International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2011) Bangkok Dec., 2011
PCR-RFLP in IGFBP-3 Gene and its
Association with Economic Traits in
Muzaffarnagari Sheep
Sharma B., Dass G. And Sharma D
production of this breed has not been exploited due to
inadequate information about genetic basis and the breeding
strategies. Considering the importance of Muzaffarnagari
sheep in production traits, the present research work was
conducted to detect polymorphism in IGFBP-3 gene and to
find out its association with body weight and wool production.
Abstract—Using two resource populations, differing for body
weight at 12 months of age (Resource population I) and for total
greasy fleece yield (Resource population II), PCR-RFLP studies
were carried out for Insulin like Growth Factor Binding Protein
(IGFBP3) in Muzaffarnagari sheep. Significant differences (P < 0.01)
between high and low 12 month body weight groups (43.22 ± 1.46
kg and 22.81 ± 0.99 kg, respectively); and between high and low
greasy fleece yield groups (1676.18 ± 55.81 g and 625.24 ± 44.13 g ,
respectively) showed the effectiveness of resource populations.
Monomorphic PCR-RFLP pattern of 654 bp fragment of IGFBP-3
(comprising of partial exon 2, complete intron 2 and exon 3, partial
intron 3) with HaeIII restriction enzyme was observed between high
and low 12 month body weight groups as well as between high and
low greasy fleece yield groups. Similar trend was observed for PCRRFLP of 333 bp fragment (comprising of intron 1, exon 2 and intron
2). Hence, no association could be established between Hae III PCRRFLP pattern of 654 bp as well as 333 bp fragment of IGFBP-3 gene
with either of the production traits, i.e. body weight or greasy fleece
yield.
II. MATERIALS AND METHODS
Muzaffarnagari sheep flock maintained at Central Institute
for Research on Goat for genetic improvement through
selective breeding using quantitative genetic tools were used.
Two resource populations were developed. For 1st resource
population, a total of 10 individuals (5 males and 5 females)
showing least and 10 individual showing maximum 12 month
body weight were selected. Similarly 2nd resource population
was comprised of 10 individuals yielding least total greasy
fleece yield and 10 individuals yielding maximum total greasy
fleece yield. Approximately 10 ml of blood sample of each
individual animal was collected from the jugular vein into a
sterile 10 ml vacutainer tube containing 0.5 ml of 2.7% EDTA
solution as an anticoagulant. Genomic DNA was isolated as
per the standard phenol-chloroform extraction protocol [4]
with some minor modifications. Two set of primers were used.
While set 1 primers, adopted from [5] i.e. Forward (P3: 5′CCA AGC GTG AGA CAG AAT AC-3′) and reverse (P4: 5′AGG AGG GAT AGG AGC AAG AT-3′) was expected to
amplify a 654 bp IGFBP3 gene fragment comprising of partial
exon 2, complete intron 2, complete exon 3 and partial intron
3; second set of primers, adopted from i.e. Forward (TAT
CAA TGA CCG TCA AGT CTG TG) and reverse (GTG
ATC TCT GGA TAC CCA GGC) was used to amplify a 333
bp fragment consisting of partial intron 1, complete exon 2
and partial intron 2. For amplification, 25 μl of PCR reaction
was prepared by adding 10 pM of each primer, 200 μM of
each dNTPs, 1.5 mM MgCl 2 , 1× PCR assay buffer, 50-75 ng
DNA template and 1.5 Unit Taq DNA polymerase. The
amplification programme used was comprised of initial
denaturation of 1 min at 94 °C followed by 40 cycles of
denaturation at 94 °C , annealing at 56 °C and extension at
72 °C each of 1 min each and lastly the final extension of
5 min at 72 °C. The PCR products were purified by using
Promega kit. An aliquot of 20 μl of PCR product was digested
overnight at 37 0 C with 0.5 units of HaeIII restriction
Keywords— Greasy fleece yield, IGFBP-3, monomorphic
pattern, and resource populations.
I. INTRODUCTION
I
NSULIN-like growth factor binding proteins (IGFBPs)
belong to a family of at least six homologous proteins that
bind insulin-like growth factors (IGFs) and modulate many of
their biological actions. IGFBP-3 is used as a marker for
different body functions such as growth, metabolism,
reproduction, in controlling body weight, immunity and
energy balance etc. Due to the key role of IGFBP-3 in growth
and development of animals, the IGFBP-3 gene is considered
as a candidate gene for its use as a marker for growth and
production traits. Hence the polymorphism in IGFBP3 has
been studied in different live stocks such as pig, cattle, buffalo
and goat for its association with economic traits [1], [2], [3].
Muzaffarnagari sheep is one of the tallest and heaviest mutton
breeds of India. The inherent potential for growth and wool
Sharma B. is Lecturer, Institute of Biomedical Education and Research,
Mangalayatan Univ., 33rd Milestone Aligarh-Mathura Highway, Beswan,
Aligarh (U.P.); Mob.: 9897288856; E mail: [email protected].
Dass G is Senior Scientist, Animal Genetics and Breeding Division,
C.I.R.G., Makhdoom, Farah, Mathura (U.P.).
Sharma D is Principal Scientist & HOD, Avian biotechnology, I.V.R.I.,
Izzatnagar, Bareilly.
380
International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2011) Bangkok Dec., 2011
enzyme, as per manufacturer instructions. The restriction
enzyme digested PCR products were electrophoresed in 4%
agarose gel containing ethidium bromide as staining agent in
1× TAE buffer for 8 hours at 50 Volts. The digested products
were visualized and documented under gel documentation
system. The data generated by electrophoresis of Hae III
digested product of each sample was used for estimating the
frequency of different restriction fragment patterns. For this,
gene and genotypic frequencies of alleles of IGFBP 3 gene
were estimated by standard procedure [7].
Total No. of individuals of a particular genotype
Genotypic Frequency =
Total No. of individuals of all genotypes
(2 D + H)
Gene Frequency =
2N
Where, D is number of homozygotes of particular gene, H
is number of heterozygotes having that gene; and N is Total
no. of individuals
654 bp in four different breeds of sheep including
Muzafarnagari with Hae III [8] as well as with MspI, TaqI and
Hae III [9]. Similar to the above findings recently, [10]
reported similar restriction pattern of IGFBP-3 gene in four
Egyptian local sheep breeds namely Rahmani, Ossimi, Awassi
and Barki in his PCR-RFLP studies. Similarly, [10] and [2]
reported monomorphic restriction pattern of IGFBP-3 gene
with Hae III in bovines. We have used more number of
samples as well as also expanded the region of IGFBP3 gene,
but still could not detect any polymorphism in this region,
which suggested that the IGFBP3 region, more particularly
exon 2 and exon 3 have very high sequence homology within
sheep, especially Muzaffarnagari sheep. However, the 333 bp
fragment of IGFBP3 gene was not explored for PCR-RFLP in
sheep. We have attempted for Hae III PCR-RFLP of the 333
bp fragment, but could not detect polymorphism in either of
the resource populations; hence no association could be
established between PCR-RFLP pattern of this fragment with
Hae III and body weight as well as GFY. [11] although
detected polymorphism in IGFBP-3 gene but could not find
any significant association of IGFBP-3 genotype with milk
traits in goat (P > 0.05).
In conclusion, the Hae III RFLP studies showed single
restriction pattern of IGFBP-3 gene or presence of only one
genotype in both the resource populations under study
indicating fixation of gene in Muzaffarnagari sheep
population maintained at C.I.R.G. However, the length of
amplified product of IGFBP-3 gene differed with that of
earlier reports of cattle and buffalo. Hence, no association
could be established between polymorphic pattern of IGFBP-3
gene and production traits.
III. RESULTS & DISCUSSION
st
For 1 resource population, average 12th month body
weight was 22.81 ± 0.99 kg in low body weight group and
43.22 ± 1.46 kg in high body weight group. Similarly, average
GFY was 625.24 ± 44.13 g in low GFY group and 1676.18 ±
55.81 g in high GFY group of second resource population.
Significant differences (P < 0.01) between high and low group
showed the marked divergence of the resource populations.
Both the sets of primers i.e. Set I and Set II amplified the
654 bp and 333 bp fragments, respectively in all the
individuals of both the resource populations. Typical
restriction enzyme profile of 654 bp band with Hae III
restriction enzyme includes eight restriction fragments of 56
bp, 7 bp, 201 bp, 19 bp, 16 bp, 201 bp, 67 bp and 87 bp. On
resolution of Hae III digested PCR products at 1.2 % agarose
gel, only 4 of the total 8 restriction fragments were visualized
as two fragments of 201 bp appeared as single band and other
three restriction fragments of 7 bp, 16 bp and 19 bp were not
visible due to their very small sizes. Hae III PCR RFLP of
654 bp fragment in resource population I as well as resource
population II yielded monomorphic restriction enzyme profile
(Fig.1). Only AA genotypes showing the restriction enzyme
profile of 201 bp, 87 bp, 67 bp and 56 bp were observed.
Similarly, typical restriction enzyme profile of 333 bp
with Hae III restriction enzymes including 4 restriction
fragments of 100 bp, 205 bp, 8 bp and 20 bp sizes were
observed. On resolution of Hae III digested PCR products at
1.2 % agarose gel, only 2 of the total 4 restriction fragments
were visualized as two fragments 8 bp and 20 bp were not
visible due to their very small sizes. Hae III PCR RFLP of
333 bp fragment in resource population I as well as resource
population II yielded monomorphic restriction enzyme profile
(Fig.2). Only AA genotype showing the restriction enzyme
profile of 205 bp and 100 bp were observed. Earlier workers
have also reported monomorphic restriction enzyme profile of
ACKNOWLEDGMENT
The authors are thankful to the Director, C.I.R.G.,
Makhdoom, Farah, Mathura for providing necessary facilities.
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bp
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600
500
400
300
200
100
Fig. 1 HaeIII PCR-RFLP of 654 bp IGFBP3 fragment in resource population I. Lane 1-10: Low body
weight group; Lane 11-20: High body weight group. Lane M: Trackit 100 bp ladder, Invitrogen)
bp
bp
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600
200
200
100
100
Fig. 2 HaeIII PCR-RFLP of 333 bp IGFBP3 fragment. Lane 11-20: Low body weight group; Lane 11-20: High body
weight group. Lane M: Trackit 100 bp ladder, Invitrogen)
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