DNA amount of X and B chromosomes in the grasshoppers Eyprepocnemis plorans and Locusta migratoria

We analyzed the DNA amount in X and B chromosomes of 2 XX/X0 grasshopper species (Eyprepocnemis plorans and Locusta migratoria), by means of Feulgen image analysis densitometry (FIAD), using previous estimates in L. migratoria as standard (5.89 pg). We first analyzed spermatids of 0B males and found a bimodal distribution of integrated optical densities (IODs), suggesting that one peak corresponded to +X and the other to -X spermatids. The difference between the 2 peaks corresponded to the X chromosome DNA amount, which was 1.28 pg in E. plorans and 0.80 pg in L. migratoria. In addition, the +X peak in E. plorans gave an estimate of the C-value in this species (10.39 pg). We next analyzed diplotene cells from 1B males in E. plorans and +B males in L. migratoria (a species where Bs are mitotically unstable and no integer B number can be defined for an individual) and measured B chromosome IOD relative to X chromosome IOD, within the same cell, taking advantage of the similar degree of condensation for both positively heteropycnotic chromosomes at this meiotic stage. From this proportion, we estimated the DNA amount for 3 different B chromosome variants found in individuals from 3 E. plorans Spanish populations (0.54 pg for B1 from Saladares, 0.51 pg for B2 from Salobre?a and 0.64 for B24 from Torrox). Likewise, we estimated the DNA amount of the B chromosome in L. migratoria to be 0.15 pg. To automate measurements, we wrote a GPL3 licensed Python program (pyFIA). We discuss the utility of the present approach for estimating X and B chromosome DNA amount in a variety of situations, and the meaning of the DNA amount estimates for X and B chromosomes in these 2 species.     

Karyotypes, constitutive heterochromatin, and genomic DNA values in the blowfly genera Chrysomya, Lucilia, and Protophormia (Diptera: Calliphoridae).

The karyotypes and C-banding patterns of Chrysomya species C. marginalis, C. phaonis, C. pinguis, C. saffranea, C. megacephala (New Guinean strain), Lucilia sericata, and Protophormia terraenovae are described. All species are amphogenic and have similar chromosome complements (2n = 12), including an XY-XX sex-chromosome pair varying in size and morphology between species. Additionally, the C-banding pattern of the monogenic species Chrysomya albiceps is presented. The DNA contents of these and of further species Chrysomya rufifacies, Chrysomya varipes, and Chrysomya putoria were assessed on mitotic metaphases by Feulgen cytophotometry. The average 2C DNA value of the male genomes ranged from 1.04 pg in C. varipes to 2.31 pg in C. pinguis. The DNA content of metaphase X chromosomes varied from 0.013 pg (= 1.23% of the total genome) in C. varipes to 0.277 pg (12.20%) in L. sericata; that of Y chromosomes ranged from 0.003 pg (0.27%) in C. varipes to 0.104 pg (5.59%) in L. sericata. In most species, the corresponding 5 large chromosome pairs showed similar relative DNA contents. The data suggest that the interspecific DNA differences in most species are mainly due to quantitative variation of (repetitive) sequences lying outside the centromeric heterochromatin blocks of the large chromosomes. The results are also discussed with regard to phylogenetic relationships of some species.     

Higher-order genome organization in platypus and chicken sperm and repositioning of sex chromosomes during mammalian evolution

In mammals, chromosomes occupy defined positions in sperm, whereas previous work in chicken showed random chromosome distribution. Monotremes (platypus and echidnas) are the most basal group of living mammals. They have elongated sperm like chicken and a complex sex chromosome system with homology to chicken sex chromosomes. We used platypus and chicken genomic clones to investigate genome organization in sperm. In chicken sperm, about half of the chromosomes investigated are organized non-randomly, whereas in platypus chromosome organization in sperm is almost entirely non-random. The use of genomic clones allowed us to determine chromosome orientation and chromatin compaction in sperm. We found that in both species chromosomes maintain orientation of chromosomes in sperm independent of random or non-random positioning along the sperm nucleus. The distance of loci correlated with the total length of sperm nuclei, suggesting that chromatin extension depends on sperm elongation. In platypus, most sex chromosomes cluster in the posterior region of the sperm nucleus, presumably the result of postmeiotic association of sex chromosomes. Chicken and platypus autosomes sharing homology with the human X chromosome located centrally in both species suggesting that this is the ancestral position. This suggests that in some therian mammals a more anterior position of the X chromosome has evolved independently.     

Molecular and cytological characterization of repetitive DNA sequences from the centromeric heterochromatin of <Emphasis Type="Italic">Sciara coprophila</Emphasis>

Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.     

Studies on rodent chromosomes

The karyotype ofTatera indica cuverii consists of 68 chromosomes with two X chromosomes in the female and an X and a Y chromosome in the male. The X chromosomes are the largest and can be distinguished from each other morphologically. Autoradiographic studies indicate that the two morphologically distinguishable X chromosomes are out-of-phase in DNA replication in approximately equal proportion and thus substantiate the random inactivation hypothesis.     

Structural characteristics of genome organization in amphibians: Differential staining of chromosomes and DNA structure

Summary Differential staining patterns on amphibian chromosomes are in some respects distinct from those on mammalian chromosomes; C-bands are best obtained, whereas G- and Q-bands are either unobtainable (on anuran chromosomes) or coincide with C-bands (chromosomes of urodeles). In amphibians, rRNA genes are located at secondary constrictions, but in urodeles they are also found at other chromosome sites, the positions of these sites being strictly heritable. DNA content in amphibian cells is tens and hundreds times higher than in mammals. DNA contents in anurans and urodeles differ within certain limits: from 2 to 25 pg/N and from 30 to over 160 pg/N respectively. Species characterized by slow morphogenesis have larger genomes. Genome growth is normally due to an increase in the amount of repetitive DNA (mostly intermediate repetitive sequences), the amount of unique sequences being almost constant (11 pg/genome in urodeles, and 1.5 pg/genome in anurans). In anurans in general no satellite DNA was found, whereas such fractions were found in manyUrodela species. Nucleosome chromatin structure in amphibians is identical to that of other eukariotes. It is postulated that differences in chromosome banding between amphibians and mammals are due to differences in chromatin packing which in turn is related to the distinct organization of DNA repetitive sequences. It is likely that fish chromosomes have a similiar structure. A comparison of such properties as the chromosome banding patterns, variations in nuclear DNA content and some genome characteristics enable us to group fishes and amphibians together as regards chromosome structure, as distinct from amniotes - reptiles, birds and mammals. It is probable that in the ancient amphibians - ancestors of reptiles - chromatin packing underwent a radical transformation, following changes in the organization of DNA repetitive sequences.     

Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes

DNA methylation patterns were studied at the chromosome level in normal and abnormal X chromosomes using an anti-5-methylcytosine antibody. In man, except for the late-replicating X of female cells, the labeled chromosome structures correspond to R- and T-bands and heterochromatin. Depending on the cell type, the species, and cell culture conditions, the late-replicating X in female cells appears to be more or less undermethylated. Under normal conditions, the only structures that remain methylated on the X chromosomes correspond to pseudoautosomal regions, which harbor active genes. Thus, active genes are usually hypomethylated but are located in methylated chromatin. Structural rearrangements of the X chromosome, such as t(X;X)(pter;pter), induce a Turner syndrome-like phenotype that is inconsistent with the resulting triple-X constitution. This suggests a position effect controlling gene inactivation. The derivative chromosomes are always late replicating, and their duplicated short arms, which harbor pseudoautosomal regions, replicate later than the normal late-replicating X chromosomes. The compaction or condensation of this segment is unusual, with a halo of chromatin surrounding a hypocondensed chromosome core. The chromosome core is hypomethylated, but the surrounding chromatin is slightly labeled. Thus, unusual DNA methylation and chromatin condensation are associated with the observed position effect. This strengthens the hypothesis that DNA methylation at the chromosome level is associated with both chromatin structure and gene expression.     

Karyotype analysis of the Russian wheat aphid, <Emphasis Type="Italic">Diuraphis noxia</Emphasis> (Kurdjumov) (Hemiptera: Aphididae) reveals a large X chromosome with rRNA and histone gene families

The Russsian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a worldwide pest of cereals. Despite its economic importance, little is known about its genome. Here we investigated physical genomic features in RWA by karyotype analysis using differential staining with AgNO₃, CMA₃, and DAPI, by chromosomal localization of ribosomal DNA (rDNA), H3 and H4 histone genes, and the “arthropod” telomeric sequence (TTAGG) _n using fluorescence in situ hybridization (FISH), and by measuring the RWA genome size using flow cytometry. The female karyotype, 2n = 10, is composed of four autosome pairs and a pair of X chromosomes, whereas the male karyotype, 2n = 9, has a single X. The X chromosome is the largest element in the karyotype. All three molecular markers used, i.e., 18S rRNA and both H3 and H4 probes are co-localized at one end of the X chromosome. The FISH probes revealed that the AgNO₃-positive bridge between two prometaphase X chromosomes of females, which is believed to be responsible for the elimination of one X chromosome in aphid oocytes determined to undergo male development, contains clusters of both histone genes, in addition to an rDNA cluster. Interestingly, RWA lacks the (TTAGG) _n telomeric sequence in its genome, in contrast to several previously investigated aphid species. Additionally, we compared female and male genome sizes. The female genome size is 2C = 0.86 pg, whereas the male genome size is 2C = 0.70 pg. The difference between the DNA content in the two genders suggests that the RWA X chromosome occupies about 35% of the female haploid genome (1C = 0.43 pg), which makes it one of the largest sex chromosomes in the animal kingdom.     

The origin of multiple sex chromosomes in the gerbil Gerbillus gerbillus (Rodentia: Gerbillinae)

The sex chromosomes of the partly sympatric species of gerbils Gerbillus pyramidum and G. gerbillus (Mammalia: Gerbillinae) were investigated by a variety of light- and electron-microscope methods, including DNA replication banding and synaptonemal complex (SC) techniques. The sex-chromosome mechanism of G. pyramidum is of the maleXY:femaleXX type, whereas that of G. gerbillus is of the less common maleXY1Y2:femaleXX system. The results include the demonstration that the X chromosomes of both species are compound. One segment is added to the X chromosome of G. pyramidum, leading to an increase in length from the standard 5% to approximately 7.3%, whereas two different extra segments increase the length of the X chromosome of G. gerbillus to approximately 11% of the length of the haploid genome. In both cases the extra material is autosomal and is also represented in the respective Y chromosomes. Classifying heterochromatin by the variation in staining quality was helpful in elucidating the possible origin of the different chromosome segments, including the pericentromeric regions. Observations on meiotic chromosome pairing and chiasma formation have confirmed the homologies established by band comparisons. The occurrence of chiasmata between the sex chromosomes supports the autosomal origin of the pairing segments. These and other findings have been interpreted in the framework of a multistep evolutionary model. This sequence starts from a hypothetical pair of sex chromosomes, the X element of which amounts to 5% of the haploid genome, and leads through three translocations involving two pairs of autosomes and one pericentric inversion to the most complex situation of this series, manifested in G. gerbillus. The adaptive value, if any, of autosome incorporation into the sex chromosomes repeatedly occurring here is unknown. It is, however, a remarkable fact that in one species, G. gerbillus, the complex sex-chromosome constitution is conserved over vast geographic distances, and in the other, G. pyramidum, the compound X and Y chromosomes withstand change in the face of extreme autosome restructuring.     

Accelerated evolution of sex chromosomes in aphids, an x0 system

Sex chromosomes play a role in many important biological processes, including sex determination, genomic conflicts, imprinting, and speciation. In particular, they exhibit several unusual properties such as inheritance pattern, hemizygosity, and reduced recombination, which influence their response to evolutionary factors (e.g., drift, selection, and demography). Here, we examine the evolutionary forces driving X chromosome evolution in aphids, an XO system where females are homozygous (XX) and males are hemizygous (X0) at sex chromosomes. We show by simulations that the unusual mode of transmission of the X chromosome in aphids, coupled with cyclical parthenogenesis, results in similar effective population sizes and predicted levels of genetic diversity for X chromosomes and autosomes under neutral evolution. These results contrast with expectations from standard XX/XY or XX/X0 systems (where the effective population size of the X is three-fourths that of autosomes) and have deep consequences for aphid X chromosome evolution. We then localized 52 microsatellite markers on the X and 351 on autosomes. We genotyped 167 individuals with 356 of these loci and found similar levels of allelic richness on the X and on the autosomes, as predicted by our simulations. In contrast, we detected higher dN and dN/dS ratio for X-linked genes compared with autosomal genes, a pattern compatible with either positive or relaxed selection. Given that both types of chromosomes have similar effective population sizes and that the single copy of the X chromosome of male aphids exposes its recessive genes to selection, some degree of positive selection seems to best explain the higher rates of evolution of X-linked genes. Overall, this study highlights the particular relevance of aphids to study the evolutionary factors driving sex chromosomes and genome evolution.     

Purification of the chromosomes of the Indian muntjac by flow sorting.

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.     

DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes.

Lycopersicon esculentum (tomato) has a small genome (2C = 1.90 pg of DNA) packaged in 2n = 2x = 24 small acrocentric to metacentric chromosomes. Like the chromosomes of other members of the family Solanaceae, tomato chromosomes have pericentromeric heterochromatin. To determine the fraction of the tomato genome found in euchromatin versus heterochromatin, we stained pachytene chromosomes from primary microsporocytes with Feulgen and analyzed them by densitometry and image analysis. In association with previously published synaptonemal complex karyotype data for tomato, our results indicate that 77% of the tomato microsporocyte genome is located in heterochromatin and 23% is found in euchromatin. If heterochromatin is assumed to contain few active genes, then the functional genes of the tomato must be concentrated in an effective genome of only 0.22 pg of DNA (1C = 0.95 pg x 0.23 = 0.22 pg). The physical segregation of euchromatin and heterochromatin in tomato chromosomes coupled with the small effective genome size suggests that tomato may be a more useful subject for chromosome walking and gene mapping studies than would be predicted based on its genome size alone. Key words : tomato, Lycopersicon esculentum, genome size, heterochromatin, euchromatin, pachytene chromosomes, synaptonemal complex.     

Chromosome banding in Amphibia. XXVI. Coexistence of homomorphic XY sex chromosomes and a derived Y-autosome translocation in Eleutherodactylus maussi (Anura,Leptodactylidae)

A 15-year cytogenetic survey on one population of the leaf litter frog Eleutherodactylus maussi in northern Venezuela confirmed the existence of multiple XXAA male symbol /XAA(Y) female symbol sex chromosomes which originated by a centric (Robertsonian) fusion between the original Y chromosome and an autosome. 95% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA(Y) sex chromosomes pair in the expected trivalent configuration. In the same population, 5% of the male animals still possess the original, free XY sex chromosomes. In a second population of E. maussi analyzed, all male specimens are characterized by these ancestral XY chromosomes which form normal bivalents in meiosis. E. maussi apparently represents the first vertebrate species discovered in which a derived Y-autosome fusion still coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA(Y) sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. DNA flow cytometric measurements show that the genome of E. maussi is among the largest in the anuran family Leptodactylidae. The present study also supplies further data on differential chromosome banding and fluorescence in situ hybridization experiments in this amphibian species.     

Karyology and nuclear DNA quantification of four species ofChaetomorpha (Cladophorales,Chlorophyta) from the western Atlantic

Chromosome numbers are given for four species ofChaetomorpha from the warm temperate and tropical western Atlantic. The basic chromosome number is six, with three median and three submedian chromosomes.Chaetomorpha species represent a polyploid series, with numbers of 12, 18 and 24 found in the present study. Microspectrophotometry data for each species were quantified by reference to standards with known DNA contents. Results indicate similar 2X =1C=12 genome sizes forC. aerea (0.20 pg) andC. brachygona (0.26 pg), and forC. antennina (0.53 pg) andC. melagonium (0.58 pg). These findings are compared with karyological features ofCladophora species to characterize the karyology of the cladophoralean genome.     

Gender in plants: sex chromosomes are emerging from the fog

Although most plants have flowers with both male and female sex organs, there are several thousands of plant species where male or female flowers form on different individuals. Surprisingly, the presence of well-established sex chromosomes in these dioecious plants is rare. The best-described example is white campion, for which large sex chromosomes have been identified and mapped partially. A recent study presented a comprehensive genetic and physical mapping of the genome of dioecious papaya. It revealed a short male specific region on the Y chromosome (MSY) that does not recombine with the X chromosome, providing strong evidence that the sex chromosomes originated from a regular pair of autosomes. The primitive papaya Y chromosome thus represents an early event in sex chromosome evolution. In this article, we review the current status of plant sex-chromosome research and discuss the advantages of different dioecious models.     

Chromosome and nuclear DNA study on Luzula - a genus with holokinetic chromosomes.

Chromosomes and nuclear DNA amount were analyzed in leaf tissues of Luzula nivea, Luzula luzuloides, and Luzula multiflora. Intra- and interspecific karyological variability was stated. Chromosome numbers in diploids ranged 2n = 8-24 in L. nivea and L. luzuloides and 2n = 12-84 in hexaploid L. multiflora. Karyological variability resulted mainly from chromosome fission (agmatoploidy) and aneusomaty; chromosome fusion (symploidy) and polyploidy were also involved. Flow cytometric determination of nuclear genome size using propidium iodide staining gave values of 1.584 pg in L. luzuloides, 1.566 pg in L. nivea, and 3.034 pg in L. multiflora. Variability in relative nuclear genome size within species was measured by 4',6-diamidino-2-phenylindole staining. In contrast with previous reports, variability was fairly small and ranged from 1.796 to 1.864 pg in L. luzuloides, from 1.783 to 1.847 pg and from 1.737 to 1.808 pg in two populations (S and F) of L. nivea, respectively, and from 3.125 to 3.271 pg in L. multiflora. An intraplant (interleaf) genome size variation was also observed and its possible causes are discussed.     

Detection of alien chromosomes from S-genome species in the addition/substitution lines of bread wheat and visualization of A-, B- and D-genomes by GISH

A modified approach based on the GISH technique for detecting introgressed chromosomes/chromosome arms from closely related S-genome species to wheat genome and for visualization of A-, B- and D-genomes of Triticum aestivum L. (genome AABBDD, 2n = 6x = 42) is presented. For detecting alien chromosomes we investigated two lines of bread wheat, one is an addition line with a pair of chromosome No. 4 short arms from Aegilops searsii (4SsS) and a wheat substitution line with a pair of chromosomes No. 6 from Ae. longissima (6S1). A hybridization mixture consists of two differently labelled DNAs, one from the line used for chromosome spread preparations, and the second from origin species of alien chromosomes. The latter adds different color in the regions of its hybridization showing the presence of alien chromosomes by creating a strong and easily detected combined signal. For discriminating A-, B-, and D-genome chromosomes, the hybridization mixture of differently labelled total DNA from Ae. tauschii--the proposed progenitor of D-genome (detected red) and T. dicoccoides (genome AABB) (detected green) were used. The high temperature of hybridization allows high precision annealing of chromosome/probe sequences and at the same time it sharpens differences between reassociation kinetics of eu- and heterochromatin revealing chromosome substructure. A pre-annealing step increases probe specificity. As a result, we observed brown chromosomes of A-genome, banded green chromosomes of B-genome and red chromosomes of D-genome. Inter genomic invasion of the sequences from A/B-genomes to D-genome has been detected.     

Similarities in the chromosomal distribution of AG and AC repeats within and between Drosophila, human and barley chromosomes

Two simple sequence repeats (SSRs), AG and AC, were mapped directly in the metaphase chromosomes of man and barley (Hordeum vulgare L.), and in the metaphase and polytene chromosomes of Drosophila melanogaster. To this end, synthetic oligonucleotides corresponding to (AG)(12) and (AC)(8) were labelled by the random primer technique and used as probes in fluorescent in situ hybridisation (FISH) under high stringency and strict washing conditions. The distribution and intensity of the signals for the repeat sequences were found to be characteristic of the chromosomes and genomes of the three species analysed. The AC repeat sites were uniformly dispersed along the euchromatic segments of all three genomes; in fact, they were largely excluded from the heterochromatin. The Drosophila genome showed a high density of AC sequences on the X chromosome in both mitotic and polytene nuclei. In contrast, the AG repeats were associated with the euchromatic regions of the polytene chromosomes (and in high density on the X chromosome), but were only seen in specific heterochromatic regions in the mitotic chromosomes of all three species. In Drosophila, the AG repeats were exclusively distributed on the tips of the Y chromosome and near the centromere on both arms of chromosome 2. In barley and man, AG repeats were associated with the centromeres (of all chromosomes) and nucleolar organizer regions, respectively. The conserved chromosome distribution of AC within and between these three phylogenetically distant species, and the association of AG in specific chromosome regions with structural or functional properties, suggests that long clusters of these repeats may have some, as yet unknown, role.