text:
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.
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.
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.
Keywords:45S rDNA sites; Chromosome regions; Secondary constrictions; NOR; Meta-analysis
When observed under an optical or electronic microscope, the chromosomes of plants and other eukaryotes seem to be uniform structures, except for the primary and secondary constrictions. However, based on some functional and molecular characteristics, at least five distinct regions can be recognized along each chromosome arm: telomere, subtelomeric region, interstitial region, proximal region and centromere. The distribution of genes, retrotransposons, satellite DNA and other sequences within these regions seems to follow general trends related to the frequency of recombination and the functional role of these sequences [1-4]. Therefore, the structural regularity does not reflect the different selection pressures that different regions of the chromosome arm suffer, creating sub-regions more permissive or more prohibitive for the establishment of a specific sequence [4-6].
Lima-de-Faria [7] showed that the secondary constrictions of mitotic chromosomes, which bear the 18S-5.8S-25S ribosomal RNA genes transcribed in the previous interphase, also called the nucleolus organizer regions (NORs) or 45S rDNA sites, were preferentially distributed on the short arms and in the subterminal region in most species of plants and animals. The author compared the frequency of secondary constrictions in the proximal, interstitial and terminal regions of the chromosome arm, taking into account the size of the arms (arm frame method). Currently, by using the fluorescence in situ hybridization (FISH) technique, it is known that the secondary constrictions represent only the expression of rRNA genes which were active during the last interphase and that other functional sites may not form secondary constrictions, especially if located too close to the terminal end of the chromosomes [8]. In Vigna unguiculata, for example, three pairs of rDNA sites not previously detected as secondary constrictions were revealed by FISH [9]. The fact that the 18S-5.8S-25S rRNA genes are arranged in hundreds or thousands of tandem repeats and are highly conserved between species made of this chromosome region the most widely investigated by FISH, being the position of these sites known in hundreds of plant species, especially in angiosperms.
In this study, the general pattern of distribution of rDNA sites in plant chromosomes was addressed to establish whether these genes are randomly distributed or occupy preferred regions on chromosome and whether certain chromosomal or karyotypic characteristics influence this distribution. Karyotypic data including information about rDNA sites of 846 species of angiosperms and gymnosperms were compiled from the literature. Other groups of plants have few representatives studied and do not allow generalizations. In pteridophytes, the rDNA sites have been observed only in four genera (Osmunda, Ceratopteris, Acrostichum[10] and Selaginella[11]), all of them with terminal or subterminal sites. In bryophytes, data were found only for Marchantia polymorpha[12] and species of Pellia[13], all of them with rDNA sites in terminal position. In a recent paper, a rDNA database containing crude data from a similar sample of plant species has been developed allowing easy access to a large amount of data [14].
In some cases, the analysis was done separately in karyotypes containing only one pair of sites and karyotypes with more than one pair of sites, considering that in those with only one pair of sites they should always be functionally active. In karyotypes with multiple sites the assumption that they had a tendency to equilocal distribution [15-17] was evaluated. As polyploid plants tend to diploidize reducing the original monoploid genome size [18,19], the effect of polyploidy in the number and position of rDNA sites in the intraspecific and intrageneric levels was also evaluated.
Results and discussion
Number of rDNA sites in angiosperms and gymnosperms
In this paper we compiled all the information about number and position of rDNA sites we had access until early 2011. A total of 846 species and 198 genera were included, representing 51 families of angiosperms and six families of gymnosperms. Among angiosperms, 42 species stand out for having holokinetic or holocentric chromosomes, i.e., chromosomes with a large kinetochore plate extended along almost their whole length [20], which will be treated in a separate topic. Since some species were polymorphic or had different cytotypes, a total sample of 984 karyotypes was analyzed, 921 and 63 of them having monocentric or holokinetic chromosomes, respectively.
The results obtained here seem to represent well the diversity in plants, because of the wide diversity of taxa and karyotypic variations sampled. The number of rDNA sites observed was very variable, ranging from just one pair of sites per chromosome complement in most angiosperms up to 45 sites in Kyllinga brevifolia[21]. In angiosperms with monocentric chromosomes, the average number of sites per somatic chromosome complement was 5.1 (3.6) (mean and standard deviation) ranging from two to 32, with mode equal to two and a median of four (Table 1). Figure 1a shows that there was a high frequency of diploid karyotypes with two or four sites and a progressive reduction in the number of karyotypes with higher numbers of sites. When plolyploids were included in the sample the modal number of sites per karyotype (2) differed from the second more common number (4) by a very small difference: 265 out of the 856 karyotypes had two sites per somatic chromosome complement, whereas 263 karyotypes had four and 328 had three or more rDNA sites. The modal number of rDNA sites in angiosperms can easily oscillate between two and four depending on the sampling.
Table 1. Number of rDNA sites per somatic chromosome complement (2n) in species of angiosperms and gymnosperms
Figure 1. Variation in the number of rDNA sites per karyotype in a sample of 665 angiosperm (a) and 64 gymnosperm (b) karyotypes. Polyploid cytotypes and species with holokinetic chromosomes were not included. The median is underlined and the modal number is indicated by #. Sample size: 729 karyotypes.
In gymnosperms, the average number of sites 10.4 (7.1) was more than twice that in angiosperms, although the modal number of sites was two, as in angiosperms (Table 1, Figure 1b). The presence of multiple sites in some genera as Picea[22], Pinus[23,24] and Zamia[25], upraised the median of this group to 10. In general, gymnosperms have a larger number of copies of these genes [26] and a larger average genomic size than angiosperms, probably meaning that they are more tolerant to amplifications [27]. However, a larger number of 45S rDNA sites does not mean more functional copies of these genes, either because the size of the sites is very variable or because the rDNA probe can also detect non-functional or permanently blocked copies of these genes [28,29]. In karyotypes with a single pair of 45S rDNA sites, here referred as single site karyotypes, both sites are necessarily functional whereas karyotypes with multiple sites may eventually include some non-functional or inactive sites. Therefore, in some cases it is important to visualize the distribution of single site and multiple sites karytotypes separately.
rDNA sites often display intraspecific heteromorphism in number and size (see e.g. [30,31]). For example, in the common bean Phaseolus vulgaris, we found that the number of 45S rDNA sites varied from six to 18 per accession and their size varied widely within chromosome pairs [32]. Some sites may be below the limit of detection by FISH and their eventual amplification in different populations may account for the numerical polymorphism. It is also possible that inactive rDNA sites are more likely to show intraspecific polymorphisms and eventually be eliminated [33]. This dynamics of inactivation and subsequent deletion of rDNA sites seem to counteract the mechanisms of duplication and dispersion of 45S rDNA repeats, leading to the observation of only a small number of sites in most of the species.
Distribution of rDNA sites along the chromosome
729 karyotypes of angiosperms and gymnosperms have been used for determining the number of rDNA sites per chromosomal arm, 509 of them displaying multiple sites (Table 2, Figure 2). The total number of rDNA sites included in this sample was 3,966. In some karyotypes the position of the site was just between the two chromosome arms and the signal seemed to coincide exactly with the position of the centromere. In these cases, the sites were referred to as "centromeric" (cen), without implying that they were really located in the functional region of the centromere. In general, 69.9% of the rDNA sites were located on the short arm. The frequency of rDNA sites in the short arm was higher among karyotypes with single sites (77.7%) than in those with multiple sites (68.9%), suggesting that when the number of sites increases, the trend to be preferentially positioned on the short arm decreases. According to Lima-de-Faria [7] 86.6% of secondary constrictions of animal and plant species occur on the short arms, a value higher than that observed here (p <0.01). This divergence is at least partially due to the techniques employed. In the case of Lima-de-Faria, he detected only secondary constrictions, that is, rDNA sites which were active in the last interphase, whereas in the present report, using FISH, all kind of 45S rDNA cluster, including non-active and very small ones, were detected.
Table 2. Frequency of rDNA sites in the short and long chromosome arms
Figure 2. Percentage frequency of rDNA sites along chromosomes of angiosperms (a) and gymnosperms (b). Total number of sites: 3.287 in a and 679 in b. In b, the only eleven karyotypes with single sites, were represented together with multiples sites. To stress that some distributions are particular for some gymnosperm families, they were shown distinctly (cen, centromeric region; p, proximal; ip, interstitial-proximal; it, interstitial-terminal; t, terminal; wa, whole arm sites).
In angiosperms, we observed that rDNA sites occupied preferentially (50.1%) the terminal position of the chromosomes, both in karyotypes with single site (36.8%) and in karyotypes with multiple sites (52.0%) (Figure 2 and Additional file 1: Table S1). It is worth mentioning that 23.0% of the single sites and 15.5% of multiple sites were represented by signals that occupied the whole chromosome arm (wa sites) and these sites were not computed as terminal ones. In many karyotypes, whole arm sites should represent a technical artifact generated by over-exposition of the signal during image acquirement whereas in other karyotypes they seem to correspond to the whole visible chromatin of the short arm, as in species of Nothoscordum and Ipheion[34]. If they were included as terminal sites, the frequency of rDNA sites in the terminal region would be more similar for single (59.8%) and multiples sites (67.5%). A higher percentage of terminal secondary constrictions (85.2%) was observed by Lima-de-Faria in a sample of 189 species of eukaryotes (p<0.01) including two families of plants [35]. One of them (Asteraceae) showed also a higher percentage of terminal rDNA sites in the present study. Whole arm sites were always located on the short arm (Figure 2), except in Solanum pennellii, where it spanned the entire slightly longer arm [36]. Therefore, considering that the chromosome arm was divided into four regions of equal size (p, ip, it, t), there seems to be a strong positive selection favoring the location of 45S rDNA sites in the terminal region. On the other hand, in gymnosperms the rDNA sites were located mainly in the interstitial-terminal (52.4%) or proximal (21.8%) regions. These frequencies were strongly influenced by members of Pinaceae and Zamiaceae families which had a large number of species investigated, each one displaying multiple sites. The terminal position of rDNA sites (18.9%) was largely dominant in the less sampled families of gymnosperms.
Additional file 1. Supplementary tables. Tables showing details about karyotypes and analyses. (XLS 1198 kb)
Format: XLS Size: 1.2MB Download file
This file can be viewed with: Microsoft Excel Viewer
The location of rDNA sites in the terminal chromosome region in most angiosperms may be the result of homologous recombination constrains. Due to its repetitive organization in tandem, rDNA sites may be subject to a higher rate of allelic and non-allelic homologous recombinations, which play a fundamental role in the homogenization of intralocus and interloci repeats [37]. Homogenization is an essential process to reduce the nuclear variability of this key molecule, ensuring the removal of non-functional units from the clusters [38]. The terminal or subterminal position of rDNA sites allows the occurrence of rearrangements without deleterious effects related to gene balance and meiotic segregation [32,39]. Nonetheless, various genera of plants and animals have interstitial or proximal 45S rDNA sites [40] and display at least partial inter-loci homogenization [41,42]. In addition, repetitive sequences of 45S rDNA can also protect the telomere, in a way similar to subtelomeric DNA [43-45]. In Allium cepa, 45S rDNA acts as a substitute of telomeric DNA at some chromosomal termini [43].
The particular case of acrocentric chromosomes
During the compilation of the data, it was often observed that acrocentric chromosomes had rDNA sites on the short arm, as for example in species of Ipheion[35], Nothoscordum[34,46], Rumex[47], and Alstroemeria[48]. To assess whether the short arm of acrocentric chromosomes is a especially preferred region for rDNA cluster location, 266 species with single site or multiple sites having at least one acrocentric chromosome per karyotype were analyzed. In this sample, 36.2% of the chromosomes were acrocentrics and 53.6% of the sites occurred on the long or short arm of these chromosomes. Considering only the acrocentric chromosomes, 64.7% of the sites occurred on the short arm and most of them (70.1%) occupied the entire short arm (wa sites). Therefore, considering that the short arm corresponds to no more than 25% of the length of the acrocentric chromosome [49], there is indeed a very high frequency of rDNA sites in the short arms of acrocentrics.
The rationale for this association is not clear. In some karyotypes the occurrence of a whole-arm site seems to be associated with a centric fission event, as observed in a rearranged karyotype of Hypochaeris radicata[50] and in species of Ipheion[35] and Nothoscordum[34,51]. Strong evidence for the association of rDNA with fission was also reported for hymenoptera and mice [42,52]. In these cases, the centric fission may have been preceded by insertion of 45S rDNA repeats in the centromeric region of a bi-armed chromosome, or both telocentrics may have acquired rDNA sequences in the new terminal regions after fission [50]. However, not all karyotypes with centric fission have extra rDNA sites and in some cases they may have the number of rDNA sites reduced [53]. In addition, though 45S rDNA are found on the short arms of acro/telocentric chromosomes in several plant and animal species, usually they are not associated with centric fissions [40]. Alternatively, the very short arms of acrocentric chromosomes may be a more accessible site for rDNA transposition and may allow a quicker interloci homogenization of rDNA sequences than the proximal region of metacentric chromosomes [54].
Distribution of rDNA sites in different taxa
An analysis of the distribution of rDNA sites by the arm frame method was carried out both in the whole sample and separately by family, aiming to identify specific trends (details of the arm frame construction are indicated in Material and Methods. 1201 rDNA bearing chromosomes from 457 karyotypes (420 species) with single site or multiple sites were included in this analysis. The graph of the arm frame revealed a higher concentration of sites in the terminal region while the proximal half of arms was particularly poor in sites (Figure 3), as described for the distribution of secondary constrictions in chromosomal arms [7]. Because the resolution of sites in the smaller arms was not very clear, the data of absolute distances of the sites to the centromeres were converted to relative distances (considering the centromere-telomere distance as 100% of the arm). Since angiosperms and gymnosperms have different distribution patterns of rDNA sites (Figures 1 and 2), these two groups of plants were distinctly represented in Figure 4. In this figure we can see that the higher occurrence of interstitial sites may be associated to gymnosperms, as previously stated, but it is also influenced by the different chromosome arm sizes. The interstitial distribution of sites on chromosome arms larger than 2 or 3 μm is characteristic of the largely sampled Poaceae [55,56] while in shorter arms the site position is more variable (Figure 4, Additional file 2: Figures S1, S2). The statement of Lima-de-Faria [7] that as the distance between centromere and telomere increases the ribosomal genes are successively displaced maintaining a relative distance to centromere and telomere seems to apply for most genera but not for every family. This is especially apparent when the position of sites is visualized in terms of percentages (Additional file 2: Figures S1, S2).
Additional file 2. Supplementary figures. Distribution of rDNA sites for the best sampled families and genera. (PDF 827 kb)
Format: PDF Size: 828KB Download file
This file can be viewed with: Adobe Acrobat Reader
Figure 3. Distribution of 45S rDNA sites in the arm frame. Chromosomes are represented as grey lines aligned by centromeres (0) and sites as short bold lines. 46 chromosomes with sites in both arms were drawn twice in opposite directions. Sample size: 419 angiosperms and 38 gymnosperms karyotypes (383 and 37 species, respectively).
Figure 4. Relative distribution of rDNA sites along the chromosome arm in 397 angiosperm (+) and 38 gymnosperm (o) karyotypes. The distance from the site to the centromere was represented as a percentage value in relation to the centromere-telomere length of the arm bearing the rDNA site. Whole arm (wa) sites were not included in this sample.
Figure 5. Percentage frequency of rDNA sites along the chromosome arm in the most sampled families of angiosperms (a) and gymnosperms (b). The sites were positioned in one of four equal-sized regions of the arm or spanning the whole arm. p, proximal; ip, interstitial-proximal; it, interstitial-terminal; t, terminal; wa, whole arm. The number of karyotypes analyzed in each family appears in brackets.
The distribution of rDNA sites may also be related to karyotype characteristics or chromosomal organization of specific taxa. For example, during the interphase, centromeres are often clustered in one pole of the cell and telomeres in the opposite pole, as a consequence of the anaphase movement. This kind of chromosome distribution in the nucleus, named Rabl orientation, seems to be more common in species with large chromosomes than in those with small ones [57,58]. In wheat, for example, with large chromosomes and large genome, telomeres are associated with the nuclear envelope in one pole of the nucleus whereas in Arabidopsis species, with much smaller chromosomes and genomes, telomeres are organized around nucleolus [57,59,60].
The distribution of sites by sub-regions of the chromosome arm (p, ip, it, t) is also not the same in different taxa. In Amaryllidaceae, Asteraceae, Cycadaceae, Ginkgoaceae, Poaceae, Podocarpaceae and Solanaceae the sites were predominantly terminal, while in Asparagaceae, Cupressaceae, Fabaceae, Pinaceae and Zamiaceae, the most common position was proximal to interstitial (Figure 5). Within some families, such as Solanaceae, the distribution of sites was similar in most genera (Additional file 2: Figure S3), while in others, such as Fabaceae, the sites were predominantly or exclusively proximal in some genera, such as Arachis[61-64] and Lens[65], and mainly terminal in others, as Phaseolus[66,67] and Vicia[68-71] (Additional file 2: Figure S3). The stability of sites in Solanaceae may be related to the karyotype stability of this group [72] although conservation in chromosome number and morphology is not enough to ensure stability of rDNA site position as observed in other taxa with apparently stable karyotype, as Citrus[73] and Pinus[74].
Distribution of rDNA sites in holokinetic chromosomes
A special case of rDNA distribution was observed in species with holokinetic chromosomes. This group is characterized by absence of a localized centromere (primary constriction) and the occurrence of a large kinetochore plate along almost its entire length [75]. Among 63 karyotypes with holokinetic chromosomes (42 species of 7 genera), the average number of sites per karyotype was 6.6 (6.6) and the mode and median were equal to 4.0, therefore, quite similar to those of angiosperms with monocentric chromosomes (Table 1). Although the number of sites was quite variable, 100% of them were located in the terminal region. In the genus Rhynchospora, for example, the number of sites ranged from 2 to ~30, but the position was always terminal [21,76]. Also in Caenorhabditis elegans and several species of insects with holokinetic chromosomes the 45S rDNA site is located terminally [77-79]. This suggests that in organisms with holokinetic chromosomes there are tighter constraints on the establishment of non-terminal sites. A possible explanation is that a secondary constriction in the interstitial region would interrupt the kinetochore plate along the holokinetic chromosome establishing a condition similar to a dicentric chromosome, with consequent errors in mitotic segregation [75]. However, this assumption has yet to be demonstrated and at least in Lepidoptera it does not seems to apply [80].
Equilocality of rDNA in karyotypes with multiple sites
The equilocality or equal positioning of rDNA sites in relation to centromere or telomere has been previously observed in several genera [15,17,25,31,34]. To assess the frequency of equilocality of rDNA sites per chromosome complement, 243 diploid karyotypes with multiple sites were analyzed and the distance of each site to centromere and telomere were measured. Two sites were considered equilocated when they were at the same distance from the centromere or telomere, or at a distance less than 1/10 of the length of the largest chromosome arm bearing rDNA site. In relation to the centromere, 67.7% of karyotypes had at least two sites equilocalized and only 38.7% of karyotypes had all sites equally located. Regarding the telomere, the frequency of equilocality was significantly higher (89.9% and 73.8%, respectively - p<0.01).
Equilocality of rDNA sites seems to be promoted by ectopic recombination between regions of different chromosomes located in close proximity during prophase or interphase [39]. This spatial distribution could be facilitated by Rabl configuration in mitosis [15] or the bouquet configuration during meiotic prophase, where all telomeres attach to one pole of the nuclear envelope [81]. However, in species in which Rabl configuration does not occur, as Arabidopsis and human [82,83], as well as in those with holokinetic chromosomes [21,84], all rDNA sites are also terminal and equilocal, suggesting that Rabl orientation during interphase is not necessary to maintain the regularity in the position of rDNA. The bouquet configuration, on the other hand, is a more universal phenomenon [85] and could better explain the equilocality since it promotes the confluence of all telomeres to a limited region of the nuclear envelope. In this case, the dispersion of rDNA sites would occur mainly among chromosome regions that are equidistant from the telomere, while centromeres are not directly co-oriented in the bouquet (see also [42]). This may explain the high frequency of karyotypes with all sites equilocated in relation to telomeres (73.8%) and a relatively low frequency of equilocality in relation to centromeres (38.7%).
Changes in the number of rDNA sites in relation to its position on chromosomes
During early meiotic prophase nuclei exhibit a highly polarized and clustered arrangement of chromosome ends, called 'bouquet'. Assuming that the dispersion of sites is mediated mainly by the bouquet configuration, the variation in the number of rDNA sites should be higher when the sites are located in the terminal region, since only these regions are clustered by the bouquet. To evaluate the role of the site position on the variation in number of sites, a sample of 56 genera was analyzed, each one having species with single sites restricted to a sinlge position. Among 34 genera having terminal single sites, 20 (58.8%) presented also species with multiple sites and in 14 of them all sites were terminal. On the other hand, 11 genera had species with proximal single sites but only two out of these 11 genera (18.2%) had species with multiple sites and in both cases the sites were not proximal (Table 3). Therefore, terminal rDNA sites seem to have higher mobility than proximal ones.
Table 3. Distribution of multiple rDNA sites in genera having species with single sites restricted to only one chromosome region
The tight proximity of the telomeres in the bouquet and the highest rate of recombination in the terminal region seem to contribute to the higher rate of changes in the number rDNA sites on this region. Certainly, other chromosomal features should also influence the dispersion of these sites, leading to species with similar karyotypes and different distribution of sites. A clear example of divergent patterns in very similar karyotypes was observed in species of Phaseolus. Among 37 accessions of P. vulgaris analyzed there was a variation of six to 18 sites of 45S rDNA per diploid complement [32] while in 17 accessions of P. lunatus the number of sites was stable - only one pair of sites [66]. This extreme contrast in two species with very similar chromosome size and morphology, geographical distribution and cultivation history, suggests that other factors, such as association with mobile elements [86,87], may play a decisive role in site dispersion.
Variability between diploids and polyploids
The variability between diploids and polyploids was analyzed at the intra-specific and intra-generic levels. In 19 out of 26 species (59 karyotypes) that had one or more polyploidy cytotypes, the number of sites per monoploid complement (SMC) was constant (Additional file 1: Table S2). In four of these 19 species there was variation in the number of sites per karyotype in different diploid cytotypes, but the tetraploid cytotype exhibited the same number found in one of the diploids. The position of the sites could be determined in only 18 out of 26 species. In 14 of them the position of the sites remained unchanged in diploids and polyploids (Additional file 1: Table S3).
To investigate the variability at the intrageneric level, 40 genera with two or more species were analyzed (species with intraspecific variability were not included). The number of SMC between different ploidy levels was stable in only 6 genera (15%). Among the remaining 34 genera the number of SMC in polyploidy species was lower than in diploids in 29 cases, and higher in only seven (Additional file 1: Table S4).
In general, polyploidy results in doubling of sites when it is of recent origin (intraspecific polyploidy), but when comparing different diploid and polyploid species of the same genus there was a clear trend to reduce the number of SMC in the latter. Considering that the period of time of evolutionary divergence is usually larger between species of the same genus than between cytotypes of the same species, it is reasonable to assume that older polyploids more often display "diploidized" numbers of rDNA sites, as observed in Aristolochia[88], Nicotiana[38], and Avena[89].
The distribution of 45S rDNA sites in animals
The number and location of rDNA sites in other eukaryotes seem to follow some of the trends observed here, although we are not aware of such a similar compilation in other large groups of organisms. A quick look at the literature showed that the modal number of rDNA sites was 2 or 4 per cell in each one of the following samples: 49 species of Orthoptera [40], 31 Scarabaeinae bettles [90], 18 Lepidoptera [80], 38 triatomine (Heteroptera) [91], 13 representatives of five tribes of cichlid fishes [92], 61 rodents [42,93], 50 bats [93], and 56 species of reptiles [94]. The number of rDNA sites in this sample was quite variable within some groups, as rodents and grasshoppers, but much more conserved in others, as triatomine and bats.
The distribution of 45S rDNA sites per arm was distinct for different taxa. For example, in bats the sites were restricted to the short arm in all 7 families and 38 genera [93] whereas in reptiles they were largely concentrated on large arms or on microchromosomes [94]. The site distribution per chromosome arm region was more difficult to assess. However, most sites were located very close to the centromere of acrocentric chromosomes or at the terminal/subterminal region of the short or long arms, but only rarely they were found in the interstitial region. Curiously, in holokinetic chromosomes of Lepidoptera the rDNA sites were not restricted to the terminal position [80], as observed in plants. All together, these data suggest that the non-random distribution of rDNA sites observed in most taxa may be influenced by other important aspects of the chromosomal structure and organization, such as the amount of active mobile elements [87], nuclear DNA amount [26], and dynamics of interphase chromosomes [57].
All these data together, indicate that: i, in angiosperms 45S rRNA genes occur preferentially on the short arms, concentrated on one or two clusters per haploid set, and in the terminal region of chromosomes; ii, in gymnosperms there are two different patterns: mainly interstitial sites in Pinaceae and mainly terminal or proximal sites in all other families; iii, the preference for the short arm is especially evident in very short arms of acrocentric chromosomes, where they often seem to span the whole arm; iiii, the location is more strictly controlled in holokinetic chromosomes where all 45S rDNA sites have been reported in the terminal position; iiiii, when the number of rDNA sites per karyotype increases, either by dispersion of repetitive units or by polyploidy, they tend to maintain the relative position of the original site. Although single rDNA loci are able to change its number and position in the genome, the establishment and dispersion of new sites appear to be constrained along the chromosome arms. Despite some general trends concerning the number and position of rDNA sites in plants, some species and even large taxonomic groups of plants deviate completely from these trends. Although there may be selection pressures favoring the terminal location, data of some groups indicate that there are no restrictions to the functioning of rDNA clusters in other positions or in higher number of sites and the forces driving the observed trends may be related to the chromosome architecture, nuclear organization and the dynamics of allelic and non-allelic recombinations.
Information on the karyotype and the number and position of the 45S rDNA signals in mitotic metaphase cells of 253 original studies were used to build a database. The complete list of these articles and genera/families to which they refer is indicated in Additional file 1: Table S5. For the analysis of different aspects of the distribution of these sites, only part of the total sample was used since not all of the available karyotypes displayed all necessary information. Statistical analysis included Chi-square, Fisher's exact test and proportion tests implemented in the software Epicalc (Brixton Health).
Estimating the chromosomal morphology, size and the position of rDNA sites
The definition of metacentric, submetacentric and acrocentric chromosomes types was based on the arm ratio (ar) values, considering only metacentric chromosomes (ar < 1.5), submetacentric (ar ≥ 1.5 < 3.0) and acrocentric (ar ≥ 3.0, including those apparently telocentric [49]. The size in micrometers of the chromosomes bearing the rDNA sites was found in only 28 of 253 articles consulted. In other 194 articles there was a scale bar in micrometers, which allowed estimating the size of chromosomes by carefully measuring the size of the bar and the chromosomes. In 31 original works, where FISH data were presented without any information on chromosome size, this measurement was based on the size of the NOR bearing chromosome found in other publications for the same species. For example, for Allium fistulosum the number and relative position of rDNA sites were obtained from the work of Ricroch et al. [95] and the size of the chromosome bearing the NOR was based on measurements reported by Gernand et al. [96].
The position of the rDNA site was determined by two distinct approaches. First, based on metaphase photographs, the chromosome arm was divided into four regions of equal size [proximal (p), interstitial-proximal (ip), interstitial-terminal (it) and terminal (t)] and the site was positioned in one of them. When the rDNA signal seemed to coincide with the position of the centromere, it was referred to as "centromeric" (cen). Some sites appeared to span the entire chromosome arm, from the proximal to the terminal region, being named "whole-arm" sites (Additional file 1: Table S5). In the second approach, considering only idiograms, the absolute distance in micrometers from the site to the centromere was evaluated. In this case, the lengths from the centromere to the beginning of the site and from the end of the site to the telomere were estimated, as well as the size of the other chromosome arm. Whenever available, measurements provided in the original paper were used, otherwise they were estimated from the available idiograms or metaphases. All measurements were performed with Adobe Professional 9.0 (Adobe Inc.) in the digital version of the papers (Additional file 1: Table S6).
Analysis of the distribution of rDNA sites by the arm frame method
The method proposed by Lima-de-Faria [7] was used aiming primarily to compare the distribution of rDNA sites with his data for secondary constrictions. Graphically, the chromosomes were represented by parallel lines, with centromeres aligned at the vertical axis and the rDNA bearing arm at the right side of this axis (Figure 6). The exact position of this arm in the graphic was given by its size, in both vertical and horizontal axes. Chromosomes with rDNA sites on both arms were represented twice in the graphic. In addition, the regions of higher and lower occurrence of these sites along the chromosome were identified by means of a graph of density of sites in the arm frame. Another analysis, similar to the arm frame, considered the size of the chromosome arm in micrometers in the Y axis and the relative distance of the site to the centromere in percentage on the X axis.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FR acquired the data and drafted the manuscript. MG designed the study and revised the manuscript. All authors read and approved the final manuscript.
The authors thank the collaboration of Dr. Isabelle Meunier, from the Federal Rural University of Pernambuco for her valuable contributions for the statistical analyses, Dr. Wolfgang Harand (Centro de Tecnologias Estratégicas do Nordeste) for the macro to draw the arm frame, and the Brazilian agency Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Federal University of Pernambuco (UFPE) for their financial support.
Sandhu D, Gill KS: Gene-containing regions of wheat and the other grass genomes.
Plant Physiol 2002, 128(3):803-811. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lippman Z, Gendrel AV, Black M, Vaughn MW, Dedhia N, McCombie WR, Lavine K, Mittal V, May B, Kasschau KB, et al.: Role of transposable elements in heterochromatin and epigenetic control.
Nature 2004, 430(6998):471-476. PubMed Abstract | Publisher Full Text
Fengler K, Allen SM, Li B, Rafalski A: Distribution of genes, recombination, and repetitive elements in the maize genome.
Tian Z, Rizzon C, Du J, Zhu L, Bennetzen JL, Jackson SA, Gaut BS, Ma J: Do genetic recombination and gene density shape the pattern of DNA elimination in rice long terminal repeat retrotransposons?
Genome Res 2009, 19(12):2221-2230. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ugarković DI: Centromere-competent DNA: structure and evolution.
Prog Mol Subcell Biol 2009, 48:53-76. PubMed Abstract | Publisher Full Text
Choulet F, Wicker T, Rustenholz C, Paux E, Salse J, Leroy P, Schlub S, le Paslier MC, Magdelenat G, Gonthier C, et al.: Megabase level sequencing reveals contrasted organization and evolution patterns of the wheat gene and transposable element spaces.
Plant Cell 2010, 22(6):1686-1701. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lima-de-Faria A: The chromosome field I. Prediction of the location of ribosomal cistrons.
Vanzela ALL, Cuadrado A, Guerra M: Localization of 45S rDNA and telomeric sites on holocentric chromosomes of Rhynchospora tenuis Link (Cyperaceae).
Genet Mol Biol 2003, 26(2):199-201. Publisher Full Text
Guerra M, Kenton A, Bennett MD: rDNA sites in mitotic and polytene chromosomes of Vigna unguiculata (L.) Walp. and Phaseolus coccineus L. revealed by in situ hybridization.
Ann Bot 1996, 78(2):157-161. Publisher Full Text
Marcon AB, Barros ICL, Guerra M: A karyotype comparison between two closely related species of Acrostichum.
Am Fern J 2003, 93(3):116-125. Publisher Full Text
Marcon AB, Barros ICL, Guerra M: Variation in chromosome numbers, CMA bands and 45S rDNA sites in species of Selaginella (Pteridophyta).
Ann Bot 2005, 95(2):271-276. PubMed Abstract | Publisher Full Text
Nakayama S, Fujishita M, Sone T, Ohyama K: Additional locus of rDNA sequence specific to the X chromosome of the liverwort, Marchantia polymorpha.
Chromosome Res 2001, 9(6):469-473. PubMed Abstract | Publisher Full Text
Orzechowska M, Siwinska D, Maluszynska J: Molecular cytogenetic analyses of haploid and allopolyploid Pellia species.
J Bryol 2010, 32(2):113-121. Publisher Full Text
Garcia S, Garnatje T, Kovařík A: Plant rDNA database: ribosomal DNA loci information goes online.
Chromosoma 2012, 121(4):389-394. PubMed Abstract | Publisher Full Text
Pedersen C, Linde-Laursen I: Chromosomal locations of four minor rDNA loci and a marker microsatellite sequence in barley.
Chromosome Res 1994, 2(1):65-71. PubMed Abstract | Publisher Full Text
Guerra M: Patterns of heterochromatin distribution in plant chromosomes.
Genet Mol Biol 2000, 23(4):1029-1041. Publisher Full Text
Fernandes T, Rego LNAA, Nardy M, Yuyama PM, Vanzela ALL: Karyotype differentiation of four Cestrum species (Solanaceae) revealed by fluorescent chromosome banding and FISH.
Genet Mol Biol 2009, 32(2):320-327. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Leitch IJ, Bennett MD: Genome downsizing in polyploid plants.
Biol J Linn Soc 2004, 82(4):651-663. Publisher Full Text
Leitch IJ, Hanson L, Lim KY, Kovarik A, Chase MW, Clarkson JJ, Leitch AR: The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae).
Ann Bot 2008, 101(6):805-814. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Guerra M, Cabral G, Cuacos M, González-García M, González-Sánchez M, Vega J, Puertas MJ: Neocentrics and holokinetics (holocentrics): Chromosomes out of the centromeric rules.
Cytogenet Genome Res 2010, 129(1–3):82-96. PubMed Abstract | Publisher Full Text
Sousa A, Barros e Silva AE, Cuadrado A, Loarce Y, Alves MV, Guerra M: Distribution of 5S and 45S rDNA sites in plants with holokinetic chromosomes and the "chromosome field" hypothesis.
Micron 2011, 42(6):625-631. PubMed Abstract | Publisher Full Text
Brown GR, Carlson JE: Molecular cytogenetics of the genes encoding 18S–5.8S–26S rRNA and 5S rRNA in two species of spruce (Picea).
Liu ZL, Zhang D, Hong DY, Wang XR: Chromosomal localization of 5S and 18S–5.8S–25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization.
Theor Appl Genet 2003, 106(2):198-204. PubMed Abstract | Publisher Full Text
Islam-Faridi MN, Nelson CD, Kubisiak TL: Reference karyotype and cytomolecular map for loblolly pine (Pinus taeda L.).
Genome 2007, 50(2):241-251. PubMed Abstract | Publisher Full Text
Tagashira N, Kondo K: Chromosome phylogeny of Zamia and Ceratozamia by means of Robertsonian changes detected by fluorescence in situ hybridization (FISH) technique of rDNA.
Ingle J, Timmis JN, Sinclair J: The relationship between satellite deoxyribonucleic acid, ribosomal ribonucleic acid gene redundancy, and genome size in plants.
Plant Physiol 1975, 55(3):496-501. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Prokopowich CD, Gregory TR, Crease TJ: The correlation between rDNA copy number and genome size in eukaryotes.
Genome 2003, 46(1):48-50. PubMed Abstract | Publisher Full Text
Komarova NY, Grabe T, Huigen DJ, Hemleben V, Volkov RA: Organization, differential expression and methylation of rDNA in artificial Solanum allopolyploids.
Plant Mol Biol 2004, 56(3):439-463. PubMed Abstract | Publisher Full Text
Vicari MR, Artoni RF, Moreira-Filho O, Bertollo LAC: Colocalization of repetitive DNAs and silencing of major rRNA genes. A case report of the fish Astyanax janeiroensis.
Cytogenet Genome Res 2008, 122(1):67-72. PubMed Abstract | Publisher Full Text
Brasileiro-Vidal AC, Dos Santos-Serejo JA, Soares Filho WDS, Guerra M: A simple chromosomal marker can reliably distinguishes Poncirus from Citrus species.
Genetica 2007, 129(3):273-279. PubMed Abstract | Publisher Full Text
Dantas LG, Guerra M: Chromatin differentiation between Theobroma cacao L. and T. grandiflorum Schum.
Genet Mol Biol 2010, 33(1):94-98. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Pedrosa-Harand A, De Almeida CCS, Mosiolek M, Blair MW, Schweizer D, Guerra M: Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution.
Theor Appl Genet 2006, 112(5):924-933. PubMed Abstract | Publisher Full Text
Hasterok R, Wolny E, Hosiawa M, Kowalczyk M, Kulak-Ksiazczyk S, Ksiazczyk T, Heneen WK, Maluszynska J: Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae.
Ann Bot 2006, 97(2):205-216. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Souza LGR, Crosa O, Guerra M: Karyological circumscription of Ipheion Rafinesque (Gilliesioideae, Alliaceae).
Plant Syst Evol 2010, 287(3):119-127. Publisher Full Text
Lima-de-Faria A: Classification of genes, rearrangements and chromosomes according to the chromosome field.
Hereditas 1980, 93(1):1-46. PubMed Abstract
Brasileiro-Vidal AC, Melo-Oliveira MB, Carvalheira GMG, Guerra M: Different chromatin fractions of tomato (Solanum lycopersicum L.) and related species.
Micron 2009, 40(8):851-859. PubMed Abstract | Publisher Full Text
Wendel JF, Schnabel A, Seelanan T: Bidirectional interlocus concerted evolution following allopolyploid speciation in cotton (Gossypium).
Proc Natl Acad Sci U S A 1995, 92(1):280-284. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kovarik A, Dadejova M, Lim YK, Chase MW, Clarkson JJ, Knapp S, Leitch AR: Evolution of rDNA in Nicotiana allopolyploids: A potential link between rDNA homogenization and epigenetics.
Ann Bot 2008, 101(6):815-823. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hanson RE, Islam-Faridi MN, Percival EA, Crane CF, Ji Y, McKnight TD, Stelly DM, Price HJ: Distribution of 5S and 18S–28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors.
Chromosoma 1996, 105(1):55-61. PubMed Abstract | Publisher Full Text
Cabrero J, Camacho JPM: Location and expression of ribosomal RNA genes in grasshoppers: Abundance of silent and cryptic loci.
Chromosome Res 2008, 16(4):595-607. PubMed Abstract | Publisher Full Text
Fukushima K, Imamura K, Nagano K, Hoshi Y: Contrasting patterns of the 5S and 45S rDNA evolutions in the Byblis liniflora complex (Byblidaceae).
J Plant Res 2011, 124(2):231-244. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Cazaux B, Catalan J, Veyrunes F, Douzery EJP, Britton-Davidian J: Are ribosomal DNA clusters rearrangement hotspots? A case study in the genus Mus (Rodentia, Muridae).
BMC Evol Biol 2011, 11(1):124. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Pich U, Fuchs J, Schubert I: How do Alliaceae stabilize their chromosome ends in the absence of TTTAGGG sequences?
Chromosome Res 1996, 4(3):207-213. PubMed Abstract | Publisher Full Text
Kuo HF, Olsen KM, Richards EJ: Natural variation in a subtelomeric region of Arabidopsis: Implications for the genomic dynamics of a chromosome end.
Genetics 2006, 173(1):401-417. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
González-García M, González-Sánchez M, Puertas MJ: The high variability of subtelomeric heterochromatin and connections between nonhomologous chromosomes, suggest frequent ectopic recombination in rye meiocytes.
Cytogenet Genome Res 2006, 115(2):179-185. PubMed Abstract | Publisher Full Text
Souza LGR, Crosa O, Speranza P, Guerra M: Cytogenetic and molecular evidence suggest multiple origins and geographical parthenogenesis in Nothoscordum gracile (Alliaceae).
Ann Bot 2012, 109(5):987-999. PubMed Abstract | Publisher Full Text
Koo DH, Hur Y, Bang JW: Variability of rDNA loci in dioecious Rumex acetosa L. detected by fluorescence in situ hybridization.
Baeza C, Schrader O, Budahn H: Characterization of geographically isolated accessions in five Alstroemeria L. species (Chile) using FISH of tandemly repeated DNA sequences and RAPD analysis.
Guerra M: Reviewing the chromosome nomenclature of Levan et al.
Hall KJ, Parker JS: Stable chromosome fission associated with rDNA mobility.
Chromosome Res 1995, 3(7):417-422. PubMed Abstract | Publisher Full Text
Souza LGR, Crosa O, Winge H, Guerra M: The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): A populational and cytomolecular analysis.
Genet Mol Biol 2009, 32(1):111-116. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rousselet J, Monti L, Auger-Rozenberg MA, Parker JS, Lemeunier FL: Chromosome fission associated with growth of ribosomal DNA in Neodiprion abietis (Hymenoptera: Diprionidae).
Proceedings of the Royal Society B: Biological Sciences 2000, 267(1455):1819-1823. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lan T, Albert VA: Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid.
BMC Plant Biol 2011, 11:126. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Hirai H, Taguchi T, Godwin AK: Genomic differentiation of 18S ribosomal DNA and β-satellite DNA in the hominoid and its evolutionary aspects.
Chromosome Res 1999, 7(7):531-540. PubMed Abstract | Publisher Full Text
Taketa S, Ando H, Takeda K, Ichii M, Von Bothmer R: Ancestry of American polyploid Hordeum species with the I genome inferred from 5S and 18S-25S rDNA.
Ann Bot 2005, 96(1):23-33. PubMed Abstract | Publisher Full Text
Winterfeld G, Röser M: Disposition of ribosomal DNAs in the chromosomes of perennial oats (Poaceae: Aveneae).
Bot J Linn Soc 2007, 155(2):193-210. Publisher Full Text
Schubert I, Shaw P: Organization and dynamics of plant interphase chromosomes.
Trends Plant Sci 2011, 16(5):273-281. PubMed Abstract | Publisher Full Text
Dong F, Jiang J: Non-Rabl patterns of centromere and telomere distribution in the interphase nuclei of plant cells.
Chromosome Res 1998, 6(7):551-558. PubMed Abstract | Publisher Full Text
Berr A, Pecinka A, Meister A, Kreth G, Fuchs J, Blattner FR, Lysak MA, Schubert I: Chromosome arrangement and nuclear architecture but not centromeric sequences are conserved between Arabidopsis thaliana and Arabidopsis lyrata.
Plant J 2006, 48(5):771-783. PubMed Abstract | Publisher Full Text
Pecinka A, Schubert V, Meister A, Kreth G, Klatte M, Lysak MA, Fuchs J, Schubert I: Chromosome territory arrangement and homologous pairing in nuclei of Arabidopsis thaliana are predominantly random except for NOR-bearing chromosomes.
Chromosoma 2004, 113(5):258-269. PubMed Abstract | Publisher Full Text
Robledo G, Lavia GI, Seijo G: Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection.
Theor Appl Genet 2009, 118(7):1295-1307. PubMed Abstract | Publisher Full Text
Robledo G, Seijo G: Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of rDNA loci and heterochromatin detection: A new proposal for genome arrangement.
Theor Appl Genet 2010, 121(6):1033-1046. PubMed Abstract | Publisher Full Text
Robledo G, Seijo G: Characterization of the Arachis (Leguminosae) D genome using fluorescence in situ hybridization (FISH) chromosome markers and total genome DNA hybridization.
Genet Mol Biol 2008, 31(3):717-724. Publisher Full Text
Seijo JG, Lavia GI, Fernández A, Krapovickas A, Ducasse D, Moscone EA: Physical mapping of the 5S and 18S–25S rRNA genes by FISH as evidence that Arachis duranensis and A. ipaensis are the wild diploid progenitors of A. hypogaea (Leguminosae).
Am J Bot 2004, 91(9):1294-1303. PubMed Abstract | Publisher Full Text
Galasso I: Distribution of highly repeated DNA sequences in species of the genus Lens Miller.
Genome 2003, 46(6):1118-1124. PubMed Abstract | Publisher Full Text
Almeida C, Pedrosa-Harand A: Contrasting rDNA evolution in lima bean (Phaseolus lunatus L.) and common bean (P. vulgaris L. Fabaceae).
Cytogenet Genome Res 2011, 132(3):212-217. PubMed Abstract | Publisher Full Text
Moscone EA, Klein F, Lambrou M, Fuchs J, Schweizer D: Quantitative karyotyping and dual-color FISH mapping of 5S and 18S-25S rDNA probes in the cultivated Phaseolus species (Leguminosae).
Genome 1999, 42(6):1224-1233. PubMed Abstract
Fuchs J, Strehl S, Brandes A, Schweizer D, Schubert I: Molecular-cytogenetic characterization of the Vicia faba genome - Heterochromatin differentiation, replication patterns and sequence localization.
Chromosome Res 1998, 6(3):219-230. PubMed Abstract | Publisher Full Text
Galasso I, Sonnante G, Tota DG, Pignone D: Comparison of molecular cytogenetic and genetic analyses in accessions of the two biotypes of Vicia benghalensis L.
Ann Bot 1997, 79(3):311-317. Publisher Full Text
Li R, Taylor S, Jenkins G: Unravelling the phylogeny of tetraploid Vicia amoena (Fabaceae) and its diploid relatives using chromosomal landmarks.
Hereditas 2001, 134(3):219-224. PubMed Abstract
Raina SN, Mukai Y, Kawaguchi K, Goel S, Jain A: Physical mapping of 18S–5.8S–26S and 5S ribosomal RNA gene families in three important vetches (Vicia species) and their allied taxa constituting three species complexes.
Wu F, Tanksley SD: Chromosomal evolution in the plant family Solanaceae.
BMC Genomics 2010, 11(1):182. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Guerra M: Chromosomal variability and the origin of Citrus species complex. In Genetic Diversity. Edited by Mahoney CL. New York: Springer DA Nova Science Publishers; 2009:51-68.
Cai Q, Zhang D, Liu ZL, Wang XR: Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus.
Ann Bot 2006, 97(5):715-722. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Heckmann S, Schroeder-Reiter E, Kumke K, Ma L, Nagaki K, Murata M, Wanner G, Houben A: Holocentric chromosomes of Luzula elegans are characterized by a longitudinal centromere groove, chromosome bending, and a terminal nucleolus organizer region.
Cytogenet Genome Res 2011, 134(3):220-228. PubMed Abstract | Publisher Full Text
Vanzela ALL, Cuadrado A, Jouve N, Luceño M, Guerra M: Multiple locations of the rDNA sites in holocentric chromosomes of Rhynchospora (Cyperaceae).
Chromosome Res 1998, 6(5):345-349. PubMed Abstract | Publisher Full Text
Albertson DG, Thomson JN: Segregation of holocentric chromosomes at meiosis in the nematode, Caenorhabditis elegans.
Chromosome Res 1993, 1(1):15-26. PubMed Abstract | Publisher Full Text
Morielle-Souza A, Azeredo-Oliveira MTV: Differential characterization of holocentric chromosomes in triatomines (Heteroptera, Triatominae) using different staining techniques and fluorescent in situ hybridization.
Genet Mol Res 2007, 6(3):713-720. PubMed Abstract | Publisher Full Text
Criniti A, Simonazzi G, Cassanelli S, Ferrari M, Bizzaro D, Manicardi GC: Distribution of heterochromatin and rDNA on the holocentric chromosomes of the aphids Dysaphis plantaginea and Melanaphis pyraria (Hemiptera: Aphididae).
Nguyen P, Sahara K, Yoshido A, Marec F: Evolutionary dynamics of rDNA clusters on chromosomes of moths and butterflies (Lepidoptera).
Genetica 2010, 138(3):343-354. PubMed Abstract | Publisher Full Text
Schweizer D, Loidl J: A model for heterochromatin dispersion and the evolution of C-band patterns.
Stults DM, Killen MW, Pierce HH, Pierce AJ: Genomic architecture and inheritance of human ribosomal RNA gene clusters.
Genome Res 2008, 18(1):13-18. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wako T, Fukui K: Higher organization and histone modification of the plant nucleus and chromosome.
Cytogenet Genome Res 2010, 129(1–3):55-63. PubMed Abstract | Publisher Full Text
Da Silva CRM, Quintas CC, Vanzela ALL: Distribution of 45S and 5S rDNA sites in 23 species of Eleocharis (Cyperaceae).
Genetica 2010, 138(9):951-957. PubMed Abstract | Publisher Full Text
Bass HW: Telomere dynamics unique to meiotic prophase: Formation and significance of the bouquet.
Cell Mol Life Sci 2003, 60(11):2319-2324. PubMed Abstract | Publisher Full Text
Schubert I, Wobus U: In situ hybridization confirms jumping nucleolus organizing regions in Allium.
Chromosoma 1985, 92(2):143-148. Publisher Full Text
Raskina O, Barber JC, Nevo E, Belyayev A: Repetitive DNA and chromosomal rearrangements: Speciation-related events in plant genomes.
Cytogenet Genome Res 2008, 120(3–4):351-357. PubMed Abstract | Publisher Full Text
Berjano R, Roa F, Talavera S, Guerra M: Cytotaxonomy of diploid and polyploid Aristolochia (Aristolochiaceae) species based on the distribution of CMA/DAPI bands and 5S and 45S rDNA sites.
Linares C, González J, Ferrer E, Fominaya A: The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S–5.8S–26S rDNA and a C genome specific DNA sequence in the genus Avena.
Genome 1996, 39(3):535-542. PubMed Abstract | Publisher Full Text
Cabral-de-Mello D, Oliveira S, de Moura R, Martins C: Chromosomal organization of the 18S and 5S rRNAs and histone H3 genes in Scarabaeinae coleopterans: insights into the evolutionary dynamics of multigene families and heterochromatin.
BMC Genet 2011, 12(1):88. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Panzera Y, Pita S, Ferreiro MJ, Ferrandis I, Lages C, Perez R, Silva AE, Guerra M, Panzera F: High dynamics of rDNA cluster location in kissing bug holocentric chromosomes (Triatominae, Heteroptera).
Cytogenet Genome Res 2012, 138(1):56-67. PubMed Abstract | Publisher Full Text
Schneider C, Gross M, Terencio M, Artoni R, Vicari M, Martins C, Feldberg E: Chromosomal evolution of neotropical cichlids: the role of repetitive DNA sequences in the organization and structure of karyotype.
Reviews in Fish Biology and Fisheries
in pressPublisher Full Text
Baker R, Maltbie M, Owen J, Hamilton M, Bradley R: Reduced number of ribosomal sites in bats: evidence for a mechanism to contain genome size.
J Mammal 1992, 73(4):847-858. Publisher Full Text
Porter CA, Haiduk MW, de Queiroz K: Evolution and phylogenetic significance of ribosomal gene location in chromosomes of squamate reptiles.
Ricroch A, Peffley EB, Baker RJ: Chromosomal location of rDNA in Allium: in situ hybridization using biotin- and fluorescein-labelled probe.
Gernand D, Golczyk H, Rutten T, Ilnicki T, Houben A, Joachimiak AJ: Tissue culture triggers chromosome alterations, amplification, and transposition of repeat sequences in Allium fistulosum.
Genome 2007, 50(5):435-442. PubMed Abstract | Publisher Full Text


text:
336 Views
By CGRundertow
185 Views
By CGRundertow
129 Views
The Beatles: Rock Band - Opening Video [HD]
9,377 Views
By Video Games
(Spoiler) The Beatles: Rock Band - Ending Cinem...
5,987 Views
By Video Games
The Beatles: Rock Band - Abbey Road Medley Trailer [HD]
2,906 Views
By Video Games
Meat Flesh - Beatles: Rock Band - Best Of...
105 Views
By Revision3
The Contract That Made the Beatles Goes Up for...
172 Views
By Muzu
Da Bacon Ai Beatles - L'arte Che Si Ascolta in...
141 Views
By Muzu
Beatles Stories - Beatles Stories
119 Views
By hulu
ArcadeMojo - 7 Things You Don't Know About the...
239 Views
By hulu
Forever / Beatles Medley - Trafficsound (Music...
166 Views
By Muzu
The Contract That Made the Beatles is to Be Auc...
84 Views
By Muzu
`Ringo Starr – 2nd Best Drummer In The Beatle...
234 Views
By Muzu
The Contract That Made the Beatles is to Be Auc...
74 Views
By Muzu
Sneak Peak at the New Beatles Rock Band Game
146 Views
By Muzu
The Enemy Announce Tour, Kylie for Glasto 2010...
44 Views
By Muzu
188 Views
By Muzu
Yoko Ono War Nicht Schuld an Trennung Der Beatles
0 Views
By Muzu
Yoko Ono War Nicht Schuld an Trennung Der Beatles
100 Views
By Muzu
66 Views
By Muzu
Addio a Ravi Shankar, Il Maestro Indiano Del Si...
116 Views
By Muzu
Covers The Beatles, 'Like Dreamers Do' - Jake B...
35 Views
By Muzu
Salute to The Beatles: Top Artists Reflect
5 Views
By CBS
Salute to The Beatles: Lady Antebellum
4 Views
By CBS
Salute to The Beatles: John Mayer
5 Views
By CBS
Salute to The Beatles: Glen Hansard Reflects
7 Views
By CBS
Beatles Fan Remembers Fab Four's Ed Sullivan Pe...
23 Views
By CBS
Imagine Dragons and the Influence of The Beatles
3 Views
By CBS
WSJ Live - The Beatles On 'Ed Sullivan': 50 Yea...
10 Views
By hulu
Joe Diffie - Bigger Than The Beatles (Official...
3,571 Views
By Music
A vid of the song strawberry fields, with pics of The Beatles.
One of the legendary places of Liverpool, Strawberry Fields, because it's a childhood haunt which Lennon immortalised on one of the greatest double-A-side singles ever released (twinned with Penny Lane, a song about a suburban street a short bus ride away).
In Lennon's youth and until very recently Strawberry Fields was a Salvation Army home. Thanks to the efforts of some visionary Salvationists it's been reborn as a 24-7 prayer centre called the Boiler Room.

subset: commoncrawl


text:
Invite Freelancer to Project
You don't seem to have an active project at the moment. Why not post a project now? It's free!Post a project
thimna2011cooray Online Offline
- N/AJobs Completed
- N/AOn Budget
- N/AOn Time
- N/ARepeat Hire Rate
Recent Reviews
dataentry operaterJan 2004 - Jul 2009 (5 years)
i will do data-entry at five years of this company i had most experience of data-entry © writing i do the best of my job
internet ,Email,e commerce (2012)amazonlanka higher educational institute
certificate of the participation was participated of the internet &Commerce program& successfully completed of the course contend of the course internet & eCommerce
diploma in softwareingineering (2006)TEC Sri Lanka (technical ingineering collage of Sri Lanka
successful completion of the prescribed course of the study and examination of the Tec Srli lanka
My Top Skills
- Data Entry 0
- Excel 0
- Photoshop 0
- Sales 0
- Marketing 0
- Sports 0
- Photo Editing 0
- eBay 0


text:
Welcome, skip to the content.
Official site for Melbourne, Victoria, Australia
Delight in being surrounded by Gippsland's picturesque lakes, beaches and national parks at Loch Sport. The town is situated east of Sale on a narrow spit of land between the sand dunes of Ninety Mile Beach and Lake Victoria.
Witness some of the most beautiful bird life in Australia at The Lakes National Park, covering more than 2300 hectares of coastal flora and fauna. Enjoy a picnic and the barbecue facilities in the park at Sperm Whale Head, and see the surrounding wildlife. Campsites are also available – pitch a tent and sleep under the stars.
Cast a line in and hook bream, mullet, skip jack and flounder. Try river fishing for some of the best bream in the area, ocean beach fishing for abundant open water fish, or launch a boat at one of the many ramps along the foreshore. The shallow sandy beach of Lake Victoria at Loch Sport is favourite spot for swimming.
Experience the largest inland network of waterways in Australia, the Gippsland Lakes. Go on board a cruiser or enjoy a range of water-based activities.
Read more | Show on map
One of Gippsland's highlights, Ninety Mile Beach is one of the longest and most beautiful stretches of sand in the world offering a wealth of activities.
Read more
The tranquil Gippsland Lakes are a system of coastal lagoons separated from the Tasman Sea by the coastal dunes of the Ninety Mile Beach. Seven...
Protected from the pounding surf of Bass Strait by barrier islands, the quiet waters of Nooramunga Marine and Coastal Park create an intricate...
The Lakes National Park is a peaceful bushland retreat in the Gippsland Lakes, fringed by the waters of Lake Victoria and Lake Reeve. The park...
Find accommodation in Gippsland
Did you not find what you are looking for? Try our specialised searches:


text:
See All Contributors »
NCAA Tournament Bracket: ESPN.com Has No Perfect Brackets Left - Barbecue Sauce - SB Nation Kansas City
No perfect March Madness entries left for ESPN.
Let Prohm's Posse Ride: Georges, Georgios, 3sus, Hallice, Deonte, Abdel, Monte, Matt, and McKaT.
As much parity as there is this year, I'm not surprised. Picking brackets was a wash this year. With about 16 teams that have a legit shot of winning it all, not shocked in the least.
There were none even after the first round...
Forum Rules
All content owned by CycloneFanatic.com - All rights reserved 2005-09. By viewing this website you agree to the Terms of Service, Site Rules and Legal Disclaimer. The words, views, images and opinions expressed or provided by users do not reflect the opinions or views of CycloneFanatic.com or Iowa State University. The names, words, symbols, and graphics representing Iowa State University are trademarks and copyrights of the University protected by the trademark and copyright laws of the United States of America and other countries and are used on this web site under license from the University. Original site design, premise & construction by Jeremy Lind.


text:
Welcome to the German Program
*Now accepting applications for German Lecturer — click for details*
The German program at the University of Tennessee seeks to provide a command and appreciation of the language, literature and culture of German-speaking peoples. Our offerings range from the beginning language, through the B.A. and M.A. in German, to the Ph.D. in Modern Foreign Languages with German as the first concentration.
Our program offers many opportunities including a:
- Stammtisch
- Film and Lecture Series
- Sprachwochenende
- Study Abroad
- Graduate Teaching Assistantships
- Honor Society Membership
- Scholarships for Graduates and Undergraduates
Why Study German?
Getting Ready for the World
German language study - actually any foreign language study, German included - creates more positive attitudes and lowers prejudice toward people who are different. You begin participating genuinely and responsibly in a multi-cultural world when you learn German.
German - the language of Berlin, Hamburg, Munich, Cologne, Frankfurt, Stuttgart, Zurich, Salzburg and Vienna - has 100 million speakers spanning Europe's heartland.
The European Central Bank, Swiss banks, Germany's economy (Europe's largest and the world's second-largest exporter) run on German. Germany hosts the world's leading trade fairs and counts among the world's largest and most technologically advanced producers of iron, steel, coal, cement, chemicals, machinery, autos, machine tools, electronics, foodstuffs, beverages, ships, and textiles. UT's German program sponsors an exchange program with Mannheim that includes a German business internship.
Job Market
Knowing German improves your employment chances in East Tennessee, where Volkswagen, Wacker Chemie and Mahle Industries are currently investing over a billion dollars and creating jobs in an otherwise tight economy.
Eighteen percent of the world's books are published in Germany, the world's third largest book publisher.
German is second only to English in scientific publications. Knowing German, you can access research findings in all areas of modern science. Forty percent of US scientists recommend that students learn German.
Cultural Enrichment
German-the language of Goethe, Nietzsche, Kant, Luther, Kafka, Mozart, Bach, Beethoven, Freud and Einstein-opens the door to one of Europe's greatest cultures.
Germans, Austrians and Swiss rank among the most numerous and important tourists. Knowing German pays off in the tourist industry.
Exploring Germany
Scores of UT students visit Germany and study there annually. Knowing German enables you to know the German people better and gain a deeper insight into their history and culture.
Scholarship Support
The German Program annually grants endowed scholarships to its best and brightest, enabling many to study abroad. Last year over 20 students received support.


text:
Got To:: World Images - Philippines Images by provinces - Images Province Province-of-South-Cotabato - City
Enjoy the best photos of Cabinuangan , his landscapes, monuments, men, women and girls. Visit other towns and cities province,
region or state Province-of-South-Cotabato Davao. See zip code, number of inhabitants, coordinates, restaurants and hotels
Join Cabinuangan group G+ 1, Please Share in facebook, twitter and Gogle +
These are photos taken by their neighbors and tourists, In the vicinity of the following coordinates, latitude and longitude 7.68333,126.03333 and in order of the most visited. It is therefore possible that no images of what you are looking for is displayed. On this same page you can access the google map of Cabinuangan and Street View Google and Other movies in Cabinuangan - Province-of-South-Cotabato
|Photos of the city and surroundings of the town of
|Pictures of landscapes and monuments in estado of
|Getting there, hotle, lodging and dining in:
|Map & satellite map of how to get
|map of Cabinuangan
|Forum Cabinuangan customs, postal zip code,
|Latitude longitude coordinates Cabinuangan
|coordinates of Cabinuangan
|Number of residents, men, women girls Cabinuangan
|See photos of other regions and peoples
- Other states philippines
Cabinuangan and Province-of-South-Cotabato province, state


text:
Land (21)
- 4x Blackcleave Cliffs
- 1x Boseiju, Who Shelters All
- 4x City of Brass
- 4x Darkslick Shores
- 4x Gemstone Mine
- 4x Steam Vents
Sorcery (12)
Creature (12)
Instant (12)
Artifact (3)
Sideboard (15)
I run a bunch of non-conventional lands to make sure I always hit my colors when I need them, the damage from City is usually irrelevant, but would the first one for me to cut. I consistently go off turn 2-3, sometimes even 1 with SSG. I also like the land because it allows to me play Abrupt Decay and Dispel in the sideboard to deal with sided in hate.
One question is why the lone Jin-Gitaxias? Because, sometimes after looting once or twice, I will have two or even more Emrakuls and I hated discarding one and resetting my graveyard too early because I needed to keep what was in my hand. I decided to cut one Emrakul for him, and I think putting in Jin on an opponent's end step unanswered is a huge setback for them.
Please login to comment
|Date added
|2 years
|Last updated
|2 years
This deck is Modern legal.
|Avg. CMC


text:
Does it really work and does anyone recommend it? I would love to do it at 39 weeks and 2 days.
Posted 11/25/2012
Comments (4)
Posted 11/26/2012
I was wondering the same thing.
DLR -- PCOS for five years now.
Still TTC after my first miscarriage back in 2009
Avoiding metformin by doing a low carb diet, exercise and vitamins
Posted 11/26/2012
It has a success rate of 70%+, whereas the success rate of a membrane strip is 50/50.
I'd do the acupuncture over the membrane stripping, tbh.
3 minions
I suffer from CRS on a daily basis. Can't Remember Shit.
Posted 11/26/2012
I did it at 39 weeks. Once on wednesday, then again on friday. We into labor that saturday. Very easy labor. I also walked a lot. Husband was weirded out by having sex with me so big, baby right there, etc. So off to the acupuncturist I went. You also could try acupressure.
Posted 11/26/2012
Forgot to tell you, I had membranes stripped that Monday, but I stopped having consistent BHs after that. To the point where I started doing kick counts just to make sure she was ok. I think the stripping pissed her(baby) off :) contactions picked up again after the acupuncture though.


text:
I have had my fair share of life which there are many ups and downs that I have gone through.
Been there, Done that. Twice!! <-- This is something which I can say for myself when it comes to life learning experiences.
I hardly have much to live on expect that I want the very best for my family, particularly for my Mom. The amount of sacrifices that she had given for me is far beyond what words can describe.
On a normal circumstances, I tend to try things which interest me or things which is worth trying. As the saying goes, Life is Short, so, just enjoy it while you can.


text:
Student Microscopes
View Item T-1050 kena™ $233.09
View Item T-12021C Cordless PrepScope $192.14
View Item T-17021C Cordless CoreScope $240.44
View Item Premiere® MS-01 Student Microscope $149.09 - $153.29
View Item Microprojector 2 X2000 $1,870.04 - $3,088.04
View Item Premiere® MS-03 Student Microscope $233.09 - $236.24
View Item The Professor ESH101 $72.44 - $594.29
Student Microscopes
Our Student Microscopes offer high-resolution viewing, intuitive operation, lightweight portability, and multi-functional versatility for young scientists of all grade levels. Available features include rechargeable batteries, high-powered magnification, adjustable platforms for multi-angle viewing of slides & samples, built-in lighting, camera connectivity, binocular and trinocular style viewing, and more. Equip your school classroom laboratory with our Student Microscopes and give your students the power to explore the natural world in a number of ways.


text:
Disclosure: This project was partially funded by a grant from McDonald's Corporation, Oak Brook, IL to Drs. Wansink and Hanks, who were responsible for data analysis and drafting this manuscript. Brian Wansink, PhD, is a member of McDonald's® Corporation's Global Advisory Council.
Brief Cutting Edge Reports
Calorie reductions and within-meal calorie compensation in children's meal combos
Article first published online: 23 DEC 2013
DOI: 10.1002/oby.20668
Copyright © 2013 The Obesity Society
Volume 22, Issue 3, pages 630–632, March 2014
Additional Information
How to Cite
Wansink, B. and Hanks, A. S. (2014), Calorie reductions and within-meal calorie compensation in children's meal combos. Obesity, 22: 630–632. doi: 10.1002/oby.20668
Author Contributions: Dr. Hanks had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Both Dr. Wansink and Dr. Hanks substantially contributed to the interpretation of the results and the writing of the manuscript.
Publication History
- Issue published online: 5 MAR 2014
- Article first published online: 23 DEC 2013
- Accepted manuscript online: 25 NOV 2013 10:30PM EST
- Manuscript Accepted: 22 NOV 2013
- Manuscript Received: 4 OCT 2013
- Abstract
- Article
- References
- Cited By
What happens if a fast-food restaurant chain lowers the calories in a children's meal bundle (CMB), mainly by reducing the portion size of French fries? This study examines how such changes may influence within-meal selections. Specifically, do lower-calorie changes lead to within-meal calorie compensation?
Item-level anonymous transaction data were collected for thirty chain-owned representative US restaurants during June, July, and August of 2011 (pre-changes) and 2012 (post-changes) with a focus on transaction records that included a CMB. Mixed-effects, repeated measures estimation techniques were used for the analysis. Outcome measures were the percentage and caloric profile of specific entrée items, side items, and beverages purchased in all children selecting meals.
The new CMB resulted in selected children's meals that had an average of 18.8% fewer calories (P < 0.001). Additionally, a greater percentage of meals had milk (P < 0.001) compared to the prior year.
Small changes in the automatic—or default—foods offered or promoted in children's meals can reduce calorie intake and improve the overall nutrition from selected foods as long as there is still an indulgence. Importantly, balancing a meal with smaller portions of favored foods might avoid reactance and overeating. Just as managers have done this in restaurants, parents can do this at home.


text:
Minter Ellison
Practice Area
- Company/Commercial (6)
- Funds (6)
- Construction (5)
- Corporate (4)
- Energy (3)
- Regulatory and compliance (3)
- Tax (3)
- Banking / Finance (2)
- Employment (2)
- Public Sector/Local Authority (2)
- Sovereign Wealth (2)
- Financial services (1)
- Healthcare (1)
- Litigation / Dispute Resolution (1)
- Planning (1)
- Real Estate (1)
- Transport (Including aviation and shipping) (1)
Search results
11 articles matched your search
Sort By: Newest first | Oldest first
Minter Ellison creates financing structures for construction of Northern Beaches Hospital
Minter Ellison advised the NSW government over the procurement of Northern Beaches Hospital in a 'first of its kind' transaction.
'Who funds infrastructure' a key question for G20 Summit in Brisbane, says Minter Ellison
Australia 'stands to become a world leader in initiatives for funding the global infrastructure deficit' following the G20 summit in Brisbane this weekend.
Minter Ellison advises Infratil on sale of Australian businesses to Snowy Hydro
NZ-listed Infratil has agreed to sell its Australian businesses to Snowy Hydro for AUD605m. Minter Ellison advised Infratil on the strategic review.
NDRC issues new rules on verification, approval and filing of foreign investment projects weblink
The NDRC of China has issued the Administrative Measures for the Verification and Approval and Filing of Foreign Investment Projects.
Port of Newcastle lease is indicative of rising wave of interest, says Minter Ellison's Rentoul
Minter Ellison's Andrew Rentoul has predicted more infrastructure deals across Australia along the lines of the successful leasing of the Port of Newcastle by the NSW government.
Minter Ellison advises New South Wales government on lease of Port of Newcastle
Minter Ellison has advised NSW Treasury on the lease of the Port of Newcastle to a consortium comprising Hastings Funds Management and China Merchants.
Best Lawyers recognises capabilities of Minter Ellison's team across Australia
The range and depth of capabilities of Minter Ellison's team across Australia has been recognised by the 2014–15 list of Best Lawyers.
More amendments in store for the Building and Construction Industry Payments Act weblink
The Queensland Government has unveiled a series of amendments to be made to the Building and Construction Industry Payments Act 2004 (BCIPA).
China Business Law recognises Minter Ellison for energy, projects and infrastructure work
Minter Ellison is one of five international law firms named as winners of a China Business Law 2013 award for energy, projects and infrastructure.
New Unsolicited Proposal Guideline in Victoria weblink
The Unsolicited Proposal Guideline provides a framework for the government's assessment and implementation of infrastructure proposals that originate directly from the private sector.
Productivity Commission inquiry announced to boost infrastructure funding weblink
The federal government has announced a Productivity Commission inquiry into the funding of public infrastructure.


text:
Okay, so I bought a liver chestnut tail extension for my horse. Grew out of that horse, and got a chestnut QH.
The tail is only used TWICE, but now does not match my new horse Is it possible to dye the hair? It is a real horse hair switch if that helps.