شناسايي جهش‌هاي FecXI، FecXH و FecXB در ژن BMP15 در گوسفند نژادهاي لك قشقايي و بهمئي در استان كهگيلويه و بوير احمد

Investigation of FecXI, FecXH and FecXB Polymorphism in BMP15 Gene in Lac Qashqai and Bahmaei Sheep Breeds at Kohkiloueh and Boyerahmad Province

نرخ باروري يكي از صفات مهم اقتصادي در گوسفند است، كه تحت تأثير ژنتيك و محيط مي­باشد. ژنBMP15 يكي از ‌ژن‌هاي موثر بر اين صفت مي‌باشد و جهش ­هاي مختلف در اين ژن باعث تغيير راندمان صفات توليدمثلي و رشد در گوسفند مي‌شود. چنانكه باروري بالا در گوسفندان به علت جهش در جايگاه BMP15 با منشأ اووسيتي (ژن فاكتور رشد) شناخته شده است. در اين مطالعه از روش PCR-RFLP براي شناسايي چند شكلي‌ جايگاه‌هاي ژني FecXI، FecXH وFecXB استفاده شد. بدين منظور از تعداد 92 رأس ميش نژاد لك قشقايي و بهمئي به طور تصادفي خون‌گيري صورت گرفت و استخراج DNA با استفاده از كيت استخراج DNA انجام شد. قطعات مورد نظر با استفاده از آغازگرهاي اختصاصي (قطعه bp 204 از FecXI، قطعه bp 235 از FecXH و قطعه bp 153 از ژن FecXB)، و توسط واكنش زنجيره پلي‌مراز (PCR) تكثير گرديد. پس از مجاورت محصولات PCR با آنزيم­هاي اختصاصي (XbaI، SpeI وDdeI)، مشاهده گرديد كه همه قطعات FecXI و FecXH فاقد جايگاه برش براي آنزيم هضم‌كننده بوده و تنها يك الگوي باندي در آن­ها مشاهده شد. اين مطالعه تنها وجود آلل ­هاي تيپ وحشي را تأييد كرد. تمامي نمونه‌ها داراي ژنوتيپ هموزيگوت بودند و جهش اينوردل و هانا (جهشي كه در حالت هتروزيگوت باعث افزايش ميزان تخمك‌گذاري مي‌شود) در آن­ها يافت نشد. براساس نتايج اين مطالعه، ژن BMP15 نمي­تواند تأثيري بر ميزان چند‌قلوزايي در گوسفندان مورد مطالعه داشته باشد، بنابراين بايد به دنبال جهش ­ها يا ژن­هاي ديگري بود.

Introduction Litter size is an important economic trait in sheep breeding which is influenced by genetics and environment factors. Application of phenotypic data in traditional breeding programs is a time consuming process. Thus, marker assisted selection (MAS) play an important role for genetic improvement of reproduction efficiency. Since, there has been great interest in the identification of major genes that influence fecundity traits in sheep. Three of these fecundities identified genes are bone morphogenetic protein receptor type IB (BMPRIB) or Activin Like Kinase 6, known as FecB located on chromosome 6; growth differentiation factor (GDF9), known as FecG located on chromosome 5 and bone morphogenetic protein 15 (BMP15), known as FecX located on the X chromosome. The BMP15) is expressed in oocytes where encodes for a mature peptide of 125 amino acids that stimulate the proliferation rate of the granulose cells and thus it seems to be essential in folliculogenesis. Some mutations in BMP15 gene are reported to increase fertility rate. Six different point mutations i.e. FecXI and FecXH in the Inverdale and Hanna breeds, respectively, FecXL in the Lacaune, FecXG and FecXB in the Cam-bridge and Beclare breeds, respectively and the FecXR in Raza Aragoneza breed have been identified in BMP15 gene. In this study, the FecXB, FecXH and FecXI mutations at the BMP15 gene were investigated by PCR–RFLP in Lac Ghashghaei and Bahmaei sheep breeds. The FecXB (G to T nucleotide change) was detected using DdeI; the wild-type strand was cleaved. FecXH (C to T nucleotide change) was detected using SpeI, the mutation-type strand was cleaved and FecXI (T to A nucleotide change) was detected using XbaI; the mutation-type strand was cleaved. Materials and Methods Blood samples were taken from 92 ewes according to data on litter size at the last lambing (Lac Ghashghaei: 24 single and 24 double lambing, Bahmaei: 22 single and 22 double lambing). The animals of the Lac Ghashghaei and Bahmaei breeds originate from two farms in Gachsaran and Dehdasht, respectively. Then, genomic DNA was isolated from whole blood using the DNA Extraction Kit according to the manufacturer’s instructions. Required parts of BMP15 gene were amplified using specific primers (fragment of 204 bp from FecXI, fragment of 235 bp of FecXH and fragment of 153 bp of FecXB) through Polymerase Chain Reaction (PCR). The PCR products of amplified fragments were digested by XbaI, SpeI and DdeI restriction enzymes and visualized by agarose gel electrophoresis, separately. Results and Discussion Sheep breeding is the major activity in the Kohkiloueh and Boyerahmad province of Iran and is very important in the rural economy of this region. Although litter size is an important trait that affects the profitability of sheep production, but the litter size of almost all sheep breeds of this province is low. In this study, PCR-RFLP method was used to identify three mutations (FecXI, FecXH and FecXB) of BMP15 gene in Lac Ghashghaei and Bahmaei ewes. As expected, the size of PCR production for FecXI, FecXH and FecXB loci were 204, 235 and 153 bp, respectively. After digestion of amplified fragments with specific restriction enzyme, the three earlier mentioned mutation sites were not detected in Lac Ghashghaei and Bahmaei sheep and only the wild-type genotype (++) was observed in both breeds for each mutation of BMP15 gene. In more detail, restriction digestion of amplified fragments for FecXB locus resulted DNA fragments with 31 and 122 bp. The wild type of this locus has one restriction site and this means that FecXB mutation was not detected in two breeds of this study and animal are homozygous for wild type allele of this locus. The resulted PCR products were digested with SpeI and DdeI restriction enzymes for FecXH, FecXI loci revealed DNA fragments with 235 and 204 bp, respectively. The mutant allele of FecXH, FecXI loci has one restriction site and enzyme digestion will be resulted in 2 DNA fragments while the wild type allele with no restriction site, resulted in one DNA fragments with the size. It means all 92 Lac Ghashghaei and Bahmaei ewes sampled were homozygous wild allele (++) for FecXH, FecXI loci. Conclusion The obtained results of this study indicated that there was an absence of three BMP15 mutations (FecXB, FecXH and FecXI) among the Lac Qashqai and Bahmaei sheep breeds sampled in the Kohkiloueh and Boyerahmad province, it seems genetic factor responsible for litter size is not related to reported mutated alleles of BMP15 gene and therefore other prolificacy related genes should be investigated in these breeds using a larger sample size.