Genetic Variability Analysis of Early Growth Response 2 (EGR2) Gene in Native Goat Breeds of Kerala 

India contributes about 15 per cent of the total goat population in the world. India is blessed to have vast genetic resources of the goat and stands second largest goat producing country across the globe (Joshi et al., 2004). Goats constitute about 26.40 per cent of the total livestock population which accounts to 137.17 million goats in the country. Goats are considered to be a main source of income to the marginal farmers and the landless labourers in the rural India (Yadav, 2019). Malabari breed is originated in Tellicherry (Thalassery) in the Malabar Coast of Kannur district in North Kerala, also popularly known as Tellicherry goat. It is a dual purpose breed famous for low fat meat and high prolificacy. These animals are well adapted to the hot and humid conditions of the state (Verma et al., 2009). Attappady Black goats were found in the hilly regions of the Palakkad district in Kerala, Attappady. The breed was phenotypically characterised by the presence of black coloured skin coat with bronze coloured eyes. Animals have medium sized ears that were drooping, the tail was curved and bunchy (Stephen et al., 2005). Attappady Black are highly adaptable and disease resistant breed. This breed is generally considered to be low prolific. This breed has a lesser frequency of multiple births (Naicy et al., 2016). Improvement in the reproductive traits was very much essential due to the increased prolificacy of the goats that will directly enhance the income of the goat rearing farmers. The economic efficiency of the goats is dependent on the total productivity of the goats (Khanum et al., 2007). The EGR2 gene (also called as krox20) is a part of multigene family which encodes Cys₂His₂ type zinc-finger proteins which is responsible for DNA binding. The EGR2 gene is located in the plus strand of caprine chromosome 28. It is 2845 bp in size starting from 26,656,206 bp to 26,659,051 bp and has 3 exons with 1651 bp size. This gene had a major role in cellular prolification. The EGR2 is a DNA binding protein involved in genetic control at transcription level. Deoxyribonucleic acid (DNA) binding is affected by mutations in this gene. It was an acetylated protein (Warner et al., 1999). During folliculogenesis, the action of gonadotropins was regulated by EGR2. For a successful reproduction, ambient growth and development of follicles in the ovary into final graffian follicles is a necessity. Survival of granulosa cells is promoted by gonadotropin induced by EGR2 (Jin et al., 2017). The present study was conducted with the objective to identify the polymorphism in EGR2 gene in native goat breeds of Kerala.

The research was conducted during the year 2018-2019 in the Department of Animal Genetics and Breeding at College of Veterinary and Animal Sciences Mannuthy, Kerala, India.
 
Experimental animals
 
To conduct the polymorphism study a total number of 282 animals belonging to the species Capra hircus (goat) were selected. Samples and data were collected from 129 Attappady Black goats and 153 Malabari goats maintained at University Goat and Sheep Farm, Mannuthy, Thrissur, Goat farm of Kottakkal Aryavaidyasala (AICRP on Goat Improvement Scheme-field centre, Malappuram), Department of Animal Husbandry Goat Farm, Attappady, Palakkad.
 
Collection of blood samples and isolation of DNA
 
Approximately, six millilitres of venous blood was collected from the jugular vein of each animal into vaccutainer tubes containing ethylene diamine tetra acetic acid (EDTA) as anticoagulant. The tubes were then kept in an ice box and were taken to the Molecular Biology Laboratory of Centre for Advanced Studies in Animal Genetics and Breeding (CASAGB) and stored at -20°C until isolation of DNA. The genomic DNA was extracted using phenol chloroform method. The concentration, purity and quality of DNA were checked by NanoDrop spectrophotometer (Thermo Scientific, USA). The purity of the DNA was verified by measuring absorbance at 260 nm and 280 nm. A 260/280 ratio of approximately 1.8 is generally accepted as “pure” for DNA (Sambrook and Russell, 2001). DNA samples having good quality, purity and integrity were taken for PCR-SSCP analysis.
 
Amplification of exons of EGR2 gene
 
All the exons of EGR2 were amplified using specific set of primers. Exon 1 and 2 were amplified as two fragments and since the size of exon 3 was 1094 bp, it was amplified as five overlapping fragments. The primers were designed from the caprine EGR2 gene sequence GenBank (Accession No: XM_018042609.1) using Primer 3 (V.0.4.0) software. The primers selected were custom synthesised (Sigma Aldrich India) and diluted to a concentration of 10 pM/μl. The primer sequences and details are given in the Table 1.
 

       
The seven fragments of EGR2 gene were amplified from genomic DNA by PCR. The PCR conditions were optimised using different concentrations of reagents and different temperatures ranging from 60-65°C and PCR was performed in a thermal cycler (Bio-Rad, USA).  PCR was performed in a total volume of 20µl. The reaction mixture was composed of 10 µl of 2X PCR master mix (Emerald Amp® GT PCR master mix), 1 µl of template DNA, 1 µl of forward and reverse primer and 7 µl of nuclease free water. The cycling parameters were one cycle at 95°C for 3 min, followed by 35 cycles (30 sec at 94°C, 45 sec temperatures mentioned in Table 1 for different fragments and 45 sec at 72°C), followed by 1 cycle at 72°C for 5 min, stopped at 4°C. The amplified PCR products were checked by gel electrophoresis using two per cent agarose gels along with 50/100 bp DNA Ladder (Thermoscientific). Electrophoresis was performed at 5 V/cm for 30 min. The gels were visualised under UV light and recorded in a gel documentation system.
               
Single strand conformation polymorphism (SSCP)
 
Genotyping of the samples was done by single stand conformation polymorphism (SSCP). The amplified fragments were mixed with SSCP loading buffer in the ratio of 1:2 (6 μl sample with 12 μl dye), denatured at 95°C for 10 min and immediately snap chilled in ice. The products were run in 12% poly-acrylamide gel at 4°C for 3 h at 120 V. The composition of poly-acrylamide gel was 30% acrylamide/bis-acrylamide (29:1)-6 ml, 10% ammonium per sulphate-75 μl, TEMED-15 μl, 10X TBE-1.5 ml and nuclease free water-7.5 ml. Gels were stained with silver nitrate as per the procedure described by Byun et al., (2009) and SSCP fragments were visualised directly. The genotypes were detected directly by observing SSCP pattern of samples in the gels. Representative samples from each fragment were sequenced to find out the nucleotide differences. The obtained sequence was subjected to BLAST analysis to retrieve similar sequences of other breeds of goats.

The present study was conducted to identify Early Growth Response 2 gene polymorphism in native goat breeds of Kerala using PCR-SSCP method. OD ratio of the extracted genomic DNA was ranging between 1.7-1.8. The quality of the genomic DNA extracted was checked by one per cent agarose gel electrophoresis and visualized under UV- trans-illuminator. DNA samples had an intact band without any shearing and was considered to be of a good quality. All the three exons and its fragments of EGR2 gene were amplified using the specific set of primers. Exon 1 and exon 2 were amplified as two separate fragments. Since the size of the exon 3 was large (1094 bp) it was amplified as five overlapping fragments. The amplified products are displayed in Fig 1, 2, 3, 4, 5, 6 and 7, respectively. Thus seven fragments of EGR2 were explored to find out the SNPs in the coding region.
 

 

 

 

 

 

 

       
For screening of allelic variations in the amplified exons of Early Growth Response 2 gene, the obtained PCR products were subjected to SSCP analysis. The SSCP pattern of Exon 1, Exon 2 and all the overlapping fragments of Exon 3 exhibited similar banding patterns for all the goat population screened and was monomorphic (Fig 8-14). The PCR products were sequenced to confirm the same. The BLAST analysis showed 100 per cent similarity. This result suggests the conserved nature of Early Growth Response 2 gene in the screened Malabari and Attappady Black goat population of Kerala.
 

 

 

 

 

 

 

       
The PCR-SSCP analysis of seven fragments of EGR2 exhibited a monomorphic pattern in all the population screened in the native goat breeds of Kerala (n=282). This is the first study conducted to characterize EGR2 gene in goats making the current study a novel one. No reports are available to compare and contrast the findings of EGR2 gene polymorphism in Goats. However, in 2016, Kwon et al., 2016 were successful in extracting four missense SNPs in EGR2 gene in pigs, but the association between the EGR2 gene and litter size were not studied.

The polymorphism was not detected in caprine population screened from two distinguishable goat breeds. Absence of polymorphism indicated that EGR2 gene is highly conserved in the native goat breeds of Kerala. For a successful reproduction, there is a need of proper growth and development of ovarian follicles into mature graffian follicles. Survival of granulosa cells are promoted by EGR2 gene. Since EGR2 plays a crucial role during folliculogensis for the survival of granulosa cells, this gene is highly conserved.

The authors are thankful to authorities of Kerala Veterinary and Animal Sciences University and Indian Council of Agricultural Research-All India Co-ordinated Research Project on Goat Improvement for providing facilities to conduct the research.