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Genetic Resources and Crop

Evolution
An International Journal

ISSN 0925-9864
Volume 59
Number 2

Genet Resour Crop Evol (2012)
59:277-288
DOI 10.1007/s10722-011-9683-8
Karyotype characterization reveals an up
and down of 45S and 5S rDNA sites in
Crotalaria (Leguminosae-Papilionoideae)
species of the section Hedriocarpae
subsection Macrostachyae
A.G.Morales, M.L.R.Aguiar-Perecin &
M.Mondin
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RESEARCH ARTI CLE
Karyotype characterization reveals an up and down
of 45S and 5S rDNA sites in Crotalaria (Leguminosae-
Papilionoideae) species of the section Hedriocarpae
subsection Macrostachyae
A. G. Morales

M. L. R. Aguiar-Perecin

M. Mondin
Received: 27 October 2010 / Accepted: 7 March 2011 / Published online: 6 April 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, ve 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 identied 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 identied 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 identied 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 oral 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 classication.
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 xation, cover the soil protect-
ing against erosion, in combat against nematodes
contamination in the elds (Polhill 1982; Hanelt and
Institute of Plant Genetics and Crop Plant Research
2001; Germani and Plenchette 2004), also because of
the bers one of the primary uses is to manufacture
twine, cord, marine cordage, shing nets, matting,
sacking, and paper (revision in Morris and Kays
2005). Moreover, Crotalaria species has been used in
sugar eld 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-inammatory 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
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Genet Resour Crop Evol (2012) 59:277288
DOI 10.1007/s10722-011-9683-8
Author's personal copy
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 owers and another without oral appendages
that confer some specialization to pollinators (in
other words non-specialized owers). The section
Hedriocarpae belongs to the non-specialized ower
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 ower specialization (Boul-
ter et al. 1970), with the non-specialized oral 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 oral 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 identication and
detections of chromosomal alterations difcult (Al-
mada et al. 2006; Oliveira and Aguiar-Perecin 1999).
Nevertheless, the presence of NOR in chromosome 1
allows its identication. 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
diversication 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 ower 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 Passiora (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 ve species
belonging to the botanic section Hedriocarpae, sub-
section Macrostachyae, a non-specialized oral group
of the genus Crotalaria, through chromosome
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banding and FISH with ribosomal DNA probes (45S
and 5S).
Materials and methods
Plant material
Five species were analyzed according to the classi-
cation received from the donors (Table 1). The
species belong to the section Hedriocarpae, subsec-
tion Machrostachyae, according to the Polhills
(1982) infrageneric classication. 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 uorescent staining
The seeds were scaried in pure sulfuric acid during
8 min. and subsequently rinsed abundantly with
water for dormancy breakage, and air-dried. The
scaried seeds were germinated at 28C 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
xed 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
(nal
concentrations) in citrate buffer (pH 4.6) at 37C 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
4
0
, 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 briey 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 modications 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 96C for
10 min, then cooled and dropped onto the slide and
together denatured in a thermocycler (M.J. Research,
Inc., USA) at 83C for 10 min and the hybridization
was performed at 37C for 16 h. The stringency
washes were made twice in 2xSSC at 37C 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 RitaSao Paulo BR 116 (Km 114) Instituto de Zootecnia de Nova OdessaSP CL-3
C. ochroleuca G. Don Indeterminate Piracicaba, ESALQSP COC-1
C. pallida Ait.
a
Rio BonitoRJ Instituto de Zootecnia de Nova OdessaSP CPA-1
C. mucronata Desv.
a
Indeterminate Piracicaba, ESALQSP CM-1
C. striata DC.
a
Indeterminate Piracicaba, ESALQSP CSR-1
a
Species synonyms of C. pallida according to Polhill (1982)
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42C (5 min each), then in 20% (v/v) formamide in
0.5 9 SSC for 10 min at 42C 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 amplication 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 uorescein 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 epiuorescence microscope with
appropriate lters. The uorochrome 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
identication of the pairs difcult. However, slight
modications 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 identied as
conspicuous primary constriction in all chromosomes being
possible a measurement to obtain the ideograms
280 Genet Resour Crop Evol (2012) 59:277288
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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 rDNAwas 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 amplication. Accordingly,
the 45S rDNA additional site of C. ochroleuca can be
interpreted as a recent event, because it is not
amplied, 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 uorescence 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 specic 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 oral 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
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282 Genet Resour Crop Evol (2012) 59:277288
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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 signicant. 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 rst columns, with
Chromomycin A3 (CMA) and DAPI respectively; CMA-DA
and DAPI-AMD staining resulted in a similar uorescent
pattern and have not been showed. Fluorescent in situ
hybridization 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 uorochromes. C.
lanceolata (ac) and C. ochroleuca (df) where the bright
bands and both FISH signals are identied 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 (gi), C. mucronata (jl) and C. striata (mo) clearly
show an amplication of the 5S rDNA sites, however no
additional uorescent bands could be identied with uoro-
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 3.622.06 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 3.472.07 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 3.971.98 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 3.331.86 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 3.431.87 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
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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 difcult 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 nal 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 ower 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
rst has been amplied 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 identied 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
identied 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
284 Genet Resour Crop Evol (2012) 59:277288
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indicate a probable inversion in this chromosome
during evolution. This inversion represents an ancient
event during the divergence between the specialized
oral species from non-specialized ones and before
the diversication 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 congurations,
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 (2012) 59:277288 285
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rDNA probes for specic understanding of chromo-
some 1 modications during the genus diversica-
tion. Most of the cytogenetic works on the genus
Crotalaria assumed that the chromosome rearrange-
ments did not have a signicant importance during
species diversication. 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 oral 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 uorescent 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 oral 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 diversication
of species within the genus Crotalaria.
Acknowledgments We are thankful to Coordenacao de
Aperfeicoamento de Pessoal de N vel Supeior (CAPES) and
Conselho Nacional de Desenvolvimento Cient co 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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