Association of BMPR-1B and GDF9 genes polymorphisms and secondary protein structure changes with reproduction traits in Mehraban ewes

Methods paper o s ran an an, I Accepted 27 March 2013 Available online xxxx BMPR-1B GDF9 Single-strand conformation polymorphism (SSCP) Reproduction traits re w trolling ovulation rate in sheep. In the present study, polymorphisms of BMPR-1B gene exon 8 and GDF9 gene ngle strand conformational polymorphism (SSCP) analysis and DNA sequencing ewes. The PCR reaction forced to amplify 140 and 380-bp fragments of BMPR-1B breeding. This trait and generally, reproductive traits have low on goats (reviewed by Hua et al., 2008; Polley et al., 2009). The Booroola Gene xxx (2013) xxx–xxx GENE-38517; No. of pages: 8; 4C: Contents lists available at SciVerse ScienceDirect Gen j ourna l homepage: www.e heritabilities and noticeably are affected by non-additive genetic effects. Application of traditional breedingmethods, based on phenotypic data, is a time consuming process. Thus, molecular genetics and marker assisted selection (MAS) have a high importance for genetic improve- ment of reproduction efficiency. Some mutations in BMPR-1B, BMP15 and GDF9 genes are reported to increase ovulation rate (Davis, 2004). Some studies have indicated that the ovulation rate and litter size can be genetically regulated by a set of different genes, called as fecundity gene (FecB) or bone morphogenetic protein receptor 1B (BMPR-1B) was the first major gene associated with reproductivity in sheep (Mulsant et al., 2001; Wilson et al., 2001). This gene has an additive effect on litter size and ovulation rate in BooroolaMerino sheep (Smith et al., 1993). The growth differentiation factor 9 (GDF9) is another major gene, affecting prolificacy of the sheep (Hanrahan et al., 2004). Mutations in this gene have different effects on ovulation rate in each estrus and even they can cause infertility in some cases (Souza et al., 2001). In Iran, in spite of phenotypic variation of litter size in sheep, in most cases, application of molecular DNA technologies such as PCR-RFLP analysis could not play a pivotal role to identification of polymorphisms in these genes in other Abbreviations: BMPR-1B, bone morphogenetic prote differentiation factor 9; SNP, single nucleotide polymor chain reaction single–stranded conformation polymorp system; Lys, lysine amino acid; Thr, threonine amino a His, histidine amino acid; MAS, marker-assisted selectio ⁎ Corresponding author at: Department of Animal Sc Hamedan, Iran. Tel.: +98 9188148718; fax: +98 811 E-mail address: [email protected] (P. Zamani). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All http://dx.doi.org/10.1016/j.gene.2013.03.133 Please cite this article as: Abdoli, R., et al., Ass reproduction traits in Mehraban ewes, Gene onomic traits for sheep to the large family of TGFβ, and are located on chromosomes 6, X and 5 in sheep, respectively (Davis, 2004). Also, several studies have been carried Litter size is one of the most important ec Secondary structure of protein 1. Introduction SSCP patterns of BMPR-1B gene (CC and CA genotypes) that deduced one amino acid exchange. Also, two SNPS were identified in three different SSCP patterns of GDF9 gene (AA, AG and GG genotypes) that deduced one amino acid exchanges. Two different secondary structures of protein were predicted for BMPR-1B exon 8, but the secondary protein structures predicted for GDF9 exon 1 were similar together. The evaluation of the associations between the SSCP patterns and the protein structure changes with reproduction traits showed that BMPR-1B exon 8 genotypes have significant effects on some of reproduction traits but the GDF9 genotypes did not have any significant effect. The CA genotype of BMPR-1B exon 8 had a significant positive effect on reproduc- tion performance and could be considered as an important and new mutation, affecting the ewes reproduction performance. Marker assisted selection using BMPR-IB gene could be noticed to improve the reproduction traits in Mehraban sheep. © 2013 Elsevier B.V. All rights reserved. (Fec) genes (Davis et al., 1982). BMPR-1B, BMP15 and GDF9 genes belong Keywords: Sheep and GDF9 genes, respectively. Two single nucleotide polymorphisms (SNPS) were identified in two different exon 1 were detected by si methods in 100 Mehraban Association of BMPR-1B and GDF9 genes p structure changes with reproduction trait R. Abdoli a, P. Zamani a,⁎, A. Deljou b, H. Rezvan c a Department of Animal Science, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, I b Department of Biotechnology, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Ir c Department of Parasitology, Faculty of Veterinary Science, Bu-Ali Sina University, Hamed a b s t r a c ta r t i c l e i n f o Article history: BMPR-1B and GDF9 genes a in receptor 1B; GDF9, growth phism; PCR–SSCP, polymerase hism; SAS, statistical analysis cid; Arg, arginine amino acid; n. ience, Bu-Ali Sina University, 4424012. rights reserved. ociation of BMPR-1B and GDF (2013), http://dx.doi.org/10. lymorphisms and secondary protein in Mehraban ewes ran ell known due to their important effects on litter size and mechanisms con- e l sev ie r .com/ locate /gene breeds of sheep (Ghaffari, 2007; Moradband et al., 2011; Zare et al., 2010). Thus, the use of PCR-SSCP technique could be considered as an alternative to other methods such as PCR-RFLP analysis. The objectives of the present study were identification of the BMPR-1B and GDF9 genes mutations by PCR-SSCP and DNA sequencing methods and evaluation of the associationbetween thesemutations andprotein structure changes with reproduction traits in Mehraban ewes. 9 genes polymorphisms and secondary protein structure changes with 1016/j.gene.2013.03.133 2. Materials and methods 2.1. Sheep and DNA sources A total of 100 Mehraban ewes were selected from different re- gions of Hamedan province in Iran. Fifteen sheep were selected based on high litter size records and 85 ewes were randomly selected, all over three different flocks. Blood samples were collected from jug- ular vein (5 ml per ewe) by venoject tubes contained EDTA (ethylene diamine tetra acetic acid) and immediately transported to the labora- tory with ice before DNA isolation. The genomic DNA was extracted from whole blood by the CinnaGen kit (CinnaGen DNP kit, Iran) and kept at −20 °C. 2.2. PCR amplification Two pairs of primers were designed to amplify a 140-bp fragment in exon 8 of BMPR-1B (GenBank accession no. GQ863578)and a 380-bp fragment in exon 1 of GDF9 (GenBank accession number 2 R. Abdoli et al. / Gene xxx (2013) xxx–xxx Fig. 1. PCR products of GDF9 and BMPR-1B genes bands. CG: template negative control of GDF9 gene (PCR without genomic DNA). CB: template negative control of BMPR-1B gene (PCR without genomic DNA). M: DNA molecular weight marker (Orange Ruler 50 bp DNA Ladder, CinnaGen, Iran). 1–6: 380 bp band of GDF9 gene. 7–12: 140 bp AF078545.2) genes, using Primer 3 software online (Rozen and Skaletsky, 1998). These primers were as follows: BMPR-1B: Forward: 5′-GTCGCTATGGGGAAGTTTGG-3′ Reverse: 5′-CAAGATGTTTTCATGCCTCATC-3′ GDF9: Forward: 5′-GAAGACTGGTATGGGGAAATG-3′ Reverse: 5′-TGTAGAGGTGGCGTCTGTTG-3′ The PCR amplification reactionswere similar in both genes andwere carried out in a 50 μL final volume and consisted of PCR Buffer 10× [50 mM KCl, 10 mM Tris–HCl (pH 8.0), 0.1% Triton X-100], MgCl2 (2.5 mM), 200 μM of each dNTPs, 10 pM of each primer, 2.5 units of Taq DNA Polymerase (CinnaGen, Iran), 50–100 ng of DNA template and distilled water. PCR reactions were run on a Mastercycler ASTEC (PC708, Japan) under the following thermal condition: Initial denatur- ation at 94 °C for 5 min, followed by 35 cycles consisting of denatur- ation at 94 °C for 30 s, annealing at 60 °C for 45 s, extension at 72 °C for 45 s and a final extension at 72 °C for 5 min. The PCR products of the 140 bp bands of BMPR-1B and 380 bp bands of GDF9 genes were separated by electrophoresis on 2% agarose gels (CinnaGen, Iran) using 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM Na2 EDTA). The gels were stained with ethidium bromide and photographed under UV light (BTS-20.M model, UVItec Ltd, UK) (Fig. 1). band of BMPR-1B gene. Please cite this article as: Abdoli, R., et al., Association of BMPR-1B and GDF reproduction traits in Mehraban ewes, Gene (2013), http://dx.doi.org/10. 2.3. Single-strand conformation polymorphism (SSCP) analysis The SSCP analysis of BMPR-1B gene was carried out as follows. A 5 μL of PCR product sample of BMPR-1B gene exon 8 was added to 10 μL of SSCP gels loading dye (0.05% bromophenol blue, 0.05% xylene cyanol, 95% formamide, 20 mM EDTA) and mixed. After heat denaturation at 97 °C for 8 min, the samples were immediately chilled on ice to prevent heteroduplex formation and then run (18 h, 300 V, 5 °C) on 15% acrylamide:bis-acrylamide gels (29:1 acrylamide to bisacrylamide), without glycerol in 1× TBE buffer on a 21 × 22 cm gel casting vertical electrophoresis (Payapajoohesh Pars, Iran). For SSCP analysis of GDF9, 4 μL of GDF9 gene exon 1 PCR sample was aliquoted into separate tubes, then 7 μL of SSCP gel loading dye was added and mixed. After heat denaturation at 98 °C for 10 min, the samples were immediately chilled on ice and then run (22 h, 300 V, 5 °C) on 8% acrylamide:bis-acrylamide gels (29:1 acrylamide to bisacrylamide), in 1× TBE buffer. DNA visualization in the polyacryl- amide gel after electrophoresis was done by silver staining (Sanguinetti et al., 1994). 2.4. DNA sequencing Two samples of each SSCP pattern were randomly selected for DNA sequencing. The primers used for sequencing were the same as those for the PCR reaction. The PCR products were sequenced by Bioneer Co., Korea. Sequence alignments, translations and comparisons were carried out using ExPASy translate tools website (http://us.expasy.org/ translatetool/) and MegAlign module of DNASTAR software (DNASTAR Inc., Madison, WI. USA) by Clustal W method. Moreover, the identified SNPs were compared with the sheep and goat NCBI dbSNP database using BLAST (http://www.ncbi.nlm.nih.gov/SNP/index.html). 2.5. Protein structure To design the secondary structure of protein in referring sequence and different alleles in BMPR-1B and GDF9 genes, the sequences of amplified fragments of the studied genes (BMPR-1B exon 8 and GDF9 exon 1) were retrieved from NCBI GenBank databases and DNASTAR, EditSeq software (DNASTAR Inc., Madison, WI. USA) was used to enter the mutations. Then, ExPASy translate tools website (http:// us.expasy.org/translatetool/) andMegAlignmodule of DNAstar, soft- ware (DNASTAR Inc., Madison, WI. USA) were used to translation of nucleotide sequences to amino acid chain. Secondary structures of proteins were predicted using Psipred section of SWISS-MODEL available in the ExPASy website (http://us.expasy.org/) and Protean section of DNASTAR software (DNASTAR Inc., Madison, WI. USA). 2.6. Statistical analysis Associations of different genotypes for both genes with some repro- duction traits, including the litter size (LS), number of lambs born until 4 years of age (NLB4), number of lambs weaned until 4 years of age (NLW4), average birth weight of lambs until 4 years of age (ABW4), total birth weight of lambs until 4 years of age (TBW4), average weaning weight of lambs until 4 years of age (AWW4) and total weaning weight of lambs until 4 years of age (TWW4) in all genotyped animals (100 ewes) were investigated using a general linear model as follows: Yijk ¼ μ þ Ai þ Bj þ ABij þ eijk where Yijk is the phenotypic value of the reproduction trait; μ is overall mean; Ai is the effects of ith genotype of BMPR-1B gene; Bj is the effects of jth genotype of GDF9 gene; ABij is the interaction between ith geno- type of BMPR-IB and jth genotype of GDF9 and eijk is the residual effects. The means were compared by Duncan's multiple range test, assuming 9 genes polymorphisms and secondary protein structure changes with 1016/j.gene.2013.03.133 error level of 0.05. General linear model analysis and comparison of means were done using GLM procedure of SAS software (SAS Inc., 2004). The frequencies of SSCP patterns and genotypes were calculated in 85 randomly selected ewes but the associations of different geno- types with the studied traits were evaluated using all 100 animals. 3. Results 3.1. SSCP analysis of BMPR-1B gene exon 8 The SSCP analysis of BMPR-1B gene exon 8 in Mehraban ewes re- vealed 2 distinct patterns (CA and CC genotypes) on polyacrylamide gels (Fig. 2). The CA genotype showed 6 major bands on the gel, but the CC genotype showed 4 bands on the gel. The frequencies of CC and CA genotypes of BMPR-1B exon 8 in 85 randomly selected Mehraban ewes were 80 % and 20 %, respectively (Table 1). 3.2. SSCP analysis of GDF9 gene exon 1 The SSCP analysis of GDF9 gene exon 1 showed three distinct pat- terns (AA, AG and GG genotypes) on polyacrylamide SSCP gel. The AG genotype exhibited 5 bands on SSCP gel. Also, the AA and GG geno- types exhibited 4 bands with different intervals on SSCP gel (Fig. 3). The frequencies of the AA, AG and GG genotypes in 85 randomly se- lected Mehraban ewes were 9.41 %, 62.35 % and 28.24 % respectively (Table 1). 3.3. Sequence analysis of BMPR-1B gene exon 8 The results obtained by DNA sequencing showed a transition of C → A at position 112 of the amplified fragment of BMPR-1B exon 8 in the allele A. Moreover, both alleles (A and C) showed a transition C → G at position of 113 in comparison to the referring sequence (GenBank accession number GQ863578). However, the replacement of both nucleotides (ACC = ACG) deduce threonine amino acid and this replacement is a neutral mutation (Fig. 4). 3.4. Sequence translation of BMPR-1B gene exon 8 The sequencing of the allele A of BMPR-1B gene exon 8 showed two transitions of C → A and C→ G at positions 112 and 113, respec- tively (Fig. 4), that will result in a deduced Thr → Lys exchange (T → K) at position 37 of the codified protein sequence. The C allele showed a transition at position of 113 in comparison to the referring sequence (GenBank accession number GQ863578)), which did not show any deduced amino acid exchange (Fig. 5). Table 1 Frequencies of BMPR-1B exon 8 and GDF9 exon 1 genotypes in randomly selected Mehraban ewes. BMPR-1B exon 8 GDF9 exon 1 Genotype Frequency Percentage Genotype Frequency Percentage CC 68 80 AA 8 9.41 CA 17 20 AG 53 62.35 GG 24 28.24 Total 85 100% 85 100% Fig. 3. Different SSCP patterns (AA, AG and GG genotypes) of 380-bp fragment of GDF9 3R. Abdoli et al. / Gene xxx (2013) xxx–xxx Fig. 2. Different SSCP patterns (CC and CA genotypes) of 140-bp fragment of BMPR-1B gene exon 8. Please cite this article as: Abdoli, R., et al., Association of BMPR-1B and GDF reproduction traits in Mehraban ewes, Gene (2013), http://dx.doi.org/10. gene exon 1. 9 genes polymorphisms and secondary protein structure changes with 1016/j.gene.2013.03.133 Gen ank 4 R. Abdoli et al. / Gene xxx (2013) xxx–xxx 3.5. Protein structure changes of BMPR-1B gene exon 8 The allele A of BMPR-1B gene exon 8, showed various predictions for secondary protein structure in comparison to the GenBank se- quence. The sequence of the C allele was similar to the referring se- quence (GenBank accession number GQ863578)). The most changes in the allele A included the alpha and beta amphipathic regions in the secondary structure of protein. Moreover, the allele A showed a difference in antigenic index and surface probability plot in compari- son to the referring sequence (Fig. 6). 3.6. Sequence analysis of GDF9 gene exon 1 Sequencing results showed a transition of G → A at positions 306 in the allele A of GDF9 gene exon 1, in comparison to GenBank acces- sion number AF078545.2 sequence (Fig. 7). This mutation is reported in Belclare and Cambridge sheep (Hanrahan et al., 2004). 3.7. Sequence translation of GDF9 gene exon 1 Translation results showed a deduced amino acid exchange, Fig. 4. The comparison between sequences of A and C alleles of BMPR-1B exon 8 and (GenBank no. GU979817.1), Capra hircus (GenBank no. JN100107), Garole sheep (GenB Arg → His (R → H) at position 87 in the allele A of GDF9 gene exon 1 in comparison to GenBank accession number AF078545.2 (Fig. 8). That this amino acid exchange is reported in Belclare and Cambridge sheep. 3.8. Protein structure changes of GDF9 gene exon 1 Two different alleles of GDF9 gene exon 1 (G and A) had different secondary protein structures in comparison to the GenBank sequence Fig. 5. The codified protein sequence and deduced amino acid exchange of different alleles ( with other sequences in NCBI for Corriedal sheep (GenBank no. GU979817.1), Capra hircus ( sheep (Mulsant et al., 2001). Please cite this article as: Abdoli, R., et al., Association of BMPR-1B and GDF reproduction traits in Mehraban ewes, Gene (2013), http://dx.doi.org/10. (GenBank accession number AF078545.2), but the secondary structures of protein predicted for G and A alleles were similar to secondary struc- ture of protein for the referring sequence (GenBank accession number AF078545.2). They did not show any major changes in structural prop- erties of protein (Fig. 9). 3.9. Association of BMPR-1B gene exon 8 and GDF9 gene exon 1genotypes with reproduction traits Evaluation of the associations between the SSCP patterns with the studied reproduction traits showed that the CA genotype of BMPR-1B exon 8 (P b 0.05) has a significant positive effect on some reproduction traits, including LS, NLB4, NLW4, TBW4, AWW4 and TWW4. The ABW4 was not significantly affected by the CA genotype of BMPR-1B exon 8 (Table 2). The results obtained on the association of different genotypes of GDF9 exon 1 with the studied reproduction traits are also presented in Table 2. As it could be seen in this table, genotypes of GDF9 gene exon 1 did not affect the studied traits. BMPR-IB and GDF9 genotype did not have any significant interaction on the studied traits (Table 2). Bank accession number GQ863578, with other sequences in NCBI for Corriedal sheep no. GQ863576.1) and Booroola merino sheep (Mulsant et al., 2001). 4. Discussion The studied genes, BMPR-1B (bone morphogenetic protein receptor 1B) and GDF9 (growth differentiation factor 9) are twomajor fecundity genes affecting prolificacy in sheep. Both studied fragments of BMPR-1B and GDF9 genes in the present study were polymorphic in the studied population of Iranian Mehraban sheep. While, in other Iranian native breeds of sheep, such as Shal, Ghezel, Baluchi, despite the phenotypic variation of litter size, the PCR-RFLP technique could not show any A and C) of BMPR-1B exon 8 and comparison to GenBank accession number GQ863578, GenBank no. JN100107), Garole sheep (GenBank no. GQ863576.1) and Booroola merino 9 genes polymorphisms and secondary protein structure changes with 1016/j.gene.2013.03.133 5R. Abdoli et al. / Gene xxx (2013) xxx–xxx polymorphism the studied fragments of these genes (Ghaffari, 2007; Moradband et al., 2011; Zare et al., 2010). Hence, it seems that the use of PCR-SSCP technique alongwith DNA sequencing could be considered as a more efficient method in comparison to PCR-RFLP. A point mutation (A→ G) at position 746 of the cDNA sequence deduced a substitution of arginine with a glutamine (Q → R) in posi- tion 87 in corresponding polypeptide in the GenBank accession num- ber AF312016 sequence (Souza et al., 2001) is correlated to increase of litter size and ovulation rate in sheep (Davis et al., 2002). The posi- tion of this mutation is equivalent to the position 36 in corresponding amino acid chain of the studied fragment of BMPR-1B gene in the present study. The codified polypeptide of the C allele of BMPR-1B gene exon 8 in the present study, with one mutation (C → G), was similar to the corresponding sequence of this fragment (GenBank Fig. 6. The changes in the secondary structure of protein and comparison between the GenBa exon 8. Differentiations are presented by ↓ symbol. Please cite this article as: Abdoli, R., et al., Association of BMPR-1B and GDF reproduction traits in Mehraban ewes, Gene (2013), http://dx.doi.org/10. accession numbers GQ863577 and GQ863578) in Muzaffarangari breed of Indian sheep (Siva Kumar and Singh, 2009). The sequences of BMPR-1B gene exon 8 in the present study and the report of Siva Kumar and Singh (2009) did not show the FecB mutation (Q249R) which is occurred in the corresponding region in Garole sheep (GenBank accession number GQ863576.1) and Australian Booroola Merino sheep (GenBank accession number AF312016). The Q249R mutation is also reported in other breeds such as small tailed Han (Chu et al., 2006; Liu et al., 2003; Yan et al., 2005), and Hu sheep (Guan et al., 2006; Yan et al., 2005). The sequence of the allele A of BMPR-1B gene exon 8 in the present study had two mutations of C → A and C → G at the positions 112 and 113, indicating a deduced amino acid exchange Thr → Lys (T → K) at position 37 of the codified protein sequence. This is very nk accession number (GQ863578) sequence and different alleles (C and A) in BMPR-1B 9 genes polymorphisms and secondary protein structure changes with 1016/j.gene.2013.03.133 Fig. 7. The comparison between sequences of G and A alleles of GDF9 exon 1 and GenBank accession number AF078545.2, with other sequence in NCBI for Norwegian white sheep (GenBank no. HE866499.1), Belclare and Cambridge sheep (Hanrahan et al., 2004). Fig. 8. The codified protein sequence and deduced amino acid exchange of different alleles (G and A) of GDF9 exon 1 and comparison to GenBank accession number AF078545.2, with other sequences in NCBI for Norwegian white sheep (GenBank no. HE866499.1), Belclare and Cambridge sheep (Hanrahan et al., 2004). 6 R. Abdoli et al. / Gene xxx (2013) xxx–xxx Please cite this article as: Abdoli, R., et al., Association of BMPR-1B and GDF9 genes polymorphisms and secondary protein structure changes with reproduction traits in Mehraban ewes, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.03.133 7R. Abdoli et al. / Gene xxx (2013) xxx–xxx close to the amino acid position of FecB mutation in GenBank acces- sion number GQ863576.1 (position 36). The mutations detected in the present study and the FecB mutation have not been detected in other Iranian breeds of sheep using PCR-RFLP method, yet. The BMPR-1B exon 8 genotypes had significant effects on most of the studied reproduction traits. As it was mentioned earlier, the ewes having the CA genotype, generally were more fertile, in comparison to the ewes having the CC genotypes (Table 2). Moreover, among 15 Mehraban ewes that were selected based on high litter size records, 11 ewes had the CA genotype second pattern with the deduced amino acid Fig. 9. The changes in the secondary structure of protein and the comparison between the refer GDF9 exon 1. Differentiations are presented by ↓ symbol. Please cite this article as: Abdoli, R., et al., Association of BMPR-1B and GDF reproduction traits in Mehraban ewes, Gene (2013), http://dx.doi.org/10. exchange (T→ K). The positive effects of the CA genotype of BMPR-1B exon 8 could be attributed to the major changes in structure of the de- duced protein (Fig. 6). By a number of non-covalent interactions such as hydrogen bonding, ionic interactions, Van derWaals forces, and hydro- phobic packing, proteins fold into one or more specific spatial conforma- tions; thus any changes in amino acid sequence could lead to the change of protein properties. The effect of BMPR-1B exon 8 genotype CA on most of the studied reproduction traits could be attributed to some changes in predicted secondary structure of protein (Fig. 6) and as a result, some changes in the third and fourth structures of the protein. ring sequence (GenBank accession number AF078545.2) and different alleles (G and A) of 9 genes polymorphisms and secondary protein structure changes with 1016/j.gene.2013.03.133 s. CC 1.055b 1.595b 1.500b a a a bs w of la in D e xxx (2013) xxx–xxx In GDF9 gene, eight point mutations of G1, G2, G3, G4, G5, G6, G7 and G8 (GenBank accession number AF078545)) have been detected in Belclare and Cambridge sheep breeds (Hanrahan et al., 2004), but only five mutations will lead to deduced amino acid exchanges as G1: R87H; G4: E241K; G6: V332I; G7: V371M and G8: S395F (Polley et al., 2010). The first mutation (G1) is occurring in exon 1 and other muta- tions (G4, G6, G7 and G8) are found in exon 2 of GDF9 gene. The first mutation (G1) with one amino acid exchange Arg→ His (R87H) was found in the allele A of the GDF9 gene exon 1 (G-1) in the present study (Figs. 7 and 8). This mutation (G1) is also reported in Ghezel and Moghani sheep, two Iranian breeds (Barzegari et al., 2010). In the present study, three genotypes of GDF9 exon 1 inMehraban sheep, did not have any significant effect on reproduction traits (Table 2). The not significant effect of GDF9 exon 1 genotypes, detected in the present study (AA, AG and GG), could be attributed to similar secondary structures and thus, structural properties of the corresponding codified proteins (Fig. 9). This in agreement with the results of Barzegari et al. (2010) and Hanrahan et al. (2004) who did not find any significant effect from the genotypes similar to those detected in the present study. This result also agrees with the report of Hanrahan et al. (2004), who found eight mutations in exons 1 and 2 of GDF9 in Cambridge and Belclare sheep and only onemutation in exon 2 (G8), with deduced S395F amino acid exchange, had a significant effect on fertility. In their study, other mutations in exon 1 ofGDF9 did not have any significant effect on reproduction traits. Themutation in the allele A of BMPR-1B gene exon 8, detected in the present study, which had highly significant effects on most of the stud- ied fertility traits (litter size, number of lambs born or weaned until 4 years of age, total birth weight of lambs until 4 years of age, average weaning weight of lambs until 4 years of age and total weaning weight of lambs until 4 years of age)means that the correspondingmutation in CA 1.319 2.900 2.428 SEM 0.049 0.204 0.206 GDF9 gene exon 1 AA 1.047 1.857 1.750 AG 1.103 1.888 1.750 GG 1.245 2.315 1.928 SEM 0.076 0.290 0.309 P value of BMPR-1B 0.0137 0.0001 0.0046 P value of GDF9 0.3040 0.6229 0.9366 P value of BMPR-1B*GDF9 0.7925 0.1447 0.4613 LS: litter size; NLB4: number of lambs born until 4 years of age; NLW4: number of lam TBW4: total birth weight of lambs until 4 years of age; AWW4: average weaning weight The means with same letter in each part of each column are not significantly different Table 2 Association of the BMPR-1B and GDF9 genes with reproduction traits in Mehraban ewe Traits LS (n) NLB4 (n) NLW4 (n) BMPR-1B gene exon 8 this allele could be considered as an important and new mutation, af- fecting reproduction performance of the ewes. Thus, marker-assisted selection using the allele A of BMPR-1B gene could be noticed to im- prove the reproduction traits in Mehraban and probably other breeds of sheep. However, the researches in the future need to evaluate the mutations in other parts of this gene or other genes such as BMP15 and ESRα etc. Due to limited sample size, the combined effects of GDF9 exon 1 and BMPR-1B exon 8 could not be evaluated in the present study. It seems that evaluation ofmore genes in a studywithmore sam- ple size and considering of their combined effects on fertility could be considered as an interesting subject for similar studies in the future. Conflict of interest statement None. Please cite this article as: Abdoli, R., et al., Association of BMPR-1B and GDF reproduction traits in Mehraban ewes, Gene (2013), http://dx.doi.org/10. Acknowledgments This research was financially supported by Bu-Ali Sina University, Hamedan, Iran. 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DNA sequencing 2.5. Protein structure 2.6. Statistical analysis 3. Results 3.1. SSCP analysis of BMPR-1B gene exon 8 3.2. SSCP analysis of GDF9 gene exon 1 3.3. Sequence analysis of BMPR-1B gene exon 8 3.4. Sequence translation of BMPR-1B gene exon 8 3.5. Protein structure changes of BMPR-1B gene exon 8 3.6. Sequence analysis of GDF9 gene exon 1 3.7. Sequence translation of GDF9 gene exon 1 3.8. Protein structure changes of GDF9 gene exon 1 3.9. Association of BMPR-1B gene exon 8 and GDF9 gene exon 1genotypes with reproduction traits 4. Discussion Conflict of interest statement Acknowledgments References