Multiple reconstruction of barley karyotype resulting in complete cytological marking of the chromosome complement

Summary A new reconstructed barley karyotype, PK88, which is a quadruple homozygote for three unequal translocations, 1–2, 3–4, 5–7, and one pericentric inversion in chromosome 6, was studied. As a result of these chromosome rearrangements, a complete cytological marking of the complement has been achieved. Due to the specific intra or interchromosomal transfer of particular bands, Giemsa staining of somatic chromosomes provided clear-cut indications about the localization of translocation and inversion breakpoints. It was established that the long arms of chromosomes 1, 2, 4, 5 and 7 and the short arm of chromosome 3 have been involved in interchanges 1–2, 3–4, and 5–7. The breakpoints of pericentric inversion proved to be located proximally to the short (satellite) arm and distally in the long arm of chromosome 6. PK-88 offers an essential gain in resolution power and extension of the areas of application in cytogenetics over other reconstructed karyotypes produced so far in barley.     

Localization of translocation breakpoints in somatic metaphase chromosomes of barley

Karyotype analyses based on staining by acetocarmine followed by Giemsa N-banding of somatic metaphase chromosomes of Hordeum vulgare L. were carried out on 61 reciprocal translocations induced by X-irradiation. By means of computer-based karyotype analyses all of the 122 breakpoints could be localized to defined sites or segments distributed over the seven barley chromosomes. The pre-definition of translocations with respect to their rearranged chromosome arms from other studies rendered it possible to define the break positions even in translocations having exchanged segments equal in size and the breakpoints located distally to any Giemsa band or other cytological marker. The breakpoints were found to be non-randomly spaced along the chromosomes and their arms. All breaks but one occurred in interband regions of the chromosomes, and none of the breaks was located directly within a centromere. However, short and long chromosome arms recombined at random. An improved tester set of translocations depicting the known break positions of most distal location is presented.     

Effects of chromosome reconstruction on deletion clustering in Hordeum vulgare

Summary The barley standard karyotype, two reconstructed karyotypes with all chromosomes interdistinguishable, and four translocation lines were treated with maleic hydrazide. A specific chromosomal site in satellite chromosome 7 (segment 44 adjacent to the nucleolus organizer region) of the standard karyotype was found to represent a deletion hot spot. A sample of specifically reconstructed karyotypes were used to check whether or not transposition of the hot spot region, or changes of its neighborhood, would affect its involvement in deletions. One of the seven karyotypes (translocation line T 505 with a pair of chromosomes having both nucleolus organizer regions and satellites in opposite arms) was without deletion clustering in segment 44. At the same time, a prominent Giemsa band close to the secondary constriction was absent from segment 44. These data show that the involvement in deletions of a certain chromosome segment is modifiable in certain cases by chromosome reconstruction. Similar observations have been made in Vicia faba.     

DNA methylation pattern in a barley reconstructed karyotype with deleted ribosomal gene cluster of chromosome 6H

A reconstructed barley karyotype (T-35) was utilised to study the influence of chromosomal rearrangements on the DNA methylation pattern at chromosome level. Data obtained were also compared with the distribution of Giemsa N-bands and high gene density regions along the individual chromosomes that have been previously described. In comparison to the control karyotype (T-1586), the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complement. Significant remodelling process of methylation pattern was found also in the residual nucleolus organiser regions (NOR) on chromosome 5H as a consequence of deletion comprising the whole NOR of chromosome 6H in T-35. Moreover, differences between corresponding segments of the homologues with respect to some other chromosome locations were also observed. Repositioning of genomic DNA methylation along the metaphase chromosomes following chromosomal reconstruction in barley seems to be essential to ensure correct chromatin organisation and function.     

Molecular Definition of Pericentric Inversion Breakpoints Occurring during the Evolution of Humans and Chimpanzees

High-resolution G-banding analysis has demonstrated remarkable morphological conservation of the chromosomes of the Hominidae family members (humans, chimpanzees, gorillas, and orangutans), with the most notable differences between the genomes appearing as changes in heterochromatin distribution and pericentric inversions. Pericentric inversions may have been important for the establishment of reproductive isolation and speciation of the hominoids as they diverged from a common ancestor. Here the previously published primate karyotype comparisons, coupled with the resources of the Human Genome Project, have been used to identify pericentric inversion breakpoints seen when comparing the human karyotype to that of chimpanzee. Yeast artificial chromosome (YAC) clones were used to detect, by fluorescencein situhybridization, five evolutionary pericentric inversion breakpoints present on the chimpanzee chromosome equivalents of human chromosomes 4, 9, and 12. In addition, two YACs from human 12p that detect a breakpoint in chimpanzee detect a similar rearrangement in gorilla.     

Spontaneous chromosomal rearrangements in cultivated and wild barleys

Summary Four of 1,240 cultivated barley lines collected from different regions of the world and 3 of 120 lines of wild barley, Hordeum spontaneum C. Koch, carry spontaneous reciprocal translocations. Break-point positions and rearrangements in the interchanged chromosomes have been examined by both test crosses and Giemsa banding techniques. The four translocation lines in cultivated barley were all of Ethiopian origin and have the same translocation involving chromosomes 2 and 4. The breakpoints are at the centromeres of both chromosomes, resulting in interchanged chromosomes 2S+4S and 2L+4L (S=short arm, L=long arm). A wild barley line, Spont.II, also has translocated chromosomes 2 and 4 which are broken at the centromeres. The resultant chromosomes are, however, 2S+4L and 2L+4S. Another wild barley line, Spont.S-4, has interchanged chromosomes with breakpoints in the short arm of chromosome 3 and the long arm of chromosome 7. In addition, this line has a paracentric inversion in the short arm of chromosome 7 that includes a part of nucleolar constriction, resulting in two tandemly arranged nucleolar constrictions. The third wild barley line, Spont.S-7, has interchanged chromosomes with breakpoints in the long arms of both chromosomes 3 and 6. The translocated chromosome 3 is metacentric and the translocated chromosome 6 has a long arm similar in length to the long arm of chromosome 7.     

Complex genome rearrangements reveal evolutionary dynamics of pericentromeric regions in the Triticeae.

Most pericentromeric regions of eukaryotic chromosomes are heterochromatic and are the most rapidly evolving regions of complex genomes. The closely related genomes within hexaploid wheat (Triticum aestivum L., 2n=6x=42, AABBDD), as well as in the related Triticeae taxa, share large conserved chromosome segments and provide a good model for the study of the evolution of pericentromeric regions. Here we report on the comparative analysis of pericentric inversions in the Triticeae, including Triticum aestivum, Aegilops speltoides, Ae. longissima, Ae. searsii, Hordeum vulgare, Secale cereale, and Agropyron elongatum. Previously, 4 pericentric inversions were identified in the hexaploid wheat cultivar 'Chinese Spring' ('CS') involving chromosomes 2B, 4A, 4B, and 5A. In the present study, 2 additional pericentric inversions were detected in chromosomes 3B and 6B of 'CS' wheat. Only the 3B inversion pre-existed in chromosome 3S, 3Sl, and 3Ss of Aegilops species of the Sitopsis section, the remaining inversions occurring after wheat polyploidization. The translocation T2BS/6BS previously reported in 'CS' was detected in the hexaploid variety 'Wichita' but not in other species of the Triticeae. It appears that the B genome is more prone to genome rearrangements than are the A and D genomes. Five different pericentric inversions were detected in rye chromosomes 3R and 4R, 4Sl of Ae. longissima, 4H of barley, and 6E of Ag. elongatum. This indicates that pericentric regions in the Triticeae, especially those of group 4 chromosomes, are undergoing rapid and recurrent rearrangements.     

Reconstruction of the female Gorilla gorilla karyotype using 25-color FISH and multicolor banding (MCB)

The origin of the human and great ape chromosomes has been studied by comparative chromosome banding analysis and, more recently, by fluorescence in situ hybridization (FISH), using human whole-chromosome painting probes. It is not always possible, however, to determine the exact breakpoints and distribution or orientation of specific DNA regions using these techniques. To overcome this problem, the recently developed multicolor banding (MCB) probe set for all human chromosomes was applied in the present study to reanalyze the chromosomes of Gorilla gorilla (GGO). While the results agree with those of most previous banding and FISH studies, the breakpoints for the pericentric inversion on GGO 3 were defined more precisely. Moreover, no paracentric inversion was found on GGO 14, and no pericentric inversions could be demonstrated on GGO 16 or 17.     

DNA methylation and chromosomal rearrangements in reconstructed karyotypes of Hordeum vulgare L.

Summary. One standard and two reconstructed barley karyotypes were used to study the influence of chromosomal rearrangements on the distribution pattern of DNA methylation detectable at the chromosome level. Data obtained were also compared with Giemsa N-bands and high gene density regions that had been previously described. The effect of chromosomal reconstruction in barley seems to be decidedly prominent in the repositioning of genomic DNA methylation along metaphase chromosomes. In comparison to the standard karyotype, the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complements not subjected to structural alterations. Moreover, differences may occur between corresponding regions of homologues. Some specific chromosomal bands, including the nucleolus-organizing regions, showed a relative constancy in the methylation pattern, but this was not the case when the two satellites were combined by translocation in chromosome 6H^5H of line T-30. Our results suggest that epigenetic changes like DNA methylation may play an important role in the overall genome reorganization following chromosome reconstruction. Correspondence: R. Cremonini, Dipartimento di Biologia, Università di Pisa, Via L. Ghini 5, 56126 Pisa, Italy.     

Electron microscope mapping of the pericentric and intercalary heterochromatic regions of the polytene chromosomes of the mutant Suppressor of underreplication in Drosophila melanogaster

Breaks and ectopic contacts in the heterochromatic regions of Drosophila melanogaster polytene chromosomes are the manifestations of the cytological effects of DNA underreplication. Their appearance makes these regions difficult to map. The Su(UR)ES gene, which controls the phenomenon, has been described recently. Mutation of this locus gives rise to new blocks of material in the pericentric heterochromatic regions and causes the disappearance of breaks and ectopic contacts in the intercalary heterochromatic regions, thereby making the banding pattern distinct and providing better opportunities for mapping of the heterochromatic regions in polytene chromosomes. Here, we present the results of an electron microscope study of the heterochromatic regions. In the wild-type salivary glands, the pericentric regions correspond to the beta-heterochromatin and do not show the banding pattern. The most conspicuous cytological effect of the Su(UR)ES mutation is the formation of a large banded chromosome fragment comprising at least 25 bands at the site where the 3L and 3R proximal arms connect. In the other pericentric regions, 20CF, 40BF and 41BC, 15, 12 and 9 new bands were revealed, respectively. A large block of densely packed material appears in the most proximal part of the fourth chromosome. An electron microscope analysis of 26 polytene chromosome regions showing the characteristic features of intercalary heterochromatin was also performed. Suppression of DNA underreplication in the mutant transforms the bands with weak spots into large single bands.     

Cytologically integrated physical restriction fragment length polymorphism maps for the barley genome based on translocation breakpoints

We have developed a new technique for the physical mapping of barley chromosomes using microdissected translocation chromosomes for PCR with sequence-tagged site primers derived from >300 genetically mapped RFLP probes. The positions of 240 translocation breakpoints were integrated as physical landmarks into linkage maps of the seven barley chromosomes. This strategy proved to be highly efficient in relating physical to genetic distances. A very heterogeneous distribution of recombination rates was found along individual chromosomes. Recombination is mainly confined to a few relatively small areas spaced by large segments in which recombination is severely suppressed. The regions of highest recombination frequency (相似文献   

inv(9)(p24q13) in three sterile brothers

Only nine non-polymorphic constitutional pericentric inversions of chromosome 9 have been described. We report on a familial inv(9)(p24q13) associated with sterility in three brothers. The mother's chromosomes were normal in blood lymphocytes (n=130); the father was already deceased and his karyotype unknown. However, the presence of any of the maternal chromosomes 9 (as assessed by C-banding) in her carrier children is inconsistent with the assumption of maternal mosaicism. Two single sisters were also carriers. The same rearranged chromosome 9 in the three sterile brothers can hardly be regarded as a fortuitous association, especially when the breakpoints are almost identical to those of the sole inversion previously found in an azoospermic male. If their father was a carrier, the observed sterility may be the result of 'chromosome anticipation', a phenomenon already invoked for certain familial chromosomal rearrangements.     

Effects of chromosome reconstruction of barley genome on tissue culture response using two different regeneration systems

Mature embryos and seedlings from mature embryos of one standard and five reconstructed karyotypes of barley (Hordeum vulgare L.) were cultured in vitro to study the influence of repositioning of particular chromosome segments of barley genome on the regeneration response. A comparative analysis of the regeneration response of a reconstructed karyotype having complete and well characterized rearrangement of the chromosome complement, and its four parental lines were used as experimental material. Depending on the source of explants two systems of in vitro culture were applied. The regeneration ability was found to be significantly influenced by both chromosome reconstruction and protocol applied. Possible reasons underlying the effects of chromosomal reconstruction on the regeneration response of karyotypes are briefly discussed.     

Karyology and karyotype analysis of diploid freshwater planarian populations of the Dugesia gonocephala group (Platyhelminthes,Tricladida) found in Sardinia

Karyology and karyotype analysis were carried out on freshwater planarian populations of the Dugesia gonocephala group. The strains studied were all diploid with chromosomic number 2n = 16; n = 8. They came from 12 sites mainly localized on the west of the island of Sardinia. Three karyotypes indicated with the letters A, B and C were found in which eight homomorphic pairs of chromosomes were easily identified. In karyotype A all chromosomes are metacentric. Ten populations of the twelve examined showed this karyotype which appears to be the most common. In karyotype B the seventh pair of chromosomes is submetacentric. This karyotype is quite common having been previously found in another eight Sardinian localities. Karyotype C differs from the others in having submetacentric third and seventh pairs of the chromosome complement. It was found in only one locality. The differences observed between these three karyotypes could be interpreted either as sign of differentiation at species level, or as an intraspecific variation due to chromosome mutations (pericentric inversions). This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic characterization of a reciprocal translocation present in a widely grown barley variety

Artificially induced translocation stocks have been used to physically map the barley genome; however, natural translocations are extremely uncommon in cultivated genotypes. Albacete is a barley variety widely grown in recent decades in Spain and carrying a reciprocal translocation which obviously does not affect its agronomical fitness. This translocation has been characterized by a combination of cytological and molecular genetic approaches. Firstly, recombination frequencies between markers on chromosomes 1H and 3H were estimated to determine the boundaries of the reciprocal interchange. Secondly, 1H-3H wheat barley telosome addition lines were used to assign selected markers to chromosome arms. Thirdly, fluorescence in situ hybridization (FISH) with rDNA probes (5S and 18S-5.8S-26S) and microsatellite probes [(ACT)(5), (AAG)(5) and (CAG)(5)] was used to determine the locations of the translocation breakpoints more precisely. Fourthly, fine-mapping of the regions around the translocation breakpoints was used to increase the marker density for comparative genomics. The results obtained in this study indicate that the translocation is quite large with breakpoints located on the long arms of chromosomes 1H and 3H, between the pericentromeric (AAG)(5) bands and above the (ACT)(5) interstitial distal bands, resulting in the reciprocal translocation 1HS.1HL-3HL and 3HS.3HL-1HL. The gene content around the translocation breakpoints could be inferred from syntenic relationships observed among different species from the grass family Poaceae (rice, Sorghum and Brachypodium) and was estimated at approximately 1,100 and 710 gene models for 1H and 3H, respectively. Duplicated segments between chromosomes Os01 and Os05 in rice derived from ancestral duplications within the grass family overlap with the translocation breakpoints on chromosomes 1H and 3H in the barley variety Albacete.     

Effect of Environmental Pollution on the Chromosomal Variability of Chironomus Riparius

Different frequencies of chromosomal alterations in salivary gland polytene chromosomes AB, CD and EF were described in larvae of Chironomus riparius (syn. Chironomus thummi) from the trace metal-polluted station of Santena on the river Banna, near Turin, and from the unpolluted station of Corio (40 Km from Turin) which was taken as a reference area. In a sample of 56 larvae from Santena, no specimen with the standard karyotype in all cells of the salivary glands was found. Different types of aberrations were found: 33 paracentric and five pericentric inversions, three deficiencies, four amplified sections and one chromatid break. Fifteen out of the 38 inversions and two amplified sections appeared to be inherited, while all the other aberrations were somatic. Most of the aberrations' breakpoints were located on both sides of the centromere regions, where constitutive heterochromatin is present. Also functional alterations were observed (mainly telomere and centromere decondensations and nine novel puffs). In a sample of 49 larvae of a population from the well-preserved area of Corio only six somatic and one inherited paracentric inversions were found. These results suggest that the strong destabilization of the genomes of C. riparius larvae from Santena could be a reaction to the activity of the toxic substances present in the polluted sediments of the river Banna. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evolution versus constitution: differences in chromosomal inversion

We compared the chromosomal breakpoints of evolutionary conserved and constitutional inversions. Multicolor banding and human-specific bacterial artificial chromosomes were applied to map the breakpoints of constitutional pericentric inversions on human chromosomes 2 and 9. For the first time, we present a high-resolution analysis of the breakpoint regions, which are characterized by gene destitution, co-localization with fragile sites, multitude repeats as well as pseudogenes and, remarkably, a large sequence homology to the opposite breakpoint. In contrast, evolutionary inversion breakpoints lack such extensive cross-hybridizing regions and are often associated with fragile sites of the genome and low-copy repeats. These molecular characteristics gave evidence for different types of inversion formation and indicate that evolutionary inversions cannot originate from constitutional inversions like those of chromosomes 2 and 9. Finally, the constitutional inversion breakpoints were investigated on three different great ape species and on four test persons each bearing the same cytogenetically determined inversion on chromosomes 2 and 9, respectively. Our data indicate the existence of different molecular breakpoints for the two variant chromosomes.     

Karyological study of the dolly vardenSalvelinus malma from Alaska

The karyotype of the dolly vardenSalvelinus malma from Alaska was analysed. A pair of huge acrocentrics, which could have resulted from either tandem fusion or centric fusion followed by pericentric inversion, was observed. Ag-NORs were observed at the terminal regions of the second largest chromosome pair, and individual size-differences in Ag-NORs and in chromosomes with Ag-bands were found. From a comparison of the karyotypes of the dolly varden from Alaska and Hokkaido (Japan), karyotype differentiation by pericentric inversion and translocation was discussed.