Association of novel polymorphisms in follicle stimulating hormone beta (FSHβ) gene with litter size in Mehraban sheep

Document Type : Original Research Article (Regular Paper)


1 Department of Animal Science, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran

2 Department of Animal Sciences, Faculty of Agriculture, Bu-Ali Sina University, Hamedan - IRAN

3 Department of Systems and Synthetic Biology, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran


Follicle-stimulating hormone (FSH) is necessary for the hypothalamic-pituitary-gonadal axis, and plays an important role in reproduction by binding to a specific receptor (FSHR) through β-subunit of FSH in the surface of the ovarian granulosa cells. This study aimed to characterize FSH β-subunit gene (FSHβ) polymorphism and its association with litter size (LS) using a sample of 118 Mehraban sheep. Polymerase chain reaction (PCR) was performed to amplify two fragments of 300 bp and 431 bp of the ovine FSHβ gene (Oar_v4.0; Chr 15, NC_019472.2). Polymorphisms in the studied fragments were then explored using single strand conformational polymorphism (SSCP) and DNA sequencing methods. A total of seven single-nucleotide polymorphisms (SNPs), including g.59078564 C>G, g.59078624 T>C, g.59078655 T>C, g.59078691 T>C, g.59078754 C>A, g.59080186 G>C and g.59080365 C>T, were found among the six detected SSCP patterns A to F. Moreover, two novel indel polymorphisms called e.g., g.59078702del8-bp−ins64-bp and g.59078726ins54-bp were identified among the three different SSCP genotypes patterns G to I. We found significant differences on prolificacy categories between SSCP genotypes patterns D, E and F (P < 0.01) that simultaneously represented SNP polymorphisms of g.59078754 C>A, g.59080186 G>C and g.59080365 C>T. Similarly, novel indel polymorphisms revealed a significant difference on prolificacy categories between SSCP genotypes patterns G, H and I (P < 0.05). Our results suggest that the FSHβ is a strong candidate gene to associate with the LS in sheep.


Main Subjects

Abdoli, R., Mirhoseini, S.Z., Ghavi Hossein-Zadeh, N., Zamani, P., Gondro, C., 2018. Genome-wide association study to identify genomic regions affecting prolificacy in Lori-Bakhtiari sheep. Animal Genetics 49, 488-491.
Abdoli, R., Zamani, P., Deljou, A., Rezvan, H., 2013. Association of BMPR-1B and GDF9 genes polymorphisms and secondary protein structure changes with reproduction traits in Mehraban ewes. Gene 524, 296-303.
Abdoli, R., Zamani, P., Mirhoseini, S.Z., Ghavi Hossein-Zadeh, N., Nadri, S., 2016. A review on prolificacy genes in sheep. Reproduction in Domestic Animals 51, 631-637.
Ahmadi, A., Afraz, F., Talebi, R., Farahavar, A., Vahidi, S.M.F., 2016. Investigation of GDF9 and BMP15 polymorphisms in mehraban sheep to find the missenses as impact on protein. Iranian Journal of Applied Animal Sciences 6, 863-872.
Ahmadi, A., Mohammadizade, M., Zamani, P., Farah-avar, A., Talebi, R., 2021. Identification of polymorphism in HSP70A1A gene in Mehraban and Romanov sheep breeds using PCR-SSCP technique. Research On Animal Production 12, 169-179.
An, X.P., Han, D., Hou, J.X., Li, G., Wang, Y.N., Li, L., Zhu, G.Q., Wang, J., Song, Y.X., Cao, B.Y., 2010. Polymorphism of exon 2 of FSHβ gene and its relationship with reproduction performance in two goat breeds. Agricultural Sciences in China 9, 880-886.
Bianco, B., Loureiro, F.A., Trevisan, C.M., Peluso, C., Christofolini, D.M., Montagna, E., Laganà, A.S., Barbosa, C.P., 2021. Effects of FSHR and FSHB variants on hormonal profile and reproductive outcomes of infertile women with endometriosis. Frontiers in Endocrinology 12, 1-10.
Gholiadeh, M., Najafi, M., 2017. Association of genetic variation in exon 1 and 3 of FSHB gene with litter size in Baluchi sheep. Research on Animal Production 8, 177-182.
Gholizadeh, M., Rahimi-Mianji, G., Nejati-Javaremi, A., De Koning, D.J., Jonas, E., 2014. Genomewide association study to detect QTL for twinning rate in Baluchi sheep. Journal of Genetics 93, 489-493.
Goodman, R.L., Inskeep, E.K., 2015. Control of the ovarian cycle of the sheep. In: Plant, T.M., Zeleznik, A.J. (Eds.), Knobil and Neill's Physiology of Reproduction (Fourth Edition). Elsevier Inc., San Diego, USA, pp. 1259–1305.   
Gootwine, E., 2020. Invited review: Opportunities for genetic improvement toward higher prolificacy in sheep. Small Ruminant Research 186, 106090.
Hediger, R., Johnson, S.E., Hetzel, D.J.S., 2009. Localization of the beta-subunit of follicle stimulating hormone in cattle and sheep by in situ hybridization. Animal Genetics 22, 237-244.
Janssens, S., Vandepitte, W., Bodin, L., 2004. Genetic parameters for litter size in sheep: Natural versus hormone-induced oestrus. Genetics Selection Evolution 36, 543-562.
Laisk, T., Kukuškina, V., Palmer, D., Laber, S., Chen, C.Y., Ferreira, T., Rahmioglu, N., Zondervan, K., Becker, C., Smoller, J.W., Lippincott, M., Salumets, A., Granne, I., Seminara, S., Neale, B., Mägi, R., Lindgren, C.M., 2018. Large-scale meta-analysis highlights the hypothalamic–pituitary–gonadal axis in the genetic regulation of menstrual cycle length. Human Molecular Genetics 27, 4323-4332.
Lameei, H., Afshari, K.P., Abozar, M., 2015. Identification of different allelic forms in exon 1 of FSHβ in Iranian Baluchi and Naeini sheep breeds by PCR-SSCP. Research Opinions in Animal & Veterinary Sciences  5, 360-365.
Liang, C., Chu, M.X., Zhang, J.H., Liu, W.Z., Fang, L., Ye, S.C., 2006. PCR-SSCP polymorphism of FSHbeta gene and its relationship with prolificacy of Jining grey goats. Hereditary 28, 1071-1077.
Liu, J.J., Ran, X.Q., Li, S., Feng, Y., Wang, J.F., 2009. Polymorphism in the first intron of follicle stimulating hormone beta gene in three Chinese pig breeds and two European pig breeds. Animal Reproduction Science 111, 369-375.
Majd, S.A., Ahmadi, A., Talebi, R., Koohi, P.M., Fabre, S., Qanbari, S., 2019. Polymorphism identification in ovine KISS1R/GPR54 gene among pure and crossbreeds of Iranian sheep. Small Ruminant Research 173, 23-29.  
Matoušek, V., Čermáková, A., Kernerová, N., Králová, P., 2005. Analysis of prolificacy in sows of hyperprolific lines of Large White breed. Czech Journal of Animal Science 50, 155-162.
Mitchell, F., 2012. Male fertility influenced by FSHB and FSHR polymorphisms. Nature Reviews Endocrinology 8, 564.
Mokhtari, M.S., Shahrbabak, M.M., Esmailizadeh, A.K., Shahrbabak, H.M., Gutierrez, J.P., 2014. Pedigree analysis of Iran-Black sheep and inbreeding effects on growth and reproduction traits. Small Ruminant Research 116, 14-20.
Nazifi, N., Rahimi-Mianji, G., Ansari-Pirsarai, Z., 2015. Polymorphism in FSHβ and FSHR genes and their relationship with productive and reproductive performance in Iran Black, Arman and Baluchi sheep breeds. Iranian Journal of Applied Animal Science 5, 361-368.
Nikbin, S., Panandam, J.M., Yaakub, H., Murugaiyah, M., 2018. Association of novel SNPs in gonadotropin genes with sperm quality traits of Boer goats and Boer crosses. Journal of Applied Animal Research 46, 459-466.
Niu, X., Martin, G.B., Liu, W., Henryon, M.A., Ren, K., 2019. Follicle-stimulating hormone (FSHβ) gene polymorphisms and associations with reproductive traits in Rex rabbits. Animal Reproduction Science 207, 36-43.
Nosrati, M., Asadollahpour Nanaei, H., Amiri Ghanatsaman, Z., Esmailizadeh, A., 2019. Whole genome sequence analysis to detect signatures of positive selection for high fecundity in sheep. Reproduction in Domestic Animals 54, 358-364.
Pedersen, L.D., Sørensen, A.C., Berg, P., 2009. Marker-assisted selection can reduce true as well as pedigree-estimated inbreeding. Journal of Dairy Science 92, 2214-2223.
Rull, K., Grigorova, M., Ehrenberg, A., Vaas, P., Sekavin, A., Nõmmemees, D., Adler, M., Hanson, E., Juhanson, P., Laan, M., 2018. FSHB -211 G>T is a major genetic modulator of reproductive physiology and health in childbearing age women. Human Reproduction 33, 954-966.
Sanguinetti, C., 1994. RAPD silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques 17, 914-921.
Talebi, R., Ahmadi, A., Afraz, F., Sarry, J., Woloszyn, F., Fabre, S., 2018. Detection of single nucleotide polymorphisms at major prolificacy genes in the Mehraban sheep and association with litter size. Annals of Animal Science 18, 685-698.
Talebi, R., Seighalani, R., Qanbari, S., 2021. A handmade DNA extraction kit using laundry powder; insights on simplicity, cost-efficiency, rapidity, safety and the quality of purified DNA. Animal Biotechnology 32, 388-394.
Trevisan, C.M., De Oliveira, R., Christofolini, D.M., Barbosa, C.P., Bianco, B., 2019. Effects of a polymorphism in the promoter region of the follicle-stimulating hormone subunit beta (FSHB) gene on female reproductive outcomes. Genetic Testing and Molecular Biomarkers 23, 39-44.
Vinet, A., Drouilhet, L., Bodin, L., Mulsant, P., Fabre, S., Phocas, F., 2012. Genetic control of multiple births in low ovulating mammalian species. Mammalian Genome 23, 727-740.
Xu, S.S., Gao, L., Xie, X.L., Ren, Y.L., Shen, Z.Q., Wang, F., Shen, M., Eypórsdóttir, E., Hallsson, J.H., Kiseleva, T., Kantanen, J., Li, M.H., 2018. Genome-wide association analyses highlight the potential for different genetic mechanisms for litter size among sheep breeds. Frontiers in Genetics 9, 1-14.
Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., Madden, T.L., 2012. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134.
Yeh, F.C., Yang, R.C., Boyle, T., 1999. POPGENE. Version 1.31. Microsoft window-based freeware for population genetic analysis. University of Alberta, Edmonton, AB, Canada.
Zamani, P., Nadri, S., Saffaripour, R., Ahmadi, A., Dashti, F., Abdoli, R., 2015. A new mutation in exon 2 of the bone morphogenetic protein 15 gene is associated with increase in prolificacy of Mehraban and Lori sheep. Tropical Animal Health and Production 47, 855-860.
Zhang, C.Y., Wu, C.J., Zeng, W.B., Huang, K.K., Li, X., Feng, J.H., Wang, D., Hua, G.H., Xu, D.Q., Wen, Q.Y., Yang, L.G., 2011. Polymorphism in exon 3 of follicle stimulating hormone beta (FSHB) subunit gene and its association with litter traits and superovulation in the goat. Small Ruminant Research 96, 53-57.