Cyto-swapping in maize by haploid induction with a cenh3 mutant.

Maize mutants of the centromeric histone H3 (CENP-A/CENH3) gene can form haploids that inherit only chromosomes of the pollinating parent but the cytoplasm from the female parent. We developed CENH3 haploid inducers carrying a dominant anthocyanin colour marker for efficient haploid identification and harbouring cytoplasmic male sterile cytoplasm, a type of cytoplasm that results in male sterility useful for efficient hybrid seed production. The resulting cytoplasmic male sterility cyto-swapping method provides a faster and cheaper way to convert commercial lines to cytoplasmic male sterile compared to conventional trait introgression.

Engineering better artificial chromosomes.

Constructing human artificial chromosomes in yeast avoids unintended multimerization.

The maize striate leaves2 (sr2) gene encodes a conserved DUF3732 domain and is homologous to the rice yss1 gene.

Maize striate leaves2 (sr2) is a mutant that causes white stripes on leaves that has been used in mapping studies for decades though the underlying gene has not been identified. The sr2 locus has been previously mapped to small regions of normal chromosome 10 (N10) and a rearranged variant called abnormal chromosome 10 (Ab10). A comparison of assembled genomes carrying N10 and Ab10 revealed only five candidate sr2 genes. Analysis of a stock carrying the sr2 reference allele (sr2-ref) showed that one of the five genes has a transposon insertion that disrupts its protein sequence and has a severe reduction in mRNA. An independent Mutator transposon insertion in the gene (sr2-Mu) failed to complement the sr2-ref mutation, and plants homozygous for sr2-Mu showed white striped leaf margins. The sr2 gene encodes a DUF3732 protein with strong homology to a rice gene with a similar mutant phenotype called young seedling stripe1 (yss1). These and other published data suggest that sr2 may have a function in plastid gene expression.

Potent pollen gene regulation by DNA glycosylases in maize.

Although DNA methylation primarily represses transposable elements (TEs) in plants, it also represses select endosperm and pollen genes. These genes, or their cis-regulatory elements, are methylated in plant body tissues but are demethylated by DNA glycosylases (DNGs) in endosperm and pollen, enabling their transcription. Activity of either one of two DNGs, MDR1 or DNG102, is essential for pollen viability in maize. Using single-pollen mRNA sequencing on pollen segregating mutations in both genes, we identified 58 candidate DNG target genes, whose expression is strongly decreased in double mutant pollen (124-fold decrease on average). These genes account for 11.1% of the wild-type pollen polyadenylated transcriptome, but they are silent or barely detectable in the plant body. They are unusual in their tendency to lack introns but even more so in their having TE-like methylation in their coding DNA sequence. Moreover, they are strongly enriched for predicted functions in cell wall modification. While some may support development of the pollen grain cell wall, expansins and pectinases in this set of genes suggest a function in cell wall loosening to support the rapid tip growth characteristic of pollen tubes as they carry the sperm cells through maternal apoplast and extracellular matrix of the pistil. These results suggest a critical role for DNA methylation and demethylation in regulating maize genes with potential for extremely high expression in pollen but constitutive silencing elsewhere.

Haplotype-resolved genome assembly of Populus tremula × P. alba reveals aspen-specific megabase satellite DNA.

Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717-1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence-sensitive analyses. Here we report a telomere-to-telomere genome for this hybrid aspen with two chromosome-scale, haplotype-resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array-based and array-less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus) but PtaM147-like sequences occur in LTR-retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis-driven research to probe into the function of the abundant and aspen-specific PtaM147 satellite DNA.

Natural methylation epialleles correlate with gene expression in maize.

DNA methylation in plants is depleted from cis-regulatory elements in and near genes but is present in some gene bodies, including exons. Methylation in exons solely in the CG context is called gene body methylation (gbM). Methylation in exons in both CG and non-CG contexts is called TE-like methylation (teM). Assigning functions to both forms of methylation in genes has proven to be challenging. Toward that end, we utilized recent genome assemblies, gene annotations, transcription data, and methylome data to quantify common patterns of gene methylation and their relations to gene expression in maize. We found that gbM genes exist in a continuum of CG methylation levels without a clear demarcation between unmethylated genes and gbM genes. Analysis of expression levels across diverse maize stocks and tissues revealed a weak but highly significant positive correlation between gbM and gene expression except in endosperm. gbM epialleles were associated with an approximately 3% increase in steady-state expression level relative to unmethylated epialleles. In contrast to gbM genes, which were conserved and were broadly expressed across tissues, we found that teM genes, which make up about 12% of genes, are mainly silent, are poorly conserved, and exhibit evidence of annotation errors. We used these data to flag teM genes in the 26 NAM founder genome assemblies. While some teM genes are likely functional, these data suggest that the majority are not, and their inclusion can confound the interpretation of whole-genome studies.

Synthetic maize centromeres transmit chromosomes across generations.

Centromeres are long, often repetitive regions of genomes that bind kinetochore proteins and ensure normal chromosome segregation. Engineering centromeres that function in vivo has proven to be difficult. Here we describe a tethering approach that activates functional maize centromeres at synthetic sequence arrays. A LexA-CENH3 fusion protein was used to recruit native Centromeric Histone H3 (CENH3) to long arrays of LexO repeats on a chromosome arm. Newly recruited CENH3 was sufficient to organize functional kinetochores that caused chromosome breakage, releasing chromosome fragments that were passed through meiosis and into progeny. Several fragments formed independent neochromosomes with centromeres localized over the LexO repeat arrays. The new centromeres were self-sustaining and transmitted neochromosomes to subsequent generations in the absence of the LexA-CENH3 activator. Our results demonstrate the feasibility of using synthetic centromeres for karyotype engineering applications.

Large haplotypes highlight a complex age structure within the maize pan-genome.

The genomes of maize and other eukaryotes contain stable haplotypes in regions of low recombination. These regions, including centromeres, long heterochromatic blocks, and rDNA arrays, have been difficult to analyze with respect to their diversity and origin. Greatly improved genome assemblies are now available that enable comparative genomics over these and other nongenic spaces. Using 26 complete maize genomes, we developed methods to align intergenic sequences while excluding genes and regulatory regions. The centromere haplotypes (cenhaps) extend for megabases on either side of the functional centromere regions and appear as evolutionary strata, with haplotype divergence/coalescence times dating as far back as 450 thousand years ago (kya). Application of the same methods to other low recombination regions (heterochromatic knobs and rDNA) and all intergenic spaces revealed that deep coalescence times are ubiquitous across the maize pan-genome. Divergence estimates vary over a broad timescale with peaks at ∼16 and 300 kya, reflecting a complex history of gene flow among diverging populations and changes in population size associated with domestication. Cenhaps and other long haplotypes provide vivid displays of this ancient diversity.

The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm.

Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.

The maize abnormal chromosome 10 meiotic drive haplotype: a review.

The maize abnormal chromosome 10 (Ab10) haplotype encodes a meiotic drive system that converts heterochromatic knobs into centromere-like bodies that are preferentially segregated through female meiosis. Ab10 was first described in the 1940s and has been intensively studied. Here I provide a comprehensive review of the literature, starting from the discovery of knobs and Ab10, preceding through the classic literature, and finishing with molecular structure and mechanisms. The defining features of the Ab10 haplotype are its two specialized kinesins, Kinesin driver and TR-1 kinesin, that activate neocentromeres at knobs containing different classes of the tandem repeat. In most Ab10 haplotypes, the two kinesin/knob systems cooperate to promote maximum meiotic drive. However, recent interpretations suggest that each kinesin/knob system can function as an independent meiotic driver and that in some cases they compete with each other. Ab10 is present at low frequencies throughout the genus Zea and has significantly expanded genome size by promoting the formation of knobs throughout the genome.

Frequent Spindle Assembly Errors Require Structural Rearrangement to Complete Meiosis in Zea mays.

The success of an organism is contingent upon its ability to faithfully pass on its genetic material. In the meiosis of many species, the process of chromosome segregation requires that bipolar spindles be formed without the aid of dedicated microtubule organizing centers, such as centrosomes. Here, we describe detailed analyses of acentrosomal spindle assembly and disassembly in time-lapse images, from live meiotic cells of Zea mays. Microtubules organized on the nuclear envelope with a perinuclear ring structure until nuclear envelope breakdown, at which point microtubules began bundling into a bipolar form. However, the process and timing of spindle assembly was highly variable, with frequent assembly errors in both meiosis I and II. Approximately 61% of cells formed incorrect spindle morphologies, with the most prevalent being tripolar spindles. The erroneous spindles were actively rearranged to bipolar through a coalescence of poles before proceeding to anaphase. Spindle disassembly occurred as a two-state process with a slow depolymerization, followed by a quick collapse. The results demonstrate that maize meiosis I and II spindle assembly is remarkably fluid in the early assembly stages, but otherwise proceeds through a predictable series of events.

QTL Map of Early- and Late-Stage Perennial Regrowth in Zea diploperennis.

Numerous climate change threats will necessitate a shift toward more sustainable agricultural practices during the 21st century. Conversion of annual crops to perennials that are capable of regrowing over multiple yearly growth cycles could help to facilitate this transition. Perennials can capture greater amounts of carbon and access more water and soil nutrients compared to annuals. In principle it should be possible to identify genes that confer perenniality from wild relatives and transfer them into existing breeding lines to create novel perennial crops. Two major loci controlling perennial regrowth in the maize relative Zea diploperennis were previously mapped to chromosome 2 (reg1) and chromosome 7 (reg2). Here we extend this work by mapping perennial regrowth in segregating populations involving Z. diploperennis and the maize inbreds P39 and Hp301 using QTL-seq and traditional QTL mapping approaches. The results confirmed the existence of a major perennial regrowth QTL on chromosome 2 (reg1). Although we did not observe the reg2 QTL in these populations, we discovered a third QTL on chromosome 8 which we named regrowth3 (reg3). The reg3 locus exerts its strongest effect late in the regrowth cycle. Neither reg1 nor reg3 overlapped with tiller number QTL scored in the same population, suggesting specific roles in the perennial phenotype. Our data, along with prior work, indicate that perennial regrowth in maize is conferred by relatively few major QTL.

Photodiagnostic services in the UK and Republic of Ireland: a British Photodermatology Group Workshop Report.

BACKGROUND: Photodiagnostic investigations are essential for the accurate diagnosis of abnormal cutaneous photosensitivity and provide important information for the management of patients with photodermatoses (cutaneous photosensitivity disorders). Although photodiagnosis has been undertaken since the early 1970s, specialist services in the United Kingdom (UK) and Republic of Ireland are limited and there is no formal guidance on diagnostic approach. Indeed, there is a limited literature in this area of methodology and diagnostic practice. OBJECTIVES: The primary objective was to undertake a British Photodermatology Group Workshop to review the role and activities of specialist centres in the UK and Republic of Ireland in order to ascertain whether there were consensus practices. Secondary objectives were to identify key priorities for service, training and research. METHODS: An initial detailed survey review of current activities was undertaken prior to the Workshop and data from this survey were used to inform discussion at the Workshop, which was attended by key photodermatology experts from the UK and Republic of Ireland. RESULTS/CONCLUSIONS: We have undertaken a detailed review of current Photodiagnostic Services in the UK and Republic of Ireland and report on our findings from the 12 centres and we have identified key areas of consensus practice. This is an important step in the process of standardising and optimising procedures and protocols and defining minimum clinical standards for photodiagnostic investigations, which are of such diagnostic importance in Dermatology.

De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes.

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The number of pan-genes in these diverse genomes exceeds 103,000, with approximately a third found across all genotypes. The results demonstrate that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres revealed additional variation in major cytological landmarks. We show that combining structural variation with single-nucleotide polymorphisms can improve the power of quantitative mapping studies. We also document variation at the level of DNA methylation and demonstrate that unmethylated regions are enriched for cis-regulatory elements that contribute to phenotypic variation.

Sequence of the supernumerary B chromosome of maize provides insight into its drive mechanism and evolution.

B chromosomes are enigmatic elements in thousands of plant and animal genomes that persist in populations despite being nonessential. They circumvent the laws of Mendelian inheritance but the molecular mechanisms underlying this behavior remain unknown. Here we present the sequence, annotation, and analysis of the maize B chromosome providing insight into its drive mechanism. The sequence assembly reveals detailed locations of the elements involved with the cis and trans functions of its drive mechanism, consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B-containing sperm. We identified 758 protein-coding genes in 125.9 Mb of B chromosome sequence, of which at least 88 are expressed. Our results demonstrate that transposable elements in the B chromosome are shared with the standard A chromosome set but multiple lines of evidence fail to detect a syntenic genic region in the A chromosomes, suggesting a distant origin. The current gene content is a result of continuous transfer from the A chromosomal complement over an extended evolutionary time with subsequent degradation but with selection for maintenance of this nonvital chromosome.

Maize centromeric chromatin scales with changes in genome size.

Centromeres are defined by the location of Centromeric Histone H3 (CENP-A/CENH3) which interacts with DNA to define the locations and sizes of functional centromeres. An analysis of 26 maize genomes including 110 fully assembled centromeric regions revealed positive relationships between centromere size and genome size. These effects are independent of variation in the amounts of the major centromeric satellite sequence CentC. We also backcrossed known centromeres into two different lines with larger genomes and observed consistent increases in functional centromere sizes for multiple centromeres. Although changes in centromere size involve changes in bound CENH3, we could not mimic the effect by overexpressing CENH3 by threefold. Literature from other fields demonstrate that changes in genome size affect protein levels, organelle size and cell size. Our data demonstrate that centromere size is among these scalable features, and that multiple limiting factors together contribute to a stable centromere size equilibrium.

Narrowband ultraviolet B phototherapy is associated with a reduction in topical corticosteroid and clinical improvement in atopic dermatitis: a historical inception cohort study.

BACKGROUND: Despite decades of use, the magnitude of efficacy of narrowband ultraviolet B (NB-UVB) phototherapy for atopic dermatitis (AD) beyond industry-sponsored trials remains unclear. AIM: To evaluate the clinical efficacy of NB-UVB in AD under real-world conditions. METHODS: We conducted a historical inception cohort study using automated recording of dispensed drugs to provide an objective treatment outcome in a large population catchment of 420 000 people over 15 years. We analysed clinical treatment outcomes, recorded multicentre and prospectively over 15 years, of a large AD treatment cohort (n = 844), along with the drugs dispensed to this cohort. RESULTS: The majority (70%) of patients with AD received significantly fewer topical corticosteroids (TCS) during the 12-month window after finishing NB-UVB compared with the 12-month window before starting the treatment (median reduction from 37.5 to 19.7 g/month). The number of patients dispensed with oral corticosteroids and antihistamines also dropped significantly (from 20% to 10% and from 69% to 31%, respectively), while all AD-unrelated drugs dispensed remained unchanged. Clinically, NB-UVB treatment achieved a 'clear' or 'almost clear' status in 48.7% of patients, while 20.4% achieved 'moderate clearance'. Treatment outcomes scores were validated by a strong correlation with reduction in AD-specific drug treatment. CONCLUSION: Our data confirm the significant efficacy of NB-UVB for AD under conditions of routine care.