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Dawe, R. K.

Publications and source records attributed to Dawe, R. K..

18 recordsLinked to original sources

TE-like DNA methylation of highly expressed endosperm genes in maize

Unlike the stable transcription of plant genes with CG gene body methylation alone, genes with methylation in TE-like CHG and CG contexts are poorly transcribed and often missanotated. In contrast, here we describe a set of TE-like methylated genes that are maternally demethylated and expressed toward the extreme high end of the spectrum in endosperm. They are enriched for short, secreted proteins, and about a third encode zeins, the major seed storage proteins of maize. Consistent with their dynamic expression in endosperm, they acquire either activating or repressive gene regulatory modifications when demethylated but are heterochromatic when methylated in other tissues. The majority are expressed both maternally and paternally, but a subset whose methylation/demethylation extends upstream of gene bodies into promoters are strongly imprinted. These and other data indicate that TE-like methylation and associated heterochromatin can be a signature of broad silencing but exceptionally high and specific gene expression in either pollen or endosperm.

molecular biology↗

Genetic and environmental influences on the distributions of three chromosomal drive haplotypes in maize

Meiotic drive elements are regions of the genome that are transmitted to progeny at frequencies that exceed Mendelian expectations, often to the detriment of the organism. In maize there are three prevalent chromosomal drive elements known as Abnormal chromosome 10 (Ab10), K10L2, and the B chromosome. There has been much speculation about how these drivers might interact with each other and the environment in traditional maize landraces and their teosinte ancestors. Here we used genotype-by-sequencing data to score more than 10,000 maize and teosinte lines for the presence or absence of each driver. Less than [~]0.5% of modern inbred lines carry chromosomal drivers. Among individuals from 5331 open-pollinated landraces, 6.32% carried Ab10, 5.16% carried K10L2, and 12.28% carried at least one B chromosome. Using a GWAS approach we identified unlinked loci that associate with the presence or absence of the selfish genetic elements. Many genetic modifiers are positively associated with the drivers, suggesting that there may have been selection for alleles that ameliorate their negative fitness consequences. We then assessed the contributions of population structure, associated loci, and the environment on the distribution of each chromosomal driver. There was no significant relationship between any chromosomal driver and altitude, contrary to conclusions based on smaller studies. Our data suggest that the distribution of the major chromosomal drivers is primarily influenced by neutral processes and the deleterious fitness consequences of the drivers themselves. While each driver has a unique relationship to genetic background and the environment, they are largely unconstrained by either. AUTHOR SUMMARYMeiotic drivers are selfish genetic elements that are transmitted to progeny at frequencies that exceed what is expected by chance. There are three such meiotic drivers in maize known as Ab10, K10L2 and the B chromosome. Prior data indicate that each driver lowers plant fitness to some degree, partially explaining the fact that they are usually observed at low frequencies, but the impact of environment and genetic modifiers has not been investigated. Here we identified the presence or absence of each driver using low coverage sequencing data from over 10,000 maize inbreds, open pollinated landraces, and accessions of teosinte -- the grass-like ancestor of maize. We found that they exist at 5-12% frequencies in open pollinated varieties, but are rare in modern inbreds. We went on to use location data to assess the impact of environmental variables on their distribution, and applied genome-wide association methods to look for genetic modifiers that might contribute to their prevalence. We found that neither the environment nor genetic modifiers have strong influences on the distribution of the three meiotic drive elements, suggesting that their low but consistent frequencies are determined primarily by the fitness consequences of the drivers themselves.

genetics↗

Two CENH3 paralogs in the green alga Chlamydomonas reinhardtii have a redundantly essential function and associate with ZeppL-LINE1 elements

Centromeres in eukaryotes are defined by the presence of histone H3 variant CENP-A/CENH3. Chlamydomonas encodes two predicted CENH3 paralogs, CENH3.1 and CENH3.2, that have not been previously characterized. We generated peptide antibodies to unique N-terminal epitopes for each of the two predicted Chlamydomonas CENH3 paralogs as well as an antibody against a shared CENH3 epitope. All three CENH3 antibodies recognized proteins of the expected size on immunoblots and had punctuated nuclear immunofluorescence staining patterns. These results are consistent with both paralogs being expressed and localized to centromeres. CRISPR-Cas9 mediated insertional mutagenesis was used to generate predicted null mutations in either CENH3.1 or CENH3.2. Single mutants were viable but cenh3.1 cenh3.2 double mutants were not recovered, confirming that the function of CENH3 is essential. We sequenced and assembled two chromosome-scale Chlamydomonas genomes from strains CC-400 and UL-1690 (a derivative of CC-1690) with complete centromere sequences for 17/17 and 14/17 chromosomes respectively, enabling us to compare centromere evolution across four isolates with near complete assemblies. These data revealed significant changes across isolates between homologous centromeres including mobility and degeneration of ZeppL-LINE1 (ZeppL) transposons that comprise the major centromere repeat sequence in Chlamydomonas. We used Cleavage Under Targets and Tagmentation (CUT&Tag) to purify and map CENH3-bound genomic sequences and found enrichment of CENH3-binding almost exclusively at predicted centromere regions. An interesting exception was chromosome 2 in UL-1690, which had enrichment at its genetically mapped centromere repeat region as well as a second, distal location, centered around a single recently acquired ZeppL insertion. The CENH3-bound regions of the 17 Chlamydomonas centromeres ranged from 63.5kb (average lower estimate) to 175kb (average upper estimate). The relatively small size of its centromeres suggest that Chlamydomonas may be a useful organism for testing and deploying artificial chromosome technologies. Significance statementHistone H3 variant CENP-A/CENH3 is a conserved centromere protein in eukaryotes but has not been well-characterized in green algae. Our data establish the function of CENH3 at Chlamydomonas centromeres and identify an unexpected strain-specific neocentromere candidate region in chromosome 2 likely caused by a single ZeppL-LINE1 insertion into a genic region that is distal to the native chromosome 2 centromere.

genetics↗

Antagonistic kinesin-14s within a single chromosomal drive haplotype

In maize, there are two meiotic drive systems that operate on large tandem repeat arrays called knobs that are found on chromosome arms. One meiotic drive haplotype, Abnormal chromosome 10 (Ab10), encodes two kinesin proteins that interact with two distinct tandem repeat arrays in a sequence-specific manner to confer meiotic drive. The kinesin KINDR associates with knob180 repeats while the kinesin TRKIN associates with TR-1 repeats. Prior data show that meiotic drive is conferred primarily by the KINDR/knob180 system, with the TRKIN/TR-1 system having little or no role. The second meiotic drive haplotype, K10L2, shows low levels of meiotic drive and only encodes the TRKIN/TR-1 system. Here we used long-read sequencing to assemble the K10L2 haplotype and showed that it has strong homology to an internal portion of the Ab10 haplotype. We also carried out CRISPR mutagenesis of Trkin to test the role of Trkin on Ab10 and K10L2. The data indicate that the Trkin gene on Ab10 does not improve drive or fitness but instead has a weak deleterious effect when paired with a normal chromosome 10. The deleterious effect is more severe when Ab10 is paired with K10L2: in this context functional Trkin on either chromosome nearly abolishes Ab10 drive. We modeled the effect of Trkin on Ab10 and found it should not persist in the population. We conclude that Trkin either confers an advantage to Ab10 in untested circumstances or that it is in the process of being purged from the Ab10 population. ARTICLE SUMMARYMendels first law states that paired chromosomes are transmitted through meiosis at equal frequencies. Some chromosome variants, however, are transmitted at higher frequencies in a process called meiotic drive. We wanted to know the function of a motor protein called TRKIN that is encoded on a maize meiotic drive chromosome. Surprisingly, we found that TRKIN provides no advantage to the meiotic driver and instead seems to be deleterious, suggesting it had a function in a wild ancestor but is now being purged from the population. The results illustrate how genomes are shaped by often-conflicting forces of selection and selfish genetic elements.

evolutionary biology↗

Higher order repeat structures reflect diverging evolutionary paths in maize centromeres and knobs

BackgroundHighly repetitive tandem repeat arrays, known as satellite DNAs, are enriched in low-recombination regions such as centromeres. Satellite arrays often contain complex internal structures called higher-order repeats (HORs), which may have functional significance. Maize is unusual in that its satellites occur in two distinct genomic contexts: centromeres, which interact with kinetochore proteins, and knobs, which undergo meiotic drive in the presence of Abnormal chromosome 10. Whether maize centromeres or knobs contain HOR patterns, and how such patterns relate to function, remains unclear. ResultsHere, we generate 13 repeat-sensitive genome assemblies of maize and its recent ancestor, teosinte. We develop a new graph-based pipeline, HiReNET, to classify HORs and demonstrate its utility in both Arabidopsis and maize. We find that HORs are ubiquitous in maize satellites but are typically low-frequency with small patterns, unlike the large, continuous HOR blocks characteristic of human centromeres. Approximately 38% of centromeric CentC monomers occur in HORs; however, no specific HOR class dominates any functional centromere as marked by centromeric histone H3. Arabidopsis centromeres have a similar HOR landscape. In contrast, maize knobs exhibit a more structured HOR distribution. Large knobs contain megabase-scale similarity blocks with repeated HOR patterns. These repeat units likely promote unequal crossing over, enabling rapid knob expansion, and may harbor motifs recognized by trans-acting factors involved in meiotic drive. ConclusionsHORs occur in all major maize satellite arrays. Specific HORs are not associated with centromere function, but knobs contain conserved HOR patterns within similarity blocks that may facilitate meiotic drive.

genomics↗

Increased maize chromosome number by engineered chromosome fission

Activation of synthetic centromeres on chromosome 4 in maize leads to its breakage and formation of trisomic fragments called neochromosomes. A limitation of neochromosomes is their low and unpredictable transmission rates due to trisomy. Here we report that selecting for dicentric recombinants through male crosses uncovers stabilized chromosome 4 fission events which split it into 4a-4b complementary chromosome pairs, where 4a carries a native centromere and 4b a synthetic one. The cells rapidly stabilized chromosome ends by de novo telomere formation and the new centromeres spread among genes without altering their expression. When both 4a and 4b chromosomes were present in a homozygous state, they segregated through meiosis indistinguishably from wild-type, and gave rise to healthy plants with normal seed set. This work leverages synthetic centromeres to engineer chromosome fission, effectively raising the diploid chromosome number of maize from 20 to 22. TEASERUsing synthetic centromeres, we divide one maize chromosome into two chromosomes, and show that both chromosomes are fully functional.

synthetic biology↗

Extensive genome evolution distinguishes maize within a stable tribe of grasses

Over the last 20 million years, the Andropogoneae tribe of grasses has evolved to dominate 17% of global land area. Domestication of these grasses in the last 10,000 years has yielded our most productive crops, including maize, sugarcane, and sorghum. The majority of Andropogoneae species, including maize, show a history of polyploidy - a condition that, while offering the evolutionary advantage of multiple gene copies, poses challenges to basic cellular processes, gene expression, and epigenetic regulation. Genomic studies of polyploidy have been limited by sparse sampling of taxa in groups with multiple polyploidy events. Here, we present 33 genome assemblies from 27 species, including chromosome-scale assemblies of maize relatives Zea and Tripsacum. In maize, the after-effects of polyploidy have been widely studied, showing reduced chromosome number, biased fractionation of duplicate genes, and transposable element (TE) expansions. While we observe these patterns within the genus Zea, 12 other polyploidy events deviate significantly. Those tetraploids and hexaploids retain elevated chromosome number, maintain nearly complete complements of duplicate genes, and have only stochastic TE amplifications. These genomes reveal variable outcomes of polyploidy, challenging simple predictions and providing a foundation for understanding its evolutionary implications in an ecologically and economically important clade.

evolutionary biology↗

Potent pollen gene regulation by DNA glycosylases in maize

Although DNA methylation primarily represses TEs, it also represses select genes that are methylated in plant body tissues but demethylated by DNA glycosylases (DNGs) in endosperm or pollen. 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 that account for 11.1% of the wild-type transcriptome but are silent or barely detectable in the plant body (sporophyte). They are unusual in their tendency to lack introns but even more so in their having TE-like methylation in their CDS. The majority have predicted functions in cell wall modification, and they likely support the rapid tip growth characteristic of pollen tubes. 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.

genetics↗

The maize striate leaves2 (sr2) gene encodes a conserved DUF3732 domain 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 the 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.

genetics↗

Natural methylation epialleles correlate with gene expression in maize

DNA methylation (5-methylcytosine) represses transposon activity and contributes to inaccessible chromatin structure of repetitive DNA in plants. It 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. To compare between genomes, we analyzed each data source relative to its own genome assembly rather than the easier but less accurate method of using one assembly as reference for all. 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 limited to specific maize stocks, and exhibit evidence of annotation errors. We used these data to flag all 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 interpretation of whole-genome studies.

plant biology↗

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 LexA-CENH3 tethering approach that activates functional centromeres at maize synthetic repeat arrays containing LexO binding sites. The synthetic centromeres are sufficient to cause chromosome breakage and release of chromosome fragments that are passed through meiosis and into progeny. Several independent chromosomes were identified, each with newly created centromeres localized over the repeat arrays where they were directed. The new centromeres were self-sustaining and stably transmitted chromosomes to progeny in the absence of the LexA-CENH3 activator. Our results demonstrate the feasibility of using synthetic centromeres for karyotype engineering applications.

genetics↗

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 non-genic 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 time scale with peaks at [~]300 kya and 16 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.

genomics↗

The maize gene maternal depression of r1 (mdr1) encodes a DNA glycosylase with maternal and paternal fertility functions

Demethylation of transposons can activate expression of nearby genes and cause imprinted gene expression in endosperm, and it is hypothesized to lead to expression of transposon siRNAs that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis 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 are its main function in endosperm.

genetics↗

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.

genomics↗

Effect of Sequence Depth and Length in Long-read Assembly of the Maize Inbred NC358

Recent improvements in the quality and yield of long-read data and scaffolding technology have made it possible to rapidly generate reference-quality assemblies for complex genomes. Still, generating these assemblies is costly, and an assessment of critical sequence depth and read length to obtain high-quality assemblies is important for allocating limited resources. To this end, we have generated eight independent assemblies for the complex genome of the maize inbred line NC358 using PacBio datasets ranging from 20-75x genomic depth and N50 read lengths of 11-21 kb. Assemblies with 30x or less depth and N50 read length of 11 kb were highly fragmented, with even the low-copy genic fraction of the genome showing degradation at 20x depth. Distinct sequence-quality thresholds were observed for complete assembly of genes, transposable elements, and highly repetitive genomic features such as telomeres, heterochromatic knobs and centromeres. This study provides a useful resource allocation reference to the community as long-read technologies continue to mature.

genomics↗

Genome-scale sequence disruption following biolistic transformation in rice and maize

We biolistically transformed linear 48 kb phage lambda and two different circular plasmids into rice and maize and analyzed the results by whole genome sequencing and optical mapping. While some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by non-homologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR.

plant biology↗

Loss of mCHH islands in maize chromomethylase and DDM1-type nucleosome remodeler mutants

Plants make use of three types of DNA methylation, each characterized by distinct DNA methyltransferases. One type, RNA-directed DNA methylation (RdDM), is guided by siRNAs to the edges of transposons that are close to genes, areas called mCHH islands in maize. Another type, chromomethylation, is guided by histone H3 lysine 9 methylation to heterochromatin across the genome. We examined DNA methylation and small RNA expression in plant tissues that were mutant for both copies of the genes encoding chromomethylases as well as mutants for both copies of the genes encoding DDM1-type nucleosome remodelers, which facilitate chromomethylation. Both sets of double mutants were nonviable but produced embryos and endosperm. RdDM was severely compromised in the double mutant embryos, both in terms of DNA methylation and siRNAs. Loss of 24nt siRNA from mCHH islands was coupled with a gain of 21, 22, and 24nt siRNAs in heterochromatin. These results reveal a requirement for both chromomethylation and DDM1-type nucleosome remodeling for RdDM in mCHH islands, which we hypothesize is due to dilution of RdDM components across the genome when heterochromatin is compromised.

genetics↗

The complex sequence landscape of maize revealed by single molecule technologies

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate elucidation of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here, we report the assembly and annotation of maize, a genetic and agricultural model species, using Single Molecule Real-Time (SMRT) sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and significant improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed over 130,000 intact transposable elements (TEs), allowing us to identify TE lineage expansions unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by SMRT sequencing. In addition, comparative optical mapping of two other inbreds revealed a prevalence of deletions in the low gene density region and maize lineage-specific genes.

genomics↗