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Gene expression and complex phenotypes are determined by the activity of cis-regulatory elements. However, an understanding of how extant genetic variants affect cis regulation remains limited. Here, we investigated the consequences of cis-regulatory diversity using single-cell genomics of more than 0.7 million nuclei across 172Zea mays(maize) inbreds. Our analyses pinpointed cis-regulatory elements distinct to domesticated maize and revealed how historical transposon activity has shaped the cis-regulatory landscape. Leveraging population genetics principles, we fine-mapped about 22,000 chromatin accessibility–associated genetic variants with widespread cell type–specific effects. Variants in TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR–binding sites were the most prevalent determinants of chromatin accessibility. Finally, integrating chromatin accessibility–associated variants, organismal trait variation, and population differentiation revealed how local adaptation has rewired regulatory networks in unique cellular contexts to alter maize flowering. 

While considerable knowledge exists about the enzymes pivotal for C₄photosynthesis, much less is known about thecis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C₄enzymes for five different grass species. This study spans four C₄species, covering three distinct photosynthetic subtypes:Zea maysandSorghum bicolor(NADP-dependent malic enzyme),Panicum miliaceum(NAD-dependent malic enzyme),Urochloa fusca(phosphoenolpyruvate carboxykinase), along with the C₃outgroupOryza sativa. We studied thecis-regulatory landscape of enzymes essential across all C₄species and those unique to C₄subtypes, measuring cell-type-specific biases for C₄enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C₄evolution. Besides promoter proximal ACRs, we found that, on average, C₄genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C₄evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution ofcis-regulation at these C₄loci. This study illuminates the dynamic and complex nature ofcis-regulatory elements evolution in C₄photosynthesis, particularly highlighting the intricatecis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C₃crop performance under changing climatic conditions. 

Abstract Gene regulation in eukaryotes is partly shaped by the 3D organization of chromatin within the cell nucleus. Distal interactions between cis-regulatory elements and their target genes are widespread, and many causal loci underlying heritable agricultural traits have been mapped to distal non-coding elements. The biology underlying chromatin loop formation in plants is poorly understood. Dissecting the sequence features that mediate distal interactions is an important step toward identifying putative molecular mechanisms. Here, we trained GenomicLinks, a deep learning model, to identify DNA sequence features predictive of 3D chromatin interactions in maize. We found that the presence of binding motifs of specific transcription factor classes, especially bHLH, is predictive of chromatin interaction specificities. Using an in silico mutagenesis approach we show the removal of these motifs from loop anchors leads to reduced interaction probabilities. We were able to validate these predictions with single-cell co-accessibility data from different maize genotypes that harbor natural substitutions in these TF binding motifs. GenomicLinks is currently implemented as an open-source web tool, which should facilitate its wider use in the plant research community. 

Abstract Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called scifi-ATAC-seq, single-cell combinatorial fluidic indexing ATAC-sequencing, which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using the 10X Genomics platform. With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples can be indexed in a single emulsion reaction, representing an approximately 20-fold increase in throughput compared to the standard 10X Genomics workflow. 

The ease and throughput of single-cell genomics have steadily improved, and its current trajectory suggests that surveying single-cell populations will become routine. We discuss the merger of quantitative genetics with single-cell genomics and emphasize how this synergizes with advantages intrinsic to plants. Single-cell population genomics provides increased detection resolution when mapping variants that control molecular traits, including gene expression or chromatin accessibility. Additionally, single-cell population genomics reveals the cell types in which variants act and, when combined with organism-level phenotype measurements, unveils which cellular contexts impact higher-order traits. Emerging technologies, notably multiomics, can facilitate the measurement of both genetic changes and genomic traits in single cells, enabling single-cell genetic experiments. The implementation of single-cell genetics will advance the investigation of the genetic architecture of complex molecular traits and provide new experimental paradigms to study eukaryotic genetics. 

Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called single-cell combinatorial fluidic indexing ATAC-sequencing (scifi-ATAC-seq), which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using a widely commercialized microfluidics platform (10X Genomics). With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples in a single emulsion reaction can be indexed, representing a ~20-fold increase in throughput compared to the standard 10X Genomics workflow. 

cis-Regulatory elements encode the genomic blueprints that ensure the proper spatiotemporal patterning of gene expression necessary for appropriate development and responses to the environment. Accumulating evidence implicates changes to gene expression as a major source of phenotypic novelty in eukaryotes, including acute phenotypes such as disease and cancer in mammals. Moreover, genetic and epigenetic variation affecting cis-regulatory sequences over longer evolutionary timescales has become a recurring theme in studies of morphological divergence and local adaptation. Here, we discuss the functions of and methods used to identify various classes of cis-regulatory elements, as well as their role in plant development and response to the environment. We highlight opportunities to exploit cis-regulatory variants underlying plant development and environmental responses for crop improvement efforts. Although a comprehensive understanding of cis-regulatory mechanisms in plants has lagged behind that in animals, we showcase several breakthrough findings that have profoundly influenced plant biology and shaped the overall understanding of transcriptional regulation in eukaryotes. 

Core Ideas Cross‐species models of chromatin state from sequence are comparable or superior to within‐species models. Model performance is highest on accessible regions open in many tissues. Transcription factor motifs can be ranked by importance to each species and chromatin state.