Ultrastructure of meiotic pairing in B chromosomes of Crepis capillaris

The meiotic pairing behaviour of four B isochromosomes of Crepis capillaris was studied by synaptonemal complex (SC) surface spreading of pollen mother cells. The four B chromosomes form a tightly associated group, separate from the standard chromosomes, throughout zygotene and pachytene. All four B chromosomes are also folded around their axis of symmetry, the centromere, and the eight homologous arms are closely aligned from the earliest prophase I stages. A high frequency of multivalent pairing of the four B chromosomes is observed at pachytene, in excess of 90%, mirroring the situation observed at metaphase I but exceeding the frequency expected (76.2%) on the assumption of random pairing among the eight B isochromosome arms with a single distal pairing initiation site per arm. The higher than expected frequency of multivalents is due to the occurrence of multiple pairing initiations along the B isochromosome arms, resulting in high frequencies of pairing partner switches. Pairing of the standard chromosome set is frequently incomplete in the presence of four B chromosomes, and abnormalities of SC structure such as thickening and splitting of axes and lateral elements are also frequently seen. Similarly, B chromosomes show partial pairing failure, the extent of which is correlated with pairing failure in the standard chromosome set. The B chromosomes themselves also show abnormalities of SC structure. Both standard and B chromosomes show non-homologous foldback pairing of regions that have failed to pair homologously.by D. Schweizer     

Meiosis in autopolyploid Crepis capillaris

Meiotic chromosome pairing of triploid and trisomic Crepis capillaris was analysed by electron microscopy in surface-spread prophase I nuclei and compared with light microscopic observations of metaphase I. This system allows identification and separate analysis of each chromosome of the C. capillaris genome. Prophase I trivalent frequencies are very high in all three trisomes and only slightly dependent on chromosome size. At metaphase I, on the other hand, trivalent frequencies are much lower and strongly dependent on chromosome size. There is no evidence for trivalent elimination during prophase I in this system, and the reduction in trivalent frequency at metaphase I can be explained by an insufficiency of appropriately placed chiasmata. The high prophase I trivalent frequencies far exceed the two-thirds expected on a simple model with two terminal independent pairing initiation sites per trisome, suggesting that multiple pairing initiation occurs. Direct observations reveal high frequencies of pairing partner switches (PPSs) in prophase I trisomes, which confirms this supposition. The numbers of PPSs per trisome shows a better fit to the Poisson than to the binomial distribution and their positional distribution along trisomes is random and non-localized. All these observations favour a model of pairing initiation in trisomes based on a large number of evenly distributed autonomous pairing sites each with a uniform and low probability of generating a PPS.by C. Heyting     

Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris

The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a Bspecific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.     

Structure of the interphase nucleus and formation of chromosome aberrations in Crepis capillaris]

A distribution of chromosome X-rays induced aberrations was studied in 2n and 4n root cells of Crepis capillaris at different stages of the mitotic cycle. Participation of various chromosomes in aberrations was found to be different from theoretically expected one found from the chromosome length. The chromosome A was characterized by excession of intrachromosome aberrations (terminal deletions, isolocuses, duplications and rings). The chromosome D very often took part in the formation of rings. The chromosome C was characterized by an excession of interchromosome aberrations and the lack of intrachromosome ones. The data obtained support the model of interphase nucleus proposed by Wagenaar. The frequency of exchanges of homologues in 2n and 4n cells well correlated with theoretically expected. The inhibition of mitosis increased the exchanges of homologues. In 2n and 4n cells asymmetrical translocations predominated over symmetrical ones.     

Repetitive DNA sequence families in Crepis capillaris

Three families of tandemly repetitive DNA from Crepis capillaris were cloned and characterized. Data obtained from in situ hybridization indicate that these families are located mainly in the heterochromatic C-bands. The pCcH32 family hybridizes at the paracentromeric C-band of the NOR (nucleolus-organized region) chromosome and along most of the long arm of the same chromosome. The pCcD29 family is located in all the remaining C-bands of the karyotype, while the third family, pCcE9, is restricted to the more proximal C-bands. Nucleotide sequence comparisons between one cloned repeating unit from each DNA family showed some significant regions of homology between the families. We discuss the sequence relationships between the three DNA families and the significance of our data in relation to models of heterochromatin evolution, emphasizing the concepts of equilocality and the differentiation of the NOR-bearing chromosome. We also examine the possible role that chromosome disposition, in either mitotic or meiotic nuclei, plays in the distribution and homogenization of heterochromatic DNA sequences.     

Chromosome-specific control of chiasma formation in Crepis capillaris

An experimental population of Crepis capillaris (2n=6) displays frequent chromosome-specific univalence affecting all three chromosome pairs of the complement independently in different plants. The frequency of univalence in the population varies from 0% in some plants to about 40% of pollen mother cells in other plants. Most commonly, affected pollen mother cells contain just one pair of univalents and wherever the frequency of cells containing univalents exceeds 10%, a chromosome-specific effect almost invariably appears. Univalence affecting the A, C and D chromosome pairs is about equally frequent in the population. The mean cell chiasma frequencies of affected plants are generally lower than those of normal plants from the same population, despite suggestions of compensating increases in the chiasma frequencies of unaffected chromosomes pairs in the presence of specific univalence of a third pair. Breeding tests have been carried out, and observations made on pachytene stages which demonstrate that the specific univalence is due to recessive genes causing desynapsis following apparently normal pairing of homologues.     

Chromosomal aberrations in Crepis capillaris cells detected by FISH

Crepis capillaris (2n=6) is an excellent plant for the assay of chromosome aberrations after mutagenic treatment. It has simple karyotype: three pairs of morphologically distinct and relatively large chromosomes. The frequency of structural chromosome aberrations and micronuclei in root meristem cells has been used for evaluation of the genotoxicity of chemicals and environmental pollutants. The introduction of fluorescence in situ hybridization method allows more detailed detection and localization of chromosomal rearrangements not only in mitotic but also in interphase nuclei. We demonstrate a few examples of the detection of chromosomal aberrations using rDNA and telomeric sequences as probes for in situ hybridization to C. capillaris chromosomes.     

Tumorous cultures of Crepis capillaris: chromosomes and growth

Different crown-gall culture strains of Crepis capillaris derived from haploid and diploid tumors were studied karyologically during the period of 18 transfers (18–20 months). The very low proportion of mitotic anomalies observed in the first transfers is in agreement with the generally accepted assumption that chromosomal changes are not involved in the initiation of tumors. However, it has been established that an intimate relationship exists between chromosome aberrations and the further development of the tumors. Morphological and physiological changes which occurred in some strains and substrains were invariably associated with cytologieal changes. The results are discussed in connection with similar phenomena described for animal tumors.     

Meiosis in allopolyploid Crepis capillaris. II. Autotetraploids.

Meiotic chromosome pairing of autotetraploid Crepis capillaris was analysed by electron microscopy of surface-spread prophase I nuclei and compared with light microscopic observations of metaphase I chromosome configurations. Prophase I quadrivalent frequencies are high in all three tetrasomes. (A, D, and C) and partially dependent on chromosome size. At metaphase I quadrivalent frequencies are much lower and strongly dependent on chromosome size. There is no evidence for multivalent elimination during prophase I in this system, and the reduction in multivalent frequency at metaphase I can be explained by an insufficiency of appropriately placed chiasmata. The high frequencies of prophase I quadrivalents far exceed the two-thirds expected on a simple model with two terminal independent pairing initiation sites per tetrasome, suggesting that multiple pairing initiation occurs. Direct observations reveal relatively high frequencies of pairing partner switches (PPSs) at prophase I, which confirms this suggestion. The numbers of PPSs per tetrasome show a good fit to the Poisson distribution, and their positional distribution along chromosomes is random and nonlocalized. These observations favour a model of pairing initiation based on a large number of evenly distributed autonomous pairing sites each with a uniform and low probability of generating a PPS.     

The effect of B chromosomes and T-DNA on chromosomal variation in callus cells and regenerated roots of Crepis capillaris

The chromosome behaviour has been compared in three Crepis capillaris callus culture lines and the roots regenerated from these calli. The calli were obtained from explants derived from plants without and with two B chromosomes and the hairy roots were obtained from plants transformed with Agrobacterium rhizogenes. Cytological studies demonstrated that the presence of additional DNA as B chromosomes or as T-DNA had an influence on the numerical and structural variability of the standard chromosome in long-term callus cultures and in regenerated organs. The callus with two B chromosomes displayed higher levels of polyploidyzation than callus without B chromosomes. The roots regenerated from both these calli were only diploid, while roots regenerated from transformed callus were also polyploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nucleotypic influences on chromosome-specific chiasma variation in Crepis capillaris. I. Responses to early colchicine treatment and chromosome doubling.

Additive and multiplicative effects of colchicine treatment at the seedling stage and of chromosome doubling on chromosome-specific chiasma frequency at metaphase I have been measured in comparisons between C0 and untreated diploids and between C0 autotetraploids and C0 diploids. Early colchicine treatment increases the frequency of chromosome C univalents to 1.8% but has no similar effect on chromosomes A and D. Colchicine treatment has little net effect on mean chiasma frequency, deducting an average of 0.204 chiasmata per set but otherwise multiplying the mean by a factor of 1.182. These additive and multiplicative effects represent averages of six phenotypes. Chromosome doubling in a tetraploid-diploid chimaera subtracts an average of 0.265 chiasmata per set but otherwise doubles the numbers of chiasmata at the diploid level (x 2.134). Comparison of six diploids and tetraploids reveals modest average additive (+ 1.103) and multiplicative effects (x 1.190). The implications of these findings are discussed in the light of new analyses of previously published data. Key words : chiasmata, Crepis, colchicine, meiosis, polyploidy.     

]Effect of kinetin on the formation of chromosome aberrations during irradiation of Crepis capillaris L. seeds and its radioprotective effect

The object of this investigation was the mutagenic effect of kinetin (6-phurphuryl-aminopurine) on chromosomes of Crepis capillaris L. Dry seeds were soaked in the 0.05% kinetin solution for 4, 6 and 8 hours before the irradiation with X-rays. It is observed that soaking of dry seeds in kinetin solution stimulates the appearance of chromatid aberrations for almost two hours earlier, than in the control (soaking seeds in water). A regular increase of the chromatid-type rearrangements with increasing the duration of soaking in the kinetin solution was observed (3.3%, 16-20% and almost 50% at 4 hours; 6 hours' and 8 hours' soaking respectively). This increase is indicative for the effect of kinetin on the processes taking place in the cell before the irradiation. Apparently kinetin stimulates the onset of the stage S for a certain proportion of cells, and thus at the moment of irradiation the cell population is at the asynchronous state. A considerable radioprotective effect of kinetin was observed in all the variants of the experiment, which is not associated with the capacity of stimulating the initiation of chromatid aberrations inherent in kinetin. Kinetin alone (without irradiation) exerts no mutagenic effect on the cells of Crepis capillaris.     

Effect of copper ions on chromosome structural mutations caused by Cs1 37 gamma rays in moist seed cells of Crepis capillaris L]

When seeds of Crepis capillaris were germinated in solutions of Cu(NO3)2 (at 0.55-10(-5) M and 1.1-10(-5) M concentrations) the frequency of structural chromosome mutations induced by gamma-rays (137Cs) at 200 and 400 r doses was higher as compared to that induced in the absence of copper ions.     

Relationship between sister-chromatid exchanges and heterochromatin or DNA replication in chromosomes of Crepis capillaris

The C-band patterns, DNA late replication patterns and distribution patterns of spontaneous and γ-ray-induced SCEs in Crepis capillaris chromosomes were studied. The fluorescence plus Giemsa (FPG) technique was used for detection of SCEs and late-replicating chromosome regions after unifilar incorporation of BrdU into DNA. An asynchronous replication of both euchromatic and heterochromatic chromosome regions was established. The frequency of SCEs is increased about 2-fold by 1.5 Gy γ-rays. The localization of the sites of SCEs was analyzed with special reference to eu- and heterochromatin and early- and late-replicating regions. The data obtained showed that SCEs were distributed nonrandomly along the chromosomes. Preferential occurrence of SCEs was observed in the following chromosome regions: at the junction between eu- and heterochromatic regions, the latter being rich in late-replicating DNA; at the junction between early- and late-replicating regions, the latter not being C-band positive. Certain heterochromatic regions were more rarely involved in SCEs than expected on the basis of their length. The lowest incidence of SCEs was found in the centromeric regions. Very similar distribution patterns of spontaneous and γ-ray-induced SCEs were observed. The possible role of the differences in the time of replication of the different chromosome regions in the formation of SCEs is discussed.     

Cytogenetics of protoplast cultures of Brachycome dichromosomatica and Crepis capillaris and regeneration of plants

Summary Callus derived protoplasts of Brachycome dichromosomatica (2n=2x=4) and Crepis capillaris (2n=2x=6) have been regenerated into karyologically normal plants, i.e. plants without visible alterations of the diploid chromosome set. However, metaphase analysis of protoplast cultures derived from both callus as well as mesophyll cells showed karyological changes in the overwhelming majority of cells in both species leading to multinucleated, polyploid and aneuploid cells. Furthermore, callus derived protoplasts sometimes exhibited changes at the chromosome level as indicated by translocations. The vast majority of aberrant karyotypes arose from failures during mitosis and cytokinesis, pointing to inadequate microtubules as a possible underlying cause. Karyological events of the kind described herein greatly affect the plating efficiency of isolated protoplasts and the viability of protoplast derived calli. Plant regeneration, although demonstrated in this study for the first time in both species, seems to be limited to rarely occurring, protoplast-derived colonies with a relatively stable genome. Our experiments, performed with chromosomal model species, emphasize the need for controlled, non-mutagenic culture conditions.