Trop Anim Health Prod (2012) 44:15871592

DOI 10.1007/s11250-012-0111-7

ORIGINAL RESEARCH

PCR-SSCP analysis of leptin gene and its association

with milk production traits in river buffalo (Bubalus bubalis)
Tushar Tanpure & Praveen Kumar Dubey &
Krishan Pal Singh & Periasamy Kathiravan &
Bishnu Prasad Mishra & Saket Kumar Niranjan &
Ranjit Singh Kataria

Accepted: 14 February 2012 / Published online: 7 March 2012

# Springer Science+Business Media B.V. 2012

Abstract Leptin gene has been found to be associated with
various economic traits including milk production and fat
quality in dairy animals. In the present study, we investigated
genetic variations in intron 1 region of leptin gene in riverine
buffaloes (Bubalus bubalis) using polymerase chain reaction-
single strand conformation polymorphism (PCR-SSCP) and
sequencing methods and associated them with milk traits. The
study revealed three SSCP variants A, B and C among a total
of 301 buffaloes from nine breeds. The frequency of variant C
was found invariably high among all the breeds except in
Marathwada buffalo. Variant A was found to be absent in
Chilika, Nili-Ravi, Nagpuri and Pandharpuri breeds and also
had the lowest frequencies in Mehsana, Jaffarabadi, Murrah
and Toda breeds. Sequencing of SSCP variants revealed a
total of five polymorphic sites, with three haplotypes.
Statistical analysis revealed significantly high fat percentage
at 150 days in SSCP variant B in Mehsana buffaloes.
However, the associations of SSCP variants of leptin gene
T. Tanpure : P. K. Dubey : P. Kathiravan : S. K. Niranjan :
R. S. Kataria (*)
Buffalo Genomics Laboratory, DNA Fingerprinting Unit,
National Bureau of Animal Genetic Resources,
P.B. No. 129, GT Road Bypass,
Karnal, Haryana 132001, India
e-mail: katariaranji@yahoo.co.in
K. P. Singh
Buffalo Breeding and Genetics,
Central Institute for Research on Buffaloes,
Sirsa Road,
Hisar, Haryana 125 001, India
e-mail: rishikps@yahoo.com
B. P. Mishra
Veterinary Biotechnology,
Indian Veterinary Research Institute,
Izatnagar, U P 243 122, India
e-mail: bpmishra_1@hotmail.com
with total milk yield, 305 days milk yield and total fat yield
were found to be non-significant. The present study is the first
report on association analysis of leptin gene polymorphisms
with milk production and milk quality traits in river buffalo.
Keywords Buffalo . Leptin . SSCP . SNPs . Association .
Milk traits
Introduction
Leptin, the fat derived protein hormone has a variety of
physiological functions like regulation of feed intake, ener-
gy balance, fertility and immune functions. Leptin down-
regulates adipose tissue deposition and has been found to be
negatively correlated with the amount of non-esterified fatty
acids (Liefers et al. 2003) in cattle. It also modulates appe-
tite, energy expenditure and reproductive axis by signaling
the status of body energy stores to brain via leptin receptors
(Giblin et al. 2010). In mammary gland, leptin plays an
important role in lactogenesis (Feuermann et al. 2004) and
its receptors have been found to be expressed in mammary
epithelial cells (Silva et al. 2002). Polymorphisms within
bovine leptin gene and their association with various traits
of economic importance have been reported in dairy as well
as beef cattle. The region adjoining exon 2 of leptin gene is
highly polymorphic in cattle with significant association to
milk yield and is considered as a potential quantitative trait
locus (Buchanan et al. 2003). Several leptin gene polymor-
phisms were found to be significantly associated with the
energetically expensive process of lactogenesis, especially
traits related to energy output and energy storage in dairy
cattle (Giblin et al. 2010). Genetic polymorphisms within
bovine leptin gene were found to be associated with growth,
fertility and milk production in Holstein cows (Clempson et
1588
al. 2011). Leptin genotypes were also found to be signifi-
cantly associated with increased prevalence of estrous cyclic
cows (Chebel and Santos 2011) and perinatal mortality
(Brickell et al. 2010) in Holstein cows and Holstein
Friesian heifers, respectively. Recently, LEP-R25C poly-
morphism has been shown to have significant association
with longevity (Szyda et al. 2011) and juvenile body meas-
urements (Clempson et al. 2010) in dairy cattle. In case of
beef cattle, traits such as body fatness (Buchanan et al. 2002;
Nkrumah et al. 2005) and drip loss (Pinto et al. 2011) were
found to be associated with leptin gene variations.
Buffalo is considered as a major dairy animal in South
Asian countries and also contributes towards draught power
as well as meat production. This species is well known for
high fat percentage in its milk; better quality of meat, low in
cholesterol and adaptability to tropical climate in Indian
sub-continent. With the initiatives taken on complete ge-
nome sequencing in India (Tantia et al. 2011), western
countries have also started showing interest in this species
now. However, the genetic potential of this livestock species
is yet to be exploited fully as there are only few reports on
characterization of candidate genes governing milk produc-
tion and quality traits in buffalo (Venkatachalapathy et al.
2008; Matteis et al. 2009). Leptin is one of the important
candidate genes whose variations need to be studied in
buffalo to understand their significant association with traits
related to milk production and milk composition. Earlier, the
leptin gene has been characterized and mapped to chromo-
some 8 in buffalo (Vallinoto et al. 2004). However, very
limited research on the identification of polymorphism in
bubaline leptin gene has been reported. Therefore, the pres-
ent study was undertaken with the objectives of detecting
the genetic effect of bubaline leptin gene polymorphism on
milk production and composition traits and to further ex-
plore the possibilities of leptin gene being used as candidate
gene marker in buffalo.
Materials and methods
Sampling and DNA extraction
A total of 301 genetically unrelated buffaloes belonging to nine
different breeds were selected from their native breeding tracts
dispersed in different agro-climatic regions of the country. The
breeds included were Mehsana (154), Marathwada (30),
Chilika (24), Jaffarabadi (19), Murrah (15), Nili-Ravi (15),
Toda (15), Pandharpuri (15) and Nagpuri (14). Genomic
DNA was isolated from blood samples collected from the
animals by using standard SDS-proteinaseK treatment fol-
lowed by phenolchloroform extraction protocol (Sambrook
and Russell 2001).
Trop Anim Health Prod (2012) 44:15871592
Single-strand conformation polymorphism analysis
and sequencing
A set of oligonucleotide primers (forward: 5-GTGGGG
GATACAGGGGGAGTTT-3 and reverse: 5-CCACG
TCTTCAGATGCGGATAA-3) were designed from the cat-
tle leptin gene sequence (Acc No. AJ512638) to amplify
290 base pair sequence of leptin intron 1, using PrimerSelect
Program of Lasergene software (DNASTAR Inc., Madison,
WI, USA). PCR was carried out in a final reaction volume
of 25 l, containing 100 ng of genomic DNA, 10 pmol of
each primer, 1 l of 10 mM dNTPs mix, 2.5 l 10 buffer
with 1.5 mM MgCl2 final concentration and 1 unit of Taq
DNA polymerase. Amplification was carried out using a
programmable thermal cycler (PTC-200, MJ Research,
USA) with initial denaturation at 95C for 2.5 min followed
by 35 cycles of 94C for 30 s, 57C for 30 s and 72C for
1 min, with a final extension for 5 min at 72C. Amplified
PCR products were resolved by single-strand conformation
polymorphism (SSCP) analysis after optimization of non-
denaturing PAGE conditions. The electrophoresis was car-
ried out in 8% PAGE gel by using Protean II xi vertical
electrophoresis unit (Bio-Rad, USA) using 1 TBE buffer.
Gels were silver stained (Sambrook and Russell 2001) and
dried by using cellophane sheet and scored manually for
SSCP variants recording. The SSCP variants were further
sequenced for the identification of single nucleotide poly-
morphisms within intron 1 region of leptin gene. PCR
products from three representative samples of each of the
identified SSCP variants were purified and sequenced using
both forward and reverse primers with the help of BigDye
Terminator Cycle Sequencing Kit, on an automated DNA
sequencer ABI 3100 (Applied Biosystems, USA). Obtained
sequences were edited using Chromas (ver.1.45, http://
www.technelysium.com.au/chromas.html) and submitted to
NCBI, GenBank (Acc. No. JN408505-7). Multiple se-
quence alignment of the buffalo sequences along with cattle
sequence was carried out using MegAlign program of
Lasergene software (DNASTAR, Inc., Madison, WI, USA).
Statistical analysis
Data related to different performance traits of 154 animals
belonging to the Mehsana breed of buffalo were collected
and classified according to different seasons, years and herd
to estimate the effect of different non-genetic factors on
the traits. Among these, 37 samples were collected
from Mehsana buffaloes maintained at Livestock Research
Station, Sardarkrushinagar Dantewada Agricultural
University, Sardarkrushinagar, Gujarat, and 117 samples
were collected from Mehsana buffaloes under field progeny
testing program of Banas Dairy, Palanpur, Gujarat. Effects
of different non-genetic factors like season (rainy, mid-June
Trop Anim Health Prod (2012) 44:15871592 1589

to September; winter, October to February; summer, March Table 1 Frequency of different SSCP variants in leptin intron 1
fragment in different breeds of buffaloes
to mid-June), year (20012005), parity and influence of
differing management practices in the farm and field con- Breeds Variants (no. of Frequency
ditions were included in the analysis. The effect of non- observations)
genetic factors on different traits were estimated by least
A B C Total A B C
squares analysis of variance for non-orthogonal data using a
complete fixed model as described by Harvey (1987). The Mehsana 15 57 82 154 0.097 0.370 0.532
data were adjusted for those non-genetic factors (herd, year, Marathwada 14 3 13 30 0.467 0.100 0.433
season and parity), which were statistically significant. The Chilika 1 23 24 0.042 0.958
adjusted records were used for subsequent analysis. The Murrah 2 1 12 15 0.133 0.067 0.800
effect of different genotypes on various production traits Nili-Ravi 3 12 15 0.200 0.800
milk yield, fat percentage and fat yield at different stages Jaffarabadi 2 4 13 19 0.105 0.211 0.684
of lactation (90 days, 150 days, 305 days and total)] were Nagpuri 2 12 14 0.143 0.857
analyzed by least squares method using a mixed model with Toda 2 3 10 15 0.133 0.200 0.667
random effect of sire and fixed effect of genotype. Pandharpuri 1 14 15 0.067 0.933
Y ijk S i Gj eijk Overall 35 75 191 301 0.116 0.249 0.635

where Yijk is the dependent trait under study (milk yield, fat
percentage and fat yield at different stages of lactation of kth
individual with jth genotype and ith sire), is the overall
population mean, Si is the random effect of the ith sire, Gj is
the fixed effect of the jth genotype, and eijk is the random
error, assumed to be normally independently distributed
with mean zero and constant variance, i.e., NID (0, e2).
Results and discussion
SSCP variants and sequence analysis
SSCP analysis revealed three variants, viz. A, B and C, in all
nine breeds of buffaloes (Fig. 1). The overall frequency of
the variant C was highest (0.635), followed by variants B
(0.249) and A (0.116). The frequency of variant C was
invariably high in all of the breeds under study except
Marathwada, in which variant A was highest (Table 1).
The frequency of variant C ranged from 0.433 to 0.958
among the different breeds with highest in Chilika buffalo.
Variant A was found to be absent in Chilika, Nili-Ravi,
Nagpuri and Pandharpuri and had lowest frequency among
three variants in Mehsana, Jaffarabadi, Murrah and Toda
SSCP single-strand conformation polymorphism
breeds. The frequency of variant A varied between 0.105
(Jaffarabadi) and 0.467 (Marathwada). The frequency of
variant B varied between 0.042 (Chilika) and 0.211
(Jaffarabadi). Variant A was found only in Marathwada,
Murrah, Jaffarabadi and Toda buffaloes. Wide variability
was found among various breeds with respect to the fre-
quency of different SSCP variants. Almost similar number
of SSCP variants was also reported in Sahiwal cattle (Dubey
et al. 2008). However, predominantly high frequency of var-
iant C across the breeds indicates the low variability of leptin
gene in buffaloes. Representative samples corresponding to
different SSCP variants of intron 1 region of bubaline leptin
gene were sequenced and analyzed. The buffalo sequences
were one nucleotide longer (291 bp long) due to insertion of G
at 188 position compared to cattle (Fig. 2). Bubaline leptin
gene revealed a total of nine changes compared to cattle,
which included three CT transitions and one CA transver-
sion. Sequence comparison of bubaline leptin gene
corresponding to three identified SSCP variants revealed a
total of five polymorphic nucleotide sites at 98, 111, 172, 209,
266 nucleotide positions (Table 2). Three haplotypes analo-
gous to SSCP variants were constructed based on these five
identified SNPs. Several SNPs have been previously reported
in leptin gene of cattle (Yoon et al. 2005). The distribution of
SNPs was found to be higher (1SNP/58 bp) in buffalo than in
cattle (Konfortov et al. 1999), which is understandable since
intronic region was the target in the present study.

Association of SSCP variants with milk traits

Least squares mean (LSM) for milk traits related to different

Fig. 1 Non-denaturing polyacrylamide gel electrophoresis, showing
different single-strand conformation polymorphism (SSCP) variants at
leptin intron 1 locus (samples from Toda buffalo: lane 1 variant A,
lanes 24 variant C, Lane 5 variant B, lanes 6 and 7 variant C)
SSCP variants obtained by statistical analysis are given in
Table 3. The mean total milk yield was found to be maxi-
mum for variant B (2,131.21 kg) followed by variant C
1590 Trop Anim Health Prod (2012) 44:15871592

Fig. 2 Multiple alignment of

nucleotide sequences of
different SSCP variants (variant
A, variant B and variant C) at
leptin intron 1 locus of buffalo,
compared with cattle

(2,100.97 kg) and A (2,064.29 kg). The 305 days milk yield polymorphisms with production traits in Holstein Friesian
also showed a similar trend without significant variation. In cattle. However, our findings were inconsistent to some of
contrast, 150 days milk yield and 90 days milk yield were the previous reports about association between leptin gene
highest for variant C followed by variants B and A. polymorphisms and milk yield in cattle (Veerkamp et al.
However, the association of these traits with different 2000; Liefers and Te Pas 2002; Clempson et al. 2011).
SSCP variants was found to be non-significant (P>0.05). Buchanan et al. (2003) reported a significant association
Madeja et al. (2004) also reported non-significant associa- between SNP in exon 2 of leptin gene and milk yield in
tion between Kpn2I (exon 2) and Sau3AI (intron 2) cattle during first 100 days of lactation. Liefers and Te Pas
(2002) did not find any effect of the HphI polymorphism in
Table 2 Nucleotide changes observed at five nucleotide positions in cattle, although they reported Sau3AI as a possible marker
different SSCP variants of bubaline leptin intron 1 region, compared for milk and protein yield. The contradictory results of the
with cattle
effect of leptin gene polymorphisms on milk production
Nucleotide B. taurus SSCP Variants in intron may reflect the presence of population specific haplotypes
position 1 of Bubaline leptin gene and/or other unknown QTL affecting milk production.
Most of the milk quality traits, like total fat yield and fat
Variant A Variant B Variant C
percentage, also showed non-significant association with
98 C C C T SSCP variants. Total fat and 305 days fat percentages were
111 G A G G found highest in variant B followed by variants A and C,
172 G A G G respectively. Similarly, total fat yield was highest in variant
209 T T T/C A B (142.23 kg), followed by variant C (138.12 kg) and A
266 G G A A
(131.57 kg), respectively. Similar trend was observed with
respect to 305 days fat yield in different SSCP variants.
Trop Anim Health Prod (2012) 44:15871592 1591

Table 3 Least squares mean (SE) of different production traits for three SSCP patterns of buffalo leptin gene. Lactation length given is in days;
milk yield and fat yield in Kg

Traits SSCP variants P value

A B C

Lactation length 282.249.56 281.735.36 277.235.25 0.674

Total milk yield 2,064.29109.16 2,131.2161.18 2,100.9759.94 0.797
305 days milk yield 2,058.46107.12 2,147.5960.04 2,100.2458.82 0.621
150 days milk yield 1,324.0286.65 1,433.7451.19 1,444.1448.81 0.366
90 day milk yield 819.2655.34 856.3033.49 867.9831.76 0.644
Total fat percentage 6.310.10 6.360.06 6.240.02 0.173
Total fat yield 131.579.29 142.235.91 138.125.54 0.473
305 days fat percentage 6.310.10 6.350.06 6.240.06 0.163
305 days fat yield 134.099.41 145.185.99 140.335.61 0.430
150 days fat percentage 6.130.11* 6.210.06* 6.060.06* 0.044*
150 days fat yield 81.665.67 89.083.35 87.903.19 0.434
90 days fat percentage 6.070.11 6.120.06 6.000.06 0.134
90 days fat yield 50.083.59 52.552.17 52.352.06 0.779

Variants showing significant effect on 150 days fat percentage (*P<0.05)

Non-significant association between different SSCP variants
and total fat percentage in our study was in agreement with
the findings of Liefers and Te Pas (2002), who reported
similar non-significant polymorphisms in intron 2 and exon
3 of leptin gene with fat percentage and fat yield in
HolsteinFriesian cattle. However, Madeja et al. (2004)
reported significant association between HphI polymor-
phisms in exon 3 and fat yield in HolsteinFriesian cattle.
Only significant (P<0.05) association of SSCP variants
observed in this study was with 150 days fat percentage. It
was highest for variant B (6.21%) and lowest in variant C
(6.06%). Least square means of 150 days fat percentage for
variant A was 6.13%. Similar differences but statistically
non-significant values were observed for 90 days fat per-
centage. In contrast, no such type of association was
reported between leptin genotype and fat percentage in early
or mid-lactation by Buchanan et al. (2003) in cattle. Fat
yield at 150 and 90 days had not shown any significant
difference for different SSCP variants. Thus our study indi-
cates that the polymorphisms observed in intron 1 of leptin
gene have no significant effect on different traits related to
milk yield, fat percentage and fat yield, except for 150 days
fat percentage. Lindersson et al. (1998) reported QTLs for
milk production traits close to the leptin gene and also
found QTLs for milk, fat and protein yield, and fat and
protein percentage. Although, SNPs in intronic regions
have apparently less importance, there may be other
factors, such as species, breed and population differen-
ces, and small number of animals in certain SSCP var-
iants, being responsible for non-significant association
between the genotype and traits.
In conclusion, the present study reports identification of
three SSCP variants within intron 1 region of bubaline leptin
gene. Sequencing of identified SSCP variants revealed five
single nucleotide polymorphic sites which corresponded to
three haplotypes. Association study of intron 1 leptin var-
iants revealed no significant association with most of the
milk production and quality traits although there were con-
siderable differences among the least squares means of
different SSCP variants. However, significant association
was observed between SSCP variants and 150 days milk
fat percentage in Mehsana buffalo.
Acknowledgements The authors wish to thank the director of the
National Bureau of Animal Genetic Resources, Karnal, India, for
providing the financial support that allowed to carry out this work.
Technical support received from Mr. Naresh Yadav is gratefully
acknowledged.
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