PCR-RFLP Analysis of Single Nucleotide Polymorphism (SNP) C-2402T at the Promoter Region of Prolactin Gene and its Association with Positively Correlated Production Traits in White Leghorn Chicken

In Indian agriculture, the poultry industry is one of the fastest-growing sectors among all livestock sectors. At present, the total poultry population in our country is about 851.81 million numbers as per the 20^th livestock census (Government of India, 2020) and the egg production of around 103.318 billion numbers (BAHS, 2019). Persistent efforts and collaborative research on poultry breeding have been carried out for the evolution of high producing layer stock which is suitable for various environment and management practices (Tomar et al., 2015). White Leghorn is a non-broody bird and produces more than 300 eggs per year. On one hand, the non-broody behaviour of White Leghorn is due to the presence of a major autosomal recessive gene on the Z chromosome (Romanov, 2001). On another hand, a 24 bp insertion polymorphism at the promoter region of the prolactin gene suppresses the expression of the prolactin gene, and broodiness is prevented (Jiang et al., 2005). Also, White Leghorn is used as a major line in the cross-breeding programmes to develop different layer strains for high egg production in the commercial poultry industry. Poultry breeding is done mainly to increase the level of egg-laying capacity, egg quality and meat quality (Kulibaba et al., 2020). Over the past few years, newer DNA technologies, marker-assisted selection and genomic selection have been efficiently employed in poultry selection and breeding (Salisu et al., 2018). The foremost objective of commercial poultry breeding is to achieve genetic improvement in age at sexual maturity, egg weight, egg number and body weight. Restriction fragment length polymorphism (RFLP) enables to detection of the large insertion and deletion within the genome sequence and to identify of the single base changes called single nucleotide polymorphism (SNP) (Fulton, 2012). The chicken prolactin gene plays a significant role in egg production (Wang et al., 2011). Since the avian prolactin gene was cloned and sequenced, most of the research has focused on detecting new polymorphic sites in this gene. The results of several studies had shown a significant association of SNPs of prolactin gene with economically important traits in different species of poultry (Wilkanowska et al., 2014). Specifically, a significant association was found between the SNP of C-2402T genotypes with egg number and laying rate in Fars native chicken (Bagheri Sarvestani et al., 2013). Thus, this study aimed to study was to identify the SNP C-2402T at the promoter region of the prolactin gene using the PCR- RFLP technique and find out the association of this SNP with production traits in White Leghorn chicken.

Experimental birds
 
A total of 200 birds of White Leghorn were randomly selected during September 2016 to October 2017 from the All India Research Co-ordinated Project (AICRP) on poultry improvement, Mannuthy, Thrissur, Kerala, India.
 
Workplace
 
The present study was carried out at Avian Biotechnology Laboratory, in the Department of Poultry Science, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur, Kerala.
 
Collection of blood samples
 
 Approx. 0.5-1 ml of blood was collected from the wing vein under aseptic conditions. The samples were brought to the laboratory at 4°C in an ice pack.
 
Isolation of genomic DNA
 
Isolation of genomic DNA was done from the whole blood using ODP304 origin genomic DNA isolation kit according to the standard procedure given by manufacturers. The yield and quality of the DNA obtained were checked by Nano-drop spectrophotometer and by 0.8% agarose gel electrophoresis, respectively. The DNA samples showing the OD260/OD280 ratio between 1.7 and 1.9 were used for further investigation.
 
PCR assay
 
The polymerase chain reaction was carried out using a specific set of forward (F-5¢AGAGGCAGCCCAGGCAT TTTAC3¢) and reverse primer (R-5¢CCTGGGTCTGGTTTGG AAATTG3¢) to amplify the 439 bp fragment of prolactin gene containing SNP C-2402T at the promoter region (Cui et al., 2006; Bagheri Sarvestani et al., 2013). Each diluted primer (10 pM/µl) was added to the template DNA (100 ng/µl) and 2X PCR Smart Mix (origin) in a PCR tube and made up to the final volume of 20 µl using ultra-filtered Millipore water. PCR was done in Bio-Rad thermal cycler and standardization was done with the following program: initial denaturation of 5 min at 94°C; 35 cycles of 94°C for 30 s, annealing at 67.7°C for 30 s and 72°C for 30 s with a final elongation of 5 min at 72°C. PCR amplicons were checked on 2% agarose gel.
 
Agarose gel electrophoresis
 
The genomic DNA and PCR products were checked in agarose gels of 0.8 per cent and 2%, respectively prepared using 1X TBE buffer. The PCR products were loaded along with a molecular weight marker (50 bp ladder) for relative sizing. Electrophoresis was carried out at 5V/cm and the gel was photographed in a Gel Doc System (Bio-Rad, USA).
 
Restriction fragment length polymorphism (RFLP) analysis
 
For restriction digestion five microlitres of the amplified PCR product (439 bp) of promoter C-2402T was added with 5 units of AluI enzyme and incubated at 37°C for 1 hr. The composition of the reaction mixture was made with the final volume of 12 μl (PCR product-5 μl, 10X buffer -1.2 μl, AluI (10 U/μl) -0.5 μl and Distilled water -5.3 μl). After restriction digestion, the digested PCR products were separated by electrophoresis in 3% agarose gel in 1X buffer with 50 bp DNA size marker. The restriction pattern was visualized under a UV trans-illuminator and documented in a gel documentation system.
       
The amplicons of the promoter region of prolactin gene 439 bp and the digested PCR products were sequenced using respective forward and reverse primers in an automated sequencer using Sanger’s dideoxy chain termination method at Agri Genome Labs Pvt. Ltd., Cochin, Kerala. Based on the results of RFLP analysis, all the birds were designated with the same genotype and allele frequency was calculated, accordingly. Bodyweight at 16^th, 40^th and 64^th weeks of age and egg weight at 28^th, 40^th and 64^th weeks of age, respectively were recorded.
 
Statistical analysis
 
The association of SNP site C-2402T of prolactin with body weight and egg weight were analyzed by one-way ANOVA by using the software SPSS (Version 21.0).

The quality of extracted DNA was checked on 0.8% agarose gel (Fig 1) PCR amplification of 439 bp amplicon of prolactin gene is represented on 2% agarose gel (Fig 2). On restriction analysis, 439 bp PCR products of prolactin with AluI restriction enzyme revealed the presence of only one genotype CC with different sizes of restriction fragments (160, 144, 81 and 54 bp) on 3% agarose gel (Fig 3). The same RFLP pattern was observed in all 200 digested products of PCR samples. Simultaneously, this AluI restriction enzyme has been widely used in most of the studies to reveal the restriction sites for the SNP position C-2402T in the chicken prolactin gene (Cui et al., 2006; Kulibaba et al., 2012; Bagheri Sarvestani et al., 2013; Kulibaba, 2015). Also, we applied the NEBcutter V2.0 tool to identify the restriction sites within our DNA sequence and found AluI restriction enzyme was suitable for the SNP site C-2402T of the prolactin gene. Therefore, we have planned to proceed with our research work further but we have not gone through any pilot study for the present work using the AluI restriction enzyme. According to the restriction digestion results, it had been confirmed that all the 200 birds of  White Leghorn chicken were monomorphic with the same genotype CC for the SNP site C-2402T of prolactin gene. Thus, no polymorphism were found in this White Leghorn chicken population which had undergone 29 generations of continuous selection. In contrary to our findings, the AluI restriction enzyme produced different sizes of fragments with three genotypes viz. CC (four fragments- 160, 144, 81 and 54 bp), CT (five fragments-304, 160, 144, 81 and 54 bp) and TT (three fragments - 304, 81 and 54 bp), respectively for the SNP site C-2402T of prolactin gene in Fars native chicken of Iran (Bagheri Sarvestani et al., 2013). Similarly, all three genotypes CC, CT and TT were observed in five different chicken populations (Yangshan, Taihe Silkies 1, White Rock, Nongdahe and Taihe Silkies 2) with six SNPs (Cui et al., 2006). Also, Chau, (2016) reported similar findings in the Noi native chicken of Vietnam. In comparison, Kulibaba et al., (2015) conducted research on polymorphism of prolactin gene in connection with egg production in Poltava clay chicken of Ukraine and found SNP at the position of C-2402T with all three possible genotypes in this population. Allele C had three restriction sites for AluI (two monomorphic and one polymorphic) and allele T had two restriction sites. Similar polymorphism (C-2402T) was reported by Kulibaba et al., (2012) in chicken lines of Ukrainian selection.
 

 

 

       
Our findings revealed that all 200 birds of White Leghorn chicken carry CC genotype. As a result, all White Leghorn chicken selected from the AICRP farm are monomorphic for the allele C and the allele frequency of C was 1.0 in this population. Similar findings were reported by Cui et al., (2006) in the White Leghorn chicken population for the SNP site C-2402T. In contrary to these findings, Bagheri Sarvestani et al., (2013) reported the allele frequency as 0.66 (C) and 0.34 (T) in Fars native chicken of Iran. In comparison, Chau., (2016) found the allele frequency as C (0.17) and T (0.83) in the Noi native chicken of Vietnam. Furthermore, Kulibaba et al., (2015) reported the allele frequency as C (0.372) and T (0.628) in the Poltava clay chicken population of Ukraine. In addition, Kulibaba et al., (2012) found the allelic frequency as C (0.745) and T (0.255) for the chicken line A in Ukrainian selection. According to these research findings, it has been observed that frequency of the C allele varies in different breeds of the chicken population. Allele frequency is a measure of the relative frequency of an allele on a genetic locus in a population and usually, it is expressed as a proportion or a percentage. Hence, the variation in allele frequency is possibly due to gene flow, genetic drift, natural selection, and mutation. These are referred to as the four fundamental forces of evolution. Besides, the mutation can create new genetic variation and the other three forces simply rearrange this variation within and among populations.
 
Association of SNP C-2402T at the promoter of prolactin gene with body weight and egg weight in White Leghorn chicken
 
Bodyweight at 16^th, 40^th and 64^th weeks of age and egg weight at 28^th, 40^th and 64^th weeks of age, respectively were recorded for all 200 birds of White Leghorn which were randomly selected from AICRP on poultry improvement farm, Mannuthy, Thrissur. By the acquired statistical results, the mean body weight at 16^th, 40^th and 64^th weeks of age were 1036.75±2.62, 1561.00±6.07 and 1544.00±6.31 g and mean egg weight at 28^th, 40^th and 64^th weeks of age were 48.00±0.01, 52.16±0.10 and 54.16±0.37 g, accordingly for the genotype CC of SNP site C-2402T. Since only CC genotype was found in this White Leghorn chicken population, association study with positively correlated traits was not possible.

Our research findings revealed the presence of only one genotype CC for the SNP site C-2402T of prolactin gene in all the 200 birds of White Leghorn chicken using PCR- RFLP analysis. It indicates that the prolactin gene is monomorphic for this chicken population and the frequency of the C allele was one. Hence, further research could be done in other breeds of the chicken population to confirm the association of SNP C-2402T of prolactin gene with production traits.

The current research was funded by the Kerala state plan project. I would like to thank the Professors, staff of the Department of Poultry Science and the Department of Animal Breeding and Genetics. Also to the Dean, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala for providing the necessary infrastructure required for the investigation of the present research.