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After the most recent tetraploidy in the Arabidopsis lineage, most gene pairs lost one, but not both, of their duplicates. We manually inspected the 3,179 retained gene pairs and their surrounding gene space still present in the genome using a custom-made viewer application. The display of these pairs allowed us to define intragenic conserved noncoding sequences (CNSs), identify exon annotation errors, and discover potentially new genes. Using a strict algorithm to sort high-scoring pair sequences from the bl2seq data, we created a database of 14,944 intragenomic Arabidopsis CNSs. The mean CNS length is 31 bp, ranging from 15 to 285 bp. There are [almost equal to]1.7 CNSs associated with a typical gene, and Arabidopsis CNSs are found in all areas around exons, most frequently in the 5' upstream region. Gene ontology classifications related to transcription, regulation, or \"response to ...\" external or endogenous stimuli, especially hormones, tend to be significantly overrepresented among genes containing a large number of CNSs, whereas protein localization, transport, and metabolism are common among genes with no CNSs. There is a 1.5% overlap between these CNSs and the 218,982 putative RNAs in the Arabidopsis Small RNA Project database, allowing for two mismatches. These CNSs provide a unique set of noncoding sequences enriched for function. CNS function is implied by evolutionary conservation and independently supported because CNS-richness predicts regulatory gene ontology categories.

Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root–nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.

Background Efforts to characterize regulatory elements in plant genomes traditionally rely on evolutionary conservation and chromatin accessibility. Recently, intergenic bi-directional nascent transcript has emerged as a putative hallmark of active enhancers. Here, we integrate these approaches to better define the cis -regulatory landscape of the rice genome. Results In juvenile leaf tissues of the Azucena rice variety, we analyze conserved noncoding sequences, intergenic bi-directional transcripts, and regions of open chromatin. These three features highlight distinct classes of regulatory targets, each exhibiting complexity and regulatory roles. Conserved noncoding sequences are associated with more complex regulatory interactions, while regions marked by chromatin accessibility or bi-directional nascent transcription tend to promote more stable regulatory activity. Some transcribed regulatory sites harbor elements linked to transposable element silencing, whereas others correlate with increased expression of nearby genes, pointing to candidate transcribed regulatory elements. We further identified molecular interactions between genic regions and intergenic transcribed regulatory elements using 3-dimensional chromatin contact data, we identify physical interactions between transcribed intergenic regions and genic regions. These interactions often co-localize with expression quantitative trait loci and coincide with increased transcription, further supporting a regulatory role. Conclusions Our integrative analysis reveals multiple distinct classes of regulatory elements in the rice genome, with overlapping but non-identical targets and signatures. Many candidate elements share features consistent with transcriptional enhancement, though the specific criteria for defining active enhancers in plants require further characterization. These findings underscore the importance of using complementary genomic signals to discover and characterize functionally diverse regulatory elements in plant genomes.

Abstract Advances in genome sequencing have accelerated the assembly of hundreds of plant genomes. Because of limited tools, current pangenome approaches overlook conserved noncoding sequences, though these sequences potentially harbor diverse regulatory elements. Here, we present panCG, an integrative pipeline for family level super-pangenome analysis across coding and noncoding sequences. PanCG enables the reference-free construction of pangene and pan-conserved noncoding sequences (panCNS) indices and integrative analyses between them. Applying this pipeline to the orange subfamily (Aurantioideae) with 22 representative genomes, we identified 167,989 panCNSs, 44.63% of which exhibited potential functionality, as evidenced by multiomic data (eg ATAC-seq, ChIP-seq). Functionally, approximately half of duplicated genes showed expression divergence, likely mediated by sequence variants in these panCNSs. Although transposable element (TE) mobilization resulted in substantial CNS loss, it also contributed to the origin of a small portion of functional CNSs. By integrating panCNS and pangene data, we identified 1,391 conserved colocalized blocks of CNSs and genes across all selected genomes, providing clear evidence for coevolution between CNSs and their target genes. Notably, leveraging these pan-genomic resources, we pinpointed numerous CNSs and gene–CNS regulatory modules as candidates underlying the domestication of citrus fruit traits from wild relatives. Our study presents a powerful tool for interspecific super-pangenome analysis, whereas shedding new light on the functional roles and evolutionary dynamics of CNSs in citrus, and provides a valuable resource of candidate regulatory elements for citrus fruit biology and breeding research.

One of the major quantitative trait loci for flowering time in maize, the Vegetative to generative transition 1 (Vgt1) locus, corresponds to an upstream (70 kb) noncoding regulatory element of ZmRap2.7, a repressor of flowering. At Vgt1, a miniature transposon (MITE) insertion into a conserved noncoding sequence was previously found to be highly associated with early flowering in independent studies. Because cytosine methylation is known to be associated with transposons and to influence gene expression, we aimed to investigate how DNA methylation patterns in wild-type and mutant Vgt1 correlate with ZmRap2.7 expression. The methylation state at Vgt1 was assayed in leaf samples of maize inbred and F1 hybrid samples, and at the syntenic region in sorghum. The Vgt1-linked conserved noncoding sequence was very scarcely methylated both in maize and sorghum. However, in the early maize Vgt1 allele, the region immediately flanking the highly methylated MITE insertion was significantly more methylated and showed features of methylation spreading. Allele-specific expression assays revealed that the presence of the MITE and its heavy methylation appear to be linked to altered ZmRap2.7 transcription. Although not providing proof of causative connection, our results associate transposon-linked differential methylation with allelic state and gene expression at a major flowering time quantitative trait locus in maize.

Abstract Recent pangenome studies have revealed a large fraction of the gene content within a species exhibits presence–absence variation (PAV). However, coding regions alone provide an incomplete assessment of functional genomic sequence variation at the species level. Little to no attention has been paid to noncoding regulatory regions in pangenome studies, though these sequences directly modulate gene expression and phenotype. To uncover regulatory genetic variation, we generated chromosome-scale genome assemblies for thirty Arabidopsis thaliana accessions from multiple distinct habitats and characterized species level variation in Conserved Noncoding Sequences (CNS). Our analyses uncovered not only PAV and positional variation (PosV) but that diversity in CNS is nonrandom, with variants shared across different accessions. Using evolutionary analyses and chromatin accessibility data, we provide further evidence supporting roles for conserved and variable CNS in gene regulation. Additionally, our data suggests that transposable elements contribute to CNS variation. Characterizing species-level diversity in all functional genomic sequences may later uncover previously unknown mechanistic links between genotype and phenotype.

Abstract We developed dbCNS (http://yamasati.nig.ac.jp/dbcns), a new database for conserved noncoding sequences (CNSs). CNSs exist in many eukaryotes and are assumed to be involved in protein expression control. Version 1 of dbCNS, introduced here, includes a powerful and precise CNS identification pipeline for multiple vertebrate genomes. Mutations in CNSs may induce morphological changes and cause genetic diseases. For this reason, many vertebrate CNSs have been identified, with special reference to primate genomes. We integrated ∼6.9 million CNSs from many vertebrate genomes into dbCNS, which allows users to extract CNSs near genes of interest using keyword searches. In addition to CNSs, dbCNS contains published genome sequences of 161 species. With purposeful taxonomic sampling of genomes, users can employ CNSs as queries to reconstruct CNS alignments and phylogenetic trees, to evaluate CNS modifications, acquisitions, and losses, and to roughly identify species with CNSs having accelerated substitution rates. dbCNS also produces links to dbSNP for searching pathogenic single-nucleotide polymorphisms in human CNSs. Thus, dbCNS connects morphological changes with genetic diseases. A test analysis using 38 gnathostome genomes was accomplished within 30 s. dbCNS results can evaluate CNSs identified by other stand-alone programs using genome-scale data.

• Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root–nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. • To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). • We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. • CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.

Background Gene regulatory evolution is a well-known source of phenotypic diversity and adaptive evolution. Although cis- regulatory elements (CREs) play a vital role in gene expression evolution, the molecular evolution of CREs remains mostly unknown due to the difficulty in identifying and characterizing these functional elements. Comparative genomic analyses of noncoding DNA can be leveraged to identify conserved noncoding sequences (CNS), many of which may harbor functional CREs conserved by purifying selection. However, purely computational inference of CREs from putative CNS can be erroneous due to the complex genomic architecture in plants. Results One promising experimental approach to identify CREs is by profiling accessible chromatin regions (ACRs) that are often associated with the location of CREs. In this study, we use comparative genomics along with the profiling of ACRs to study the molecular evolution of putative functional noncoding regulatory regions in Panicoid grasses. We identified sets of CNS that varied in relationship to the degree of evolutionary divergence among the studied taxa, including identifying core-Panicoid-CNS. We augmented this analysis by profiling ACRs in Panicum hallii ecotypes using ATAC-seq. ACRs had low SNP density at the summit, harbored a high frequency of core-Panicoid-CNS, and were enriched with expression QTL. These data help to annotate the P. hallii genome for putative functional elements and suggest that a large proportion of these ACRs are evolving under purifying selection. Turnover in CNS and ACR between ecotypes of P. hallii identifies a small set of putatively divergent CREs that may underlie differences in gene regulation between genotypes from inland and coastal habitats. Conclusions In summary, we profiled ACRs in Panicoid grasses and integrated this data with our putative CNS prediction framework, which provides unique insight into patterns of polymorphism and divergence in CREs in C4 perennial grasses.

Experimental studies have found the involvement of certain conserved noncoding sequences (CNSs) in the regulation of the proximal protein-coding genes in mammals. However, reported cases of long range enhancer activities and inter-chromosomal regulation suggest that proximity of CNSs to protein-coding genes might not be important for regulation. To test the importance of the CNS genomic location, we extracted the CNSs conserved between chicken and four mammalian species (human, mouse, dog, and cattle). These CNSs were confirmed to be under purifying selection. The intergenic CNSs are often found in clusters in gene deserts, where protein-coding genes are in paucity. The distribution pattern, ChIP-Seq, and RNA-Seq data suggested that the CNSs are more likely to be regulatory elements and not corresponding to long intergenic noncoding RNAs. Physical distances between CNS and their nearest protein coding genes were well conserved between human and mouse genomes, and CNS-flanking genes were often found in evolutionarily conserved genomic neighborhoods. ChIP-Seq signal and gene expression patterns also suggested that CNSs regulate nearby genes. Interestingly, genes with more CNSs have more evolutionarily conserved expression than those with fewer CNSs. These computationally obtained results suggest that the genomic locations of CNSs are important for their regulatory functions. In fact, various kinds of evolutionary constraints may be acting to maintain the genomic locations of CNSs and protein-coding genes in mammals to ensure proper regulation.