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Trop Anim Health Prod (2012) 44:1587 1592 DOI 10.1007/s11250-012-0111-7



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 si tes, 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


Tanpure : P. K. Dubey : P. Kathiravan : S. K. Niranjan :


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


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

bovine leptin gene were found to be associated with growth, fertility and milk production in Holstein


Trop Anim Health Prod (2012) 44:1587 1592

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).

extraction protocol (Sambrook and Russell 2001 ). Single-strand conformation polymorphism analysis and

Single-strand conformation polymorphism analysis and sequencing

A set of oligonucleotide primers (forward: 5 -GTGGGG


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 μ l, containing 100 ng of genomic DNA, 10 pmol of

each primer, 1 μ l of 10 mM dNTPs mix, 2.5 μ l 10× buffer

with 1.5 mM MgCl 2 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 95°C for 2.5 min followed

by 35 cycles of 94°C for 30 s, 57°C for 30 s and 72°C for

1 min, with a final extension for 5 min at 72°C. 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) 44:1587 1592


to September; winter, October to February; summer, March to mid-June), year (2001 2005), parity and influence of differing management practices in the farm and field con- ditions were included in the analysis. The effect of non- genetic factors on different traits were estimated by least squares analysis of variance for non-orthogonal data using a complete fixed model as described by Harvey ( 1987 ). The data were adjusted for those non-genetic factors (herd, year, season and parity), which were statistically significant. The adjusted records were used for subsequent analysis. The effect of different genotypes on various production traits milk yield, fat percentage and fat yield at different stages of lactation (90 days, 150 days, 305 days and total)] were analyzed by least squares method using a mixed model with random effect of sire and fixed effect of genotype.

Y ijk ¼

μ þ S i þ G j þ e ijk

where Y ijk is the dependent trait under study (milk yield, fat percentage and fat yield at different stages of lactation of k th individual with j th genotype and ith sire), μ is the overall population mean, S i is the random effect of the i th sire, G j is the fixed effect of the jth genotype, and e ijk is the random error, assumed to be normally independently distributed with mean zero and constant variance, i.e., NID (0, σ e 2 ).

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

three variants in Mehsana, Jaffarabadi, Murrah and Toda Fig. 1 Non-denaturing polyacrylamide gel electrophoresis,

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)

Table 1 Frequency of different SSCP variants in leptin intron 1 fragment in different breeds of buffaloes


Variants (no. of observations)



















Marathwada 14































































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 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

given in Table 3 . The mean total milk yield was found to be maxi- mum


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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

C) at leptin intron 1 locus of buffalo, compared with cattle (2,100.97 kg) and A (2,064.29

(2,100.97 kg) and A (2,064.29 kg). The 305 days milk yield also showed a similar trend without significant variation. In contrast, 150 days milk yield and 90 days milk yield were highest for variant C followed by variants B and A. However, the association of these traits with different SSCP variants was found to be non-significant ( P >0.05). Madeja et al. ( 2004 ) also reported non-significant associa- tion between Kpn 2I (exon 2) and Sau 3AI (intron 2)

Table 2 Nucleotide changes observed at five nucleotide positions in different SSCP variants of bubaline leptin intron 1 region, compared with cattle


B. taurus

SSCP Variants in intron 1 of Bubaline leptin gene



Variant A

Variant B

Variant C


























polymorphisms with production traits in Holstein Friesian cattle. However, our findings were inconsistent to some of the previous reports about association between leptin gene polymorphisms and milk yield in cattle (Veerkamp et al. 2000 ; Liefers and Te Pas 2002 ; Clempson et al. 2011 ). Buchanan et al. ( 2003 ) reported a significant association between SNP in exon 2 of leptin gene and milk yield in cattle during first 100 days of lactation. Liefers and Te Pas ( 2002 ) did not find any effect of the Hph I polymorphism in cattle, although they reported Sau 3AI as a possible marker for milk and protein yield. The contradictory results of the effect of leptin gene polymorphisms on milk production may reflect the presence of population specific haplotypes and/or other unknown QTL affecting milk production. Most of the milk quality traits, like total fat yield and fat percentage, also showed non-significant association with SSCP variants. Total fat and 305 days fat percentages were found highest in variant B followed by variants A and C, respectively. Similarly, total fat yield was highest in variant B (142.23 kg), followed by variant C (138.12 kg) and A (131.57 kg), respectively. Similar trend was observed with respect to 305 days fat yield in different SSCP variants.

and A (131.57 kg), respectively. Similar trend was observed with respect to 305 days fat yield

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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


SSCP variants

P value



Lactation length





Total milk yield





305 days milk yield





150 days milk yield





90 day milk yield





Total fat percentage 6.31±0.10




Total fat yield





305 days fat percentage





305 days fat yield





150 days fat percentage





150 days fat yield





90 days fat percentage





90 days fat yield





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 Holstein Friesian cattle. However, Madeja et al. ( 2004 ) reported significant association between Hph I polymor- phisms in exon 3 and fat yield in Holstein Friesian 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 clos e 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 M r. Naresh Yadav is gratefully acknowledged.


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