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The Relationship of POU1F1-HinfI Gene Polymorphisms on Milk Yields in Simmental Cattle

Year 2023, Volume: 33 Issue: 4, 513 - 521, 31.12.2023
https://doi.org/10.29133/yyutbd.1271873

Abstract

As the global population grows, there is a need to produce higher yields in food, agriculture, and livestock. To achieve this, scientists are exploring new techniques and methods. However, it is crucial to select the right candidate genes and markers, especially in techniques like QTL and MAS in livestock, to ensure success. We conducted a study to determine allele frequencies and their association with milk yield in 70 Simmental cattle breeds in two lactations using the PCR-RFLP technique. The statistical analysis was conducted using the general linear model procedure with the least square method The study focused on the allele frequencies for the POU1F1-HinfI gene in Simmental cattle. The dominant B allele frequency for POU1F1/Hinf1 was 0.58, while the A allele frequency was 0.42. Simmental population was under HardyWeinberg Equilibrium (HWE) for the POU1F1-HinfI genotypes (p>0.05) The study found that genotype frequencies were in balance for POU1F1/Hinf1. No significant correlation between POU1F1-HinfI gene polymorphisms and milk yield was found, but they have been associated with growth and reproductive traits in various cattle breeds. The results could provide useful information for breeding programs aimed at improving the performance traits of Simmental cattle.

References

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  • Akers, R. M. (2006). Major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows. Journal of Dairy Science, 89(4), 1222-1234.
  • Aytekin, İ., & Fadhıl, M. (2022). Pit-1 Gene Polymorphisms in Anatolian Black, Holstein Friesian, Brown Swiss and Simmental Cattle Reared in Turkey. Journal of Agricultural Sciences, 28(1), 139-144.
  • Cañizares-Martínez, M. A., Parra-Bracamonte, G. M., Segura-Correa, J. C., & Magaña-Monforte, J. G. (2021). Effect of Leptin, Pituitary Transcription Factor and Luteinizing Hormone Receptor Genes Polymorphisms on Reproductive Traits and Milk Yield in Holstein Cattle. Brazilian Archives of Biology and Technology, 64, 1–11. https://doi.org/10.1590/1678-4324-2021190643
  • Carrijo, S. M., Alencar, M. M. D., Toral, F. L. B., & Regitano, L. C. D. A. (2008). Association of PIT1 genotypes with growth traits in Canchim cattle. Scientia Agricola, 65, 116-121.
  • Chauhan, A., Tiwari, M., Singh, S. P., Sharma, D., Kumar, S., Goel, R., & Singh, V. (2015). Association of PIT-1 gene polymorphism with milk production traits in Sahiwal cattle. Indian Journal of Animal Sciences, 85(6), 610-612.
  • Çobanoğlu, Ö., & Ardicli, S. (2022). Genetic variation at the OLR1, ANXA9, MYF5, LTF, IGF1, LGB, CSN3, PIT1, MBL1, CACNA2D1, and ABCG2 loci in Turkish Grey Steppe, Anatolian Black, and EastAnatolian Red cattle. Turkish Journal of Veterinary & Animal Sciences, 46(3), 494-504. https://doi.org/10.55730/1300-0128.4198
  • Das, D. N., Karuthadurai, T., & Gnanasekaran, S. (2021). Genomic selection: a molecular tool for genetic improvement in livestock. In Advances in Animal Genomics (pp. 141-163).
  • Dierkes, B., Kriegesmann, B., Baumgartner, B. G., & Brenig, B. (1998). Partial genomic structure of the bovine PIT1 gene and characterization of a HinfI transition polymorphism in exon 6. Animal Genetics. 29(5), 405-405.
  • Ebrahimi Hoseinzadeh, Z., Mohammadabadi, M. R., Esmailizadeh, A. K., & Khezri, A. (2015). Association of PIT1 gene and milk protein percentage in Holstein cattle. Journal of Livestock Science and Technologies, 3(1), 40-49.
  • Fındık, I., & Özdemir, M. (2022). Genetic polymorphism of Pit-1 and CSN3 genes in Holstein calves and its associations with calf birth weight. Archives Animal Breeding, 65(3), 285–292. https://doi.org/10.5194/aab-65-285-2022
  • Francis, C. Y., Rong, C. Y., & Boyle, T. (1999). POPGENE, Microsoft Window-based freeware for population genetic analysis. Version 1.31. Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, Canada.
  • Grossi, D. D. A., Buzanskas, M. E., Grupioni, N. V., de Paz, C. C. P., Regitano, L. C. D. A., de Alencar, M. M., . & Munari, D. P. (2015). Effect of IGF1, GH, and PIT1 markers on the genetic parameters of growth and reproduction traits in Canchim cattle. Molecular Biology Reports, 42, 245-251. https://doi.org/10.1007/s11033-014-3767-4
  • Hartati, H., Anwar, S., & Soewandi, B. D. P. (2018). Genetic polymorphism of Pit-1- HinfI gene in Grati-Ongole Grade cattle at Indonesian Beef Cattle Research Station. Journal of the Indonesian Tropical Animal Agriculture, 43(4), 315-322. https://doi.org/10.14710/jitaa.43.4.315-322
  • Heidari, M., Azari, M., Hasani, S., Khanahmadi, A., & Zerehdaran, S. (2012). Effect of polymorphic variants of GH, Pit-1, and β-LG genes on milk production of Holstein cows. Russian Journal of Genetics, 48(4), 417–421. https://doi.org/10.1134/S1022795412040060
  • IBM Corp. Released (2017). IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.
  • Jakaria, J., & Noor, R. R. (2015). Identification of a single nucleotide polymorphism at Hinf-1 enzyme restriction site of Pit-1 gene on Indonesian Bali cattle population. Media Peternakan, 38(2), 104-109. https://doi.org/10.5398/medpet.2015.38.2.104
  • Kai, X. U. E., Hong, C. H. E. N., Shan, W. A. N. G., Xin, C. A. I., Bo, L. I. U., Zhang, C. F., & Hui, N. I. U. (2006). Effect of genetic variations of the POU1F1 gene on growth traits of Nanyang cattle. Acta Genetica Sinica, 33(10), 901-907.
  • Khatib, H., Huang, W., Wang, X., Tran, A. H., Bindrim, A. B., Schutzkus, V., ... & Yandell, B. S. (2009). Single gene and gene interaction effects on fertilization and embryonic survival rates in cattle. Journal of Dairy Science, 92(5), 2238-2247. https://doi.org/10.3168/jds.2008-1767
  • Laporta, J., Driver, A., & Khatib, H. (2011). Expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos. Journal of Dairy Science, 94(8), 4220-4223.
  • Li, S., Crenshaw III, E. B., Rawson, E. J., Simmons, D. M., Swanson, L. W., & Rosenfeld, M. G. (1990). Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature, 347(6293), 528-533.
  • Mattos, K. K. D., Del Lama, S. N., Martinez, M. L., & Freitas, A. F. (2004). Association of bGH and Pit-1 gene variants with milk production traits in dairy Gyr bulls. Pesquisa Agropecuária Brasileira, 39, 147-150. https://doi.org/10.1590/s0100-204x2004000200007
  • Moravčíková, N., Trakovická, A., Miluchová, M., Bujko, J., & Navrátilová, A. (2021). HinfI polymorphism of PIT-1 gene in Slovak spotted cattle. Journal of Microbiology, Biotechnology and Food Sciences, 2(1), 1883-1890.
  • Mullis, P. E. (2007). Genetics of growth hormone deficiency. Endocrinology and Metabolism Clinics, 36(1), 17-36.
  • Oprzadek, J., & Flisikowski, K. (2003). Polymorphisms at loci of leptin (LEP), Pit1 and STAT5A and their association with growth, feed conversion. Animal Science Papers and Reports, 21(3), 135-145.
  • Ozdemir M, Sonmez Z, Aksakal V. (2021). Associations between PRL/RsaI polymorphism and some performance traits in Holstein cattle reared under organic condition. J Anim Plant Sci, 31(3), 900-905. doi: 10.36899/ JAPS. 3.0279
  • Ozdemir, M. (2012). Determination of Pit-1/Hinf1 polymorphism in Holstein and Native Ear cattle raised as genetic resource in Turkey. J. Anim. Plant Sci, 22(1), 25-28.
  • Ozdemir, M., Topal, M., & Aksakal, V. (2018). The relationships between performance traits and the bGH/Alu I and Pit-1/Hinf I polymorphisms in Holstein cows. Indian Journal of Animal Research, 52(2), 186-191. https://doi.org/10.18805/ijar.v0iOF.8495
  • Pytlewski, J., Antkowiak, I. R., & Czerniawska-Piątkowska, E. (2022). Polymorphism of PIT-1 Genes and its Relationship with Traits in the Limousine Cattle Breed. Pakistan Journal of Zoology, 54(2), 599–605. https://doi.org/10.17582/journal.pjz/20191116151125
  • Pytlewski, J., Antkowiak, I., & Czerniawska-Piatkowska, E. (2018). Relationship of PIT-1 gene polymorphism with breeding parameters and body weights of cows and calves. Pakistan Journal of Zoology, 50(1), 183–187. https://doi.org/10.17582/journal.pjz/2018.50.1.183.187
  • Renaville, R., Gengler, N., Vrech, E., Prandi, A., Massart, S., Corradini, C., & Portetelle, D. (1997). Pit-1 gene polymorphism, milk yield, and conformation traits for Italian Holstein-Friesian bulls. Journal of Dairy Science, 80(12), 3431-3438.
  • Reshma, R. S., & Das, D. N. (2021). Molecular markers and its application in animal breeding. In Advances in Animal Genomics, (pp. 123-140). Academic Press.
  • Sadeghi, M., Mokhber, M., & Shahrbabak, M. M. (2022). Genetic Variation in Hypothalamic-Pituitary Axis Candidate Genes and Their Effects on Milk Production Traits in Iranian Holstein Cattle. Russian Journal of Genetics, 58(11), 1393-1400. https://doi.org/10.1134/S1022795422110096
  • Sakar, Ç. M., & Zülkadir, U. (2022). Determination of the relationship between Anatolian black cattle growth properties and myostatin, GHR and Pit-1 gene. Animal Biotechnology, 33(3), 536-545. https://doi.org/10.1080/10495398.2021.1884566
  • Silva, M. V. G. B. D., Martinez, M. L., Machado, M. A., Nascimento, C. S. D., Campos, A. L., Guimarães, M. F. M., & Lui, J. F. (2006). Genes do eixo somatotrófico e características de crescimento numa população F2 de bovinos. Pesquisa Agropecuária Brasileira, 41, 981-986. Sjaunja, K. S. & Olsson, K. (2005). Endocrinology of milk production. Domestic Animal Endocrinology, 29(2), 241-258.
  • Thuy, N. T. D., Thu, N. T., Cuong, N. H., Ty, L. V., Nguyen, T. T. B., & Khoa, D. V. A. (2018). Polymorphism of PIT-1 and prolactin genes and their effects on milk yield in Holstein Frisian dairy cows bred in Vietnam. Russian Journal of Genetics, 54, 346-352. https://doi.org/10.1134/S1022795418030146
  • Trakovická, A., Moravčíková, N., Minarovič, T., & Navrátilová, A. (2015). SNPs analyses of the bovine LEP and PIT-1 genes by multiplex PCR-RFLP method and their effect on milk performance traits in Slovak Simmental cattle. Journal of Central European Agriculture. 16(1), 65-75. https://doi.org/10.5513/JCEA01/16.1.1542
  • Zghairand, Z. S., & Hassooni, H. A. (2021). The Relationship Between The PIT-1 Gene at The Rate of Daily Milk Production and Some of Its Components in Buffaloes. In IOP Conference Series: Earth and Environmental Science 923(1), 012-029. https://doi.org/10.1088/1755-1315/923/1/012029
  • Zhang, C., Liu, B., Chen, H., Lan, X., Lei, C., Zhang, Z., & Zhang, R. (2009). Associations of a Hinf I PCR-RFLP of POU1F1 gene with growth traits in Qinchuan cattle. Animal Biotechnology, 20(2), 71-74. https://doi.org/10.1080/10495390802640462, 2009
Year 2023, Volume: 33 Issue: 4, 513 - 521, 31.12.2023
https://doi.org/10.29133/yyutbd.1271873

Abstract

References

  • Xue, K., Chen, H., Wang, S., Cai, X., Liu, B., Zhang, C.F., Lei, C. Z., Wang, X.Z., Wang, Y.M., and Niu, H. (2006). Effect of genetic variations of the POU1F1 gene on growth traits of Nanyang cattle. Acta Genetica Sinica, 33, 901–907. https://doi.org/10.1016/S0379-4172(06)60124-8
  • Akers, R. M. (2006). Major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows. Journal of Dairy Science, 89(4), 1222-1234.
  • Aytekin, İ., & Fadhıl, M. (2022). Pit-1 Gene Polymorphisms in Anatolian Black, Holstein Friesian, Brown Swiss and Simmental Cattle Reared in Turkey. Journal of Agricultural Sciences, 28(1), 139-144.
  • Cañizares-Martínez, M. A., Parra-Bracamonte, G. M., Segura-Correa, J. C., & Magaña-Monforte, J. G. (2021). Effect of Leptin, Pituitary Transcription Factor and Luteinizing Hormone Receptor Genes Polymorphisms on Reproductive Traits and Milk Yield in Holstein Cattle. Brazilian Archives of Biology and Technology, 64, 1–11. https://doi.org/10.1590/1678-4324-2021190643
  • Carrijo, S. M., Alencar, M. M. D., Toral, F. L. B., & Regitano, L. C. D. A. (2008). Association of PIT1 genotypes with growth traits in Canchim cattle. Scientia Agricola, 65, 116-121.
  • Chauhan, A., Tiwari, M., Singh, S. P., Sharma, D., Kumar, S., Goel, R., & Singh, V. (2015). Association of PIT-1 gene polymorphism with milk production traits in Sahiwal cattle. Indian Journal of Animal Sciences, 85(6), 610-612.
  • Çobanoğlu, Ö., & Ardicli, S. (2022). Genetic variation at the OLR1, ANXA9, MYF5, LTF, IGF1, LGB, CSN3, PIT1, MBL1, CACNA2D1, and ABCG2 loci in Turkish Grey Steppe, Anatolian Black, and EastAnatolian Red cattle. Turkish Journal of Veterinary & Animal Sciences, 46(3), 494-504. https://doi.org/10.55730/1300-0128.4198
  • Das, D. N., Karuthadurai, T., & Gnanasekaran, S. (2021). Genomic selection: a molecular tool for genetic improvement in livestock. In Advances in Animal Genomics (pp. 141-163).
  • Dierkes, B., Kriegesmann, B., Baumgartner, B. G., & Brenig, B. (1998). Partial genomic structure of the bovine PIT1 gene and characterization of a HinfI transition polymorphism in exon 6. Animal Genetics. 29(5), 405-405.
  • Ebrahimi Hoseinzadeh, Z., Mohammadabadi, M. R., Esmailizadeh, A. K., & Khezri, A. (2015). Association of PIT1 gene and milk protein percentage in Holstein cattle. Journal of Livestock Science and Technologies, 3(1), 40-49.
  • Fındık, I., & Özdemir, M. (2022). Genetic polymorphism of Pit-1 and CSN3 genes in Holstein calves and its associations with calf birth weight. Archives Animal Breeding, 65(3), 285–292. https://doi.org/10.5194/aab-65-285-2022
  • Francis, C. Y., Rong, C. Y., & Boyle, T. (1999). POPGENE, Microsoft Window-based freeware for population genetic analysis. Version 1.31. Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, Canada.
  • Grossi, D. D. A., Buzanskas, M. E., Grupioni, N. V., de Paz, C. C. P., Regitano, L. C. D. A., de Alencar, M. M., . & Munari, D. P. (2015). Effect of IGF1, GH, and PIT1 markers on the genetic parameters of growth and reproduction traits in Canchim cattle. Molecular Biology Reports, 42, 245-251. https://doi.org/10.1007/s11033-014-3767-4
  • Hartati, H., Anwar, S., & Soewandi, B. D. P. (2018). Genetic polymorphism of Pit-1- HinfI gene in Grati-Ongole Grade cattle at Indonesian Beef Cattle Research Station. Journal of the Indonesian Tropical Animal Agriculture, 43(4), 315-322. https://doi.org/10.14710/jitaa.43.4.315-322
  • Heidari, M., Azari, M., Hasani, S., Khanahmadi, A., & Zerehdaran, S. (2012). Effect of polymorphic variants of GH, Pit-1, and β-LG genes on milk production of Holstein cows. Russian Journal of Genetics, 48(4), 417–421. https://doi.org/10.1134/S1022795412040060
  • IBM Corp. Released (2017). IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.
  • Jakaria, J., & Noor, R. R. (2015). Identification of a single nucleotide polymorphism at Hinf-1 enzyme restriction site of Pit-1 gene on Indonesian Bali cattle population. Media Peternakan, 38(2), 104-109. https://doi.org/10.5398/medpet.2015.38.2.104
  • Kai, X. U. E., Hong, C. H. E. N., Shan, W. A. N. G., Xin, C. A. I., Bo, L. I. U., Zhang, C. F., & Hui, N. I. U. (2006). Effect of genetic variations of the POU1F1 gene on growth traits of Nanyang cattle. Acta Genetica Sinica, 33(10), 901-907.
  • Khatib, H., Huang, W., Wang, X., Tran, A. H., Bindrim, A. B., Schutzkus, V., ... & Yandell, B. S. (2009). Single gene and gene interaction effects on fertilization and embryonic survival rates in cattle. Journal of Dairy Science, 92(5), 2238-2247. https://doi.org/10.3168/jds.2008-1767
  • Laporta, J., Driver, A., & Khatib, H. (2011). Expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos. Journal of Dairy Science, 94(8), 4220-4223.
  • Li, S., Crenshaw III, E. B., Rawson, E. J., Simmons, D. M., Swanson, L. W., & Rosenfeld, M. G. (1990). Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature, 347(6293), 528-533.
  • Mattos, K. K. D., Del Lama, S. N., Martinez, M. L., & Freitas, A. F. (2004). Association of bGH and Pit-1 gene variants with milk production traits in dairy Gyr bulls. Pesquisa Agropecuária Brasileira, 39, 147-150. https://doi.org/10.1590/s0100-204x2004000200007
  • Moravčíková, N., Trakovická, A., Miluchová, M., Bujko, J., & Navrátilová, A. (2021). HinfI polymorphism of PIT-1 gene in Slovak spotted cattle. Journal of Microbiology, Biotechnology and Food Sciences, 2(1), 1883-1890.
  • Mullis, P. E. (2007). Genetics of growth hormone deficiency. Endocrinology and Metabolism Clinics, 36(1), 17-36.
  • Oprzadek, J., & Flisikowski, K. (2003). Polymorphisms at loci of leptin (LEP), Pit1 and STAT5A and their association with growth, feed conversion. Animal Science Papers and Reports, 21(3), 135-145.
  • Ozdemir M, Sonmez Z, Aksakal V. (2021). Associations between PRL/RsaI polymorphism and some performance traits in Holstein cattle reared under organic condition. J Anim Plant Sci, 31(3), 900-905. doi: 10.36899/ JAPS. 3.0279
  • Ozdemir, M. (2012). Determination of Pit-1/Hinf1 polymorphism in Holstein and Native Ear cattle raised as genetic resource in Turkey. J. Anim. Plant Sci, 22(1), 25-28.
  • Ozdemir, M., Topal, M., & Aksakal, V. (2018). The relationships between performance traits and the bGH/Alu I and Pit-1/Hinf I polymorphisms in Holstein cows. Indian Journal of Animal Research, 52(2), 186-191. https://doi.org/10.18805/ijar.v0iOF.8495
  • Pytlewski, J., Antkowiak, I. R., & Czerniawska-Piątkowska, E. (2022). Polymorphism of PIT-1 Genes and its Relationship with Traits in the Limousine Cattle Breed. Pakistan Journal of Zoology, 54(2), 599–605. https://doi.org/10.17582/journal.pjz/20191116151125
  • Pytlewski, J., Antkowiak, I., & Czerniawska-Piatkowska, E. (2018). Relationship of PIT-1 gene polymorphism with breeding parameters and body weights of cows and calves. Pakistan Journal of Zoology, 50(1), 183–187. https://doi.org/10.17582/journal.pjz/2018.50.1.183.187
  • Renaville, R., Gengler, N., Vrech, E., Prandi, A., Massart, S., Corradini, C., & Portetelle, D. (1997). Pit-1 gene polymorphism, milk yield, and conformation traits for Italian Holstein-Friesian bulls. Journal of Dairy Science, 80(12), 3431-3438.
  • Reshma, R. S., & Das, D. N. (2021). Molecular markers and its application in animal breeding. In Advances in Animal Genomics, (pp. 123-140). Academic Press.
  • Sadeghi, M., Mokhber, M., & Shahrbabak, M. M. (2022). Genetic Variation in Hypothalamic-Pituitary Axis Candidate Genes and Their Effects on Milk Production Traits in Iranian Holstein Cattle. Russian Journal of Genetics, 58(11), 1393-1400. https://doi.org/10.1134/S1022795422110096
  • Sakar, Ç. M., & Zülkadir, U. (2022). Determination of the relationship between Anatolian black cattle growth properties and myostatin, GHR and Pit-1 gene. Animal Biotechnology, 33(3), 536-545. https://doi.org/10.1080/10495398.2021.1884566
  • Silva, M. V. G. B. D., Martinez, M. L., Machado, M. A., Nascimento, C. S. D., Campos, A. L., Guimarães, M. F. M., & Lui, J. F. (2006). Genes do eixo somatotrófico e características de crescimento numa população F2 de bovinos. Pesquisa Agropecuária Brasileira, 41, 981-986. Sjaunja, K. S. & Olsson, K. (2005). Endocrinology of milk production. Domestic Animal Endocrinology, 29(2), 241-258.
  • Thuy, N. T. D., Thu, N. T., Cuong, N. H., Ty, L. V., Nguyen, T. T. B., & Khoa, D. V. A. (2018). Polymorphism of PIT-1 and prolactin genes and their effects on milk yield in Holstein Frisian dairy cows bred in Vietnam. Russian Journal of Genetics, 54, 346-352. https://doi.org/10.1134/S1022795418030146
  • Trakovická, A., Moravčíková, N., Minarovič, T., & Navrátilová, A. (2015). SNPs analyses of the bovine LEP and PIT-1 genes by multiplex PCR-RFLP method and their effect on milk performance traits in Slovak Simmental cattle. Journal of Central European Agriculture. 16(1), 65-75. https://doi.org/10.5513/JCEA01/16.1.1542
  • Zghairand, Z. S., & Hassooni, H. A. (2021). The Relationship Between The PIT-1 Gene at The Rate of Daily Milk Production and Some of Its Components in Buffaloes. In IOP Conference Series: Earth and Environmental Science 923(1), 012-029. https://doi.org/10.1088/1755-1315/923/1/012029
  • Zhang, C., Liu, B., Chen, H., Lan, X., Lei, C., Zhang, Z., & Zhang, R. (2009). Associations of a Hinf I PCR-RFLP of POU1F1 gene with growth traits in Qinchuan cattle. Animal Biotechnology, 20(2), 71-74. https://doi.org/10.1080/10495390802640462, 2009
There are 39 citations in total.

Details

Primary Language English
Subjects Zootechny (Other)
Journal Section Articles
Authors

Zeynep Sönmez 0000-0003-2696-9138

Hamiye Ünal 0000-0003-3099-8142

Early Pub Date December 15, 2023
Publication Date December 31, 2023
Acceptance Date July 28, 2023
Published in Issue Year 2023 Volume: 33 Issue: 4

Cite

APA Sönmez, Z., & Ünal, H. (2023). The Relationship of POU1F1-HinfI Gene Polymorphisms on Milk Yields in Simmental Cattle. Yuzuncu Yıl University Journal of Agricultural Sciences, 33(4), 513-521. https://doi.org/10.29133/yyutbd.1271873
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Yuzuncu Yil University Journal of Agricultural Sciences by Van Yuzuncu Yil University Faculty of Agriculture is licensed under a Creative Commons Attribution 4.0 International License.