Origin and evolution of highly polymorphic rDNA sites in Alstroemeria longistaminea (Alstroemeriaceae) and related species.

Alstroemeria (Alstroemeriaceae) displays a conserved and highly asymmetric karyotype, where most rDNA sites can be properly recognized by the size and morphology of the chromosomes. We analyzed the intraspecific variation of rDNA sites in A. longistaminea and compared with their distribution in other species (A. caryophyllaea and A. piauhyensis) and a representative of a sister genus, Bomarea edulis. All three species of Alstroemeria presented 2n = 16, and one to six B chromosomes were found in some individuals of A. longistaminea. There was a set of 12 conserved rDNA sites (four 5S and eight 35S) and up to 11 variable sites. B chromosomes were almost entirely covered by 35S signals, coupled with tiny 5S sites. Noteworthy, most rDNA sites found in A. caryophyllaea and A. piauhyensis were localized in chromosome positions similar to those in A. longistaminea, suggesting the existence of conserved hotspots for rDNA accumulation. Some of these hotspots were absent in Chilean Alstromeria as well in B. edulis. We propose that insertions of rDNA sequences on chromosomes do not occur randomly but rather on preferential sites or hotspots for insertions. The maintenance of these arrays, however, may be favored/constrained by different factors, resulting in stable or polymorphic sites.

The karyotype of Adenia and the origin of the base number x = 12 in Passifloroideae (Passifloraceae).

Adenia is an Old World genus of Passifloroideae closely related to Passiflora. The two genera comprise the large majority of Passifloroideae species, although most studies are concentrated on Passiflora. Cytological analyses reveal that changes in chromosome numbers played an important role in the evolution of Passiflora, whereas in the remaining genera little is known, hindering the identification of the base number of the family. Here we analyzed the chromosome number and the 35S rDNA sites of three species of Adenia and reevaluated the base number (x) of the subfamily Passifloroideae and the family Passifloraceae, including chromosome data for Turneroideae and Malesherbioideae. The chromosome number of Adenia species seemed to be stable with 2n = 24 or 48 and one or two pairs of rDNA sites, very similar to Passiflora subgenus Astrophea, suggesting a common ancestral karyotype with x = 12. Differently, Turneroideae and Malesherbioideae present x = 7. A whole genomic duplication detected after the separation of Passifloroideae and Malesherbioideae suggests that the base number of Passifloraceae most probably was x = 7, which by dysploidy and polyploidy generated x = 12 for the subfamily Passifloroideae.

Does the chromosomal position of 35S rDNA sites influence their transcription? A survey on Nothoscordum species (Amaryllidaceae).

35S ribosomal DNA (rDNA) sites are the regions where the ribosomal genes 18S, 5.8S and 25S, responsible for the formation of the nucleoli, are found. The fact that rDNA sites have non-random distribution on chromosomes suggests that their positions may influence their transcription. To identify if the preferentially transcribed rDNA sites occupy specific position, six species (nine cytotypes) of the genus Nothoscordum were analyzed using two different techniques to impregnate the nucleolar organizer regions (NORs) with silver nitrate. Both techniques strongly stained NORs, but one of them also stained the proximal region of all chromosomes, suggesting the existence of another group of argentophilic proteins in this region. In species with rDNA sites in acrocentric and metacentric chromosomes, sites located on the short arms of the acrocentric chromosomes were preferentially activated. On the other hand, in species with rDNA sites restricted to the short arms of the acrocentrics, all of them were activated, whereas in those species with sites restricted to the terminal region of metacentric chromosomes, the frequency of active sites was always lower than expected. This indicate that, at least in Nothoscordum, the transcription of an rDNA site is influenced by its chromosomal position, and may explain, at least partially, the strongly non-random distribution of these sites in plant and animal chromosomes.

Intense proliferation of rDNA sites and heterochromatic bands in two distantly related Cuscuta species (Convolvulaceae) with very large genomes and symmetric karyotypes.

The genome size varies widely among angiosperms but only a few clades present huge variation at a low phylogenetic level. Among diploid species of the genus Cuscuta the genome size increased enormously in at least two independent lineages: in species of subgenus Monogynella and in at least one species (C. indecora) of the subgenus Grammica. Curiously, the independent events lead to similar karyotypes, with 2n = 30 mostly metacentric chromosomes. In this paper we compared the patterns of heterochromatic bands and rDNA sites of C. indecora and C. monogyna, aiming to evaluate the role of these repetitive fractions in these karyotypes. We found out that the large genomes of these species were incremented by a huge number of small heterochromatic CMA+ and DAPI+ bands and 5S and 35 rDNA sites, most of them clearly colocalized with CMA+ bands. Silver nitrate impregnation revealed that the maximum number of nucleoli per nucleus was low in both species, suggesting that some of these sites may be inactive. Noteworthy, the tandem repeats did not generate large bands or sites but rather dozens of small blocks dispersed throughout the chromosomes, apparently contributing to conserve the original karyotype symmetry.

Allopolyploid origin and genome differentiation of the parasitic species Cuscuta veatchii (Convolvulaceae) revealed by genomic in situ hybridization.

Interspecific hybridization and genome duplication to form allopolyploids are major evolutionary events in angiosperms. In the parasitic genus Cuscuta (Convolvulaceae), molecular data suggested the existence of species of hybrid origin. One of them, C. veatchii, has been proposed as a hybrid between C. denticulata and C. nevadensis, both included in sect. Denticulatae. To test this hypothesis, a cytogenetic analysis was performed with CMA/DAPI staining and fluorescent in situ hybridization using 5S and 35S rDNA and genomic probes. Chromosomes of C. denticulata were small with a well-defined centromeric region, whereas C. nevadensis had larger, densely stained chromosomes, and less CMA+ heterochromatic bands. Cuscuta veatchii had 2n = 60 chromosomes, about 30 of them similar to those of C. denticulata and the remaining to C. nevadensis. GISH analysis confirmed the presence of both subgenomes in the allotetraploid C. veatchii. However, the number of rDNA sites and the haploid karyotype length in C. veatchii were not additive. The diploid parentals had already diverged in their chromosomes structure, whereas the reduction in the number of rDNA sites more probably occurred after hybridization. As phylogenetic data suggested a recent divergence of the progenitors, these species should have a high rate of karyotype evolution.

Interstitial telomeric sites and Robertsonian translocations in species of Ipheion and Nothoscordum (Amaryllidaceae).

The genera Nothoscordum and Ipheion (Allioideae, Amaryllidaceae) are cytologically characterized by a dysploid series with variable numbers of metacentric and acrocentric chromosomes typical of karyotypes rearranged by Robertsonian translocations (RT). Since they have large chromosomes, low diploid numbers, and possess two telomeric motifs [the vertebrate-type (TTAGGG) n and the Arabidopsis-type (TTTAGGG) n ] they are suitable for investigating the occurrence and possible role of interstitial telomeric sites (ITS) associated with RT. We analyzed the distributions of telomeric sites in 12 species of Nothoscordum and Ipheion and found that both telomeric probes colocalized in all chromosome termini. Cloning and sequencing PCR products obtained using both telomeric primers simultaneously revealed long stretches of (TTAGGG) n and (TTTAGGG) n sequences together with degenerated telomeric sequences. Most acrocentric chromosomes have a 45S rDNA site at the terminal region of the short arms adjacent to the most distal telomeric sites. Telomeric signals were found at all chromosome ends, but ITS were also detected in a few proximal and subterminal regions in some Nothoscordum species. Although RT are common in this group of plants, our findings suggest that proximal positioning of telomeric motifs are not necessarily related to that kind of rearrangement. Rather, transposition of telomeric sequences followed by amplification, could better explain the presence of (TTAGGG) n and (TTTAGGG) n repeats at those sites. Furthermore, a few small interstitial sites found in some Nothoscordum species indicate that dispersion of these sequences was not restricted to the proximal region.

Agmatoploidy and symploidy: a critical review.

Agmatoploidy is a type of chromosome rearrangement that involves the fragmentation of an entire chromosome complement, generating a diploid with double its original chromosome number. Agmatoploidy and other related karyotype changes, such as symploidy (the opposite change, promoted by chromosome fusion), partial agmatoploidy, polyagmatoploidy, etc., are restricted to species with holokinetic chromosomes and are assumed to play an important role in their karyotype evolution. However, a critical review of the literature shows that examples of chromosome number doubling by agmatoploidy are rare and not clearly demonstrated, while partial agmatoploidy and partial symploidy seem to be the same as ascending and descending disploidy, respectively. It is therefore proposed here that these terms should be avoided or even abolished.

Non-Random Distribution of 5S rDNA Sites and Its Association with 45S rDNA in Plant Chromosomes.

5S and 45S rDNA sites are the best mapped chromosome regions in eukaryotic chromosomes. In this work, a database was built gathering information about the position and number of 5S rDNA sites in 784 plant species, aiming to identify patterns of distribution along the chromosomes and its correlation with the position of 45S rDNA sites. Data revealed that in most karyotypes (54.5%, including polyploids) two 5S rDNA sites (a single pair) are present, with 58.7% of all sites occurring in the short arm, mainly in the proximal region. In karyotypes of angiosperms with only 1 pair of sites (single sites) they are mostly found in the proximal region (52.0%), whereas in karyotypes with multiple sites the location varies according to the average chromosome size. Karyotypes with multiple sites and small chromosomes (<3 µm) often display proximal sites, while medium-sized (between 3 and 6 µm) and large chromosomes (>6 µm) more commonly show terminal or interstitial sites. In species with holokinetic chromosomes, the modal value of sites per karyotype was also 2, but they were found mainly in a terminal position. Adjacent 5S and 45S rDNA sites were often found in the short arm, reflecting the preferential distribution of both sites in this arm. The high frequency of genera with at least 1 species with adjacent 5S and 45S sites reveals that this association appeared several times during angiosperm evolution, but it has been maintained only rarely as the dominant array in plant genera.

The efficacy of two oral hygiene regimens in reducing oral malodour: a randomised clinical trial.

OBJECTIVES: This study compared the efficacy of two oral hygiene regimens in reducing oral malodour and the proportions of bacterial species involved in the production of volatile sulphur compounds. MATERIAL AND METHODS: Seventy subjects who participated in a halitosis-induction phase and achieved an organoleptic score of ≥ 3.0 [time point 0 (T0)] randomised into two groups: brushing with regular fluoride toothpaste alone (control group) or brushing with regular fluoride toothpaste followed by rinsing with a 0.075% cetylpyridinium chloride (CPC) mouthwash (CPC group). Subjects followed their assigned oral hygiene regimen for 21 days. Then, they underwent an organoleptic examination and measurement of volatile sulphur compounds (VSCs) using a portable gas chromatograph, 12 hours after their last oral hygiene procedure (T1) and 4 hours after an on-site oral hygiene (T2). Microbiological samples (supragingival biofilm, tongue coating and saliva) were analysed using checkerboard DNA-DNA hybridisation. RESULTS: Both therapies statistically significantly improved the organoleptic scores (P < 0.05), but the VSC levels and/or concentrations were reduced only in the CPC group (P < 0.05). In subjects rinsing with CPC, oral malodour scores were reduced by 49% at the 4-hour assessment (T2) compared with those not rinsing (P < 0.05). Red-complex pathogens were reduced more effectively in the CPC group than in the control group. CONCLUSIONS: Brushing followed by rinsing with a 0.075% CPC mouthwash provided statistically significantly greater reductions in oral malodour, measured organoleptically and instrumentally, and in the proportions of red-complex species when compared with brushing alone.

Different patterns of chromosomal histone H3 phosphorylation in land plants.

The chromosomal phosphorylation of histone H3 during mitosis and meiosis seems to play a fundamental role in the control of cell division in all eukaryotes. In plants, the temporal and spatial distribution of H3S10 phosphorylated (H3S10ph) is currently known only for chromosomes of a few angiosperms. In the present study, we analyzed the pattern of H3S10ph in mitotic chromosomes of 14 plant species, including 2 bryophytes and 12 tracheophytes. In all species, the phosphorylation of H3S10 was cytologically detected first in prophase and disappeared in late anaphase. Two patterns of chromosomal phosphorylation were observed: (1) only the pericentromeric regions were hyperphosphorylated, whereas the remaining chromosome arms appeared weakly and diffusely immunolabeled, and (2) the whole chromosomes appear uniformly phosphorylated, increasing the labeling intensity at metaphase. The first pattern was observed in all tracheophytes with monocentric chromosomes, whereas the second one was restricted to the bryophytes and angiosperms with holokinetic chromosomes. In both cases, no particular kind of chromatin such as NORs or heterochromatic bands were differentially labeled. Based on this data and previous analyses in other eukaryotes, we suggest that hyperphosphorylation of the whole mitotic metaphase chromosome represents the ancestral condition for eukaryotic chromosomes, and the change to the pericentromeric pattern occurred in the transition from bryophytes to tracheophytes. The meaning and possible implications of these results are discussed in the light of recent and classical experiments.

Evaluation of micro-energy dispersive X-ray fluorescence and histochemical tests for aluminium detection in plants from High Altitude Rocky Complexes, Southeast Brazil.

The soils developed under High Altitude Rocky Complexes in Brazil are generally of very low chemical fertility, with low base saturation and high exchangeable aluminium concentration. This stressful condition imposes evolutionary pressures that lead to ecological success of plant species that are able to tolerate or accumulate high amounts of aluminium. Several analytical methods are currently available for elemental mapping of biological structures, such as micro-X-ray fluorescence (µ-EDX) and histochemical tests. The aim of this study was to combine µ-EDX analysis and histochemical tests to quantify aluminium in plants from High Altitude Rocky Complexes, identifying the main sites for Al-accumulation. Among the studied species, five showed total Al concentration higher than 1000 mg kg-1. The main Al-hyperaccumulator plants, Lavoisiera pectinata, Lycopodium clavatum and Trembleya parviflora presented positive reactions in the histochemical tests using Chrome Azurol and Aluminon. Strong positive correlations were observed between the total Al concentrations and data obtained by µ-EDX analysis. The µ-EDX analysis is a potential tool to map and quantify Al in hyperaccumulator species, and a valuable technique due to its non-destructive capacity. Histochemical tests can be helpful to indicate the accumulation pattern of samples before they are submitted for further µ-EDX scrutiny.

B chromosomes of rye are highly conserved and accompanied the development of early agriculture.

BACKGROUND AND AIMS: Supernumerary B chromosomes (Bs) represent a specific type of selfish genetic element. As Bs are dispensable for normal growth, it is expected to observe B polymorphisms among populations. To address whether Bs maintained in geographically distinct populations of cultivated and weedy rye are polymorphic, the distribution patterns and the transcriptional activity of different B-located repeats were analysed. METHODS: Bs of cultivated and weedy rye from seven origins were analysed by fluorescence in situ hybridization (FISH) with probes specific for the pericentromeric and interstitial regions as well as the B-specific non-disjunction control region. The DNA replication, chromatin composition and transcription behaviour of the non-disjunction regions were determined. To address whether the B-marker repeats E3900 and D1100 have diverged genotypes of different origin at the sequence level, the genomic sequences of both repeats were compared between cultivated rye and weedy rye from five different origins. KEY RESULTS: B chromosomes in cultivated and weedy rye have maintained a similar molecular structure at the level of subspecies. The high degree of conservation of the non-disjunction control region regarding its transcription activity, histone composition and replication underlines the functional importance of this chromosome region for the maintenance of Bs. The conserved chromosome structure suggests a monophyletic origin of the rye B. As Bs were found in different countries, it is likely that Bs were frequently present in the seed material used in early agriculture. CONCLUSIONS: The surprisingly conserved chromosome structure suggests that although the rye Bs experienced rapid evolution including multiple rearrangements at the early evolutionary stages, this process has slowed significantly and may have even ceased during its recent evolution.

CMA/DAPI Banding of Plant Chromosomes.

Chromosome banding based on base-specific fluorochromes, mainly double staining with chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI), has been widely used since the 1970s. This technique allows the differential staining of distinct types of heterochromatin. Afterward, the fluorochromes can be easily removed and leave the preparation ready for sequential procedures such as FISH or immunodetection. Interpretations of similar bands obtained with different techniques, however, merit certain caution. Here we present a detailed protocol for CMA/DAPI staining optimized for plant cytogenetics and call attention to the most common sources of misinterpretation of DAPI bands.

Distribution of 45S rDNA sites in chromosomes of plants: structural and evolutionary implications.

BACKGROUND: 45S rDNA sites are the most widely documented chromosomal regions in eukaryotes. The analysis of the distribution of these sites along the chromosome in several genera has suggested some bias in their distribution. In order to evaluate if these loci are in fact non-randomly distributed and what is the influence of some chromosomal and karyotypic features on the distribution of these sites, a database was built with the position and number of 45S rDNA sites obtained by FISH together with other karyotypic data from 846 plant species. RESULTS: In angiosperms the most frequent numbers of sites per diploid karyotype were two and four, suggesting that in spite of the wide dispersion capacity of these sequences the number of rDNA sites tends to be restricted. The sites showed a preferential distribution on the short arms, mainly in the terminal regions. Curiously, these sites were frequently found on the short arms of acrocentric chromosomes where they usually occupy the whole arm. The trend to occupy the terminal region is especially evident in holokinetic chromosomes, where all of them were terminally located. In polyploids there is a trend towards reduction in the number of sites per monoploid complement. In gymnosperms, however, the distribution of rDNA sites varied strongly among the sampled families. CONCLUSIONS: The location of 45S rDNA sites do not vary randomly, occurring preferentially on the short arm and in the terminal region of chromosomes in angiosperms. The meaning of this preferential location is not known, but some hypotheses are considered and the observed trends are discussed.

Cytogenetic and molecular evidence suggest multiple origins and geographical parthenogenesis in Nothoscordum gracile (Alliaceae).

BACKGROUND AND AIMS: Nothoscordum gracile is an apomitic tetraploid widely distributed throughout the Americas and naturalized in many temperate regions of other continents. It has been suggested to form a species complex with sexual and apomictic N. nudicaule and N. macrostemon. Tetraploids of these species also share a structurally heterozygous chromosome complement 2n = 19 (13M + 6A). In this work, the origin of N. gracile and its relationships with its related species was investigated based on cytological and molecular data. METHODS: Cytogenetic analyses were based on meiotic behaviour, CMA bands, localization of 5S and 45S rDNA sites, and genomic in situ hybridization (GISH). Nuclear ITS and plastidial trnL-trnF sequences were also obtained for most individuals. KEY RESULTS: Proximal CMA bands were observed in the long arms of all acrocentrics of 2x and 4x N. macrostemon but not in diploid and some tetraploid cytotypes of N. nudicaule. Samples of N. gracile showed a variable number of CMA bands in the long arms of acrocentrics. Analysis of ITS sequences, dot-blot, GISH, and 5S and 45S rDNA sites, revealed no differentiation among the three species. The trnL-trnF cpDNA fragment showed variation with a trend to geographical structuring irrespective of morphospecies and fully congruent with karyotype variation. CONCLUSIONS: The 2n = 19 karyotype was probably formed by a centric fusion event occurring in N. nudicaule and later transmitted to tetraploid cytotypes of N. macrostemon. Diploids of N. nudicaule and N. macrostemon appeared as consistent recently diverged species, whereas tetraploid apomicts seem to constitute an assemblage of polyploid hybrids originating from multiple independent hybridization events between them, part of which are morphologically recognizable as N. gracile.

Diversification of the American bulb-bearing Oxalis (Oxalidaceae): dispersal to North America and modification of the tristylous breeding system.

PREMISE OF THE STUDY: The American bulb-bearing Oxalis (Oxalidaceae) have diverse heterostylous breeding systems and are distributed in mountainous areas from Patagonia to the northeastern United States. To study the evolutionary processes leading to this diversity, we constructed the first molecular phylogeny for the American bulb-bearing Oxalis and used it to infer biogeographic history and breeding system evolution. METHODS: We used DNA sequence data (nuclear ribosomal internal transcribed spacer, trnL-trnL-trnF, trnT-trnL, and psbJ-petA) to infer phylogenetic history via parsimony, likelihood, and Bayesian analyses. We used Bayes Multistate to infer ancestral geographic distributions at well-supported nodes of the phylogeny. The Shimodaira-Hasegawa (SH) test distinguished among hypotheses of single or multiple transitions from South America to North America, and tristyly to distyly. KEY RESULTS: The American bulb-bearing Oxalis include sampled members of sections Ionoxalis and Pseudobulbosae and are derived from a larger clade that includes members of sections Palmatifoliae, Articulatae, and the African species. The American bulb-bearing Oxalis comprise two clades: one distributed in SE South America and the other in the Andes and North America. An SH test supports multiple dispersals to North America. Most sampled distylous species form a single clade, but at least two other independent distylous lineages are supported by the topologies and SH tests. CONCLUSIONS: Phylogenetic results suggest the American bulb-bearing Oxalis originated in southern South America, dispersed repeatedly to North America, and had multiple transitions from tristyly to distyly. This study adds to our understanding of biogeographic history and breeding system evolution and provides a foundation for more precise inferences about the study group.

Satellite DNA probes of Alstroemeria longistaminea (Alstroemeriaceae) paint the heterochromatin and the B chromosome, reveal a G-like banding pattern, and point to a strong structural karyotype conservation.

Alstroemeria species present a well-conserved and asymmetric karyotype. The genus is divided into a Chilean clade, rich in heterochromatin, and a Brazilian clade, poor in heterochromatin. We investigated the distribution of the main repetitive sequences in the chromosomes of the Brazilian species A. longistaminea (2n = 16 + 0-6B) aiming to evaluate the role played by these sequences on the structural organization of the karyotype. In situ hybridization of the three most abundant retrotransposons, corresponding to ~ 45% of the genome, was uniformly distributed. Three satellite DNA sequences, representing near half of the whole satellite fraction (1.93% of the genome), were mainly concentrated on the heterochromatin and one of them painted the whole B chromosome. Noteworthy, some satellites were located on euchromatin, either dispersed or concentrated in clusters along the chromosomes, revealing a G-band-like pattern. The two satellites that presented more C-band- and G-band-like labeling were also hybridized in situ in two other Alstroemeria species. They revealed astonishing similar patterns of distribution, indicating an unusually structural karyotype conservation among Brazilian species.

Low serum levels of vitamin D significantly increase the risk of death in older adults with hip fractures: a prospective cohort.

OBJECTIVE: to evaluate the relationship between 25(OH)D3 levels and fatal outcome in patients over 60 years of age undergoing surgical repair of hip fractures. METHODS: prospective cohort of patients undergoing surgical repair of hip fractures. At admission, 25(OH)D3 levels were measured, among other parameters. Patients were followed for at least 1 year, and incident mortality was recorded. RESULTS: 209 patients were included in the study, with a mean age of 79.5 ± 7.6 years among survivors and 80.7 ± 8.2 years among those who died in the first postoperative year (p=0.346). The 25(OH)D3 levels of survivors were significantly higher than those of patients who died (p=0.003). After adjusting for confounding variables, 25(OH)D3 levels below 12.5ng/mL were significant risk factors regardless of mortality (adjusted OR: 7.6; 95% CI: 2.35 to 24.56). CONCLUSIONS: our data show that serum 25(OH)D3 levels below 12.5ng/mL significantly and independently increased the risk of mortality in the first year after surgical repair of low-energy hip fracture in patients over 60 years of age in the geographic region where this study was conducted. Low albumin also showed a significant association with mortality in these patients. All other factors had no significant associations.

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA.

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.

The evolution of CMA bands in Citrus and related genera.

Most species of Citrus and related genera display a similar karyotype with 2n = 18 and a variable number of terminal heterochromatic blocks positively stained with chromomycin A(3) (CMA(+) bands). Some of these blocks are 45S rDNA sites, whereas others may correspond to the main GC-rich satellite DNA found in several Citrus species. In the present work, the distribution of the 45S rDNA and the main satellite DNA isolated from C. sinensis (CsSat) were investigated by in situ hybridization in seven species of Citrus, two species of closely related genera (Fortunella obovata and Poncirus trifoliata) and four species of the subfamily Aurantioideae, which were less related to Citrus (Atalantia monophylla, Murraya paniculata, Severinia buxifolia, and Triphasia trifolia). In Citrus, Fortunella, and Poncirus, most CMA(+) bands colocalized only with CsSat sites, whereas others colocalized only with rDNA sites. However, some of these species displayed a few CMA(+) bands that colocalized with sites of both probes and other CMA(+) bands that did not colocalized with any of the probes. On the other hand, in the four species less related to Citrus, no CsSat signal was found on chromosomes. On Southern blot, the CsSat probe hybridized with genomic DNA from Citrus, Fortunella, and Poncirus at high stringency only, while under the less stringent conditions, it also hybridized with distantly related species. Therefore, CsSat sequences are the principal component of the heterochromatic blocks of Citrus, Poncirus, and Fortunella, whereas CsSat-like sequences seem to be widespread in the subfamily Aurantioideae. These data further suggest that the variable number of terminal CMA(+) bands observed on chromosomes of Citrus and related genera are probably the consequence of amplification or reduction in the number of CsSat-like sequences distributed on chromosome termini, paralleled by mutation and homogenization events, as proposed by the library hypothesis.