Knob Heterochromatin Research Articles

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.

Genome size and repetitive sequences are driven by artificial selection on the length of the vegetative cycle in maize landraces from Northeastern Argentina

Abstract Variation in genome size and knob heterochromatin content was explored in relationship to altitudinal cline and length of the vegetative cycle in northern Argentina, USA and Mexico landraces. It was considering that the decrease in DNA and heterochromatin content could be an adaptation to a shorter growing season and the result of artificial selection by man. Guaraní landraces from Northeastern Argentina (NEA) show similar variation in genome size (3.81pg to 7.56pg) and knob heterochromatin content than maize growing across an altitudinal cline. The present analysis offers an overview of the genetic variability of NEA maize to explain why Guaraní landraces and those along an altitudinal cline share this similar variation. Karyotype and flow cytometry data were employed. The DNA content of Guaraní landraces which lacking B chromosomes, showed no significant relationship with knob heterochromatin, suggesting differences in the amount of interspersed DNA. A significant positive relationship was found between the length of the vegetative cycle and both number and percentage of knob heterochromatin. No significant correlation was found between genome size and vegetative cycle. All these results allow us to conclude that the variation in heterochromatin content among Guaraní maize is driven by the selection of farmers for flowering time.

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.

Meiotic Crossing Over in Maize Knob Heterochromatin.

There is ample evidence that crossing over is suppressed in heterochromatin associated with centromeres and nucleolus organizers (NORs). This characteristic has been attributed to all heterochromatin, but the generalization may not be justified. To investigate the relationship of crossing over to heterochromatin that is not associated with centromeres or NORs, we used a combination of fluorescence in situ hybridization of the maize 180-bp knob repeat to show the locations of knob heterochromatin and fluorescent immunolocalization of MLH1 protein and AFD1 protein to show the locations of MLH1 foci on maize synaptonemal complexes (SCs, pachytene chromosomes). MLH1 foci correspond to the location of recombination nodules (RNs) that mark sites of crossing over. We found that MLH1 foci occur at similar frequencies per unit length of SC in interstitial knobs and in the 1 µm segments of SC in euchromatin immediately to either side of interstitial knobs. These results indicate not only that crossing over occurs within knob heterochromatin, but also that crossing over is not suppressed in the context of SC length in maize knobs. However, because there is more DNA per unit length of SC in knobs compared to euchromatin, crossing over is suppressed (but not eliminated) in knobs in the context of DNA length compared to adjacent euchromatin.

Intra-specific variation in genome size in maize: cytological and phenotypic correlates.

Genome size variation accompanies the diversification and evolution of many plant species. Relationships between DNA amount and phenotypic and cytological characteristics form the basis of most hypotheses that ascribe a biological role to genome size. The goal of the present research was to investigate the intra-specific variation in the DNA content in maize populations from Northeastern Argentina and further explore the relationship between genome size and the phenotypic traits seed weight and length of the vegetative cycle. Moreover, cytological parameters such as the percentage of heterochromatin as well as the number, position and sequence composition of knobs were analysed and their relationships with 2C DNA values were explored. The populations analysed presented significant differences in 2C DNA amount, from 4.62 to 6.29 pg, representing 36.15 % of the inter-populational variation. Moreover, intra-populational genome size variation was found, varying from 1.08 to 1.63-fold. The variation in the percentage of knob heterochromatin as well as in the number, chromosome position and sequence composition of the knobs was detected among and within the populations. Although a positive relationship between genome size and the percentage of heterochromatin was observed, a significant correlation was not found. This confirms that other non-coding repetitive DNA sequences are contributing to the genome size variation. A positive relationship between DNA amount and the seed weight has been reported in a large number of species, this relationship was not found in the populations studied here. The length of the vegetative cycle showed a positive correlation with the percentage of heterochromatin. This result allowed attributing an adaptive effect to heterochromatin since the length of this cycle would be optimized via selection for an appropriate percentage of heterochromatin.

Diversity of Chromosomal Karyotypes in Maize and Its Relatives

Maize is a highly diverse species on the gene sequence level. With the recent development of methods to distinguish each of the 10 pairs of homologues in somatic root tip spreads, a wide collection of maize lines was subjected to karyotype analysis to serve as a reference for the community and to examine the spectrum of chromosomal features in the species. The core nested association mapping progenitor collection and additional selections of diversity lines were examined. Commonly used inbred lines were included in the analysis. The centromere 4 specific repeat and ribosomal RNA loci were invariant. The CentC centromere repeat exhibited extensive differences in quantity on any particular chromosome across lines. Knob heterochromatin was highly variable with locations at many sites in the genome. Lastly, representative examples from other species in the genus Zea (teosintes) were examined, which provide information on the evolution of chromosomal features.

Stability of Repeated Sequence Clusters in Hybrids of Maize as Revealed by FISH

Previous studies of genome size in inbreds and hybrids had revealed that in some combinations the genome size in hybrids deviates from the midparent value of the parents. We examined whether repetitive sequence arrays such as knob heterochromatin, centromere repeats and ribosomal RNAs, as visualized cytologically in root tip chromosome spreads, would reveal any changes in copy number in such hybrids. The results indicate that no obvious changes in copy number are observed. Thus, the mechanisms by which repetitive arrays change copy number seem unrelated to the hybrid effect. Moreover, the hybrid genome effect is not manifested in gross changes in the most common repetitive sequences in the genome as determined in root tip spreads.

PRINS-Labeled Knobs Are Not Associated with Increased Chromosomal Stickiness in the Maize st1 Mutant

In maize, the st1 mutant has been observed to result in chromosomes that stick together during both mitotic and meiotic anaphase. These sticky chromosomes result in abnormal chromosome separation at anaphase. Although the mechanism producing the st1 mutant phenotype is unknown, delayed replication of knob heterochromatin has been implicated in similar phenomena that result in sticky chromosomes. Primed in situ labeling (PRINS) was used to locate the 180-bp knob DNA sequences on mitotic metaphase chromosomes of several maize lines. The chromosomal regions labeled by PRINS corresponded to the reported C bands found in these lines. Additionally, PRINS was used to identify knob-bearing regions in anaphase spreads of a line carrying the st1 mutant and a nonmutant line having a similar number of chromosome knobs. The increase in abnormal anaphase figures in the st1 mutant was not accompanied by an increase in association of knob DNA with abnormal anaphases. Thus, the increase in chromosomal stickiness appears to be due to an increase in stickiness of knob and nonknob chromosomal regions. The mechanism responsible for the st1 mutant, therefore, is hypothesized to be different from that implicated in the other previously described sticky chromosomes situations.

Characterization of a maize isochromosome 8S·8S

An isochromosome was found in the maize HiII Parent B line during somatic karyotyping with a multiprobe fluorescence in situ hybridization (FISH) system. Cytological analyses showed that it pairs with the short arm of chromosome 8 during the pachytene stage of meiosis. The chromosome 8 short arm origin of this isochromosome was also confirmed by FISH at mitotic metaphase. Knob heterochromatin signals were present at the short arms of chromosome 8 when subjected to prolonged exposure and also observed at both ends of the isochromosome. This isochromosome can be a univalent or a trivalent by pairing with the normal chromosome 8 short arms during meiosis. At anaphase and telophase, the isochromosome lagged behind other chromosomes. It had a transmission rate of 17%-20% from both male and female gametes. One plant homozygous for the isochromosome contained 2 isochromosomes that differed in the quantity of their CentC centromere repeat sequence. Both variations of the isochromosome were transmitted to the next generation. Because the 2 isochromosomes should be identical by descent, these observations document a radical change in copy number of the centromere repeat array within 1 generation. Plants with 1 isochromosome were not normal as compared with the original HiII Parent B plants. Those that contained a pair of this isochromosome (6 total copies of 8S) were even more abnormal and had reduced fertility. The results indicate the ability of the somatic karyotyping system to recognize and characterize chromosomal aberrations.

Fluorescence in situ hybridization analysis reveals multiple loci of knob-associated DNA elements in one-knob and knobless maize lines.

Fluorescence in situ hybridization analyses were conducted to examine the presence or absence of the 180- and 350-bp knob-associated tandem repeats in maize strains previously defined as "one-knob" or "knobless." Multiple loci were found to hybridize to these two repeats in all maize lines analyzed. Our results show that the number of 180- and 350-bp repeat loci do not correlate with the number of knobs in maize and that these tandem repeats are not independently sufficient to confer knob heterochromatin, even when present at megabase sizes.

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.

Involvement of knob heterochromatin in mitotic abnormalities in germinating aged seeds of maize

Seeds of three inbred lines of maize, with contrasting numbers of heterochromatic knobs and stored under two different ageing treatments, were analyzed for the occurrence of abnormalities at mitotic anaphase in root meristems 3, 7, 21,42, and 56 days after germination, and in root meristems of their freshly harvested selfed progeny. The largest category of detectable aberrations involved breakage of knobbed chromosome arms. We have obtained evidence that knob heterochromatin plays a central role in the origin of primary chromosome bridges. The initial event responsible for the occurrence of breakages and lagging chromosomes was characterized by the nondisjunction of newly replicated sister chromatids, which was observed to occur preferentially at the knob level. Such configurations, and all the other types of abnormalities (as for example, lagging chromosomes, typical chromosome bridges, fragments, and micronuclei), were observed at decreasing frequencies throughout root growth. Nevertheless, we have detected the occurrence of breakage-fusion-bridge cycles that were initiated by broken chromosomes. The relationship between late-replicating DNA in maize knob heterochromatin and the vulnerability of such regions to breakage is discussed. Our observations suggest a similarity between the mechanisms involved or associated with the origin of the described abnormalities and those reported to occur in cultured maize cells.

Determining relative abundance of specific DNA sequences in flow cytometrically sorted maize nuclei

A wide breadth of DNA content variation has been reported among maize lines. While the extent of this variation has been well documented, few studies have focused on its cause. Some of the nuclear DNA content variation has been explained by the presence of B chromosomes or knobs. However, variation in these two structures does not account for all of the observed variation. In order to identify other fluctuating DNA sequences, a rapid and reliable method of estimating relative abundance of DNA sequences needed to be developed. The potential of flow cytometry in conjunction with spot hybridization for determining relative abundance of specific DNA sequences in maize was studied. Different numbers of G1 phase nuclei were sorted on nitrocellulose filters and non-radioactive hybridization and signal detection performed. Results from these experiments revealed a significant, positive linear correlation between the amount of target sequence and signal density using both knob (R = 0.98) and ribosomal spacer (R = 0.99) DNA sequences. In addition, G1 phase nuclei of eight inbred lines differing in the amount of knob heterochromatin, were sorted on to filters and the non-radioactive hybridization and signal detection performed. A significant, positive linear correlation between C-band number and signal density (R = 0.83; P = 0.0051) as well as between per cent heterochromatin and signal density (R = 0.96; P = 0.0002) was observed. These results indicate the usefulness of flow cytometry for spot hybridization in determining the relative abundance of DNA sequences in the maize genome

Meiotic chromosome pairing in maize is associated with a novel chromatin organization

Three-dimensional light microscopy and the excellent cytological features of maize meiotic chromosomes are used to analyze the early events of chromosome synapsis. We demonstrate that the chromosomes undergo a dramatic structural reorganization prior to synapsis in zygotene. The unique features of prezygotene chromosomes are a partial separation of sister chromatids, an elongation of knob heterochromatin, an increase in surface complexity, a 50% increase in total chromosome volume, and a peripheral localization and alignment of telomeric, but not proximal, loci. At zygotene, chromosome volume decreases and chromosomes appear as single fibers. The specialized prezygotene chromosome morphology may facilitate homology recognition once the homologs have been brought together.

Distribution and organization of a tandemly repeated 352-bp sequence in the oryzae family

A 352-bp EcoRI fragment from rice DNA was cloned and shown to be a member of a tandem repeat. Sequence determination revealed homologies with human alpha satellite DNA and maize knob heterochromatin specific repeat. This 352-bp sequence is highly specific for the AA genome of rice. However, copy number and sequence organization are variable, depending on the accession analyzed. Several examples of amplification were observed in O. rufipogon and O. longistaminata. Use of resolutive polyacrylamide gel electrophoresis and 4-bp cutter enzymes allowed one to distinguish between the Indica and Japonica subtypes of O. sativa. The same method also discriminates between two groups of O. rufipogon, the presumed ancestor of O. sativa, suggesting that the present day Indica and Japonica subtypes originated independently from two O. rufipogon distinct populations.

Genome size variation in Zea mays ssp. mays adapted to different altitudes

Previous studies have indicated a positive correlation between genome size and altitude among plant species. It has been hypothesized that increasing genome size occurs due to increasing C-banded heterochromatin. In corn, increasing altitude has been correlated with decreasing knob (C-banded) heterochromatin, suggesting that DNA content may decrease with increasing altitude. In this study, nuclear DNA content of 12 southwestern United States Indian maize populations, collected at various altitudes, was determined. The significant positive correlation observed between genome size and altitude suggests that corn follows the trend of increasing DNA content with increasing altitude observed in other plant species. Whether this correlation is due to increasing knob heterochromatin or additional intra- or supernumerary chromosomal DNA sequences has yet to be determined.

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T4 phage DNA and the base sequence divergence is low.3H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.

Cytogenetic stability of maize tissue cultures: a cell line pedigree analysis

Cytogenetic instability in maize plants regenerated from tissue culture is a commonly observed phenomenon. In an attempt to understand the origin of this instability meiotic analyses were performed on 370 regenerated plants that were initiated from cell lines of 22 immature embryos of 9 maize genotypes. Cell lineage pedigrees were maintained on these cultures to record the familial relationship between regenerated plants. Overall, 12.4% of the 370 régénérants contained cytological aberrations. The largest category of aberrations involved chromosome breakage (translocations, deletions, and an inversion) between a heterochromatic knob or knob site and the centromere. Late replicating knob heterochromatin might have been responsible for the chromosome breakage events. Cytologically identical aberrations were observed in plants regenerated at various time points in the pedigreed cultures, indicating an early occurrence of the aberration during culture maintenance. Heterogeneity of cell karyotypes also was observed within embryo cell lines. Multiple independent aberrations occurred within a single embryo cell line. The pedigree of the cultures provided evidence that the sectoring of regenerated plants was due to a multicellular origin of meristematic areas. The frequency of cytogenetically abnormal plants recovered increased with culture age. The data presented are consistent with the age effect not being due to an increased mutation rate but due to mutational events that occurred throughout culture development with a subsequent maintenance and accumulation of aberrant cells over time.Key words: Zea mays, cytogenetic stability, tissue culture, somaclonal variation.

Differential staining of mitotic chromosomes from inbred lines and hybrids ofZea mays L.

The Giemsa banding patterns of four inbred lines (DB1113, DB1116, WIR44 and WIR27) ofZea mays and their hybrids have been compared. The polymorphism of knob heterochromatin of 4 chromosome pairs allowed to distinguish the 4 inbred lines on the basis of their banding patterns, reflected the evolutionary distance of the inbred lines and provided evidence as to the origin of these chromosomes in hybrids.

Distribution and clustering of two highly repeated sequences in the A and B chromosomes of maize

Clones from a family of highly repeated sequences present in a heterochromatin rich maize line have been characterized by sequencing and chromosome location. The repeats differ from each other in length and degree of sequence homology, and show areas rich in purine and pyrimidine. In "situ" hybridization experiments indicate that the repeats are mainly located in the knob heterochromatin of the A chromosomes and the centromeric heterochromatin of the B chromosome. However, in addition to previously published data, some copies are also distributed in euchromatic regions of the A chromosomes and in the distal heterochromatic block of the B chromosome. The results are discussed in relation to the centromeric activity of maize heterochromatin.