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Abstract Zoologists have adduced morphological convergence among embryonic stages of closely related taxa, which has been called the phylotypic stage of embryogenesis. Transcriptomic analyzes reveal an hourglass pattern of gene expression during plant and animal embryogenesis, characterized by the accumulation of evolutionarily older and conserved transcripts during mid-embryogenesis, whereas younger less-conserved transcripts predominate at earlier and later embryonic stages. In contrast, comparisons of embryonic gene expression among different animal phyla describe an inverse hourglass pattern, where expression is correlated during early and late stages but not during mid-embryo development. Here, multiplexed spatial-transcriptomic analyzes is used to investigate embryogenesis and homology in maize, which has grass-specific morphology. A set of shared, co-expressed genes is identified during initiation of maize embryonic organs, replete for ancient/conserved genes manifesting an hourglass pattern during mid-embryogenesis. Transcriptomic comparisons of maize and Arabidopsis embryogenesis with that of the moss Physcomitrium patens identify an inverse hourglass pattern across plant phyla, as in animals. The data suggest that the phylotypic stages in plants and animals are characterized by expression of ancient and conserved genes during histogenesis, organization of embryonic axes, and initial morphogenesis. We propose a mechanism for gene evolution during the innovation of morphological novelty.

Social Impact Statement Although tremendously successful at feeding humanity, row crop agriculture based on annuals contributes to numerous ecosystem dis‐services, ranging from soil degradation and aquatic eutrophication to greenhouse gas production. In contrast, perennial grain crops (which produce harvests for multiple seasons from single plantings) have the potential to provide valuable regulating and supporting ecosystem services in addition to food production. In particular, losses of ecological capital that threaten permanent food insecurity such as ~1% of global soil per year are expected to be mitigated or even reversed by crops that combine the high yield realized by scientific breeding via multiple cropping cycles from single plantings. Summary Perennial herbaceous may provide food and biomass while preserving ecological capital and reducing energy inputs. Sorghum has two perennial relatives and rich morphological diversity being used to breed for perenniality. We elucidate genetic determinants of rhizomatousness and survival, in two BC1F2 populations totaling 246 genotypes derived from backcrossing different annual Sorghum bicolor X perennial S. halepense F1 plants to a tetraploidized S. bicolor. RNA‐seq assisted in identifying candidate genes for rhizomatousness. Correspondence of rhizomatousness quantitative trait loci (QTLs) with those from two populations derived from crosses between S. halepense progenitors S. bicolor X S. propinquum suggests either the preservation of interspecific polymorphism or the formation of novel alleles following polyploid S. halepense formation. Correspondence of tillering and branching QTLs further supports their developmental. Identification of genes from RNA‐seq study within QTL intervals provides insight toward discovery of causal rhizomatous genes. An unexpected finding from both S. halepense‐ and S. propinquum‐derived populations is that alleles contributing to late flowering are related to reduced rhizomatousness. Twelve of 16 QTL regions conferring rhizomatousness fall in paleo‐duplicated regions tracing to single ancestral regions 96 million years ago, indicating that corresponding genes in these regions have retained similar functions since the duplication event.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed at night and under water-limited conditions. Elucidating the genetic architecture of natural variation for leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we conducted a genome-wide association study of gc of adult leaves in a maize inbred association panel that was evaluated in four environments (Maricopa, AZ, and San Diego, CA, in 2016 and 2017). Five genomic regions significantly associated with gc were resolved to seven plausible candidate genes (ISTL1, two SEC14 homologs, cyclase-associated protein, a CER7 homolog, GDSL lipase, and β-D-XYLOSIDASE 4). These candidates are potentially involved in cuticle biosynthesis, trafficking and deposition of cuticle lipids, cutin polymerization, and cell wall modification. Laser microdissection RNA sequencing revealed that all these candidate genes, with the exception of the CER7 homolog, were expressed in the zone of the expanding adult maize leaf where cuticle maturation occurs. With direct application to genetic improvement, moderately high average predictive abilities were observed for whole-genome prediction of gc in locations (0.46 and 0.45) and across all environments (0.52). The findings of this study provide novel insights into the genetic control of gc and have the potential to help breeders more effectively develop drought-tolerant maize for target environments.

Bulliform cells comprise specialized cell types that develop on the adaxial (upper) surface of grass leaves, and are patterned to form linear rows along the proximodistal axis of the adult leaf blade. Bulliform cell patterning affects leaf angle and is presumed to function during leaf rolling, thereby reducing water loss during temperature extremes and drought. In this study, epidermal leaf impressions were collected from a genetically and anatomically diverse population of maize inbred lines. Subsequently, convolutional neural networks were employed to measure microscopic, bulliform cell-patterning phenotypes in high-throughput. A genome-wide association study, combined with RNAseq analyses of the bulliform cell ontogenic zone, identified candidate regulatory genes affecting bulliform cell column number and cell width. This study is the first to combine machine learning approaches, transcriptomics, and genomics to study bulliform cell patterning, and the first to utilize natural variation to investigate the genetic architecture of this microscopic trait. In addition, this study provides insight toward the improvement of macroscopic traits such as drought resistance and plant architecture in an agronomically important crop plant.

Abstract Background Plant domestication provides a unique model to study genome evolution. Many studies have been conducted to examine genes, genetic diversity, genome structure, and epigenome changes associated with domestication. Interestingly, domesticated accessions have significantly higher [A] and [T] values across genome-wide polymorphic sites than accessions sampled from the corresponding progenitor species. However, the relative contributions of different genomic regions to this genome divergence pattern and underlying mechanisms have not been well characterized. Results Here, we investigate the genome-wide base-composition patterns by analyzing millions of SNPs segregating among 100 accessions from a teosinte-maize comparison set and among 302 accessions from a wild-domesticated soybean comparison set. We show that non-genic part of the genome has a greater contribution than genic SNPs to the [AT]-increase observed between wild and domesticated accessions in maize and soybean. The separation between wild and domesticated accessions in [AT] values is significantly enlarged in non-genic and pericentromeric regions. Motif frequency and sequence context analyses show the motifs (PyCG) related to solar-UV signature are enriched in these regions, particularly when they are methylated. Additional analysis using population-private SNPs also implicates the role of these motifs in relatively recent mutations. With base-composition across polymorphic sites as a genome phenotype, genome scans identify a set of putative candidate genes involved in UV damage repair pathways. Conclusions The [AT]-increase is more pronounced in genomic regions that are non-genic, pericentromeric, transposable elements; methylated; and with low recombination. Our findings establish important links among UV radiation, mutation, DNA repair, methylation, and genome evolution.

From noble beginnings as a prospective forage, polyploid Sorghum halepense (‘Johnsongrass’) is both an invasive species and one of the world’s worst agricultural weeds. Formed by S. bicolor x S. propinquum hybridization, we show S. halepense to have S. bicolor-enriched allele composition and striking mutations in 5,957 genes that differentiate it from representatives of its progenitor species and an outgroup. The spread of S. halepense may have been facilitated by introgression from closely-related cultivated sorghum near genetic loci affecting rhizome development, seed size, and levels of lutein, a photochemical protectant and abscisic acid precursor. Rhizomes, subterranean stems that store carbohydrates and spawn clonal propagules, have growth correlated with reproductive rather than other vegetative tissues, and increase survival of both temperate cold seasons and tropical dry seasons. Rhizomes of S. halepense are more extensive than those of its rhizomatous progenitor S. propinquum, with gene expression including many alleles from its non-rhizomatous S. bicolor progenitor. The first surviving polyploid in its lineage in ∼96 million years, its post-Columbian spread across six continents carried rich genetic diversity that in the United States has facilitated transition from agricultural to non-agricultural niches. Projected to spread another 200–600 km northward in the coming century, despite its drawbacks S. halepense may offer novel alleles and traits of value to improvement of sorghum.

Forward genetics remains a powerful method for revealing the genes underpinning organismal form and function, and for revealing how these genes are tied together in gene networks. In maize, forward genetics has been tremendously successful, but the size and complexity of the maize genome made identifying mutant genes an often arduous process with traditional methods. The next generation sequencing revolution has allowed for the gene cloning process to be significantly accelerated in many organisms, even when genomes are large and complex. Here, we describe a bulked-segregant analysis sequencing (BSA-Seq) protocol for cloning mutant genes in maize. Our simple strategy can be used to quickly identify a mapping interval and candidate single nucleotide polymorphisms (SNPs) from whole genome sequencing of pooled F2 individuals. We employed this strategy to identify narrow odd dwarf as an enhancer of teosinte branched1, and to identify a new allele of defective kernel1. Our method provides a quick, simple way to clone genes in maize.

Abstract Background There are significant limitations in existing methods for the genome-wide identification of genes whose expression patterns affect traits. Results The transcriptomes of five tissues from 27 genetically diverse maize inbred lines were deeply sequenced to identify genes exhibiting high and low levels of expression variation across tissues or genotypes. Transcription factors are enriched among genes with the most variation in expression across tissues, as well as among genes with higher-than-median levels of variation in expression across genotypes. In contrast, transcription factors are depleted among genes whose expression is either highly stable or highly variable across genotypes. We developed a Bayesian-based method for genome-wide association studies (GWAS) in which RNA-seq-based measures of transcript accumulation are used as explanatory variables (eRD-GWAS). The ability of eRD-GWAS to identify true associations between gene expression variation and phenotypic diversity is supported by analyses of RNA co-expression networks, protein–protein interaction networks, and gene regulatory networks. Genes associated with 13 traits were identified using eRD-GWAS on a panel of 369 maize inbred lines. Predicted functions of many of the resulting trait-associated genes are consistent with the analyzed traits. Importantly, transcription factors are significantly enriched among trait-associated genes identified with eRD-GWAS. Conclusions eRD-GWAS is a powerful tool for associating genes with traits and is complementary to SNP-based GWAS. Our eRD-GWAS results are consistent with the hypothesis that genetic variation in transcription factor expression contributes substantially to phenotypic diversity.

Leaves are initiated as lateral outgrowths from shoot apical meristems throughout the vegetative life of the plant. To achieve proper developmental patterning, cell-type specification and growth must occur in an organized fashion along the proximodistal (base-to-tip), mediolateral (central-to-edge), and adaxial−abaxial (top-bottom) axes of the developing leaf. Early studies of mutants with defects in patterning along multiple leaf axes suggested that patterning must be coordinated across developmental axes. Decades later, we now recognize that a highly complex and interconnected transcriptional network of patterning genes and hormones underlies leaf development. Here, we review the molecular genetic mechanisms by which leaf development is coordinated across leaf axes. Such coordination likely plays an important role in ensuring the reproducible phenotypic outcomes of leaf morphogenesis.

The maize (Zea mays subsp. mays L.) shoot apical meristem (SAM) is a self-replenishing pool of stem cells that produces the above-ground plant. Improvements in image acquisition and processing techniques have allowed high-throughput, quantitative genetic analyses of SAM morphology. As with other large-scale phenotyping efforts, meaningful descriptions of genetic architecture depend on the collection of relevant measures. In this study, we tested two quantitative image processing methods to describe SAM morphology within the genus Zea, represented by 33 wild relatives of maize and 841 lines from a domesticated maize by wild teosinte progenitor (MxT) backcross population, along with previously-reported data from several hundred diverse maize inbred lines. Approximating the MxT SAM as a paraboloid derived eight parabolic estimators of SAM morphology that identified highly-overlapping QTL on eight chromosomes, which implicated previously-identified SAM morphology candidate genes along with new QTL for SAM morphological variation. Using a Fourier-transform related method of comprehensive shape analysis, we detected cryptic SAM shape variation that identified QTL on six chromosomes. We found that Fourier transform shape descriptors and parabolic estimation measures are highly correlated and identified similar QTL. Analysis of shoot apex contours from 73 anciently-diverged plant taxa further suggested that parabolic shape may be a universal feature of plant SAMs, regardless of evolutionary clade. Future high-throughput examinations of SAM morphology may benefit from the ease of acquisition and phenotypic fidelity of modeling the SAM as a paraboloid.

The original version [1] of this article unfortunately contained a mistake. The additive effects of the eQTLs of lncRNAs were flipped, meaning that the base allele in the contrast to derive the additive effects should have been B73, rather than Mo17, due to the original coding of biallele SNPs as “0s” and “1s”. Going through the entire analysis procedure, it was determined that the mistake was made while tabulating the eQTL results from QTL Cartographer.

The embryo and endosperm are the products of double fertilization and comprise the clonally distinct products of angiosperm seed development. Recessive mutations in the maize gene DISCOLORED1 (DSC1) condition inviable seed that are defective in both embryo and endosperm development. Here, detailed phenotypic analyses illustrate that dsc1 mutant kernels are able to establish, but fail to maintain, differentiated embryo and endosperm structures. Development of the dsc1-R mutant embryo and endosperm is normal albeit delayed, prior to the abortion and subsequent degeneration of all differentiated kernel structures. Using a genomic fragment that was previously isolated by transposon tagging, the full length DSC1 transcript is identified and shown to encode an ADP-RIBOSYLATION FACTOR-GTPase ACTIVATING PROTEIN (ARF-GAP) that co-localizes with the trans-Golgi network/early endosomes and the plasma membrane during transient expression assays in N. benthamiana leaves. DSC1 function during endomembrane trafficking and the maintenance of maize kernel differentiation is discussed.

[This corrects the article DOI: 10.1371/journal.pgen.1003202.].

Salinity and drought stress are the primary cause of crop losses worldwide. In sodic saline soils sodium chloride (NaCl) disrupts normal plant growth and development. The complex interactions of plant systems with abiotic stress have made RNA sequencing a more holistic and appealing approach to study transcriptome level responses in a single cell and/or tissue. In this work, we determined the Petunia transcriptome response to NaCl stress by sequencing leaf samples and assembling 196 million Illumina reads with Trinity software. Using our reference transcriptome we identified more than 7,000 genes that were differentially expressed within 24 h of acute NaCl stress. The proposed transcriptome can also be used as an excellent tool for biological and bioinformatics in the absence of an available Petunia genome and it is available at the SOL Genomics Network (SGN) http://solgenomics.net. Genes related to regulation of reactive oxygen species, transport, and signal transductions as well as novel and undescribed transcripts were among those differentially expressed in response to salt stress. The candidate genes identified in this study can be applied as markers for breeding or to genetically engineer plants to enhance salt tolerance. Gene Ontology analyses indicated that most of the NaCl damage happened at 24 h inducing genotoxicity, affecting transport and organelles due to the high concentration of Na+ ions. Finally, we report a modification to the library preparation protocol whereby cDNA samples were bar-coded with non-HPLC purified primers, without affecting the quality and quantity of the RNA-seq data. The methodological improvement presented here could substantially reduce the cost of sample preparation for future high-throughput RNA sequencing experiments.

Transcriptome variation plays an important role in affecting the phenotype of an organism. However, an understanding of the underlying mechanisms regulating transcriptome variation in segregating populations is still largely unknown. We sought to assess and map variation in transcript abundance in maize shoot apices in the intermated B73 × Mo17 recombinant inbred line population. RNA-based sequencing (RNA-seq) allowed for the detection and quantification of the transcript abundance derived from 28,603 genes. For a majority of these genes, the population mean, coefficient of variation, and segregation patterns could be predicted by the parental expression levels. Expression quantitative trait loci (eQTL) mapping identified 30,774 eQTL including 96 trans-eQTL "hotspots," each of which regulates the expression of a large number of genes. Interestingly, genes regulated by a trans-eQTL hotspot tend to be enriched for a specific function or act in the same genetic pathway. Also, genomic structural variation appeared to contribute to cis-regulation of gene expression. Besides genes showing Mendelian inheritance in the RIL population, we also found genes whose expression level and variation in the progeny could not be predicted based on parental difference, indicating that non-Mendelian factors also contribute to expression variation. Specifically, we found 145 genes that show patterns of expression reminiscent of paramutation such that all the progeny had expression levels similar to one of the two parents. Furthermore, we identified another 210 genes that exhibited unexpected patterns of transcript presence/absence. Many of these genes are likely to be gene fragments resulting from transposition, and the presence/absence of their transcripts could influence expression levels of their ancestral syntenic genes. Overall, our results contribute to the identification of novel expression patterns and broaden the understanding of transcriptional variation in plants.

Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA-dependent RNA polymerase 2 (RDR2) is a component of the RNA-directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA-seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, >6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2-sensitive and RDR2-resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant.

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection-microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize.

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial-abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

Microarrays enable comparative analyses of gene expression on a genomic scale, however these experiments frequently identify an abundance of differentially expressed genes such that it may be difficult to identify discrete functional networks that are hidden within large microarray datasets. Microarray analyses in which mutant organisms are compared to nonmutant siblings can be especially problematic when the gene of interest is expressed in relatively few cells. Here, we describe the use of laser microdissection microarray to perform transcriptional profiling of the maize shoot apical meristem (SAM), a ~100-microm pillar of organogenic cells that is required for leaf initiation. Microarray analyses compared differential gene expression within the SAM and incipient leaf primordium of nonmutant and narrow sheath mutant plants, which harbored mutations in the duplicate genes narrow sheath1 (ns1) and narrow sheath2 (ns2). Expressed in eight to ten cells within the SAM, ns1 and ns2 encode paralogous WUSCHEL1-like homeobox (WOX) transcription factors required for recruitment of leaf initials that give rise to a large lateral domain within maize leaves. The data illustrate the utility of laser microdissection-microarray analyses to identify a relatively small number of genes that are differentially expressed within the SAM. Moreover, these analyses reveal potentially conserved WOX gene functions and implicate specific hormonal and signaling pathways during early events in maize leaf development.