The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm.

Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.

Prediction of conserved and variable heat and cold stress response in maize using cis-regulatory information.

Changes in gene expression are important for responses to abiotic stress. Transcriptome profiling of heat- or cold-stressed maize genotypes identifies many changes in transcript abundance. We used comparisons of expression responses in multiple genotypes to identify alleles with variable responses to heat or cold stress and to distinguish examples of cis- or trans-regulatory variation for stress-responsive expression changes. We used motifs enriched near the transcription start sites (TSSs) for thermal stress-responsive genes to develop predictive models of gene expression responses. Prediction accuracies can be improved by focusing only on motifs within unmethylated regions near the TSS and vary for genes with different dynamic responses to stress. Models trained on expression responses in a single genotype and promoter sequences provided lower performance when applied to other genotypes but this could be improved by using models trained on data from all three genotypes tested. The analysis of genes with cis-regulatory variation provides evidence for structural variants that result in presence/absence of transcription factor binding sites in creating variable responses. This study provides insights into cis-regulatory motifs for heat- and cold-responsive gene expression and defines a framework for developing models to predict expression responses across multiple genotypes.

A novel active transposon creates allelic variation through altered translation rate to influence protein abundance.

Protein translation is tightly and precisely controlled by multiple mechanisms including upstream open reading frames (uORFs), but the origins of uORFs and their role in maize are largely unexplored. In this study, an active transposition event was identified during the propagation of maize inbred line B73. The transposon, which was named BTA for 'B73 active transposable element hAT', creates a novel dosage-dependent hypomorphic allele of the hexose transporter gene ZmSWEET4c through insertion within the coding sequence in the first exon, and results in reduced kernel size. The BTA insertion does not affect transcript abundance but reduces protein abundance of ZmSWEET4c, probably through the introduction of a uORF. Furthermore, the introduction of BTA sequence in the exon of other genes can regulate translation efficiency without affecting their mRNA levels. A transposon capture assay revealed 79 novel insertions for BTA and BTA-like elements. These insertion sites have typical euchromatin features, including low levels of DNA methylation and high levels of H3K27ac. A putative autonomous element that mobilizes BTA and BTA-like elements was identified. Together, our results suggest a transposon-based origin of uORFs and document a new role for transposable elements to influence protein abundance and phenotypic diversity by affecting the translation rate.

Maize decrease in DNA methylation 1 targets RNA-directed DNA methylation on active chromatin.

DNA methylation plays vital roles in repressing transposable element activity and regulating gene expression. The chromatin-remodeling factor Decrease in DNA methylation 1 (DDM1) is crucial for maintaining DNA methylation across diverse plant species, and is required for RNA-directed DNA methylation (RdDM) to maintain mCHH islands in maize (Zea mays). However, the mechanisms by which DDM1 is involved in RdDM are not well understood. In this work, we used chromatin immunoprecipitation coupled with high-throughput sequencing to ascertain the genome-wide occupancy of ZmDDM1 in the maize genome. The results revealed that ZmDDM1 recognized an 8-bp-long GC-rich degenerate DNA sequence motif, which is enriched in transcription start sites and other euchromatic regions. Meanwhile, 24-nucleotide siRNAs and CHH methylation were delineated at the edge of ZmDDM1-occupied sites. ZmDDM1 co-purified with Argonaute 4 (ZmAGO4) proteins, providing further evidence that ZmDDM1 is a component of RdDM complexes in planta. Consistent with this, the vast majority of ZmDDM1-targeted regions co-localized with ZmAGO4-bound genomic sites. Overall, our results suggest a model that ZmDDM1 may be recruited to euchromatic regions via recognition of a GC-rich motif, thereby remodeling chromatin to provide access for RdDM activities in maize.

The genomic ecosystem of transposable elements in maize.

Transposable elements (TEs) constitute the majority of flowering plant DNA, reflecting their tremendous success in subverting, avoiding, and surviving the defenses of their host genomes to ensure their selfish replication. More than 85% of the sequence of the maize genome can be ascribed to past transposition, providing a major contribution to the structure of the genome. Evidence from individual loci has informed our understanding of how transposition has shaped the genome, and a number of individual TE insertions have been causally linked to dramatic phenotypic changes. Genome-wide analyses in maize and other taxa have frequently represented TEs as a relatively homogeneous class of fragmentary relics of past transposition, obscuring their evolutionary history and interaction with their host genome. Using an updated annotation of structurally intact TEs in the maize reference genome, we investigate the family-level dynamics of TEs in maize. Integrating a variety of data, from descriptors of individual TEs like coding capacity, expression, and methylation, as well as similar features of the sequence they inserted into, we model the relationship between attributes of the genomic environment and the survival of TE copies and families. In contrast to the wholesale relegation of all TEs to a single category of junk DNA, these differences reveal a diversity of survival strategies of TE families. Together these generate a rich ecology of the genome, with each TE family representing the evolution of a distinct ecological niche. We conclude that while the impact of transposition is highly family- and context-dependent, a family-level understanding of the ecology of TEs in the genome can refine our ability to predict the role of TEs in generating genetic and phenotypic diversity.

Widespread imprinting of transposable elements and variable genes in the maize endosperm.

Fertilization and seed development is a critical time in the plant life cycle, and coordinated development of the embryo and endosperm are required to produce a viable seed. In the endosperm, some genes show imprinted expression where transcripts are derived primarily from one parental genome. Imprinted gene expression has been observed across many flowering plant species, though only a small proportion of genes are imprinted. Understanding how imprinted expression arises has been complicated by the reliance on single nucleotide polymorphisms between alleles to enable testing for imprinting. Here, we develop a method to use whole genome assemblies of multiple genotypes to assess for imprinting of both shared and variable portions of the genome using data from reciprocal crosses. This reveals widespread maternal expression of genes and transposable elements with presence-absence variation within maize and across species. Most maternally expressed features are expressed primarily in the endosperm, suggesting that maternal de-repression in the central cell facilitates expression. Furthermore, maternally expressed TEs are enriched for maternal expression of the nearest gene, and read alignments over maternal TE-gene pairs indicate that these are fused rather than independent transcripts.

Plant height heterosis is quantitatively associated with expression levels of plastid ribosomal proteins.

The use of hybrids is widespread in agriculture, yet the molecular basis for hybrid vigor (heterosis) remains obscure. To identify molecular components that may contribute to trait heterosis, we analyzed paired proteomic and transcriptomic data from seedling leaf and mature leaf blade tissues of maize hybrids and their inbred parents. Nuclear- and plastid-encoded subunits of complexes required for protein synthesis in the chloroplast and for the light reactions of photosynthesis were expressed above midparent and high-parent levels, respectively. Consistent with previous reports in Arabidopsis, ethylene biosynthetic enzymes were expressed below midparent levels in the hybrids, suggesting a conserved mechanism for heterosis between monocots and dicots. The ethylene biosynthesis mutant, acs2/acs6, largely phenocopied the hybrid proteome, indicating that a reduction in ethylene biosynthesis may mediate the differences between inbreds and their hybrids. To rank the relevance of expression differences to trait heterosis, we compared seedling leaf protein levels to the adult plant height of 15 hybrids. Hybrid/midparent expression ratios were most positively correlated with hybrid/midparent plant height ratios for the chloroplast ribosomal proteins. Our results show that increased expression of chloroplast ribosomal proteins in hybrid seedling leaves is mediated by reduced expression of ethylene biosynthetic enzymes and that the degree of their overexpression in seedlings can quantitatively predict adult trait heterosis.

Genome-wide loss of CHH methylation with limited transcriptome changes in Setaria viridis DOMAINS REARRANGED METHYLTRANSFERASE (DRM) mutants.

The DOMAINS REARRANGED METHYLTRANSFERASEs (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element (TE) content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. Double mutant (drm1ab) plants exhibit some morphological abnormalities but are fully viable. Whole-genome methylation profiling provided evidence for the widespread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. Evidence was also found for the locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified genes with altered expression in the drm1ab mutants. However, the majority of genes with high levels of CHH methylation directly surrounding the transcription start site or in nearby promoter regions in wild-type plants do not have altered expression in the drm1ab mutant, even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of TEs identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons are likely to require active RdDM for the maintenance of transcriptional repression.

Epigenetic features drastically impact CRISPR-Cas9 efficacy in plants.

CRISPR-Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR-Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR-Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR-Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR-Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.

Meta Gene Regulatory Networks in Maize Highlight Functionally Relevant Regulatory Interactions.

The regulation of gene expression is central to many biological processes. Gene regulatory networks (GRNs) link transcription factors (TFs) to their target genes and represent maps of potential transcriptional regulation. Here, we analyzed a large number of publically available maize (Zea mays) transcriptome data sets including >6000 RNA sequencing samples to generate 45 coexpression-based GRNs that represent potential regulatory relationships between TFs and other genes in different populations of samples (cross-tissue, cross-genotype, and tissue-and-genotype samples). While these networks are all enriched for biologically relevant interactions, different networks capture distinct TF-target associations and biological processes. By examining the power of our coexpression-based GRNs to accurately predict covarying TF-target relationships in natural variation data sets, we found that presence/absence changes rather than quantitative changes in TF gene expression are more likely associated with changes in target gene expression. Integrating information from our TF-target predictions and previous expression quantitative trait loci (eQTL) mapping results provided support for 68 TFs underlying 74 previously identified trans-eQTL hotspots spanning a variety of metabolic pathways. This study highlights the utility of developing multiple GRNs within a species to detect putative regulators of important plant pathways and provides potential targets for breeding or biotechnological applications.

Exploiting induced and natural epigenetic variation for crop improvement.

Plant breeding has traditionally relied on combining the genetic diversity present within a species to develop combinations of alleles that provide desired traits. Epigenetic diversity may provide additional sources of variation within a species that could be captured or created for crop improvement. It will be important to understand the sources of epigenetic variation and the stability of newly formed epigenetic variants over generations to fully use the potential of epigenetic variation to improve crops. The development and application of methods for widespread epigenome profiling and engineering may generate new avenues for using the full potential of epigenetics in crop improvement.

Improved maize reference genome with single-molecule technologies.

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

Stable unmethylated DNA demarcates expressed genes and their cis-regulatory space in plant genomes.

The genomic sequences of crops continue to be produced at a frenetic pace. It remains challenging to develop complete annotations of functional genes and regulatory elements in these genomes. Chromatin accessibility assays enable discovery of functional elements; however, to uncover the full portfolio of cis-elements would require profiling of many combinations of cell types, tissues, developmental stages, and environments. Here, we explore the potential to use DNA methylation profiles to develop more complete annotations. Using leaf tissue in maize, we define ∼100,000 unmethylated regions (UMRs) that account for 5.8% of the genome; 33,375 UMRs are found greater than 2 kb from genes. UMRs are highly stable in multiple vegetative tissues, and they capture the vast majority of accessible chromatin regions from leaf tissue. However, many UMRs are not accessible in leaf, and these represent regions with potential to become accessible in specific cell types or developmental stages. These UMRs often occur near genes that are expressed in other tissues and are enriched for binding sites of transcription factors. The leaf-inaccessible UMRs exhibit unique chromatin modification patterns and are enriched for chromatin interactions with nearby genes. The total UMR space in four additional monocots ranges from 80 to 120 megabases, which is remarkably similar considering the range in genome size of 271 megabases to 4.8 gigabases. In summary, based on the profile from a single tissue, DNA methylation signatures provide powerful filters to distill large genomes down to the small fraction of putative functional genes and regulatory elements.

Single-parent expression drives dynamic gene expression complementation in maize hybrids.

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.

Genetic and epigenetic variation in transposable element expression responses to abiotic stress in maize.

Transposable elements (TEs) pervade most eukaryotic genomes. The repetitive nature of TEs complicates the analysis of their expression. Evaluation of the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress reveals no evidence for genome-wide activation of TEs; however, some specific TE families generate transcripts only in stress conditions. There is substantial variation for which TE families exhibit stress-responsive expression in the different genotypes. In order to understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. The stress-responsive activation of a TE family can often be attributed to a small number of elements in the family that contains regions lacking DNA methylation. Comparisons of the expression of TEs in different genotypes revealed both genetic and epigenetic variation. Many of the specific TEs that are activated in stress in one inbred are not present in the other inbred, explaining the lack of activation. Among the elements that are shared in both genomes but only expressed in one genotype, we found that many exhibit differences in DNA methylation such that the genotype without expression is fully methylated. This study provides insights into the regulation of expression of TEs in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression variation. The highly repetitive nature of many TEs complicates the analysis of their expression. Although most TEs are not expressed, some exhibits expression in certain tissues or conditions. We monitored the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress. While genome-wide activation of TEs did not occur, some TE families generated transcripts only in stress conditions with variation by genotype. To better understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. In most cases, stress-responsive activation of a TE family was attributed to a small number of elements in the family. The elements that contained small regions lacking DNA methylation regions showed enriched expression while fully methylated elements were rarely expressed in control or stress conditions. The cause of varied expression in the different genotypes was due to both genetic and epigenetic variation. Many specific TEs activated by stress in one inbred were not present in the other inbred. Among the elements shared in both genomes, full methylation inhibited expression in one of the genotypes. This study provides insights into the regulation of TE expression in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression.

Identification of the expressome by machine learning on omics data.

Accurate annotation of plant genomes remains complex due to the presence of many pseudogenes arising from whole-genome duplication-generated redundancy or the capture and movement of gene fragments by transposable elements. Machine learning on genome-wide epigenetic marks, informed by transcriptomic and proteomic training data, could be used to improve annotations through classification of all putative protein-coding genes as either constitutively silent or able to be expressed. Expressed genes were subclassified as able to express both mRNAs and proteins or only RNAs, and CG gene body methylation was associated only with the former subclass. More than 60,000 protein-coding genes have been annotated in the reference genome of maize inbred B73. About two-thirds of these genes are transcribed and are designated the filtered gene set (FGS). Classification of genes by our trained random forest algorithm was accurate and relied only on histone modifications or DNA methylation patterns within the gene body; promoter methylation was unimportant. Other inbred lines are known to transcribe significantly different sets of genes, indicating that the FGS is specific to B73. We accurately classified the sets of transcribed genes in additional inbred lines, arising from inbred-specific DNA methylation patterns. This approach highlights the potential of using chromatin information to improve annotations of functional genes.

Monitoring the interplay between transposable element families and DNA methylation in maize.

DNA methylation and epigenetic silencing play important roles in the regulation of transposable elements (TEs) in many eukaryotic genomes. A majority of the maize genome is derived from TEs that can be classified into different orders and families based on their mechanism of transposition and sequence similarity, respectively. TEs themselves are highly methylated and it can be tempting to view them as a single uniform group. However, the analysis of DNA methylation profiles in flanking regions provides evidence for distinct groups of chromatin properties at different TE families. These differences among TE families are reproducible in different tissues and different inbred lines. TE families with varying levels of DNA methylation in flanking regions also show distinct patterns of chromatin accessibility and modifications within the TEs. The differences in the patterns of DNA methylation flanking TE families arise from a combination of non-random insertion preferences of TE families, changes in DNA methylation triggered by the insertion of the TE and subsequent selection pressure. A set of nearly 70,000 TE polymorphisms among four assembled maize genomes were used to monitor the level of DNA methylation at haplotypes with and without the TE insertions. In many cases, TE families with high levels of DNA methylation in flanking sequence are enriched for insertions into highly methylated regions. The majority of the >2,500 TE insertions into unmethylated regions result in changes in DNA methylation in haplotypes with the TE, suggesting the widespread potential for TE insertions to condition altered methylation in conserved regions of the genome. This study highlights the interplay between TEs and the methylome of a major crop species.

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis.

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.

CHH DNA methylation increases at 24-PHAS loci depend on 24-nt phased small interfering RNAs in maize meiotic anthers.

Plant phased small interfering RNAs (phasiRNAs) contribute to robust male fertility; however, specific functions remain undefined. In maize (Zea mays), male sterile23 (ms23), necessary for both 24-nt phasiRNA precursor (24-PHAS) loci and Dicer-like5 (Dcl5) expression, and dcl5-1 mutants unable to slice PHAS transcripts lack nearly all 24-nt phasiRNAs. Based on sequence capture bisulfite-sequencing, we find that CHH DNA methylation of most 24-PHAS loci is increased in meiotic anthers of control plants but not in the ms23 and dcl5 mutants. Because dcl5-1 anthers express PHAS precursors, we conclude that the 24-nt phasiRNAs, rather than just activation of PHAS transcription, are required for targeting increased CHH methylation at these loci. Although PHAS precursors are processed into multiple 24-nt phasiRNA products, there is substantial differential product accumulation. Abundant 24-nt phasiRNA positions corresponded to high CHH methylation within individual loci, reinforcing the conclusion that 24-nt phasiRNAs contribute to increased CHH methylation in cis.

Variation and Inheritance of Small RNAs in Maize Inbreds and F1 Hybrids.

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.