Heterozygosity of Knob-Associated Tandem Repeats and Knob Instability in Mitotic Chromosomes of Zea (Zea mays L. and Z. diploperennis Iltis Doebley)

Knobs are blocks of heterochromatin present on chromosomes of maize (Zea mays L.) and its relatives that have effects on the frequency of genetic recombination, as well as on chromosome behavior.Knob heterozygosity and instability in six maize inbred lines and one Z. diploperennis Iltis Doebley line were investigated using the fluorescence in situ hybridization (FISH) technique with knob-associated tandem repeats (180 bp and 350 bp (TR-1)) as probes. Signals of seven heterozygous knobs containing 180-bp repeats and of one heterozygous knob containing TR- 1 were captured in chromosomes of all materials tested according to the results of FISH, which demonstrates that the 180-bp repeat is the main contributor to knob heterozygosity compared with the TR-1 element. In addition, one target cell with two TR-1 signals on one homolog of chromosome 2L, which was different from the normal cells in the maize inbred line GB57,was observed, suggesting knob duplication and an instability phenomenon in the maize genome.     

Heterozygosity of Knob-Associated Tandem Repeats and Knob Instability in Mitotic Chromosomes of Zea （Zea mays L. and Z. diploperennis Iltis Doebley）

Knobs are blocks of heterochromatin present on chromosomes of maize （Zea mays L.） and its relatives that have effects on the frequency of genetic recombination, as well as on chromosome behavior. Knob heterozygosity and instability in six maize inbred lines and one Z. diploperennis Iltis Doebley line were investigated using the fluorescence in situ hybridization （FISH） technique with knob-associated tandem repeats （180 bp and 350 bp （TR- 1）） as probes. Signals of seven heterozygous knobs containing 180- bp repeats and of one heterozygous knob containing TR- 1 were captured in chromosomes of all materials tested according to the results of FISH, which demonstrates that the 180-bp repeat is the main contributor to knob heterozygosity compared with the TR- 1 element. In addition, one target cell with two TR- 1 signals on one homolog of chromosome 2L, which was different from the normal cells in the maize inbred line GB57, was observed, suggesting knob duplication and an instability phenomenon in the maize genome.     

Molecular characterization of a family of tandemly repeated DNA sequences,TR-1, in heterochromatic knobs of maize and its relatives

Two families of tandem repeats, 180-bp and TR-1, have been found in the knobs of maize. In this study, we isolated 59 clones belonging to the TR-1 family from maize and teosinte. Southern hybridization and sequence analysis revealed that members of this family are composed of three basic sequences, A (67 bp); B (184 bp) or its variants B' (184 bp), 2/3B (115 bp), 2/3B' (115 bp); and C (108 bp), which are arranged in various combinations to produce repeat units that are multiples of approximately 180 bp. The molecular structure of TR-1 elements suggests that: (1) the B component may evolve from the 180-bp knob repeat as a result of mutations during evolution; (2) B' may originate from B through lateral amplification accompanied by base-pair changes; (3) C plus A may be a single sequence that is added to B and B', probably via nonhomologous recombination; and (4) 69 bp at the 3' end of B or B', and the entire sequence of C can be removed from the elements by an unknown mechanism. Sequence comparisons showed partial homologies between TR-1 elements and two centromeric sequences (B repeats) of the supernumerary B chromosome. This result, together with the finding of other investigators that the B repeat is also fragmentarily homologous to the 180-bp repeat, suggests that the B repeat is derived from knob repeats in A chromosomes, which subsequently become structurally modified. Fluorescence in situ hybridization localized the B repeat to the B centromere and the 180-bp and TR-1 repeats to the proximal heterochromatin knob on the B chromosome.     

Karyotype of maize (Zea mays L.) mitotic metaphase chromosomes as revealed by fluorescencein situ hybridization (FISH) with cytogenetic DNA markers

Here we demonstrate fluorescencein situ hybridization (FISH) of chromosome-specific cytogenetic DNA markers for chromosome identification in maize using repetitive and single copy probes. The fluorescently labeled probes, CentC and pZm4–21, were shown to be reliable cytogenetic markers in the maize inbred line KYS for identification of mitotic metaphase chromosomes. The fluorescent strength of CentC signal, relative position, knob presence, size and location were used for the karyotyping. Based on direct visual analysis of chromosome length and position of FISH signals, a metaphase karyotype was constructed for maize inbred line KYS. All chromosomes could be identified unambiguously. The knob positions in the karyotype agreed well with those derived from traditional cytological analyses except chromosomes 3, 4 and 8. One chromosome with a telomeric knob on the short arm was assigned to 3. A chromosome with a knob in the middle of the long arm was assigned number 4 by simultaneous hybridization with a knob-specific probe pZm4–21 and a chromosome 4-specific probe Cent 4. On chromosome 8, we found an additional small telomeric knob on the short arm. In addition, chromosome-specific probes were employed to identify chromosome 6 (45S rDNA) and chromosome 9 (single-copy probeumc105a cosmid).     

The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences

A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize. Received: 29 June 1999 / Accepted: 10 November 1999     

High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice

Large-scale physical mapping has been a major challenge for plant geneticists due to the lack of techniques that are widely affordable and can be applied to different species. Here we present a physical map of rice chromosome 10 developed by fluorescence in situ hybridization (FISH) mapping of bacterial artificial chromosome (BAC) clones on meiotic pachytene chromosomes. This physical map is fully integrated with a genetic linkage map of rice chromosome 10 because each BAC clone is anchored by a genetically mapped restriction fragment length polymorphism marker. The pachytene chromosome-based FISH mapping shows a superior resolving power compared to the somatic metaphase chromosome-based methods. The telomere-centromere orientation of DNA clones separated by 40 kb can be resolved on early pachytene chromosomes. Genetic recombination is generally evenly distributed along rice chromosome 10. However, the highly heterochromatic short arm shows a lower recombination frequency than the largely euchromatic long arm. Suppression of recombination was found in the centromeric region, but the affected region is far smaller than those reported in wheat and barley. Our FISH mapping effort also revealed the precise genetic position of the centromere on chromosome 10.     

Molecular Cytogenetic Identification of Triticum aestivum-Secale cereale Substitution and Addition Lines

Two substitution lines, designated as 930498 and 930483, and one addition line, designated as 930029, via Fo immature embryo culture of Triticum aestivum x octoploid triticale ( x Triti-cosecale Wittmack) were identified. Fluorescence in situ hybridization (FISH) using total genomic DNA of rye ( Secale cereale L. ) as probe corroborated the existence of rye chromosomes, further confirmed through chromosome paring at meiotic metaphase 1, C-banding and glutenin SDS- PAGE. The results demonstrated that the two substitution lines are ID/IR, and the addition line is also IR addition. Rye chromosomes that are distinct to the red-colored wheat chromosomes appear yellow-green at mitotic metaphase after FISH.     

Complex structure of knobs and centromeric regions in maize chromosomes

The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of individual maize chromosomes via the isolation and characterization of chromosome-specific cosmid clones. Restriction fragment fingerprinting, sequencing, and in situ hybridization were applied to discover a new family of knob associated tandem repeats, the TR1, which are capable of forming fold-back DNA segments, as well as a new family of centromeric tandem repeats, CentC. Analysis of knob and centromeric DNA segments revealed a complex organization in which blocks of tandemly arranged repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration/association of certain retrotransposable elements into knobs or centromere regions as well as for integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. DNA hybridization to a blot panel of eight individual maize chromosome addition lines revealed that CentC, TR1, and 180-bp tandem repeats are found in each of these maize chromosomes, but the copy number of each can vary significantly from about 100 to 25,000. In situ hybridization revealed variation among the maize chromosomes in the size of centromeric tandem repeats as well as in the size and composition of knob regions. It was found that knobs may be composed of either 180-bp or TR1, or both repeats, and in addition to large knobs these repeated elements may form micro clusters which are detectable only with the help of in situ hybridization. The association of the fold-back elements with knobs, knob polymorphism and complex structure suggest that maize knob may be consider as megatransposable elements. The discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes.     

Cohesion and centromere activity are required for phosphorylation of histone H3 in maize

Haspin‐mediated phosphorylation of histone H3 at threonine 3 (H3T3ph) promotes proper deposition of Aurora B at the inner centromere to ensure faithful chromosome segregation in metazoans. However, the function of H3T3ph remains relatively unexplored in plants. Here, we show that in maize (Zea mays L.) mitotic cells, H3T3ph is concentrated at pericentromeric and centromeric regions. Additional weak H3T3ph signals occur between cohered sister chromatids at prometaphase. Immunostaining on dicentric chromosomes reveals that an inactive centromere cannot maintain H3T3ph at metaphase, indicating that a functional centromere is required for H3T3 phosphorylation. H3T3ph locates at a newly formed centromeric region that lacks detectable CentC sequences and strongly reduced CRM and ZmBs repeat sequences at metaphase II. These results suggest that centromeric localization of H3T3ph is not dependent on centromeric sequences. In maize meiocytes, H3T3 phosphorylation occurs at the late diakinesis and extends to the entire chromosome at metaphase I, but is exclusively limited to the centromere at metaphase II. The H3T3ph signals are absent in the afd1 (absence of first division) and sgo1 (shugoshin) mutants during meiosis II when the sister chromatids exhibit random distribution. Further, we show that H3T3ph is mainly located at the pericentromere during meiotic prophase II but is restricted to the inner centromere at metaphase II. We propose that this relocation of H3T3ph depends on tension at the centromere and is required to promote bi‐orientation of sister chromatids.     

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.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

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.     

Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa

We describe the morphology and molecular organization of heterochromatin domains in the interphase nuclei, and mitotic and meiotic chromosomes, of Brassica rapa, using DAPI staining and fluorescence in situ hybridization (FISH) of rDNA and pericentromere tandem repeats. We have developed a simple method to distinguish the centromeric regions of mitotic metaphase chromosomes by prolonged irradiation with UV light at the DAPI excitation wavelength. Application of this bleached DAPI band (BDB) karyotyping method to the 45S and 5S rDNAs and 176 bp centromere satellite repeats distinguished the 10 B. rapa chromosomes. We further characterized the centromeric repeat sequences in BAC end sequences. These fell into two classes, CentBr1 and CentBr2, occupying the centromeres of eight and two chromosomes, respectively. The centromere satellites encompassed about 30% of the total chromosomes, particularly in the core centromere blocks of all the chromosomes. Interestingly, centromere length was inversely correlated with chromosome length. The morphology and molecular organization of heterochromatin domains in interphase nuclei, and in mitotic and meiotic chromosomes, were further characterized by DAPI staining and FISH of rDNA and CentBr. The DAPI fluorescence of interphase nuclei revealed ten to twenty conspicuous chromocenters, each composed of the heterochromatin of up to four chromosomes and/or nucleolar organizing regions.     

Genetic induction of chromosomal rearrangements in barley chromosome 7H added to common wheat

Chromosome 2C of Aegilops cylindrica induces chromosomal rearrangements in alien chromosome addition lines, as well as in euploid lines, of common wheat. To induce chromosomal rearrangements in barley chromosome 7H, reciprocal crosses were made between a mutation-inducing common wheat line that carries a pair of 7H chromosomes and one 2C chromosome and a 7H disomic addition line of common wheat. Many shrivelled seeds were included in the progeny, which was an indication of the occurrence of chromosome mutations. The chromosomal constitution of the viable progeny was examined by FISH (fluorescence in situ hybridization) using the barley subterminal repeat HvT01 as a probe. Structural changes of chromosome 7H were found in about 15% of the progeny of the reciprocal crosses. The aberrant 7H chromosomes were characterized by a combination of N-banding, FISH and genomic in situ hybridization. Mosaicism for aberrant 7H chromosomes was observed in seven plants. In total, 89 aberrant 7H chromosomes were identified in 82 plants, seven of which had double aberrations. More than half of the plants carried a simple deletion: four short-arm telosomes, one long-arm telosome, and 45 terminal deletions (23 in the short arm, 21 in the long arm, and one involving both arms). About 40% of the aberrations represented translocations between 7H and wheat chromosomes. Twenty of the translocations had wheat centromeres, 12 the 7H centromere, with translocation points in the 7HS (five) and in the 7HL (seven), and the remaining four were of Robertsonian type, three involving 7HS and one with 7HL. In addition, one translocation had a barley segment in an intercalary position of a wheat chromosome, and two were dicentric. The breakpoints of these aberrations were distributed along the entire length of chromosome 7H.     

Minichromosomes derived from the B chromosome of maize

Fourteen minichromosomes derived from the B chromosome of maize are described. The centromeric region of the B chromosome contains a specific repetitive DNA element called the B repeat. This sequence was used to determine the transmission frequency of the different types of minichromosomes over several generations via Southern blot analysis at each generation. In general, the minichromosomes have transmission rates below the theoretical 50% frequency of a univalent chromosome. The gross structure of each minichromosome was determined using fluorescence in situ hybridization (FISH) on root tip chromosome spreads. The presence of the B centromeric repeat and of the adjacent heterochromatic knob sequences was determined for each minichromosome. In two cases, the amount of the centromeric knob repeat is increased relative to the progenitor chromosome. Other isolates have reduced or undetectable levels of the knob sequence. Potential uses of the minichromosomes are discussed.     

Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyces pombe chromosome I.

We have determined the structural organization and functional roles of centromere-specific DNA sequence repeats in cen1, the centromere region from chromosome I of the fission yeast Schizosaccharomyces pombe. cen1 is composed of various classes of repeated sequences designated K', K"(dgl), L, and B', arranged in a 34-kb inverted repeat surrounding a 4- to 5-kb nonhomologous central core. Artificial chromosomes containing various portions of the cen1 region were constructed and assayed for mitotic and meiotic centromere function in S. pombe. Deleting K' and L from the distal portion of one arm of the inverted repeat had no effect on mitotic centromere function but resulted in greatly increased precocious sister chromatid separation in the first meiotic division. A centromere completely lacking K' and L, but containing the central core, one copy of B' and K" in one arm, and approximately 2.5 kb of the core-proximal portion of B' in the other arm, was also fully functional mitotically but again did not maintain sister chromatid attachment in meiosis I. However, deletion of K" from this minichromosome resulted in complete loss of centromere function. Thus, one copy of at least a portion of the K" (dgl) repeat is absolutely required but is not sufficient for S. pombe centromere function. The long centromeric inverted-repeat region must be relatively intact to maintain sister chromatid attachment in meiosis I.     

An asymptotic determination of minimum centromere size for the maize B chromosome

The maize B chromosome is a dispensable chromosome and therefore serves as a model system to study centromere function. The B centromere region is estimated to be approximately 9,000 kb in size and contains a 1.4 kb repeat that is specific to this centromere. When maintained as a univalent, the B chromosome occasionally undergoes centric misdivision. Consecutive misdivision analysis of the maize B chromosome centromere has generated a collection of functional centromeres that are greatly reduced in complexity. These small centromeres are often correlated with strongly reduced meiotic transmission. Molecular analyses of the misdivision collection have revealed that the smallest functional maize B centromere is a minimum of 110 kb in size. Considering the collection as a whole, meiotic transmission becomes severely compromised when the estimated centromere size is reduced to a few hundred kilobases.     

Differentiation between wheat chromosomes 4B and 4D.

A linkage map based on homoeologous recombination, induced by the absence of the Ph1 locus, between chromosome 4D of Triticum aestivum L. (genomes AABBDD) and chromosome 4B of T. turgidum L. (genomes AABB) was compared with a linkage map of chromosome 4Am of T. monococcum L. and a consensus map of chromosomes 4B and 4D of T. aestivum based on homologous recombination. The 4D/4B homoeologous map was only one-third the length of the homologous maps and all intervals were reduced relative to the 4B-4D consensus map. After the homoeologous map was corrected for this overall reduction in recombination, the distribution of recombination in the short arm was similar in both types of maps. In the long arm, homoeologous recombination declined disproportionally in the distal to proximal direction. This gradient was shown to be largely caused by severe segregation distortion reflecting selection against 4D genetic material. The segregation distortion had a maximum that coincided with the centromere and likely had a polygenic cause. Chromosomes 4D and 4B were colinear and recombination between them occurred in almost all intervals where homologous recombination occurred. These findings suggest that these chromosomes are not differentiated structurally and that the differentiation is not segmental. In the presence of Ph1, metaphase I chromosome pairing between chromosomes composed of homologous and differentiated regions correlated with the lengths of the homologous regions. No compensatory allocation of crossovers into the homologous regions was detected. In this respect, the present results are in dramatic contrast with the crossover allocation into the pseudoautosomal region in the mammalian male meiosis.     

Purification of the chromosomes of the Indian muntjac by flow sorting.

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.