Characterization of different types of condensed chromatin inCostus (Zingiberaceae)

The chromatin structure of six diploids species ofCostus was analysed using conventional Giemsa staining, C-banding and DAPI/CMA fluorochromes. The interphase nuclei in all the species show an areticulate structure and the prophase chromosomes show large blocks of proximal condensed chromatin. After banding procedures, each chromosome exhibits only centromeric dot-like DAPI⁺/CMA^– C-bands whereas the satellites (one pair at each karyotype) are weakly stained after C-banding and show a DAPI^–/CMA⁺ fluorescence. Two chromocentres show bright fluorescence with CMA and weak staining after C-banding whereas the others chromocentres show only a small fraction of DAPI⁺ heterochromatin. These results were interpreted to mean that the greater part of the condensed chromatin has an euchromatic nature whereas two types of well localized heterochromatin occur in a small proportion. The Z-stage analysis suggests that heterochromatin and condensed euchromatin decondense at different times. The chromosome number and morphology of all species are given and the implications of the condensed euchromatin are discussed.Dedicated to Prof.Elisabeth Tschermak-Woess on the occasion of her 70th birthday.     

Karyological features and banding patterns in Arachis species belonging to the Heteranthae section

The section Heteranthae of Arachis is endemic to Brazil, occurring mainly in the semi-arid northeastern region. The section is considered derived within the genus and includes only annual herbs. Most previous cytological evaluations were restricted to chromosome numbers and morphology. The present approach comprised karyomorphological evaluation in 10 accessions from five species of this section, including standard staining and fluorochrome banding [chromomycin A3 (CMA)/4′,6-diamidino-2-phenylindole (DAPI)]. All accessions presented diploid chromosome numbers (2n = 20) with a prevalence of metacentric to submetacentric chromosome morphology. Arachis dardani, Arachis pusilla, and Arachis interrupta presented karyotypic formula 18m + 4sm and satellite type 2, while Arachis sylvestris and Arachis giacomettii presented 16m + 4sm and satellite type 10. Despite the conserved morphological features, higher diversity was detected in terms of size and number of GC-rich (CMA⁺) heterochromatic blocks among the species; however, all of them were located in the pericentromeric regions. The species A. pusilla presented the highest number of GC-rich blocks, present in all chromosomes of the complement. Based on the data obtained and considering literature data, we suggest that A. dardani and A. interrupta occupy a basal position in the group due to their moderate asymmetry and satellite type. At least in A. pusilla, the constitutive heterochromatin seems to have suffered recent modifications of its constitution, in contrast to other species that present pericentromeric CMA⁺ blocks in all chromosomes. A. giacomettii and A. sylvestris are closely related to each other and also similar to the previously studied Arachis seridoensis, revealing two clear-cut subgroups within the section from the karyological point of view.     

The meaning of DAPI bands observed after C-banding and FISH procedures

Abstract

Under specific technical conditions chromosome staining with 4′,6-diamidino-2-phenylindole (DAPI) permits characterization of heterochromatic regions as AT-rich (DAPI⁺) or AT-poor (DAPI^?), especially when the chromosomes are counterstained with chromomycin A₃ (CMA), which preferentially binds to GC-rich DNA. DAPI⁺ bands also often have been observed after C-banding or FISH. In these cases, however, it is not clear whether only AT-rich regions stain positively with DAPI or other heterochromatins with different base compositions also are stained. We evaluated the meaning of DAPI bands observed after C-banding and FISH using three plant species bearing different types of heterochromatin: DAPI⁺/CMA^?, DAP^?/CMA⁺ and DAPI⁰/CMA⁰ (neutral bands). Additional tests were performed using propidium iodide, a fluorochrome without preferential affinity for AT or GC. Our results indicate that AT-rich heterochromatin stains as DAPI⁺ bands after C-banding or FISH, but other kinds of heterochromatin also may be stained by DAPI.     

Heterosynapsis and suppression of chiasmata within heterozygous pericentric inversions of the Sitka deer mouse

The patterns of chromosomal pairing and chiasma distribution were analyzed in male Sitka deer mice (Peromyscus sitkensis) polymorphic for terminally positioned pericentric inversions of chromosomes 6 and 7. Gand C-banding of somatic metaphases indicated that the inversions involved 30% and 40% of chromosomes 6 and 7, respectively. Analysis of silver-stained synaptonemal complexes in surface-spread zygotene and pachytene nuclei from heterozygous individuals revealed that inversion loops were not formed. The inverted segments proceeded directly to heterosynapsis without an intervening homosynaptic phase, and the heteromorphic bivalents remained straight-paired throughout pachynema. C-banded pachytene nuclei corroborated the occurrence of heterosynapsis, as the heteromorphic bivalents exhibited nonaligned centromeres. Analysis of diplonema and diakinesis indicated that crossing over had not occurred within the heterosynapsed inverted segments. The observation of chiasma suppression within the inversions indicates that pericentric inversion heterozygosity does not lead to the production of unbalanced gametes. Heterosynapsis of the inverted segments during zygonema and pachynema and the resulting chiasma suppression therefore represent a meiotic mechanism for the maintenance of pericentric inversion polymorphisms in this population of P. sitkensis.     

Evidence for a role of the synaptonemal complex in provision for normal chromosome disjunction at meiosis II in maize

The meiotic behavior of heterozygotes from three different maize pericentric inversion stocks was quantitatively observed at a variety of stages throughout meiosis I and II. With heterozygosity for either of two of these inversions, the usual mode of pairing observed at pachytene involved synapsis of the centromere containing inverted region, and synaptic failure of the centromere region was rarely found. Abnormal chromosome behavior at subsequent meiotic stages was rare in these cases. With heterozygosity for the third inversion, however, homologous synapsis was generally found in the distal regions of the chromosome involved, the inverted region was often non-homologously synapsed, and a substantial frequency of cells apparently showed synaptic failure in the centromere containing inverted region. A substantial frequency of cells at anaphase II in this case contained two lagging monads in the plate region of the spindle. Where cells could be identified as sisters, sister cells showed identical behavior at anaphase II. Findings seem to be most simply explained by the supposition that pachytene synapsis of the centromere region is important to provision for sister centromere association until anaphase II.     

Karyotype analysis for diploid and polyploid species of the Solanum L.

Solanum species were cytogenetically analysed to localize species-specific markers. Solanum atropurpureum Schrank, S. dulcamara L., S. gilo Raddi., S. melongena L., S. nitidibaccatum Bitter and S. paniculatum L. showed 2n = 24, whereas S. luteum Mill., S. nigrum L. and S. laciniatum Ait. showed 2n = 48, 2n = 72 and 2n = 92, respectively. All species demonstrated a symmetrical karyotype. The average chromosome size varied between diploid (1.95 μm) and polyploid (1.31 μm) species. Application of the CMA₃/DAPI technique showed two CMA₃ ⁺/DAPI^? terminal blocks in S. dulcamara, S. atropurpureum and S. luteum, indicating one homologous pair. In S. nitidibaccatum two large CMA₃ ⁺/DAPI^? segments were observed apart from several terminal blocks in almost all chromosomes. The same CMA₃ ⁺ telomeric standard was also found in prometaphases of S. luteum. Similar results were obtained with FISH with 45S rDNA probes: fluorochrome CMA₃ ⁺ telomeric blocks associated with satellites, with two blocks in S. atropurpureum, S. dulcamara, S. nitidibaccatum and S. luteum and four blocks in S. nigrum and S. lacinatum. Localization of species-specific markers was successful, allowing recognition of particular cytological features in most of the species analysed.     

Sequential meiotic prophase development in the pubertal Indian pygmy field mouse: synaptic progression of the XY chromosomes, autosomal heterochromatin, and pericentric inversions.

Sequential meiotic prophase development has been followed in the pubertal male pygmy mouse Mus terricolor, with the objective to identify early meiotic prophase stages. The pygmy mouse differs from the common mouse by having large heterochromatic blocks in the X and Y chromosomes. These mice also show various chromosomal mutations; for example, fixed variations of autosomal short arms heterochromatin among different chromosomal species and pericentric inversion polymorphism. Identification of prophase stages was crucial to analyzing effects of heterozygosity for these chromosomal changes on the process of homologous synapsis. Here we describe identification of the prophase stages in M. terricolor, especially the pachytene substages, on the basis of morphology of the XY bivalent. Based on this substaging, we show delayed pairing of the heterochromatic short arms, which may be the reason for their lack of chiasmata. The identification of precise pachytene substages also reveals an early occurrence of "synaptic adjustment" in the pericentric inversion heterobivalents, a mechanism that would prevent chiasma formation in the inverted segment and thereby would abate adverse effects of such heterozygosity. The identification of pachytene substages would serve as the basis to analyze the nature of synaptic anomalies met in M. terricolor hybrids (which will be the basis of a subsequent paper).     

Classical and molecular cytogenetics and DNA content in Maihuenia and Pereskia (Cactaceae)

We studied cacti species of the subfamilies Pereskioideae (five species of the southern clade) and both species of Maihuenioideae using molecular cytogenetic techniques and DNA content. Mitotic chromosomes were analyzed for Pereskia aculeata, P. bahiensis, P. grandifolia, P. nemorosa, P. sacharosa, Maihuenia poeppigii, and M. patagonica, using the Feulgen stain, CMA/DAPI fluorescent chromosome banding, fluorescence in situ hybridization (FISH, probes of 5S rDNA and pTa71 for 18-5.8-26S rDNA), and DNA content by flow cytometry technique. The karyotypes were highly symmetrical, most of the pairs being metacentric (m). CMA/DAPI banding revealed the presence of CMA⁺/DAPI^? bands associated with NORs in the first m pair of all species. The co-localization of 18-5.8-26S rDNA loci with CMA⁺/DAPI^?/NORs blocks allowed the identification of homeologous chromosome pairs between species of both subfamilies. FISH using probe 5S rDNA was applied for the first time in both subfamilies. Diploid species had always one m pair carrying 5S rDNA genes, with pericentromeric location in different chromosome pairs. In the tetraploid cytotype of M. patagonica, the 5S rDNA probe hybridized to two pairs. The 2C DNA content obtained by FC varied twofold (from 1.85 to 2.52 pg), with significant differences between species. Mean chromosome length, karyotype formula, percentage of heterochromatin position of 5S rDNA locus, and nuclear Cx DNA content vary among Maihuenia and Pereskia species and allowed to differentiate them. Both genera are closely related and that the differences found are not strong enough to separate Maihuenioideae from Pereskioideae.     

Heterochromatin and rDNA patterns in Solanum species of the Morelloid and Dulcamaroid clades (Solanaceae)

The heterochromatin distribution and the position of 18-5.8-26S, and 5S rDNA loci were determined in 13 species of Solanum of the Morelloid and Dulcamaroid clades. The CMA/DAPI staining and FISH were employed. Two types of constitutive heterochromatin were determined: CMA⁺/DAPI^? associated to NOR and CMA⁺/DAPI^? distributed as terminal bands. In the Morelloid clade, CMA⁺/DAPI^? bands were found in five species while in the Dulcamaroid clade, only S. angustifidum presented this feature. In the Morelloid clade, two to four 18-5.8-26S rDNA loci occupied terminal positions and two rDNA 5S loci were found with variable positions (terminal, intercalary, and centromeric). In the Dulcamaroid clade, two terminal 18-5.8-26S rDNA loci were detected with the exception of S. salicifolium which possessed four such loci and two to four 5S rDNA loci. Solanum crispum is the only species possessing the 5S in synteny with 18-5.8-26S rDNA loci. Karyotype features chromosome banding pattern as well as the location of ribosomal genes which varied among the species, reflecting the chromosome differentiation and evolutionary divergence. The findings obtained contributed to the development of tools that can be used for establishing chromosomic homeologies among species and hence to clarify their taxonomic relationships.     

All paired up with no place to go: pairing, synapsis, and DSB formation in a balancer heterozygote

The multiply inverted X chromosome balancer FM7 strongly suppresses, or eliminates, the occurrence of crossing over when heterozygous with a normal sequence homolog. We have utilized the LacI-GFP: lacO system to visualize the effects of FM7 on meiotic pairing, synapsis, and double-strand break formation in Drosophila oocytes. Surprisingly, the analysis of meiotic pairing and synapsis for three lacO reporter couplets in FM7/X heterozygotes revealed they are paired and synapsed during zygotene/pachytene in 70%–80% of oocytes. Moreover, the regions defined by these lacO couplets undergo double-strand break formation at normal frequency. Thus, even complex aberration heterozygotes usually allow high frequencies of meiotic pairing, synapsis, and double-strand break formation in Drosophila oocytes. However, the frequencies of failed pairing and synapsis were still 1.5- to 2-fold higher than were observed for corresponding regions in oocytes with two normal sequence X chromosomes, and this effect was greatest near a breakpoint. We propose that heterozygosity for breakpoints creates a local alteration in synaptonemal complex structure that is propagated across long regions of the bivalent in a fashion analogous to chiasma interference, which also acts to suppress crossing over.     

The centric region of the X chromosome rDNA functions in male meiotic pairing in Drosophila melanogaster

In Drosophila melanogaster males, sex chromosome pairing at meiosis is ensured by so-called pairing site(s) located discretely in the centric heterochromatin. The property of the pairing sites is not well understood. Recently, an hypothesis has been proposed that 240 bp repeats in the nontranscribed spacer region of rDNA function as the pairing sites in male meiosis. However, considerable cytogenetic evidence exists that is contrary to this hypothesis. Hence, the question is whether the chromosomal rDNA clusters, in which a high copy number of 240 bp repeats exists, are involved in the pairing. In order to resolve the problem we X-rayed Drosophila carrying the X chromosome inversion In(1)sc ^V2L sc ^8R and generated free, mini-X chromosomes carrying a substantial amount of rDNA. We defined cytogenetically the size of the mini-chromosomes and studied their meiotic behavior. Our results demonstrate that the heterochromatin at the distal end of the inversion, whose length is approximately 0.4 times that of the fourth chromosome, includes a meiotic pairing site in the male. We discuss the cytological location of the pairing site and the possible role of rDNA in meiotic pairing.     

The ultrastructural meiotic phenotype of the radiation sensitive mutant rad 6-1 in yeast

An ultrastructural analysis of three yeast rad 6-1/rad 6-1 diploids on sporulation medium for 0, 6, 10, and 24 h shows that arrest occurs at meiotic prophase. Two strains, CL 139 and PU 6, fail to complete chromosome synapsis based on the continued presence of single chromosomal cores in arrested nuclei. A clone derived from CL 139, however, showed complete pairing as evident from the presence of 17 synaptonemal complexes. All three strains underwent spindle pole body duplication but the poles failed to form a proper metaphase I spindle. A revertant Rad 6⁺ isolated from CL 139 showed normal chromosome behaviour and normal kinetic functions. It is concluded that the absence of meiotic recombination in some Rad 6^– strains may result from asynapsis, but that in other strains (e.g., CL 139s) recombination fails in spite of complete synapsis. In all cases the lack of sporulation is adequately explained by failure of the kinetic apparatus to form a metaphase I spindle.     

Synapsis of a chromosomal pair heterozygous for a pericentric inversion and the presence of a heterochromatic short arm

Analysis of meiotic pairing configurations in a deer mouse heterozygous for both a pericentric inversion and the presence of a heterochromatic short arm at chromosome 15 revealed straight-paired synaptonemal complexes with equal axial lengths in a majority of the pachytene nuclei. Nonhomologous pairing in this bivalent occurs by direct heterosynapsis of the inverted segments followed by synaptic adjustment of the heterochromatin heteromorphism.     

Molecular analysis of recombination in a family with Duchenne muscular dystrophy and a large pericentric X chromosome inversion.

It has been demonstrated in animal studies that, in animals heterozygous for pericentric chromosomal inversions, loop formation is greatly reduced during meiosis. This results in absence of recombination within the inverted segment, with recombination seen only outside the inversion. A recent study in yeast has shown that telomeres, rather than centromeres, lead in chromosome movement just prior to meiosis and may be involved in promoting recombination. We studied by cytogenetic analysis and DNA polymorphisms the nature of meiotic recombination in a three-generation family with a large pericentric X chromosome inversion, inv(X)(p21.1q26), in which Duchenne muscular dystrophy (DMD) was cosegregating with the inversion. On DNA analysis there was no evidence of meiotic recombination between the inverted and normal X chromosomes in the inverted segment. Recombination was seen at the telomeric regions, Xp22 and Xq27-28. No deletion or point mutation was found on analysis of the DMD gene. On the basis of the FISH results, we believe that the X inversion is the mutation responsible for DMD in this family. Our results indicate that (1) pericentric X chromosome inversions result in reduction of recombination between the normal and inverted X chromosomes; (2) meiotic X chromosome pairing in these individuals is likely initiated at the telomeres; and (3) in this family DMD is caused by the pericentric inversion.     

Cytological heterozygosity and the hybrid origin of sweet orange [Citrus sinensis (L.) Osbeck]

Citrus sinensis chromosomes, although small in size, present a remarkable differentiation of bands with the fluorochromes CMA and DAPI. These bands suggest that some heteromorphisms are fixed in this species. To investigate the extension of these heteromorphisms, ten cultivars of C. sinenesis were analysed with CMA/DAPI staining and, in some of them, the 18S–5.8S–25S rRNA and 5S rRNA genes were located by in situ hybridization. CMA/DAPI staining showed exactly the same CMA⁺/DAPI⁻ banding pattern for all cultivars. In situ hybridization revealed three 18S–5.8S–25S rRNA gene sites, two proximally located on two similar chromosomes and one terminally located on a third non-related chromosome. Two 5S rRNA gene sites were observed in this species, with one located proximal to the telomeric 18S–5.8S–25S rDNA site. Both cytological approaches revealed an invariable, heterozygotic karyotype among sweet orange cultivars. Based on these data, the putative hybrid origin of the species is discussed. Received: 9 April 1999 / Accepted: 22 June 1999     

美味猕猴桃原生质体再生植株细胞遗传学研究 Ⅲ .母株减数分裂前期Ⅰ染色体配对的光镜观察

首次报道在光镜下观察美味猕猴桃 (品种 :No.2 6原生质体植株的母株 )花粉母细胞( PMC)染色体在减数分裂前期的配对 ,发现其配对和凝缩有明显不同步性。不同细胞间染色体配对形式变化较大 ,一般以二价联会为主 ,其次由其它多种配对方式 (包括有复合配对、重复配对、着丝点或端粒处联合和多价联会 )形成多价体 ,还有少数未配对或发生内配对 (偶见 )的单价体和几条二价体之间的次级配对。粗线期观察到少数染色体有缺失 (或重复 )、倒位、易位和疏松配对等结构性改变。表明该植株是一个复杂的区段异源六位体 ,少数染色体在结构上累积有变异。还认为该植株是研究减数分裂染色体配对和联会机制的好材料。     

Karyomorphology and GC-rich heterochromatin pattern in Passiflora (Passifloraceae) wild species from Decaloba and Passiflora subgenera

Thirteen wild species of Passiflora were analyzed using conventional and CMA/DA/DAPI staining to evaluate the karyotype diversity between and within the subgenus Decaloba and Passiflora. The karyotypic features indicate that both subgenera have a conserved chromosome number, as reported before for several species. Submetacentric (sm) chromosomes were found in species from both subgenera, suggesting that sm chromosomes are not restricted to a particular subgenus. The analysis of the karyotypic heterogeneity enabled to distribute the species in three groups, but with no support to phylogenetic and taxonomic levels. The application of fluorochromes allowed for the visualization of CMA⁺/DAPI⁻ blocks, which in our studies always correlated with the occurrence of satellites, showing that occurrence of two chromosome pairs with satellites per cell is a characteristic shared by some species from both subgenera. This feature does not always have relationship with the basic chromosome number. The data found in this study will help to understand the phylogeny, cytotaxonomy, and evolution of the genus Passiflora showing that karyotypic variation can be seen between and within the subgenus Decaloba and Passiflora.     

Chromosome mapping of ribosomal genes and histone H4 in the genus Radacridium (Romaleidae)

In this study, two species of Romaleidae grasshoppers, Radacridium mariajoseae and R.nordestinum, were analyzed after CMA₃/DA/DAPI sequential staining and fluorescence in situ hybridization (FISH) to determine the location of the 18S and 5S rDNA and histone H4 genes. Both species presented karyotypes composed of 2n = 23, X0 with exclusively acrocentric chromosomes. CMA₃⁺ blocks were detected after CMA₃/DA/DAPI staining in only one medium size autosome bivalent and in the X chromosome in R. mariajoseae. On the other hand, all chromosomes, except the L1 bivalent, of R. nordestinum presented CMA₃⁺ blocks. FISH analysis showed that the 18S genes are restricted to the X chromosome in R. mariajoseae, whereas these genes were located in the L₂, S₉ and S₁₀ autosomes in R. nordestinum. In R. mariajoseae, the 5S rDNA sites were localized in the in L₁ and L₂ bivalents and in the X chromosome. In R. nordestinum, the 5S genes were located in the L₂, L₃, M₄ and M₅ pairs. In both species the histone H4 genes were present in a medium size bivalent. Together, these data evidence a great variability of chromosome markers and show that the 18S and 5S ribosomal genes are dispersed in the Radacridium genome without a significant correlation.     

Karyology and cytotaxonomy of the genus Passiflora L. (Passifloraceae)

 The chromosomes of 31 species of Passiflora, distributed throughout the subgenera Astrophea, Calopathanthus, Distephana, Dysosmia, Passiflora, Plectostemma and Tacsonia were analysed. Three different karyotypes were observed: 2n = 12, 24, 36; 2n = 18, 72 and 2n = 20. The karyotype of these species was almost always constituted of metacentric and submetacentric chromosomes with variable karyotype symmetry. In the group with x = 6, represented by the subgenus Plectostemma, six diploid species with 2n = 12, one tetraploid with 2n = 24 (P. suberosa) and an intraspecific polyploid with 2n = 12, 36 (P. misera) were analysed. P. pentagona (subgenus Astrophea) may also be included in this karyological group since it presents 2n = 24 and may be of polyploid origin, with x = 6. The interphase nuclei in this group were areticulate, except those of P. morifolia and P. pentagona with semi-reticulate characteristics. Two small terminal heterochromatic blocks, positive for chromomycin A₃, were identified in the largest chromosome pair of P. capsularis and P. rubra, species very closely related, while P. tricuspis displayed four chromosomes with proximal blocks. In the group with x = 9, represented mainly by subgenus Passiflora, 20 species with 2n = 18 and one with 2n = 72 were studied. They presented chromosomes larger than those species with x = 6 and interphase nuclei of semi-reticulate type, except for P. mixta with areticulate nuclei. Four terminal CMA⁺ blocks were observed in P. edulis, six blocks in P. caerulea and P. racemosa, while five blocks were observed in the single P. amethystina plant analysed. P. foetida (subgenus Dysosmia), the only species with 2n = 20, exhibited six chromosomes with CMA⁺ blocks and interphase nuclei of the areticulate type. The meiotic analysis of representatives of the three groups (P. foetida, P. suberosa, P. cincinnata and P. racemosa) always presented regular pairing and regular chromosome segregation, except in P. jilekii where a tetravalent was observed. The analysis of the chromosome variation within the genus and the family suggests that the base number of Passiflora may be x₁ = 6 or x₁ = 12, whereas x₂ = 9 is only an important secondary base number. Received April 11, 2000 Accepted October 5, 2000     

Meiosis in haploid barley — An interpretation of non-homologous chromosome associations

Detailed meiotic studies were conducted on ten haploid plants representing six different genotypes of barley (Hordeum vulgare, 2n=14). At pachytene stages the non-homologous chromosomes were observed to pair as intimately as homologous chromosomes in many cells. Foldback pairing, involving single chromosomes, and multivalent associations were common. At diplotene, up to 4 chiasmatalike structures were observed in paired chromosomes but it is not likely that they resulted from crossing over. At diakinesis the bivalent frequency mean was from 1 to 1.3 per cell whereas by metaphase I the paired associations were rare with a single rod bivalent being observed in 3 to 5% of the cells. The frequencies of various types of secondary associations at metaphase were also recorded. — The origin and significance of bivalents and secondary associations in haploids is reviewed and discussed. Caution is urged in the interpretation that low levels of chromosome pairing in haploids is evidence of homology. It is concluded that very little chromosome duplication is likely to be found within the haploid set of barley chromosomes and that the basic chromosome number is seven.