Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

Summary Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.     

The karyotype ofFestucopsis serpentini (Poaceae Triticeae) from Albania studied by banding techniques and in situ hybridization

The karyotypes of two populations ofFestucopsis serpentini (2n = 2x = 14) endemic to Albania were investigated in detail by Giemsa C- and N-banding, AgNO₃ staining, and in situ hybridization with an rDNA probe. The complements consisted of 14 large chromosomes, 10 metacentric and 4 SAT-chromosomes, a metacentric and a submetacentric pair. SAT-chromosomes from one population carried exclusively minute satellites, whereas SAT-chromosomes from another population also carried larger polymorphic satellites, suggesting a geographical differentiation. The existence of four chromosomes with nucleolus forming activity was established through AgNO₃ staining; however, the rDNA probe additionally hybridized to intercalary positions in the short arms of two metacentric chromosomes revealing two inactive rDNA sites. C-banding patterns comprised from zero and up to four very small to larger, generally telomeric bands per chromosome giving low levels of constitutive heterochromatin. Similarities in chromosome morphology and C-banding patterns identified the homologous relationships of all chromosomes in one population, but of three pairs only in the other. Reliable identification of homologous chromosomes between plants was only possible for the SAT-chromosomes. A comparison between the C-banded karyotypes ofF. serpentini andPeridictyon sanctum supports their position in two genera.     

Inter- and intraplant C-banding polymorphism in one population ofAegilops comosa subsp.comosa var.comosa (Poaceae)

The Giemsa C-banding pattern of the chromosomes of the native self-pollinatedAegilops comosa subsp.comosa var.comosa was studied. Six of the seven chromosomes of the haploid genome were found to be polymorphic for C-banding patterns. Chromosome A had four variants, chromosome E three variants and each of the chromosomes B, D, and F two variants. Chromosomes E and G were polymorphic for arm length and arm ratio.This paper is part of the doctoral dissertation ofA. Georgiou.     

C-band distribution,DNA content and base composition inAdoxa moschatellina (Adoxaceae), a plant with cold-sensitive chromosome segments

The chromosomes ofAdoxa moschatellina (2n = 36, paleo-4x) contain mostly terminal, occasionally intercalary, negatively heteropycnotic cold-induced regions which correspond to all major C-bands including the satellites, as revealed by sequential analysis. Positively C-stained are also centromeres, the dotlike arms of the 7 telocentric chromosome pairs, and some very narrow intercalary bands; their cold-sensitivity is hardly traceable. There exists a fraction of condensed interphase chromatin, at least after chilling, which is virtually not C-banded (possibly condensed euchromatin).The DNA amount is 14.3 pg (1 C). The heterochromatin content is 13.0%. The thermal melting profile (Tm corresponding to 38.6% GC) does not reveal a particular AT- or GC-rich fraction. Significantly, the heterochromatin respond to the Hy-banding procedure is neutral.The distribution of cold-sensitive regions in plants was analysed with the arm-frame method: Intercalary positions, clearly, are not especially favoured regions. The obvious deficiency at centromeric positions may depend on the action of natural selection against mechanically labile centromeric regions.Dedicated to Univ.-Prof. Dr.Lothar Geitler on the occasion of his 80th birthday.     

Comparison of the Giemsa C-banded and N-banded karyotypes of twoElymus species,E. dentatus andE. glaucescens (Poaceae: Triticeae)

The karyotypes ofElymus dentatus from Kashmir andE. glaucescens from Tierra del Fuego, both carrying genomesS andH, were investigated by C- and N-banding. Both taxa had 2n = 4x = 28. The karyotype ofE. dentatus was symmetrical with large chromosomes. It had 18 metacentric, four submetacentric and six satellited chromosomes. The karyotype ofE. glaucescens resembled that ofE. dentatus, but a satellited chromosome pair was replaced by a morphologically similar, non-satellited pair. The C-banding patterns of both species had from one to five conspicuous and a few inconspicuous bands per chromosome. N-banding differentiated the chromosomes of the constituent genomes by producing bands in theH genome only. TheS genomes of both species were similar with five metacentric and two satellited chromosomes having most conspicuous C-bands at telomeric and distal positions. They resembled theS genome of the genusPseudoroegneria. TheH genomes had four similar metacentric and two submetacentric chromosomes. The seventhH genome chromosome ofE. dentatus was satellited, that ofE. glaucescens nonsatellited, but otherwise morphologically similar. The C-bands were distributed at no preferential positions. TheH genome ofE. dentatus resembles theH genomes of some diploidHordeum taxa.     

Systematics and karyoevolution inMagnoliidae:Tetrameranthus as compared with otherAnnonaceae genera of the same chromosome number

First generic chromosome counts reveal the base number x=7 for the generaTetrameranthus andRollinia. T. umbellatus from the Peruvian Amazon is diploid (2n=14),T. duckei from Brazil (Manaus) is tetraploid (2n=28). In the NeotropicsRollinia (7 species counted) has developed diploid to octoploid taxa (2n=14, 28, 42, 56). Counts of 7 South AmericanAnnona species are presented for comparison (2n=14, 28). The West AfricanCleistopholis patens has 2n=14. The Asian genusMezettia: 2n=14 and the neotropicalGuatteria tribe: 2n=28 are also revised. A detailed karyomorphological comparison, including karyotypes, banding patterns, condensing behaviour of chromosomes and structure of interphase nuclei reveals that the closely related generaAnnona andRollinia are almost identical in their diploid genomes, whereas the polyploid ones differ in their heterochromatin (=hc) composition and number of NO-chromosomes.Cleistopholis, Mezettia and theGuatteria tribe are karyologically and systematically distinct from each other and fromAnnona/Rollinia. Tetrameranthus as compared with the karyomorphology of about 60 other Annonaceous genera has a very peculiar and unusual karyomorphology which underlines its isolated position. Nuclear structures are almost identical in the African genusUvariopsis (2n = 16) and partly similar in theGuatteria tribe; both also share some morphological similarities and possibly are related. From a comparison ofTetrameranthus with several nuclear types within theMagnoliidae, a new model of chromosome evolution in primitive Angiosperms is suggested. In respect to their eco-morphological differentiation the genera investigated differ strongly from each other.Dedicated to Prof. Dr. K.-H.Rechinger on the occasion of His 80th birthday.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

Comparison of the karyotypes ofPsathyrostachys juncea andP. huashanica (Poaceae) studied by banding techniques

The karyotypes ofP. juncea (Elymus junceus) andP. huashanica (both outbreeders) were investigated by Feulgen-staining and by C-, N-, and Agbanding, based on a single plant in cach case. Both species have 2n=2x=14 and large chromosomes, possibly a generic character. The karyotype ofP. juncea has 8 metacentrics and 6 SAT-chromosomes with minute, heterochromatic satellites while that ofP. huashanica has 9 metacentrics and 5 SAT-chromosomes only, 2 of which with small, heterochromatic satellites. The C-banding patterns ofP. juncea chromosomes comprise from one to five, mostly small, bands at distal, and terminal positions, while those ofP. huashanica chromosomes are characterized by large telomeric bands in most arms. Banding patterns and chromosome morphology allow identification of the homologues of the seven chromosome pairs inP. juncea, but of two pairs inP. huashanica only. The patterns of both taxa are polymorphic, supporting that both taxa are outbreeders. The karyotypic characters suggest thatP. juncea is more closely related toP. fragilis than either is toP. huashanica. N-banding stains weakly. Silver nitrate staining demonstrates that nucleolus organizers of both species have different nucleolus forming capacities. The presence of micronucleoli suggests that both species have an extra unidentified chromosome with nucleolus forming capacity.     

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.     

The cytology of cotyledon cells and the induction of giant polytene chromosomes inPisum sativum

Summary The cotyledon cells ofPisum sativum have high DNA contents. By appropriate culture techniques, some of these cells can be triggered into division. Two types of dividing nuclei were seen. Firstly those that were polyploid with metaphases containing chromosome numbers ranging in value from 4 x to 32 x. Included among these were unexpected numbers equivalent to 12 x and 14 x. Secondly there were cells containing giant polytene chromosomes and these progressed from prophase to a metaphase where the polytene chromosomes separated into constituent single chromosomes.     

The absence of the somatic association of centromeres of homologous chromosomes in grass mitotic metaphases

Root-tip metaphases from Hordeum vulgare (19 cells), H. marinum (11 cells), Aegilops umbellulata (10 cells) and Zea mays (10 cells) were completely reconstructed from electron micrographs of serially sectioned nuclei. The identity of each chromosome was found by measuring the volumes of its two arms and the presence or absence of a secondary constriction at the nucleolar organising region. With the position of the centromere in three dimensions, these data were used to analyse the relative positions of homologous and heterologous centromeres. In 31 out of the 50 cells analysed, homologues were on average further apart than heterologues. Except for two nucleolar organising chromosomes, there was no evidence of any tendency for the distances between different homologue types to be differently distributed from distances between heterologues. Average distances between homologues of the single nucleolar organising chromosome (linkage group 6) of Zea (2n = 20) were lower than the average for heterologues and the interhomologue distances were distributed significantly differently from the separation distances of chromosome 6 to other chromosomes. Presumably this association occurred because of nucleolar fusion in the previous interphase. Homologues of one of the two nucleolar organising chromosomes of A. umbellulata were also distributed significantly differently from heterologues, with a tendency for homologues to lie farther apart than the average heterologous pair. These results do not support previous work using squashed and spread metaphase preparations (some including abnormal, marked chromosomes) for these species.     

Patterns of replication in the neo-sex chromosomes ofDrosophila nasuta albomicans

Drosophila nasuta albomicans (with 2n = 6), contains a pair of metacentric neo-sex chromosomes. Phylogenetically these are products of centric fusion between ancestral sex (X, Y) chromosomes and an autosome (chromosome 3). The polytene chromosome complement of males with a neo-X- and neo-Y-chromosomes has revealed asynchrony in replication between the two arms of the neo-sex chromosomes. The arm which represents the ancestral X-chromosome is faster replicating than the arm which represents ancestral autosome. The latter arm of the neo-sex chromosome is synchronous with other autosomes of the complement. We conclude that one arm of the neo-X/Y is still mimicking the features of an autosome while the other arm has the features of a classical X/Y-chromosome. This X-autosome translocation differs from the other evolutionary X-autosome translocations known in certain species ofDrosophila.     

Taxonomic studies ofAsarum sensu lato

The karyotype and the C-banding pattern in two species ofHexastylis andAsarum epigynum were analysed in detail, and the results obtained were compared with those of the other species ofAsarum, Asiasarum andHeterotropa previously reported. The present results were partially different from the previous reports related to the karyotypes of these species. The karyotype observed in two species ofHexastylis (2n=26) was represented by ten pairs of metacentric chromosomes and three pairs of small subtelocentric chromosomes, which is very similar to that ofAsiasarum in eastern Asia. The C-banding patterns ofHexastylis andAsiasarum, however, were clearly different from each other. A striking difference was found in one of the three pairs of small subtelocentric chromosomes. A Formosan speciesAsarum epigynum had the somatic chromosome number 2n=12 and a highly asymmetrical karyotype composed of mainly subtelocentric chromosomes. These karyological features were remarkably different from those of the other groups inAsarum s.l.     

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.     

Ribosomal DNA distribution in somatic chromosomes of Stangeria eriopus (Stangeriaceae, Cycadales) and molecular-cytotaxonomic relationships to some other cycad genera

Somatic chromosomes ofStangeria eriopus (Stangeriaceae, Cycadales) were investigated by fluorescentin situ hybridization (FISH) using an 18S ribosomal DNA (rDNA) probe.Stangeria eriopus showed a chromosome number of 2n=16 with a karyotype of 12 median-, 2 subterminal-, and 2 terminal-centromeric chromosomes. FISH study ofS. eriopus revealed 16 signals made up of rDNA sites located on the terminal regions of the long arms of the 7 median- and 2 subterminal-centromeric chromosomes, on terminal region of the short arm of the 1 median-centromeric chromosome, on the terminal regions of the long and the short arms of 1 median- and 2 terminal-centromeric chromosomes. This result suggests that, not only karyomorphologically but also molecular-cytologically, the genusStangeria may be more closely related to the genusCeratozamia than the genusBowenia or the genusMicrocycas previously hypothesized.     

Revision of SAT chromosome number in Triticum monococcum with respect to nucleolar organizers activity

Summary Six varieties of Triticum monococcum were analysed by means of the nucleolar test; i.e., estimation of the maximum number of primary nucleoli per nucleus. All of the varieties exhibited 4 primary nucleoli in telophase and early interphase. Following detailed karyological analysis four SAT chromosomes in all six karyotypes were found in accordance with the maximum nucleolar number. Secondary constrictions and microsatellites were localised on the short arms of chromosome pairs 3 and 5. A new order of the chromosomes in the idiogram of Tr. monococcum is proposed.     

A new pollen type,C-banded and fluorochrome counterstained chromosomes,and evolution inGuatteria and related genera (Annonaceae)

Guatteria, Guatteriopsis, Guatteriella andHeteropetalum share the same conspicuous pollen type which is new for theSpermatophyta. It is zonoaperturate with a folded aperture region and an extremely reduced exine. First chromosome counts and karyotype analyses forGuatteriopsis (4 species investigated) andGuatteriella (1 species) are identical with those ofGuatteria (19 species seen): 2n = 28. The genome is characterized by diploidization and partly telocentric chromosomes. Sequentially Giemsa C- and fluorochrome banded chromosomes and interphase nuclei are described. The cuticular folding pattern is distinct forHeteropetalum only. Growth forms and ecology are reported for many species. The evolutionary pattern of theGuatteria group is discussed and compared with other genera and families.     

Detection of 5S rDNA and other repeated DNA on supernumerary B chromosomes ofTriticum species (Poaceae)

The supernumerary B chromosomes ofTriticum speltoides andT. tripsacoides were analyzed in mitotic metaphases of spike primordium cells by C-banding and in situ hybridization (ISH) analyzes. B chromosomes ofT. speltoides have larger telomeric and interstitial C-bands, whereas those ofT. tripsacoides are almost completely devoid of C-bands. A prominent ISH site of rye related DNA sequences (using probe pSc119) was detected on B chromosomes ofT. tripsacoides and only a minor ISH site was observed on theT. speltoides B chromosomes. However, two ISH sites of 5S rRNA loci were detected at a terminal and an interstitial location of theT. speltoides B chromosomes. These sites were absent on B chromosomes ofT. tripsacoides. The results are discussed with respect to the phylogenetic origin of these B chromosomes.     

Localization of repetitive DNA in cereals byin situ hybridization: Cross hybridization among wheat,rye, barley,and oat

³H-RNA, complementary to repetitive DNA of wheat, rye, barley, and oat, was hybridizedin situ to root tip or pollen mother cells of the species mentioned. The cRNAs hybridized best with the DNA in cell nuclei of the species from which they were prepared. Cross hybridization with cells of the other related species resulted in a significant but diminished labelling. Wheat, rye, and barley hybridized better to each other than to oat, andvice versa, in agreement with the usual taxonomical classification. Over the interphase nuclei the label was distributed unevenly; not all regions of dense chromatin were labelled, and little label was found over the nucleoli. On chromosomes, the repetitive DNA was located somewhere along the chromosome arms or near the centromers in wheat, barley, and oat. Only in rye, most of the label was located near the telomers, probably over the large heterochromatin areas.     

Invasion of thehobo transposable element studied byin situ hybridization on polytene chromosomes ofDrosophila melanogaster

The invasion kinetics ofhobo transposable element in theDrosophila melanogaster genome was studied byin situ hybridization on the polytene chromosomes. Six independent lines ofDrosophila melanogaster flies that had been previously transformed by microinjection of the pHFL1 plasmid containing a completehobo element were followed over 50 generations. We observed thathobo elements were scattered on each of the chromosome arms, with more insertion sites on the 3R arm. The total number of insertion sites remains quite small, between four and six, at generation 52. On the 2R arm, a short inversion appeared once at generation 52. Most of the integration sites reported here were already described for several transposons but some of them appear to be hotspots forhobo elements.