Annals of Warsaw University of Life Sciences – SGGW. Animal ...

40 J. Gruszczyńska, M. Łukasiewicz
(29 cocks and 24 hens) from the experimental
flock of Ayam Cemani hens. The
experiment was conducted at the Poultry
Farm of the Warsaw University of Live
Sciences in Obory. Genomic DNA was
extracted from the blood by phenol-chloroform
extraction. The presence and the
purity of isolated DNA was checked on a
NanoDropp spectrophotometer (Thermo
Scientific).
Five microsatellite sequences were
used as a tool to determine genetic diversity
of the bird population examined. Owing to
the specific character of the experiment,
in molecular analysis the microsatellite
sequences were selected so as to be
linked with the body weight of the hens.
On the basis of literature, 5 microsatellite
sequences were chosen as follows:
MCW0145, MCW0184, MCW0210,
LEI0071, and ADL0306 (Atzmon et al.,
1998; Weissmann et al., 1998).
PCR amplification
Five pairs of primers of microsatellite
sequences were selected from the Roslin
Institute chicken mapping database
ArkDB (http://www.thearkdb.org).
Forward primers were labelled with Cy5
marker. The reaction mixture comprised
primers each at 20ppmol, dNTP MIX,
1.0 unit of Taq polymerase (Sigma) per
24 μl reaction mixture, 1 ng template
genomic DNA (prepared from red blood
cells) in each reaction mixture, and 5 μl
of 10x reaction buffer (100 mM Tris
HCl, 15mM MgCl2, 50mM KCl, 0.01%
gelatin). The cycling temperature was as
follows: denaturation was run at 95 o C
for 30 s; annealing was run for 60 s at
46 o C for LEI0071, 47 o C for ADL0306,
51 o C for MCW0210 and MCW0145,
and 56 o C for MCW0184, depending on
primer composition; extension war run
at 72 o C for 60s.The reaction was carried
out for 30 cycles. The final extension
was run at 72 o C for 10 min. The conditions
of polymerase chain reaction (PCR)
were employed as earlier established by
Gruszczyńska and Michalska (2005) and
by Gruszczyńska et al. (2007).
The birds were genotyped, using an
automatic sequencer ALF Express (Pharmacia
LKB).
Statistical analysis
Results of the molecular analysis were
used to estimate the frequency of alleles of
the analyzed loci, frequency of genotypes
(homo- and heterozygotes), expected
heterozygosity H E (Ott, 1992), and to
determine the Polymorphic Information
Content PIC (Botstein et al., 1980). The
frequency of alleles, H O (observed heterozygosity),
H E (expected heterozygosity)
and PIC (polymorphic information
content) were computed using Cervus
3.0.3. software (Kalinowski et al., 2007),
whereas the frequency of genotypes – with
the use of an original ALFREQ software
(Tereba Z., Tereba A., 2005).
RESULTS AND DISCUSSION
In the analyzed population of Ayam
Cemani breed hens the polymorphism
within all of 5 selected microsatellite
sequences. The lengths of the sequences
were: 156–168bp for MCW0210; 240 and
260bp for MCW0184; 280–300bp for
LEI0071; 206–218bp for MCW0145, and
119–125bp for ADL0306 (Tab. 1). The
number of alleles was ranging from 2 for
MCW0184 sequence to 4 for MCW0145
sequence. Within particular microsatellite
loci we observed the following