Genetic Polymorphism BMP15 and Myostatin Gene in the Arabic Sheep Using PCR-SSCP

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Genetic Polymorphism BMP15 and Myostatin gene in the Arabic sheep using PCR-SSCP

Zeinab Davoodi[1]

Hedayatolah Roshanfek[2]

Jamal Fayazi[3]

Mohammad Taghi Beigi Nasiri[4]

ABSTRACT

Myostatin (also called growth and differentiation factor 8, GDF8) and BMP15genethis super-family includes enormous number of growth and differentiation factors those play essential roles in regulating embryonic development and maintaining tissue homeostasis in adult animals. In order to evaluate the BMP15 and myostatin gene polymorphism blood samples collected in EDTA coated tube from jugular vein of 140 Arabic breed sheep and genomic DNA was isolated using the DNA kit .One primer pairs were used to amplify genomic DNA of BMP15 gene and the PCR products were separated on 1% agarose gels. The results showed that products had good specificity and banding patterns shown by 8% acril amid. The allele frequency of M and N BMP15 and myostatin respectively, 0.79, 0.21 and 0.62, 0.38and the genotype frequencies MM and MN 0.58, 0.41 and 0.25, 0.75were obtained. These loci were not in Hardy-Weinberg equilibrium. It seems that these genes can be used as a marker which had a major effect on increasing the twin rate and muscling.

Keyword: BMP15, myostatin, Arabic sheep, Fecundity, PCR-SSCP

INTRODUCTION

Arabic sheep is a light weight fat tail measured to be of main economic importance because Iranian breed of its meat well modified to a wide range of severe environmental conditions in southern Iran. The body size differs between 35 and 40 kg in adult ewes. This breed has resistance to disease, perfect flexibility to the severe environmental conditions. Sheep is among the first mammalian species that have been domesticated. The population of sheep in the Khuzestan was approximately4 million. In Iran, sheep meat is a major source of animal protein and examination for meat produce and associated genes is important Meat quality is one of the important economic characters in domestic animals. Determination of meat quality needs examination and sorting of, structure,fat content, tenderness, water-holding capacity, color, stability, oxidative, and uniformity. Meat quality is affected by numerous factors such as type, genotype, nourishing, fasting, pre slaughter handling, stunning, slaughter systems chilling, and storage conditions (Rosenvold and Anderson, 2003). recently a lot of works have been achieved in this ground to discovery potential genes or chromosome regions related with the meat quality characters indifferent farm animals, including sheep, cattle, and chicken.

Myostatin gene

Myostatinencodes a negative regulator of skeletal-muscle growth invertebrate Myostatin (MSTN) block myogenesis, hematogenesis and enhance chondrogenesis.According to chromosome map, this gene is placed on chromosome 2 and contains three exons and two introns (Charlier et all.,1995).

·  The regulation of muscularity, adiposity and tendon

·  Structures possibly have important implications for sheepmeat production.

In mice, null mutants are significantly larger than wild-type animals, with >200% more skeletal-musclemass, because of mass because of an increase in the number of myocytes (hyperplasy) and an increase in the size of muscle fibers (hypertrophy) (McPherron et all., 1997). double-muscling in cattlethathad less bone,less fat, and more muscle on an average (Kobolak and Gocza,2002).Mutationswithin myostatin gene was reported recently also in dogs, sheep and a child (Kambadur et all., 1997; Mosher et all., 2007; Clop et all., 2006).

BMP15 gene

The bone morphogenetic protein 15 (BMP15) identified as growth differentiation factor9B (GDF9B) on X choromosome (Dube et al, 1998) which is a member of the transforming growth factor beta super family (TGFB) expressed in human, rodent and sheep (Vitt et al., 2001). According to chromosome map, this gene is placed on chromosome X and contains two exone and one intron that create a protein with 125 Amino Acid, (Hanrahan et al., 2004). BMP15 acts as a paracrine /autocrine factor to simulate follicle development and have a major role in regulating ovulation rate, oocyte quality and corpus lutein formation (Moore et al., 2005; davis et all., 2005). In previous studies Reported that mRNAs and proteins of BMP-15 are expressed in sheep ovarian follicles at all phases of their growth. Mutation in BMP15 gene in ewes has been detected to cause defects in folliculogenesis. Natural occurring mutation in sheep BMP15 such as FecXG, B.I.H.L (Chu et al., 2005; Hanrahan et al., 2004; McNatty et al., 2005; Bodin et al., 2006) cause infertility in homozygous ewes due to deficiencies in early folliculogenesis whereas heterozygous ewes have increased ovulation rate.

Materials and methods

In this study blood samples were primarily taken from 140 sheep. Approximately 3 ml of blood was collected from the jugular vein and the collected samples were transported to the laboratory at 4 c before DNA isolation. The DNA was isolate according to the procedure described by a routine protocol. One pair of PCR primers used for intron 1 of myostatin (forward 5′-GAAACGGTCATTACCATGC-3′ and reverse 5′-CATATTTCAGGCAACCAAATG-3′ (Hycfordet all, 2009) and for exon 2 of BMP15 (forward 5-TACAGACCCTGGACTTTCCTCT-3 and reverse 5-GCCCAACATGTTCCATGATATCC-3). The PCR reaction was performed in a 25µl reaction volume containing about 3 µl genomic DNA, 1 µl of each primer, 2.5 µl PCR buffer 10-X, 0.75 µl Mgcl2, 0.5 µl dNTP and 0.2 units of Taq DNA Polymerase, initial denaturation at 95 C for 5 min 35 cycles of denaturation at 95 for 30 s, annealing at 60 for 30 s, extension at 72 C for 30 s, then holding at 72 C for 10 min and for BMP15 3 µl genomic DNA, 1 µl of each primer, 2.5 µl PCR buffer 10-X, 0.75 µl Mgcl2, 0.5 µl dNTP and 0.2 units of Taq DNA Polymerase, initial denaturation at 95 C for 5 min 35 cycles of denaturation at 95 for 30 s, annealing at 56 for 30 s, extension at 72 C for 30 s, then holding at 72 C for 10 min.

Single-Stranded conformation polymorphism

Aliquots of 5 µl PCR products were mixed with 5 µl denaturing solution (98% formamide, 0.025% xylenecyanoleand, 0.025%, bromophenol blue and 10 mM EDTA), incubated at 98 c for 10 min and then chilled on ice Denatured DNA was loaded on 8% PAGE gel and constant voltage 160 v for 3-4 h. The gel was stained with 0.1% silver nitrate solution. PCR products of myostatinand BMP15 Homozygous and heterozygous genotypes recognized from different SSCP patterns in Arabic sheep. The genotype and allele frequencies of myostatin gene polymorphisms determine the populations are in Hardy-Weinberg equilibrium using GenAlex software.

Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) is one further analysis method that utilizes PCR product. PCR-SSCP method is a reliable method of quickly detecting a mutation. This method is based on the assumption that the nucleotide acid changes would lead to change in migration patterns on polyacrylamide gel. Mutation was detected from the differences in migration patterns from conformation of single strand DNA on polyacrylamide gels. The mutation recognition rate of SSCP differs depending on the parameters, such as size of the fragment, base composition of the sequence, electrophoresis temperature and/or gel composition.

Result and discussion

Genomic DNA was isolated using the DNA kit (Figure 1). A 235 and 414bp generated DNA fragment BMP15 and myostatinwas subjected to 1% agarose (Figure 2, 3).

Gel electrophoresis PCR products of myostatin gene intron1 and BMP15 Exone 2 were polymorphic in Arabic breeds.

All samples were assessed with PCR- SSCP technique using 8%Acril amid gel by silver nitrate. Using of PCR-SSCP to identify new mutation and amplified samples show distinct banding pattern in Acril amid gels. In Figure 4 are shown MM and MN genotypes frequencies 0.25 and 0.75in Arabic sheep. Resulted polymorphismin myostatin established previous studies that were reported by Hicford et al. 2009, Ansari et al.2011had a major effecton musclingmyostatin gene and were polymorphic in Ramny and baluchi breeds. In contrast, the deficiency myostatin mutation is reported many sheep breeds such as dehnaviet all 2012 whichThis locus was not in Hardy-Weinberg equilibrium. M (suitable) Allelfrecuency MSTN locus in Zel sheepare very lowObserved.studies showed that myostatin had a major effecton muscling and fat depth in New Zealand Texel siresand Charollais sheep (McRae et all., 2005; Johnson et all., 2005). We report deletion (1-bp deletion atposition 960have been found in Norwegian White sheep.

The haplotypes were named M and N, respectively, M alleles were 0.62 and N alleles were .0.38, and the Ne was 1.88, and Na 2 andHe0.469and Ho 0.750, in Arabic sheep. Was not in Hardy-Weinberg equilibrium buthigh genetic diversity in this population wasobservedwhich contrastGenetic diversity MSTN locus in Indonesia local sheepare very lowObserved heterozygosity for this locus was very low showing high level of homozygosity in the herd.This confirmed that factors leading to disequilibrium, especially selection, may affect the genetic structure of the population.

BMP15 Gene

In Figure 5 are shown MM and MN genotypes frequencies 0.58 and 0.42in Arabic sheep. Resulted polymorphism in BMP15 established previous studies that were reported by Wang et al., 2011; Soleimaniet al.,1390; Shebir et al., 2012; Hanrahan et al., 2004. In contrast, the deficiency BMP15 mutation is reported many sheep breeds such as north florina shall and sang sari. The haplotypes were named M and N, respectively, M alleles were 0.79 and N alleles were .0.21, and the Ne was 1.48, and Na 2 andHe0.327 and Ho 0.413, in Arabic sheep. Chi square (X2) in these populations for exon 2 showed Hardy – Weinberg equilibrium but (X2) not observed average genetic diversity in this population wasobserved. The With attention the role of BMP15 in increasing rate ovulation it seems this gene can be used as marker for increasing multiparty rate. Studies on BMP15 gene as candidate genes for fertility in sheep were recognized in the Belclare/Cambridge sheep, the Lacaune sheep, and Small Tailed Han sheep (Bodin et al., 2003; Kumar et al., 2006). Ovulation rates in BMP15 mutants in the homozygous are more than heterozygous in Arabic sheep and all sample shown fecundity which agreement with result in Soleimani et al., 1390.With respect to the positive effect of increased lambing rate on meat production and decreased the number of breeder ewes other pasture, discovery major genes affecting on twining trait is of great importance from the economic point of view therefore establishing.

CONCLUSION

Based on these results, it can be concludedthat the diversity of Myostatin and BMP15 locust in Arabic sheep is high and showed polymorphisms. All ofsheep’s were tested had MM and MN genotype for Myostatinand BMP15 locust.This locust was not in Hardy-Weinberg equilibrium. Identifying genes of major effect offers the opportunity to improve production efficiency, product quality and product diversity in livestock industry.

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