PCR- RFLP Polymorphism at Exon 3 and Promoter Region of Prolactin Gene in Gaolao Cattle

DNA markers of category candidate genes have been largely used in livestock species to dissect the complexity of quantitative trait variation. Candidate genes have been usually selected according to their function based on the target traits. A quite large list of candidate gene markers has been already investigated in dairy cattle to identify polymorphisms associated with milk production and quality traits (Ogorevc et al., 2009). Bovine PRL consists of 199 amino acids (Wallis, 1974) and is associated with the initiation and maintenance of lactation. The hormone acts on its target tissue (mammary alveoli) and is primarily responsible for the synthesis of milk proteins, lactose and lipids, all major components of milk (Leprovost et al., 1994). Bovine Prolactin (PRL) gene is mapped to chromosome 23 (Barendse et al., 1997) and consists of five exons (Camper et al., 1984). A silent A-G transition mutation at the nucleotide position 103 in exon 3 of bovine PRL gene introduces a polymorphic RsaI site (Lewin et al., 1992; Thuy et al., 2018; Karuthadurai et al., 2019). Chung et al., (1996) described that the PRL- RsaI locus had a significant effect on milk yield and fat % in dairy cattle. Dybus (2002) reported that the cows of AA genotype of the PRL gene had higher milk protein content than AB individuals.
       
Gaolao is an indigenous cattle breed from Vidarbha region of Maharashtra currently reared for fast transport and milk production. The Gaolao cattle are resistant to diseases, heat tolerance and are able to sustain in adverse ecological conditions with sustainable milking potential. The PCR RFLP polymorphism in the candidate gene and its association with various milk components facilitates identification of superior genotypes for the trait. Hence the present study was under taken with the aim of elucidating polymorphism through PCR RFLP in PRL-RsaI and PRLGIII-Tsp45I loci and to correlate milk component traits in Gaolao cows. This type of strategy may help in revealing marker for productivity which can be exploited in implementing future marker/gene assisted selection, conservation and genetic improvement strategies of indigenous Gaolao cattle.

Experimental animals
 
 50 purebred Gaolao cows from various sources viz. Cattle Breeding Farm, Hetikundi and farmer’s herds of Arvi, Karanja villages and Wardha tehsils of Wardha district were used for polymorphism studies.
 
Blood collection and milk components data
 
08-10 ml of blood was collected aseptically from 50 Gaolao cows in centrifuge tubes containing 2.5 ml EDTA (Merck Specialties Pvt. Ltd.) for DNA isolation. 20 ml of milk collected from the 50 purebred Gaolao cows in centrifuge tubes for estimation of various milk components (Fat%, Protein%, SNF% and Lactose %) through milk analyzer (Citizon instrument).
 
DNA isolation, quality and quantity check
 
High quality genomic DNA was isolated through standard Phenol, chloroform-isoamyl alcohol extraction protocol (Sambrook and Russel, 2001) with some modifications. After assessing the quality and quantity of DNA through nano drop (Eppendorf) and agarose gel electrophoresis the DNA (60 ng/µl) was used as template to amplify genes by PCR.
 
Amplification of prolactin gene through PCR
 
Amplification of exon 3 and part of promoter region of Prolactin gene was done with two sets of specific primers (Table 1)  available  in literature ( Chung et al., 1996 and  Lu et al., 2010) viz PRLG-I and PRLG-III primers in thermal cycler (SimpliAmp-Applied Biosystems and Eppendorf). The  PCR was carried out with 200ul PCR tube  with  25 µl reaction mix consisted of 5 pM/µl and10 pM/µl of each primer, 1.5 to 2.5 µl of 1X PCR buffer (Sigma-Aldrich),1 µl quantity (60 ng/µl) of template DNA and Molecular biology (Himedia) grade water.
 

 
Thermal cycling conditions
 
Each primer pair was judiciously optimized for number of variables such as concentration of DNA, Taq polymerase, dNTPs, MgCl₂ and temperature profiles. Cyclic conditions used for amplification were an initial denaturation at 95°C for 5 minutes followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 59°C for 40 seconds, extension at 72°C for 20 seconds and final extension at 72°C for 3 minutes. The amplified products were confirmed through electrophoresis on 2.5% agarose gel and visualised in GelDoc system (Bio-Era, Syngene). 
 
Restriction digestion and analysis
 
The PCR products of (0.1-0.5 μg) prolactin gene were digested with 5 units of RsaI (PRLG-I) and 2 units of Tsp45I (PRLG-III, Fermentas) at 37°C temperature for 12 to 15 hours without inactivation at 65°C for 1 hours. The total volume (20-22 μl) of restriction digested PCR products were loaded for gel electrophoresis on 3.5 to 2.5% agarose gel along with 100 bp DNA ladder and electrophoresed for 30 and 120 minutes at 50-70 Volts on TAE/TBE buffer system and band patterns resolved were observed under GelDoc system.
 
Statistical analysis
 
Genotype and allele frequencies were estimated using POPGENE Ver­sion 1.31 (Yeh and Boyle, 1997). The obtained gene and genotype frequencies were tested for deviation from Hardy-Weinberg equilibrium (HWE) by using a Chi-square test (Devlin and Risch, 1995; Nielsen et al., 1998). To test the associations between each PRL genotypes and estimated milk components were quanti­fied using Chi Square test for goodness of fit. The significant effect of genotypes on milk components was analyzed using one way Analysis of variance by Least Square method.

 
                                Y_ijkl= μ+F_i+P_j+S_k+L_l + e_ijkl

 
Where,
Y_ijkl - Genotypes on milk components traits;
μ - General mean effect of milk components;
F_i - Fixed effect of the i^thFat % on genotypes;
P_j - Fixed effect of Protein %,
S_k - Fixed effect of SNF %, 
L_l - Fixed effect of Lactose,
e_ijkl - random residual effect of each milk components
 
All the data analysis was performed using SPSS Version 20.0 software (SPSS Inc., Chicago, IL, USA).

PCR-RFLP of exon 3 (156 bp) region of prolactin gene (Fig 1) with Rsa-I enzyme reveled three genotype patterns in Gaolao cattle. One of the patterns observed was with a single band of 156 bp designated as AA genotype. The second pattern with two fragments (82 and 74 bp) was referred as BB and 3^rd pattern with (Fig 2) with three fragments (156, 82 and 74 bp) was AB Similarly the promoter region (311bp) of prolactin gene (Fig 3) was digested using Tsp45I restriction enzyme and resolved was showed single band (Fig 4) indicating no polymorphism.
 

 

 

 

         
The different genotypic frequencies observed for the PCR- RFLP polymorphism in exon 3 of prolactin gene with RsaI. Genotype frequency of AA genotype was 0.865 and that of AB was 0.135 where as the BB was absent. The allele frequencies of PRL^A and PRL^B were 0.933 and 0.067 respectively and  the chi square  test (P<0.05) revealed that they were not in  Hardy-Weinberg law in equilibrium. These findings with respect to allele frequency and genotype frequency were in agreement with the  reports  of  Alfonso et al., (2012) for allele A = 0.8765 and B = 0.1235 for in American Swiss cattle. The genotype frequencies of AA, AB and BB were 0.776, 0.174 and 0.026, respectively and were not in the Hardy-Weinberg equilibrium (P<0.05). Similarly, Ghasemi et al., (2009) observed that the frequency of PRL A allele was 0.89 and genotype frequencies for AA, AB and BB were 0.81, 0.15 and 0.04 respectively in Montebeliard cows. Paramitasarim et al., (2015) noticed that in Bali cattle the allele frequency for A was 0.953 and for B it was 0.047. The genotype frequencies were 0.913, 0.080 and 0.006 for AA, AB and BB, respectively. The gene frequencies found for allele A and B were 0.854 and 0.146 with genotype frequencies of AA, AB and BB were 0.708, 0.292 and 0.0, respectively in NTB cattle (Paramitasari et al., 2015). The gene frequency of allele A = 0.945 and B = 0.55 and the genotype frequencies of AA, AB and BB were 0.891, 0.109 and 0.0, respectively in South Sulawesi cattle (Paramitasari et al., 2015). In American Swiss cattle, Edy et al., (2012) reported allele frequencies as A = 0.8765 and B =0.1235. The genotype frequencies of AA, AB and BB were 0.776, 0.174 and 0.026, respectively. Masoud et al., (2007) studied Prolactin gene polymorphism in exon 3 region of American Swiss cattle and reported allele frequencies as A= 0.794 and B = 0.206. The frequencies of AA, AB and BB genotypes were 0.598, 0.392 and 0.01, respectively. Unal et al., (2015) studied prolactin gene polymorphism in exon 3 region of Turkish native cattle breedsand reported allele frequencies in range as A= 0.713-0.683 and B= 0.287- 0.476. The genotype frequencies of AA, AB and BB were 0.262-0.532, 0.362-0.634 and 0.00-0.214, respectively. In Frieswal cattle, Singh et al., (2015) reported allele frequencies in range as A= 0.750 and B= 0.250. The genotype frequencies of AA, AB and BB were 0.595, 0.310 and 0.095, respectively. Bilal and Cinar (2014) analyzed Prolactin gene polymorphism in four cattle breeds (East Anatolian Red, Zavot, Brown Swiss and Simmental) and reported highest frequency for PRL-A and PRL-B alleles in SIM breed (0.801) and BS breed (0.315), respectively. The Chi-square test among the investigated cattle breeds showed that only the Zavot breed was in Hardy-Weinberg equilibrium (HWE) for both loci. Kumari et al., (2008) reported AA-genotype frequency (0.55) was predominant over the AB-(0.39) and BB-genotype frequency (0.06) in exotic and zebu cattle breeds. Sonmez and Ozdemir (2017) studied Prolactin-RsaI gene polymorphism in East Anatolian Red cattle in Turkey and reported G allele frequency (0.76) and genotype frequencies of AA, AG and GG genotypes as 0.07, 0.34 and 0.59, respectively. Bukhari et al., (2013) investigated Prolactin gene polymorphism and reported genotype frequency of AA, AB and BB genotypes as 0.315, 0.629 and 0.056 respectively in Frieswal cows. Allele frequencies of A and B were 0.630 and 0.370, respectively. The significant (P<0.05) chi-square value in Frieswal cattle breeds indicated that the studied population was not in Hardy-Weinberg equilibrium. Sodhi et al., (2011) studied PRL-RsaI locus polymorphism in Indian native cattle breeds (Bos indicus) reported that there is pre- dominance of the heterozygous AB genotype (mean frequency 0.58), homozygous AA (0.22) and BB (0.20) genotypes were in a similar range. The PRL^A and PRL^B alleles exhibited similar gene frequencies (means 0.52 and 0.48, respectively). They reported allele frequency for PRL^A and PRL^B was 0.62 and 0.38 respectively and genotype frequency for AA, AB and BB was 0.53, 0.20 and 0.27 in Gaolao cattle. However in the present study, allele frequency for PRL^A and PRL^B was 0.93 and 0.07 respectively and genotype frequency for AA, AB and BB was 0.87,0. 13 and 0.00, respectively.
       
However, some researchers have reported lower frequency of PRL^A and PRL^B genes at PRL-RsaI locus. Patel and Chauhan (2017) studied polymorphism of the Prolactin Gene in Gir and Kankrej Cattle and found that the allele frequencies in the studied breed were A= 0.52 and B= 0.48.  Mitra et al., (1995) studied polymorphisms at Prolactin loci in cattle and buffaloes and reported that in Sahiwal cattle, gene frequencies for A and B allele were 0.49/0.51 (PRL /RsaI). In Egyptian buffalo, PRL/RsaI frequencies for A and B alleles were 0.93/0.07 and 0.84/0.16 in Murrah and Nili-Ravi buffalo, respectively. Das et al., (2012) revealed three genotypes AA, AB, BB in the frequencies of 0.097, 0.58 and 0.32 respectively, thus frequencies of A and B alleles were 0.39 and 0.61 respectively in Deoni cattle. The promoter region of the prolactin gene screened at PRLG-III-Tsp45I Locus exhibited monomorphism.
       
The significant Chi-square value (P<0.05) indicated the animals differ in their genotyping distribution with respect to Fat % and Protein % frequency, but non-significant Chi-square value (P≤0.05) indicated the animals did not differ in their genotyping distribution with respect to SNF % and Lactose % frequency at PRLGI - RsaI locus (Table 2). The non-significant deviation of observed and expected genotype frequencies at PRLGI – RsaI locus in the studied Gaolao population may also be due to small population size. The result of analysis variance in the present study revealed significant (P≤0.05) effect of AA genotype (4.4267%) on fat % as compared to AB genotype (4.1714%) however the non-significant effect of these genotypes was found on protein %, SNF % and lactose % which may be due to small sample resulting in higher error variance (Table 3).
 

 

       
In American Swiss cattle, Alfonso et al., (2012) reported that animals with genotype AA had a greater milk production during lactation than genotypes AB and BB (P<0.05) with genotype BB being the one that had the lowest production (P<0.05). Edy et al., (2012) reported that animals with genotype AA had a greater milk production during lactation than genotypes AB and BB (P<0.05), with genotype BB being the one that had the lowest production (P<0.05). Masoud et al., (2007) reported that BB genotype had higher milk yield than AA and AB individuals (P<0.05). BB genotype showed higher milk fat yield than AA and AB individuals (P< 0.05). With respect to milk fat content (%), the AB genotype had higher levels than the AA and BB individuals (P<0.05). They showed that the highest milk and milk fat yields were obtained by cows with the genotype PRL-RsaI BB. Patel and Chauhan (2017) studied polymorphism of the Prolactin Gene in Gir and Kankrej Cattle and found that means and standard deviations for fat content (%) were 3.99 ± 0.18 for the AA genotype, 4.16±0.33 for the AB genotype and 4.34 ± 0.11 for the BB genotype, respectively, in Gir cattle. Similarly, for Kankrej cattle, means and standard deviations for fat content (%) were 4.05±0.16 for the AA genotype, 4.10±0.156 for the AB genotype and 4.30±0.178 for the BB genotype, respectively, in Kankrej cattle. This study showed differences in milk traits among PRL genotypes of Gir and Kankrej cattle. Das et al., (2012) revealed no significant differences between estimated least square means of milk production traits in relation to PRL gene exon 3, in which genotype BB was associated with highest lactation milk yield (1007.354 ± 92.328 kg) whereas heterozygotic genotype AB was associated with highest fat % (4.780±0.126) and highest protein percent (3.290±0.033).  Khatami et al., (2005) studied the association of DNA polymorphisms of Prolactin genes with milk productivity in Yaroslavl and Black-and-White cattle. The heterozygosity at the RsaI marker was low (9.4%) in the Russian Black-and-White breed. Genotype BB of RsaI polymorphism of the bPRL gene tended to show a negative association with the fat content in milk.

Prolactin gene is traditionally regarded as a good candidate gene and statistically significant genetic polymorphisms identified within it will aid in revealing SNPs related with economic traits for their use in marker-assisted selection. The gene regions of Prolactin viz; exon-3 (PRLG-I) and Part of promoter (PRLG-III) were screened for variation using PCR RFLP analysis  revealed polymorphism (two alleles A, B) in exon3 portion of gene but no polymorphism was detected in promoter region. The allele frequency for the PRL^A alleles was found 0.933 and for allele PRL^B was 0.067 for exon 3. The ANOVA of genotypes for various milk  components revealed that the genotypes differed for only fat % but not in other  three (protein, SNF and lactose) which might be  due to small sample size resulting in higher error variance. The identified Prolactin gene polymorphisms after validation in large breeding population data with precise data records may prove as important candidate gene markers for selection and breeding decisions in genetic improvement of indigenous cattle.

The help rendered by Dr. Satish Raju, Dr. Surendra Parate and Mr. Prasanna Bamb in locating and collection of blood and milk samples of Gaolao cattle from the breeding tract is duely acknowledged.