RESEARCH ARTICLE

Karyotype characterization reveals an up and downof 45S and 5S rDNA sites in Crotalaria (Leguminosae-Papilionoideae) species of the section Hedriocarpaesubsection Macrostachyae

A. G. Morales • M. L. R. Aguiar-Perecin •

M. Mondin

Received: 27 October 2010 / Accepted: 7 March 2011

� Springer Science+Business Media B.V. 2011

Abstract The genus Crotalaria is one of the largest

within the family Leguminosae-Papilionoideae, with

more than 600 species. However, few karyotypes have

been described. In the present paper, five species

belonging to the section Hedriocarpae were studied

(subsection Machrostachyae), in order to better under-

stand chromosomal evolution in Crotalaria. The

results reveals that all species presented 2n = 2x =

16 with symmetrical karyotypes, and slight differences

in the chromosome morphology. A secondary con-

striction was identified at short arm of the pair 1. The

45S rDNA was mapped in the secondary constriction

and adjacent heterochromatin (NOR-heterochromatin)

and a minor site was identified in C. ochroleuca. The

5S rDNA was mapped linked to 45S rDNA at

chromosome 1 short arm in all species. Additional

sites for 5S rDNA were identified in C. pallida,

C. striata and C. mucronata. Heterochromatin blocks

around the centromeres are not CMA? neither DAPI?.

The karyotypes of the subsection Macrostachyae are

characterized by an inversion at chromosome pair one

in relation to previous specialized floral species

analyzed. Additional sites of 45S and 5S rDNA were

assumed to be a result of transposition events by

different ways. The results suggest heterochromatin

differentiation and the position of ribosomal genes

indicates chromosomal rearrangements during evolu-

tion. Karyotype characteristics corroborate the mor-

phological infrageneric classification.

Keywords Crotalaria � Karyotype � Ribosomal

variability � Transposition � 45S rDNA � 5S rDNA

Introduction

The genus Crotalaria is an important group of

species of the Leguminosae-Papilionoideae family,

including crops those have been using mainly to

atmospheric nitrogen fixation, cover the soil protect-

ing against erosion, in combat against nematodes

contamination in the fields (Polhill 1982; Hanelt and

Institute of Plant Genetics and Crop Plant Research

2001; Germani and Plenchette 2004), also because of

the fibers one of the primary uses is to manufacture

twine, cord, marine cordage, fishing nets, matting,

sacking, and paper (revision in Morris and Kays

2005). Moreover, Crotalaria species has been used in

sugar field rotation to improve the production,

reducing fertilizer usage and promoting a more

sustainable production (Dinardo-Miranda and Gil

2005). Other potential usage include soil remediation

(Pereira et al. 2002), anti-inflammatory and anti-

A. G. Morales � M. L. R. Aguiar-Perecin �M. Mondin (&)

Departamento de Genetica, Escola Superior de

Agricultura ‘‘Luiz de Queiroz’’, Universidade de Sao

Paulo, Av Padua Dias 11 Caixa Postal 83, Piracicaba, SP

13400-970, Brazil

e-mail: mmondin@esalq.usp.br

123

Genet Resour Crop Evol

DOI 10.1007/s10722-011-9683-8

hepatotoxic production (Ahmed et al. 2006), as

functional food for preventing and treating large

intestinal cancer (Seong et al. 2008) and others.

About 600 species of the genus Crotalaria have

been described and organized into sections and

subsections according to their morphological traits.

The sections can be divided into two major groups,

one with species presenting some specialized features

in the flowers and another without floral appendages

that confer some specialization to pollinators (in

other words non-specialized flowers). The section

Hedriocarpae belongs to the non-specialized flower

group and is subdivided into two subsections, Hed-

riocarpae and Machrostachyae, both of African origin

(Polhill 1982). However the Hedriocarpae species are

versatile and widespread in the tropical and subtrop-

ical regions.

Most of the cytogenetic studies in the genus

Crotalaria have been concentrated on chromosome

counting (Boulter et al. 1970; Flores et al. 2006),

karyotype description (Almada et al. 2006; Gupta and

Gupta 1978; Oliveira and Aguiar-Perecin 1999;

Raina and Verma 1979;), and karyotype variability

among populations (Palomino and Vazquez 1991;

Tapia-Pastrana et al. 2005). Cytogenetically, the

genus presents a decrease in chromosome size

according to the level of flower specialization (Boul-

ter et al. 1970), with the non-specialized floral species

having bigger chromosomes than specialized species.

Some karyotype morphometric parameters are pre-

served across the species from the botanic sections

(Almada et al. 2006; Oliveira and Aguiar-Perecin

1999). Most African species are diploids with

2n = 2x = 16, while polyploids are predominantly

restricted to specialized floral unifoliate species from

the native American section Calycinae (Almada et al.

2006; Flores et al. 2006; Oliveira and Aguiar-Perecin

1999; Windler 1974). The karyotype symmetry,

evidenced by the presence of metacentric or sub-

metacentric chromosomes, is characteristic of the

genus, what makes the individual identification and

detections of chromosomal alterations difficult (Al-

mada et al. 2006; Oliveira and Aguiar-Perecin 1999).

Nevertheless, the presence of NOR in chromosome 1

allows its identification. The analyses of male meiosis

revealed a high stability of the chromosomes with

mainly bivalents formed even in polyploid species

(Almada et al. 2006; Palomino and Vazquez 1991;

Verma and Raina 1983). Many authors have proposed

that mutation and recombination of individual genes,

or complexes of genes, played a major role in the

diversification of the genus (Gupta and Gupta 1978;

Raina and Verma 1979; Verma et al. 1984). How-

ever, Mondin et al. (2007a) pointed out that these

conclusions could be the result of the lack of

adequate markers to detect major chromosomal

rearrangements and heterochromatin block variations

among the species.

Recently, the presence of GC rich heterochromatic

blocks distributed around the centromere and the

NOR was detected in species of the sections

Calycinae and Crotalaria subsections Crotalaria and

Longirostres (Mondin 2003). However the NOR-

heterochromatin appears brighter when stained with

CMA/DA while the centromeric-heterochromatin

quenched (Mondin et al. 2007b). The physical

mapping of 45S and 5S rDNA showed a high

correspondence with CMA/DA bright regions. A

non-specialized flower species, C. incana, showed a

distinct pattern of chromosome banding without GC

rich regions around the centromere (Mondin 2003).

The use of chromosome banding and FISH with

45S and 5S rDNA or other repetitive sequences are

powerful approaches to understand the structure and

evolution of the karyotypes and have been applied

successfully to different groups of species, for

instance Passiflora (Cuco et al. 2005), Hypochaeris

(Ruas et al. 2005), Maxillaria (Cabral et al. 2006),

Cestrum (Fregonezi et al. 2006, 2007), Nicotiana

(Lim et al. 2000), Aegilops (Raskina et al. 2004a,

2004b), Vicia (Raina et al. 2001), Lathyrus (Ali et al.

2000), Hordeum (Taketa et al. 1999, 2000) and

Arabidopsis (Maluszynska and Heslop-Harrison

1993).

Typically, a species present in its karyotype at

least one locus of 45S rDNA and another of 5S

rDNA, that could be linked or not in the same

chromosome or be present in different chromosome

pairs. Additional locus for ribosomal genes could

have resulted from polyploidy events, unequal

recombination, rDNA movement associated to En/

Spm transposons or extrachromosomal circular DNA

(Cohen and Segal 2009; Schubert 2007; Schubert and

Wobus 1985).

Herein, we studied the karyotype of five species

belonging to the botanic section Hedriocarpae, sub-

section Macrostachyae, a non-specialized floral group

of the genus Crotalaria, through chromosome

Genet Resour Crop Evol

123

banding and FISH with ribosomal DNA probes (45S

and 5S).

Materials and methods

Plant material

Five species were analyzed according to the classi-

fication received from the donors (Table 1). The

species belong to the section Hedriocarpae, subsec-

tion Machrostachyae, according to the Polhill’s

(1982) infrageneric classification. All species occur

in natural environments in Brazil; however these

species were originated in the African continent and

have been introduced in the Americas.

Chromosome preparation, Feulgen

and fluorescent staining

The seeds were scarified in pure sulfuric acid during

8 min. and subsequently rinsed abundantly with

water for dormancy breakage, and air-dried. The

scarified seeds were germinated at 28�C and for

metaphase accumulation the roots were pretreated in

a solution containing 300 mg l-1 8-hydroxyquinoline

and 6.25 mg l-1 cycloheximide for 90 min and then

fixed in 3:1 (v/v) ethanol:acetic acid. The roots were

stained by the Feulgen staining described by Cuco

et al. (2003) and the karyotypes were established

selecting metaphases with the same condensation

level. At least ten selected metaphases were used to

morphometric analysis with standard deviations for

short and long arm presented as a bar in the

ideograms. For slide preparations root tips were

digested in a mixture of cellulase at 9.2 units ml-1

combined with pectinase at 14.7 units ml-1 (final

concentrations) in citrate buffer (pH 4.6) at 37�C for

1 h and then squashed in 60% (v/v) acetic acid. The

coverslip was removed in liquid nitrogen and the

slides were air-dried. Based on Mondin et al. (2007b)

the slides were stained with chromomycin A3 (CMA)

and counterstained either with distamycin A (DA) or

40, 6-diamidino-2-phenylindole (DAPI), or actinomy-

cin D (AMD). The slides were mounted in Vecta-

shield (Vector, USA).

FISH analysis

The methodology is the same as previously described

by Mondin et al. (2007b) and presented briefly below.

The 45S rDNA probe consisting of a 9.1 kb sequences

isolated from maize rDNA and cloned in a pUC18

plasmid (McMullen et al. 1986) and the 5S rDNA

probe, consisting of a fragment about 150 bp long,

was isolated through PCR from genomic total DNA

extracted from leaves of C. striata seedlings by

DNeasy Plant kit (Qiagen, Germany) using the

primers described in Cuco et al. (2005). The 45S

rDNA probe was labeled with biotin-14-dATP by nick

translation (Bionick Labelling System, Gibco BRL)

while the 5S rDNA was labeled with digoxigenin-11-

dUTP by random primer labeling (Roche, Germany).

The pre-hybridization treatment followed the protocol

described in Schwarzacher and Heslop-Harrison

(2000) with slight modifications described by Cuco

et al. (2005). The 45S rDNA (5 ng ll-1) and 5S rDNA

(6 ng ll-1) probes in the hybridization mixture were

denatured by heating in a water-bath at 96�C for

10 min, then cooled and dropped onto the slide and

together denatured in a thermocycler (M.J. Research,

Inc., USA) at 83�C for 10 min and the hybridization

was performed at 37�C for 16 h. The stringency

washes were made twice in 2xSSC at 37�C and at

Table 1 Crotalaria species of the section Hedriocarpae, subsection Machrostachyae descriptions and accessions in the germplasm

bank

Species Origin Donor Germplasm Code

C. lanceolata E. Mey. Santa Rita—Sao Paulo BR 116 (Km 114) Instituto de Zootecnia de Nova Odessa—SP CL-3

C. ochroleuca G. Don Indeterminate Piracicaba, ESALQ—SP COC-1

C. pallida Ait.a Rio Bonito—RJ Instituto de Zootecnia de Nova Odessa—SP CPA-1

C. mucronata Desv.a Indeterminate Piracicaba, ESALQ—SP CM-1

C. striata DC.a Indeterminate Piracicaba, ESALQ—SP CSR-1

a Species synonyms of C. pallida according to Polhill (1982)

Genet Resour Crop Evol

123

42�C (5 min each), then in 20% (v/v) formamide in

0.5 9 SSC for 10 min at 42�C and then in 0.5 9 SSC

for 5 min at the same temperature. The biotin-labeled

probe was detected by applying mouse anti-biotin

antibody followed by an amplification with rabbit

anti-mouse (DAKO, A/S, Denmark) conjugated with

tetramethyl rhodamine isothiocyanate (TRITC) and

the digoxigenin-labeled probe was detected with

sheep anti-digoxigenin antibody (Roche) conjugated

with fluorescein isothiocyanate (FITC). The slides

were counterstained with DAPI (2 lg ml-1), air-dried

and then mounted in Vectashield H1000 (Vector,

USA). Chromosome spreads were analyzed using a

Zeiss Axiophot-2 epifluorescence microscope with

appropriate filters. The fluorochrome banding and

FISH images were acquired by a CCD using the ISIS

software (Meta Systems, Germany), and the color

channels were separated and the images adjusted as a

whole and superimposed in Adobe Photoshop.

Results

The karyotypes are presented in Fig. 1 and the

ideograms based on morphometric data are show in

Fig. 4. The species are 2n = 2x = 16 and most of the

chromosomes are metacentrics, with few submeta-

centrics. Such high symmetry makes individual

identification of the pairs difficult. However, slight

modifications can be seen in arm ratios (Table 2) and

Fig. 1 Karyotypes of Crotalaria species. a C. lanceolata, b. C.ochroleuca, c C. pallida, d C. mucronata, e C. striata.

Secondary constriction may be clearly seem associated to short

arm of chromosome 1 and centromeres position are identified as

conspicuous primary constriction in all chromosomes being

possible a measurement to obtain the ideograms

Genet Resour Crop Evol

123

the ideograms show the standard deviation in each

arm (Fig. 4). Only, chromosome pair 1 contains a

secondary constriction in the short arm.

Crotalaria lanceolata presented the larger haploid

complement length (haploid set) (Table 2), followed

by C. pallida and C. ochroleuca. The two smallest

haploid sets were C. striata and C. mucronata, both C.

pallida synonyms (Table 1). Comparing the species

that are described as synonyms, we were able to detect

an apparent difference in the haploid set. Moreover,

differences in chromosome morphology are clearly

seen in the arm ratio, for instance in chromosome pairs

5 and 6, where the relative length is almost not altered.

C. pallida presented the highest arm ratio on chromo-

some 3 and 5 among the species studied. Despite the

presence of some discrete chromosome morphometric

differences in the three species, they are not enough to

discriminate them; however these differences could

indicate some rearrangements in the genome in the

molecular level. Physical mapping of the 45S and 5S

rDNA revealed a conserved site in the chromosome

pair 1 among the species. The 45S rDNA was localized

at secondary constriction and NOR-heterochromatin,

as expected (Fig. 2). No additional site could be

observed. However, C. ochroleuca showed a minor

site at chromosome 4 (Fig. 3). This minor site could be

observed in most of the metaphase plates, where

42,85% showed one and 14,3% two additional sites.

Such variability could be explained by the reduced

locus size and the reduced sensitivity of FISH to sites

smaller than 10 Kbp. The presence of other signals

smaller than 10 Kbp could not be disregarded,

however to cytogenetic analysis only signals visible

must be considered. The minor 45S rDNA site at

chromosome 4 of C. ochroleuca has been interpreted

as a recent transposition event, due to its small size and

because other species of the subsection analyzed so far

did not show additional sites. The 45S rDNA was

localized at chromosome 1 short arm and is always

associated to a bright CMA? or CMA-DA? band and

to quenched DAPI- or DAPI-AMD- (Fig. 2).

The 5S rDNA was mapped at the short arm of

chromosome 1 adjacent to the 45S rDNA. C. pallida,

C. mucronata and C. striata showed 5S rDNA

additional sites at chromosomes 5 and 6 on the short

arm. Differences in size could also be observed. For

instance, the site of chromosome 5 is larger than in

chromosome 6; and a reduction in the 5S rDNA of the

chromosome 1 was detected when comparing the

signal among these three species to the site mapped in

C. lanceolata and C. ochroleuca. The additional 5S

rDNA sites and the reducing in the main site at

chromosome 1 of C. pallida and synonyms have been

attributed to transposition events. None of the 5S rDNA

sites was associated to CMA or CMA-DA neither to

DAPI or DAPI-AMD bright or quenched bands.

Even though additional 45S and 5S rDNA could be

attributed to transposition activity, unequal recombi-

nation could not be discarded, mainly because a

reduction was detected in signal size among the

species suggesting transference of DNA sequences to

another site followed by amplification. Accordingly,

the 45S rDNA additional site of C. ochroleuca can be

interpreted as a recent event, because it is not

amplified, as described above.

After the FISH procedures, bright heterochromatic

regions were detected around the centromere in all

species analyzed. However, those regions did not

present any kind of fluorescence using different

combination of dyes, CMA, CMA-DA, DAPI and

DAPI-AMD (Fig. 2). The lack of bright or quenched

bands is probably due to the nature and organization

of repetitive sequences, which could have affected

the specific ligation of the dyes, turning these regions

to neither GC or AT rich.

Discussion

The cytogenetic studies on the Crotalaria genus have

been restricted only to chromosome counting and a

few karyotype descriptions. About 30% of the species

had their chromosome number determined (Almada

et al. 2006; Flores et al. 2006) and the basic

chromosome number is clearly x = 8 for African

species, with one variant x = 7 in a restricted number

of species, for instance C. incana; and tetraploids

species (2n = 4x = 32) occurring in the New World

(Windler 1974).

Karyotype studies have shown a high symmetry

and a decrease in chromosome length following the

level of floral specialization (Boulter et al. 1970;

Almada et al. 2006; Oliveira and Aguiar-Perecin

1999). Chromosomal morphometric descriptions

showed the maintenance of karyotype features in

species from one section, in accordance with the plant

morphological phylogenetic relationships established

by Flores (2004), Polhill (1982), and Bisby and

Genet Resour Crop Evol

123

Genet Resour Crop Evol

123

Polhill (1973) (see Oliveira and Aguiar-Perecin

1999). Our present results based on Feulgen-staining

are in accordance with these preview works.

The species are diploid with 2n = 2x = 16 and

the karyotype is symmetric, with most chromosomes

being metacentric and few submetacentric. Morpho-

metric analysis revealed a slight difference among

chromosomes from different species and the haploid

length varies from 20 lm in C. mucronata to

22.91 lm in C. lanceolata. However, big differences

among the karyotype of these species have been

shown by Gupta and Gupta (1978) and Verma et al.

(1984). Some authors have attributed these differ-

ences to the qualities of chromosome preparations

(Almada et al. 2006; Cuco et al. 2003; Oliveira and

Aguiar-Perecin 1999). In fact Oliveira and Aguiar-

Perecin (1999), using a large number of well spread

and high quality morphological metaphase plates to

measurements, were able to establish karyotypes with

a high precision and this strategy was subsequently

followed by other authors (Almada et al. 2006;

Mondin et al. 2007b and the karyotypes therein).

An interesting difference was found in haploid

length, where C. pallida was 22.48 lm and C.

mucronata and C. striata showed 20.00 and

20.89 lm, respectively. A similar difference in

magnitude was observed by Gupta (1976) determin-

ing the nuclear DNA content, but the differences

were not statistically significant. As reported before,

the three species are in fact only one: C. pallida

(Polhill 1982). However these species have been

studied cytogenetically under different species names

(Gupta and Gupta 1978; Verma et al. 1984). In the

present case, they could represent different popula-

tions (germplasm accessions) with peculiar morpho-

logical variations and can be accessed to possible

chromosome variations.

Crotalaria lanceolata and C. ochroleuca have a

higher copy number of 5S rDNA repeats than other

species, adjacent to 45S rDNA in the short arm of

chromosome pair 1, while the C. pallida showed

additional sites at chromosomes 5 and 6 followed by

a reduction in the signal at chromosome 1. In the

other two species, C. mucronata and C. striata the

signals are identical in position but vary slightly in

brightness compared to C. pallida. We are interpret-

ing the additional sites as an event of transpositions,

Fig. 2 Fluorescent banding, in two first columns, with

Chromomycin A3 (CMA) and DAPI respectively; CMA-DA

and DAPI-AMD staining resulted in a similar fluorescent

pattern and have not been showed. Fluorescent in situhybridization of 45S (red) and 5S (green) rDNA are showed

in last column. The arrowheads in red indicate de 45S rDNA

FISH signals and their correspondence to secondary constric-

tions and NOR-heterochromatins when CMA staining and as

quenched region when DAPI staining.The 5S rDNA has been

indicated as a green arrowhead and has not been associated to

any kind of bright bands stained with the fluorochromes. C.lanceolata (a–c) and C. ochroleuca (d–f) where the bright

bands and both FISH signals are identified only at chromosome

pair 1; to the last species in some cases additional 45S rDNA

could be observed, as have been showing in the Fig. 3. C.pallida (g–i), C. mucronata (j–l) and C. striata (m–o) clearly

show an amplification of the 5S rDNA sites, however no

additional fluorescent bands could be identified with fluoro-

chrome staining, mainly by CMA. Bar = 5 lm

b

Table 2 Karyotype morphometric analysis of the Crotalaria species

Species 2n Karyotype features Chromosome features

Range (lm) Haploid Set (lm) 1 2 3 4 5 6 7 8

C. lanceolata 16 3.62–2.06 22.91 AR 1.15 1.06 1.23 1.45 1.62 1.28 1.56 1.18

RL 14.32 14.60 13.75 12.83 11.99 11.42 10.75 9.12

C. ochroleuca 16 3.47–2.07 22.31 AR 1.07 1.36 1.15 1.50 1.18 1.39 1.10 1.39

RL 15.42 14.26 13.93 13.43 12.66 10.83 9.99 9.25

C. pallida 16 3.97–1.98 22.48 AR 1.18 1.20 1.71 1.53 1.95 1.30 1.28 1.16

RL 17.34 14.62 13.81 12.56 11.81 11.32 10.36 8.93

C. mucronata 16 3.33–1.86 20.00 AR 1.43 1.13 1.49 1.45 1.61 1.17 1.41 1.25

RL 16.66 14.53 13.59 12.39 11.73 11.19 10.67 9.31

C. striata 16 3.43–1.87 20.89 AR 1.27 1.20 1.35 1.66 1.39 1.58 1.23 1.22

RL 16.43 14.52 13.43 12.84 11.89 11.22 11.01 8.94

AR arm ratio, RL relative length

Genet Resour Crop Evol

123

since these events have been demonstrated for

Aegilops (Raskina et al. 2004a, 2004b). Alternatively,

the reduction in the signal of chromosome 1 could be

a consequence of unequal recombination, which has

been considered as an important mechanism of

intragenomic mobility of the rDNAs (Schubert

2007). The 5S rDNA mobility occurred during the

divergence of C. pallida and before its radiation

around the tropical regions. C. pallida is an African

species, however its cultivation as a green manure

and sometimes as a fodder crop, make it difficult to

establish its natural area of distribution (Polhill

1982).

In the case of C. ochroleuca, where a minor 45S

rDNA signal was detected, we have interpreted this

as a transposition event similar to that in 5S rDNA,

with a recent origin because of the signal size and its

probable inactive nature. Mobility of 45S rDNA has

been demonstrated by Altinkut et al. (2006), Datson

and Murray (2006), Raskina et al. (2004a, 2004b),

Schubert and Wobus (1985), Shishido et al. (2000)

and others. The probably inactive state of the 45S

rDNA is inferred by the small size of the signal. As

discussed by Winterfeld and Roser (2007) working

with in perennial oat species, the signal of 45S rDNA

signals smaller than 0.2 lm suggest inactivity. On the

other hand, the presence of a minor site could

represent the final stage of DNA loss for this locus.

The ribosomal loss during the evolution is well

established to polyploids (Kotseruba et al. 2003,

Kotseruba et al. 2010). For diploid species a great

variability in loci number of 45S and 5S has been

detected among related species (Heslop-Harrison and

Schwarzacher 2011, Castilho and Heslop-Harrison

1995, Leitch and Heslop-Harrison 1992) and a

decreasing number to ribosomal sites could occur.

The reduction in genome size has been proposed and

observed in Brassica (Lysak et al. 2009) and Festuca

(Smarda et al. 2008) for instance; and a mechanism to

DNA loss has been proposed at least to retranspos-

able elements (Devos et al. 2002). Despite the fact of

any kind of mechanism had been proposed to

ribosomal loss, it could not be disregarded. In fact,

a decrease in chromosome size has been described to

the genus Crotalaria from non-specialized to spe-

cialized flower species (Boulter et al. 1970; Almada

et al. 2006; Oliveira and Aguiar-Perecin 1999) and

this way loss of 45S or 5S rDNA could play a role in

this process.

Accordingly to the discussion above, we can infer

that C. lanceolata has the most original karyotype

structure among the species studied since no addi-

tional sites of 45S and 5S rDNA were detected.

Moreover the chromosome 1 structure is highly

conserved among the species. Other works have been

showing a secondary constriction always in chromo-

some one even among species of different botanic

sections (Almada et al. 2006; Oliveira and Aguiar-

Perecin 1999). At least for C. juncea, 45S and 5S

rDNA have been mapped by FISH in chromosome 1,

differing from our results because the 5S rDNA is

located at short arm (Mondin et al. 2007) what could

Fig. 3 C. ochroleuca showing 45S rDNA (arrowheads in red)

and 5S rDNA (arrowheads in green) FISH signals where the

first has been amplified increasing the number of signals.

a Metaphase plate where only one chromosome of the pair 4

shows a 45S rDNA FISH signal and the chromosome 1 has

been identified by both 45S and 5S rDNA signals in adjacent

position. b In this case both chromosomes of the pair 4 show

45S rDNA signals and the chromosome pair one may be

identified as described above. In both cases the 45S rDNA

additional sites of chromosome pair 4 have been mapped

adjacent to a pericentromeric heterochromatin and have been

interpreted as inactive by their size and position. Bar = 5 lm

Genet Resour Crop Evol

123

indicate a probable inversion in this chromosome

during evolution. This inversion represents an ancient

event during the divergence between the specialized

floral species from non-specialized ones and before

the diversification of the Machrostachyae subsection

species. Despite the fact of the change in the position

of 5S rDNA from the short to the long arm or vice

versa, both arrays of ribosomal genes were main-

tained in the same chromosome at least in the species

studied so far. A similar condition was observed in

the genus Achyrocline (Asteraceae) where both 45S

and 5S rDNA were mapped linked and near to the

pericentromeric region of chromosome 10 and the

karyotypes were quite similar. However, the ribo-

somal sites could appear in different configurations,

in the same or in different chromosomes, making

important a comparison with phylogenetic related

species (Mazzella et al. 2010).

Other species from different botanic sections of

the genus Crotalaria could be explored with this two

Fig. 4 Ideograms of Crotalaria species. The chromosomes

were constructed based on the morphometric analysis of

metaphases with the same contraction level and with clearly

distinguishable primary and secondary constriction. a C.

lanceolata, b C. ochroleuca, the asterisk indicates the additional

site of 45S rDNA, c C. pallida, d C. mucronata and e C. striata.

The statistical deviations of the chromosome arms are indicated

in both arms

Genet Resour Crop Evol

123

rDNA probes for specific understanding of chromo-

some 1 modifications during the genus diversifica-

tion. Most of the cytogenetic works on the genus

Crotalaria assumed that the chromosome rearrange-

ments did not have a significant importance during

species diversification. However it has been demon-

strated recently that some chromosomal alterations

played a role in Crotalaria genus (Mondin 2003;

Mondin et al. 2007b and results herein).

Most of the specialized floral species showed

CMA? bands at centromeric position, while a unique

non-specialized species, C. incana did not; instead,

this species showed dispersed AT rich regions

(DAPI?). It was suggested that this late organization

would be a characteristic of non-specialized species

(Mondin 2003; Mondin et al. 2007a, 2007b). In fact,

we did not identify, in the present species, any kind of

GC or AT-rich regions around the centromere

detected by fluorescent dye combinations. However,

after the FISH procedures, it was clearly possible to

identify heterochromatin blocks at centromeric posi-

tions. This phenomenon is common in plants after

chromosome denaturation by FISH and counterstain-

ing with DAPI. However, it does not indicate the

nature of DNA staining both DAPI? and other kinds

of heterochromatins (Barros e Silva and Guerra

2010). The changes in the nature of the pericentro-

meric heterochromatin could be a consequence of

events of turnover involving repetitive sequences

(reviewed in Kuhn et al. 2008) during the divergence

between floral specialized and non-specialized

species.

The results presented here represent an important

contribution to the understanding of the phylogenetic

relationships among the species of the genus Crota-

laria. Moreover the results are in accordance with the

morphological relationships established previously

by Polhill (1982). Finally, we showed that chromo-

some rearrangements represented an important mech-

anism of chromosome evolution and diversification

of species within the genus Crotalaria.

Acknowledgments We are thankful to Coordenacao de

Aperfeicoamento de Pessoal de Nıvel Supeior (CAPES) and

Conselho Nacional de Desenvolvimento Cientıfico e

Tecnologico (CNPq) for supporting AGM. MM was a

PRODOC/CAPES fellowship. Research was also supported by

Fundacao de Apoio a Pesquisa do Estado de Sao Paulo (FAPESP

Proc. 98/01170-5). We are thankful to Gustavo C.S. Kuhn for

critical reading of the manuscript.

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