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Short Communication · Volume 52, Issue 1, 21 November 2019 · Open Access

A Prl/RsaI Polymorphism in Exon 3 and 4 of Prolactin Gene in Dairy Cattle

Affiliations & Notes ⌄
  1. 1Department of Animal Science, Faculty of Agriculture, Ataturk University, 25240 Erzurum, Turkey , * Corresponding author: ozdemirm@atauni.edu.tr
Article Info ⌄
Journal:
Pakistan Journal of Zoology
Published:
21 November 2019
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Abstract

Prolactin is a quantitative trait locus and is a potential genetic marker that can be used in the improvement of production traits in dairy cattle, because prolactin has an important regulatory role in the development of the mammary glands, the secretion of milk and the expression of milk protein genes. It has been seen many studies about Prl/RsaI polymorphism found in the exon 3 or exon 4 region of bovine prolactin gene in the literature. The aim of this study was to determine whether the DNA sequence of the Prl gene exon 3 or exon 4 in cattle breeds has the RsaI polymorphic digestion site. As a result of the study, it has been seen that the Prl/RsaI specificpolymorphic site is on exon 4 in Bos taurus cattle breeds, but have been not detected the RsaI restriction enzyme digestion site in Prl gene exon 3 region reported by many literature and, these results also have been approved by blasted at the NCBI Genbank.

Materials and methods

Blood samples of 50 Holstein cows reared at the Research and Application Farm Faculty of Agriculture, Ataturk University in Turkey were collected from the jugular vein in 10 ml vacuum tube containing K3EDTA. Genomic DNA was extracted from whole blood samples using Purgene kit (Gentra Systems, Plymouth, MN, USA). For Prl/RsaI polymorphism, the primers used for exon 3 were : Prl/RsaI Forward: 5’-CGA GTC CTT ATG AGC TTG ATT CTT-3’, Reverse: 5’- GCCTTCCAGAAGTCGTTTGTTTTC-3’, and the primers used for exon 4 were : Prl/RsaI Forward: 5’-CAT GGT GAC CTG CAT CCT C-3’, Reverse: 5’-ACC CTC ATG CCT CTC ACA TC-3’ primers were used. The amplified PCR products of both regions were digested by using RsaI at 37°C overnight. To genotype animals for the RFLP, each 15 μl digestion mix was electrophoresed in 2.5% agarose gel at 40 V for 2.5 h and DNA was visualized by staining with ethidium bromide under UV light. For each animal, Prolactin allele frequencies were calculated by counting the alleles.

The specificity of the above primers revealed by the literature to be specific to the region defined as Prl exon 3 or exon 4 in NCBI Genbank site was investigated by making BLAST.

Results and discussion

The PCR-RFLP results of Prl/RsaI polymorphism (Prl/RsaI +/-) in exon 3 and 4 are shown in Table I.

Brym et al. (2005) and Mitra et al. (1995) had reported 156 bp band for exon 3 and 294 bp band for exon 4 product, respectively (Table II).

Table I.- The allelic frequencies and genotype numbers of the Prl/RsaI Polymorphism for both primers pairs.

Prl

Genotype

Allele frequency

(Rsa+/+)

(Rsa+/-)

(Rsa-/-)

BB

AB

AA

B

A

Exon 3

3

19

28

0.25

0.75

AA

AG

GG

A

G

Exon 4

3

19

28

0.25

0.75

Note that RsaI enzyme was applied to the same examples.

As a result, while it has been observed that the both primer pairs in the test results indicate the same polymorphism, the same DNA sequence (NCBI, GenBank No: AF426315.1) was obtained when these primers were blasted with nucleotide sequences for specificity checking separately (Fig. 1). The Figure 1 shows exon 4 nucleotide sequences, using primers which had restriction site for RsaI enzyme.

The most important polymorphism of the Prl gene has been found on exon 4, and this SNP named as RsaI polymorphism, no matter which primer pair is used, must be called as A/G polymorphism or RsaI+/- in order to avoid misunderstandings (Fig. 1). Moreover, in the bovine Prl gene, exon 3 region certainly do not have the RsaI restriction enzyme digestion site and RsaI polymorphism. Halabian et al. (2008) have reported 156 bp fragment by PCR-SSCP method in exon 3, and reported that they had identified 4 SNP. However, it is detected that the DNA sequence of the region examined is similar to exon 4. Furthermore, in the similar studies conducted by different researchers, the exon 3 and exon 4 regions have been shown to produce 156 bp and 294 bp fragment of the Prl gene, respectively, but different genotype frequencies have been

Table II.- bPrl locus region and fragment size digested by RsaI endonuclease.

Region

Primers used for amplification

Fragment size

References

Exon 3

F: 5'-CGAGTCCTTATGAGCTTGATTCTT-3'

R: 5'-GCCTTCCAGAAGTCGTTTGTTTTC-3'

AA: 156 nt

AB: 156-82-74 nt

BB: 82-74 nt

Lewin et al. (1992)

Mitra et al. (1995)

Exon 4

F: 5’-CCAAATCCACTGAATTATGCTT-3’

R: 5’-ACAGAAATCACCTCTCTCATTCA-3

GG: 294 nt

AA: 162, 132 nt

AG: 294, 162, 132 nt

Brym et al. (2005)

determined (Dayal Das et al., 2012; ; . Although the both primer pairs indicating the same region have been studied together in these studies, the observation that the gene and genotype frequencies are found to be different can probably be explained as an experimental or sampling error.

Conclusion

In Bos taurus cattle breeds showsRsaI polymorphism in Prl exon 4 and not in exon 3. It is recommended that similar studies should indicate that the Prl/RsaI polymorphism is only in the exon 4 region, and the correct way to define these different polymorphisms in the A/G or A/B format should be defined as Prl/RsaI+/- polymorphism with the presence or absence of the RsaI restriction enzyme recognition site in the Prl gene region.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/2020.52.1.sc7

Statement of conflict of ınterest

The author confirms that he has no conflict of interest with reference to this article.

Figures

Fig. 1.
Fig. 1..

Prl gene exon 4 nucleotide sequences, studied both primer pairs and digested site by the RsaI enzyme (for DNA sequence, GenBank: AF426315.1; note that single underlined primer pairs produce 294 bp fragment, and doubled underlined primer pairs produce 156 bp fragment. GTˆAC: RsaI restriction enzyme digestion site).

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