Heterochromatic Knobs Research Articles

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.

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.

CAUSAS Y CONSECUENCIAS DE LA VARIACIÓN DEL CONTENIDO DE ADN EN ZEA

Cytogenetic evidence indicates that Zea, which comprises maize (Z. mays ssp. mays) and its wild relatives, is an allopolyploid genus. Our research group has carried out numerous cytogenetic studies on Zea species, mainly focused on native Argentinian and Bolivian maize landraces. We found a wide inter- and intraspecific genome size variation in the genus, with mean 2C-values ranging between 4.20 and 11.36 pg. For the maize landraces studied here, it varied between 4.20 and 6.75 pg. The objectives of this work are to analyze the causes of genome size variation and to discuss their adaptive value in Zea. This variation is mainly attributed to differences in the heterochromatin located in the knobs and to the amount of interspersed DNA from retrotransposons. Polymorphisms in presence or absence of B-chromosomes (Bs) and the population frequency of Bs are also a source of genome size variation, with doses ranging between one and eight in the landraces analyzed here. Correlation analysis revealed that the percentage of heterochromatin is positively correlated with genome size. In addition, populations cultivated at higher altitudes, which are known to be precocious, have smaller genome sizes than do those growing at lower altitudes. This information, together with the positive correlation observed between the length of the vegetative cycle and the percentage of heterochromatin, led us to propose that it has an adaptive role. On the other hand, the negative relationship found between Bs and heterochromatic knobs allowed us to propose the existence of an intragenomic conflict between these elements. We hypothesize that an optimal nucleotype may have resulted from such intranuclear conflict, where genome adjustments led to a suitable length of the vegetative cycle for maize landraces growing across altitudinal clines. Key words: B chromosomes, heterochromatin, intragenomic conflict, knobs, maize landraces

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.

A Heterochromatic Knob Reducing the Flowering Time in Maize.

Maize flowering time is an important agronomic trait, which has been associated with variations in the genome size and heterochromatic knobs content. We integrated three steps to show this association. Firstly, we selected inbred lines varying for heterochromatic knob composition at specific sites in the homozygous state. Then, we produced homozygous and heterozygous hybrids for knobs. Second, we measured the genome size and flowering time for all materials. Knob composition did not affect the genome size and flowering time. Finally, we developed an association study and identified a knob marker on chromosome 9 showing the strongest association with flowering time. Indeed, modelling allele substitution and dominance effects could offer only one heterochromatic knob locus that could affect flowering time, making it earlier rather than the knob composition.

Intragenomic Conflict between Knob Heterochromatin and B Chromosomes Is the Key to Understand Genome Size Variation along Altitudinal Clines in Maize.

In maize, we studied the causes of genome size variation and their correlates with cultivation altitude that suggests the existence of adaptive clines. To discuss the biological role of the genome size variation, we focused on Bolivian maize landraces growing along a broad altitudinal range. These were analyzed together with previously studied populations from altitudinal clines of Northwestern Argentina (NWA). Bolivian populations exhibited numerical polymorphism for B chromosomes (Bs) (from 1 to 5), with frequencies varying from 16.6 to 81.8 and being positively correlated with cultivation altitude. The 2C values of individuals 0B (A-DNA) ranged between 4.73 and 7.71 pg, with 58.33% of variation. The heterochromatic knobs, detected by DAPI staining, were more numerous and larger in individuals 0B than in those with higher doses of Bs. Bolivian and NWA landraces exhibited the same pattern of A-DNA downsizing and fewer and smaller knobs with increasing cultivation altitude, suggesting a mechanistic link among heterochromatin, genome size and phenology. The negative association between the two types of supernumerary DNA (knob heterochromatin and Bs), mainly responsible for the genome size variation, may be considered as an example of intragenomic conflict. It could be postulated that the optimal nucleotype is the result of such conflict, where genome adjustment may lead to an appropriate length of the vegetative cycle for maize landraces growing across altitudinal clines.

Changing local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering

Chromosomal inversions are recurrent rearrangements that occur between different plant isolates or cultivars. Such inversions may underlie reproductive isolation in evolution and represent a major obstacle for classical breeding as no crossovers can be observed between inverted sequences on homologous chromosomes. The heterochromatic knob (hk4S) on chromosome 4 is the most well-known inversion of Arabidopsis. If a knob carrying accession such as Col-0 is crossed with a knob-less accession such as Ler-1, crossovers cannot be recovered within the inverted region. Our work shows that by egg-cell specific expression of the Cas9 nuclease from Staphylococcus aureus, a targeted reversal of the 1.1 Mb long hk4S-inversion can be achieved. By crossing Col-0 harbouring the rearranged chromosome 4 with Ler-1, meiotic crossovers can be restored into a region with previously no detectable genetic exchange. The strategy of somatic chromosome engineering for breaking genetic linkage has huge potential for application in plant breeding.

Mixed knobs in corn cobs.

Maize heterochromatic knobs cheat female meiosis by forming neocentromeres that bias their segregation into the future egg cell. In this issue of Genes & Development, Swentowsky and colleagues (pp. 1239-1251) show that two types of knobs, those composed of 180-bp and TR1 sequences, recruit their own novel and divergent kinesin-14 family members to form neocentromeres.

European maize genomes highlight intraspecies variation in repeat and gene content

The diversity of maize (Zea mays) is the backbone of modern heterotic patterns and hybrid breeding. Historically, US farmers exploited this variability to establish today’s highly productive Corn Belt inbred lines from blends of dent and flint germplasm pools. Here, we report de novo genome sequences of four European flint lines assembled to pseudomolecules with scaffold N50 ranging from 6.1 to 10.4 Mb. Comparative analyses with two US Corn Belt lines explains the pronounced differences between both germplasms. While overall syntenic order and consolidated gene annotations reveal only moderate pangenomic differences, whole-genome alignments delineating the core and dispensable genome, and the analysis of heterochromatic knobs and orthologous long terminal repeat retrotransposons unveil the dynamics of the maize genome. The high-quality genome sequences of the flint pool complement the maize pangenome and provide an important tool to study maize improvement at a genome scale and to enhance modern hybrid breeding.

Effect of sequence depth and length in long-read assembly of the maize inbred NC358

Improvements in long-read data and scaffolding technologies have enabled rapid generation of reference-quality assemblies for complex genomes. Still, an assessment of critical sequence depth and read length is important for allocating limited resources. To this end, we have generated eight assemblies for the complex genome of the maize inbred line NC358 using PacBio datasets ranging from 20 to 75 × genomic depth and with N50 subread lengths of 11–21 kb. Assemblies with ≤30 × depth and N50 subread length of 11 kb are highly fragmented, with even low-copy genic regions showing degradation at 20 × depth. Distinct sequence-quality thresholds are observed for complete assembly of genes, transposable elements, and highly repetitive genomic features such as telomeres, heterochromatic knobs, and centromeres. In addition, we show high-quality optical maps can dramatically improve contiguity in even our most fragmented base assembly. This study provides a useful resource allocation reference to the community as long-read technologies continue to mature.

Preparing Maize Synaptonemal Complex Spreads and Sequential Immunofluorescence and Fluorescence In Situ Hybridization.

Immunofluorescence and fluorescence in situ hybridization (FISH) can be used to locate specific proteins and DNA sequences, respectively, in chromosomes by light microscopy. Here we describe sequential use of these techniques on spreads of maize synaptonemal complexes (SCs) to determine whether crossing over can occur in knob heterochromatin. We used antibodies to AFD1, an SC protein, and MLH1, a class I (interference-sensitive) crossover protein found in most recombination nodules (RNs) to identify crossovers (COs) along SCs. Next, we used FISH to localize a 180bp knob-specific tandem repeat. Combining immunofluorescence and FISH images of the same SC spreads showed that heterochromatic knobs do not prohibit class I COs. This technique is broadly applicable to investigations of plant prophase I chromosomes where meiotic recombination takes place.

Two jatropha karyotypes constructed from meiotic pachytene chromosomes: Pericentric distribution of heterochromatin and variation in repetitive DNAs.

Jatropha (Jatropha curcas) is an oil-bearing plant used for biodiesel production. Construction of its standard karyotype and identification of the euchromatin/heterochromatin distribution associated with gene expression and meiotic recombination are essential to fully characterize its genome. Here, we developed a J. curcas karyotype based on meiotic pachytene chromosomes. In addition, a karyotype of J. integerrima, a useful species for jatropha breeding, was also constructed. Five out of eleven J. curcas chromosomes were metacentric, but only two were metacentric in J. integerrima. Almost all of the heterochromatin was distributed around the pericentric regions. The interstitial and distal regions were euchromatic without heterochromatic knobs, except for small heterochromatin regions associated with the subtelomeric repeat sequence JcSat1. These pericentric heterochromatin distribution patterns, together with chromosome structure data and the results of FISH probing with rDNA and JcSat1, allowed us to classify all chromosomes of both species. The two species had two 35S rDNA loci and one 5S rDNA locus; one 35S rDNA locus in J. integerrima was located on the interstitial region of the short arms. In addition, JcSat1 was found at only the heterochromatic ends of the J. curcas chromosome, not the J. integerrima chromosome. Despite the same chromosome number, the two pachytene chromosome-based karyotypes suggest variation in chromosome structure and distribution of repetitive DNAs in these two species.

Parallel altitudinal clines reveal trends in adaptive evolution of genome size in Zea mays.

While the vast majority of genome size variation in plants is due to differences in repetitive sequence, we know little about how selection acts on repeat content in natural populations. Here we investigate parallel changes in intraspecific genome size and repeat content of domesticated maize (Zea mays) landraces and their wild relative teosinte across altitudinal gradients in Mesoamerica and South America. We combine genotyping, low coverage whole-genome sequence data, and flow cytometry to test for evidence of selection on genome size and individual repeat abundance. We find that population structure alone cannot explain the observed variation, implying that clinal patterns of genome size are maintained by natural selection. Our modeling additionally provides evidence of selection on individual heterochromatic knob repeats, likely due to their large individual contribution to genome size. To better understand the phenotypes driving selection on genome size, we conducted a growth chamber experiment using a population of highland teosinte exhibiting extensive variation in genome size. We find weak support for a positive correlation between genome size and cell size, but stronger support for a negative correlation between genome size and the rate of cell production. Reanalyzing published data of cell counts in maize shoot apical meristems, we then identify a negative correlation between cell production rate and flowering time. Together, our data suggest a model in which variation in genome size is driven by natural selection on flowering time across altitudinal clines, connecting intraspecific variation in repetitive sequence to important differences in adaptive phenotypes.

Alterations in Heterochromatic Knobs in Maize Callus Culture by Breakage-Fusion-Bridge Cycle and Unequal Crossing Over

The meiotic and mitotic behavior of regenerated plants derived from a long-term callus culture, designated 12-F, was analyzed. This culture was heterozygous for an amplification of the heterochromatic knob on the long arm of chromosome 7 (K7L). We aimed to investigate if the amplification resulted from a breakage-fusion-bridge (BFB) cycle or from unequal sister chromatid recombination. Therefore, C-banded mitotic metaphases and pachytene, diakinesis, and anaphase I of regenerated plants were analyzed. Additionally, the occurrence of alterations in K7L was investigated in C-banded metaphases from short-term callus cultures derived from lines related to the donor genotype of the 12-F culture. As a result, plants homozygous and heterozygous for the amplification were detected. Meiosis was normal with few abnormalities, such as a low frequency of univalents at diakinesis. In the callus cultures a chromosome 7 with knobs of different sizes in the sister chromatids was detected and interpreted as a result of unequal crossing over. Other chromosomal alterations were consistent with the occurrence of BFB cycles. The finding of unequal crossing over in the cultures supports the conclusion that the amplification in the culture 12-F would be derived from this mechanism. If the amplification was derived from a BFB cycle, the terminal euchromatic segment between knob and the telomere would be deleted, and possibly, homozygous plants would not be viable.

Are cytological parameters of maize landraces (Zea mays ssp. mays) adapted along an altitudinal cline?

The Northwestern Argentina (NWA) highland region is one of the southernmost areas of native maize cultivation. We studied variations of different cytological parameters, such as DNA contents, presence/absence of B chromosomes (Bs), and number and sequence composition of heterochromatic knobs in ten accessions of four maize landraces growing along a broad altitudinal cline in NWA. The aim of this work was to assess variations in cytological parameters and their relationship with the crop altitude of cultivation, in an adaptive context. The A-DNA content of the A chromosome complements showed 40% of difference between the lowest (4.5pg) and the highest (6.3pg) 2C value. This variation could be attributed to differences in number and size of heterochromatic knobs. Fluorescent in situ hybridization studies revealed the sequence composition of each knob, with a higher proportion of knobs composed of 180-bp repeats rather than TR-1 repeats, in all accessions. We also found numerical polymorphisms and the highest frequency of Bs reported in maize to this date. These results lead us to propose that the frequencies and doses of Bs are influenced by the landrace genotypical make-up. The Bs might be maintained in higher frequencies in those accessions having lower heterochromatin content, so as to preserve an optimal nucleotype. Furthermore, selective forces acting along the altitudinal gradient might be modulating the cytological parameters studied, as suggested by the significant correlations found among them.

Breakage-fusion-bridge cycles and de novo telomere formation on broken chromosomes in maize callus cultures.

Breakpoints involved in chromosome alterations associated with heterochromatin have been detected in maize plants regenerated from callus culture. A cytogenetic analysis of plants regenerated from a maize callus was performed aiming to analyze the stability of a chromosome 7 bearing a deficiency-duplication (Df-Dp), which was interpreted as derived from a chromatid type breakage-fusion-bridge (BFB) cycle. The Df-Dp chromosome 7 was stable in mitotic and meiotic cells of the regenerated plants. Fluorescence in situ hybridization showed signals of telomeric sequences on the broken chromosome arm and provided evidence of de novo telomere formation. The stability of two types of altered chromosome 7 was investigated in C-banded metaphases from samples of the original callus that were collected during a period of 30-42 months after culture initiation. New alterations involving heterochromatic knobs of chromosomes 7 and 9 were observed. The aberrant chromosomes were stable in the subcultures, thus providing evidence of broken chromosome healing. The examination of anaphases showed the presence of bridges, which was consistent with the occurrence of BFB cycles. De novo telomere formation occurred in euchromatic and heterochromatic chromosome termini. The results point to events of chromosomal evolution that might occur in plants.

Karyotype variability in tropical maize sister inbred lines and hybrids compared with KYS standard line.

Maize karyotype variability has been extensively investigated. The identification of maize somatic and pachytene chromosomes has improved with the development of fluorescence in situ hybridization (FISH) using tandemly repeated DNA sequences as probes. We identified the somatic chromosomes of sister inbred lines that were derived from a tropical flint maize population (Jac Duro [JD]), and hybrids between them, using FISH probes for the 180-bp knob repeat, centromeric satellite (CentC), centromeric satellite 4 (Cent4), subtelomeric clone 4-12-1, 5S ribosomal DNA and nucleolus organizing region DNA sequences. The observations were integrated with data based on C-banded mitotic metaphases and conventional analysis of pachytene chromosomes. Heterochromatic knobs visible at pachynema were coincident with C-bands and 180-bp FISH signals on somatic chromosomes, and most of them were large. Variation in the presence of some knobs was observed among lines. Small 180-bp knob signals were invariant on the short arms of chromosomes 1, 6, and 9. The subtelomeric 4-12-1 signal was also invariant and useful for identifying some chromosomes. The centromere location of chromosomes 2 and 4 differed from previous reports on standard maize lines. Somatic chromosomes of a JD line and the commonly used KYS line were compared by FISH in a hybrid of these lines. The pairing behavior of chromosomes 2 and 4 at pachytene stage in this hybrid was investigated using FISH with chromosome-specific probes. The homologues were fully synapsed, including the 5S rDNA and CentC sites on chromosome 2, and Cent4 and subtelomeric 4-12-1 sites on chromosome 4. This suggests that homologous chromosomes could pair through differential degrees of chromatin packaging in homologous arms differing in size. The results contribute to current knowledge of maize global diversity and also raise questions concerning the meiotic pairing of homologous chromosomes possibly differing in their amounts of repetitive DNA.

Intragenomic Conflict Between the Two Major Knob Repeats of Maize

Examples of meiotic drive, the non-Mendelian segregation of a specific genomic region, have been identified in several eukaryotic species. Maize contains the abnormal chromosome 10 (Ab10) drive system that transforms typically inert heterochromatic knobs into centromere-like domains (neocentromeres) that move rapidly poleward along the spindle during meiosis. Knobs can be made of two different tandem repeat sequences (TR-1 and 180-bp repeat), and both repeats have become widespread in Zea species. Here we describe detailed studies of a large knob on chromosome 10 called K10L2. We show that the knob is composed entirely of the TR-1 repeat and is linked to a strong activator of TR-1 neocentromere activity. K10L2 shows weak meiotic drive when paired with N10 but significantly reduces the meiotic drive exhibited by Ab10 (types I or II) in Ab10/K10L2 heterozygotes. These and other data confirm that (1) there are two separate and independent neocentromere activities in maize, (2) that both the TR-1 and knob 180 repeats exhibit meiotic drive (in the presence of other drive genes), and (3) that the two repeats can operate in competition with each other. Our results support the general concept that tandem repeat arrays can engage in arms-race-like struggles and proliferate as an outcome.

Characterization of chromosomal architecture in Arabidopsisby chromosome conformation capture

BackgroundThe packaging of long chromatin fibers in the nucleus poses a major challenge, as it must fulfill both physical and functional requirements. Until recently, insights into the chromosomal architecture of plants were mainly provided by cytogenetic studies. Complementary to these analyses, chromosome conformation capture technologies promise to refine and improve our view on chromosomal architecture and to provide a more generalized description of nuclear organization.ResultsEmploying circular chromosome conformation capture, this study describes chromosomal architecture in Arabidopsis nuclei from a genome-wide perspective. Surprisingly, the linear organization of chromosomes is reflected in the genome-wide interactome. In addition, we study the interplay of the interactome and epigenetic marks and report that the heterochromatic knob on the short arm of chromosome 4 maintains a pericentromere-like interaction profile and interactome despite its euchromatic surrounding.ConclusionDespite the extreme condensation that is necessary to pack the chromosomes into the nucleus, the Arabidopsis genome appears to be packed in a predictive manner, according to the following criteria: heterochromatin and euchromatin represent two distinct interactomes; interactions between chromosomes correlate with the linear position on the chromosome arm; and distal chromosome regions have a higher potential to interact with other chromosomes.

Integration of Cytological Features with Molecular and Epigenetic Properties of Rice Chromosome 4

It has been reported that rice chromosome 4 has eight major heterochromatic knobs within the heterochromatic half and that this organization correlates with chromosomal-level transcriptional activity. To better understand this chromosomal organization, we created a model based on the statistical distribution of various types of gene models to divide chromosome 4 into 17 euchromatic and heterochromatic regions that correspond with the cytological staining. Fluorescence in-situ hybridization (FISH) experiments using a set of bacterial artificial chromosome (BAC) clones from chromosome 4 placed all 18 clones in the region predicted by the model. Elevated levels of H3K4 di- and tri-methylation detected by chromatin-immunoprecipitation (ChIP) on chip were correlated with euchromatic regions whereas lower levels of these two modifications were detected in heterochromatic regions. Small RNAs were more abundant in the heterochromatic regions. To validate these findings, H3K4 trimethylation, H3K9 acetylation, H4K12 acetylation, and H3K9 di- and tri-methylation of 19 individual genes were measured by ChIP-PCR. Genes in heterochromatic regions had elevated H3K9 di- and tri-methylation while genes in euchromatic regions had elevated levels of the other three modifications. We also assayed cytosine methylation of these genes using the restriction enzymes McrBC, HapII, and Msp I. This analysis indicated that cytosines of transposable elements and some genes located in heterochromatic regions were methylated while cytosines of the other genes were unmethylated. These results suggest that local transcriptional activity may reflect the organization of the corresponding part of the chromosome. They also indicate that epigenetic regulation plays an important role in correlating chromosomal organization with transcriptional activity.