DNA polymorphism of leptin gene in Bos indicus and Bos taurus cattle

Leptin plays an important role in the regulation of feed intake, energy metabolism, growth and reproduction of cattle. We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique to screen for DNA polymorphisms of the leptin gene in 403 cattle belonging to various breeds of Bos indicus (Hariana, Sahiwal, Gir and Nimari cattle), Bos taurus (Holstein Friesian (HF) and Jersey cattle) as well as Bos taurus x Bos indicus crossbreds (1⁄2 HF x 1⁄2 Hariana). In all the cattle we amplified two regions of the leptin gene, a 522 bp fragment comprising the partial intron 2 and exon 3 and another 94 bp fragment consisting of part of exon 2. Digestion of 522 bp PCR products with the BsaAI restriction enzyme revealed three genotypes in all the breeds of cattle studied. This is the first report of the presence of leptin gene polymorphism in purebred Bos indicus cattle of Indian origin (indicine cattle). Almost similar gene and genotype frequencies were observed in all the breed groups, while the frequency of mutant homozygotes (AA) was very low (0.03 to 0.07). On digestion of the 94 bp fragment with the Kpn2I restriction enzyme, three genotypes were observed in HF, Jersey and crossbred cattle. The CC genotype had the highest frequency (0.68) in crossbreds whereas the frequency of CT heterozygotes was highest (0.69) in HF cattle. This mutation was absent in all the breeds of indicine cattle. The results suggest that the BsaAI-RFLP mutation has occurred far back in evolution before the divergence of taurine and indicine cattle while the Kpn2I mutation has occurred recently as indicated by the fact that this mutation was only detected in taurine cattle.

Variations at DNA level contribute to the genetic characterization of livestock populations and this may help to identify possible hybridization events as well as past evolutionary trends.Variation in the exonic region of a gene may lead to changes in amino acids which alter the expressed protein, and although intronic variation does not change the amino acid sequence of the protein it may play a significant role in gene splicing or the binding of regulatory proteins during transcription.In livestock, such variations in DNA may also be associated with, or linked to, economic traits, which are governed by many genes each having a small effect (Gelderman, 1997).However, the major gene model suggests that only a few genes may account for relatively large proportion of the genetic variation (Lande, 1981), such major genes being the genes usually involved in the biology of a trait and are the candidate genes for marker identification.There is also the possibility that major genes may be linked with some quantitative trait loci (QTL) contributing to a major part of the variation in traits.Leptin is a protein involved intricately in the growth and metabolism of animals and which plays an important role in the regulation of feed intake, energy metabolism, growth and reproduction of cattle (Ramsay and Cranwell, 1999) and thus the leptin gene is a potential candidate gene for QTL studies.Although many polymorphic studies on the bovine leptin gene have been reported (Pomp et al., 1997;Lien et al., 1997;Wilkins and Davey, 1997;Konfortov et al. 1999;Haegeman et al., 2000;Buchanan et al., 2002), to our knowledge there has been no reported polymorphic study involving cattle originating in the Indian subcontinent (indicine cattle, Bos indicus).The objective of the present investigation was to study genetic variations in the leptin gene of the Bos indicus, Bos taurus and their crossbred cattle.From each animal, about 15 mL of venous blood was collected from the jugular vein and placed into a sterile 15 mL polypropylene centrifuge tube containing 0.5 mL of 2.7% EDTA solution as an anticoagulant and transported in an icebox to the laboratory and stored at 4 °C until needed for DNA extraction.Genomic DNA was isolated by phenol-chloroform extraction method (Sambrook and Russell, 2001) and the purity assessed by spectrophotometry; samples showing an optical density (OD) ratio (260 nm/ 280 nm) of between 1.7 and 1.9 being used for further analysis while samples outside this range were reprocessed.From the purified genomic DNA, a 522 bp region spanning over a part of intron 2 and exon 3 and a 94 bp region of exon 2 of the leptin gene was amplified using two different sets of primers (Table 1).PCR was carried out for both fragments in a final volume of 25 mL containing 100 mM dNTPs mix, 10 pmol of each primer, 2.5 mL of 10x PCR assay buffer containing 1.5 mM MgCl 2 , 1.0 unit of Taq DNA polymerase and 80-100 ng of the purified bovine genomic DNA.The amplification of the 522 bp region was carried out using a thermal cycler (PTC-200, M J Research) pre-programmed for the following conditions: initial denaturation for 5 min at 94 °C followed by 35 cycles (denaturation at 94 °C for 15 s, annealing at 64 °C for 30 s and extension at 72 °C for 1 min) and a final extension at 72 °C for 5 min.The amplification program for the 94 bp fragment consisted of initial denaturation at 94 °C for 5 min, followed by 35 cycles (denaturation at 94 °C for 30 s, annealing at 54 °C for 45 s and extension at 72 °C for 30 s) and a final extension at 72 °C for 5 min.The PCR products were separated by 2% (w/v) agarose gel electrophoresis at 6 V cm -1 for one hour.A 20 mL aliquot of the 522 bp PCR product was digested with 5 Units of BsaAI restriction enzyme and 15 mL of 94 bp PCR product was digested with 5 Units of Kpn2I restriction enzyme at 37 °C overnight in a water bath, the reaction being stopped by adding 6x loading dye (bromophenol blue 0.25% (w/v) in 40% (w/v) sucrose) to the digested product.The digested PCR products were subjected to 3.5% (w/v) agarose gel electrophoreses and stained with ethidium bromide, the banding being visualized and documented using a gel documentation system.
The gene and genotype frequencies were calculated according to the method of Falconer and Mackay (1996).
For the 522 bp PCR leptin fragment we found three BsaAI digestion patterns in all the breed groups (B.indicus, B. taurus and crossbreds) indicating three genotypes; an intact 522 bp fragment as AA genotype; 441 and 81 bp fragments as GG genotype; and 522, 441 and 81 bp fragments as GA genotype.Our results agree with those of Lien et al. (1997) who for the first time described a guanine (G) to adenine (A) substitution (G ® A substitution) in intron 2 of the leptin gene of Norwegian cattle.Although Konfortov et al. (1999) have reported that some of the B. indicus crossbred cattle have leptin gene polymorphisms that are not found in B. taurus breeds, our findings are the first report of leptin gene polymorphisms in purebred B. indicus cattle.Taken together, the present results suggest that leptin gene mutation might have occurred far back in evolution before the divergence of cattle into taurine (B.taurus) and indicine (B.indicus) cattle.Buchanan et al. (2002) described a cytosine (C) to thymine (T) substitution (C ® T substitution) in intron 2 of the leptin gene of the B. taurus breeds i.e.Angus, Charolais, Hereford and Simmental, suggesting the existence of C and T alleles and therefore CC, TT and CT genotypes.In our work involving the 94 bp leptin fragment we identified three Kpn2I digestion patterns in the Bos taurus and crossbreds indicating three genotypes: an intact 94 bp fragment as TT genotype; 75 and 19 bp fragments as CC genotype; and 94, 75 and 19 bp fragments as CT genotype.Konfortov et al. (1999) also reported this mutation in both B. taurus (Limousin, Charolais, Charolais x Friesian, Simmental x Friesian, Limousin x Friesian, Charolais x Hereford/Friesian, Jersey, Simmental breeds) and its crossbreds with Bos indicus.In our study, the Hariana, Sahiwal, Gir and Nimari breeds of B. indicus cattle showed only the 75 and 19 bp (CC genotype) restriction pattern, indicating absence of the C ® T mutation.We also know that the TT polymorphism observed in our Holstein Friesian x Hariana crossbred population was inherited from Holstein Friesian sires.This C ® T mutation may be used as a marker to differentiate the two subspecies of cattle along with many other markers.
The genotype frequencies of the BsaAI-RFLP genotypes are presented in Table 2, from which it can be seen that the AA homozygote genotype frequency was very low (0.03 to 0.07) in all the indicine and exotic cattle populations.Of all the breeds, the Holstein Friesian population showed the highest GG homozygote genotype frequency It can be seen that in all the populations the frequency of the A allele was low (0.18 to 0.34), suggesting that there may be a selection force acting against the A allele or favoring the G allele in these populations.
In case of the Kpn2I-RFLP, the CC, CT and TT genotype frequency was 0.68CC, 0.27CT and 0.05TT in the crossbreds, 0.25CC, 0.69CT and 0.06TT in the Holstein Friesian cattle and 0.18CC, 0.52CT and 0.30TT in Jersey cattle.The C and T allele frequency was 0.82C and 0.18T for crossbreds, 0.60C and 0.40T for Holstein Friesian cattle and 0.44C and 0.56T in Jersey cattle.The TT genotype was present at very low frequency in crossbred and Holstein Friesian cattle but was comparatively higher in Jersey cattle.The T allele frequency was comparatively low in crossbred population as compared to the two pure breeds of taurine cattle, possibly due to the absence of this mutation in Hariana cattle which contributed only the C allele to the crossbred population.Our findings for taurine cattle are similar to those of Buchanan et al. (2002) who reported T allele frequencies of 0.58 in Angus cattle, 0.34 in Charolais cattle, 0.55 in Hereford cattle and 0.32 in Simmental cattle (all taurine cattle) and Konfortov et al. (1999) who found the T allele frequency to be 0.41 in taurine cattle.In all the four breeds of indicine cattle, the C allele frequency was 1.0, possibly due to the absence of the V allele (valine) in these populations.
Summarizing, we found that the 522 bp leptin gene fragment is polymorphic in all the breeds studied, while the 94 bp leptin gene was polymorphic only in taurine cattle and B. taurus x B. indicus crossbreds.In all the polymor-phic breeds the frequencies of mutant homozygotes were very low for both the fragments.The results also suggested that the BsaAI-RFLP mutation has occurred far back in evolution before the divergence of taurine and indicine cattle while the Kpn2I mutation has occurred recently as indicated by the fact that it was only detected in taurine cattle.
Hariana crossbred cattle maintained at Cattle and Buffalo Farm, Indian Veterinary Research Institute, Izatnagar.Hariana, Sahiwal, Gir and Nimari cattle are Bos indicus (indicine) breeds while Holstein Friesian and Jersey cattle are Bos taurus (taurine) breeds.

Table 1 -
Primer sequences used to amplify fragments of the leptin gene.