Research Article |
Corresponding author: Margarida L. R. Aguiar-Perecin ( mlrapere@usp.br ) Academic editor: Puneet Puneet
© 2019 Daniel Pizzaia, Vanessa M. Oliveira-Maekawa, Aline R. Martins, Mateus Mondin, Margarida L. R. Aguiar-Perecin.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Pizzaia D, Oliveira-Maekawa VM, Martins AR, Mondin M, Aguiar-Perecin MLR (2019) Karyotype structure and NOR activity in Brazilian Smilax Linnaeus, 1753 species (Smilacaceae). Comparative Cytogenetics 13(3): 245-263. https://doi.org/10.3897/CompCytogen.v13i3.35775
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The genus Smilax Linnaeus, 1753 (Smilacaceae) is a large genus of dioecious plants distributed in tropical, subtropical and temperate regions. Some Smilax species have medicinal importance and their identification is important for the control of raw material used in the manufacture of phytotherapeutical products. The karyotypes of seven Brazilian Smilax species were investigated. Mitotic metaphases of roots from young plants were analysed in Feulgen-stained preparations. The karyotypes were asymmetric and modal with 2n = 2x = 32 chromosomes gradually decreasing in size. In S. goyazana A De Candolle & C De Candolle, 1878, a polyploid species, 2n = 4x = 64. In all the species, the large and medium-sized chromosomes were subtelocentric and submetacentric and the small chromosomes were submetacentric or metacentric. Their karyotypes were quite similar, with differences in the arm ratio of some chromosomes. S. fluminensis Steudel, 1841 differed from the other species by having a large metacentric chromosome 1. These findings suggest that evolution occurred without drastic changes in the chromosomal structure in the species analyzed. Terminal secondary constrictions were visualized on the short arm of some chromosomes, but they were detected only in one homologue of each pair. Due to the terminal location and the degree of chromosome condensation, secondary constrictions were not visualized in some species. The nucleolus organizer regions (NORs) were mapped by silver-staining and fluorescent in situ hybridization (FISH) in S. rufescens Grisebach, 1842 and S. fluminensis. Silver-staining and FISH signals were colocalized on the short arms of six chromosomes in S. rufescens and four chromosomes in S. fluminensis. In FISH preparations, one of the largest chromosomes had the secondary constrictions highly decondensed in some cells. This finding and the heteromorphism observed in Feulgen-stained chromosomes suggest that differential rRNA gene expression between homologous rDNA loci can occur in some cells, resulting in different degrees of ribosomal chromatin decondensation. The presence of a heteromorphic chromosome pair in S. rufescens, S. polyantha Grisebach, 1842 and S. goyazana suggests a chromosomal sex determination in these dioecious species.
Smilax, karyotype, chromosomal evolution, nucleolus organizer region (NOR), Silver-staining, FISH, 45S rDNA
The genus Smilax Linnaeus, 1753 (Smilacaceae) is a large genus of dioecious plants distributed in tropical, subtropical and temperate regions. The genus has approximately 300 species, and their classification has been controversial (see
Some Smilax species have medicinal importance. Roots have been used as anti-syphilitic, anti-inflammatory and antimicrobial remedies or as antioxidant agents (
The characterization of karyotypes in higher plants has evolutionary and taxonomic significance. Some studies on Smilax cytogenetics have reported chromosome numbers and karyotype characterization. Chromosome numbers of n = 16 were described for most species, but n = 13 and n = 15 were also recorded (
In the present study, we investigated the karyotype characteristics of seven species of Brazilian species of Smilax using conventional techniques. We compared the positions of 45S rDNA sites of S. rufescens with the sites in S. fluminensis. Procedures to germinate wild-collected seeds to obtain plants providing roots for cytogenetic research were also developed. We aimed to analyze aspects of karyotype evolution in these species and to contribute to their taxonomic treatment.
Seeds from wild plants collected from southern, southeastern, northeastern and western central Brazil were used (Table
Origin of collection, chromosome number, chromosome and haploid set length (µm) and ratio of the largest/smallest chromosomes of the Smilax species.
Species | Origin† | 2n | Chromosome length | ||
---|---|---|---|---|---|
Range (µm) | Ratio (largest/ smallest) | Haploid set (µm) | |||
S. rufescens Grisebach, 1842 | Ilha do Cardoso (SP) | 32 | 5.62–1.84 | 3.05 | 54.24 |
S. fluminensis Steudel, 1841 | Uruana (GO) | 32 | 6.41–1.33 | 4.82 | 43.47 |
Itirapina (SP) | 32 | 6.48–1.31 | 4.95 | 43.32 | |
S. polyantha Grisebach, 1842 | Botucatu (SP) | 32 | 5.85–1.92 | 3.05 | 44.09 |
Mogi Guaçu (SP) | 32 | 5.36–1.94 | 2.80 | 41.03 | |
S. brasiliensis Sprengel, 1825 | Itapagipe (MG) | 32 | 5.04–1.77 | 2.85 | 38.02 |
S. campestris Grisebach, 1842 | Caçapava do Sul (RS) | 32 | 6.20–1.95 | 3.18 | 43.44 |
S. cissoides Martius ex Grisebach, 1842 | Feira de Santana (BA) | 32 | 5.51–2.00 | 2.75 | 40.17 |
S. goyazana A. de Candolle & C de Candolle, 1878 | Brasilia (DF) | 64 | 4.85–1.61 | 3.01 | 79.25 |
Experiments to germinate seeds to obtain plants were evaluated as reported by
Roots excised from young plants were pretreated with 8-hydroxyquinoline combined with cycloheximide, a protein synthesis inhibitor that induces chromosome condensation. Some treatments were evaluated, and in most cases, two treatments were used: combinations of 300 mg/L 8-hydroxyquinoline with 1.25 mg/L or 20 mg/L cycloheximide at 28 °C for 3 h and 2.5 h, respectively. The roots were then fixed in 3:1 ethanol:acetic acid and kept at 4 °C. The roots were Feulgen-stained as previously described (
Active NORs were detected in metaphase chromosomes of S. rufescens and S. fluminensis, by employing the silver-staining technique according to
Fluorescent in situ hybridization was used to detect 45S rDNA sites as previously described (
All preparations were examined with a Zeiss Axiophot-2-epifluorescence microscope with appropriate filters. The images were acquired by a CCD camera using the IKAROS software to analyze the Feulgen-stained metaphases and the ISIS software for the FISH images (Meta-Systems, Germany). Silver-stained chromosomes were photographed using the Fujicolor Superia 100 film (Fuji Photo Film, Brazil). All images were processed with Adobe Photoshop 6.0.
The experiments to germinate seeds from wild plants were successful for obtaining plants and roots with high index of mitosis. The pretreatments used also provided metaphases suitable for karyotype analysis.
All of the species analyzed had 2n = 2x = 32, except the tetraploid S. goyazana with 2n = 4x = 64. The karyotypes were asymmetric and modal with the chromosomes gradually decreasing in size, as shown in Fig.
Karyotypes of Smilax species: Feulgen-stained metaphase chromosomes. A S. rufescens B S. fluminensis (Uruana access) C S. fluminensis (Itirapina access) D S. polyantha (Botucatu access) E S. polyantha (Mogi Guaçu access) F S. brasiliensis G S. campestris H S. cissoides I S. goyazana. Arrows indicate secondary constrictions and satellites. Note the heteromorphic pair 10 in S. rufescens, pair 11 in S. polyantha (Mogi Guaçu) and pair 27 in S. goyazana. Scale bar: 10 µm.
Karyological data of Smilax species including chromosome relative length, arm ratio, karyotype type.
Chromosomes | 1 | |||||||||||||||||
Species | ||||||||||||||||||
Relative length (%) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | ||
S. rufescens | 10.37 | 9.64 | 8.70 | 8.23 | 7.55 | 7.08 | 6.36 | 6.03 | 5.82 | 5.06/5.06 | 4.75 | 4.57 | 4.35 | 4.09 | 3.87 | 3.41 | ||
S. fluminensis (Uruana) | 15.08 | 11.32 | 9.25 | 8.84 | 8.26 | 6.50 | 5.87 | 5.59 | 5.11 | 4.81 | 4.03 | 4.04 | 3.95 | 3.77 | 3.53 | 3.33 | ||
S. fluminensis (Itirapina) | 15.79 | 10.87 | 8.67 | 8.35 | 8.16 | 7.00 | 6.31 | 5.96 | 5.43 | 5.04 | 4.26 | 4.24 | 4.09 | 4.01 | 3.77 | 3.13 | ||
S. polyantha (Botucatu) | 11.80 | 10.29 | 9.68 | 9.01 | 8.29 | 7.88 | 6.99 | 6.76 | 5.94 | 5.21 | 5.00 | 4.75 | 4.56 | 4.43 | 4.05 | 3.90 | ||
S. polyantha (Mogi Guaçu) | 10.52 | 9.71 | 8.86 | 8.15 | 7.90 | 6.95 | 6.85 | 6.07 | 5.95 | 5.59 | 4.91/491 | 4.37 | 4.34 | 3.88 | 3.06 | 3.02 | ||
S. brasiliensis | 11.82 | 10.50 | 10.00 | 9.24 | 8.74 | 8.17 | 7.61 | 6.94 | 6.44 | 5.41 | 5.19 | 4.89 | 4.89 | 4.80 | 4.65 | 4.17 | ||
S. campestris | 11.06 | 9.32 | 8.54 | 8.18 | 7.47 | 7.25 | 6.70 | 6.40 | 5.65 | 5.32 | 4.63 | 4.14 | 4.14 | 3.85 | 3.81 | 3.47 | ||
S. cissoides | 10.33 | 9.39 | 8.18 | 7.97 | 7.70 | 7.24 | 6.56 | 6.11 | 5.82 | 5.28 | 4.69 | 4.23 | 4.19 | 3.57 | 3.57 | 3.50 | ||
S. goyazana | 5.51 | 5.51 | 5.00 | 4.50 | 4.48 | 4.38 | 4.29 | 4.28 | 3.79 | 3.69 | 3.64 | 3.59 | 3.45 | 3.28 | 3.18 | 3.09 | ||
Chromosomes | ||||||||||||||||||
17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 | |||
S. goyazana (cont) | 2.94 | 2.85 | 2.85 | 2.73 | 2.69 | 2.17 | 2.09 | 2.05 | 1.97 | 1.94 | 1.89/1.89 | 1.89 | 1.85 | 1.77 | 1.71 | 1.67 | ||
Chromosomes | ||||||||||||||||||
Arm ratio / Chromosome types | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | Karyotypes | Stebbins karyotype classification |
S. rufescens | 5.28 | 4.13 | 4.46 | 3.41 | 3.28 | 3.00 | 3.30 | 2.56 | 2.82 | 2.17/1.67 | 2.15 | 1.99 | 1.92 | 1.84 | 1.53 | 1.24 | 7st+6.5sm+2.5m | 3B |
st | st | st | st | st | st | st | sm | sm | sm/m | sm | sm | sm | sm | m | m | |||
S. fluminensis (Uruana) | 1.02 | 2.02 | 3.17 | 2.91 | 3.03 | 4.00 | 2.50 | 2.70 | 2.98 | 2.55 | 2.31 | 3.00 | 1.77 | 1.49 | 1.70 | 1.85 | 4st+10sm+2m | 3C |
m | sm | st | sm | st | st | sm | sm | sm | sm | sm | st | sm | m | sm | sm | |||
S. fluminensis (Itirapina) | 1.07 | 2.08 | 3.00 | 2.68 | 3.03 | 3.03 | 1.60 | 3.0 | 2.24 | 2.49 | 2.03 | 3.00 | 1.84 | 1.49 | 1.56 | 1.78 | 5st+7sm+4m | 3C |
m | sm | st | sm | st | st | m | st | sm | sm | sm | st | sm | m | m | sm | |||
S. polyantha (Botucatu) | 6.10 | 6.00 | 5.00 | 4.00 | 4.00 | 3.8 | 3.10 | 3.00 | 2.11 | 2.43 | 1.59 | 3.00 | 2.50 | 1.45 | 1.35 | 1.24 | 9st+3sm+4m | 3B |
st | st | st | st | st | st | st | st | sm | sm | m | st | sm | m | m | m | |||
S. polyantha (Mogi Guaçu) | 6.00 | 6.00 | 4.00 | 3.50 | 3.50 | 3.17 | 3.17 | 3.00 | 3.00 | 3.00 | 2.00/3.00 | 2.04 | 2.00 | 1.60 | 1.50 | 1.24 | 10.5st+2.5sm+3m | 3B |
st | st | st | st | st | st | st | st | st | st | sm/st | sm | sm | m | m | m | |||
S. brasiliensis | 6.00 | 5.10 | 4.00 | 3.90 | 3.38 | 3.17 | 3.00 | 2.72 | 2.54 | 2.74 | 3.00 | 1.89 | 2.00 | 1.78 | 1.47 | 1.50 | 8st+6sm+2m | 3B |
st | st | st | st | st | st | st | sm | sm | sm | st | sm | sm | sm | m | m | |||
S. campestris | 4.51 | 4.1 | 3.23 | 2.94 | 3.18 | 2.27 | 3.00 | 2.7 | 1.99 | 2.17 | 1.5 | 1.50 | 1.38 | 1.5 | 1.62 | 1.4 | 5st+5sm+6m | 3B |
st | st | st | sm | st | sm | st | sm | sm | sm | m | m | m | m | m | m | |||
S. cissoides | 4.60 | 4.54 | 3.00 | 3.82 | 2.79 | 3.44 | 2.4 | 3.00 | 3.00 | 2.00 | 2.00 | 1.70 | 1.87 | 2.00 | 1.49 | 1.50 | 7st+7sm+2m | 3B |
st | st | st | st | sm | st | sm | st | st | sm | sm | sm | sm | sm | m | m | |||
S. goyazana | 8.00 | 7.5 | 5.75 | 4.35 | 4.89 | 5.01 | 4.16 | 4.00 | 3.00 | 3.17 | 3.00 | 2.7 | 3.44 | 3.10 | 3.03 | 3.00 | 2t+15.5st+ 8.5sm+6m | 3B |
t | t | st | st | st | st | st | st | st | st | st | sm | st | st | st | st | |||
Chromosomes | ||||||||||||||||||
17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 | |||
S. goyazana (cont) | 3.00 | 2.96 | 2.96 | 2.7 | 3.00 | 1.83 | 1.5 | 1.78 | 2.50 | 2.60 | 3.00/2.09 | 1.17 | 1.18 | 1.32 | 1.43 | 1.40 | ||
st | sm | sm | sm | st | sm | m | sm | sm | sm | st/sm | m | m | m | m | m |
This species with 2n = 2x = 32 showed a karyotype with 7 pairs of st-type, 6 pairs of sm-type and 2 pairs of m-type chromosomes, and the heteromorphic pair 10 with sm-and m-type chromosomes with similar sizes, probably sex chromosomes. The size of the chromosomes varied from 1.84 to 5.62 µm with a largest/smallest ratio of 3.05 and the Stebbins karyotype classifications was 3B. The total haploid length was 54.24 µm. Secondary constrictions were detected on the short arms of chromosomes 7, 11 and 14 in some metaphases. These constrictions were visible only in one homologue of each chromosome pair. (Fig.
Specimens collected from Uruana (GO) and Itirapina (SP) were analyzed. Both specimens had 2n= 2x = 32. The karyotypes were quite similar with slight differences in the arm ratio of chromosomes 7, 8 and 15. The plants from Uruana showed a karyotype with 4 pairs of st-type, 10 pairs of sm-type and 2 pairs with m-type chromosomes. The size of the chromosomes varied from 1.33 to 6.41 µm with a largest/ smallest ratio of 4.82 and the Stebbins karyotype classification was 3C. The total haploid length was 43.47 µm. Secondary constrictions were not detected (Fig.
Specimens collected from Botucatu (SP) and Mogi Guaçu (SP) were analyzed. Both had 2n = 2x = 32. The karyotypes were quite similar with slight differences in the arm ratio of chromosomes 9, 10, 11 and 12. The plants from Botucatu showed a karyotype with 9 pairs of st-type, 3 pairs of sm-type and 4 pairs of m-type chromosomes. The size of chromosomes varied from 1.92 to 5.85 µm with a largest/smallest ratio of 3.05 and the Stebbins karyotype classification was 3B. The total haploid length was 44.09. Secondary constrictions were not detected (Fig.
This species with 2n = 2x = 32 showed a karyotype with 8 pairs of st-type, 6 pairs of sm-type and 2 pairs of m-type chromosomes. The size of chromosomes varied from 1.77 to 5.04 µm with a largest/smallest ratio of 2.85 and the Stebbins karyotype classification was 3B. The total haploid length was 38.02 µm. A satellite and secondary constriction were observed on chromosome 11 and they were visible only in one homologue of the chromosome pair (Fig.
This species with 2n = 2x = 32 showed a karyotype with 5 pairs of st-type, 5 pairs of sm-type and 6 pairs of m-type chromosomes. The size of chromosomes varied from 1.95 to 6.20 µm with a largest/smallest ratio of 3.18 and the Stebbins karyotype classification was 3B. The total haploid length was 43.44 µm. Satellites and secondary constrictions were detected on the chromosomes 10 and 14, visible only in one homologue of each pair (Fig.
This species with 2n = 2x = 32 showed a karyotype with 7 pairs of st-type, 7 pairs of sm-type and 2 pairs of m-type chromosomes. The size of chromosomes varied from 2.00 to 5.51 µm with a largest/smallest ratio of 2.75 and the Stebbins karyotype classification was 3B. The total haploid length was 40.17 µm. Satellites were not detected (Fig.
This polyploid species with 2n = 4x = 64 showed a karyotype with 2 pairs of t-type, 15 pairs with st-type, 8 pairs with sm-type, 6 pairs with m-type chromosomes and the heteromorphic pair 27 with st-type and sm-type chromosomes with similar sizes. The size of chromosomes varied from 1.61 µm to 4.85 with a largest/smallest ratio of 3.01 and the Stebbins karyotype classification was 3B. The total haploid length was 79.25 µm. A satellite and secondary constriction were detected on the largest and t-type chromosome 1, and it was visible only in one of the homologues (Fig.
As previously reported (
In the silver-stained metaphases of S. rufescens, six chromosomes showed positive signals on the termini of the short arms (Fig.
In S. fluminensis, only four chromosomes showed 45S rDNA sites. In Fig.
In the silver-stained metaphases of S. fluminensis, four chromosomes showed positive signals on the termini of short arms (Fig.
FISH signals of 45S rDNA (red) (A, C), silver staining (B, D) in S. rufescens (A, B) and S. fluminensis (C, D). Arrows in A and C indicate distended secondary constrictions, and arrowheads show condensed rDNA sites. Arrows in B and D indicate larger silver signals and arrowheads in B shows small sites. Scale bar: 10 µm.
All of the diploid species studied here had 2n = 2x = 32, except the polyploid S. goyazana with 2n = 4x= 64. Most diploid species from East Asia and India also have 2n = 2x = 32 (
The karyotypes of the species analyzed were asymmetric, and the absolute size of the chromosomes was rather similar, gradually decreasing in size, except in S. fluminensis, in which the metacentric chromosome 1 was larger than in the other species. This finding was clearly emphasized with the evaluation of the ratio between the largest and smallest chromosomes that varied from 2.75 to 3.18 in most species compared with the values of S. fluminensis that were larger (4.82–4.95). The relative chromosome lengths were also quite similar with clear differences concerning the relative lengths of chromosome 1 in most species (10.33 to 11.82) compared with S. fluminensis (15.08–15.79). In a general sense, these data were similar to those reported for species from East Asia (
The presence of a large metacentric chromosome 1 in the karyotype of S. fluminensis is a special feature that is unusual in Smilax species.
In general, the species analyzed had asymmetric karyotypes with chromosomes gradually decreasing in size and showing variability in centromere position. The classification of the karyotypes according to
The genus Smilax has been assigned to the family Smilacaceae and to the order Liliales sensu APG III thus, it is a sister family of Liliaceae.
Significant differences in karyotype asymmetry are apparent within Liliales, in which two different aspects can be observed, such as variation in chromosome length and variation in centromere position. Variation in chromosome lengths such as the observed in Smilax, was observed in some Liliaceae genera with small genome sizes such as Streptopus and Prosartes, while in some genera, such as Lilium and Fritillaria, the asymmetry is mainly due to variation in the position of centromeres (two large metacentric chromosomes and subtelocentric and telocentric chromosomes with rather similar lengths (see
Secondary constrictions were not described for the karyotypes of most Smilax species reported in the literature.
In the present study, in situ hybridization of 45S rDNA probes showed six sites of ribosomal DNA in S. rufescens and four sites in S. fluminensis. In addition to differing from S. rufescens and the other species analyzed by the presence of a large metacentric chromosome 1, S. fluminensis has a different number of ribosomal DNA sites. The number of positive silver-staining signals was correlated with the FISH signals in both species. Terminal secondary constrictions were observed in three chromosome pairs of S. rufescens. As it was unclear if satellites were visible, the structures visualized were considered terminal secondary constrictions. In more condensed metaphases, only two chromosomes displayed secondary constrictions (
Secondary constrictions have been considered to be the organization pattern of active ribosomal chromatin on metaphase chromosomes. Silver-staining on secondary constriction allows the visualization of ribosomal genes that were transcribed in the previous interphase. It has been shown that silver binds to proteins that are components of the transcription machinery and remain at NOR regions throughout metaphase and anaphase (see revision in
Details on the structure and function of the NOR chromosomes, as well as the quantification of observed events were not the scope of this study, but the detection of some features allows some discussion. The presence of positive-silver staining signals corresponding with the number of rDNA sites revealed by FISH showed that all these loci were active in both species analyzed. However, in some Feulgen-stained metaphases of S. rufescens only two chromosomes bearing secondary constriction were observed (
In the present study, heteromorphic secondary constrictions were detected in FISH preparations of S. rufescens and S. fluminensis, but metaphases with both homologues of the largest NOR chromosome pair displaying distended secondary constrictions were also observed (not shown).Therefore, we conclude that in these species, differential expression of rDNA loci occurs only in some cells. The observation of homologous NOR chromosomes showing differences in the presence of secondary constrictions in Feulgen-stained metaphases of S. rufescens, S. fluminensis and also in S. campestris, S. cissoides and S. goyazana suggests that differential expression of rDNA loci is frequent in the genus Smilax.
Interestingly, the treatment of roots of S. cereale with the methyltransferase inhibitor 5-azacytidine (5-AC) resulted in an increase of rRNA gene transcription and then in a reduction in the number of cells showing a significant difference in the size of silver-stained domains in the two NORs (
The differential expression between homologous NOR chromatin is a different phenomenon in relation to nucleolar dominance. Nucleolar dominance has been characterized as an epigenetic phenomenon that occurs in plant allopolyploids and hybrids, in which only one ancestral set of ribosomal genes retains the ability to organize the nucleolus, while the rDNA loci derived from the other progenitor are silenced. For instance, in Atropa belladonna Linnaeus, 1753 derived from a tetraploid and a diploid ancestor species, only four out of six rDNA sites are transcriptionally active, as revealed by silver-staining (
The karyotypes of seven Brazilian Smilax species investigated were asymmetric and modal with 2n = 2x = 32 chromosomes gradually decreasing in size. In S. goyazana, a polyploid species, 2n = 4x = 64. In all the species, the large and medium-sized chromosomes were subtelocentric and submetacentric and the small chromosomes were submetacentric or metacentric. Their karyotypes were quite similar, with slight differences in the arm ratio of some chromosomes and belong to class 2B according with Stebbins classification (1971). S. fluminensis differed from the other species by having a large metacentric chromosome 1 and belonging to class 3C. These findings suggest that evolution occurred without drastic changes in karyotype structure in the species analyzed, except S. fluminenesis. Terminal secondary constrictions were visualized on the short arm of some chromosomes, but they were detected only in one homologue of each pair. Due to the terminal location and the degree of condensation of the chromosomes, secondary constrictions were not visualized in some species. In S. rufescens and S. fluminensis all the rDNA loci were active as demonstrated by silver-staining signals colocalized with the FISH signals. We concluded that differential expression of rDNA loci occurs in these species based on the observation of a distended secondary constriction in the largest NOR chromosome visualized in FISH preparations of some cells in both species. Distended secondary constrictions were not observed in the smallest chromosomes, probably due to their small size and the degree of metaphase condensation. In this connection, it is interesting to note that the absence of secondary constriction on an active locus was observed in Crotalaria juncea Linnaeus, 1753, in which two silver-stained rDNA loci were observed, that is, one major locus showing secondary constriction and one minor locus in which a secondary constriction was not detected (
We acknowledge the support of the Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP, Project BIOTA, #05/58964-9), of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), of Dr. Beatriz Appezzato-da-Glória (University of São Paulo) and Dr. Eliana R. Forni-Martins (The University of Campinas) for cooperation in this research, and Mrs. S. C. M. Molina for technical assistance.