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Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 53 issue 12 (december 2019) : 1551-1558,   Doi: 10.18805/ijar.B-3706

Genotyping of MHC class II DRB3 gene using PCR-RFLP and DNA sequencing in small ruminant breeds of Western Himalayan state of Himachal Pradesh, India

V
Varun Sankhyan
Y
Y.P. Thakur
P
P.K. Dogra
1<div style="text-align:justify">Department of Animal Genetics and Breeding, College of Veterinary and Animal Sciences,&nbsp;<br />CSK&nbsp;Himachal&nbsp;Pradesh Krishi Vishvavidyalaya, Palampur-176 062, Himachal&nbsp;Pradesh, India.</div>
Cite article:- Sankhyan Varun, Thakur Y.P., Dogra P.K. (2019). Genotyping of MHC class II DRB3 gene using PCR-RFLP and DNA sequencing in small ruminant breeds of Western Himalayan state of Himachal Pradesh, India. Indian Journal of Animal Research. 53(12): 1551-1558. doi: 10.18805/ijar.B-3706.

ABSTRACT

Sheep and goat farming especially under migratory system is an important activity in state of Himachal Pradesh. DRB locus of major histocompatibility complex is widely studied for its association with disease resistance. Present study was planned to analyse genetic diversity of DRB3 locus in small ruminant breeds of Himachal Pradesh using PCR-RFLP and DNA sequencing. 63, 68, 80 and 73 true to the breed animals belonging to Gaddi sheep, Rampur-Bushair sheep, Gaddi goats and Chegu goats, respectively were utilized. Amplification of exon 2 of DRB3 gene yielded 285bp amplified product in different breeds. Three different digestion patterns corresponding to 2 alleles and seven digestion patterns corresponding to 4 alleles were observed with PstI and HaeIII restriction digestion. For Pst 1 digestion frequency of AA, AB and BB genotypes ranged from 0.09 to 0.22, 0.40 to 0.53 and 0.28 to 0.46 in different breeds studied. For HaeIII digestion frequency of five genotypes (AA, BB, CC, AB and AC) which were detected in all breeds varied from 0.18 to 0.32, 0.11 to 0.13, 0.07 to 0.17, 0.18 to 0.30 and 0.09 to 0.3 in different breeds, while frequency of CD and AD genotype unique to Rampur-Bushair sheep population was 0.09 and 0.06, respectively. The observed allele number (No) ranged from 5 to 6 in different breeds while effective allele number (Ne) ranged from 4.46 to 5.15 in different populations. Hobs and Hexp values ranged 0.42 to 0.49 and 0.55 to 0.59, respectively in different breeds. The nucleotide variability was detected using sequences from different breeds which was found at 36 places. In the present study the specific amplification of the exon 2 of DRB3 gene of native sheep and goat populations demonstrated marked polymorphism. Further association studies need to be carried out to investigate the association of these SNPs of DRB region with parasitic diseases.

REFERENCES

  1. Ahmed, S. and Othman, O.E. (2006). A PCR-RFLP method for the analysis of Egyptian Goat. MHC Class II DRB gene. Biotech., 5: 58-61.
  2. Amills, M., Francino, O. and Sanchez, A. (1995). Nested PCR allows the characterization of TaqI and PstI RFLPs in the second exon of the caprine MHC class II DRB gene. Vet. Immunol. Immunop., 48: 313-321.
  3. Amills, M., Francino, O. and Sanchez, A. (1996). A PCR- RFLP typing method for the caprine MHC class II DRB gene. Vet. Immunol. Immunop., 55: 255-260.
  4. Ashrafi, F., Mardani, K., Hashemi, A., Darvishzadeh, R. and Farhadian, M. (2014). Association of molecular polymorphism in exon 2 of Ovar-DRB1 gene with weight traits in Iranian Makooei sheep breed. Turkish. J. Vet. Anim. Sci., 38: 126-132.
  5. Baghizadeh, A., Bahhaddini, M., Mohamadabadi, M.R. and Askari, N. (2009). Allelic Variations in Exon 2 of Caprine MHC Class II DRB3 Gene in Raeini Cashmere Goat. Ameri. Eura. J. Agric. Environ. Sci., 6(4): 454-59. 
  6. Brujeni, G.H.N., Emam, M., Mahmoudzadeh, H., Hamedmonfared, E., Talebnia, J.R. and Rezaei, H. (2009). Typing of ovar-DRB1 second exon with PCR-RFLP technique in Iranian Shaul Sheep. Iran. J. Vet. Res., 10(3:28): 250-254.
  7. Dongxiao, S. and Yuan, Z. (2004). Polymorphisms of the second exon of MHC-DRB gene in Chinese local sheep and goat. Biochem. Genet., 42: 385-390.
  8. Gruszczynska, J., Brokowska, K., Charon, K.M. and Swiderek, W.P. (2004). Restriction fragment length polymorphism of exon 2 Ovar-DRB1 gene in Polish Heath Sheep and Polish Lowland Sheep. J. App. Genet., 46: 311-314.
  9. Hernandez, T. S. (2011). DRB molecular gene polymorphism: MHC II in goats of the central highlands of Veracruz. Ph.D Thesis submitted to Faculty of Veterinary Medicine, Universidad Veracruzana Veracruz, Mexico.
  10. Hohenhaus, M.A and Outteridge, P.M. (1995). The immunogenetics of resistance to Trichostrongylus colubriformis and Haemonchus contortus parasites in sheep. Br. Vet. J., 151(2): 119-140.
  11. Jamshidi, R., Brujeni, N., Derakhshandeh, A. and Talebnia, R. (2011). Exon2 gene polymorphism in the Iranian Sangsari sheep. Int. J. Vet. Res., 5(1): 59-62.
  12. Jugo, B.M. and Vicario, A. (2000). Single-strand conformational polymorphism and sequence polymorphism of MHC-DRB in Latxa and Karrantzar sheep: Implications for Caprine phylogeny. Immunogenetics., 51: 887-897.
  13. Khobra, V., Gupta, S.C., Gopal, G.R., Narayanan, K., Prasad, A., Kumar, A., Saravanan, B.C., et al., (2013). Major Histocompatibility class II DRB3 exon 2 polymorphism and resistance to gastrointestinal nematodes in Jamunapari goats. J. Vet. Parasitology., 26(2): 108-111.
  14. Konnai, S., Nagaoka, Y., Takeshima, S., Onuma, M. and Aida, Y. (2003). Sequences and diversity of 17 new Ovar-DRB1 alleles from three breeds of sheep. Eur. J. Immunogenetics; 30: 275-282.
  15. Li, M and Zhao, S. (2005). Allelic variations in exon 2 of caprine MHC class II DRB3 gene in Chinese indigenous goats. Small Ruminant Res., 66: 236-243.
  16. Li, Y.R., Jia, B., Zhang, W.J., Zhao, Z.S., Shi, G.Q., Shen, H., Peng, Q., Lv, L.M., Zhou, Q.W. and Du, Y.C. (2010). Analysis of the relationship between MHC-DRB1 gene polymorphism and hydatidosis in Kazakh Sheep. Asian-Australas. J. Anim. Sci., 23(9): 1145-1151. 
  17. Livestock Census, (2012). 19th Livestock Census, All India Report. Ministry of Agriculture Department of Animal Husbandry, Dairying and Fisheries Krishi Bhawan, New Delhi.
  18. Outteridge, P.M., Andersson, L. and Douch, P.G.C. (1996). The PCR typing of MHC-DRB genes in the sheep using primers for an intronic microsatellite: application to nematode parasite resistance. Immunol. Cell Biol., 74(4): 330-336.
  19. Prakash, O., Kumar, P., Shrivastava, K., Thirunavukkarasu, S.B., Sahoo, N.R., Kumar, A., et al., (2014). Genetic Diversity in MHC Class II DRB3.2 Gene in Marwari Goats. Indian J. Anim. Genet. Breed. 33-34: 32-36. 
  20. Riggio, V., Matika, O., Pong-Wong, R., Stear, M.J. and Bishop, S.C. (2013). Genome-wide association and regional heritability mapping to identify loci underlying variation in nematode resistance and body weight in Scottish Blackface lambs. Heredity; 110: 420-429.
  21. Sambrook, J. and Russell, W.D. (2001). Molecular Cloning. A Laboratory Manual. 3rd edn. Vol.1 Cold Spring Harbor Laboratory Press, Cold Spring, New York. pp. 6.4-6.12.
  22. Sayers, G., Good, B., Hanrahan, J.P., Ryan, M., Angles, J.M and Sweeney, T. (2005). Major Histocompatibility Complex DRB1 gene: its role in nematode resistance in Suffolk and Texel sheep breeds. Parasito, 131(3): 403–409. 
  23. Sheikh, F.D., Bhattacharya, T.K., Kumar, P. and Sharma, A. (2006). DRB3.2 gene polymorphism and its association with pashmina production in Changthangi goat. Eur. J. Immunogenetics., DOI: 10.1111/j.1744-313X.2006.00612.x. 
  24. Shrivastava, K., Kumar, P., Sahoo, N.R, Kumar, A., Khan, M.F., Kumar, A., Prasad, A., et al., (2015). Genotyping of major histocompatibility complex Class II DRB gene in Rohilkhandi goats by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing. Vet. World., 8(10): 1183-1188.
  25. Shrivastava, K., Kumar, P., Khan, M.F, Sahoo, N.R, Sinha, R., Prakash, O., Kumar, A., et al., (2018a). Sequence Characterization of Ovine MHC Class II (DRB I Gene) in Indian Sheep Breeds. Int .J. Curr. Microbiol. App. Sci., 7(7): 490-499.
  26. Shrivastava, K., Kumar, P., Khan, M.F., Sahoo, N.R, Prakash, O., Kumar, A., Panigrahi, M., et al., (2018b). Exploring the molecular basis of resistance/ susceptibility to mixed natural infection of Haemonchus contortus in tropical Indian goat breed. Vet. Parasitol., 262:6-10
  27. Singh, P.K., Singh, S.V, Singh, M.K, Saxena, V.K, Singh, A.V. and Sohal, J.S. (2012). Genetic analysis of MHC Class II DRB gene in an endangered Jamunapari breed of goats. Indian J. Biotech., 1: 220-223. 
  28. Traherne, J.A, Horton, R., Roberts, A.N, Miretti, M.M., Hurles, M.E. and Stewart, C.A. (2006). Genetic analysis of completely sequenced disease associated MHC haplotypes identifies shuffling of segments in recent human history. PLoS Genetics: 2e9.
  29. Yadav, A.K., Tomar, S.S., Kumar, A. and Thakur, M.S. (2016). Association of caprine lymphocyte antigen-DRB3 gene with gastrointestinal nematode resistance in Sirohi and Barbari breeds of goat. Indian J. Anim. Res., 50(6): 958-963. 
  30. Yeh, F.C, Yang, R.C, Boyle-Timothy, B.J, Ye, Z.H and Mao-Judy, X. (1999). POPGENE 32 version 1.32, the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Canada. http://www.ualberta.ca/~fyeh/ fyeh. 
  31. Zhao, Y., Xu, H., Shi, L. and Zhang, J. (2011). Polymorphisms in Exon 2 of MHC Class II DRB3 Gene of 10 Domestic goats in Southwest China. Asian-Australas. J. Anim. Sci., 24(6): 752-756 
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    Research Article

    volume 53 issue 12 (december 2019) : 1551-1558,   Doi: 10.18805/ijar.B-3706

    Genotyping of MHC class II DRB3 gene using PCR-RFLP and DNA sequencing in small ruminant breeds of Western Himalayan state of Himachal Pradesh, India

    V
    Varun Sankhyan
    Y
    Y.P. Thakur
    P
    P.K. Dogra
    1<div style="text-align:justify">Department of Animal Genetics and Breeding, College of Veterinary and Animal Sciences,&nbsp;<br />CSK&nbsp;Himachal&nbsp;Pradesh Krishi Vishvavidyalaya, Palampur-176 062, Himachal&nbsp;Pradesh, India.</div>
    Cite article:- Sankhyan Varun, Thakur Y.P., Dogra P.K. (2019). Genotyping of MHC class II DRB3 gene using PCR-RFLP and DNA sequencing in small ruminant breeds of Western Himalayan state of Himachal Pradesh, India. Indian Journal of Animal Research. 53(12): 1551-1558. doi: 10.18805/ijar.B-3706.

    ABSTRACT

    Sheep and goat farming especially under migratory system is an important activity in state of Himachal Pradesh. DRB locus of major histocompatibility complex is widely studied for its association with disease resistance. Present study was planned to analyse genetic diversity of DRB3 locus in small ruminant breeds of Himachal Pradesh using PCR-RFLP and DNA sequencing. 63, 68, 80 and 73 true to the breed animals belonging to Gaddi sheep, Rampur-Bushair sheep, Gaddi goats and Chegu goats, respectively were utilized. Amplification of exon 2 of DRB3 gene yielded 285bp amplified product in different breeds. Three different digestion patterns corresponding to 2 alleles and seven digestion patterns corresponding to 4 alleles were observed with PstI and HaeIII restriction digestion. For Pst 1 digestion frequency of AA, AB and BB genotypes ranged from 0.09 to 0.22, 0.40 to 0.53 and 0.28 to 0.46 in different breeds studied. For HaeIII digestion frequency of five genotypes (AA, BB, CC, AB and AC) which were detected in all breeds varied from 0.18 to 0.32, 0.11 to 0.13, 0.07 to 0.17, 0.18 to 0.30 and 0.09 to 0.3 in different breeds, while frequency of CD and AD genotype unique to Rampur-Bushair sheep population was 0.09 and 0.06, respectively. The observed allele number (No) ranged from 5 to 6 in different breeds while effective allele number (Ne) ranged from 4.46 to 5.15 in different populations. Hobs and Hexp values ranged 0.42 to 0.49 and 0.55 to 0.59, respectively in different breeds. The nucleotide variability was detected using sequences from different breeds which was found at 36 places. In the present study the specific amplification of the exon 2 of DRB3 gene of native sheep and goat populations demonstrated marked polymorphism. Further association studies need to be carried out to investigate the association of these SNPs of DRB region with parasitic diseases.

    REFERENCES

    1. Ahmed, S. and Othman, O.E. (2006). A PCR-RFLP method for the analysis of Egyptian Goat. MHC Class II DRB gene. Biotech., 5: 58-61.
    2. Amills, M., Francino, O. and Sanchez, A. (1995). Nested PCR allows the characterization of TaqI and PstI RFLPs in the second exon of the caprine MHC class II DRB gene. Vet. Immunol. Immunop., 48: 313-321.
    3. Amills, M., Francino, O. and Sanchez, A. (1996). A PCR- RFLP typing method for the caprine MHC class II DRB gene. Vet. Immunol. Immunop., 55: 255-260.
    4. Ashrafi, F., Mardani, K., Hashemi, A., Darvishzadeh, R. and Farhadian, M. (2014). Association of molecular polymorphism in exon 2 of Ovar-DRB1 gene with weight traits in Iranian Makooei sheep breed. Turkish. J. Vet. Anim. Sci., 38: 126-132.
    5. Baghizadeh, A., Bahhaddini, M., Mohamadabadi, M.R. and Askari, N. (2009). Allelic Variations in Exon 2 of Caprine MHC Class II DRB3 Gene in Raeini Cashmere Goat. Ameri. Eura. J. Agric. Environ. Sci., 6(4): 454-59. 
    6. Brujeni, G.H.N., Emam, M., Mahmoudzadeh, H., Hamedmonfared, E., Talebnia, J.R. and Rezaei, H. (2009). Typing of ovar-DRB1 second exon with PCR-RFLP technique in Iranian Shaul Sheep. Iran. J. Vet. Res., 10(3:28): 250-254.
    7. Dongxiao, S. and Yuan, Z. (2004). Polymorphisms of the second exon of MHC-DRB gene in Chinese local sheep and goat. Biochem. Genet., 42: 385-390.
    8. Gruszczynska, J., Brokowska, K., Charon, K.M. and Swiderek, W.P. (2004). Restriction fragment length polymorphism of exon 2 Ovar-DRB1 gene in Polish Heath Sheep and Polish Lowland Sheep. J. App. Genet., 46: 311-314.
    9. Hernandez, T. S. (2011). DRB molecular gene polymorphism: MHC II in goats of the central highlands of Veracruz. Ph.D Thesis submitted to Faculty of Veterinary Medicine, Universidad Veracruzana Veracruz, Mexico.
    10. Hohenhaus, M.A and Outteridge, P.M. (1995). The immunogenetics of resistance to Trichostrongylus colubriformis and Haemonchus contortus parasites in sheep. Br. Vet. J., 151(2): 119-140.
    11. Jamshidi, R., Brujeni, N., Derakhshandeh, A. and Talebnia, R. (2011). Exon2 gene polymorphism in the Iranian Sangsari sheep. Int. J. Vet. Res., 5(1): 59-62.
    12. Jugo, B.M. and Vicario, A. (2000). Single-strand conformational polymorphism and sequence polymorphism of MHC-DRB in Latxa and Karrantzar sheep: Implications for Caprine phylogeny. Immunogenetics., 51: 887-897.
    13. Khobra, V., Gupta, S.C., Gopal, G.R., Narayanan, K., Prasad, A., Kumar, A., Saravanan, B.C., et al., (2013). Major Histocompatibility class II DRB3 exon 2 polymorphism and resistance to gastrointestinal nematodes in Jamunapari goats. J. Vet. Parasitology., 26(2): 108-111.
    14. Konnai, S., Nagaoka, Y., Takeshima, S., Onuma, M. and Aida, Y. (2003). Sequences and diversity of 17 new Ovar-DRB1 alleles from three breeds of sheep. Eur. J. Immunogenetics; 30: 275-282.
    15. Li, M and Zhao, S. (2005). Allelic variations in exon 2 of caprine MHC class II DRB3 gene in Chinese indigenous goats. Small Ruminant Res., 66: 236-243.
    16. Li, Y.R., Jia, B., Zhang, W.J., Zhao, Z.S., Shi, G.Q., Shen, H., Peng, Q., Lv, L.M., Zhou, Q.W. and Du, Y.C. (2010). Analysis of the relationship between MHC-DRB1 gene polymorphism and hydatidosis in Kazakh Sheep. Asian-Australas. J. Anim. Sci., 23(9): 1145-1151. 
    17. Livestock Census, (2012). 19th Livestock Census, All India Report. Ministry of Agriculture Department of Animal Husbandry, Dairying and Fisheries Krishi Bhawan, New Delhi.
    18. Outteridge, P.M., Andersson, L. and Douch, P.G.C. (1996). The PCR typing of MHC-DRB genes in the sheep using primers for an intronic microsatellite: application to nematode parasite resistance. Immunol. Cell Biol., 74(4): 330-336.
    19. Prakash, O., Kumar, P., Shrivastava, K., Thirunavukkarasu, S.B., Sahoo, N.R., Kumar, A., et al., (2014). Genetic Diversity in MHC Class II DRB3.2 Gene in Marwari Goats. Indian J. Anim. Genet. Breed. 33-34: 32-36. 
    20. Riggio, V., Matika, O., Pong-Wong, R., Stear, M.J. and Bishop, S.C. (2013). Genome-wide association and regional heritability mapping to identify loci underlying variation in nematode resistance and body weight in Scottish Blackface lambs. Heredity; 110: 420-429.
    21. Sambrook, J. and Russell, W.D. (2001). Molecular Cloning. A Laboratory Manual. 3rd edn. Vol.1 Cold Spring Harbor Laboratory Press, Cold Spring, New York. pp. 6.4-6.12.
    22. Sayers, G., Good, B., Hanrahan, J.P., Ryan, M., Angles, J.M and Sweeney, T. (2005). Major Histocompatibility Complex DRB1 gene: its role in nematode resistance in Suffolk and Texel sheep breeds. Parasito, 131(3): 403–409. 
    23. Sheikh, F.D., Bhattacharya, T.K., Kumar, P. and Sharma, A. (2006). DRB3.2 gene polymorphism and its association with pashmina production in Changthangi goat. Eur. J. Immunogenetics., DOI: 10.1111/j.1744-313X.2006.00612.x. 
    24. Shrivastava, K., Kumar, P., Sahoo, N.R, Kumar, A., Khan, M.F., Kumar, A., Prasad, A., et al., (2015). Genotyping of major histocompatibility complex Class II DRB gene in Rohilkhandi goats by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing. Vet. World., 8(10): 1183-1188.
    25. Shrivastava, K., Kumar, P., Khan, M.F, Sahoo, N.R, Sinha, R., Prakash, O., Kumar, A., et al., (2018a). Sequence Characterization of Ovine MHC Class II (DRB I Gene) in Indian Sheep Breeds. Int .J. Curr. Microbiol. App. Sci., 7(7): 490-499.
    26. Shrivastava, K., Kumar, P., Khan, M.F., Sahoo, N.R, Prakash, O., Kumar, A., Panigrahi, M., et al., (2018b). Exploring the molecular basis of resistance/ susceptibility to mixed natural infection of Haemonchus contortus in tropical Indian goat breed. Vet. Parasitol., 262:6-10
    27. Singh, P.K., Singh, S.V, Singh, M.K, Saxena, V.K, Singh, A.V. and Sohal, J.S. (2012). Genetic analysis of MHC Class II DRB gene in an endangered Jamunapari breed of goats. Indian J. Biotech., 1: 220-223. 
    28. Traherne, J.A, Horton, R., Roberts, A.N, Miretti, M.M., Hurles, M.E. and Stewart, C.A. (2006). Genetic analysis of completely sequenced disease associated MHC haplotypes identifies shuffling of segments in recent human history. PLoS Genetics: 2e9.
    29. Yadav, A.K., Tomar, S.S., Kumar, A. and Thakur, M.S. (2016). Association of caprine lymphocyte antigen-DRB3 gene with gastrointestinal nematode resistance in Sirohi and Barbari breeds of goat. Indian J. Anim. Res., 50(6): 958-963. 
    30. Yeh, F.C, Yang, R.C, Boyle-Timothy, B.J, Ye, Z.H and Mao-Judy, X. (1999). POPGENE 32 version 1.32, the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Canada. http://www.ualberta.ca/~fyeh/ fyeh. 
    31. Zhao, Y., Xu, H., Shi, L. and Zhang, J. (2011). Polymorphisms in Exon 2 of MHC Class II DRB3 Gene of 10 Domestic goats in Southwest China. Asian-Australas. J. Anim. Sci., 24(6): 752-756 
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