Comparison of the chromosomes of Triticum timopheevi with related wheats using the techniques of C-banding and in situ hybridization

Summary The chromosomes of the tetraploid wheats Triticum timopheevi (Genome AAGG) and T. araraticum (Genome AAGG) were C-banded at mitosis. The identity of the banded and unbanded chromosomes was then established by firstly making comparisons with the hexaploid species T. zhukovskyi which has the genome formula AAAAGG. Secondly, the meiotic pairing in F₁ hybrids between T. timopheevi and diploid wheats was examined by means of C-banding. The results showed that the banded chromosomes belonged to the G genome, while the unbanded chromosomes belonged to the A genome. Only one of the two pairs of satellited chromosomes had strong heterochromatic bands. The relationship between the genomes of T. timopheevi and T. dicoccum (Genome AABB) was then assessed at meiosis in hybrids between these species, using the techniques of C-banding and in situ hybridisation of a cloned ribosomal RNA gene probe. It was concluded that there were differences both in the amount and distribution of heterochromatin and also translocation differences between the species.     

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes

"Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal. As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation. t(1;7)(1qter----1p36::7q11----7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to 1p31. Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.     

Chromosomal distribution of rDNA in Pan paniscus,Gorilla gorilla beringei,and Symphalangus syndactylus: Comparison to related primates

Hybridization in situ was used to identify rDNA in chromosomes of the pygmy chimpanzee, mountain gorilla, and siamang gibbon. In contrast to other Pongids, and man, the gorilla has only two pairs of rDNA-containing chromosomes. The single pair in the siamang bears no resemblance to the nucleolar chromosome of the closely related lar gibbon. Pan paniscus and P. troglodytes have the same rDNA distribution, and similar karyotypes except in the structure of chromosome 23p. Grain counts over unbanded preparations show that the human, orangutan, and both chimpanzees have about the same total rDNA multiplicity.     

Human cells in suspension 2

Summary When PHA stimulated human lymphoid cells are allowed to proliferate in vitro and chromosomal slides are prepared from the culture after 48,72, 96,120, and 144h of growth, gradual changes in chromosome morphology can be observed after traditional Giemsa staining of the slides. Keeping culture conditions, colcemid exposure, and fixation procedure constant for all samples, it is found that average chromosome length decreases with increasing culture time. A shift from high frequencies of subbanded chromosomes (sample 48 h and sample 72 h) to high frequencies of unbanded and G banded chromosomes (sample 120 h and sample 144 h) takes place simultaneously with the general compaction of the chromosomes. Examination of trypsin-induced G bands as well as examination of untreated G banded chromosomes from all samples clearly indicate that the basic G band pattern is not altered during proliferation and differentiation, although the progressive compaction of the chromosome observed with increasing culture time results in a phenomenon similar to that observed during mitosis, where the compact late metaphase chromosome after trypsin treatment exhibits fewer but more prominent bands than the prophase/prometaphase chromosomes. Thus the progressive compaction of metaphase chromosomes observed during in vitro aging seems to resemble the condensation processes during the G₂ phase and mitosis.It has been suggested that the chromomeres serve as centers for chromosome condensation during mitosis, probably mediated by a sulphydryl-disulphide transition in chromosomal proteins. The data presented here further suggest that the chromomeres may also serve as centers for chromosomal differentiation, presumable by a mechanism similar to that acting during chromosome condensation in mitosis.     

Intravarietal variation in satellites and C-banded chromosomes of Agropyron intermedium ssp. trichophorum cv. Greenleaf.

A high amount of intravarietal variation in satellites and C-banded chromosomes was observed in the hexaploid wheatgrass synthetic cultivar 'Greenleaf' (Agropyron intermedium ssp. trichophorum (Link) A. &Gr., 2n = 6x = 42, genome E1E1E2E2SS). The cultivar is an open-pollinated perennial that shows extensive interplant polymorphism for many biological characters. Maximum number of satellites detected varied among plants from zero to six. In 61% of the plants, we observed two large satellites in association with zero, one, or two small ones. Chromosome constitution differed significantly among plants as revealed by analysis of variance based on the total number of banded chromosomes and the number of banded chromosomes with telomeric bands at either one or both ends. Heteromorphism in C-banding patterns between homologues was found in most of the chromosomes and was classified into four types: (i) difference in band size, (ii) difference in presence/absence of one or two bands, (iii) completely different banding patterns, and (iv) banded versus unbanded. Homologous chromosomes having types iii and iv heteromorphism could only be matched by their relative length and arm ratio instead of C-banding patterns. Deletions were detected in two chromosomes. Overall, C-banded chromosomes of this cultivar were characterized by the presence of large telomeric bands and were quite different from the previously reported karyotypes of the supposed diploid ancestor Agropyron elongatum (Host) P. Beauv. (genome EE) and an Ag. intermedium (Host) P. Beauv. accession (E1E1E2E2SS) The results suggest that dramatic chromosome modifications have occurred in this species during the course of evolution. The study sheds light on the extent of intrapopulation polymorphism present in the karyotypes of outcrossing polyploids and synthetic cultivars and has implications regarding strategies for chromosomal manipulation involving open-pollinated species.     

Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissected chromosomes

A new procedure for determining the chromosomal origin of marker chromosomes has been carried out. The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes microdissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). A number of marker chromosomes prepared from unbanded and from GTG-banded lymphocyte chromosomes were collected with microneedles and transferred to a collection drop. The chromosomal material was amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products were labelled by nick-translation with biotin-11-dUTP and used as probes for FISH. They were hybridized onto normal metaphase spreads in order to determine the precise regional chromosomal origin of the markers. Following this approach, we tested 2–14 marker chromosomes in order to determine how many are necessary for reverse chromosome painting. As few as two marker chromosomes provided sufficient material to paint the appropriate chromosome of origin, regardless of whether the marker contained heterochromatic or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,XY, +mar₁,+mar₂] and an aberrant Y chromosome of a mentally retarded boy [karyotype: 46,X, der(Y)].     

Chiasma formation in duplicated segments of the haploid rye genome

In meiosis of haploid rye associations of two or more chromosomes are observed. In order to investigate whether these associations are chiasmate, metaphase I and anaphase I associations were analysed after Giemsa banding. — At anaphase I chromatid exchanges between differently marked chromosome arms were observed, which proved the presence of real chiasmata. The association between banded and unbanded arms shows that the heterochromatic telomeres do not act as secondary pairing sources. Different statistical approaches were used to test randomness of chiasma formation. It appeared to be non-random, which showed that the segments involved were non-randomly located and probably limited in number. The nature of these segments is discussed.     

Lake Erie water snakes revisited: Morph- and age-specific variation in relative crypsis

Summary Populations of water snakes (Nerodia sipedon insularum) on islands in western Lake Erie are variable in colour pattern, consisting of unbanded, intermediate, and banded morphs. In contrast, mainland populations (N. s. sipedon) consist solely of banded morphs. Previous investigators hypothesized that natural selection favoured unbanded morphs on exposed island shorelines and banded morphs in overgrown mainland habitats and that gene flow from mainland populations was responsible for the persistence of banded morphs on islands. To clarify the potential role of natural selection, I quantified relative crypsis among morphs and age classes of water snakes by comparing the size of patches making up their colour patterns with the size of patches in island and mainland backgrounds. This analysis reveals that if unbanded morphs are more cryptic than intermediate and banded morphs on islands, it is only in the young-of-the-year age class. For older snakes on islands and for all snakes on the mainland, unbanded morphs are consistently less cryptic than intermediate and banded morphs. Given these results, the net direction of selection in island populations should depend on the intensity of predation on different age classes of snakes. Overall, selection may favour unbanded morphs (e.g. if predation occurs primarily on young-of-the-year), intermediate and banded morphs (e.g. if predation occurs primarily on older snakes), or be weak or absent (e.g. certain combinations of predation on young-of-the-year and older snakes). Using estimates of relative crypsis to guide reanalysis of morph frequency data, I find support for the hypothesis that unbanded morphs are favoured by natural selection in island populations.     

Distribution patterns of morph frequencies in the snail Cepaea hortensis in Iceland

In Iceland Cepaea hortensis Müll. reaches its northern limit of distribution occurring in restricted areas along the south coast. Samples of snails collected along steep cliffs in two such restricted areas were effectively monomorphic for shell colour (yellow), but were polymorphic for banding pattern and revealed strong area effects. In both localities samples from sites facing east contained varying frequencies of unbanded snails whereas in ecologically similar sites facing south unbanded were absent. In an experimental population released at a south-facing site unbanded snails failed to become established whereas banded individuals survived and bred. It is suggested that the area effects are maintained through non-visual (most likely climatic) selection though other possibilities cannot be ruled out.     

The mouse IFN-alpha (Ifa) locus: correlation of physical and linkage maps by in situ hybridization

The physical location of the mouse IFN-alpha locus (Ifa) on chromosome 4 was defined by in situ hybridization of a cloned mouse IFN-alpha probe to metaphase spreads in which one chromosome 4 was present as part of a single metacentric chromosome, all other chromosomes being acrocentric. (This approach greatly facilitates analysis and can be used even when it is difficult to obtain good banding). Using unbanded chromosomes, the grains were localized over the chromosome 4 part of the metacentric, in a region 0.61 +/- 0.07 (SD) of the distance from the centromere to the telomere. In Giemsa-banded spreads, the majority of the grains were in the region 4C3----C6. Consideration of these results and of the known linkage maps for mouse and man indicates that the Galt - Aco-1 - Ifa syntenic group spans a distance of approximately 14 cM and suggests that the same group on human 9p will also occupy a similarly sized region, with GALT proximal and IFL distal to the centromere.     

Temperature effects on energy metabolism in banded and unbanded morphs of the snail Cepaea hortensis Müll

Summary Energy metabolism (oxygen consumption) in acclimated juvenile yellow banded and unbanded Cepaea hortensis Müll. was measured at 5°, 10°, 15°, 20°, and 25°C with Englemann respirometers. Metabolic rates were also measured after abrupt temperature changes from 5° to 20° and from 25° to 10°C.Energy metabolism in the two morphs was significantly different at 5° and 20°C. The metabolism/temperature curve in the banded morph was relatively flat with reduced regulating power at 20°C. The yellow unbanded form had a lower metabolism at 5°C, but between 15° and 25°C the curve was flat and indicated good regulating ability. A sudden decrease in experimental temperature gave significant response in both morphs. The effect of abrupt temperature increase was significant only in unbanded snails. Banded and unbanded snails thus displayed different adaptation strategies.Differential heating because of different pigmentation is discussed. The increase in metabolic rate with increased temperature was highest in the yellow unbanded morph. The effects were small and probably overridden by the influences of acclimation and behavior.The use of energetic parameters in ecological genetics is considered. It is suggested that the cohort producing most progeny with lowest energetic cost per energy unit of offspring is the better adapted to the local environment.     

Zonal Fractionation of Mammalian Metaphase Chromosomes and Determination of Their DNA Content

Chinese hamster cells in culture were synchronized, collected at metaphase, homogenized to release the chromosomes, and the chromosomes fractionated in a sucrose gradient using a zonal centrifuge with an A12 zonal rotor. Chromosomes in the separated fractions as well as in control metaphase spreads were quantitatively classified into five easily distinguished groups, according to individual measurements of length and centromeric index. For each zonal fraction, chemical determinations were made of the amount of DNA per average chromosome. Using the group compositional data for each fraction, the amount of DNA per average chromosome in each of the groups was then calculated to be: Group I (chromosomes 1, 2)= 1.00 ± 0.14 pgm/chromatid; Group II (chromosomes 4, X, 5) -0.39 ± 0.05 pgm/chromatid; Group III (chromosomes Y, 6, 7, 8)=0.24 ± 0.04 pgm/chromatid; Group IV (chromosomes 9, 10, 11)=0.13 ± 0.004 pgm/ chromatid; and Group V (a small marker in this cell line)=0.06 pgm/ chromatid. These values are in good agreement with the literature values for relative chromosomal DNA content derived from cytospectrophotometric measurements of fuelgen stained hamster metaphase spreads. They indicate that unlike the case for human chromatids the amount of DNA found in hamster chromatids is not directly proportional to the chromatid length.

The larger chromosomes contain more DNA per unit length than smaller chromosomes. The magnitude of this effect is considerably greater than that which may be ascribable to centromeric constriction.     

Polymorphism in Cepaea Hortensis in marginal populations in Spain

Cepaea hortensis (Mull) is found in Spain only in the western part of the Iberian mountain range. This is the southernmost limit of its range. The most frequent colour is yellow, brown being very rare. The most frequent band patterns are 12345, fused banded and unbanded. There are associations between maximum and minimum temperatures and pink and effectively unbanded yellow phenotypes. The populations may be grouped into two zones, one located in the north and the other in the south of the sampled area. Visual predation was not observed. The marginal populations studied by us were compared with others from Northern Europe, and it was observed that pink snails are more frequent at the southern limit, and unbanded ones at the northern limit. This was interpreted as a climatic selection.     

Cytological identification of two X-chromosome types in the wood lemming (Myopus schisticolor)

In the wood lemming (Myopus schisticolor) three genetic types of sex chromosome constitution in females are postulated: XX, X^*X and X^*Y (X^*=X with a mutation inactivating the male determining effect of the Y chromosome). Males are all XY. It is shown in the present paper that the two types of X chromosomes, X and X^*, exhibit differences in the G-band patterns of their short arms. In addition, it was demonstrated in unbanded chromosomes that the short arm in X^* is shorter than in X. The origin of these differences is still obscure; but they allow to identify and to distinguish the individual types of sex chromosome constitution, as of XX versus X^*X females and of X^*Y females versus XY males, on the basis of G-banded chromosome preparations from somatic cells.     

ISOLATION OF METAPHASE CHROMOSOMES FROM HELA CELLS

The authors have developed a method for large-scale isolation of metaphase chromosomes from HeLa cells. The distinguishing feature of this method is the use of a pH sufficiently low (about 3) to stabilize the chromosomes against mechanical damage. Many milligrams of fairly pure, morphologically intact chromosomes can be isolated in 8 hr or less of total working time. The isolated chromosomes contain about 2.0 mg of acid-soluble protein, 2.7 mg of acid-insoluble protein and 0.66 mg of RNA for each milligram of DNA. The RNA bound to the isolated chromosomes consists mainly of ribosomal RNA, but there is also a significant amount of 45S RNA.     

Thirty-four years of climatic selection in the land snail Theba pisana

Few continuous, long-term studies have measured the intensity and variability of natural selection within a framework of clear adaptive hypotheses. In the snail Theba pisana, the proportion of effectively unbanded shells is higher in exposed habitats than in adjacent acacia thickets, which has been explained by microclimatic selection. Comparisons across an ecotone for 34 consecutive years determined the combined effects on morph frequencies of habitat and changes in weather conditions during summer. The long-term average (±s.e.) frequency of effectively unbanded shells was 0.577±0.011 in the open habitat when compared with 0.353±0.005 in the acacia. The persistent association of shell banding with habitat accounted for 34% of the variation in morph frequencies. Differences among years were also large, representing 23% of the variation. Higher proportions of effectively unbanded snails were associated with hotter, sunnier summers. Thus, temporal variation supports the hypothesis of microclimatic selection, consistent with the spatial association with habitat. Based on observed rates of change, the mean annual selection on this polymorphism was about 0.13, but with a large variance: s was as high as 0.5, but ⩽0.05 in about 40% of the years. The large variance and frequent reversals in direction of selection indicate a potential for rapid genetic change, but with little net change in morph frequencies over three decades, highlighting the value of long-term continuous studies of populations facing natural environmental variation.     

Isolating individual wheat (Triticum aestivum) chromosome arms by flow cytometric analysis of ditelosomic lines

 We are reporting the successful isolation of wheat chromosome arm 1DS by flow cytometry. A chromosome suspension was prepared for the 1DS ditelosomic line and the normal ‘Chinese Spring’ (CS) by chopping 2-day-old root tip meristems, synchronized by hydroxyurea, in HEPES-magnesium sulfate buffer containing propidium iodide. Chromosomes were analyzed and sorted with a FACS Vantage flow cytometer and cell sorter. An extra peak was observed in the flow karyotype of the ditelosomic line that was absent in ‘CS’. The estimated size of chromosomes from the extra peak matched with the expected size of chromosome 1DS. Chromosomes from the putative 1DS peak were analyzed by both fluorescent microscopy and N-banding analysis. A total of 571 chromosomes from two separate experiments were analyzed, and all were observed to be telosomics except for 2 which were broken. About 82% of these telosomics showed the diagnostic N-band of 1DS, the remaining were unbanded and are probably also 1DS. This strategy can also be used to sort other wheat arms. Received: 30 September 1998 / Accepted: 2 November 1998     

Survival,behaviour and spatial distribution of shell morphs in a population of the snail Brephulopsis bidens (Pulmonata)

Summary Microspatial variation of banded and unbanded shell morphs frequencies as well as number of individuals per m², mortality, migration and burrowing into the ground were examined in a population of snail Brephulopsis bidens found in South Crimea mountains (USSR). Differential values of relative survival of morphs were determined by their thermotolerance. The relative survival of the banded morph was lower at the west sites of population area (W=0.273), and increased gradually up to 1 at the east sites. Survival of the banded morph was dependent on its burrowing activity. Differences in relative survival of morphs decreased parallel with increasing general mortality of snails.Burrowing activity and intensity of migration of the banded morph were significantly higher than that in unbanded. In experiments with artificial shaded sections, the banded morph preferred shaded sections, whereas unbanded chose illuminated sites. All these differences in behaviour probably form the main factors for microspatial variation of morph frequencies.     

On the sequential packaging of bacteriophage P22 DNA.

Bacteriophage P22 is thought to package daughter chromosomes serially along concatemeric DNA. We present experiments which show that the average DNA packaging series length increases with time after infection, which supports this model. In addition, we have analyzed the effect on average series length of lowering the amount of the various individual proteins involved in DNA packaging. These results support the notion that the protein products of gene 2 and gene 3 are both more stringently required for initiation of sequential DNA packaging series than for their extension, and they are compatible with a model for the control of series length in which that length is determined, at least in part, by a competition between series initiation events and extension events.     

Whole-genome and chromosome evolution associated with host adaptation and speciation of the wheat pathogen Mycosphaerella graminicola

The fungus Mycosphaerella graminicola has been a pathogen of wheat since host domestication 10,000-12,000 years ago in the Fertile Crescent. The wheat-infecting lineage emerged from closely related Mycosphaerella pathogens infecting wild grasses. We use a comparative genomics approach to assess how the process of host specialization affected the genome structure of M. graminicola since divergence from the closest known progenitor species named M. graminicola S1. The genome of S1 was obtained by Illumina sequencing resulting in a 35 Mb draft genome sequence of 32X. Assembled contigs were aligned to the previously sequenced M. graminicola genome. The alignment covered >90% of the non-repetitive portion of the M. graminicola genome with an average divergence of 7%. The sequenced M. graminicola strain is known to harbor thirteen essential chromosomes plus eight dispensable chromosomes. We found evidence that structural rearrangements significantly affected the dispensable chromosomes while the essential chromosomes were syntenic. At the nucleotide level, the essential and dispensable chromosomes have evolved differently. The average synonymous substitution rate in dispensable chromosomes is considerably lower than in essential chromosomes, whereas the average non-synonymous substitution rate is three times higher. Differences in molecular evolution can be related to different transmission and recombination patterns, as well as to differences in effective population sizes of essential and dispensable chromosomes. In order to identify genes potentially involved in host specialization or speciation, we calculated ratios of synonymous and non-synonymous substitution rates in the >9,500 aligned protein coding genes. The genes are generally under strong purifying selection. We identified 43 candidate genes showing evidence of positive selection, one encoding a potential pathogen effector protein. We conclude that divergence of these pathogens was accompanied by structural rearrangements in the small dispensable chromosomes, while footprints of positive selection were present in only a small number of protein coding genes.