Brief Communications
Salmon: A New Autosomal
Mutation Demonstrating
Incomplete Dominance in the
Boine Snake Boa constrictor
R. N. Ihle, G. W. Schuett, and K. A.
Hughes
An unusual and attractive pigmentation
pattern mutation termed ‘‘salmon’’ has
been identified in the United States in several captive colonies of the common neotropical boine snake boa constrictor [Boa
constrictor (Boidae)]. Boa constrictors expressing the Sa pigmentation pattern appear to be restricted to regions of Panama.
Animals with the Sa phenotype exhibit a
sharp decrease in melanophore pigments
(e.g., melanin) and an increase of xanthophore pigments (e.g., pteridines and
carotenoids) throughout the body, including ventral and caudal regions. Moreover,
the dorsal saddles (blotches) and lateral
diamond patterns are greatly reduced
and/or absent. Our study was initiated using a female B. constrictor born in captivity
and expressed the Sa pigmentation pattern. Results from breeding experiments
indicated an inheritance mode of autosomal incomplete dominance for the Sa and
Wt alleles.
Snakes are a remarkably diverse group of
reptiles in terms of variation in pigmentation pattern ( Bechtel 1995). Nonetheless,
few studies on the inheritance (e.g., Zweifel 1981) and development of pigmentation patterns (e.g., Murray and Myerscough 1991) have been conducted. The
majority of research on pigmentation patterns in snakes has been performed by either zoo personnel or private breeders
[reviewed by Bechtel (1995)].
The mode of inheritance of melanism
(an all-black pigmentation pattern) in eastern garter snakes (Thamnophis s. sirtalis)
from populations of the western Lake Erie
254
region ( Blanchard and Blanchard 1941)
and albinism (amelanism) in the red rat
snake (Elaphe g. guttata) ( Bechtel 1995;
Bechtel and Bechtel 1962) and San Diego
gopher snake (Pituophis melanoleucus annectens) [discussed in Bechtel (1995)]
were the first to be studied. In the above
cases, and in nearly all subsequent ones
( Bechtel 1995), pigmentation pattern mutations have been found to behave as a
simple two-allele Mendelian trait, with the
mutant allele recessive to the wild-type
(Wt) allele. Two notable exceptions are
the California kingsnake (Lampropeltis getula californiae) and reticulated python
(Python reticulatus). In L. g. california, the
pigmentation pattern termed ‘‘striping’’
(common in several populations) is dominant to the typical pattern termed ‘‘banding’’ ( Klauber 1944; Zweifel 1981). A newly
described pattern mutation termed ‘‘tiger’’
in P. reticulatus involves tendencies for the
Wt body pattern ( blotched) to be replaced by elongated blotches in heterozygotes and by longitudinal stripes in homozygotes, a condition termed ‘‘super
tiger’’ [reviewed in Barker and Barker
(1997); see pp. 49, 53, and 64 for color
plates of Wt, tiger, and super tiger, respectively]. Several breeders of P. reticulatus
have reported that the tiger mutation is
autosomal and shows incomplete dominance or codominance ( Barker and Barker
1997).
In this article we report a new and unusual pigmentation pattern mutation,
termed ‘‘hypomelanistic’’ or ‘‘salmon,’’ in
the boine snake Boa constrictor ( Kluge
1991; Henderson et al. 1995; de Vosjoli et
al. 1998:38–39). A female boa constrictor
with the salmon (Sa) mutation was acquired by one of us (R.N.I.) as a juvenile
and used in breeding experiments to determine the inheritance mode of this trait.
When compared to animals expressing the
Wt pigmentation pattern ( Figure 1A), the
Sa mutation ( Figure 1B) is characterized
by a sharp decrease in the expression of
melanophore pigments (e.g., melanin) and
a sharp increase in the expression of xanthophore pigments (e.g., pteridines and
carotenoids) throughout the body, including ventral and caudal regions. Moreover,
the dorsal saddles ( blotches) and lateral
diamond patterns are greatly reduced or
absent.
Materials and Methods
The Sa mutant female described above
was crossed to a Wt male (obtained from
a commercial source) and produced F1
progeny in 1994, 1995, and 1996 ( Table 1).
In 1996 and 1997, crosses between the
above F1 progeny were performed to determine the mode of inheritance of the Sa
mutation. In the F1 crosses that involved
only Sa individuals (i.e., Sa ⫻ Sa), another
novel phenotype occurred, which appeared to be an amplified version of the
Sa phenotype. This new morph showed
extreme loss of melanism and increased
salmon (sometimes orange) coloration
throughout the body, including the eye
(iris) and tongue. Furthermore, the dorsal
blotches were further reduced in size and
the lateral pattern was frequently absent
( Figure 1C,D). This phenotype will be referred to as ‘‘super salmon’’ (Ss) in this
article. Also, from 1996 to 1999, several
other different F1 crosses (dam ⫻ sire)
were performed and include Wt ⫻ Wt, Sa
⫻ Al (albino mutant; Kahl strain, and is
autosomal recessive inheritance), Wt ⫻ Ss
(performed by G. Hobbs, private breeder),
Ss ⫻ Wt, and Sa ⫻ Ss.
All animals in our facility were housed
individually in commercial Neodesha plastic cages with a sliding glass front (122 cm
⫻ 81 cm ⫻ 46 cm). A commercial heat pad
(Mylar) was positioned beneath one end
of each cage to provide a basking site
(35⬚C). Water was provided in ceramic
bowls and available ad libitum. Domestic
rodents (rats) were offered weekly. All experimental matings were performed from
Figure 1. Comparison of (A) Wt, (B) Sa, and (C,D) Ss Boa constrictor progeny produced in this study ( Table 1).
Note in (D) that the iris is nearly orange (versus gray and tan in Wt), the tongue is pale (versus black in Wt), and
the dorsal blotches show extreme fading and size reduction.
November through February, and all
births occurred from April through May.
Results and Discussion
We present in Table 1 the results of parental crosses and crosses of their F1 progeny,
along with results of chi-square tests ( Zar
1996) of the fit to a model of incomplete
dominance. Chi-square values ⱕ0.05 were
scored to deviate significantly from the expected model. The parental crosses in-
volving the Sa female and Wt male were
successful in three consecutive years
(1994–1996), and both Wt and Sa F1 progeny were produced in each of the three
litters ( Table 1). Ten different pairs of F1
progeny from our parental stock were
crossed (Sa ⫻ Sa, Wt ⫻ Sa, and Sa ⫻ Ss),
yielding Wt, Sa, and Ss F2 progeny ( Table
1). Moreover, we performed three additional crosses involving F1 animals. One
cross, Wt ⫻ Wt, yielded 100% Wt progeny,
and two other crosses, Wt ⫻ Ss (per-
Table 1. Test of the incomplete dominance model
Progeny
Parents
Cross
Dam
Sire
Wild type Salmon
(Wt)
(Sa)
F11
F12
F13
F21
F22
F23
F24
F25
F26
F27
F28
F29
F210
Albino
Crosses
Test
Crosses
Sa
Sa
Sa
Sa
Sa
Sa
Sa
Sa
Sa
Sa
Wt
Sa
Wt
Sa
Sa
Wt
Ss
Wt
Wt
Wt
Sa
Sa
Sa
Sa
Sa
Sa
Sa
Sa
Ss
Wt
Al
Al
Ss
Wt
16
16
15
10
9
9
4
9
5
3
14
0
9
12
14
0
0
7
7
15
7
8
6
12
8
9
16
9
13
0
13
14
24
17
Supersalmon
(Ss)
Test of genetic model
0
0
0
5
4
4
8
6
7
5
0
12
0
0
0
0
0
3.522
3.522a
0.033a
5.182
3.571
5.211
1.333
2.913
0.810
3.000
1.130a
0.080a
—b
0.080a
0.036a
—b
—b
␹2
a
df
P
1
1
1
2
2
2
2
2
2
2
1
1
—b
1
1
—b
—b
.07
.07
.90
.08
.17
.07
.52
.23
.67
.22
.30
.79
—b
.79
.86
—b
—b
Shown are crosses producing F1 and F2 progeny, and crosses of F1 salmon (Sa) females to an albino (Al) male.
Tests with one degree of freedom were performed using Yates’ correction for continuity.
b
Only one phenotypic class is expected in the progeny, thus no association tests could be performed. Note that
the progeny phenotypes match those expected under the incomplete dominance model.
a
formed in another facility, G. Hobbs; permission was granted to publish the data)
and Ss ⫻ Wt, yielded 100% Sa progeny
( Table 1).
To test for allelism, two additional
crosses were performed using two F1 Sa
females and a single, unrelated albino (Al)
male ( Kahl strain). The Kahl strain Al trait
is autosomal recessive (de Vosjoli et al.
1998:37). Both of the Al ⫻ Sa crosses yielded expected ratios of Wt and Sa progeny
( Table 1).
In summary, a total of 17 crosses were
performed, producing 381 healthy progeny (145 Wt, 185 Sa, and 51 Ss). The sex
ratio was close to one in all litters, with
no evidence of sex-limited expression of
the Sa trait.
Preliminary inspection of the results
suggested an autosomal model of incomplete dominance for the inheritance of the
Sa trait. The data were fit to this model by
conducting chi-square tests ( Zar 1996).
For all tests having a single degree of freedom we used Yates’ correction for continuity ( Zar 1996:464).
The chi-square tests did not reveal significant deviation from the expected ratios
under the model of incomplete dominance
( Table 1). The first two litters of the parental cross ( F11 and F12) were marginally
nonsignificant ( both at P ⫽ .07). However,
litter F13 of the parental cross showed a
strong fit to the model (P ⫽ .90). We attribute the tendency for deviation from
the model in the first two litters to errors
in scoring offspring. It is possible that we
scored some of the Sa progeny as Wt because in some situations Sa neonates are
difficult to distinguish from Wt until after
several ecdyses. Unfortunately at least 10
of the Wt animals from the first two litters
were not retained; thus we cannot rescore
those individuals. In all but two cases,
subsequent litters produced ratios close
to expected values. The two Sa ⫻ Al crosses demonstrated no interaction between
the Sa mutation and the recessive allele
for the Al mutation. These results are consistent with the traits being determined by
alleles at different loci.
We therefore propose that the Sa mutation in B. constrictor is autosomal and
shows incomplete dominance, with the alleles behaving as a pair and segregating in
predicted Mendelian fashion (e.g., Russell
1992). Individuals with a single copy of the
Sa allele ( heterozygotes) deviate from Wt
in appearance ( Figure 1B), and those with
two copies of the Sa allele (Ss, homozygotes) deviate substantially more, in some
cases bearing little resemblance to the Wt
Brief Communications 255
pigmentation pattern of the species ( Figure 1C,D; de Vosjoli et al. 1998). There is,
however, a range of phenotypes exhibited
by snakes possessing Sa and Ss alleles.
Primarily affected are the size and pattern
of dorsal and lateral body blotches, and
the degree to which melanin is expressed
in scales. Undoubtedly pigmentation pattern in Sa and Ss individuals is influenced
by modifiers and/or the environment. Future work includes performing crosses between individuals possessing both the
salmon (Sa and Ss) and albinism (Al) trait,
and those that are homozygotes, Ss ⫻ Ss.
It is known that the Sa trait we report in
captive B. constrictor occurs in certain
populations from Panama (Porras 1999;
Hardy D Sr, personal communication). According to Porras (1999, personal communication), B. constrictor from Isla Taboguilla ( Bahia de Panama) show the Sa trait
(p. 60), and some individuals bear a
strong resemblance to the Ss phenotype
(pp. 51–52). D. Hardy, Sr. (personal communication) collected a juvenile female B.
constrictor from the region of Gamboa,
Panama, that showed the Sa condition. Because we have limited information on the
Sa trait in B. constrictor, the extent of its
geographic distribution remains to be established. Nonetheless, at present we are
aware of no other localities than those
provided above (Porras L, personal communication), and thus tentatively conclude that the Sa mutation has a limited
geographic distribution in B. constrictor. Of
importance, these data provide information that will be of utility to systematists
and taxonomists working on this species.
Based on our findings, populations of B.
constrictor expressing the Sa or Ss pigmentation pattern should not be considered as
distinct geographic races or other type of
evolutionary unit. Rather it is our opinion
that these populations show polymorphism with respect to pigmentation pattern.
From 1030 W. Pecos Ave., Mesa, Arizona ( Ihle), and Department of Life Sciences, Arizona State University
West, P.O. Box 37100, Phoenix, AZ 85069-7100. G. Hobbs
kindly supplied data (Wt ⫻ Ss cross) and granted permission for its inclusion in this article. L. Clarke and K.
Hanley photographed the animals. K. Dixon made helpful suggestions in the initial analysis. D. Hardy Sr. and
L. Porras provided information on the occurrence of
the salmon trait in B. constrictor from certain Panamanian populations. Address correspondence to G. W.
Schuett at the address above or e-mail: gschuett@asu.
edu.
2000 The American Genetic Association
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Received April 26, 1999
Accepted December 31, 1999
Corresponding Editor: Rodney Honeycutt
Physical Mapping of rRNA
Genes in Medicago sativa
and M. glomerata by
Fluorescent in situ
Hybridization
E. Falistocco
Fluorescent in situ hybridization (FISH)
was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to
investigate the distribution of rRNA genes
and to utilize the sites of 18S-5.8S-25S
rDNA and 5S rDNA sequences as markers
for studying the genome evolution within
the species. Medicago glomerata Balb.,
the species considered to be the ancestor
of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ
hybridization was performed with the
probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the
diploid taxa, M. glomerata, M. sativa ssp.
coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences
were mapped to two sites corresponding
to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA
appeared to be distributed in two pairs of
sites. Chromosomes carrying 5S loci
could be distinguished on the basis of
their morphological characteristics. The
number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number
found in the respective diploid ssp. falcata
and ssp. coerulea. The results of this
study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to
the cultivated alfalfa. Modifications of the
number of rRNA loci were not observed.
The importance of in situ hybridization for
improving karyotype analysis in M. sativa
L. is discussed.
Medicago sativa L., the most studied species of the genus Medicago, includes forms
with different morphological traits and
chromosome numbers. Diploid (2n ⫽ 2x ⫽
16) and tetraploid (2n ⫽ 4x ⫽ 32) taxa
with violet flowers and coiled legumes are
classified as M. sativa ssp. coerulea ( Less.
ex Ledeb.) Schmalh and M. sativa ssp. sativa ( L.) L. & L., respectively. Cytogenetic
characteristics have revealed that these
two forms are very closely related species
(Gillies 1970; Ho and Kasha 1972; Lesins
1957; Stanford 1959). Meiotic studies on
micro- and macrosporogenesis have demonstrated that ssp. sativa originated by
sexual polyploidization following the production of 2n gametes in ssp. coerulea
(McCoy and Bingham 1988; Pfeiffer and
Bingham 1983). The original area of distribution of the primitive diploids probably
included present-day Anatolia, Armenia,
Iran, and the areas around the Caspian
Sea. It was in these localities that the tetraploids appeared, which through domestication gave rise to the cultivated form of
alfalfa. At present alfalfa is considered the
most important forage legume in both Europe and North America (Michaud et al.
1988).
M. sativa ssp. falcata ( L.) Arcangeli differs from the previous taxa in that it bears
yellow flowers and produces straight
pods. It has both diploid (2n ⫽ 2x ⫽ 16)
and tetraploid (2n ⫽ 4x ⫽ 32) forms, both
of which are widely distributed over eastern Europe. The tetraploids occur with
greater frequency than the diploids, which
are considered evolutionary relics. Despite the morphological differences, many
cytological characteristics have demonstrated a close genetic relatedness be-
tween ssp. falcata and ssp. sativa (Cleveland and Stanford 1959; Lesins 1957). At
the same ploidy level they intercross easily and produce viable hybrids with normal meiotic behavior ( Ho and Kasha
1972). Furthermore, pachytene studies
have demonstrated that their chromosomes are almost identical (Gillies 1970).
Recently RFLP maps have shown that
there is a perfect collinearity between the
genomes of the two species ( Tavoletti et
al. 1996). It is well documented that they
share a common ancestry, most probably
identifiable with M. glomerata Balb., a diploid species with yellow flowers and coiled
pods (Gillies 1971; Lesins and Lesins
1964). Differentiation of ssp. coerulea and
ssp. falcata has been proposed as the consequence of the spatial isolation of the ancestral populations of M. glomerata. Under
different selection pressures they developed morphological traits, such as violet
flowers and straight pods, which are characteristic of ssp. coerulea and ssp. falcata,
respectively (Quiros and Bauchan 1988).
Cytogenetic studies have been carried
out to understand the phylogenetics of alfalfa and to analyze the genetic similarity
with the related taxa (Gillies 1971). However, the mitotic chromosomes have a limited usefulness because they are small and
very similar in size and are generally considered extremely difficult for detailed cytogenetic studies. These limitations have
been partially overcome in recent years
by the application of the C-banding technique, which was used to characterize the
somatic chromosomes of M. sativa CADL
(cultivated alfalfa at the diploid level)
(Masoud et al. 1991) and tetraploid alfalfa
( Falistocco et al. 1995). Recently Bauchan
and Hossain (1997) applied it to M. sativa
ssp. coerulea and ssp. falcata. The C-banding pattern revealed a larger amount of
constitutive heterochromatin in the chromosomes of tetraploid ssp. sativa and ssp.
coerulea than in those of ssp. falcata,
which exhibited bands only at the centromeric regions.
Molecular cytogenetic techniques are
currently being applied to study genomic
organization and determine the evolutionary process. DNA:DNA fluorescent in situ
hybridization ( FISH) used in conjunction
with fluorochrome staining has turned out
to be an extremely valuable method for
studying species with small or uniform
chromosomes (Maluszynska and HeslopHarrison 1993; Schmidt et al. 1994). It is
possible to analyze in detail the chromosome structure and to detect the genomic
changes that occurred during the evolu-
Figure 1. Fluorescent in situ hybridization of rDNA probes, pTa71 (green) and pXVI (red), to mitotic chromosomes of diploid M. glomerata (a–c), M. sativa ssp. coerulea (d–f ), and M. sativa ssp. falcata (g–i). (a) Metaphase
of M. glomerata showing DAPI staining, (b) sites of 18S-5.8S-25S rDNA, and (c) sites of 5S rDNA. (d) Metaphase of
ssp. coerulea showing DAPI staining, (e) sites of 18S-5.8S-25S rDNA, and (f) sites of 5S rDNA. (g) Metaphase of ssp.
falcata showing DAPI staining, (h) sites of 18S-5.8S-25S rDNA, and (i) sites of 5S rDNA. Large and small arrows
indicate 18S-5.8S-25S sites and 5S sites, respectively. The bar represents 5 ␮m.
tion of the species by means of physical
mapping of repetitive genes or other sequences directly on metaphase chromosomes (Cuadrado and Jouve 1997; Fominaya et al. 1994; Pedersen et al. 1995).
Physical mapping of the chromosomes
also provides useful information for breeding programs ( Heslop-Harrison and
Schwarzacher 1996).
Identification of the sites of the ribosomal genes (rDNA) is generally the first
step toward the physical mapping of chromosomes. Such genes constitute the most
important multigene families in the eukaryotic genome. In plants they are organized in separated clusters: 18S-5.8S-25S
and 5S genes, usually located in different
chromosomes ( Lapitan 1992). The 18S5.8S-25S ribosomal genes are present in
many hundreds of repeated units arranged
in tandem arrays corresponding to the nucleolar organizer regions ( NORs) ( Lapitan
1992). Genes coding for the 5S ribosomal
RNA have a similar organization, however,
their sites along the chromosome are not
revealed by any morphological characteristics.
FISH to chromosomes of diploid subspecies of M. sativa and tetraploid alfalfa has
been utilized for determining the sites of
the ribosomal 18S-5.8S-25S gene cluster
(Calderini et al. 1996), however, loci of 5S
genes have never been identified. This
study was undertaken to determine the
complete physical mapping of rRNA genes
in diploid ssp. coerulea, ssp. falcata, tetraploid ssp. falcata and ssp. sativa, and to
use the ribosomal gene sites as cytogenetic markers for studying the genome
evolution within the species. Investigations were also conducted on M. glomerata
Balb., the species related to M. sativa and
considered its ancestor, to obtain more information on the evolution of the ribosomal gene sites.
Simultaneous in situ hybridization was
Brief Communications 257
Figure 2. Fluorescent in situ hybridization of rDNA probes, pTa71 (green) and pXVI (red), to mitotic chromosomes of tetraploid M. sativa ssp. falcata (a–c) and M. sativa ssp. sativa (d–f ). (a) Metaphase of ssp. falcata showing
DAPI staining, (b) sites of 18S-5.8S-25S rDNA, and (c) sites of 5S rDNA. (d) Metaphase of ssp. sativa showing DAPI
staining, (e) sites of 18S-5.8S-25S rDNA, and (f) sites of 5S rDNA. Large and small arrows indicate 18S-5.8S-25S sites
and 5S sites, respectively. The bar represents 5 ␮m.
performed with probes pTa71 and pXVI labeled differently to determine the localization of both 18S-5.8S-25S and 5S rDNA sequences in the same experiment.
Materials and Methods
Plant Material
The materials used for this study consisted of the accessions 2956 and 3401 of M.
sativa ssp. coerulea from the Institute of
Plant Breeding of Perugia ( Italy), M. sativa
ssp. falcata 2x WISFAL-1 and 4x WISFAL-1
( Bingham 1990), kindly provided by Professor E. T. Bingham, University of Wisconsin, Madison, M. sativa ssp. sativa cv. Turrena, a synthetic variety from the Institute
of Plant Breeding of Perugia, and the accessions PI 516906, PI 577567, and PI
577568 of M. glomerata from the Western
Regional Plant Introduction Station, Pullman, Washington.
Chromosome Preparation and FISH
Seeds of each accession were germinated
in petri dishes at room temperature. Actively growing root tips were excised when
they were about 1 cm in length, pretreated
in a saturated aqueous solution of ␣-bromonaphthalene for 4 h, and then fixed in
ethanol:acetic acid (3:1) overnight.
Chromosome preparation was carried
out in the following way. Fixed root tips
were washed in enzyme buffer (10 mM citric acid/sodium citrate pH 4.6) for 30 min
then placed on poly-L-lysine-coated slides
with 1–2 drops of enzyme solution (4% cel-
258 The Journal of Heredity 2000:91(3)
lulase Onozuka R10 and 1% pectolyase Sigma in distilled water) for 2 h at 37⬚C. For
each slide, three root tips were used. After
removing the enzyme with distilled water
and eliminating excessive water, 1–2 drops
of ethanol:acetic acid (3:1) were added.
Root tips were broken with a thin needle
to give a fine emulsion and spread on the
slide. Preparations were air dried. FISH
was accomplished by pretreating slides
with 100 ␮g/ml of RNase A in 2⫻SSC (0.3
M NaCl, 0.03 M sodium citrate) for 1 h at
37⬚C and washed three times in 2⫻SSC. After incubation with 80 U/ml of pepsin (Sigma) in 10 mM HCl for 15 min at 37⬚C, the
chromosome preparations were stabilized
by immersion at room temperature for 10
min in freshly depolymerized 4% (w/v)
paraformaldehyde in water, washed in
2⫻SSC, dehydrated in a graded ethanol series, and air dried. Heterologous probes
were used for in situ hybridization. Clone
pTa71 contains a 9 kb EcoRI fragment of
Triticum aestivum L. consisting of the 18S5.8S-25S rRNA genes and nontranscribed
spacer sequences (Gerlach and Bedbrook
1979). Clone pXVI contains the complete
gene of 5S rRNA and the spacer region of
Beta vulgaris L. (Schmidt et al. 1994).
Clone pTa71 was labeled with digoxigenin11-dUTP ( Boehringer Mannheim) by nick
translation, while pXVI was labeled with
biotin-11-dUTP (Sigma) using the polymerase chain reaction (PCR).
The hybridization solution consisting of
150 ng/␮l of DNA probe, 50% (v/v) formamide, 10% (w/v) dextran sulfate, 0.1%
(w/v) SDS (sodium dodecyl sulfate), and
300 ng/␮l sheared salmon sperm DNA, was
incubated for 10 min at 70⬚C and chilled
on ice. Forty microliters of hybridization
mixture were applied to each chromosome preparation and covered with a plastic coverslip. The hybridization mixture
and the chromosomes were denaturated
together at 70⬚C in a modified thermocycler for 5 min, then the temperature was
gradually decreased to 37⬚C. The hybridization was carried out overnight at 37⬚C.
The one-day in situ hybridization was also
performed; in this case the hybridization
time was reduced to 3 h. Since both methods gave good results, they were used
without distinction. After hybridization
the coverslips were floated off in 2⫻SSC at
42⬚C, then the slides were washed for 10
min each in 20% formamide (v/v) in
0.1⫻SSC at 42⬚C, 2⫻SSC at 42⬚C, and
2⫻SSC at room temperature. Detection of
the probes labeled with digoxigenin and
biotin was carried out simultaneously. The
slides were transferred to detection buffer
(4⫻SSC/0.1% Tween 20) for 5 min, treated
with 5% (w/v) bovine serum albumin
( BSA) in detection buffer for 5 min, and
incubated in 20 ␮g/ml of sheep antidigoxigenin antibody conjugated with FITC
( Boehringer Mannheim) and 5 ␮g/ml of
streptavidin conjugated with Cy3 in detection buffer containing 5% (w/v) BSA for 1
h at 37⬚C. After incubation the slides were
washed in detection buffer three times for
8 min each at 37⬚C. The slides were counterstained with 2 ␮g/ml of DAPI (4⬘,6-diamidino-2-phenylindole) and then mounted
in antifade solution Vectashield ( Vector
Laboratories). Slides were examined with
a Microphot Nikon epifluorescence micro-
Figure 3. Homologous chromosome pairs carrying
5S sites. A representative example of the physical location of 5S rRNA genes in the diploid taxa; the chromosomes are of ssp. falcata.
scope. About 20 slides from each accession were selected for the study. Analysis
was performed on 5–10 metaphases per
slide. Photographs were taken using Fujichrome 400 color slide film.
Results
Simultaneous in situ hybridization with
probe pTa71 labeled with digoxigenin and
probe pXVI labeled with biotin allowed the
sites of the ribosomal genes 18S-5.8S-25S
and 5S to be identified in the same metaphase plates. Figures 1 and 2 show metaphase chromosomes from the root tips of
diploid and tetraploid taxa, respectively,
after FISH with the two probes and counterstaining with DAPI.
Localization of 18S-5.8S-25S and 5S
rRNA Genes in M. glomerata, M. sativa
ssp. coerulea, and M. sativa ssp.
falcata
After in situ hybridization with probe
pTa71 labeled with digoxigenin detected
with antibody conjugated with FITC and
clone pXVI labeled with biotin detected
with streptavidin Cy3 the two clusters of
18S-5.8S-25S and 5S rRNA genes were
mapped to the chromosomes of diploid M.
glomerata, ssp. coerulea and ssp. falcata
( Figure 1). Two hybridization sites of
probe pTa71 were observed in all the accessions of the three taxa. The green fluorescent signals showed that loci of 18S5.8S-25S sequences are localized in the
satellited chromosome pair in correspondence to the regions of the nucleolar organizers ( Figure 1b,e,h). No other sites
were detected. The two hybrid sites were
clearly detected because signals were
large and of high intensity. The size of the
mapped region did not show detectable
variation among accessions or taxa when
chromosomes with an equal degree of
contraction were compared.
Two pairs of 5S rDNA sites were identified in each diploid taxa ( Figure 1c,f,i).
The strength of the signals allowed the
number of the hybrid sites to be ascertained with accuracy. In some metaphases
the signals appeared as two bright red
spots corresponding to the loci of the 5S
genes on each of the chromatids. The
chromosome pairs carrying the 5S genes
could be identified in each taxa by selecting metaphases with less contracted chromosomes; they could be distinguished
from each other by means of their different lengths: the largest pair showed the
hybridization signal in the short arm,
while in the smaller pair it was in the long
arm. In both cases signals appeared quite
near the centromeres ( Figure 3). In each
of the diploid taxa the clusters of 5S and
18S-5.8S-25S ribosomal genes are located
on different chromosomes.
Localization of 18S-5.8S-25S and 5S
rRNA Genes in M. sativa ssp. falcata
(4x) and M. sativa ssp. sativa
In situ hybridization carried out in the tetraploid ssp. falcata and ssp. sativa revealed two pairs of 18S-5.8S-25S sites.
Probe pTa71 mapped to the secondary
constrictions of the four nucleolar chromosomes ( Figure 2b,e). No signals were
detected outside these regions. Differences in the strength of hybridization signals of the four sites were not observed
either in ssp. falcata or ssp. sativa.
Loci of 5S rRNA genes were found in
four pairs of chromosomes ( Figure 2c,f ).
The identification of the hybrid sites was
difficult, however, good preparations without overlapping chromosomes or background allowed the number of sites to be
accurately determined. Simultaneous utilization of probe pTa71 and pXVI showed
that in the tetraploid complements 5S and
18S-5.8S-25S genes are also localized on
different chromosomes.
Discussion
Investigations of rRNA genes are of great
importance to phylogenetic studies because their number may change substantially during evolution. Variations may affect the number of loci within the genome
and the copy number of the repeat units
at single loci ( Flavell 1989). Analysis of the
number and distribution of the sites of
rDNA sequences provides important information for understanding the genome organization of related species and for evaluating genomic modifications that occurred
during the evolution of the species. Identification of ribosomal gene sites on plant
chromosomes by FISH has been achieved
in many species including Hordeum vulgare L. ( Leitch and Heslop-Harrison 1992;
Pedersen and Linde-Laursen 1994), Beta
vulgaris L. (Schmidt et al. 1994), Festuca
arundinacea Schreb. ( Thomas et al. 1997),
Brassica spp.(Maluszynska and HeslopHarrison 1993), and Populus species (Prado et al. 1996). This research offers the
first report of the physical mapping of the
18S-5.8S-25S and 5S rRNA genes in diploid
and tetraploid subspecies of M. sativa and
in its putative ancestor M. glomerata. FISH
to chromosomes of these forms enabled
the sites of 18S-5.8S-25S and 5S rDNA se-
quences to be examined at different evolutionary phases, that is, from the early
diploid to the cultivated tetraploid form.
The sites of 18S-5.8S-25S rDNA in the
diploid taxa correspond to the secondary
constriction of the satellited chromosomes, while those of 5S rDNA are present
in two pairs of chromosomes. Such results
indicate that the distribution of ribosomal
gene sites detected in the putative ancestral genome of M. glomerata was conserved during the evolution of the diploid
ssp. coerulea and ssp. falcata. Moreover,
the morphology of the chromosomes with
the 5S loci and the position of this cluster
of ribosomal genes seems to suggest that
structural rearrangements involving the
two chromosome pairs did not occur in
the diploid taxa.
The number of rDNA sites detected in
tetraploid ssp. falcata and ssp. sativa were
twice the number found in the respective
diploid forms. This situation demonstrates that in M. sativa, the number of ribosomal gene sites did not change during
evolution. Consistent variations of the
copy number at single loci also appear unlikely since the fluorescence intensity did
not show significant differences among
the 18S-5.8S-25S sites and the 5S sites.
This study provides further information
about the genome structure and phylogenetic relationships of M. sativa and shows
that FISH is also a valuable method for improving the karyotyping analysis in this
species. The physical mapping of the 5S
rDNA genes, for example, may be used as
a marker for identification of the two chromosomes. This is of particular interest for
investigating chromosomes in ssp. falcata
in which C- and N-banding techniques are
not useful for karyological analysis ( Bauchan and Hossain 1997, 1998). Since the
distinction of the two chromosomes carrying the 5S genes relies on morphological
characteristics that cannot always be unequivocally ascertained, in situ hybridization experiments can be carried out using
different types of sequences as probes. By
means of physical location of 5S ribosomal
genes and other sequences it will be possible to identify these chromosomes in
diploid and tetraploid metaphases with
certainty and to verify if possible structural rearrangements occurred after evolution.
From the Istituto di Miglioramento Genetico Vegetale,
Facoltà di Agraria, Università degli Studi di Perugia,
Borgo XX Giugno, 06100 Perugia, Italy. This research
was funded by NRC strategic project ‘‘Biodiversity.’’
Brief Communications 259
The author wishes to thank Professor Fabio Veronesi
for the critical reading of the manuscript, Professor
Mario Pezzotti for the helpful suggestions in the in situ
hybridization technique, and Dr. Mary Traynor for the
revision of the English form of the manuscript. Address
correspondence to E. Falistocco at the address above
or e-mail: [email protected].
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( Hanson AA, Barnes DK, and Hill RR, eds). Madison,
WI: American Society of Agronomy; 737–776.
2000 The American Genetic Association
Michaud R, Lehman WF, and Rumbaugh MD, 1988.
World distribution and historical development. In: Alfalfa and alfalfa improvement ( Hanson AA, Barnes DK,
and Hill RR, eds). Madison, WI: American Society of
Agronomy; 25–91.
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Received June 3, 1999
Accepted October 18, 1999
Corresponding Editor: Prem P. Jauhar
Karyotype Analysis,
Banding, and Fluorescent in
situ Hybridization in the
Scarab Beetle Gymnopleurus
sturmi McLeay (Coleoptera
Scarabaeoidea:
Scarabaeidae)
M. S. Colomba, R. Vitturi, and M.
Zunino
Conventional staining, differential banding, and in situ hybridization with both ribosomal and telomeric probes to mitotic
chromosomes of Gymnopleurus sturmi
(Scarabaeoidea: Scarabaeidae) are described. The karyotype is distinguished by
a pericentric inversion polymorphism in
chromosome 3, which is either acrocentric
or subtelocentric. Silver staining (Ag-NOR)
and chromomycin A3 (CMA3), failed to
study the detection of nucleolar organizer
regions (NORs), due to the extensive silver
and CMA3 stainability of all GC-rich heterochromatin. Fluorescent in situ hybridization (FISH) using a Paracentrotus lividus (Echinodermata) rDNA probe mapped
the ribosomal RNA genes (rDNA). FISH
with the all-human telomeric sequences
(TTAGGG)n revealed a lack of homology
between the telomeric probe and the telomeres of G. sturmi. This suggests that
the telomeric hesanucleotide (TTAGGG)n
is not so conserved within eukaryotes as
it has been hypothesized.
The cosmopolitan superfamily Scarabaeoidea (Coleoptera) is an ancient group
whose origins can be traced back at least
to the late Mesozoic (more than 200 million years ago) and comprises about
35,000 different species. Details regarding
the morphology, body size, habitat, feeding behavior, and reproductive biology of
these interesting organisms have been
compiled by many authors [see Halffter
and Matthews (1966), Morón (1984), Hanski and Cambefort (1991), and the authors
quoted by them] who have pointed out
the impressive diversity among species
and groups. On the contrary, data available about the chromosomes of Scarabaeoidea are very scanty, comprising the
haploid and/or diploid numbers of about
200 species and the karyotype description
of some of them (Salamanna 1965, 1972;
Vidal et al. 1977; Yadav et al. 1979, 1989).
Conventional banding techniques have
been attempted on chromosomes of four
Scarabaeoidea species—the Dynastinae
Enema pan ( F.) ( Vidal and Giacomozzi
1979), the Geotrupidae Thorectes intermedius (Costa) (Colomba et al. 1995), and the
Scarabaeidae Bubas bison L. and Glyphoderus sterquilinus (Westwood) (Colomba et
al. 1996). Recently, for the first time in
Scarabaeoidea, fluorescent in situ hybridization ( FISH) has been used to map rDNA
encoding for ‘‘large’’ rRNA molecules in
the geotrupid Thorectes intermedius ( Vitturi et al. 1999).
The genus Gymnopleurus Illiger, 1803
(Scarabaeidae: Scarabaeinae), whose distribution is Palearctic, Afrotropical, and
Oriental ( Halffter and Edmonds 1972),
comprises about 50 species ( Balthasar
1963), but only three of them have been
partly investigated from a karyologic point
of view. Dasgupta (1963) reported the haploid number n ⫽ 10 for G. koenigii, Manna
performed according to Schmid et al.
(1983). Chromosomes were classified according to the criteria proposed by Levan
et al. (1964).
FISH was performed on fixed metaphases of six males using a Paracentrotus
lividus ( Echinodermata) 4.3 kb rDNA
probe (prR14) consisting of sequences
from the 3⬘ end of the 18S to the 3⬘ end of
the 26S rDNA, provided by R. Barbieri
( University of Palermo), FISH was also carried out on chromosomes of two males using the all-human telomeric repeat
( TTAGGG)n (P4097-DG.5, Oncor).
Nick translation labeling with digoxygenin and hybridization experiments were
performed according to manufacturers’ instructions ( Boehringer Mannheim and Oncor). Slides were mounted in an antifade
solution containing propidium iodide (5
␮g/ml) and viewed under a Leica I3 filter
set ( BP 450–490; LP 515). Photographs
were taken using a Leica microscope on
Kodak Agfaortho 25 ASA and Kodak Ektacolor 1000 ASA films.
Results
Figure 1. (a) Giemsa-stained male karyotype of G. sturmi showing pair 3 with the homozygous condition for the
subtelocentric type (3a3a). (b) C-banded female karyotype showing pair 3 with the heterozygous condition for the
subtelocentric and the acrocentric type (3a3b). (c) Diagramatic representation of the average karyotype.
and Lahiri (1972) established the diploid
number 2n ⫽ 18 for G. sinuatus, and Kacker (1976) found 2n ⫽ 20 for G. cyaneus.
This study on Gymnopleurus sturmi
chromosomes by means of Giemsa staining, a number of banding techniques, and
FISH is the first advanced cytologic analysis of the subtribe Gymnopleurina,
whose phylogeny is still controversial (cfr.
Cambefort 1991; Zunino M, unpublished
data).
Materials and Methods
A total of 120 sexually mature specimens
(64 male and 56 females) of G. sturmi
McLeay (Scarabaeoidea: Scarabaeidae),
collected in the field at Bellolampo (Palermo, northwest Sicily, Italy) from May 1996
to September 1998 were used for this
study. All animals, identified following the
guidelines laid down by Baraud (1992),
were reared in breeding boxes according
to Zunino and Monteresino (1994). Due to
the scantiness of cells actively dividing,
only a few specimens (9 males and 2 females) provided high-quality metaphase
spreads that could be utilized successfully
in this study.
Chromosome preparations were obtained, using the air-drying method ( Vitturi et al. 1996), either from male gonads
or the intestinal epithelium of individuals
of both sexes after in vivo colchicine treatment. Conventional Giemsa staining followed current methods ( Vitturi et al.
1996). C-banding was performed according to Sumner (1972). Silver staining was
carried out following the one-step method
described by Howell and Black (1980).
Staining with the fluorescent dyes CMA3
(GC specific) and DAPI (AT specific) was
Counts of 10 Giemsa-stained metaphases
per specimen gave a diploid number of 2n
⫽ 20. We arranged the chromosomes, according to their dimension and centromere position, into nine pairs of autosomes and one sex chromosome pair that
was homomorphic ( XX) in the females
and heteromorphic ( XY) in the males. Autosome pairs range from 7.68 to 1.78 ␮m,
and comprise two metacentric (pairs 1
and 4), five subtelocentric (pairs 2 and 6–
9), and one acrocentric (pair 5), pair 3 is
heteromorphic, consisting either of two
subtelocentric chromosomes or one subtelocentric and one acrocentric. These
chromosomes are equal in length and the
subtelocentric type is designated as 3a
while the acrocentric type is designated as
3b. The X is a small subtelocentric of
about 1.5 ␮m and the Y is a metacentric
microchromosome ( Figure 1a,b). A haploid average karyogram was obtained
from 10 high-quality spreads ( Figure 1c).
Application of C-banding revealed the
occurrence of large heterochromatic
blocks, corresponding to the centromeric
and pericentromeric regions of chromosomes 1, 2, 3a, and 4. In the subtelocentric
pairs 6–9 and in the X, C-positive material
was spread over the paracentromeric area
of the long arm and on the whole short
arm. Chromosomes 5 and 3b showed a
centromeric C band. No C-positive reac-
Brief Communications 261
Figure 2. G. sturmi metaphase spreads after (A) silver impregnation, (B) CMA3, and (C) DAPI staining; rDNA FISH
showing (D) 4 and (E) 5 hybridization signals; (F) 5 FISH signals very small in size; (G) FISH with the telomeric
probe ( TTAGGG)n; and (H) E. foetida metaphases clearly showing hybridization signals at the telomeric region of
all chromosomes.
tion was observed in the Y, probably due
to its minute dimension ( Figure 1b).
All heterochromatic material was
stained by silver ( Figure 2A) and fluoresced brightly after CMA3 treatment ( Figure 2B). Chromosomes were homogeneously DAPI stained ( Figure 2C).
rDNA FISH performed on 40 spermatogonial metaphases of five males allowed
localization of the major ribosomal clusters on the heterochromatic regions of
four ( Figure 2D) and five ( Figure 2E) medium-sized chromosomes in 18 and in 22
spreads, respectively. Hybridization signals were also polymorphic in size ( Figure
2F). No evident hybridization signals were
detected by using the all-human telomeric
repeat ( TTAGGG)n as a probe ( Figure 2G),
whereas in the same experiments, the
telomeric probe hybridized to the telomeres of Eisenia foetida (Annelida: Lumbricidae) mitotic chromosomes ( Figure
2H).
Discussion
The present study is the first detailed
chromosome analysis of one of the 50 spe-
262 The Journal of Heredity 2000:91(3)
cies of the genus Gymnopleurus. As reported for the majority of Scarabaeoidea
studied so far (cfr. Colomba 1998; Yadav et
al. 1979) G. sturmi shows a diploid number
of 20 chromosomes. This value is consistent with that found for G. cyaneus ( Kacker 1976) and G. koenigii ( Dasgupta 1963),
but it does not match with the diploid
number of G. sinuatus (Manna and Lahiri
1972). Possibly this numerical difference is
not real, but rather is due to a miscount
of spermatogonial chromosomes of G. sinuatus for which Manna and Lahiri (1972)
reported 2n ⫽ 18, in spite of n ⫽ 10.
Two kinds of interindividual chromosome polymorphism have been observed
in the G. sturmi population under study.
The first type deals with the occurrence of
different numbers of rDNA copies, revealed by FISH signals differing in number
and size. This kind of polymorphism, however, is a very common finding in animals
(see Schmid 1982; Suzuki et al. 1990).
Another type of interindividual chromosome variation consists of two different configurations of chromosome 3, in-
cluding the homozygous condition for the
subtelocentric chromosome 3a3a and the
heterozygous for the subtelocentric 3a
and the acrocentric 3b. The lack of the homozygous condition for the acrocentric
chromosome 3b3b is likely due to the limited number of specimens analyzed. Since
3a3a and 3a3b configurations were found
both in males and females, it is clear that
this chromosome rearrangement is inherited but not linked to the sex determination mechanism. Furthermore, on the basis of a general karyoevolutionary trend
toward an increase in karyotype simmetry
(Stebbins 1950), we suggest that in G. sturmi, a transformation from the original acrocentric into the neosubtelocentric occurred through a pericentric inversion.
Pericentric inversion polymorphism has
been described in mammals ( Davis et al.
1986; Hale 1986), birds (Ansari and Kaul
1979; Christidis 1986), reptiles (Moritz
1984), Amphibia (Schmid et al. 1995), and
some fish species ( Turner et al. 1985; Vitturi et al. 1993), whereas, as far as we
know, within invertebrates, it has been reported for Orthoptera [see Cabrero and
Camacho (1982), and authors quoted by
them] and for different populations of the
genus Drosophila ( Norry et al. 1997; Paik
et al. 1998; Singh 1998).
After CMA3 staining, G. sturmi chromosomes selectively fluoresce in correspondence to all heterochromatic material.
This strongly indicates that heterochromatic DNA is compartmentalized in GC
base pairs. The lack of complementarity,
revealed after treatment with the AT-specific fluorochrome DAPI, is an unusual reaction whose explanation remains unclear.
In most fish (Mayr et al. 1986; Phillips
and Hartley 1988) and amphibians
(Schmid et al. 1995; Schmid and Guttenbach 1988), CMA3 individualizes both active and inactive NORs as a consequence
possibly of the high GC content of the
rDNA. In G. sturmi, although NORs are
found to be enriched in GC base pairs,
CMA3 is unable to locate them as a result
of the presence of additional GC-rich chromosome regions besides the ribosomal
clusters.
As in other organisms (Pendás et al.
1993a,b; Phillips and Reed 1996), as well
as G. sturmi, the most appropriate method
for detection of rDNA cistrons remains
rDNA FISH. In fact, this method of investigation revealed rRNA genes corresponding to the terminal areas of the heterochromatic regions of a maximum of three
subtelocentric chromosome pairs, indicat-
ing that just a small portion of the GC-rich
C-positive material consists of rDNA.
We also used silver nitrate to assess the
rRNA gene activity, as silver binds acidic
protein(s) associated with the DNA of the
decondensed chromatin of the NORs (Jordan 1987; Medina et al. 1983). In G. sturmi,
silver is unable to individuate active rDNA
clusters, since it stains both constitutive
heterochromatin and heterochromatin associated with the NORs.
Within Scarabaeoidea, this unusual heterochromatin reaction is also shown by
the geotrupid Thorectes intermedius ( Vitturi et al. 1999), the melolonthid Phyllognatus excavatus (Colomba 1998), and the
lucanid Dorcus parallelopipedus (personal,
unpublished data), which represent three
phyletic branches of the superfamily, thus
suggesting that such a feature is quite
common within this taxon. On the contrary, among other animal groups, a similar result is reported only for the hedgehog [Erinaceus (Aethechinus) algirus]
(Sánchez et al. 1995). Moreover, the extensive silver stainability of the heterochromatin within Scarabaeoidea is quite independent from its base pair composition. In
fact, the AT-rich heterochromatin of Bubas
bison (Scarabaeidae) is as silver positive
(Colomba et al. 1996) as the GC-rich heterochromatin of the other scarabeoid species studied so far ( Vitturi et al. 1999, this
article).
Finally, results arising from in situ hybridization using the all-human telomeric
sequence ( TTAGGG)n reveal the lack of homology between the telomeric probe and
the telomeres of G. sturmi. Taking into account that, in the same hybridization experiments, the chromosomes of the earthworm (Eisenia foetida) gave a positive
reaction, the possibility that the results
obtained for G. sturmi may be due to a
technical shortcoming can be discarded.
This assumption seems to be further supported by the fact that no evident hybridization signals have been detected in Drosophila (Mason and Brissmann 1995). On
the other hand, Okazaki et al. (1993) isolated, from the Bombyx mori genomic library, a telomeric sequence ( TTAGG)n
which does not hybridize to vertebrate
DNAs, but, on the contrary, seems to be
conserved among insects. In fact, this pentanucleotide hybridized to DNAs from 8 of
11 orders of tested insect species. All this
might suggest that the hesanucleotide
telomeric sequence ( TTAGGG)n is not as
widely conserved within eukaryotes as it
has been stressed so far ( Blackburn 1991).
From the Dipartimento di Biologia Animale, Università
di Palermo, Via Archirafi 18, 90123 Palermo, Italy. This
work was supported by a research grant from MURST.
Address correspondence to R. Vitturi at the address
above or e-mail: [email protected].
Manna GK and Lahiri M, 1972. Chromosome complement and meiosis in forty-six species of Coleoptera.
Chromosome Inform Serv 13:9–11.
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Received June 8, 1999
Accepted November 30, 1999
Corresponding Editor: Martin Tracey