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Genome-wide association study (GWAS) with single nucleotide polymorphisms (SNPs) has been widely used to explore genetic controls of phenotypic traits. Alternatively, GWAS can use counts of substrings of length k from longer sequencing reads, k-mers, as genotyping data. Using maize cob and kernel color traits, we demonstrated that k-mer GWAS can effectively identify associated k-mers. Co-expression analysis of kernel color k-mers and genes directly found k-mers from known causal genes. Analyzing complex traits of kernel oil and leaf angle resulted in k-mers from both known and candidate genes. A gene encoding a MADS transcription factor was functionally validated by showing that ectopic expression of the gene led to less upright leaves. Evolution analysis revealed most k-mers positively correlated with kernel oil were strongly selected against in maize populations, while most k-mers for upright leaf angle were positively selected. In addition, genomic prediction of kernel oil, leaf angle, and flowering time using k-mer data resulted in a similarly high prediction accuracy to the standard SNP-based method. Collectively, we showed k-mer GWAS is a powerful approach for identifying trait-associated genetic elements. Further, our results demonstrated the bridging role of k-mers for data integration and functional gene discovery., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Root metaxylems are phenotypically diverse structures whose function is particularly important under drought stress. Significant research has dissected the genetic machinery underlying metaxylem phenotypes in dicots, but that of monocots are relatively underexplored. In maize (Zea mays), a robust pipeline integrated a genome-wide association study (GWAS) of root metaxylem phenes under well-watered and water-stress conditions with a gene co-expression network to prioritize the strongest gene candidates. We identified 244 candidate genes by GWAS, of which 103 reside in gene co-expression modules most relevant to xylem development. Several candidate genes may be involved in biosynthetic processes related to the cell wall, hormone signaling, oxidative stress responses, and drought responses. Of those, six gene candidates were detected in multiple root metaxylem phenes in both well-watered and water-stress conditions. We posit that candidate genes that are more essential to network function based on gene co-expression (i.e., hubs or bottlenecks) should be prioritized and classify 33 essential genes for further investigation. Our study demonstrates a new strategy for identifying promising gene candidates and presents several gene candidates that may enhance our understanding of vascular development and responses to drought in cereals., (© 2024 The Author(s). The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

The US standard for maize commercially grown for grain specifies that yellow corn can contain at maximum 5% corn of other colors. Inbred parents of commercial hybrids typically have clear pericarp, but transgressive segregants in breeding populations can display variation in pericarp pigmentation. We identified 10 doubled haploid biparental populations segregating for pigmented pericarp and evaluated qualitative genetic models using chi-square tests of observed and expected frequencies. Pigmentation ranged from light to dark brown color, and pigmentation intensity was quantitatively measured across 1,327 inbred lines using hue calculated from RGB pixel values. Genetic mapping was used to identify loci associated with pigmentation intensity. For 9 populations, pigmentation inheritance best fit a hypothesis of a 2- or 3-gene epistatic model. Significant differences in pigment intensity were observed across populations. W606S-derived inbred lines with the darkest pericarp often had clear glumes, suggesting the presence of a novel P1-rw allele, a hypothesis supported by a significant quantitative trait locus peak at P1. A separate quantitative trait locus region on chromosome 2 between 221.64 and 226.66 Mbp was identified in LH82-derived populations, and the peak near p1 was absent. A genome-wide association study using 416 inbred lines from the Wisconsin Diversity panel with full genome resequencing revealed 4 significant associations including the region near P1. This study supports that pericarp pigmentation among dent maize inbreds can arise by transgressive segregation when pigmentation in the parental generation is absent and is partially explained by functional allelic variation at the P1 locus., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Zeins are commercially important proteins found in corn endosperms. The objective of this study was to evaluate the effect of altering zein levels in corn inbred lines carrying endosperm mutations with differential allelic dosage and analyze the effects on the composition, nutritive value, and starch digestibility of whole-plant corn silage (WPCS) at 5 storage lengths. Three inbred lines carrying 3 different endosperm modifiers (opaque-2 [o2], floury-2 [fl2], and soft endosperm-1 [h1]) were pollinated with 2 pollen sources to form pairs of near-isogenic lines with either 2 or 3 doses of the mutant allele for each endosperm modifier. The experiment was designed as a split-plot design with 3 replications. Pollinated genotype was the main plot factor, and storage length was the subplot-level factor. Agronomic precautions were taken to mimic hybrid WPCS to the extent possible. Samples were collected at approximately 30% dry matter (DM) using a forage harvester and ensiled in heat-sealed plastic bags for 0, 30, 60, 120, and 240 d. Thus, the experiment consisted of 30 treatments (6 genotypes × 5 storage lengths) and 90 ensiling units (3 replications per treatment). Measurements included nutrient analysis, including crude protein, soluble crude protein, amylase-treated neutral detergent fiber, acid detergent fiber, lignin, starch, fermentation end products, zein concentration, and in vitro starch digestibility (ivSD). The nutritional profile of the inbred-based silage samples was similar to hybrid values reported in literature. Significant differences were found in fresh (unfermented) sample kernels for endosperm vitreousness and zein profiles between and within isogenic pairs. The o2 homozygous (3 doses of mutant allele) had the highest reduction in vitreousness level (74.5 to 38%) and zein concentration (6.2 to 4.7% of DM) compared with the heterozygous counterpart (2 doses of mutant allele). All genotypes showed significant reduction of total zeins and α-zeins during progressive storage length. In vitro starch digestibility increased with storage length and had significant effects of genotype and storage length but not for genotype by storage length interaction, which suggests that the storage period did not attenuate the difference in ivSD between near-isogenic pairs caused by zeins in WPCS. Both total zeins and α-zeins showed a strong negative correlation with ivSD, which agrees with the general hypothesis that the degradation of zeins increases ruminal starch degradability. Homozygous o2 was the only mutant with significantly higher ivSD compared with the heterozygous version, which suggests that, if all other conditions remain constant in a WPCS systems, substantial reductions in endosperm α-zeins are required to significantly improve ivSD in the silo., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Source and sink interactions play a critical but mechanistically poorly understood role in the regulation of senescence. To disentangle the genetic and molecular mechanisms underlying source-sink-regulated senescence (SSRS), we performed a phenotypic, transcriptomic, and systems genetics analysis of senescence induced by the lack of a strong sink in maize (Zea mays). Comparative analysis of genotypes with contrasting SSRS phenotypes revealed that feedback inhibition of photosynthesis, a surge in reactive oxygen species, and the resulting endoplasmic reticulum (ER) stress were the earliest outcomes of weakened sink demand. Multienvironmental evaluation of a biparental population and a diversity panel identified 12 quantitative trait loci and 24 candidate genes, respectively, underlying SSRS. Combining the natural diversity and coexpression networks analyses identified 7 high-confidence candidate genes involved in proteolysis, photosynthesis, stress response, and protein folding. The role of a cathepsin B like protease 4 (ccp4), a candidate gene supported by systems genetic analysis, was validated by analysis of natural alleles in maize and heterologous analyses in Arabidopsis (Arabidopsis thaliana). Analysis of natural alleles suggested that a 700-bp polymorphic promoter region harboring multiple ABA-responsive elements is responsible for differential transcriptional regulation of ccp4 by ABA and the resulting variation in SSRS phenotype. We propose a model for SSRS wherein feedback inhibition of photosynthesis, ABA signaling, and oxidative stress converge to induce ER stress manifested as programed cell death and senescence. These findings provide a deeper understanding of signals emerging from loss of sink strength and offer opportunities to modify these signals to alter senescence program and enhance crop productivity., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genotype-by-environment (G×E) interactions can significantly affect crop performance and stability. Investigating G×E requires extensive data sets with diverse cultivars tested over multiple locations and years. The Genomes-to-Fields (G2F) Initiative has tested maize hybrids in more than 130 year-locations in North America since 2014. Here, we curate and expand this data set by generating environmental covariates (using a crop model) for each of the trials. The resulting data set includes DNA genotypes and environmental data linked to more than 70,000 phenotypic records of grain yield and flowering traits for more than 4000 hybrids. We show how this valuable data set can serve as a benchmark in agricultural modeling and prediction, paving the way for countless G×E investigations in maize. We use multivariate analyses to characterize the data set's genetic and environmental structure, study the association of key environmental factors with traits, and provide benchmarks using genomic prediction models., (© 2023. The Author(s).)

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Root anatomical phenotypes present a promising yet underexploited avenue to deliver major improvements in yield and climate resilience of crops by improving water and nutrient uptake. For instance, the formation of root cortical aerenchyma (RCA) significantly increases soil exploration and resource capture by reducing the metabolic costs of root tissue. A key bottleneck in studying such phenotypes has been the lack of robust high-throughput anatomical phenotyping platforms. We exploited a phenotyping approach based on laser ablation tomography, termed Anatomics , to quantify variation in RCA formation of 436 diverse maize lines in the field. Results revealed a significant and heritable variation for RCA formation. Genome-wide association studies identified a single-nucleotide polymorphism mapping to a root cortex-expressed gene-encoding transcription factor bHLH121. Functional studies identified that the bHLH121 Mu transposon mutant line and CRISPR/Cas9 loss-of-function mutant line showed reduced RCA formation, whereas an overexpression line exhibited significantly greater RCA formation when compared to the wild-type line. Characterization of these lines under suboptimal water and nitrogen availability in multiple soil environments revealed that bHLH121 is required for RCA formation developmentally as well as under studied abiotic stress. Overall functional validation of the bHLH121 gene's importance in RCA formation provides a functional marker to select varieties with improved soil exploration and thus yield under suboptimal conditions.

Large-scale investigations of maize kernel traits important to researchers, breeders, and processors require high throughput methods, which are presently lacking. To address this bottleneck, we developed a novel flatbed platform that automatically acquires and analyzes multiwavelength near-infrared (NIR hyperspectral) images of maize kernels precisely enough to support robust predictions of protein content, density, and endosperm vitreousness. The upward facing-camera design and the automated ability to analyze the embryo or abgerminal sides of each individual kernel in a sample with the appropriate side-specific model helped to produce a superior combination of throughput and prediction accuracy compared to other single-kernel platforms. Protein was predicted to within 0.85% (root mean square error of prediction), density to within 0.038 g/cm 3 , and endosperm vitreousness percentage to within 6.3%. Kernel length and width were also accurately measured so that each kernel in a rapidly scanned sample was comprehensively characterized., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Stiff Stalk heterotic pool is a foundation of US maize seed parent germplasm and has been heavily utilized by both public and private maize breeders since its inception in the 1930s. Flowering time and plant height are critical characteristics for both inbred parents and their test crossed hybrid progeny. To study these traits, a 6-parent multiparent advanced generation intercross population was developed including maize inbred lines B73, B84, PHB47 (B37 type), LH145 (B14 type), PHJ40 (novel early Stiff Stalk), and NKH8431 (B73/B14 type). A set of 779 doubled haploid lines were evaluated for flowering time and plant height in 2 field replicates in 2016 and 2017, and a subset of 689 and 561 doubled haploid lines were crossed to 2 testers, respectively, and evaluated as hybrids in 2 locations in 2018 and 2019 using an incomplete block design. Markers were derived from a practical haplotype graph built from the founder whole genome assemblies and genotype-by-sequencing and exome capture-based sequencing of the population. Genetic mapping utilizing an update to R/qtl2 revealed differing profiles of significant loci for both traits between 635 of the DH lines and 2 sets of 570 and 471 derived hybrids. Genomic prediction was used to test the feasibility of predicting hybrid phenotypes based on the per se data. Predictive abilities were highest on direct models trained using the data they would predict (0.55-0.63), and indirect models trained using per se data to predict hybrid traits had slightly lower predictive abilities (0.49-0.55). Overall, this finding is consistent with the overlapping and nonoverlapping significant quantitative trait loci found within the per se and hybrid populations and suggests that selections for phenology traits can be made effectively on doubled haploid lines before hybrid data is available., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Crops with reduced nutrient and water requirements are urgently needed in global agriculture. Root growth angle plays an important role in nutrient and water acquisition. A maize diversity panel of 481 genotypes was screened for variation in root angle employing a high-throughput field phenotyping platform. Genome-wide association mapping identified several single nucleotide polymorphisms (SNPs) associated with root angle, including one located in the root expressed CBL-interacting serine/threonine-protein kinase 15 (ZmCIPK15) gene (LOC100285495). Reverse genetic studies validated the functional importance of ZmCIPK15, causing a approximately 10° change in root angle in specific nodal positions. A steeper root growth angle improved nitrogen capture in silico and in the field. OpenSimRoot simulations predicted at 40 days of growth that this change in angle would improve nitrogen uptake by 11% and plant biomass by 4% in low nitrogen conditions. In field studies under suboptimal N availability, the cipk15 mutant with steeper growth angles had 18% greater shoot biomass and 29% greater shoot nitrogen accumulation compared to the wild type after 70 days of growth. We propose that a steeper root growth angle modulated by ZmCIPK15 will facilitate efforts to develop new crop varieties with optimal root architecture for improved performance under edaphic stress., (© 2021 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The stiff-stalk heterotic group in Maize (Zea mays L.) is an important source of inbreds used in U.S. commercial hybrid production. Founder inbreds B14, B37, B73, and, to a lesser extent, B84, are found in the pedigrees of a majority of commercial seed parent inbred lines. We created high-quality genome assemblies of B84 and four expired Plant Variety Protection (ex-PVP) lines LH145 representing B14, NKH8431 of mixed descent, PHB47 representing B37, and PHJ40, which is a Pioneer Hi-Bred International (PHI) early stiff-stalk type. Sequence was generated using long-read sequencing achieving highly contiguous assemblies of 2.13-2.18 Gbp with N50 scaffold lengths >200 Mbp. Inbred-specific gene annotations were generated using a core five-tissue gene expression atlas, whereas transposable element (TE) annotation was conducted using de novo and homology-directed methodologies. Compared with the reference inbred B73, synteny analyses revealed extensive collinearity across the five stiff-stalk genomes, although unique components of the maize pangenome were detected. Comparison of this set of stiff-stalk inbreds with the original Iowa Stiff Stalk Synthetic breeding population revealed that these inbreds represent only a proportion of variation in the original stiff-stalk pool and there are highly conserved haplotypes in released public and ex-Plant Variety Protection inbreds. Despite the reduction in variation from the original stiff-stalk population, substantial genetic and genomic variation was identified supporting the potential for continued breeding success in this pool. The assemblies described here represent stiff-stalk inbreds that have historical and commercial relevance and provide further insight into the emerging maize pangenome., (© 2021 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

High winter mortality limits biomass yield of lowland switchgrass (Panicum virgatum L.) planted in the northern latitudes of North America. Breeding of cold tolerant switchgrass cultivars requires many years due to its perennial growth habit and the unpredictable winter selection pressure that is required to identify winter-hardy individuals. Identification of causal genetic variants for winter survivorship would accelerate the improvement of switchgrass biomass production. The objective of this study was to identify allelic variation associated with winter survivorship in lowland switchgrass populations using bulk segregant analysis (BSA). Twenty-nine lowland switchgrass populations were evaluated for winter survival at two locations in southern Wisconsin and 21 populations with differential winter survivorship were used for BSA. A maximum of 10% of the individuals (8-20) were bulked to create survivor and nonsurvivor DNA pools from each population and location. The DNA pools were evaluated using exome capture sequencing, and allele frequencies were used to conduct statistical tests. The BSA tests revealed nine quatitative trait loci (QTL) from tetraploid populations and seven QTL from octoploid populations. Many QTL were population-specific, but some were identified in multiple populations that originated across a broad geographic landscape. Four QTL (at positions 88 Mb on chromosome 2N, 115 Mb on chromosome 5K, and 1 and 100 Mb on chromosome 9N) were potentially the most useful QTL. Markers associated with winter survivorship in this study can be used to accelerate breeding cycles of lowland switchgrass populations and should lead to improvements in adaptation within USDA hardiness zones 4 and 5., (© 2021 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Background: The maize inbred line A188 is an attractive model for elucidation of gene function and improvement due to its high embryogenic capacity and many contrasting traits to the first maize reference genome, B73, and other elite lines. The lack of a genome assembly of A188 limits its use as a model for functional studies., Results: Here, we present a chromosome-level genome assembly of A188 using long reads and optical maps. Comparison of A188 with B73 using both whole-genome alignments and read depths from sequencing reads identify approximately 1.1 Gb of syntenic sequences as well as extensive structural variation, including a 1.8-Mb duplication containing the Gametophyte factor1 locus for unilateral cross-incompatibility, and six inversions of 0.7 Mb or greater. Increased copy number of carotenoid cleavage dioxygenase 1 (ccd1) in A188 is associated with elevated expression during seed development. High ccd1 expression in seeds together with low expression of yellow endosperm 1 (y1) reduces carotenoid accumulation, accounting for the white seed phenotype of A188. Furthermore, transcriptome and epigenome analyses reveal enhanced expression of defense pathways and altered DNA methylation patterns of the embryonic callus., Conclusions: The A188 genome assembly provides a high-resolution sequence for a complex genome species and a foundational resource for analyses of genome variation and gene function in maize. The genome, in comparison to B73, contains extensive intra-species structural variations and other genetic differences. Expression and network analyses identify discrete profiles for embryonic callus and other tissues.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jarquin, de Leon, Romay, Bohn, Buckler, Ciampitti, Edwards, Ertl, Flint-Garcia, Gore, Graham, Hirsch, Holland, Hooker, Kaeppler, Knoll, Lee, Lawrence-Dill, Lynch, Moose, Murray, Nelson, Rocheford, Schnable, Schnable, Smith, Springer, Thomison, Tuinstra, Wisser, Xu, Yu and Lorenz.)

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., (Published by Oxford University Press on behalf of Genetics Society of America 2021. This work is written by US Government employees and is in the public domain in the US.)

Single-parent expression (SPE) is defined as gene expression in only one of the two parents. SPE can arise from differential expression between parental alleles, termed non-presence/absence (non-PAV) SPE, or from the physical absence of a gene in one parent, termed PAV SPE. We used transcriptome data of diverse Zea mays (maize) inbreds and hybrids, including 401 samples from five different tissues, to test for differences between these types of SPE genes. Although commonly observed, SPE is highly genotype and tissue specific. A positive correlation was observed between the genetic distance of the two inbred parents and the number of SPE genes identified. Regulatory analysis showed that PAV SPE and non-PAV SPE genes are mainly regulated by cis effects, with a small fraction under trans regulation. Polymorphic transposable element insertions in promoter sequences contributed to the high level of cis regulation for PAV SPE and non-PAV SPE genes. PAV SPE genes were more frequently expressed in hybrids than non-PAV SPE genes. The expression of parentally silent alleles in hybrids of non-PAV SPE genes was relatively rare but occurred in most hybrids. Non-PAV SPE genes with expression of the silent allele in hybrids are more likely to exhibit above high parent expression level than hybrids that do not express the silent allele, leading to non-additive expression. This study provides a comprehensive understanding of the nature of non-PAV SPE and PAV SPE genes and their roles in gene expression complementation in maize hybrids., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Key Message: Significant introgression-by-environment interactions are observed for traits throughout development from small introgressed segments of the genome. Relatively small genomic introgressions containing quantitative trait loci can have significant impacts on the phenotype of an individual plant. However, the magnitude of phenotypic effects for the same introgression can vary quite substantially in different environments due to introgression-by-environment interactions. To study potential patterns of introgression-by-environment interactions, fifteen near-isogenic lines (NILs) with > 90% B73 genetic background and multiple Mo17 introgressions were grown in 16 different environments. These environments included five geographical locations with multiple planting dates and multiple planting densities. The phenotypic impact of the introgressions was evaluated for up to 26 traits that span different growth stages in each environment to assess introgression-by-environment interactions. Results from this study showed that small portions of the genome can drive significant genotype-by-environment interaction across a wide range of vegetative and reproductive traits, and the magnitude of the introgression-by-environment interaction varies across traits. Some introgressed segments were more prone to introgression-by-environment interaction than others when evaluating the interaction on a whole plant basis throughout developmental time, indicating variation in phenotypic plasticity throughout the genome. Understanding the profile of introgression-by-environment interaction in NILs is useful in consideration of how small introgressions of QTL or transgene containing regions might be expected to impact traits in diverse environments.

Invited for this month's cover is the research team from the D.O.E. Great Lake Bioenergy Research Center (GLBRC) at the University of Wisconsin-Madison. The cover image shows how a diverse team with expertise in many different fields works together in an integrated fashion to address complex problems. Only when the whole system, from field to the liquid fuels and co-products, is assessed, can we identify the key parameters needed to design an economically viable biorefinery-based economy. Cover art by Chelsea Mamott. The Full Paper itself is available at 10.1002/cssc.201903345., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The hydroxycinnamic acids p-coumaric acid (pCA) and ferulic acid (FA) add diversity to the portfolio of products produced by using grass-fed lignocellulosic biorefineries. The level of lignin-bound pCA in Zea mays was modified by the alteration of p-coumaroyl-CoA monolignol transferase expression. The biomass was processed in a lab-scale alkaline-pretreatment biorefinery process and the data were used for a baseline technoeconomic analysis to determine where to direct future research efforts to couple plant design to biomass utilization processes. It is concluded that future plant engineering efforts should focus on strategies that ramp up accumulation of one type of hydroxycinnamate (pCA or FA) predominantly and suppress that of the other. Technoeconomic analysis indicates that target extraction titers of one hydroxycinnamic acid need to be >50 g kg -1 biomass, at least five times higher than observed titers for the impure pCA/FA product mixture from wild-type maize. The technical challenge for process engineers is to develop a viable process that requires more than 80 % reduction of the isolation costs., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Objectives: Advanced tools and resources are needed to efficiently and sustainably produce food for an increasing world population in the context of variable environmental conditions. The maize genomes to fields (G2F) initiative is a multi-institutional initiative effort that seeks to approach this challenge by developing a flexible and distributed infrastructure addressing emerging problems. G2F has generated large-scale phenotypic, genotypic, and environmental datasets using publicly available inbred lines and hybrids evaluated through a network of collaborators that are part of the G2F's genotype-by-environment (G × E) project. This report covers the public release of datasets for 2014-2017., Data Description: Datasets include inbred genotypic information; phenotypic, climatic, and soil measurements and metadata information for each testing location across years. For a subset of inbreds in 2014 and 2015, yield component phenotypes were quantified by image analysis. Data released are accompanied by README descriptions. For genotypic and phenotypic data, both raw data and a version without outliers are reported. For climatic data, a version calibrated to the nearest airport weather station and a version without outliers are reported. The 2014 and 2015 datasets are updated versions from the previously released files [1] while 2016 and 2017 datasets are newly available to the public.

Small RNAs (sRNAs) regulate gene expression, play important roles in epigenetic pathways, and are hypothesized to contribute to hybrid vigor in plants. Prior investigations have provided valuable insights into associations between sRNAs and heterosis, often using a single hybrid genotype or tissue, but our understanding of the role of sRNAs and their potential value to plant breeding are limited by an incomplete picture of sRNA variation between diverse genotypes and development stages. Here, we provide a deep exploration of sRNA variation and inheritance among a panel of 108 maize ( Zea mays ) samples spanning five tissues from eight inbred parents and 12 hybrid genotypes, covering a spectrum of heterotic groups, genetic variation, and levels of heterosis for various traits. We document substantial developmental and genotypic influences on sRNA expression, with varying patterns for 21-nucleotide (nt), 22-nt, and 24-nt sRNAs. We provide a detailed view of the distribution of sRNAs in the maize genome, revealing a complex makeup that also shows developmental plasticity, particularly for 22-nt sRNAs. sRNAs exhibited substantially more variation between inbreds as compared with observed variation for gene expression. In hybrids, we identify locus-specific examples of nonadditive inheritance, mostly characterized as partial or complete dominance, but rarely outside the parental range. However, the global abundance of 21-nt, 22-nt, and 24-nt sRNAs varies very little between inbreds and hybrids, suggesting that hybridization affects sRNA expression principally at specific loci rather than on a global scale. This study provides a valuable resource for understanding the potential role of sRNAs in hybrid vigor., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Image analysis methods for measuring crop phenotypes may replace traditional measurements if they more efficiently and reliably capture similar or superior information. This study used a recreational-grade unmanned aerial vehicle carrying a spectrally-modified consumer-grade camera to collect images in which each pixel value is a vegetation index based on the normalized difference between the blue and near infrared wavelength bands (BNDVI). The subjects of the study were Zea mays hybrids with good yield potential grown in 4-row plots. Flights were conducted at least once per week during three successive growing seasons in south-central Wisconsin. Average BNDVI for each plot (genotype) rose steadily through June, peaked in July, and then declined as plants matured. BNDVI histograms changed shape over the season as the canopy concealed soil, became more uniformly green, then senesced. Principal Components Analysis (PCA) captured the change in histogram shape. PC1 represented canopy closure. PC2 represented the mean of the BNDVI distribution. PC3 represented the spread of the distribution. Correlation analysis showed that flowering time correlated with PC2 and PC3 best (r ≈ 0.5) a few days before the event (day in which 50% of the plants exhibited tassels). Three ears were picked from each plot to quantify kernel dimensions by image analysis before each plot was mechanically harvested to determine grain weight per plot. Correlations between this measurement of yield and PC2 were low in June but exceeded 0.4 within 10 days after flowering. Kernel length correlated similarly with PC2. The correlation between PC2 and kernel thickness displayed a similar but inverted time course. These results indicate that greater mid-season BNDVI values correlate positively with yield comprised of tall, thin kernels. Partial least squares regression performed on the BNDVI time courses predicted flowering time (r = 0.54-0.79) and yield (r = 0.4-0.69). This three-year experiment demonstrated that readily available hardware and software can create a phenotyping platform capable of predicting maize flowering time, yield, and kernel dimensions to a useful degree., (Copyright © 2019 Wu, Miller, de Leon, Kaeppler and Spalding.)

sugary enhancer1 ( se1 ) is a naturally occurring mutant allele involved in starch metabolism in maize endosperm. It is a recessive modifier of sugary1 ( su1 ) and commercially important in modern sweet corn breeding, but its molecular identity and mode of action remain unknown. Here, we developed a pair of near-isogenic lines, W822Gse ( su1-ref/su1-ref se1/se1 ) and W822GSe ( su1-ref/su1-ref Se1/Se1 ), that Mendelize the se1 phenotype in an su1-ref background. W822Gse kernels have lower starch and higher water soluble polysaccharide and sugars than W822GSe kernels. Using high-resolution genetic mapping, we found that wild-type Se1 is a gene Zm00001d007657 on chromosome 2 and a deletion of this gene causes the se1 phenotype. Comparative metabolic profiling of seed tissue between these 2 isolines revealed the remarkable difference in carbohydrate metabolism, with sucrose and maltose highly accumulated in the mutant. Se1 is predominantly expressed in the endosperm, with low expression in leaf and root tissues. Differential expression analysis identified genes enriched in both starch biosynthesis and degradation processes, indicating a pleiotropic regulatory effect of se1 Repressed expression of Se1 and Su1 in RNA interference-mediated transgenic maize validates that deletion of the gene identified as Se1 is a true causal gene responsible for the se1 phenotype. The findings contribute to our understanding of starch metabolism in cereal crops., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Premature senescence in annual crops reduces yield, while delayed senescence, termed stay-green, imposes positive and negative impacts on yield and nutrition quality. Despite its importance, scant information is available on the genetic architecture of senescence in maize ( Zea mays ) and other cereals. We combined a systematic characterization of natural diversity for senescence in maize and coexpression networks derived from transcriptome analysis of normally senescing and stay-green lines. Sixty-four candidate genes were identified by genome-wide association study (GWAS), and 14 of these genes are supported by additional evidence for involvement in senescence-related processes including proteolysis, sugar transport and signaling, and sink activity. Eight of the GWAS candidates, independently supported by a coexpression network underlying stay-green, include a trehalose-6-phosphate synthase, a NAC transcription factor, and two xylan biosynthetic enzymes. Source-sink communication and the activity of cell walls as a secondary sink emerge as key determinants of stay-green. Mutant analysis supports the role of a candidate encoding Cys protease in stay-green in Arabidopsis ( Arabidopsis thaliana ), and analysis of natural alleles suggests a similar role in maize. This study provides a foundation for enhanced understanding and manipulation of senescence for increasing carbon yield, nutritional quality, and stress tolerance of maize and other cereals., (© 2019 American Society of Plant Biologists. All rights reserved.)

The lowland ecotype of switchgrass has generated considerable interest because of its higher biomass yield and late flowering characteristics compared to the upland ecotype. However, lowland ecotypes planted in northern latitudes exhibit very low winter survival. Implementation of genomic selection could potentially enhance switchgrass breeding for winter survival by reducing generation time while eliminating the dependence on weather. The objectives of this study were to assess the potential of genomic selection for winter survival in lowland switchgrass by combining multiple populations in the training set and applying the selected model in two independent testing datasets for validation. Marker data were generated using exome capture sequencing. Validation was conducted using (1) indirect indicators of winter adaptation based on geographic and climatic variables of accessions from different source locations and (2) winter survival estimates of the phenotype. The prediction accuracies were significantly higher when the training dataset comprising all populations was used in fivefold cross validation but its application was not useful in the independent validation dataset. Nevertheless, modeling for population heterogeneity improved the prediction accuracy to some extent but the genetic relationship between the training and validation populations was found to be more influential. The predicted winter survival of lowland switchgrass indicated latitudinal and longitudinal variability, with the northeast USA the region for most cold tolerant lowland populations. Our results suggested that GS could provide valuable opportunities for improving winter survival and accelerate the lowland switchgrass breeding programs toward the development of cold tolerant cultivars suitable for northern latitudes., (Copyright © 2019 Poudel et al.)

Use of a single reference genome for genome-wide association studies (GWAS) limits the gene space represented to that of a single accession. This limitation can complicate identification and characterization of genes located within presence-absence variations (PAVs). In this study, we present the draft de novo genome assembly of 'PHJ89', an 'Oh43'-type inbred line of maize ( L.). From three separate reference genome assemblies ('B73', 'PH207', and PHJ89) that represent the predominant germplasm groups of maize, we generated three separate whole-seedling gene expression profiles and single nucleotide polymorphism (SNP) matrices from a panel of 942 diverse inbred lines. We identified 34,447 (B73), 39,672 (PH207), and 37,436 (PHJ89) transcripts that are not present in the respective reference genome assemblies. Genome-wide association studies were conducted in the 942 inbred panel with both the SNP and expression data values to map (SCMV) resistance. Highlighting the impact of alternative reference genomes in gene discovery, the GWAS results for SCMV resistance with expression values as a surrogate measure of PAV resulted in robust detection of the physical location of a known resistance gene when the B73 reference that contains the gene was used, but not the PH207 reference. This study provides the valuable resource of the Oh43-type PHJ89 genome assembly as well as SNP and expression data for 942 individuals generated from three different reference genomes., (© 2019 The Author(s).)

Low-temperature related abiotic stress is an important factor affecting winter survival in lowland switchgrass when grown in northern latitudes in the United States. A better understanding of the genetic architecture of freezing tolerance in switchgrass will aid the development of lowland switchgrass cultivars with improved winter survival. The objectives of this study were to conduct a freezing tolerance assessment, generate a genetic map using single nucleotide polymorphism (SNP) markers, and identify QTL (quantitative trait loci) associated with freezing tolerance in a lowland × upland switchgrass population. A pseudo-F 2 mapping population was generated from an initial cross between the lowland population Ellsworth and the upland cultivar Summer. The segregating progenies were screened for freezing tolerance in a controlled-environment facility. Two clonal replicates of each genotype were tested at six different treatment temperatures ranging from -15 to -5°C at an interval of 2°C for two time periods. Tiller emergence (days) and tiller number were recorded following the recovery of each genotype with the hypothesis that upland genotype is the source for higher tiller number and early tiller emergence. Survivorship of the pseudo-F 2 population ranged from 89% at -5°C to 5% at -15°C with an average LT 50 of -9.7°C. Genotype had a significant effect on all traits except tiller number at -15°C. A linkage map was constructed from bi-allelic single nucleotide polymorphism markers generated using exome capture sequencing. The final map consisted of 1618 markers and 2626 cM, with an average inter-marker distance of 1.8 cM. Six significant QTL were identified, one each on chromosomes 1K, 5K, 5N, 6K, 6N, and 9K, for the following traits: tiller number, tiller emergence days and LT 50 . A comparative genomics study revealed important freezing tolerance genes/proteins, such as COR47, DREB2B, zinc finger-CCCH, WRKY, GIGANTEA, HSP70, and NRT2, among others that reside within the 1.5 LOD confidence interval of the identified QTL.

Background: Maize stover is an important source of crop residues and a promising sustainable energy source in the United States. Stalk is the main component of stover, representing about half of stover dry weight. Characterization of genetic determinants of stalk traits provide a foundation to optimize maize stover as a biofuel feedstock. We investigated maize natural genetic variation in genome-wide association studies (GWAS) to detect candidate genes associated with traits related to stalk biomass (stalk diameter and plant height) and stalk anatomy (rind thickness, vascular bundle density and area)., Results: Using a panel of 942 diverse inbred lines, 899,784 RNA-Seq derived single nucleotide polymorphism (SNP) markers were identified. Stalk traits were measured on 800 members of the panel in replicated field trials across years. GWAS revealed 16 candidate genes associated with four stalk traits. Most of the detected candidate genes were involved in fundamental cellular functions, such as regulation of gene expression and cell cycle progression. Two of the regulatory genes (Zmm22 and an ortholog of Fpa) that were associated with plant height were previously shown to be involved in regulating the vegetative to floral transition. The association of Zmm22 with plant height was confirmed using a transgenic approach. Transgenic lines with increased expression of Zmm22 showed a significant decrease in plant height as well as tassel branch number, indicating a pleiotropic effect of Zmm22., Conclusion: Substantial heritable variation was observed in the association panel for stalk traits, indicating a large potential for improving useful stalk traits in breeding programs. Genome-wide association analyses detected several candidate genes associated with multiple traits, suggesting common regulatory elements underlie various stalk traits. Results of this study provide insights into the genetic control of maize stalk anatomy and biomass.

Background: Imbibition (uptake of water by a dry seed) initiates the germination process. An automated method for quantifying imbibition would enable research on the genetic elements that influence the underlying hydraulic and biochemical processes. In the case of crop research, a high throughput imbibition assay could be used to investigate seed quality topics or to improve yield by selecting varieties with superior germination characteristics., Results: An electronic force transducer measured imbibition of single maize kernels with very high resolution but low throughput. An image analysis method was devised to achieve high throughput and sufficient resolution. A transparent fixture held 90 maize kernels in contact with water on the imaging window of a flatbed document scanner that produced an image of the kernels automatically every 10 min for 22 h. Custom image analysis software measured the area A of each indexed kernel in each image to produce imbibition time courses. The ultimate change in area (ΔA) ranged from 19.3 to 23.4% in a population of 72 hybrids derived from 9 inbred parents. Kernel area as a function of time was fit well by A t = A f 1 - e - k t where A f is the final kernel area. The swelling coefficient, k , ranged from 0.098 to 0.159 h -1 across the genotypes. The full diallel structure of the population enabled maternal genotype effects to be assessed. In a separate experiment, measurements of kernels of the same 25 inbreds produced in three different years demonstrated that production and storage variables affected imbibition much less than genotype. In a third experiment, measurements of 30 diverse inbred lines showed that k varied inversely with germination time (r = - 0.7) and directly with germination percentage (r = 0.7)., Conclusions: Nonspecialized imaging hardware and custom analysis software running on public cyber infrastructure form a low-cost platform for measuring seed imbibition with high resolution and throughput. We measured imbibition of thousands of kernels to determine that genotype influenced imbibition of maize kernels much more than seed production and storage environments. In some hybrids, k depended on which inbred parent was maternal. Quantitative relationships between k and germination traits were discovered.

Switchgrass ( L.) is a promising herbaceous energy crop, but further gains in biomass yield and quality must be achieved to enable a viable bioenergy industry. Developing DNA markers can contribute to such progress, but depiction of genetic bases should be reliable, involving simple additive marker effects and also interactions with genetic backgrounds (e.g., ecotypes) or synergies with other markers. We analyzed plant height, C content, N content, and mineral concentration in a diverse panel consisting of 512 genotypes of upland and lowland ecotypes. We performed association analyses based on exome capture sequencing and tested 439,170 markers for marginal effects, 83,290 markers for marker × ecotype interactions, and up to 311,445 marker pairs for pairwise interactions. Analyses of pairwise interactions focused on subsets of marker pairs preselected on the basis of marginal marker effects, gene ontology annotation, and pairwise marker associations. Our tests identified 12 significant effects. Homology and gene expression information corroborated seven effects and indicated plausible causal pathways: flowering time and lignin synthesis for plant height; plant growth and senescence for C content and mineral concentration. Four pairwise interactions were detected, including three interactions preselected on the basis of pairwise marker correlations. Furthermore, a marker × ecotype interaction and a pairwise interaction were confirmed in an independent switchgrass panel. Our analyses identified reliable candidate variants for important bioenergy traits. Moreover, they exemplified the importance of interactive effects for depicting genetic bases and illustrated the usefulness of preselecting marker pairs for identifying pairwise marker interactions in association studies., (Copyright © 2018 Crop Science Society of America.)

DNA methylation can contribute to the maintenance of genome integrity and regulation of gene expression. In most situations, DNA methylation patterns are inherited quite stably. However, changes in DNA methylation can occur at some loci as a result of tissue culture resulting in somaclonal variation. To investigate heritable epigenetic changes as a consequence of tissue culture, a sequence-capture bisulfite sequencing approach was implemented to monitor context-specific DNA methylation patterns in ∼15 Mb of the maize genome for a population of plants that had been regenerated from tissue culture. Plants that have been regenerated from tissue culture exhibit gains and losses of DNA methylation at a subset of genomic regions. There was evidence for a high rate of homozygous changes to DNA methylation levels that occur consistently in multiple independent tissue culture lines, suggesting that some loci are either targeted or hotspots for epigenetic variation. The consistent changes inherited following tissue culture include both gains and losses of DNA methylation and can affect CG, CHG, or both contexts within a region. Only a subset of the tissue culture changes observed in callus plants are observed in the primary regenerants, but the majority of DNA methylation changes present in primary regenerants are passed onto offspring. This study provides insights into the susceptibility of some loci and potential mechanisms that could contribute to altered DNA methylation and epigenetic state that occur during tissue culture in plant species., (Copyright © 2018 by the Genetics Society of America.)

Embryogenic and regenerable tissue cultures are widely utilized in plant transformation, clonal propagation, and biological research applications. Germplasm utilized in those applications are limited, however, due to genotype-dependent culture response. The goal of this study was to identify genomic regions controlling embryogenic and regenerable tissue culture response in the globally important crop, maize ( L.), toward the long-term objective of developing approaches for genotype-independent plant genetic engineering and clonal propagation systems. An inbred maize line, WCIC2, nearly-isogenic to reference inbred B73, was developed by phenotypic selection and molecular marker analysis. WCIC2 has over 50x increase in tissue culture response relative to the recurrent parent, B73. This line was used to genetically fine-map a region on chromosome 3 controlling embryogenic and regenerable tissue culture response to a 23.9 Mb region. WCIC2 and derivatives will be useful materials to enable maize research in a genetic background similar to B73, and our genetic mapping results will advance research to identify causal genes controlling somatic embryo formation and plant regeneration in maize., (Copyright © 2018 Crop Science Society of America.)

Flowering is an important developmental event in switchgrass (), as the time to complete the life cycle affects overall biomass accumulation. The objective of this study was to generate a linkage map using single nucleotide polymorphism (SNP) markers to identify quantitative trait loci (QTL) associated with flowering time. A pseudo-F population was created by crossing two siblings derived from an initial cross between the lowland population Ellsworth and the upland cultivar Summer. Heading and anthesis dates were collected for 2 yr at two locations: DeKalb, IL and Lafayette, IN. Nine QTL for flowering time were detected, two of which were heading-associated, four anthesis-associated, and three associated with both heading and anthesis. One QTL on linkage group (LG) 2a was detected for heading and anthesis in each location and year when environments were analyzed separately, and in a combined analysis across both locations and years. The effect on heading and anthesis of the QTL on LG 2a ranged from 4 to 13 and 5 to 9 d, respectively, depending on environment. Our findings validate QTL for switchgrass flowering time from previous research and identified additional QTL. Based on the switchgrass reference genome version 1.1, flowering time gene homologs reside near the LG 2a QTL and include PSEUDO RESPONSE REGULATOR 5, SUPPRESSOR OF FRIGIDA 4, and APETALA 1, respectively involved in the circadian clock, vernalization, and floral meristem identity. Markers linked to the QTL can be used to improve the efficiency of breeding switchgrass for delayed flowering to increase biomass yield., (Copyright © 2018 Crop Science Society of America.)

Switchgrass ( is a perennial native North American grass present in two ecotypes: upland, found primarily in the northern range of switchgrass habitats, and lowland, found largely in the southern reaches of switchgrass habitats. Previous studies focused on a diversity panel of primarily northern switchgrass, so to expand our knowledge of genetic diversity in a broader set of North American switchgrass, exome capture sequence data were generated for 632 additional, primarily lowland individuals. In total, over 37 million single nucleotide polymorphisms (SNPs) were identified and a set of 1.9 million high-confidence SNPs were obtained from 1169 individuals from 140 populations (67 upland, 65 lowland, 8 admixed) were used in downstream analyses of genetic diversity and population structure. Seven separate population groups were identified with moderate genetic differentiation [mean fixation index (Fst) estimate of 0.06] between the lowland and the upland populations. Ecotype-specific and population-specific SNPs were identified for use in germplasm evaluations. Relative to rice ( L.), maize ( L.), soybean [ (L.) Merr.], and Gaertn., analyses of nucleotide diversity revealed a high degree of genetic diversity (0.0135) across all individuals, consistent with the outcrossing mode of reproduction and the polyploidy of switchgrass. This study supports the hypothesis that repeated glaciation events, ploidy barriers, and restricted gene flow caused by flowering time differences have resulted in distinct gene pools across ecotypes and geographic regions. These data provide a resource to associate alleles with traits of interest for forage, restoration, and biofuel feedstock efforts in switchgrass., (Copyright © 2018 Crop Science Society of America.)

The environment can influence heterosis, the phenomena in which the offspring of two inbred parents exhibits phenotypic performance beyond the inbred parents for specific traits. In this study we measured 25 traits in a set of 47 maize hybrids and their inbred parents grown in 16 different environments with varying levels of average productivity. By quantifying 25 vegetative and reproductive traits across the life cycle we were able to analyze interactions between the environment and multiple distinct instances of heterosis. The magnitude and rank among hybrids for better-parent heterosis (BPH) varied for the different traits and environments. Across the traits, a higher within plot variance was observed for inbred lines compared to hybrids. However, for most traits, variance across environments was not significantly different for inbred lines compared to hybrids. Further, for many traits the correlations of BPH to hybrid performance and BPH to better parent performance were of comparable magnitude. These results indicate that inbred lines and hybrids show similar trends in environmental response and both are contributing to observed genotype-by-environment interactions for heterosis. This study highlights the degree of heterosis is not an inherent trait of a specific hybrid, but varies depending on the trait measured and the environment where that trait is measured. Studies that attempt to correlate molecular processes with heterosis are hindered by the fact that heterosis is not a consistent attribute of a specific hybrid.

Background: The cell wall polymer lignin provides structural support and rigidity to plant cell walls, and therefore to the plant body. However, the recalcitrance associated with lignin impedes the extraction of polysaccharides from the cell wall to make plant-based biofuels and biomaterials. The cell wall digestibility can be improved by introducing labile ester bonds into the lignin backbone that can be easily broken under mild base treatment at room temperature. The FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme, which may be naturally found in many plants, uses feruloyl-CoA and monolignols to synthesize the ester-linked monolignol ferulate conjugates. A mutation in the first lignin-specific biosynthetic enzyme, CINNAMOYL-CoA REDUCTASE (CCR), results in an increase in the intracellular pool of feruloyl-CoA., Results: Maize ( Zea mays ) has a native putative FMT enzyme, and its ccr mutants produce an increased pool of feruloyl-CoA that can be used for conversion to monolignol ferulate conjugates. The decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue., Conclusions: Together, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops.

Background: The maize male inflorescence (tassel) produces pollen necessary for reproduction and commercial grain production of maize. The size of the tassel has been linked to factors affecting grain yield, so understanding the genetic control of tassel architecture is an important goal. Tassels are fragile and deform easily after removal from the plant, necessitating rapid measurement of any shape characteristics that cannot be retained during storage. Some morphological characteristics of tassels such as curvature and compactness are difficult to quantify using traditional methods, but can be quantified by image-based phenotyping tools. These constraints necessitate the development of an efficient method for capturing natural-state tassel morphology and complementary automated analytical methods that can quickly and reproducibly quantify traits of interest such as height, spread, and branch number., Results: This paper presents the Tassel Image-based Phenotyping System (TIPS), which provides a platform for imaging tassels in the field immediately following removal from the plant. TIPS consists of custom methods that can quantify morphological traits from profile images of freshly harvested tassels acquired with a standard digital camera in a field-deployable light shelter. Correlations between manually measured traits (tassel weight, tassel length, spike length, and branch number) and image-based measurements ranged from 0.66 to 0.89. Additional tassel characteristics quantified by image analysis included some that cannot be quantified manually, such as curvature, compactness, fractal dimension, skeleton length, and perimeter. TIPS was used to measure tassel phenotypes of 3530 individual tassels from 749 diverse inbred lines that represent the diversity of tassel morphology found in modern breeding and academic research programs. Repeatability ranged from 0.85 to 0.92 for manually measured phenotypes, from 0.77 to 0.83 for the same traits measured by image-based methods, and from 0.49 to 0.81 for traits that can only be measured by image analysis., Conclusions: TIPS allows morphological features of maize tassels to be quantified automatically, with minimal disturbance, at a scale that supports population-level studies. TIPS is expected to accelerate the discovery of associations between genetic loci and tassel morphology characteristics, and can be applied to maize breeding programs to increase productivity with lower resource commitment.

Flowering time is a major determinant of biomass yield in switchgrass (Panicum virgatum), a perennial bioenergy crop, because later flowering allows for an extended period of vegetative growth and increased biomass production. A better understanding of the genetic regulation of flowering time in switchgrass will aid the development of switchgrass varieties with increased biomass yields, particularly at northern latitudes, where late-flowering but southern-adapted varieties have high winter mortality. We use genotypes derived from recently published exome-capture sequencing, which mitigates challenges related to the large, highly repetitive and polyploid switchgrass genome, to perform genome-wide association studies (GWAS) using flowering time data from a switchgrass association panel in an effort to characterize the genetic architecture and genes underlying flowering time regulation in switchgrass. We identify associations with flowering time at multiple loci, including in a homolog of FLOWERING LOCUS T and in a locus containing TIMELESS, a homolog of a key circadian regulator in animals. Our results suggest that flowering time variation in switchgrass is due to variation at many positions across the genome. The relationship of flowering time and geographic origin indicates likely roles for genes in the photoperiod and autonomous pathways in generating switchgrass flowering time variation., (No claim to original US government works. New Phytologist © 2016 New Phytologist Trust.)

Grain yield of the maize plant depends on the sizes, shapes, and numbers of ears and the kernels they bear. An automated pipeline that can measure these components of yield from easily-obtained digital images is needed to advance our understanding of this globally important crop. Here we present three custom algorithms designed to compute such yield components automatically from digital images acquired by a low-cost platform. One algorithm determines the average space each kernel occupies along the cob axis using a sliding-window Fourier transform analysis of image intensity features. A second counts individual kernels removed from ears, including those in clusters. A third measures each kernel's major and minor axis after a Bayesian analysis of contour points identifies the kernel tip. Dimensionless ear and kernel shape traits that may interrelate yield components are measured by principal components analysis of contour point sets. Increased objectivity and speed compared to typical manual methods are achieved without loss of accuracy as evidenced by high correlations with ground truth measurements and simulated data. Millimeter-scale differences among ear, cob, and kernel traits that ranged more than 2.5-fold across a diverse group of inbred maize lines were resolved. This system for measuring maize ear, cob, and kernel attributes is being used by multiple research groups as an automated Web service running on community high-throughput computing and distributed data storage infrastructure. Users may create their own workflow using the source code that is staged for download on a public repository., (© 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Switchgrass is undergoing development as a dedicated cellulosic bioenergy crop. Fermentation of lignocellulosic biomass to ethanol in a bioenergy system or to volatile fatty acids in a livestock production system is strongly and negatively influenced by lignification of cell walls. This study detects specific loci that exhibit selection signatures across switchgrass breeding populations that differ in in vitro dry matter digestibility (IVDMD), ethanol yield, and lignin concentration. Allele frequency changes in candidate genes were used to detect loci under selection. Out of the 183 polymorphisms identified in the four candidate genes, twenty-five loci in the intron regions and four loci in coding regions were found to display a selection signature. All loci in the coding regions are synonymous substitutions. Selection in both directions were observed on polymorphisms that appeared to be under selection. Genetic diversity and linkage disequilibrium within the candidate genes were low. The recurrent divergent selection caused excessive moderate allele frequencies in the cycle 3 reduced lignin population as compared to the base population. This study provides valuable insight on genetic changes occurring in short-term selection in the polyploid populations, and discovered potential markers for breeding switchgrass with improved biomass quality., Competing Interests: The authors have declared that no competing interests exist.

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison of these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools., (© 2016 American Society of Plant Biologists. All rights reserved.)

Carbohydrates stored in vegetative organs, particularly stems, of grasses are a very important source of energy. We examined carbohydrate accumulation in adult sorghum and maize hybrids with distinct phenology and different end uses (grain, silage, sucrose or sweetness in stalk juice, and biomass). Remarkable variation was observed for non-structural carbohydrates and structural polysaccharides during three key developmental stages both between and within hybrids developed for distinct end use in both species. At the onset of the reproductive phase (average 65 days after planting, DAP), a wide range for accumulation of non-structural carbohydrates (free glucose and sucrose combined), was observed in internodes of maize (11-24%) and sorghum (7-36%) indicating substantial variation for transient storage of excess photosynthate during periods of low grain or vegetative sink strength. Remobilization of these reserves for supporting grain fill or vegetative growth was evident from lower amounts in maize (8-19%) and sorghum (9-27%) near the end of the reproductive period (average 95 DAP). At physiological maturity of grain hybrids (average 120 DAP), amounts of these carbohydrates were generally unchanged in maize (9-21%) and sorghum (16-27%) suggesting a loss of photosynthetic assimilation due to weakening sink demand. Nonetheless, high amounts of non-structural carbohydrates at maturity even in grain maize and sorghum (15-18%) highlight the potential for developing dual-purpose (grain/stover) crops. For both species, the amounts of structural polysaccharides in the cell wall, measured as monomeric components (glucose and pentose), decreased during grain fill but remained unchanged thereafter with maize biomass possessing slightly higher amounts than sorghum. Availability of carbohydrates in maize and sorghum highlights the potential for developing energy-rich dedicated biofuel or dual-purpose (grain/stover) crops.

Switchgrass is a relatively high-yielding and environmentally sustainable biomass crop, but further genetic gains in biomass yield must be achieved to make it an economically viable bioenergy feedstock. Genomic selection (GS) is an attractive technology to generate rapid genetic gains in switchgrass, and meet the goals of a substantial displacement of petroleum use with biofuels in the near future. In this study, we empirically assessed prediction procedures for genomic selection in two different populations, consisting of 137 and 110 half-sib families of switchgrass, tested in two locations in the United States for three agronomic traits: dry matter yield, plant height, and heading date. Marker data were produced for the families' parents by exome capture sequencing, generating up to 141,030 polymorphic markers with available genomic-location and annotation information. We evaluated prediction procedures that varied not only by learning schemes and prediction models, but also by the way the data were preprocessed to account for redundancy in marker information. More complex genomic prediction procedures were generally not significantly more accurate than the simplest procedure, likely due to limited population sizes. Nevertheless, a highly significant gain in prediction accuracy was achieved by transforming the marker data through a marker correlation matrix. Our results suggest that marker-data transformations and, more generally, the account of linkage disequilibrium among markers, offer valuable opportunities for improving prediction procedures in GS. Some of the achieved prediction accuracies should motivate implementation of GS in switchgrass breeding programs., (Copyright © 2016 Ramstein et al.)

Seed size is an important component of grain yield and a key determinant trait for crop domestication. The Krug Yellow Dent long-term selection experiment for large and small seed provides a valuable resource to dissect genetic and phenotypic changes affecting seed size within a common genetic background. In this study, inbred lines derived from Krug Large Seed (KLS) and Krug Small Seed (KSS) populations and reciprocal F1 crosses were used to investigate developmental and molecular mechanisms governing seed size. Seed morphological characteristics showed striking differences between KLS and KSS inbred lines, and the reciprocal cross experiment revealed a strong maternal influence on both seed weight and seed size. Quantification of endosperm area, starchy endosperm cell size, and kernel dry mass accumulation indicated a positive correlation between seed size, endosperm cell number, and grain filling rate, and patterns of grain filling in reciprocal crosses mirrored that of the maternal parent. Consistent with the maternal contribution to seed weight, transcriptome profiling of reciprocal F1 hybrids showed substantial similarities to the maternal parent. A set of differentially expressed genes between KLS and KSS inbreds were found, which fell into a broad number of functional categories including DNA methylation, nucleosome assembly, and heat stress response. In addition, gene co-expression network analysis of parental inbreds and reciprocal F1 hybrids identified co-expression modules enriched in ovule development and DNA methylation, implicating these two processes in seed size determination. These results expand our understanding of seed size regulation and help to uncover the developmental and molecular basis underlying maternal control of seed size in maize., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Comprehensive and systematic transcriptome profiling provides valuable insight into biological and developmental processes that occur throughout the life cycle of a plant. We have enhanced our previously published microarray-based gene atlas of maize ( L.) inbred B73 to now include 79 distinct replicated samples that have been interrogated using RNA sequencing (RNA-seq). The current version of the atlas includes 50 original array-based gene atlas samples, a time-course of 12 stalk and leaf samples postflowering, and an additional set of 17 samples from the maize seedling and adult root system. The entire dataset contains 4.6 billion mapped reads, with an average of 20.5 million mapped reads per biological replicate, allowing for detection of genes with lower transcript abundance. As the new root samples represent key additions to the previously examined tissues, we highlight insights into the root transcriptome, which is represented by 28,894 (73.2%) annotated genes in maize. Additionally, we observed remarkable expression differences across both the longitudinal (four zones) and radial gradients (cortical parenchyma and stele) of the primary root supported by fourfold differential expression of 9353 and 4728 genes, respectively. Among the latter were 1110 genes that encode transcription factors, some of which are orthologs of previously characterized transcription factors known to regulate root development in (L.) Heynh., while most are novel, and represent attractive targets for reverse genetics approaches to determine their roles in this important organ. This comprehensive transcriptome dataset is a powerful tool toward understanding maize development, physiology, and phenotypic diversity., (Copyright © 2016 Crop Science Society of America.)

Panicum virgatum L. (switchgrass) is a polyploid, perennial grass species that is native to North America, and is being developed as a future biofuel feedstock crop. Switchgrass is present primarily in two ecotypes: a northern upland ecotype, composed of tetraploid and octoploid accessions, and a southern lowland ecotype, composed of primarily tetraploid accessions. We employed high-coverage exome capture sequencing (~2.4 Tb) to genotype 537 individuals from 45 upland and 21 lowland populations. From these data, we identified ~27 million single-nucleotide polymorphisms (SNPs), of which 1 590 653 high-confidence SNPs were used in downstream analyses of diversity within and between the populations. From the 66 populations, we identified five primary population groups within the upland and lowland ecotypes, a result that was further supported through genetic distance analysis. We identified conserved, ecotype-restricted, non-synonymous SNPs that are predicted to affect the protein function of CONSTANS (CO) and EARLY HEADING DATE 1 (EHD1), key genes involved in flowering, which may contribute to the phenotypic differences between the two ecotypes. We also identified, relative to the near-reference Kanlow population, 17 228 genes present in more copies than in the reference genome (up-CNVs), 112 630 genes present in fewer copies than in the reference genome (down-CNVs) and 14 430 presence/absence variants (PAVs), affecting a total of 9979 genes, including two upland-specific CNV clusters. In total, 45 719 genes were affected by an SNP, CNV, or PAV across the panel, providing a firm foundation to identify functional variation associated with phenotypic traits of interest for biofuel feedstock production., (© 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA) content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. This indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)