كليدواژه :
PCR-SSCP , ژن GDF9 , نژاد كرماني , چندقلوزايي , چند شكلي
چكيده لاتين :
چندشكلي
نيمه دوم اگزون
2
ژن
GDF
9
گوسفند
نژاد كرماني
به روش
PCR
-
SSCP
و توالي
يابي
5
9
Polymorphism at second half of exon 2 of GDF9 gene in Kermani sheep breed
using
PCR
-
SSCP and sequencing
R Khodabakhshzadeh
1
and MR Mohammadabadi
2*
Received:
J
une 3, 2016
Accepted:
February 18, 2017
1
MSc Student, Department of Animal Science, Faculty of Agriculture, College of Agriculture, Shahid
Bahonar University of Kerman, Kerman, Iran
2
Professor, Depart
ment of Animal Science,
Faculty
of Agriculture, Shahid Bahonar University of
Kerman, Kerman, Iran.
*
Corresponding Author:
mmohammadabadi@yahoo.ca
Introduction:
Applications of molecular genetics have many important advantages (
Mousavizadeh
et al. 2009
).
Using genetic markers in animal selection and breeding may also dramatically expedite
genetic improvement
(Javanmard et al. 2008).
Study of native breeds is necessary for conservation
of genetic resource in livestock (Mohammadabadi et al. 2010).
There are
more than 26 sheep breeds
in Iran adapted to different environmental circumstances (Zamani et al. 2015).
One of the most
important breeds of Iranian sheep is Kermani sheep. This local breed lives in the south
-
eastern of Iran
and is a fat
-
tail breed and wel
l adapted to a wide range of harsh environmental conditions in Kerman
province (Mohammadabadi 2017). Growth differentiation factor (GDF) 9 is a member of the
transforming growth factor β superfamily that is secreted from oocytes during folliculogenesis
(Aa
ltonen et al. 1999) and is essential for folliculogenesis and female fertility (Khodabakhshzadeh et
al. 2016). Hanrahan et al. (2004) revealed eight single nucleotide polymorphisms across the entire
coding region (G1
–
G8) and these differences correspond to
one SNP in exon 1, one SNP in the intron,
and five SNPs in exon 2. It is proven that exon 2 is more important than exon 1 and intron.
Material and
methods:
The blood samples were randomly
collected from Kermani sheep (102
animals) from both sexes and wit
h different ages (Kerman, Iran), using vacuum tubes with 0.25%
ethylene diamine tetra acetic acid (EDTA). The blood samples were transferred in dry ice to the
laboratory and stored at
-
20 °C pending assays. Blood samples of the animals were used to extract
genomic DNA using the salting out procedure described by Abadi et al. (2009). The quality of DNA
was checked by spectrophotometry taking ratio of optical density (OD) value at 260 and 280 nm. The
sheep GDF9 gene was amplified using the polymerase chain re
action (PCR) with designed specific
primers.
These primers were used to amplify fragment 647 bp
of
the exon 2 for the sheep GDF9
gene
.
The PCR reaction was performed in a 25 μL reaction volume containing 2 μL of genomic DNA (50
ng/μL), 1 μL of MgCl2 (3 mM)
, 1μL of each forward and reverse primers (10 pmol each), 0.5μL of
dNTPs (500 μM each), 0.3 unit of Taq DNA polymerase (Cinna Gene, Iran) and 10X PCR buffer.
DNA amplifications were performed using thermo cycler (CLEMENS, Germany) programmed for a
prelimin
ary step of 5 min at 94°C, followed by 33 cycles of 30 s at 94°C, 50 s at 62.5°C for the first
primer pair and 63.6°C for the second primer pair and 50 s at 72°C, with a final extension of 8 min at
72°C. Amplification was verified by electrophoresis on 1%
(w/v) agarose gel in 1 x TBE buffer (2
mM of EDTA, 90 mM of Tris
-
Borate, pH 8.3), using a 100bp ladder as a molecular weight marker
for confirmation of the length of the PCR products. Gels were stained with ethidium bromide (1
μg/mL).
The SSCP technique wa
s used to allow the sequence variants to be detected from the
migration shift in PCR amplified fragments of the gene. For SSCP analysis, 6 μL of each PCR product
was mixed with 12 μL of denaturing loading buffer (19 mL formamide, 0.98 gr NaOH (3% NaOH
solu
tion), 0.01 gr xylene cyanol and 0.01gr bromophenol blue). The samples were denatured by
heating at 95°C for 10 min, then immediately chilled on ice and loaded onto 8% polyacrylamide gel
(37.5:1). Gels were run at 170
-
180 V for 7
-
8 hours at 4°C. The electr
ophoresis was carried out in a
vertical unit in 1x TBE buffer (Tris 100 mM, boric acid 9 mM, EDTA 1mM). The gels were stained
with silver nitrate to observe the conformational patterns.
After revealing the single stranded conformation polymorphism (SSCP) p
atterns for this locus, from each of the ovine GDF9 variants
identified by PCR
–
SSCP, one sample was sequenced (Mahan Gene, Iran).
The raw sequence data
were edited using Bioedit 7.0 software. Multiple sequence alignments were performed with Bioedit
7.0 and
DNAMAN software to identify single nucleotide polymorphisms (SNPs) in the exon 2 of the
GDF9 gene in Kermani sheep. The nucleotide sequence of exon 2 was translated to amino acid
sequence for each particular allelic variant. The BLAST algorithm was u
sed t
o search the NCBI
GenBank
databases for comparison of the ovine GDF9 sequences with homologous sequences of
other animals to determine similarity percentage and detect the novel SNPs in the studied locus.
Population genetic parameters were obtained using G
enAlex6.41 software.
Results and discussion:
As expected, PCR amplification of the ovine
GDF9
gene for Kermani sheep
gave uniform fragment 647 bp by running on 1% agarose gel and the amplified fragment size were
consistent with the expected size and subsequently sequencing of the ovine
GDF9
amplicons
confirmed them to be 647 bp in size
(Fig 1)
.
The SSCP analysis revealed four unique banding patterns
for the second half of the exon representing different allelic va
riants
(Fig 2)
.
In the studied population,
four different genotypes and three haplotypes were observed for the second half of the exon 2 (Table
1). Frequencies of the detected genotypes and haplotypes in the studied population are provided in
Table 2.
In t
otal, in this population, genotype 2 in the second half of the exon 2 were most common
with a frequency of
0.411
.
The sequencing results were representative of the point mutations at
nucleotide positions 994 and 978 in exon 2 of the GDF9. The analysis resu
lts of the GenAlex software
in the studied position revealed the lack of Hardy
-
Weinberg equilibrium in single nucleotide
polymorphism (SNP) at position 978. The high level of Shannon index in both positions of the
identified mutations indicated that the le
vel of Biodiversity in GDF9 gene position associated with
the sample population was high.
Hanrahan et al. (2004) discovered eight variants (G1 to G8) of
GDF9
gene in Cambridge and Belclare sheep breeds using PCR
-
SSCP and sequencing. However, G8 variant
cau
sed serine to phenylalanine substitution at residue 395 which replaced an uncharged polar amino
acid with a nonpolar one at residue 77 of the mature coding region and may change the function of
GDF9
in sheep (Hanrahan et al. 2004). Nikol et al. (2009) disc
overed 4 variants (G3, G4, G5 and G6)
of
GDF9
gene in Icelandic Thoka sheep that is in agreement with the result of the present study.
The
high level of genetic variability observed in the coding region of the ovine
GDF
9 gene in this study
suggests that th
is region of the
GDF9
gene probably affects folliculogenesis and female fertility in
sheep; hence further association studies using appropriate populations are needed to identify genetic
variants that can be used as markers related to fertility.
Conclusion:
Regarding the estimated criteria and relatively high level of heterozygosity, it can be
concluded that the studied population has a relatively high polymorphism in the examined locus.
The
discovered alleles and genotypes can also be used as mar
kers in marker
-
assisted selection of sheep
for economic traits in future.
