Arch.Geflügelk., 72 (5). S. 207–212, 2008, ISSN 0003-9098. © Verlag Eugen Ulmer, Stuttgart
The karyotype of domestic waterfowl: Ducks – RBG chromosome
pattern
Der Karyotyp des heimischen Wassergeflügels: Enten – RBG Muster der Chromosomen
Ewa Wojcik and Elzbieta Smalec
Manuskript eingegangen am 2. Oktober 2007, angenommen am 8. Dezember 2007
Introduction
Cytogenetic studies on domestic birds have been carried
out mainly on chickens and turkeys. The standard of
banded pattern has been obtained only for eight pairs of
macrochromosomes and sex chromosomes of Gallus
domesticus (LADJALI-MOHAMMEDI et al., 1999). APITZ et al.
(1995), DENJEAN et al. (1997) and DUCOS et al. (1997) have
carried out recent studies on duck karyotypes presenting
the G and C banding pattern, whereas the genetic and
cytogenetic map for the Pekin duck was created by HUANG
et al., 2006. Little interest in cytogenetic research on
waterfowl is due to the karyotype specificity, that is a large
diploid number and the occurrence of microchromosomes
(CHRISTIDIS, 1989).
The RBG technique makes it possible to reveal R-bands
at places rich in genes (BICKMORE and SUMNER, 1989) and of
the highest transcription activity (HOLMQUIST, 1992; SACCONE et al., 1993). R bands are replicated in an early stage of
phase S of the cell cycle. They contain many Cp-G islands
and are rich in guanine and cytosine. The RBG banding is
classified as one of standard methods of chromosome
examination. Scant knowledge about the genome of waterfowl (VIGNAL et al., 1999) has stimulated the cytogenetic
research which included ducks of the breeds important
from the economic point of view.
The research aimed at describing the karyotypes of two
duck species: the Pekin duck descending from Anas platyrhynchos and the Muscovy duck Cairina moschata by
means of the RBG technique of chromosome banding.
Materials and methods
Samples for examination were taken from 30 birds, 10
from each species representing Pekin and Muscovy ducks.
Ten Mule ducks as a cross between Pekin and Muscovy
were examined for the comparison of the parental species.
It was sampled from eight-week-old Pekin ducks,
eleven-week-old Muscovy ducks and their crosses. In each
group the proportion of males and females was 50:50%.
Chromosomal preparations were obtained from an in vitro
Dept. of Genetics and Horse Breeding, Institute of Bioengineering and Animal
Breeding, University of Podlasie, Poland
Arch.Geflügelk. 5/2008
lymphocyte culture by means of the RBG technique (PERRY
and WOLFF, 1974) which included the incorporation of
BrdU and Hoechst 33258 in 65th hour of incubation, and
EB and colchicine in the 69th hour of incubation. Ten
metaphase plates were analysed for each bird. The first
eight largest pairs of autosomes and sex ZW chromosomes
were examined. The localisation of R-bands was determined on selected chromosomes in each duck species and
ideograms of the banded patterns of the chromosomes
were drawn based on average observations and measurements from separate metaphases. An analysis was done on
separate chromosomes cut from metaphase plates. The
observations of dark – light bands were connected with the
absorption profile. The profiles of separate chromosomes
were done so many times as to obtain the number of
analysed chromosomes for a given pair as presented in the
table. Finally all the schematic pictures were averaged to
get one standardised chromosome ideogram. Figure 1
presents an example of the manner of obtaining the final
ideogram.
Results
The applied technique of banding enabled determining the
pattern of R-bands on the largest chromosomes of Pekin
duck (Anas platyrhynchos) and Muscovy duck (Cairina
moschata). The interspecies crosses were used in the comparative studies. A partial ideogram of eight largest pairs of
autosomes and sex chromosome ZW was drawn. In order
to facilitate the description of the chromosomes, regions
were determined on their arms and positive R (dark) and
negative (light) bands were counted. The results of observations and analyses are presented in the form of metaphase plates, karyograms and ideograms of R-bands (see
Figures 2ab, 4ab and Figures 3, 5).
Pekin duck (Anas platyrhynchos)
Chromosome 1. 1-1. P arm: Two regions. 9 R-bands. The first
proximal region included two positive bands (11, 13) and
two negative bands (12, 14). In the second region of the p
arm of the chromosome, three positive (21, 23, 25) and
two negative (22, 24) bands were identified.
1-2. Q arm: Four regions. 23 R-bands. In the first, second
and third region of the q arm, three dark bands (11, 13, 15;
21, 23, 25; 31, 33, 35 respectively) were followed by three
light bands (12, 14, 16; 22, 24, 26; 32, 34, 36 respectively).
The fourth distal region of the q arm consisted of three
dark bands (41, 43 and 45) evenly distributed and separated by two light bands (42 and 44).
208
Wojcik and Smalec: Ducks – RBG chromosome pattern.
Figure 1. An example of the
manner of obtaining the
final ideogram
Beispiel für die Ermittlung
des endgültigen Ideogramms
a)
24) were separated by three dark bands (21, 23, 25). The
first narrow band was located in the central part of the
arm. The remaining two positive R-bands were wide.
2-2. Q arm: Two regions. 11 R-bands. The first region
contained two positive bands (11, 13) and two negative
bands (12, 14). In the second region of the q arm, four dark
bands (21, 23, 25, 27) were separated by three light bands
(22, 24, 26).
Chromosome 3. 3-1. P arm: One region. One positive band
(11) was found.
3-2. Q arm: Two regions. 15 R-bands. The first region
had four positive bands (11, 13, 15, 17) and four negative
bands (12, 14, 16, 18). The second region of the q arm included four positive bands (21, 23, 25, 27) and three alternating negative bands (22, 24, 26).
b)
Chromosome 4. 4-1. P arm: One region. One positive band
(11) was found.
4-2. Q arm: One region. 11 R-bands. Six positive bands
were observed, including two outermost ones: a centromeric (11) and a telomeric (111) band; and four central
dark bands which were grouped in pairs (13, 15; 17, 19).
The positive R-bands were separated by light R-bands (12,
14, 16, 18, 110). Sometimes, narrow light bands (14 and
18) were not observed, especially when the chromosomes
on the metaphase plates were short.
Chromosome 5. 5-1. P arm: One region. One positive band
was observed (11).
5-2. Q arm: One region with nine R-bands. Five dark
bands (11, 13, 15, 17, 19) and four light bands (12, 14, 16, 18).
Figure 2. Image of the metaphase plate (a) and the partial
karyogram of the chromosomes of the Pekin duck (b)
Bild der Metaphasenplatte (a) und des partiellen Karyogramms
der Chromosomen der Pekingente (b)
Chromosome 2. 2-1. P arm: Two regions. 11 R-bands. In the
first region of the p arm of the chromosome, three dark
bands (11, 13, 15) were followed by three light bands (12,
14, 16). In the second region, two narrow light bands (22,
Chromosome 6. 6-1. Q arm: One region. 9 R-bands. Five
dark bands (11, 13, 15, 17, 19) were separated by four light
bands (12, 14, 16, 18).
Chromosome 7. 7-1. Q arm: One region. 5 R-bands. Three
positive (11, 13, 15) and two negative bands (12, 14) were
noticed.
Chromosome 8. 8-1. Q arm: One region. 5 R-bands. Three
positive (11, 13, 15) were followed by two negative (12,
14) bands.
Arch.Geflügelk. 5/2008
Wojcik and Smalec: Ducks – RBG chromosome pattern.
209
Figure 3. Ideogram of the
chromosomes of the Pekin
duck
Ideogramm der Chromosomen der Pekingente
Chromosome Z. Z-1. P arm: One region. 3 R-bands. Two
dark bands (11, 13) were separated by a light band (12).
Z-2. Q arm: One region. 7 R-bands. Four positive
R-bands (11, 13, 15, 17) were followed by three negative
bands (12, 14, 16).
Chromosome W. W-1. Q arm: One region. 5 R-bands. Three
dark (11, 13, 15) and two light (12, 14) bands were found.
Muscovy duck (Cairina moschata)
Chromosome 1. 1-1. P arm: Two regions. 9 R-bands. The first
proximal region contained two positive bands (11, 13) and
two negative bands (12, 14). In the second region of the p
arm of the chromosome three positive bands (21, 23, 25)
and two negative bands (22, 24) were found.
1-2. Q arm: Four regions. 23 R-bands. The first, second
and third region of the q arm of the chromosome had three
dark bands (11, 13, 15; 21, 23, 25; 31, 33, 35, respectively)
and three light bands (12, 14, 16; 22, 24, 26; 32, 34, 36).
The fourth distal region of the q arm included three evenly
distributed dark bands (41, 43, 45) separated by two light
bands (42 and 44).
Chromosome 2. 2-1. P arm: Two regions. 9 R-bands. The
first region was marked by four alternating dark (11, 13)
and light (12, 14) bands. In the second region, a wide central positive R-band (23) was between two bands – a narrow interstitial (21) and a wide distal (25) band. Narrow
negative R-bands (22, 24) separated the positive bands.
2-2. Q arm: Two regions. 11 R-bands. In the first region,
two negative bands (12, 14) followed two positive bands (11,
13) whereas the second region of the q arm contained four
dark bands (21, 23, 25, 27) and three light bands (22, 24, 26).
Chromosome 3. 3-1. P arm: One region. One positive band
(11) was observed.
3-2. Q arm: Two regions. 19 R-bands. Ten dark positive
R-bands belonged to two regions. Region 1 was marked by
five dark bands (11, 13, 15, 17, 19) and five light bands (12,
14, 16, 18, 110). The second region of the q arm included
Arch.Geflügelk. 5/2008
five dark bands (21, 23, 25, 27, 29) and four light bands
(22, 24, 26, 28).
Chromosome 4. 4-1. P arm: One region. One positive band
(11) was observed.
4-2. Q arm: One region. 11 R-bands. Six dark positive
(11, 13, 15, 17, 19, 111) and five negative R-bands (12, 14,
16, 18, 110) situated in one region were observed. The
interstitial bands were concentrated in pairs (13, 15 and
17, 19) and separated by the narrow light negative R-bands
(24, 28). The narrow negative bands (24, 28) were detected only on long chromosomes.
Chromosome 5. 5-1. P arm: One region. One positive band
(11) was observed.
5-2. Q arm: One region. 11 R-bands. Six dark bands of
different width (11, 13, 15, 17, 19, 111) were separated by
five light bands (12, 14, 16, 18, 110).
Chromosome 6. 6-1. Q arm: One region. 9 R-bands. Five
dark bands (11, 13, 15, 17, 19) were separated by four light
bands (12, 14, 16, 18).
Chromosome 7. 7-1. Q arm: One region. 7 R-bands. Four
positive (11, 13, 15 and 17) and three negative bands (12,
14 and 16) were noticed.
Chromosome 8. 8-1. Q arm: One region. 5 R-bands. Three
positive (11, 13, 15) were followed by two negative (12,
14) bands.
Chromosome Z. Z-1. Q arm: One region. 7 R-bands. On the
q arm two bands were observed – one dark (11) and one
light band (12) in the proximal part of the chromosome. In
the interstitial part, two wide positive bands (13, 15) were
followed by a wide central negative R-band (14). The distal
part included one light (16) and one dark band (17).
Chromosome W. W-1. Q arm: One region. 5 R-bands. Three
dark (11, 13, 15) bands were alternately separated by two
(12, 14) light bands.
210
Wojcik and Smalec: Ducks – RBG chromosome pattern.
a)
b)
some denoted with letter “b” had the arrangement of bands
typical of the sequence described previously for the Muscovy duck. The third pair of chromosomes differed in the
number of bands. On the chromosome labelled with letter
“a”, there were eight bands whose arrangement was the
same as for the Pekin duck. The homological chromosome
denoted with letter “b” had ten bands distributed similarly
to that on the Muscovy duck chromosome. Acrocentric
chromosomes of the fifth pair differed in the number of
bands. The chromosome designated with letter “a” had one
band less than the corresponding homologous chromosome denoted with letter “b”. The banded pattern of the
“a” chromosome of the crossbreed was the same as in the
Pekin duck and the pattern of the “b” chromosome closely
resembled the pattern of the Muscovy duck. Homologues
of the seventh pair differ in the number of bands (7 vs 5).
Sex Z chromosomes differed both in the morphological
make up and the number of bands. Pekin duck Z chromosome was acrocentric and had ten R-bands whereas
telometric Z chromosome of Muscovy duck included seven
R-bands.
Discussion and conclusion
Figure 4. Image of the metaphase plate (a) and the partial
karyogram of the chromosomes of the Muscovy duck (b)
Bild der Metaphasenplatte (a) und des partiellen Karyogramms
der Chromosomen der Moschusente (b)
Mule duck – comparison between species
In the case of the crossbreed chromosomes, letters “a” and
“b” denoted a pair of homological chromosomes. Slightly
bigger chromosomes whose banded pattern resembled the
chromosomes of the Pekin duck were designated with
letter “a” whereas letter “b” denoted the chromosomes
with the banded pattern similar to that of the Muscovy
duck chromosomes. There were no differences between
the banded pattern of the chromosomes of the first and
fourth pair. Differences in this pattern were observed on
the chromosomes of the second, third and fifth pair of
autosomes. Comparison of parental sex Z chromosomes
revealed the differences between the banding patterns of Z
chromosome of the parents. The ideogram (Figure 6)
presents only those chromosomes of Mule ducks that differed between pairs of homologues.
Differences in the arrangement of bands on the chromosomes of the second pair referred to the number of bands
in the first region of arm p. The chromosome designated
with letter “a” was characterised by the arrangement of
bands described previously for the Pekin duck. The chromo-
Organization of most avian karyotypes comprises a few
macrochromosome pairs and many microchromosomes.
The standard for chicken – eight macrochromosomes plus
the Z and W sex chromosomes – has been described by
LADJALI-MOHAMMEDI et al. (1999). The other chromosomes
for most of poultry species are identified unambiguously
and classified by size.
Molecular tags allowed identifying 22 of all chicken microchromosomes (FILLON et al., 1998; SCHMID et al., 2000;
MASABANDA et al., 2004). HUANG et al. (2006) assigned 20
FISH markers to the biggest 10 duck chromosomes and 4
markers to microchromosomes. In spite of the strong
homology between macrochromosomes in different bird
species (SCHMID et al., 2000) 4 orthologous loci that were
localized on the chicken chromosomes were uncertain in
the duck genome or located on different chromosomes
(HUANG et al., 2006).
The ability to karyotype an individual or a species is
fundamental for any genome-mapping attempt as both
genetic and physical maps are made with reference to the
chromosome position (MASABANDA et al., 2004). It was
recommended by LADJALI-MOHAMMEDI et al. (1999) and
SCHMID et al. (2000) to apply general guidelines developed
for chicken to other avian species.
Analysis of the duck karyotype was done in a limited
number of papers. Two of them presented GTG banding
pattern for 5 (APITZ et al., 1995) and 12 chromosomes
(DENJEAN et al., 1997) of two duck species (A. plathyrynchos and C. moschata). Both teams described the Z and W
heterochromosomes. There were some divergences in the
banding pattern of duck chromosomes proposed (No. 3
and 2) that could be attributed to a different contraction
during the cell cycle. The differences between duck species
the authors restricted to the 2nd and Z chromosomes (APITZ
et al., 1995) or to the 3rd, 5th, 7th and Z chromosomes
(DENJEAN et al., 1997). The ideogram of eight GTG banded
macrochromosomes and Z chromosome from the paper by
DENJEAN et al. (1997) cited in the First Report on Chicken
Genes and Chromosomes 2000 differ from the ideogram
presented in the original papers in regard to the number of
G positive bands (68 in the original paper vs 62 in the
paper of SCHMID et al. (2000)).
The RBG banding structure is not exactly the inverse of
that described with GTG pattern (LADJALI et al., 1995). The
Arch.Geflügelk. 5/2008
Wojcik and Smalec: Ducks – RBG chromosome pattern.
211
Figure 5. Ideogram of the
chromosomes of the Muscovy duck
Ideogramm der Chromosomen der Moschusente
Figure 6. Ideogram of the
chromosomes of the Mule
duck. Differences between
breeds only (a – Anas
platyrhynchos; b – Cairina
moschata)
Ideogramm der Chromosomen der Mularde. Nur
Unterschiede zwischen den
Gattungen (a – Anas platyrhynchos; b – Cairina moschata).
standard of the chicken karyotype allows counting 70
R-positive bands, 73 G-positive and 78 G-negative bands.
Our investigation shows that the total number of R-positive
bands of analogous chromosomes of Mullard duck reached
71, while that of Muscovy was 72. The karyotype comparison between species reflects differences of the 2nd, 3rd,
5th, 7th and Z chromosomes. APITZ et al. (1995), HAILU et al.
(1995) and DUCOS et al. (1997) observed chromosome size
differences between duck species.
In conclusion, while for the species Gallus the karyotype
standard of R banding has already been described, there is
lack of comparable studies on R banding chromosomes in
ducks.
Arch.Geflügelk. 5/2008
Summary
The objective of the study was to describe the karyotype of
two duck species: the Pekin (Anas platyrhynchos) and the
Muscovy (Cairina moschata) utilising the RBG banding
technique of chromosome staining. The comparison between the species was done on Mule crosses. Chromosomal preparations obtained from an in vitro culture of
blood lymphocytes were stained with the RBG technique.
The first eight largest pairs of autosomes and ZW sex
chromosomes were analysed. The localisation of R-bands
was determined and the ideograms of the banded patterns
of the analysed chromosomes were drawn. No differences
212
Wojcik and Smalec: Ducks – RBG chromosome pattern.
were found in the banded pattern of the chromosomes in
the first, fourth, sixth pair of autosomes and W sex chromosome. Differences in the arrangement of bands were observed on the second, third, fifth and seventh pair of autosomes and the sex Z chromosome.
Key words
Duck, Anas platyrhynchos, Cairina moschata, karyotype,
mitotic chromosomes, R-bands
Zusammenfassung
Der Karyotyp des heimischen Wassergeflügels:
Enten – RBG Muster der Chromosomen
Das Ziel der Untersuchung war die Beschreibung des
Karyotyps von zwei Entenspezies, Peking-Ente (Anas platyrhynchos) und Moschusente (Cairina moschata), unter
Verwendung der RBG Färbungstechnik für Chromosomen.
Der Vergleich zwischen den beiden Spezies wurde anhand
der Interspezieskreuzung Mularde vorgenommen. Chromosomenpräparate aus einer In-vitro-Kultur von Blutlymphozyten wurden mit Hilfe der RBG Technik gefärbt. Die
ersten acht größten Autosomenpaare und die Geschlechtschromosomen Z und W wurden analysiert. Hierzu wurde
die Lage der R-Bänder bestimmt und das Ideogramm der
Bandenmuster der untersuchten Chromosomen angefertigt. Das Bandenmuster des ersten, vierten und sechsten
Autosomenpaars sowie des Chromosoms W unterschied
sich nicht zwischen den Spezies. Dagegen wurden für das
zweite, dritte, fünfte und siebte Autosomenpaar sowie das
Z-Chromosom Unterschiede in der Bandenanordnung festgestellt.
Stichworte
Ente, Anas platyrhynchos, Cairina moschata, mitotische
Chromosomen, Karyotyp, R-Bänder
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Correspondence: Dr. Ewa Wojcik, Department of Genetics and Horse Breeding
Institute of Bioengineering and Animal Breeding, University of Podlasie, Prusa
14, 08-110 Siedlce, Poland; e-mail: [email protected]
Arch.Geflügelk. 5/2008