Polymorphism Study of POU1F1 Gene in Mandya and NARI-Suwarna Sheep

Mandya sheep is considered as a best meat breed with respect to its conformation (Acharya, 1982). Its meat is regarded as the most nutritious and high quality mutton in human diets at national and international spectrum of sheep products (Dinakar et al., 2019). These animals are predominantly distributed in Mandya and adjacent districts of Karnataka. NARI-Suwarna sheep with more than 60 per cent ewes having twin was developed by Nimbkar Agriculture Research Institute (NARI), Phalton, through introgression of FecB gene from the prolific Garole breed of Sunderban, West Bengal into non-prolific locally adapted Deccani breed of Maharashtra (Nimbkar et al., 2002).

Identification of genetic markers in relation to growth rate and its possible use in Marker Assisted Selection (MAS) will speed up the genetic progress. In general, the crucial step to ascertain a MAS system is to fishing out and validating the genetic markers of growth traits (Allan et al., 2007). For growth traits, growth hormone (GH), growth hormone receptor (GHR), Insulin like growth factor 1 (IGF1), leptin, pituitary specific transcription factor - 1 (POU1F1), myostatin (MSTN) and bone morphogenetic protein (BMP) genes are necessary for bone formation, birth weight, weaning weight, body condition and muscle growth (Supakorn, 2009).

The POU1F1 belongs to the large family of POU domain proteins. The POU1F1 (also known as PIT-1 or GHF-1) is a tissue-specific transcription factor which is mainly expressed in the anterior pituitary. This protein has a certain role in the transcriptional adjustment of growth hormone (GH) and prolactin (PRL) genes. Additionally, POU1F1 is also involved in the activation of thyroid-stimulating hormone, POU1F1 itself and growth hormone-releasing hormone receptor (GHRH-R) genes. Moreover, besides its role in gene activation, POU1F1 is essential for differentiation, reproduction and survival of somatotrope and lactotrope as well as thyrotrope cells. The ovine POU1F1 gene contains five introns and six exons and is located on chromosome 1 (Bastos et al., 2006).

Ozmen et al. (2014) studied the association of SNP in exon 6 region of POU1F1 gene with milk productive traits in Turkish sheep breeds (Sakiz, White Karaman and Awassi). They reported a significant association of milk yield, fat and lactose with TT and CC genotypes in Sakiz sheep. Jalil-Sarghale et al., (2014) investigated the SNPs at exon 3 region of POU1F1 gene and its association with growth and biometric traits in two Iranian sheep breeds (Zel and Lori-Bakhtiari). They found that AA genotype had a higher weaning weight than those with GG genotype and opined that POU1F1 polymorphism can be used as molecular marker for production traits.

The present study was conducted to find out and compare the polymorphism of POU1F1 gene in Mandya and NARI-Suwarna sheep, if any.

Blood samples were collected from 50 Mandya sheep from farmers’ flock distributed over villages of Malavally taluk, Mandya district, Karnataka and 50 NARI-Suwarna sheep from the flock maintained at Department of Veterinary Gynaecology and Obstetrics, Veterinary College, KVAFSU, Hebbal, Bengaluru. Genomic DNA was isolated from venous blood by following high salt method as described by Miller et al., (1988) with a few modifications.

The primers previously published by Ekegbu et al., (2018) and Al-Khuzai and Al-Anbari, (2018) were utilized for amplification of exon 3 and 4 regions of POU1F1 gene, respectively. Whereas, for amplification of exon 6 region of POU1F1 gene, the primer was designed using PRIMER 3 PLUS software by referring the Ovis aries sequences available in NCBI website. The details of the primers used for amplification of POU1F1 gene are presented in Table 1.



The amplification of exon 3, 4 and 6 regions of POU1F1 gene were performed in a total volume of 25 µl which consisted of 12.5 µl of Red PCR master mix, 1 µl (10 pmol/ µl) each of forward and reverse primer, 9.5 µl of PCR grade water and 1 µl of template DNA. The PCR reaction was carried out with an initial denaturation temperature of 95°C (4 min), 35 cycles of 94°C (45 sec), 55.8°C (45 sec) and 72°C (60 sec) for exon 3 and 4 regions and 34 cycles of 94°C (60 sec), 60°C (60 sec) and 72°C (60 sec) for exon 6 region of POU1F1 gene followed by final extension at 72°C (10 min). The PCR amplified products were resolved on 1.5 per cent agarose in parallel with 100 bp DNA ladder and photographed under gel documentation system (Bio rad Molecular imager Gel Doc XR+, USA).

The PCR products of the exon 3 and 4 regions of POU1F1 gene were digested with AluI and EcoRI restriction enzymes, respectively. The digestion was done in a total volume of 30 µl which consisted of 2 µl of 10X buffer, 10 µl of PCR amplicon, 1 µl of RE and 17 µl of Nuclease free water (NFW) with incubation at 65°C for 3 hours. The RE digested products were resolved on two per cent agarose gel agarose in parallel with 100 bp DNA ladder and photographed under gel documentation system (Bio rad Molecular imager Gel Doc XR+,USA). Based on visualization of different band patterns, genotypes were determined.

The PCR product of exon 6 region of POU1F1 gene was subjected to single-strand conformation polymorphism (SSCP) by using 12 per cent polyacrylamide gel. The 12 per cent polyacrylamide gel comprised of 15 ml of Acrylamide:Bisacrylamide (29:1), 5 ml of 10x TBE, 32.6 ml of autoclaved triple distilled water, 330 µl of 10 per cent ammonium per sulphate and 70 µl of TEMED. Approximately 12 µl of PCR product was mixed with 8 µl of formamide dye and properly mixed. The mixture was denatured at 95°C for 10 minutes and snap cooled on ice for 3 minutes. The product was carefully loaded into the gel. The electrophoresis was performed for 5 hours at 150 volts at 16°C. The gel was stained with silver staining after the run in order to visualize the banding pattern.

The PCR products showing different patterns in RFLP and SSCP were custom sequenced by double pass sequencing method using primers used for amplification of different products.

 About 200 - 300 μg of good quality DNA per 10 ml of venous blood was obtained in the present study by following Miller’s high salt method.

The PCR products of sizes 365, 508 and 501 bp (Fig 1, 2 and 3) were successfully amplified for exon 3, exon 4 and exon 6 region of POU1F1 gene, respectively in the studied population.



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The restriction enzyme (RE) digested products of exon 3 region of POU1F1 gene resolved on agarose gel electrophoresis revealed fragments of sizes of 112, 105 and 95 bp in one band and 31 and 22 bp fragments in another band. The RE have cut the PCR amplicons to give the above mentioned fragments indicating AA genotype in both Mandya and NARI-Suwarna Sheep (Fig 4). Contrast to the present study, Bastos et al., (2006) observed polymorphism in ‘Churra da Terra Quente’ sheep. Sumantri et al., (2009) reported polymorphism in local sheep from Jonggol Animal Science Teaching and Research Unit (JASTRU) farm in Bogor using PCR-RFLP with Hinf1 restriction enzyme. By employing PCR-RFLP with AciI restriction enzyme, polymorphism was reported in Iranian sheep breeds (Zel and Lori-Bakhtiari) by Jalil-Sarghale et al. (2014). But, by following PCR-SSCP analysis many authors viz., Ekegbu et al., (2018) in New Zealand sheep breeds (NZ Romeny and Merino), Bahrami et al., (2014) in Mehraban sheep, Sadeghi et al., (2014) in Iranian sheep breeds (Zel and Lori-Bakhtiari) and Negahdary et al., (2013) in Makooei sheep reported polymorphism in exon 3 region of POU1F1 gene.

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The restriction enzyme digested products of exon 4 region of POU1F1 gene resolved on agarose gel electrophoresis revealed fragments of sizes 275 and 233 bp. The RE have cut the PCR amplicons to give rise to two bands indicating AA genotype in both Mandya and NARI-Suwarna Sheep (Fig  5). This is in agreement with the reports of Sudhakar (2009) in Nilagiri and Mecheri sheep breeds and Bastos et al., (2006) in ‘Churra da Terra Quente’ sheep, who also reported absence of polymorphism in exon 4 region of POU1F1 gene. Contrast to the present study, AL-Khuzai and Al-Anbari, (2018) reported polymorphism in Iraqi Awassi sheep by following PCR-RFLP. They reported two genotypes, MM and NN with frequency of 0.58 and 0.42, respectively and two alleles with frequency of 0.58 and 0.42, respectively. Similarly, Ansari et al., (2008) reported three genotypes (AA, AB and BB) in Baluchi Sheep.

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In the present study, PCR-SSCP technique was employed to determine the polymorphism at exon 6 region of POU1F1 gene. The PCR-SSCP analysis revealed two patterns, P1 and P2 both in Mandya and NARI-Suwarna sheep (Fig 6). The frequency of P1 and P2 in Mandya was 98 and 2 per cent, respectively whereas, in NARI-Suwarna it was 90 and 10 per cent, respectively. The proportion of animals with P1 was more in both Mandya and NARI-Suwarna population. In agreement to the current study, Ozmen et al., (2014) reported two genotypes (TT and CC) in Sakiz sheep by PCR-RFLP technique with AluI restriction enzyme. Similarly, Bai et al., (2016) conducted PCR-RFLP analysis and reported polymorphism in five sheep breeds (large-tailed Han, small-tailed Han, Yuxi fat-tailed, Lanzhou large-tailed and Mongolian sheep) of China. In contrast to the present study, Bastos et al., (2006) reported no polymorphism at exon 6 region of POU1F1 gene in ‘Churra da Terra Quente’ sheep.

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The alignment of P1 and P2 pattern sequences of Mandya sheep revealed two SNPs, G to C transversion at 109 bp position and T to A transversion at 112 bp position (Fig  7). Whereas, alignment of P1 and P2 pattern sequences of NARI-Suwarna sheep revealed 3 SNPs, T to G transversion at 218 bp, G to A transition at 225 bp and T to A transversion at 264 bp positions (Fig 8). In concurrence to the present study, Bai et al. (2016) conducted PCR-RFLP analysis and detected three SNPs at exon 6 region of POU1F1 gene in five sheep breeds (large-tailed Han, small-tailed Han, Yuxi fat-tailed, Lanzhou large-tailed and Mongolian sheep) of China. The three SNPs were C355T (C/T), C71G (C/G) and C330G (C/G). C allele of C355T locus was the dominant allele in all the five sheep populations.

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In the present study, PCR-RFLP analysis of exon 3 and exon 4 regions of POU1F1 gene using AluI and EcoRI, respectively showed monomorphism in both Mandya and NARI-Suwarna sheep. Polymorphism was established at exon 6 region of POU1F1 gene in both Mandya and NARI-Suwarna sheep by PCR-SSCP. Mandya sheep revealed two SNPs, G to C transversion at 109 bp position and T to A transversion at 112 bp position. NARI-Suwarna sheep revealed 3 SNPs, T to G transversion at 218 bp, G to A transition at 225 bp and T to A transversion at 264 bp positions.

The authors are thankful to Karnataka Veterinary, Animal and Fisheries Sciences University for providing the funds to carry out the work.