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Cis-regulatory elements are specific DNA sequences that control gene expression in a spatiotemporal manner, and variation within these elements represents a major source of phenotypic diversity and evolutionary innovation. Nevertheless, how regulatory elements evolve and shape gene expression remains poorly understood, particularly in plants. The well-resolved phylogeny of allopolyploid peanut (Arachis hypogaea) and its diploid progenitors,Arachis duranensisandArachis ipaensis, provides an ideal system to investigate the regulatory evolution at a lineage-specific level. By integrating comparative analyses of sequence similarity, chromatin accessibility, histone modifications, conserved noncoding sequences (CNSs), and gene expression, we reconstructed the evolutionary trajectories of Accessible Chromatin Regions (ACRs), where regulatory elements typically reside, and revealed their distinct contributions to homoeolog expression bias, unequal expressions between homoeologs. Most ACRs exhibited high sequence similarity, comparable chromatin accessibility, and conserved states for H3K4me3, H3K56ac, and H3K36me3, indicating regulatory stability after hybridization and polyploidization. However, a subset of novel ACRs emerged de novo from previously nonregulatory regions or through sequence mutations in preexisting ACRs, arising at different rates and evolutionary stages. Notably, even highly sequence-conserved ACRs exhibited substantial variation in chromatin accessibility, consistent with CNS composition differences and minor sequence variation, although causal relationships remain to be demonstrated. Our analyses further revealed a complex spectrum of CNS dynamics within the diploid-polyploid framework. Overall, our study provides empirical insights into the fine-scale evolution of plant regulatory landscapes and complements previous large-scale comparisons across distant lineages. 

Abstract Understanding the mechanisms underlying key agricultural traits remains a central challenge in crop research, but recent advances in technologies are providing powerful tools to address this issue. Among these, single-cell and spatial transcriptomics have revealed tissue heterogeneity and spatial organization, offering unique insights into cellular gene expression dynamics and the coordinated activity of multiple cell types. These approaches help uncover how specific cell types contribute to agricultural traits and refine candidate loci lists through integration with trait-associated loci. Additionally, single-cell and spatial transcriptomics have the potential to serve as cell-level readout platforms integrating cellular perturbations, enabling high-throughput discovery of causal relationships between genotype and gene expression at the cellular level in plants. Successful implementation will accelerate the identification of key genetic variants for crop improvement. Here we review lessons learned from application of single-cell screening in mammalian cells, highlight major technical and biological barriers to its use in plants, and outline potential strategies to overcome these challenges. Together, the widespread application and integration of single-cell and spatial transcriptomics with other technologies enable not only the descriptive cataloging of cell states but also the causal interrogation of sequence functions and regulatory networks at cell type resolution, ultimately advancing gene function studies and accelerating crop improvement. 

Abstract Gene duplication is a major source of evolutionary innovation, enabling the emergence of novel expression patterns and functions. Leveraging single-cell genomics, we investigated the transcriptional and cis-regulatory landscapes of duplicated genes in cultivated soybean (Glycine max), which has undergone 2 rounds of whole-genome duplication. Our analysis revealed extensive diversity of transcriptional profiles within and across tissues among duplicated gene pairs. Within-tissue divergence was largely attributable to genetic variation in their associated accessible chromatin regions (ACRs), where cis-regulatory elements reside, whereas cross-tissue divergence was more likely shaped by dynamics in ACR chromatin accessibility profiles across tissues. Distinct duplication mechanisms also likely give rise to different types of cis-regulatory variants, contributing variably to transcriptional divergence. By comparing ACRs associated with gene sets derived from 2 rounds of whole-genome duplication and sharing a common ancestral gene, we found that most ACRs retained one or multiple corresponding duplicated sequences in which mutations gradually accumulated over time, while a subset likely arose de novo. Finally, we traced the evolution of cell-type-specific expression and cell-type-specific ACRs within duplicated gene sets, illustrating a powerful framework for identifying candidate regulatory regions driving cell-type-specific expression. Collectively, our findings highlight the important role of cis-regulatory evolution in shaping transcriptional divergence in a spatiotemporal manner, uncovered with the resolution of single-cell genomics. 

The rate and spectrum of somatic mutations can diverge from that of germline mutations. This is because somatic tissues experience different mutagenic processes than germline tissues. Here, we use nanorate sequencing (NanoSeq) to identify somatic mutations inArabidopsisshoots with high sensitivity. We report a somatic mutation rate of 3.6 × 10⁻⁸mutations/bp, ~2 to 7× measured germline mutation rates. Somatic mutations displayed elevated signatures consistent with oxidative damage, UV damage, and transcription-coupled nucleotide excision repair. Both somatic and germline mutations were enriched in transposable elements and depleted in genes, but this depletion was greater in germline mutations. Somatic mutation rate correlated with proximity to the centromere, DNA methylation, chromatin accessibility, and gene/TE content, properties which were also largely true of germline mutations. We note that DNA methylation and chromatin accessibility have different predicted effects on mutation rate for genic and nongenic regions; DNA methylation associates with a greater increase in mutation rate when in nongenic regions, and accessible chromatin associates with a lower mutation rate in nongenic regions but a higher mutation rate in genic regions. Together, these results characterize key differences and similarities in the genomic distribution of somatic and germline mutations. 

The rate and spectrum of somatic mutations can diverge from that of germline mutations. This is because somatic tissues experience different mutagenic processes than germline tissues. Here, we use nanorate sequencing (NanoSeq) to identify somatic mutations in Arabidopsis shoots with high sensitivity. We report a somatic mutation rate of 3.6x10^-8 mutations/bp, ~4-5x the germline mutation rate. Somatic mutations displayed elevated signatures consistent with oxidative damage, UV damage, and transcription-coupled nucleotide excision repair. Both somatic and germline mutations were enriched in transposable elements and depleted in genes, but this depletion was greater in germline mutations. Somatic mutation rate correlated with proximity to the centromere, DNA methylation, chromatin accessibility, and gene/TE content, properties which were also largely true of germline mutations. We note DNA methylation and chromatin accessibility have different predicted effects on mutation rate for genic and non-genic regions; DNA methylation associates with a greater increase in mutation rate when in non-genic regions, and accessible chromatin associates with a lower mutation rate in non-genic regions but a higher mutation rate in genic regions. Together, these results characterize key differences and similarities in the genomic distribution of somatic and germline mutations. 

Spatial transcriptomics is a disruptive technology that enables the identification of cell-type-specific transcripts en masse. However, the use of spatial transcriptomics for plant tissues has been challenging due to issues related to tissue preparation. Here, we present a protocol for preparing fresh frozen soybean tissues for spatial transcriptomics. We describe the steps for embedding, cryosectioning, fixation, staining, and imaging of soybean tissues.We then detail procedures for library preparation and sequencing. 

Abstract BackgroundGenetic and epigenetic perturbation of cis-regulatory sequences can shift patterns of gene expression and result in novel phenotypes. Phased genome assemblies now enable the local dissection of linkages between cis-regulatory sequences, including their epigenetic state, and allele-specific gene expression to further characterize gene regulation and resulting phenotypes in heterozygous genomes. ResultsWe assembled a locally phased genome for a mandarin hybrid named ‘Fairchild’ to explore the molecular signatures of allele-specific gene expression. With local genome phasing, genes with allele-specific expression were paired with haplotype-specific chromatin states, including levels of chromatin accessibility, histone modifications, and DNA methylation. We found that 30% of variation in allele-specific expression could be attributed to haplotype associated factors, with allelic levels of chromatin accessibility and three histone modifications in gene bodies having the most influence. Structural variants in promoter regions were also associated with allele-specific expression, including specific enrichments of hAT and MULE-MuDR DNA transposon sequences. Integration of haplotype-resolved genetic and epigenetic landscapes with high-throughput phenotypic analysis of fruit traits in a panel of 154 accessions with mandarin and pummelo ancestry revealed that trait-associated variants were enriched in regions of open chromatin. Mining of trait-associated variants uncovered a Gypsy retrotransposon insertion in a gene that regulates potassium transport and may contribute to the reduction in fruit size that is observed in mandarins. Conclusions​​Using a locally phased assembly of a heterozygous cultivar of citrus, we dissected the interplay between genetic variants and molecular phenotypes to reveal cis-regulatory sequences with potential functional effects on phenotypes relevant for genetic improvement. 

DNA methylation is important to maintain genome stability, but alterations in genome-wide methylation patterns can produce widespread genomic effects, which have the potential to facilitate rapid adaptation. We investigate DNA methylation evolution in Arabidopsis thaliana during its colonization of the drought-prone Cape Verde Islands (CVI). We identified three high impact changes in genes linking histone modification to DNA methylation that underlie variation in DNA methylation within CVI. Gene body methylation is reduced in CVI relative to the Moroccan outgroup due to a 2.7-kb deletion between two VARIANT IN METHYLATION genes (VIM2 and VIM4) that causes aberrant expression of the VIM2/4 homologs. Disruptions of CHROMOMETHYLASE 2 (CMT2) and a newly identified DNA methylation modulator, F-BOX PROTEIN 5 (FBX5), which we validated using CRISPR mutant analysis, contribute to DNA methylation of transposable elements (TEs) within CVI. Overall, our results reveal rapid methylome evolution driven largely by high impact variants in three genes.