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Retinal TUNEL-Positive Cells and
High Glutamate Levels in
Vitreous Humor of Mutant Quail
with a Glaucoma-like Disorder
Ouria Dkbissi,1'2 Evelyne Chanut,5
Marguerite Wasowicz,2 Michelle Savoldelli,4
Jeanine Nguyen-Legros,1 Francis Minvielle,5 and
Claudine Versaux-Botteri1'2
investigate whether retinal cell death observed in an avian glaucoma-like disorder occurs by apoptosis and whether an increase in excitotoxic amino acid
concentration in the vitreous humor is associated temporally with cell death in the retina.
PURPOSE. TO
METHODS. Presumptive
retinal apoptotic nuclei were identified by histochemical detection of DNA fragmentation
(by TdT-dUTP terminal nick-end labeling [TUNEL]), and
vitreal concentrations of glutamate and several other
amino acids were determined by high-pressure liquid
chromatography with fluorometric detection in the al
mutant quail (Coturnix coturnix japonicci) in which a
glaucoma-like disorder develops spontaneously.
RESULTS. TUNEL-labeled nuclei were located mostly in the
ganglion cell layer (GCL) in the retina of mutant quails 3
months after hatching. However, labeled nuclei were also
observed in the inner and outer nuclear layers. At 7
months, most TUNEL-positive nuclei were detected in the
inner nuclear layer, whereas labeled cells in the GCL were
reduced in number. No TUNEL-labeled nuclei were detected in the retina of control quails at any age. Vitreal
concentrations of glutamate and aspartate were significantly increased in 1-month-old mutant quails compared
with control animals. Concentrations decreased at 3
months, and no significant differences were observed between strains at 7 months.
CONCLUSIONS. Presumptive apoptotic cell death is detected
from 3 months after hatching in mutant quails and is not
restricted to retinal ganglion cells. Cell death appears just
after a significant increase in excitotoxic amino acid concentrations in the vitreous humor, suggesting a correlation
From the 'Laboratoire cle NeuroCytologie Oculaire, Institut
National cle la Sante et de la Recherche Medicale ONSERM), Paris;
2
Laboratoire d'Anatomie Comparee, Museum National d'Histoire Naturelle, Paris; 3Laboratoire de Pharmacologie, Faculte de Pharmacie,
Chatenay-Malabry; ^Service d'Opthalmologie, Hopital de l'Hotel-Dieu,
Paris; and 5Laboratoire de Genetique Factorielle, Institut National de la
Recherche Agronomique, Jouy-en-Josas, France.
Submitted for publication July 20, 1998; revised October 22, 1998;
accepted November 4, 1998.
Proprietary interest category: N.
Reprint requests: Claudine Versaux-Botteri, Laboratoire de NeuroCytologie Oculaire, INSERM U 86, 15, rue de l'Ecole de Medecine,
75270, Paris cedex 06, France.
990
between both events. (Invest Ophthalmol Vis Sci. 1999;
40:990-995)
G
laucoma is a widespread human ocular disease, characterized by retinal ganglion cell degeneration, excavation of
the optic nerve head, and in most cases, increase of intraocular
pressure (IOP). Its cause remains unclear, and although increased IOP has long been thought to be the primary cause of
ganglion cell degeneration and optic disc cupping, evidence
from the studies of low-tension glaucoma suggests that it is
neither necessary nor sufficient to induce the disease. Other
mechanisms have been proposed that involve the neurosensory retina directly.1 Results in recent studies suggest that
apoptotic mechanisms could be involved in the degeneration
of ganglion cells2'3 and emphasize the potential neurotoxic
role of glutamate.4'5 Abnormally high levels of glutamate have
been detected in the vitreous of glaucomatous eyes of dogs,
monkeys, and humans.67
The purpose of this work was to determine whether such
particularities were also found in a hypopigmented mutant of
the Japanese quail, Coturnix coturnix japonica, in which an
ocular disease develops that has been described as resembling
human closed-angle glaucoma, with increasing IOP (measured
by cannulation), closure of the iridocorneal angle, and progressive degeneration of the ganglion cells. 8 "" However, several
morphologic characteristics such as enlargement of the retinal
surface, abnormal corneal endothelium with degenerating
cells, poorly differentiated cells with collapsed trabecular
meshwork, and attachment of the anterior face of the iris to the
posterior cornea, suggest that it also possesses some characteristics of human congenital glaucoma.1213 Therefore, we
searched for the presence and distribution of apoptotic nuclei
and determined the vitreous concentration of glutamate during
the development of glaucoma-like disease in mutant quail retina. Our results show that an increase of excitotoxic amino
acid concentrations in vitreous could be temporally associated
with the appearance of degenerating cells, suggested by the
presence of nuclei positive for TdT-dUTP terminal nick-end
labeling (TUNEL) in the mutant quail retina.
MATERIALS AND METHODS
Fifty-two mutant quails, with the mutant gene al, and 52
control quails Coturnix coturnix japonica were provided by
the Institut National de la Recherche Agronomique (Jouy-enJosas, France). Animals were maintained in a l4-hour-light-10hour-dark photoperiod from hatching to death. Mutant quails
were killed by decapitation at three stages of the disease
determined previously9: at 1 month, before the appearance of
any morphologic signs of glaucoma; at 3 months, at the onset
of the first pathologic signs of the disease, such as the appearance of buphthalmy; and at 7 months, when glaucoma, evidenced by marked buphthalmy, was well established. Control
subjects were killed according to the same schedule. The
experiments were performed in compliance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision
Research.
Investigative Ophthalmology & Visual Science, April 1999, Vol. 40, No. 5
Copyright © Association for Research in Vision and Ophthalmology
Reports
IOVS, April 1999, Vol. 40, No. 5
991
FIGURE 1. Transverse sections of 1-month-old control (A) and mutant (B) qu;iil retinas. Retinas were
processed by the TUNEL method and coimterstained with methyl green. No labeling was observed at 1
month in either type of quail. Scale bar, 20 /win.
TUNEL Staining
Four retinas from four quails of each strain killed at each of the
three stages (i.e., 12 mutant and 12 control quails) were prepared for TUNEL analysis14 by use of an in situ apoptosis
detection kit (Apoptag; Oncor, Gaithersburg, MD). The eyes
were fixed in Bouinfixativefor 48 hours at 4°C, embedded in
paraffin, and 10-^arn thick vertical sections were cut. Endogenous peroxidase was inactivated by incubating sections with
2% H2O2 for 5 minutes. Sections were preincubated in the
equilibration buffer (provided in the kit) for 30 seconds at
room temperature and were then treated with terminal deoxynucleotidyl transferase (TdT) and digoxigenin deoxyuridine
triphosphate (dUTP) for 1 hour at 37°C. They were rinsed in
buffer provided in the kit (Stop/WA; Oncor) for 30 minutes at
37°C. Retinas were incubated with a peroxidase-coupled antidigoxigenin antibody for 30 minutes at room temperature. The
3'-OH DNA tail was detected by incubating retinas with a
diaminobenzidine-H2O2 solution and counterstained with
methyl green for 10 minutes. Controls were made by omitting
TdT during the first step of the labeling procedure. No labeling
was observed in control sections. We estimated the proportion
of TUNEL-positive ganglion cells during the time course of the
disease by expressing the results as a percentage of the number
of labeled cells in all sections from one stage, compared with
the total number of cells located within the ganglion cell layer
(GCL) observed in the same sections at the same stage.
Semithin Sections
Degeneration of retinal ganglion cells was estimated by comparing the thickness of the nerve fiber layer in mutant and
control semithin sections. Eight eyes of 7-month-old animals,
four from different mutant quails and four from different control quails, were removed and immersed for 2 hours in 0.1 M
cacodylate buffer (pH 7.3) containing 2.5% glutaraldehyde.
After a rinse in 0.2 M cacodylate buffer for 24 hours, retinas
were dissected and postfrxed in 1% osmium tetroxide for 1
hour. After dehydration and embedding in epoxy resin, l-/xm
semithin sections were collected in both strains from the same
region of the retina near the optic nerve head. To measure the
optic nerve layer thickness, 20 semithin sections at 5-/xm intervals were examined in each eye in both strains. The mean
thickness of each strain was compared using the Student's
J-test.
Ghitamate and Other Amino Acid Assays
Twelve vitreous bodies, one vitreous body per quail, were
collected at each stage from each strain (i.e., 36 from mutant
and 36 from control quails). Glutamate concentrations were
determined by high-pressure liquid chromatography (HPLC)
with a fluorescence monitor (RF-551; Shimadzu, Touzart and
Matignon, Courtaboeuf, France) using a slightly modified
method described previously.15 Individual vitreous bodies
were sonicated in 0.2 M perchloric acid containing 0.1%
Na2S205 and 0.1% EDTA. Homogenates were centrifuged at
l5,000g for 5 minutes at 4°C and the supernatant used for
glutamate determination. The stock reagent contained 27 nig
o-phthaldialdehyde, 1 ml methanol, 5 jul jB-mercaptoethanol,
and 9 ml 0.1 M sodium tetraborate. The working solution was
prepared by diluting the stock solution with 0.1 M sodium
tetraborate (1:5 v/v). Precolumn derivation was performed by
mixing 10 /xl sample (1:4 dilution in perchloric mixture as
described above) with 90 yA working o-phthaldialdehyde reagent. This final mixture (10 /xl) was injected into the HPLC
system. Separation of glutamate, glutamine, •y-aminobutyric
acid (GABA), asparagine, aspaitate, threonine, taurine, and
tyrosine was achieved by reversed-phase liquid chromatography (Hypersil C18, 3-^tm column, 150 X 4.6 mm; Touzard and
Matignon, Courtaboeuf, France). The mobile phase (pH 7.8),
flow rate (1 ml/min), consisted of water-acetonitrile-methanol
(70:5:25 vol/vol/vol) containing 0.1 M dibasic sodium phosphate buffer. Excitation and emission wavelengths were set at
335 nm and 425 nm.
For precise evaluation of the within- and between-assay
coefficients of variation (CV) for amino acid quantification,
samples of pooled vitreous bodies were prepared and analyzed
three times the same clay (within CV) and on 4 different days
(between CV). The CVs were never greater than 13%. Results
were expressed in nanograms per milligram vitreous humor.
992
Reports
IOVS, April 1999, Vol. 40, No. 5
OPL
FIGURE 2. Transverse sections of 3-month-old control (A) and mutant (B, C, D) quail retinas. There is no
labeling in control animals (A). Most of the TUNEL-positive nuclei are in the GCL, but labeled nuclei are
also observed in the inner and outer nuclear layers (B). At high magnification (inset D), we observed DNA
fragmentation (white arroiv) of ganglion cell nuclei, characteristic of apoptosis. Scale bar, (A, B, C) 20 jxm;
(D) 7 jim. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer
plexiform layer; ONL outer nuclear layer.
Individual data were analyzed by global Kruskal-Wallis analysis, followed when significant (P < 0.05) by individual interstage comparisons with the nonparametric Mann-Whitney
test.
RESULTS
TUNEL Method
No TUNEL-positive cells were observed in the retina of control
quails at any of the three stages studied (Figs. 1A, 2A and 3A)
and in the retina of 1-month-old mutant quail (Fig. IB). TUNELpositive nuclei were detected mainly in the GCL in 3-month-old
mutant quails (Tigs. 2B, 2C). Labeled cell bodies were esti-
mated at almost 50% (49%) of the total number of cells located
in the GCL in retinal sections examined (Table 1). TUNELlabeled cells were also found in the inner and outer nuclear
layers (Fig. 2B). DNA fragments were observed as dark aggregates in many nuclei at the highest magnification (Fig. 2D). At
7 months, the number of ganglion cells appeared reduced and
more spaced in the mutant than in the control quails. At this
stage, TUNEL-positive nuclei were still observed in the GCL
(estimated at almost 20% (18.7%) of total cells remaining in the
GCL in retinal sections examined), but many more labeled
nuclei were located in the inner nuclear layer than were seen
in previous stages, and the outer nuclear layer was unlabeled
(Fig- 3B).
FIGURE 3. Transverse sections of 7-month-old control (A) and mutant (B) quail retinas. As at the other
stages, no labeling is observed in control animals (A). A few TUNEL-positive nuclei are still observed in the
GCL (black arrow), whereas the TUNEL-positive nuclei are more numerous in the inner nuclear layer
(INL). The INL is thicker, and the density of ganglion cells in the GCL appears less in mutant quails than
in control animals. Scale bar, 20 pt.ni. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear
layer; OPL, outer plexiform layer; ONL outer nuclear layer.
JOVS, April 1999, Vol. 40, No. 5
Reports
993
TABLE 1. TUNEL-Positive Cells in Mutant Quails
Age of Mutant
Quails
3 months
7 months
Number
TUNEL-Positive
Total Number
of Cells
% of TUNELPositive Cells
Number of
Samples
944
214
1926
1141
49
18.7
20
20
No data is reported for 1 month because no TUNEL-positive cells were observed.
Observation of the Nerve Fiber Layer
DISCUSSION
The loss of ganglion cells during the disease was confirmed by
a statistically significant (P < 0.001) 39% reduction of the
thickness of the nerve fiber layer, which contains axons of
retinal ganglion cells, in 7-month-old mutant quails (92 ± 2.4
/j,m) compared with the thickness in control quails (15 ± 0.7
jLtm; Figs. 4A, 4B)
Amino Acid Assays
Results of assays are reported in Table 2. Although no significant changes were found in control quails at any age, an
elevation in glutamate and aspartate concentrations was detected in 1-month-old mutant quails that was approximately 1.5
times that in 1-month-old control quails. A progressive decrease in vitreous glutamate and aspartate was observed at 3
months in the mutant quail, attaining control levels at 7
months. The increase in amino acid vitreous concentrations
observed in 1-month-old mutant quails was found only for
glutamate and aspartate. The time course of other amino acid
concentrations varied according to the amino acid in question.
There was no significant difference in asparagine and tyrosine
levels between the two strains at any age, and levels of GABA
and glutamine decreased from 3 months in mutant quails to
particularly low levels at 7 months. Taurine and threonine
increased significantly at 3 months, followed by a decrease at 7
months in mutant quails.
4b
FIGURE 4. Semithin sections of 7-month-old control (A) and mutant
(B) quail retinas. One-micrometer sections were obtained from the
same region of the retina, near the optic nerve head in both types of
quail. Note the large decrease (mean decrease, 39%) of the thickness of
the nerve fiber layer in 7-month-old mutant quails. Scale bar, 13 /Lim.
NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform
layer; INL, inner nuclear layer.
Glaucoma is a frequently occurring ocular disease that causes
degeneration of ganglion cells, and subsequently, impairment
of the visual field. The causes of this disease remain unclear,
but some ocular dysfunctions (principally an increase in IOP)
are often associated with it. In the present work, we have
observed presumptive apoptotic nuclei (detected by TUNEL)
in the retina of the mutant quails which, from 3 months after
hatching, manifest a glaucoma-like disease, with an increase in
IOP and degeneration of ganglion cells. 9 "" Apoptosis, which
is characterized morphologically by condensation and aggregation of chromatin and nuclear shrinkage, is described as a
process of programmed cell death that occurs naturally during
development.l6 In adulthood, it is found in a variety of diseases
including glaucoma, in which labeled nuclei are generally
found exclusively in the GCL.23
In mutant quail, however, the TUNEL-positive nuclei were
observed not only in tlie GCL but also in the outer and inner
nuclear layers. These results are not seen commonly in this
disease, although a photoreceptor loss has sometimes been
associated with spontaneous glaucoma in humans,17 and a
reduction of cells located in the inner nuclear layer has been
observed in the retina of mutant quails'3 and in the retina of
monkeys with induced glaucoma.18 Moreover, because the
percentage of degenerating cells seemed especially elevated in
our animal model, we cannot exclude that the retinas of hypopigmented mutant quails are particularly susceptible to cellular death. The reasons for such dramatic cellular susceptibility remain unknown, but it would be interesting, in that
connection, to study the proteins involved in apoptosis.
Among these, Bcl-2 is a particularly interesting candidate, because the inactivation of the bcl-2 gene leads to excessive
apoptotic cellular death and depigmentation subsequent to a
dysfunction in melanin synthesis.19
A variety of stimuli are thought to initiate or activate
apoptosis in glaucoma. Thus, increased IOP and/or organelle
accumulation at the optic nerve head could block axoplasmic
flow, hindering the circulation of trophic factors.20"22 Further
studies must be undertaken to determine the degree of involvement of increased IOP in cellular degeneration in mutant quail.
However, some data suggest that glutamate neurotoxicity
could be also involved in cellular degeneration in glaucoma. It
has been shown that an intravitreal injection of glutamate
agonists such as kainic acid23 or yV-methyl-o-aspartate can induce apoptosis-like cell death,4'5 and glutamate seems to be in
excess in the vitreous humor of dogs, monkeys, and humans
with glaucoma.6'7 We observed also an elevation of vitrcal
concentrations of glutamate and aspartate (an excitatory amino
acid resembling closely glutamate and exhibiting excitotoxic
994
Reports
TABLE
2. Amino Acid Concentrations in the Vitreous Bodies of Mutant and Control Quails
IOVS, April 1999, Vol. 40, No. 5
Control Quails
Amino
Acid
Glutamate
Aspartate
Asparagine
GABA
Glutamine
Taurine
Threonine
Tyrosine
1 Month
26.3
14.6
4.9
29.5
231.7
205.8
4.4
7.3
±
±
±
±
±
±
±
±
3.8
2
0.5
6.6
25.7
28
0.3
0.7
3 Months
26.8 ± 3.6
9.2 ± It
4
10.3
204.4
253.2
2.8
5.4
±
±
±
±
±
±
0.5
1.7f
15.6
15.7
0.3t
0.5
Mutant Quails
1 Month
7 Months
29.3
10.6
22.8
40.6
228.2
220.6
4.2
7.3
±
±
±
±
±
±
±
±
7.1
1.6
3.6f
4.6f
24.7
25.9
0.3t
1.4
40.3
20.9
5.3
25.6
227.2
194.8
3.6
7.1
±
±
±
±
±
±
±
±
4.6*
2.3*
0.4
4.9
23.5
24.7
0.2
0.6
3 Months
35.6
12.1
4.2
6.9
159-8
325.4
4.6
6.9
±
±
±
±
±
±
±
±
3.7*
l.lf
0.4
2.1t
11.6
24.2*t
0.4*
0.8
7 Months
22.3 ± 3t
8 ± 0.9
22.3 ± 3t
6.2
158.7
177.6
3.4
4.6
±
±
±
±
±
1.2*
21.9*
lit
0.5
0.8
* Statistical comparison between control quails and mutant quails at the same age (P < 0.05). Comparisons by Kruskall-Wallis test; n = 12
for each age.
t Statistical comparison between the age considered and the previous age (P < 0.05). Comparisons by Kruskall-Wallis test; n = 12 for each
age.
properties 24 ) just before the appearance of apoptotic cells in
mutant quails.
The source of the vitreal glutamate and aspartate remains
unknown, but these amino acids are synthesized and released
by all categories of retinal cells. As for ischemia, it could be
proposed that the vitreal accumulation of glutamate (and aspartate) is caused by a dysfunction in their uptake by retinal
glial cells. 25 ' 26 The retinal ubiquity of these neurotransmitters
could be one of the reasons for the degeneration of cells
located not only in the GCL, but also in the inner and outer cell
layers.
Moreover, it has been reported in recent studies that
glutamate-induced indirect neurotoxicity could be propagated
by unknown toxin released from damaged cells. 27 The absence
of changes of the vitreal content of other examined amino
acids at 1 month supports the glutamate-aspartate hypothesis."
The decrease in glutamate/aspartate content at 7 months is
probably caused by a dysfunction in their metabolism, suggested by the decrease of the vitreous content of their precursors and degradative products, GABA and glutamine.
Acknowledgments
The authors thank David Hicks and Howard Cooper for critically
reading the manuscript and correcting the English.
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Bothnia Dystrophy Caused by
Mutations in the Cellular
Retinaldehyde-Binding
Protein Gene (RLBPl^ on
Chromosome 15q26
Marie S. I. Burstedt,1 Ola Sandgren,1
Gosta Holmgren,2'5 and
Kristina Forsman-Semb23'4
determine the chromosomal location and to
identify the gene causing a type of retinitis punctata albescens, called Bothnia dystrophy, found in a restricted
geographic area in northern Sweden.
PURPOSE. TO
METHODS. Twenty patients from seven families originating
from a restricted geographic area in northern Sweden
were clinically examined. Microsatellite markers were analyzed in all affected and unaffected family members.
Direct genomic sequencing of the gene encoding cellular
retinaldehyde-binding protein was performed after the
linkage analysis had been completed.
Affected individuals showed night blindness from
early childhood with features consistent with retinitis
punctata albescens and macular degeneration. The responsible gene was mapped to 15q26, the same region to
which the cellular retinaldehyde-binding protein gene
has been assigned. Subsequent analysis showed all affected patients were homozygous for a C to T substitution
in exon 7 of the same gene, leading to the missense
mutation Arg234Trp. Analysis of marker haplotypes suggested that all cases had a common ancestor who carried
the mutation.
RESULTS.
From the 2Department of Clinical Genetics, University Hospital,
Umea, Sweden; and the Departments of 3Applied Cell and Molecular
Biology and 'Ophthalmology, University of Umea, Sweden.
Supported by grants from the Swedish Medical Research Council
G5rojects No. 09745 and 10866), the County Council of Vasterbotten
and Carmen and Bertil Regners foundation for research in the area of
eye diseases.
Submitted for publication October 14, 1998; accepted December
17, 1998.
Proprietary interest category: N.
''Present address: Department of Molecular Biology, Astra Hassle
AB, S-431 83 Molndal, Sweden.
Reprint requests: Ola Sandgren, Department of Ophthalmology,
University of Umea, S-901 85 Umea, Sweden.
Reports 995
25. Vorwerk CK, Lipton SA, Zurakowski D, Hyman BT, Sabel BA,
Dreyer AB. Chronic low-dose glutamate is toxic to retinal ganglion
cells. Toxicity blocked by memantine. Invest Ophthalmol Vis Sci.
1996;37:l6l8-l624.
26. Perez MT, Davanger S. Distribution of GABA immunoreactivity in
kainic acid treated rabbit retina. Exp Brain Res. 1994; 100:227238.
27. Yoon KW, Fuse T, Shah PT, Nguyen S, Klein ML. Indirect glutamate neurotoxicity. / Neurotrauma. 1998; 15:141-147.
CONCLUSIONS. A missense mutation in die cellular retinaldehyde- binding protein gene is the cause of Bothnia dystrophy. The disease is a local variant of retinitis punctata
albescens that is common in northern Sweden due to a
founder mutation. (Invest Ophthalmol Vis Sci. 1999;40:
995-1000)
R
etinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal dystrophies characterized by
early night blindness and later loss of peripheral and central
vision. Pigment deposition in the retina and attenuation of the
retinal blood vessels are observed. The diagnosis is confirmed
by an abnormal or extinguished electroretinogram (ERG). Retinitis pigmentosa loci have been mapped to numerous chromosomal locations, and mutations in many different genes
have been found in patients.1 An updated list of disease loci is
available on the RetNet web site: http://utsph.sph.utli.tmc.
edu/retnet.
A unique atypical variant of RP, known among clinicians
in northern Sweden for decades as Vasterbotten dystrophy or
Bothnia dystrophy (refers to the area adjacent to the Gulf of
Bothnia), is described in this report. Affected individuals have
had night blindness since early childhood, retinitis punctata
albescens (RPA), and macular degeneration. Retinitis punctata
albescens is associated with RP and characterized by numerous
punctate whitish-yellow spots in the fundus.2 We demonstrate
by linkage analysis that the disease gene is localized to 15q26.
Subsequently we show that a missense mutation in the cellular
retinaldehyde-binding protein gene (RLBPl^ is present in a
homozygous state in 20 patients from seven Vasterbotten families with Bothnia dystrophy.
METHODS
Ophthalmologic Examinations
Inclusion criteria for the families in this study were ophthalmologic records of retinal disease and more than one family
member showing early onset night blindness, fundus appearance similar to RPA with small white dots in central fundus,
macular degeneration, and lack of for RP typical bone spicules
in peripheral retina. Standard ophthalmologic examination and
fundus photography were carried out in all affected individuals
and selected siblings. Dark adaptation tests and full-field ERGs
were performed in selected cases. The study followed the
tenets of the Declaration of Helsinki, and informed consent
was obtained from all subjects.
Genotyping
Extraction o genomic DNA and analysis of microsatellite markers were performed as described by Balciuniene et al.3