Evolutionary Dynamics of Chromatin Structure and Duplicate Gene Expression in Diploid and Allopolyploid Cotton.

Polyploidy is a prominent mechanism of plant speciation and adaptation, yet the mechanistic understandings of duplicated gene regulation remain elusive. Chromatin structure dynamics are suggested to govern gene regulatory control. Here, we characterized genome-wide nucleosome organization and chromatin accessibility in allotetraploid cotton, Gossypium hirsutum (AADD, 2n = 4X = 52), relative to its two diploid parents (AA or DD genome) and their synthetic diploid hybrid (AD), using DNS-seq. The larger A-genome exhibited wider average nucleosome spacing in diploids, and this intergenomic difference diminished in the allopolyploid but not hybrid. Allopolyploidization also exhibited increased accessibility at promoters genome-wide and synchronized cis-regulatory motifs between subgenomes. A prominent cis-acting control was inferred for chromatin dynamics and demonstrated by transposable element removal from promoters. Linking accessibility to gene expression patterns, we found distinct regulatory effects for hybridization and later allopolyploid stages, including nuanced establishment of homoeolog expression bias and expression level dominance. Histone gene expression and nucleosome organization are coordinated through chromatin accessibility. Our study demonstrates the capability to track high-resolution chromatin structure dynamics and reveals their role in the evolution of cis-regulatory landscapes and duplicate gene expression in polyploids, illuminating regulatory ties to subgenomic asymmetry and dominance.

Integrating SNVs and CNAs on a phylogenetic tree from single-cell DNA sequencing data.

Single-cell DNA sequencing enables the construction of evolutionary trees that can reveal how tumors gain mutations and grow. Different whole-genome amplification procedures render genomic materials of different characteristics, often suitable for the detection of either single-nucleotide variation or copy number aberration, but not ideally for both. Consequently, this hinders the inference of a comprehensive phylogenetic tree and limits opportunities to investigate the interplay of SNVs and CNAs. Existing methods such as SCARLET and COMPASS require that the SNVs and CNAs are detected from the same sets of cells, which is technically challenging. Here we present a novel computational tool, SCsnvcna, that places SNVs on a tree inferred from CNA signals, whereas the sets of cells rendering the SNVs and CNAs are independent, offering a more practical solution in terms of the technical challenges. SCsnvcna is a Bayesian probabilistic model using both the genotype constraints on the tree and the cellular prevalence to search the optimal solution. Comprehensive simulations and comparison with seven state-of-the-art methods show that SCsnvcna is robust and accurate in a variety of circumstances. Particularly, SCsnvcna most frequently produces the lowest error rates, with ability to scale to a wide range of numerical values for leaf nodes in the tree, SNVs, and SNV cells. The application of SCsnvcna to two published colorectal cancer data sets shows highly consistent placement of SNV cells and SNVs with the original study while also supporting a refined placement of ATP7B, illustrating SCsnvcna's value in analyzing complex multitumor samples.

Differential nuclease sensitivity profiling uncovers a drought responsive change in maize leaf chromatin structure for two large retrotransposon derivatives, Uloh and Vegu.

Plant chromatin dynamics are generally recognized as playing a role in the genomic response to environmental stress. Although stress-induced transcriptional activities of LTR-retrotransposons have been reported, whether the stress response can be detected at the level of chromatin structure for LTR-retrotransposons is largely unknown. Using differential nuclease sensitivity profiling, we identified that two out of 29 maize LTR-retrotransposon families change their chromatin structure in response to drought stress in leaf tissue. The two LTR-retrotransposon families, uloh and vegu, are classified as nonautonomous LTR-retrotransposons. Differently from other families, the chromatin structure of these two families shifted from more open in normal conditions to more closed following drought stress. Although uloh and vegu lack sequence similarity, most of them shared an intriguing feature of having a new and uncharacterized insertion of a DNA sequence near one side of an LTR. In the uloh family, nine members with a strong drought response also exhibited a drought-induced reduction of published H3K4me3 histone modification in the inserted DNA region, implicating this modification in the chromatin structural changes. Our results provide new insight into how LTR-retrotransposons can alter their chromatin structure following stress response in plants.

The native cistrome and sequence motif families of the maize ear.

Elucidating the transcriptional regulatory networks that underlie growth and development requires robust ways to define the complete set of transcription factor (TF) binding sites. Although TF-binding sites are known to be generally located within accessible chromatin regions (ACRs), pinpointing these DNA regulatory elements globally remains challenging. Current approaches primarily identify binding sites for a single TF (e.g. ChIP-seq), or globally detect ACRs but lack the resolution to consistently define TF-binding sites (e.g. DNAse-seq, ATAC-seq). To address this challenge, we developed MNase-defined cistrome-Occupancy Analysis (MOA-seq), a high-resolution (< 30 bp), high-throughput, and genome-wide strategy to globally identify putative TF-binding sites within ACRs. We used MOA-seq on developing maize ears as a proof of concept, able to define a cistrome of 145,000 MOA footprints (MFs). While a substantial majority (76%) of the known ATAC-seq ACRs intersected with the MFs, only a minority of MFs overlapped with the ATAC peaks, indicating that the majority of MFs were novel and not detected by ATAC-seq. MFs were associated with promoters and significantly enriched for TF-binding and long-range chromatin interaction sites, including for the well-characterized FASCIATED EAR4, KNOTTED1, and TEOSINTE BRANCHED1. Importantly, the MOA-seq strategy improved the spatial resolution of TF-binding prediction and allowed us to identify 215 motif families collectively distributed over more than 100,000 non-overlapping, putatively-occupied binding sites across the genome. Our study presents a simple, efficient, and high-resolution approach to identify putative TF footprints and binding motifs genome-wide, to ultimately define a native cistrome atlas.

Maize (Zea mays L.) Nucleoskeletal Proteins Regulate Nuclear Envelope Remodeling and Function in Stomatal Complex Development and Pollen Viability.

In eukaryotes, the nuclear envelope (NE) encloses chromatin and separates it from the rest of the cell. The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex physically bridges across the NE, linking nuclear and cytoplasmic components. In plants, these LINC complexes are beginning to be ascribed roles in cellular and nuclear functions, including chromatin organization, regulation of nuclei shape and movement, and cell division. Homologs of core LINC components, KASH and SUN proteins, have previously been identified in maize. Here, we characterized the presumed LINC-associated maize nucleoskeletal proteins NCH1 and NCH2, homologous to members of the plant NMCP/CRWN family, and MKAKU41, homologous to AtKAKU4. All three proteins localized to the nuclear periphery when transiently and heterologously expressed as fluorescent protein fusions in Nicotiana benthamiana. Overexpression of MKAKU41 caused dramatic changes in the organization of the nuclear periphery, including nuclear invaginations that stained positive for non-nucleoplasmic markers of the inner and outer NE membranes, and the ER. The severity of these invaginations was altered by changes in LINC connections and the actin cytoskeleton. In maize, MKAKU41 appeared to share genetic functions with other LINC components, including control of nuclei shape, stomatal complex development, and pollen viability. Overall, our data show that NCH1, NCH2, and MKAKU41 have characteristic properties of LINC-associated plant nucleoskeletal proteins, including interactions with NE components suggestive of functions at the nuclear periphery that impact the overall nuclear architecture.

The regulatory landscape of early maize inflorescence development.

BACKGROUND: The functional genome of agronomically important plant species remains largely unexplored, yet presents a virtually untapped resource for targeted crop improvement. Functional elements of regulatory DNA revealed through profiles of chromatin accessibility can be harnessed for fine-tuning gene expression to optimal phenotypes in specific environments. RESULT: Here, we investigate the non-coding regulatory space in the maize (Zea mays) genome during early reproductive development of pollen- and grain-bearing inflorescences. Using an assay for differential sensitivity of chromatin to micrococcal nuclease (MNase) digestion, we profile accessible chromatin and nucleosome occupancy in these largely undifferentiated tissues and classify at least 1.6% of the genome as accessible, with the majority of MNase hypersensitive sites marking proximal promoters, but also 3' ends of maize genes. This approach maps regulatory elements to footprint-level resolution. Integration of complementary transcriptome profiles and transcription factor occupancy data are used to annotate regulatory factors, such as combinatorial transcription factor binding motifs and long non-coding RNAs, that potentially contribute to organogenesis, including tissue-specific regulation between male and female inflorescence structures. Finally, genome-wide association studies for inflorescence architecture traits based solely on functional regions delineated by MNase hypersensitivity reveals new SNP-trait associations in known regulators of inflorescence development as well as new candidates. CONCLUSIONS: These analyses provide a comprehensive look into the cis-regulatory landscape during inflorescence differentiation in a major cereal crop, which ultimately shapes architecture and influences yield potential.

Identification of tandem repeat families from long-read sequences of Humulus lupulus.

Hop (Humulus lupulus L.) is known for its use as a bittering agent in beer and has a rich history of cultivation, beginning in Europe and now spanning the globe. There are five wild varieties worldwide, which may have been introgressed with cultivated varieties. As a dioecious species, its obligate outcrossing, non-Mendelian inheritance, and genomic structural variability have confounded directed breeding efforts. Consequently, understanding the hop genome represents a considerable challenge, requiring additional resources. In order to facilitate investigations into the transmission genetics of hop, we report here a tandem repeat discovery pipeline developed using k-mer filtering and dot plot analysis of PacBio long-read sequences from the hop cultivar Apollo. From this we identified 17 new and distinct tandem repeat sequence families, which represent candidates for FISH probe development. For two of these candidates, HuluTR120 and HuluTR225, we produced oligonucleotide FISH probes from conserved regions of and demonstrated their utility by staining meiotic chromosomes from wild hop, var. neomexicanus to address, for example, questions about hop transmission genetics. Collectively, these tandem repeat sequence families represent new resources suitable for development of additional cytogenomic tools for hop research.

Arabidopsis DNA Replication Initiates in Intergenic, AT-Rich Open Chromatin.

The selection and firing of DNA replication origins play key roles in ensuring that eukaryotes accurately replicate their genomes. This process is not well documented in plants due in large measure to difficulties in working with plant systems. We developed a new functional assay to label and map very early replicating loci that must, by definition, include at least a subset of replication origins. Arabidopsis (Arabidopsis thaliana) cells were briefly labeled with 5-ethynyl-2'-deoxy-uridine, and nuclei were subjected to two-parameter flow sorting. We identified more than 5500 loci as initiation regions (IRs), the first regions to replicate in very early S phase. These were classified as strong or weak IRs based on the strength of their replication signals. Strong initiation regions were evenly spaced along chromosomal arms and depleted in centromeres, while weak initiation regions were enriched in centromeric regions. IRs are AT-rich sequences flanked by more GC-rich regions and located predominantly in intergenic regions. Nuclease sensitivity assays indicated that IRs are associated with accessible chromatin. Based on these observations, initiation of plant DNA replication shows some similarity to, but is also distinct from, initiation in other well-studied eukaryotic systems.

MLKS2 is an ARM domain and F-actin-associated KASH protein that functions in stomatal complex development and meiotic chromosome segregation.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an essential multi-protein structure spanning the eukaryotic nuclear envelope. The LINC complex functions to maintain nuclear architecture, positioning, and mobility, along with specialized functions in meiotic prophase and chromosome segregation. Members of the LINC complex were recently identified in maize, an important scientific and agricultural grass species. Here we characterized Maize LINC KASH AtSINE-like2, MLKS2, which encodes a highly conserved SINE-group plant KASH protein with characteristic N-terminal armadillo repeats (ARM). Using a heterologous expression system, we showed that actively expressed GFP-MLKS2 is targeted to the nuclear periphery and colocalizes with F-actin and the endoplasmic reticulum, but not microtubules in the cell cortex. Expression of GFP-MLKS2, but not GFP-MLKS2ΔARM, resulted in nuclear anchoring. Genetic analysis of transposon-insertion mutations, mlks2-1 and mlks2-2, showed that the mutant phenotypes were pleiotropic, affecting root hair nuclear morphology, stomatal complex development, multiple aspects of meiosis, and pollen viability. In male meiosis, the mutants showed defects for bouquet-stage telomere clustering, nuclear repositioning, perinuclear actin accumulation, dispersal of late prophase bivalents, and meiotic chromosome segregation. These findings support a model in which the nucleus is connected to cytoskeletal F-actin through the ARM-domain, predicted alpha solenoid structure of MLKS2. Functional conservation of MLKS2 was demonstrated through genetic rescue of the misshapen nuclear phenotype of an Arabidopsis (triple-WIP) KASH mutant. This study establishes a role for the SINE-type KASH proteins in affecting the dynamic nuclear phenomena required for normal plant growth and fertility. Abbreviations: FRAP: Fluorescence recovery after photobleaching; DPI: Days post infiltration; OD: Optical density; MLKS2: Maize LINC KASH AtSINE-like2; LINC: Linker of nucleoskeleton and cytoskeleton; NE: Nuclear envelope; INM: Inner nuclear membrane; ONM: Outer nuclear membrane.

Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays.

The linker of nucleoskeleton to cytoskeleton (LINC) complex is an essential multi-protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots, which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. By using bioinformatic, biochemical and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with Zea mays (Zm)SUN2 in vivo FRAP experiments using domain deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research.This article has an associated First Person interview with the first author of the paper.

3D Molecular Cytology of Hop (Humulus lupulus) Meiotic Chromosomes Reveals Non-disomic Pairing and Segregation, Aneuploidy, and Genomic Structural Variation.

Hop (Humulus lupulus L.) is an important crop worldwide, known as the main flavoring ingredient in beer. The diversifying brewing industry demands variation in flavors, superior process properties, and sustainable agronomics, which are the focus of advanced molecular breeding efforts in hops. Hop breeders have been limited in their ability to create strains with desirable traits, however, because of the unusual and unpredictable inheritance patterns and associated non-Mendelian genetic marker segregation. Cytogenetic analysis of meiotic chromosome behavior has also revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including those involving autosomes in late prophase. To explore the role of meiosis in segregation distortion, we undertook 3D cytogenetic analysis of hop pollen mother cells stained with DAPI and FISH. We used telomere FISH to demonstrate that hop exhibits a normal telomere clustering bouquet. We also identified and characterized a new sub-terminal 180 bp satellite DNA tandem repeat family called HSR0, located proximal to telomeres. Highly variable 5S rDNA FISH patterns within and between plants, together with the detection of anaphase chromosome bridges, reflect extensive departures from normal disomic signal composition and distribution. Subsequent FACS analysis revealed variable DNA content in a cultivated pedigree. Together, these findings implicate multiple phenomena, including aneuploidy, segmental aneuploidy, or chromosome rearrangements, as contributing factors to segregation distortion in hop.

Chromatin structure profile data from DNS-seq: Differential nuclease sensitivity mapping of four reference tissues of B73 maize (Zea mays L).

Presented here are data from Next-Generation Sequencing of differential micrococcal nuclease digestions of formaldehyde-crosslinked chromatin in selected tissues of maize (Zea mays) inbred line B73. Supplemental materials include a wet-bench protocol for making DNS-seq libraries, the DNS-seq data processing pipeline for producing genome browser tracks. This report also includes the peak-calling pipeline using the iSeg algorithm to segment positive and negative peaks from the DNS-seq difference profiles. The data repository for the sequence data is the NCBI SRA, BioProject Accession PRJNA445708.

The maize W22 genome provides a foundation for functional genomics and transposon biology.

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

Review: Plant G-quadruplex (G4) motifs in DNA and RNA; abundant, intriguing sequences of unknown function.

DNA sequences capable of forming G-quadruplex (G4) structures can be predicted and mapped in plant genomes using computerized pattern search programs. Non-telomeric G4 motifs have recently been found to number in the thousands across many plant species and enriched around gene promoters, prompting speculation that they may represent a newly uncovered and ubiquitous family of cis-acting elements. Comparative analysis shows that monocots exhibit five to ten times higher G4 motif density than eudicots, but the significance of this difference has not been determined. The vast scale and complexity of G4 functions, actual or theoretical, are reviewed in relation to the multiple modes of action and myriad genetic functions for which G4s have been implicated in DNA and RNA. Future experimental strategies and opportunities include identifying plant G4-interactomes, resolving the structures of G4s with and without their binding partners, and defining molecular mechanisms through reporter gene, genetic, or genome editing approaches. Given the global importance of plants for food, clothing, medicine, and energy, together with the potential role of G4 motifs as a widely conserved set of DNA sequences that could coordinate gene regulation, future plant G4 research holds great potential for use in plant improvement strategies.

iSeg: an efficient algorithm for segmentation of genomic and epigenomic data.

BACKGROUND: Identification of functional elements of a genome often requires dividing a sequence of measurements along a genome into segments where adjacent segments have different properties, such as different mean values. Despite dozens of algorithms developed to address this problem in genomics research, methods with improved accuracy and speed are still needed to effectively tackle both existing and emerging genomic and epigenomic segmentation problems. RESULTS: We designed an efficient algorithm, called iSeg, for segmentation of genomic and epigenomic profiles. iSeg first utilizes dynamic programming to identify candidate segments and test for significance. It then uses a novel data structure based on two coupled balanced binary trees to detect overlapping significant segments and update them simultaneously during searching and refinement stages. Refinement and merging of significant segments are performed at the end to generate the final set of segments. By using an objective function based on the p-values of the segments, the algorithm can serve as a general computational framework to be combined with different assumptions on the distributions of the data. As a general segmentation method, it can segment different types of genomic and epigenomic data, such as DNA copy number variation, nucleosome occupancy, nuclease sensitivity, and differential nuclease sensitivity data. Using simple t-tests to compute p-values across multiple datasets of different types, we evaluate iSeg using both simulated and experimental datasets and show that it performs satisfactorily when compared with some other popular methods, which often employ more sophisticated statistical models. Implemented in C++, iSeg is also very computationally efficient, well suited for large numbers of input profiles and data with very long sequences. CONCLUSIONS: We have developed an efficient general-purpose segmentation tool and showed that it had comparable or more accurate results than many of the most popular segment-calling algorithms used in contemporary genomic data analysis. iSeg is capable of analyzing datasets that have both positive and negative values. Tunable parameters allow users to readily adjust the statistical stringency to best match the biological nature of individual datasets, including widely or sparsely mapped genomic datasets or those with non-normal distributions.

Take a look at plant DNA replication: Recent insights and new questions.

Recent advances in replicative DNA labeling technology have allowed new ways to study DNA replication in living plants. Temporal and spatial aspects of DNA replication programs are believed to derive from genomic structure and function. Bass et al. (2015) recently visualized DNA synthesis using 3D microscopy of nuclei at three sub-stages of S phase: early, middle and late. This addendum expands on that study by comparing plant and animal DNA replication patterns, by considering implications of the two-compartment model of euchromatin, and by exploring the meaning of the DNA labeling signals inside the nucleolus. Finally, we invite the public to explore and utilize 300 image data sets through OMERO, a teaching and research web resource for visualization, management, or analysis of microscopic data.

Non-Mendelian Single-Nucleotide Polymorphism Inheritance and Atypical Meiotic Configurations are Prevalent in Hop.

Hop ( L.) breeding programs seek to exploit genetic resources for bitter flavor, aroma, and disease resistance. However, these efforts have been thwarted by segregation distortion including female-biased sex ratios. To better understand the transmission genetics of hop, we genotyped 4512 worldwide accessions of hop, including cultivars, landraces, and over 100 wild accessions using a genotyping-by-sequencing (GBS) approach. From the resulting ∼1.2 million single-nucleotide polymorphisms (SNPs), prequalified GBS markers were validated by inferences in population structures and phylogeny. Analysis of pseudo-testcross (Pt) mapping data from F families revealed mixed patterns of Mendelian and non-Mendelian segregation. Three-dimensional (3D) cytogenetic analysis of late meiotic prophase nuclei from two wild and two cultivated hop revealed conspicuous and prevalent occurrences of multiple, atypical, nondisomic chromosome complexes including autosomes. We used genome-wide association studies (GWAS) and fixation index (F) analysis to demonstrate selection mapping of genetic loci for key traits including sex, bitter acids, and drought tolerance. Among the possible mechanisms underlying the observed segregation distortion from the genomic data analysis, the cytogenetic analysis points to meiotic chromosome behavior as one of the contributing factors. The findings shed light on long-standing questions on the unusual transmission genetics and phenotypic variation in hop, with major implications for breeding, cultivation, and the natural history of .

Open chromatin reveals the functional maize genome.

Cellular processes mediated through nuclear DNA must contend with chromatin. Chromatin structural assays can efficiently integrate information across diverse regulatory elements, revealing the functional noncoding genome. In this study, we use a differential nuclease sensitivity assay based on micrococcal nuclease (MNase) digestion to discover open chromatin regions in the maize genome. We find that maize MNase-hypersensitive (MNase HS) regions localize around active genes and within recombination hotspots, focusing biased gene conversion at their flanks. Although MNase HS regions map to less than 1% of the genome, they consistently explain a remarkably large amount (∼40%) of heritable phenotypic variance in diverse complex traits. MNase HS regions are therefore on par with coding sequences as annotations that demarcate the functional parts of the maize genome. These results imply that less than 3% of the maize genome (coding and MNase HS regions) may give rise to the overwhelming majority of phenotypic variation, greatly narrowing the scope of the functional genome.

Defining multiple, distinct, and shared spatiotemporal patterns of DNA replication and endoreduplication from 3D image analysis of developing maize (Zea mays L.) root tip nuclei.

Spatiotemporal patterns of DNA replication have been described for yeast and many types of cultured animal cells, frequently after cell cycle arrest to aid in synchronization. However, patterns of DNA replication in nuclei from plants or naturally developing organs remain largely uncharacterized. Here we report findings from 3D quantitative analysis of DNA replication and endoreduplication in nuclei from pulse-labeled developing maize root tips. In both early and middle S phase nuclei, flow-sorted on the basis of DNA content, replicative labeling was widely distributed across euchromatic regions of the nucleoplasm. We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals. Instead, the early versus middle S phase patterns in maize could be distinguished cytologically by correlating two quantitative, continuous variables, replicative labeling and DAPI staining. Early S nuclei exhibited widely distributed euchromatic labeling preferentially localized to regions with weak DAPI signals. Middle S nuclei also exhibited widely distributed euchromatic labeling, but the label was preferentially localized to regions with strong DAPI signals. Highly condensed heterochromatin, including knobs, replicated during late S phase as previously reported. Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei. These results revealed that maize euchromatin exists as an intermingled mixture of two components distinguished by their condensation state and replication timing. These different patterns might reflect a previously described genome organization pattern, with "gene islands" mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.