The research team, led by investigators at the University of Chicago, collected pinch biopsies from the terminal ileum and the ascending colon of 23 patients with Crohn’s disease, some with active inflammation and some without, alongside 16 healthy controls. From 71 biopsies, they generated single-cell assay for transposase-accessible chromatin with sequencing, or scATAC-seq, data. After rigorous quality control, the dataset retained 178,030 individual cells, which the researchers clustered into 29 distinct cell types spanning the epithelial, immune and stromal lineages of the intestinal wall. By annotating these clusters with reference gene-expression data, they built a cell-type-resolved catalog of 557,310 candidate cis-regulatory elements, or cCREs, each representing a region of open, potentially active chromatin.

The scale of the atlas matters because Crohn’s disease is not a disease of a single cell type. It is a chronic inflammatory condition of the gastrointestinal tract that arises from an interplay of host genetics, environmental triggers and the gut microbiota. Genome-wide association studies have linked more than 320 genetic loci to inflammatory bowel disease risk, but most of those risk variants sit in noncoding regions of the genome, far from any obvious gene, and their function has remained opaque. Knowing which cell types open or close which regulatory elements provides exactly the kind of functional annotation needed to interpret those variants.

The cellular composition of the atlas reflects the biology of the gut. Epithelial cells dominated the dataset, accounting for 69.3 percent of all cells, followed by immune cells at 26.3 percent and stromal cells at 4.3 percent. Among the epithelial compartment, the researchers identified mature absorptive cells specific to the ileum, known as enterocytes, and their colonic counterparts, colonocytes, along with goblet cells, Paneth cells, enteroendocrine cells and Tuft cells. The immune compartment included CD4-positive and CD8-positive T cells, gamma-delta T cells, natural killer cells, innate lymphoid cells, plasma cells, several B cell subsets, macrophages, dendritic cells and mast cells. Notably, the team also captured neutrophils, innate immune cells that are notoriously difficult to profile with standard single-cell RNA sequencing and have consequently remained understudied despite their suspected role in inflammatory disease.

Because discrete cell-type clusters cannot fully capture dynamic processes such as inflammation or continuous differentiation, the researchers complemented their cluster analysis with a computational approach called topic modeling, which assigns each cell partial membership in multiple regulatory programs simultaneously. This revealed programs shared across related cell types as well as programs tied to specific contexts. One program, topic 7, stood out as a signature of inflammation in epithelial cells: its activity was high in biopsies taken directly from inflamed regions, moderate in adjacent noninflamed tissue from the same patients, and absent in healthy controls. The regulatory elements within this program were enriched for binding motifs of AP-1 family factors and interferon regulatory factors, implicating interferon and JAK-STAT signaling as central drivers of the epithelial inflammatory response. Putative target genes included well-known inflammatory markers such as LCN2, NOS2, DUOX2 and major histocompatibility complex class II genes.

Inflammation left its mark across the entire mucosa. Comparing inflamed tissue with healthy controls, the researchers identified inflammation-associated differentially accessible regions in 21 cell types, with epithelial cells yielding the largest number. Endothelial cells, which line blood vessels and gate the movement of cells and molecules into inflamed tissue, showed 1,702 such regions enriched for proinflammatory transcription factor motifs. CD8-positive T cells displayed accessibility changes linked to the effector regulator EOMES, plasma cells showed changes involving the maturation factor POU2F2, and macrophages exhibited broad remodeling involving AP-1, IRF and STAT family factors. In goblet cells and Paneth cell-associated programs, the data pointed to plasticity in the secretory lineage, suggesting that these mucus- and antimicrobial-producing cells reprogram their regulatory landscape in response to injury and inflammation.

To connect DNA sequence directly to chromatin state, the team exploited natural genetic variation among their 39 donors. By imputing genotypes from the sequencing data and mapping chromatin accessibility quantitative trait loci, or caQTLs, in 19 cell types, they identified thousands of genetic variants that alter chromatin accessibility in a cell-type-specific manner. Critically, they showed that variants predicted to disrupt transcription factor binding motifs correlate with reduced accessibility at the affected elements. Disruption of TCF3 motifs in plasma cells and CDX2 motifs in colonocytes were among the clearest examples, providing direct evidence that the regulatory logic inferred from motif enrichment reflects real functional relationships between sequence, protein binding and chromatin state.

The atlas then delivered its most consequential finding: a cell-type-resolved view of Crohn’s disease heritability. Using stratified linkage disequilibrium score regression on GWAS summary statistics, the researchers found that 10 of the 29 cell types showed significant enrichment of inflammatory bowel disease genetic risk, and all 10 were immune cells. Adaptive immune cells, particularly T cells, carried the strongest signal, but innate immune cells, including macrophages, dendritic cells and neutrophils, were also enriched for Crohn’s disease risk specifically. Strikingly, ulcerative colitis risk was not enriched in those innate cells, a result consistent with known differences in genetic architecture between the two major forms of inflammatory bowel disease and a demonstration of the specificity of the approach. Epithelial and stromal cells, despite their dramatic inflammatory responses, showed no enrichment for disease risk, and neither did the inflammation-associated regulatory programs themselves.

That separation between genetic risk and inflammatory consequence is one of the study’s most provocative implications. The data suggest that the genetic predisposition to Crohn’s disease operates primarily through immune cells, while the epithelial and stromal changes visible in inflamed tissue are largely downstream effects of the inflammatory process rather than primary causes. The team also linked body mass index heritability to enteroendocrine cells, confirming a previous observation and hinting that part of the genetic contribution to metabolism is mediated through these hormone-producing gut cells.

Finally, the researchers fine-mapped Crohn’s disease GWAS signals and overlaid the resulting variants onto their atlas. Variants with high posterior inclusion probabilities were 3.5-fold enriched within cCREs, and the team prioritized 30 candidate regulatory variants across 22 loci. Most fell in elements active in innate and adaptive immune cells, but several implicated epithelial contexts. Near the key risk gene NOD2, a fully prioritized variant sat in a neutrophil-associated regulatory element. Two variants near CIITA, a co-activator of MHC class II genes, occupied adjacent regulatory elements with strikingly different specificities, one active across immune and endothelial cells and the other strongly associated with epithelial inflammation, suggesting that multiple variants within a single locus can act through different cell types. The authors caution that their moderate cohort size limited power for finer clinical stratification and for detecting caQTLs in rare cell types, and larger cohorts will be needed to dissect disease subtypes and treatment responses. Even so, the atlas stands as a public resource that transforms a list of noncoding risk variants into testable hypotheses about which cells, which switches and which genes set the stage for Crohn’s disease.

Subject of Research: Single-cell chromatin accessibility mapping of the human intestinal mucosa to identify regulatory programs and cell types underlying Crohn's disease genetic risk

Article Title: Single-cell analysis of chromatin accessibility in the human intestine identifies regulatory programs and clarifies genetic associations in Crohn’s disease

Article References: Zhao, Y., Zhou, R., Mu, Z., Carbonetto, P., Zhong, X., Xie, B., Luo, K., Jiang, Z., Liu, J., Cham, C. M., Koval, J., He, X., Dahl, A. W., Liu, X., Chang, E. B., Basu, A., & Pott, S. (2026). Single-cell analysis of chromatin accessibility in the human intestine identifies regulatory programs and clarifies genetic associations in Crohn’s disease. Nature Genetics, 58(10), 2535-2549. https://doi.org/10.1038/s41588-026-02755-z

Image Credits: AI Generated

DOI: 10.1038/s41588-026-02755-z

Keywords: Crohn's disease, single-cell ATAC-seq, chromatin accessibility, regulatory elements, inflammatory bowel disease, GWAS, intestinal mucosa, T cells, neutrophils, epigenomics, fine-mapping, topic modeling

The research, led by Chen Chen and Geng Zhou of the Hunan Vegetable Research Institute at the Hunan Academy of Agricultural Sciences, together with colleagues at Yuelushan Laboratory and Hunan Agricultural University, focused on the gene CsMYB60. This gene encodes an R2R3-MYB transcription factor, a type of DNA-binding protein that acts as a master switch for plant pigment production. Earlier work had already established that CsMYB60 drives the biosynthesis of flavonoids and proanthocyanidins, two classes of plant secondary metabolites, in cucumber fruit spines. More recent studies extended that role to the mature fruit skin, showing that the gene helps control flavonoid accumulation and, with it, the color the peel takes on as the fruit ripens.

To dissect the genetic basis of peel color with precision, the team turned to a powerful experimental resource: a pair of near-isogenic lines. These are two cucumber inbred lines, L14 and L19, that are genetically almost identical except at the region governing peel color. L14 develops a yellow mature peel, while L19, a mutant derived from it, remains white. Because the two lines share nearly all of their genetic background, any difference in peel color can be attributed with high confidence to the small stretch of DNA where they differ, eliminating the noise that usually complicates genetic analysis in diverse varieties.

The researchers combined bulked segregant analysis with high-resolution fine mapping. In bulked segregant analysis, scientists cross the two contrasting lines, then pool the DNA of many offspring that share the same trait, in this case yellow peel, and sequence those pools alongside pools from offspring with the opposite trait. Regions of the genome where the pools diverge sharply are candidate homes for the causal gene. This approach, originally developed for rapid mapping in rice, allowed the team to narrow the peel color locus to a 121-kilobase interval on chromosome 4. Within that interval sat exactly one compelling candidate: CsMYB60.

What the team found when they compared the DNA sequences of the two alleles was the study’s central surprise. The yellow-peel line L14 carries not one but two Mutator-like element insertions, known as MULE1 and MULE2, within the second intron of CsMYB60. The white-peel mutant L19, by contrast, retains only MULE1. Mutator-like elements are a family of transposable elements, mobile DNA sequences that can copy or cut themselves out of the genome and reinsert elsewhere. Far from being genomic junk, transposable elements are now recognized as major drivers of plant evolution, frequently rewiring gene regulation by inserting into promoters, introns, or nearby regulatory regions and altering when, where, and how strongly a gene is expressed.

Here, the double insertion appeared to matter. During the peel color-transition stage, when the fruit’s skin is actively changing, the MULE1 plus MULE2 haplotype showed higher CsMYB60 transcript abundance than the MULE1-only haplotype in the near-isogenic materials examined. In other words, the line carrying both insertions produced more messenger RNA from the pigment-regulating gene, and that line was the one that turned yellow. The authors are careful to note that the causal regulatory contribution of MULE2 itself remains to be determined; the association between the structural haplotype and both expression level and peel color is clear, but precisely how the second insertion boosts transcription is a question for future work.

The team did not stop at correlation. Using CRISPR/Cas9 genome editing, they knocked out CsMYB60 directly in the L14 background. The result was unambiguous: the edited plants lost their yellow pigmentation entirely, confirming that CsMYB60 is required for yellow mature peel formation in this genetic background. This kind of functional validation is the gold standard in plant genetics, because it demonstrates necessity rather than mere association. Delete the gene, and the trait disappears; restore or retain it with the right structural haplotype, and the trait appears.

The findings fit into a broader and rapidly evolving picture of how fruit color is controlled in cucurbits. Cucumber peel and flesh color are governed by a constellation of loci affecting chloroplast development, carotenoid accumulation, and flavonoid biosynthesis. Previous studies have implicated genes such as CsMYB36 in yellow-green immature peel, APRR2 in green immature fruit color, and various chloroplast-related genes in light green phenotypes. The R2R3-MYB family, meanwhile, is famous across the plant kingdom for its role in the MYB-bHLH-WDR transcriptional complexes that regulate flavonoid pathways. What makes the new study distinctive is that it identifies a structural haplotype, a specific physical arrangement of DNA, rather than a simple single-nucleotide change, as the molecular signature associated with the trait.

That distinction has practical consequences for breeding. Structural variation like transposon insertions is often invisible to standard SNP-based genotyping, which means breeders can inadvertently lose or overlook valuable haplotypes when crossing varieties. The authors point out that the molecular marker associated with the MULE1 plus MULE2 haplotype may facilitate tracking of this structural variant in relevant cucumber breeding materials. In practice, that means seed companies and public breeding programs could screen seedlings for the double-insertion haplotype and confidently predict which plants will produce the yellow mature peel that certain markets demand, without waiting for the fruit to ripen.

The study also adds to a growing appreciation of transposable elements as engines of agricultural diversity. In crops ranging from maize to grapevine to tomato, transposon insertions have been shown to create or modulate traits as varied as seed coat color, fruit shape, and ripening behavior. The cucumber peel story is a textbook example of how a mobile element can act as a regulatory rheostat: by landing inside an intron, the MULE insertions appear to tune the output of a transcription factor that sits atop an entire pigment biosynthesis pathway. Whether MULE2 acts by altering chromatin structure, changing intron splicing, or providing novel regulatory sequences remains open, and answering it will likely require additional experiments such as reporter assays and splicing analyses.

For now, the work delivers a complete arc from field observation to molecular mechanism. Two nearly identical cucumber lines, one yellow and one white, led researchers to a 121-kilobase window on chromosome 4, then to a single MYB gene, then to two nested transposon insertions, and finally to a CRISPR experiment that sealed the case. The identification of a previously unrecognized MULE1 plus MULE2 structural haplotype associated with distinct CsMYB60 transcript abundance and mature peel coloration gives cucumber geneticists a new landmark on their maps and gives breeders a practical tool. It also serves as a reminder that some of the most important instructions in a crop’s genome are not written in its genes at all, but in the restless, mobile DNA that has been reshaping those genomes for millions of years.

Subject of Research: Transposon-mediated structural haplotypes at the CsMYB60 gene and their association with mature peel coloration in cucumber

Article Title: Transposon-mediated structural haplotypes at CsMYB60 are associated with peel coloration in cucumber

Article References: Chen, C., Chen, J., Chen, H., Liu, X., Lu, X., Tian, Y., & Zhou, G. (2026). Transposon-mediated structural haplotypes at CsMYB60 are associated with peel coloration in cucumber. Theoretical and Applied Genetics, 139(10), Article 285. https://doi.org/10.1007/s00122-026-05388-2

Image Credits: AI Generated

DOI: 10.1007/s00122-026-05388-2

Keywords: cucumber, peel color, CsMYB60, transposable elements, MULE, structural haplotype, flavonoid biosynthesis, R2R3-MYB transcription factor, CRISPR/Cas9, fine mapping, bulked segregant analysis, plant genetics

The challenge for geneticists has always been that Verticillium wilt resistance is a quantitative trait. Rather than being governed by a single gene, it emerges from the combined effects of multiple quantitative trait loci, or QTL, scattered across the genome. Detecting these loci reliably, let alone pinpointing the causal genes within them, demands dense genetic maps, replicated disease evaluations across environments, and systematic functional validation. Earlier mapping efforts in cotton had identified resistance regions on chromosomes D7 and D9, and genome-wide association studies added further candidate intervals, but the leap from a broad QTL to a specific gene has remained difficult, particularly in the large and complex allotetraploid cotton genome.

To narrow that gap, the team, led by Yunlei Zhao, Baimei Cheng and Jinfa Zhang with corresponding authors Jianhua Lu and Hongmei Wang of the Institute of Cotton Research at the Chinese Academy of Agricultural Sciences, constructed high-density genetic maps of the regions harboring major resistance QTL. The key tool was a genotyping technology called Kompetitive Allele-Specific PCR, or KASP, which allows thousands of individual plants to be scored rapidly and cheaply at specific single-nucleotide polymorphisms. By saturating the target QTL intervals with KASP markers, the researchers were able to fine-map the loci, shrinking previously vague genomic neighborhoods down to precise physical intervals on the reference genome.

The mapping effort identified six QTL for Verticillium wilt resistance, five of which qualified as major QTL because they were detected in at least two independent environments, a critical test of whether a locus genuinely contributes to resistance rather than reflecting environmental noise. Four of these loci, named qVW-D05-1, qVW-D05-2, qVW-D05-3 and qVW-A01-1, were fine mapped to remarkably compact physical regions of just 15.2 to 241.3 kilobases on chromosomes D05 and A01. In a genome spanning roughly 2.5 billion base pairs, intervals of that size contain only a handful of genes, transforming an intractable search into a manageable candidate list.

With the intervals defined, the researchers deployed a multi-layered strategy to find the genes that matter. They combined gene annotation of the reference genome with whole-genome resequencing of resistant and susceptible lines, transcriptome sequencing to capture differences in gene activity after pathogen challenge, and quantitative reverse-transcription PCR to confirm expression patterns independently. A gene only earned candidate status if it showed both DNA sequence variation between resistant and susceptible germplasm and a measurable difference in expression. This double filter proved powerful: fifteen genes emerged that carried both genetic variants and differential expression, making them the strongest current leads for the molecular basis of wilt resistance in upland cotton.

The functional work went further. Four candidate genes associated with the qVW-D05-1 locus were cloned and subjected to functional analysis using virus-induced gene silencing, a technique that temporarily suppresses a target gene’s activity in the plant, together with overexpression studies that boost it. These complementary approaches allowed the team to observe what happens to disease resistance when each gene’s function is dialled down or up. The results indicated that the genes associated with the major QTL do contribute to resistance, strengthening the case that the fine-mapped intervals harbor genuine resistance determinants rather than statistical artifacts of mapping.

Perhaps the most mechanistically revealing finding concerns the nature of the sequence variation itself. The researchers found that variation in functional gene sequences, especially variations that change amino acids within protein domains, appears to be the underlying molecular mechanism distinguishing resistant from susceptible cotton lines. In other words, resistance is not simply a matter of genes being switched on or off; the resistant alleles encode subtly altered proteins whose functional domains differ in ways that presumably improve recognition of the pathogen or the execution of defense responses. This kind of allelic diversity is exactly what marker-assisted breeding can exploit, because DNA markers linked to the favorable variants can be tracked through generations without waiting years for field evaluations.

The study also delivered a result with immediate practical implications for breeding programs: the power of pyramiding. When the team assembled lines that carried different combinations of the major resistance QTL, a clear dosage effect appeared. The more resistant QTL a line accumulated, the stronger and more durable its Verticillium wilt resistance became. Lines that polymerized all four major resistance loci showed markedly higher and more stable resistance across every environment tested. Because quantitative resistance built from multiple loci is generally harder for pathogens to overcome than single-gene resistance, these findings suggest that stacking the favorable alleles from qVW-D05-1, qVW-D05-2, qVW-D05-3 and qVW-A01-1 could produce cotton cultivars whose resistance endures in the field.

The candidate genes themselves connect to well-established pillars of plant immunity. Prior research in cotton has shown that lignin deposition in vascular tissue, driven by phenylpropanoid metabolism, physically reinforces the xylem vessels the fungus colonizes, while hormones such as jasmonic acid and brassinosteroids orchestrate defense signaling. Receptor-like kinases, including the wall-associated kinase GhWAK7A, have been shown to sense fungal chitin and trigger immune responses, and transcription factors of the MYB and bHLH families regulate defense gene networks. The newly identified candidates, evaluated against this background, fit into pathways that plausibly shape how cotton detects Verticillium dahliae and walls it off before the fungus can spread through the vascular stream.

For a crop grown on tens of millions of hectares and facing a pathogen that chemical fungicides struggle to reach, the study offers a concrete toolkit. The fine-mapped intervals and the KASP markers developed for them give breeders the ability to select resistant plants at the seedling stage, accelerating the introgression of resistance alleles into elite cultivars. The fifteen candidate genes and four functionally validated qVW-D05-1 genes provide starting points for deeper mechanistic study, potentially including genome editing of favorable alleles directly into susceptible varieties. As Verticillium wilt continues to constrain cotton production across major growing regions, the convergence of fine mapping, multi-omics screening and functional validation demonstrated here offers a template for dissecting other quantitative disease-resistance traits, moving cotton breeding decisively closer to durable, genetically anchored resistance.

Subject of Research: Identification of candidate genes within major quantitative trait loci controlling Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.).

Article Title: Identification of candidate genes associated with major QTL controlling Vertcillium wilt resistance in cotton (Gossypium hirsutum L.)

Article References: Identification of candidate genes associated with major QTL controlling Vertcillium wilt resistance in cotton (Gossypium hirsutum L.). (n.d.). https://doi.org/10.1007/s00122-026-05355-x

Image Credits: AI Generated

DOI: 10.1007/s00122-026-05355-x

Keywords: cotton, Verticillium wilt, Gossypium hirsutum, QTL mapping, candidate genes, KASP markers, fine mapping, virus-induced gene silencing, plant disease resistance, Theoretical and Applied Genetics, marker-assisted selection, plant immunity