exhibit statistically significant parent-of-origin effects in maize endosperm tissue, but focused our analyses on a subset of these genes that had >90% expression from the maternal allele (69 genes) or from the paternal allele (108 genes) in at least one reciprocal cross. Over 10% of imprinted genes show evidence of allelic variation for imprinting. A comparison of imprinting in maize and rice reveals that 13% of genes with syntenic orthologs in both species exhibit conserved imprinting. Genes that exhibit conserved imprinting between maize and rice have elevated nonsynonymous to synonymous substitution ratios compared with other imprinted genes, suggesting a history of more rapid evolution. Together, these data suggest that imprinting only has functional relevance at a subset of loci that currently exhibit imprinting in maize.

Epigenetic and genetic influences on DNA methylation variation in maize populations.

DNA methylation is a chromatin modification that is frequently associated with epigenetic regulation in plants and mammals. However, genetic changes such as transposon insertions can also lead to changes in DNA methylation. Genome-wide profiles of DNA methylation for 20 maize (Zea mays) inbred lines were used to discover differentially methylated regions (DMRs). The methylation level for each of these DMRs was also assayed in 31 additional maize or teosinte genotypes, resulting in the discovery of 1966 common DMRs and 1754 rare DMRs. Analysis of recombinant inbred lines provides evidence that the majority of DMRs are heritable. A local association scan found that nearly half of the DMRs with common variation are significantly associated with single nucleotide polymorphisms found within or near the DMR. Many of the DMRs that are significantly associated with local genetic variation are found near transposable elements that may contribute to the variation in DNA methylation. Analysis of gene expression in the same samples used for DNA methylation profiling identified over 300 genes with expression patterns that are significantly associated with DNA methylation variation. Collectively, our results suggest that DNA methylation variation is influenced by genetic and epigenetic changes that are often stably inherited and can influence the expression of nearby genes.

Genomic distribution of maize facultative heterochromatin marked by trimethylation of H3K27.

Trimethylation of histone H3 Lys-27 (H3K27me3) plays a critical role in regulating gene expression during plant and animal development. We characterized the genome-wide distribution of H3K27me3 in five developmentally distinct tissues in maize (Zea mays) plants of two genetic backgrounds, B73 and Mo17. There were more substantial differences in the genome-wide profile of H3K27me3 between different tissues than between the two genotypes. The tissue-specific patterns of H3K27me3 were often associated with differences in gene expression among the tissues and most of the imprinted genes that are expressed solely from the paternal allele in endosperm are targets of H3K27me3. A comparison of the H3K27me3 targets in rice (Oryza sativa), maize, and Arabidopsis thaliana provided evidence for conservation of the H3K27me3 targets among plant species. However, there was limited evidence for conserved targeting of H3K27me3 in the two maize subgenomes derived from whole-genome duplication, suggesting the potential for subfunctionalization of chromatin regulation of paralogs. Genomic profiling of H3K27me3 in loss-of-function mutant lines for Maize Enhancer of zeste-like2 (Mez2) and Mez3, two of the three putative H3K27me3 methyltransferases present in the maize genome, suggested partial redundancy of this gene family for maintaining H3K27me3 patterns. Only a portion of the targets of H3K27me3 required Mez2 and/or Mez3, and there was limited evidence for functional consequences of H3K27me3 at these targets.

Parent-of-origin effects on gene expression and DNA methylation in the maize endosperm.

Imprinting describes the differential expression of alleles based on their parent of origin. Deep sequencing of RNAs from maize (Zea mays) endosperm and embryo tissue 14 d after pollination was used to identify imprinted genes among a set of ~12,000 genes that were expressed and contained sequence polymorphisms between the B73 and Mo17 genotypes. The analysis of parent-of-origin patterns of expression resulted in the identification of 100 putative imprinted genes in maize endosperm, including 54 maternally expressed genes (MEGs) and 46 paternally expressed genes (PEGs). Three of these genes have been previously identified as imprinted, while the remaining 97 genes represent novel imprinted maize genes. A genome-wide analysis of DNA methylation identified regions with reduced endosperm DNA methylation in, or near, 19 of the 100 imprinted genes. The reduced levels of DNA methylation in endosperm are caused by hypomethylation of the maternal allele for both MEGs and PEGs in all cases tested. Many of the imprinted genes with reduced DNA methylation levels also show endosperm-specific expression patterns. The imprinted maize genes were compared with imprinted genes identified in genome-wide screens of rice (Oryza sativa) and Arabidopsis thaliana, and at least 10 examples of conserved imprinting between maize and each of the other species were identified.

Heritable epigenetic variation among maize inbreds.

Epigenetic variation describes heritable differences that are not attributable to changes in DNA sequence. There is the potential for pure epigenetic variation that occurs in the absence of any genetic change or for more complex situations that involve both genetic and epigenetic differences. Methylation of cytosine residues provides one mechanism for the inheritance of epigenetic information. A genome-wide profiling of DNA methylation in two different genotypes of Zea mays (ssp. mays), an organism with a complex genome of interspersed genes and repetitive elements, allowed the identification and characterization of examples of natural epigenetic variation. The distribution of DNA methylation was profiled using immunoprecipitation of methylated DNA followed by hybridization to a high-density tiling microarray. The comparison of the DNA methylation levels in the two genotypes, B73 and Mo17, allowed for the identification of approximately 700 differentially methylated regions (DMRs). Several of these DMRs occur in genomic regions that are apparently identical by descent in B73 and Mo17 suggesting that they may be examples of pure epigenetic variation. The methylation levels of the DMRs were further studied in a panel of near-isogenic lines to evaluate the stable inheritance of the methylation levels and to assess the contribution of cis- and trans- acting information to natural epigenetic variation. The majority of DMRs that occur in genomic regions without genetic variation are controlled by cis-acting differences and exhibit relatively stable inheritance. This study provides evidence for naturally occurring epigenetic variation in maize, including examples of pure epigenetic variation that is not conditioned by genetic differences. The epigenetic differences are variable within maize populations and exhibit relatively stable trans-generational inheritance. The detected examples of epigenetic variation, including some without tightly linked genetic variation, may contribute to complex trait variation.

The effects of diet on blood glucose, insulin, gastrin and the serum tryptophan: large neutral amino acid ratio in foals.

High carbohydrate diets can affect the health and behaviour of foals, but the mechanisms are not always fully understood. The objective of this study was to compare the effects of feeding a starch and sugar (SS), or a fat (oil) and fibre (FF) rich diet to two groups of eight foals. Diets were fed from 4 to 42 weeks of age, alongside ad libitum forage. Faecal pH levels did not differ significantly between groups and endoscopic examination showed that the gastric mucosa was healthy in both groups at 25 and 42 weeks of age. At 40 weeks of age, SS foals had significantly higher total blood glucose and lower total blood gastrin than FF foals during the 6h period following ingestion of their respective diets, but insulin levels did not differ significantly. The ratio between serum tryptophan and other large neutral amino acids showed a trend towards an interaction between diet and sampling time. The results provide preliminary information about the effects of diet on the physiology of young horses.