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Research Article

volume 53 issue 9 (september 2019) : 1172-1180,   Doi: 10.18805/ijar.B-3594

Identification of Single Nucleotide Polymorphism in GnRHR gene in Murrah bulls

J
J.K. Reen
K
K.P. Ramesha
P
Preeti
D
D. Revanasiddu
M
M. Ahirwar
1Genetics Laboratory, Dairy Production Section, ICAR - National Dairy Research Institute, Southern Regional Station, Adugodi, Bengaluru-560 030, Karnataka, India.
Cite article:- Reen J.K., Ramesha K.P., Preeti, Revanasiddu D., Ahirwar M. (2018). Identification of Single Nucleotide Polymorphism in GnRHR gene in Murrah bulls. Indian Journal of Animal Research. 53(9): 1172-1180. doi: 10.18805/ijar.B-3594.

ABSTRACT

The present study was undertaken with the objectives of molecular characterization and detection of genetic polymorphism in the GnRHR gene in Murrah bulls. Blood samples were collected from 109 Murrah bulls maintained at three different organized semen stations viz., Centralized Semen Collection Centre of Livestock Breeding and Training Centre, Dharwad; Nandini Sperm Station and State Livestock Breeding and Training Centre, Hessarghatta, Bengaluru, Karnataka, India. Genomic DNA was isolated from the blood samples within 24 hours of collection . Mutations were screened using Polymerase Chain Reaction – Single Stranded Conformational Polymorphism (PCR-SSCP) technique followed by Sanger Sequencing. PCR-SSCP method revealed similar band pattern within Murrah bulls. To confirm monomorphism in the studied population with respect to GnRHR gene, duplicate samples from each primer fragment were custom sequenced. PCR-SSCP and sequence analysis revealed monomorphism within the studied population that is coding region as well as exon-intron boundaries of GnRHR gene is highly conserved among Murrah bulls. However, a total of 28 Single Nucleotide variations (SNV’s) have been found when compared with Bos Taurus reference sequence for GnRHR gene.

REFERENCES

  1. Albertson, A.J., Navratil, A., Mignot, M., Dufourny, L., Cherrington, B. and Skinner, D.C. (2008). Immunoreactive GnRH type I receptors in the mouse and sheep brain. J. Chem. Neuroanat. 35(4): 326-333.
  2. BAHS, (2017). Basic Animal Husbandry Statistics.
  3. Bedecarrats, G.Y., Linher, K.D., Janovick, J.A., Beranova, M., Kada, F., Seminara, S.B. and Kaiser, U.B. (2003). Four naturally occurring mutations in the human GnRH receptor affect ligand binding and receptor function. Mol. Cell. Endocrinol 205(1): 51-64.
  4. Ferreire, L.F.G., Trarbach, E.B. and Latronico, A.C. (2010). Genetics basis for GnRH-dependent pubertal disorders in humans. Mol. Cell. Endocrinol. 324(1): 30-38.
  5. Huang, S.Y., Chen, M.Y., Lin, E.C., Tsou, H.L., Kuo, Y.H., Ju, C.C. and Lee, W.C. (2002). Effects of single nucleotide polymorphisms in the 5-flanking region of heat shock protein 70.2 gene on semen quality in boars. Anim. Reprod. Sci. 70: 99-109.
  6. Huhtaniemi, I. and Alevizaki, M. (2007). Mutations along the hypothalamic–pituitary–gonadal axis affecting male reproduction. Reprod. Biomed. Online. 15(6): 622-632.
  7. Kottler, M.L., Counis, R. and Bouchard, P. (1999). Mutations of the GnRH receptor gene: a new cause of autosomal-recessive hypogonadotropic hypogonadism. Med. Res. Arch. 30(6): 481-485.
  8. Kumar, S., Ganguly, I., Deb, R., Singh, U., Mandal, D.K., Mann, S. and Sharma, A. (2015).Elucidating the role of bovine GnRHR on semen quality traits among crossbred bull. J. Anim. Sci. 85(12): 1314–1317.
  9. Lin, C.L., Ponsuksili, S., Tholen, E., Jennen, D.G., Schellander, K. and Wimmers, K. (2006) Candidate gene markers for sperm quality and fertility of boar. Anim. Reprod. Sci. 92(3): 349-363.
  10. Milazzotto, M.P., Rahal, P., Nichi, M., MirandaNeto, T., Teixeira, L.A., Ferraz, J.B.S., Eler, J.P., Campagnari, F. and Garcia, J.F. (2008). New molecular variants of hypothalamus–pituitary-gonad axis genes and their association with early puberty phenotype in Bos taurus indicus (Nellore). Livest. Sci. 114: 274–79.
  11. Miller, S.A., Dykes, D., Polesky, H.F. (1988). A sample salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16(3): 1215.
  12. Noar, Z. (2009). Signaling by G-protein-coupled receptor (GPCR): studies on the GnRH receptor. Front. Neuroendocrinol. 30(1): 10-29.
  13. Rosenthal, I.M., Refetoff, S., Rich, B.A., Barnes, R.B., Sunthornthepvarakul, T.H., Parma, J. and Rosenfield, R.L. (1996). Response to challenge with gonadotropin-releasing hormone agonist in a mother and her two sons with a constitutively activating mutation of the luteinizing hormone receptor-a clinical research center study. J. Clin. Endocrinol. Metab. 81(10): 3802-    3806.
  14. Sambrook, J. and Russell, D.W. (2001). Molecular Cloning. A Laboratory Manual. 3rd ed. Cold Spring Harbor NY, USA : Cold Spring Laboratory Press.
  15. Tamaya, T. (2002). Normosmic idiopathic hypogonadotropic hypogonadism with GnRH receptor mutation (review). Nippon. Rinsho. 60(2): 319-324.
  16. Wang, L., Fan, J., Yu, M., Zheng, S. and Zhao, Y. (2011). Association of goat (Capra hircus) CD4 gene exon 6 polymorphisms with ability of sperm internalizing exogenous DNA. Mol. Biol. Rep, 38(3): 1621-1628.
  17. Wimmers, K., Lin C.L., Tholen, E. and Jennen, D.G. (2005). Polymorphisms in candidate genes as markers for sperm quality and boar fertility. Anim. Genet. 36: 152-155.
  18. Wise, M.E., Nieman, D., Stewart, J. and Nett, T.M. (1984). Effect of number of receptors for gonadotropin-releasing hormone on the release of luteinizing hormone. Biol. Reprod. 31: 1007-1013.
  19. Yang, W.C., Li, S.J., Xie, Y.H., Tang, K.Q., Hua, G.H., Zhang, C.Y. and Yang, L.G. (2011). Two novel SNPs of the type I gonadotropin releasing hormone receptor gene and their associations with superovulation traits in Chinese Holstein cows. Livest. Sci. 136(2): 164-168. 
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Indian Journal of Animal Research
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    Research Article

    volume 53 issue 9 (september 2019) : 1172-1180,   Doi: 10.18805/ijar.B-3594

    Identification of Single Nucleotide Polymorphism in GnRHR gene in Murrah bulls

    J
    J.K. Reen
    K
    K.P. Ramesha
    P
    Preeti
    D
    D. Revanasiddu
    M
    M. Ahirwar
    1Genetics Laboratory, Dairy Production Section, ICAR - National Dairy Research Institute, Southern Regional Station, Adugodi, Bengaluru-560 030, Karnataka, India.
    Cite article:- Reen J.K., Ramesha K.P., Preeti, Revanasiddu D., Ahirwar M. (2018). Identification of Single Nucleotide Polymorphism in GnRHR gene in Murrah bulls. Indian Journal of Animal Research. 53(9): 1172-1180. doi: 10.18805/ijar.B-3594.

    ABSTRACT

    The present study was undertaken with the objectives of molecular characterization and detection of genetic polymorphism in the GnRHR gene in Murrah bulls. Blood samples were collected from 109 Murrah bulls maintained at three different organized semen stations viz., Centralized Semen Collection Centre of Livestock Breeding and Training Centre, Dharwad; Nandini Sperm Station and State Livestock Breeding and Training Centre, Hessarghatta, Bengaluru, Karnataka, India. Genomic DNA was isolated from the blood samples within 24 hours of collection . Mutations were screened using Polymerase Chain Reaction – Single Stranded Conformational Polymorphism (PCR-SSCP) technique followed by Sanger Sequencing. PCR-SSCP method revealed similar band pattern within Murrah bulls. To confirm monomorphism in the studied population with respect to GnRHR gene, duplicate samples from each primer fragment were custom sequenced. PCR-SSCP and sequence analysis revealed monomorphism within the studied population that is coding region as well as exon-intron boundaries of GnRHR gene is highly conserved among Murrah bulls. However, a total of 28 Single Nucleotide variations (SNV’s) have been found when compared with Bos Taurus reference sequence for GnRHR gene.

    REFERENCES

    1. Albertson, A.J., Navratil, A., Mignot, M., Dufourny, L., Cherrington, B. and Skinner, D.C. (2008). Immunoreactive GnRH type I receptors in the mouse and sheep brain. J. Chem. Neuroanat. 35(4): 326-333.
    2. BAHS, (2017). Basic Animal Husbandry Statistics.
    3. Bedecarrats, G.Y., Linher, K.D., Janovick, J.A., Beranova, M., Kada, F., Seminara, S.B. and Kaiser, U.B. (2003). Four naturally occurring mutations in the human GnRH receptor affect ligand binding and receptor function. Mol. Cell. Endocrinol 205(1): 51-64.
    4. Ferreire, L.F.G., Trarbach, E.B. and Latronico, A.C. (2010). Genetics basis for GnRH-dependent pubertal disorders in humans. Mol. Cell. Endocrinol. 324(1): 30-38.
    5. Huang, S.Y., Chen, M.Y., Lin, E.C., Tsou, H.L., Kuo, Y.H., Ju, C.C. and Lee, W.C. (2002). Effects of single nucleotide polymorphisms in the 5-flanking region of heat shock protein 70.2 gene on semen quality in boars. Anim. Reprod. Sci. 70: 99-109.
    6. Huhtaniemi, I. and Alevizaki, M. (2007). Mutations along the hypothalamic–pituitary–gonadal axis affecting male reproduction. Reprod. Biomed. Online. 15(6): 622-632.
    7. Kottler, M.L., Counis, R. and Bouchard, P. (1999). Mutations of the GnRH receptor gene: a new cause of autosomal-recessive hypogonadotropic hypogonadism. Med. Res. Arch. 30(6): 481-485.
    8. Kumar, S., Ganguly, I., Deb, R., Singh, U., Mandal, D.K., Mann, S. and Sharma, A. (2015).Elucidating the role of bovine GnRHR on semen quality traits among crossbred bull. J. Anim. Sci. 85(12): 1314–1317.
    9. Lin, C.L., Ponsuksili, S., Tholen, E., Jennen, D.G., Schellander, K. and Wimmers, K. (2006) Candidate gene markers for sperm quality and fertility of boar. Anim. Reprod. Sci. 92(3): 349-363.
    10. Milazzotto, M.P., Rahal, P., Nichi, M., MirandaNeto, T., Teixeira, L.A., Ferraz, J.B.S., Eler, J.P., Campagnari, F. and Garcia, J.F. (2008). New molecular variants of hypothalamus–pituitary-gonad axis genes and their association with early puberty phenotype in Bos taurus indicus (Nellore). Livest. Sci. 114: 274–79.
    11. Miller, S.A., Dykes, D., Polesky, H.F. (1988). A sample salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16(3): 1215.
    12. Noar, Z. (2009). Signaling by G-protein-coupled receptor (GPCR): studies on the GnRH receptor. Front. Neuroendocrinol. 30(1): 10-29.
    13. Rosenthal, I.M., Refetoff, S., Rich, B.A., Barnes, R.B., Sunthornthepvarakul, T.H., Parma, J. and Rosenfield, R.L. (1996). Response to challenge with gonadotropin-releasing hormone agonist in a mother and her two sons with a constitutively activating mutation of the luteinizing hormone receptor-a clinical research center study. J. Clin. Endocrinol. Metab. 81(10): 3802-    3806.
    14. Sambrook, J. and Russell, D.W. (2001). Molecular Cloning. A Laboratory Manual. 3rd ed. Cold Spring Harbor NY, USA : Cold Spring Laboratory Press.
    15. Tamaya, T. (2002). Normosmic idiopathic hypogonadotropic hypogonadism with GnRH receptor mutation (review). Nippon. Rinsho. 60(2): 319-324.
    16. Wang, L., Fan, J., Yu, M., Zheng, S. and Zhao, Y. (2011). Association of goat (Capra hircus) CD4 gene exon 6 polymorphisms with ability of sperm internalizing exogenous DNA. Mol. Biol. Rep, 38(3): 1621-1628.
    17. Wimmers, K., Lin C.L., Tholen, E. and Jennen, D.G. (2005). Polymorphisms in candidate genes as markers for sperm quality and boar fertility. Anim. Genet. 36: 152-155.
    18. Wise, M.E., Nieman, D., Stewart, J. and Nett, T.M. (1984). Effect of number of receptors for gonadotropin-releasing hormone on the release of luteinizing hormone. Biol. Reprod. 31: 1007-1013.
    19. Yang, W.C., Li, S.J., Xie, Y.H., Tang, K.Q., Hua, G.H., Zhang, C.Y. and Yang, L.G. (2011). Two novel SNPs of the type I gonadotropin releasing hormone receptor gene and their associations with superovulation traits in Chinese Holstein cows. Livest. Sci. 136(2): 164-168. 
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