Document Type : Research Paper

Authors

1 Ph.D student of genetics & animal Biotechnology, Department of Animal science, Ferdowsi University of Mashhad Khorasan Razavi - Iran

2 Associate Professor of Animal Genetics and Breeding, Khorasan Razavi Agricultural and Natural Resources Research and Education center, AREEO, Mashhad, Iran

3 Ph.D student of genetics & animal biotechnology, Departman of animal science, Ferdowsi university of Mashhad,, Khorasan Razavi , Iran.

Abstract

The FecB gene is one one of the major genes associated with the twinning trait in sheep. AvaII enzyme has a restriction site containing FecB gene mutation is used for genotyping by PCR-RFLP method. Due to toxicity and expensive price, using this enzyme is not considered reasonable and economical. So, this research was performed to design specific primers for rapid and economical detection of FecB gene. Tetra ARMs PCR technique, caucuses elimination of AvaII enzyme, replacement of acrylamide gel by safe agarose gel, and of course saving time and money for genotyping. Four primers were designed, based of Tetra ARMs PCR method, and were tested on 3000 samples. After PCR and electrophoresis, three fragments with a length of 108, 213 and 276 bp were amplified and finally three genotypes were observed for FecB gene. In order to validate the designed primers, 20 samples were randomly sequenced. After validation, the result of this research indicates that the final obtained genotype in Tetra ARMs PCR method is same as the results from PCR-RFLP method. Tetra ARMs PCR method is rapid and economical for detection of FecB in a larger number of samples and can replace by enzyme digestion method which is all of more expensive.

Keywords

خان احمدی، ع.ر.، رحیمی میانجی، ق.، حافظیان، ح.، خاتمی نژاد، ر.، مامی زاده، ن.ب.ب. و موسوی، م. (1395). بررسی اثر چند شکلی برخی از ژن‌های کاندید بر چند قلوزایی در گوسفندان آمیخته حاصل از تلاقی نژادهای شال ایرانی و رومانوف روسی. پژوهشهای تولیدات دامی, 7(14), 192-186.‎
رشیدی، ا. (1393)، ارزیابی استراتژی های اصلاح نژاد در گوسفند و بز،  ششمین کنگره علوم دامی ایران، ایران، تبریز.
Ahlawat, S., R., Sharma, A., Maitra, M., Roy, and M. S, Tantia, (2014). Designing, optimization and validation of tetra-primer ARMS PCR protocol for genotyping mutations in caprine Fec genes. Meta gene, 2, 439-449.
Davis, G. H., Galloway, S. M., Ross, I. K., Gregan, S. M., Ward, J., Nimbkar, B.V. and Tiesnamurti, B. (2002). DNA tests in prolific sheep from eight countries provide new evidence on origin of the Booroola (FecB) mutation. Biology of reproduction, 66(6), 1869-1874.
Davis, G. H., Montgomery, G. W., Allison, A. J., Kelly, R. W. and Bray, A. R. (1982). Segregation of a major gene influencing fecundity in progeny of Booroola sheep. New Zealand Journal of Agricultural Research, 25(4), 525-529
Edwards, S. J., Reader, K. L., Lun, S., Western, A., Lawrence, S., McNatty, K. P. and Juengel, J. L. (2008). The cooperative effect of growth and differentiation factor-9 and bone morphogenetic protein (BMP)-15 on granulosa cell function is modulated primarily through BMP receptor II. Endocrinology, 149(3), 1026-1030.
Etlik, O., Koksal, V., Arican-Baris, S. T. and Baris, I. (2011). Development and validation of a cost-effective in-house method, tetra-primer ARMS PCR assay, in genotyping of seven clinically important point mutations. Molecular and cellular probes, 25(4), 177-181.
Findlay, J. K., Drummond, A. E., Dyson, M. L., Baillie, A. J., Robertson, D. M. and Ethier, J. F. (2002). Recruitment and development of the follicle; the roles of the transforming growth factor-β superfamily. Molecular and cellular endocrinology, 191(1), 35-43.
Guan, F., Shi, G., Wan, P., Dai, R., Tang, H., Wang, H. and Luo, Y. (2014). Development of cost-effective tetra-ARMS PCR for detection of FecB genotype in sheep. Animal Science Papers and Reports, 32(3), 229-237.
Kumar, S., Kolte, A. P., Mishra, A. K., Arora, A. L. and Singh, V. K. (2006). Identification of the FecB mutation in Garole× Malpura sheep and its effect on litter size. Small Ruminant Research, 64(3), 305-310.
Lajin, B., Alachkar, A. and Sakur, A. A. (2012). Triplex tetra-primer ARMS-PCR method for the simultaneous detection of MTHFR c. 677C> T and c. 1298A> C, and MTRR c. 66A> G polymorphisms of the folate-homocysteine metabolic pathway. Molecular and cellular probes, 26(1), 16-20.
Medrano, R. F. V. and de Oliveira, C. A. (2014). Guidelines for the tetra-primer ARMS–PCR technique development. Molecular biotechnology, 56(7), 599-608.
Montgomery, G. W., Crawford, A. M., Penty, J. M., Dodds, K. G., Ede, A. J., Henry, H. M. and Sise, J. A. (1993). The ovine Booroola fecundity gene (FecB) is linked to markers from a region of human chromosome 4q. Nature genetics, 4(4), 410-414.
Souza, C. J. H., MacDougall, C., Campbell, B. K., McNeilly, A. S. and Baird, D. T. (2001). The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type 1 B (BMPR1B) gene. Journal of Endocrinology, 169(2), R
Ye, S., Dhillon, S., Ke, X., Collins, A. R. and Day, I. N. (2001). An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic acids research, 29(17), e88-e88.
Zhang, C., Liu, Y., Ring, B. Z., Nie, K., Yang, M., Wang, M. and Ma, X. (2013). A novel multiplex tetra-primer ARMS-PCR for the simultaneous genotyping of six single nucleotide polymorphisms associated with female cancers. PloS one, 8(4), e62126