بررسی چند شکلی تک نوکلئوتیدی واقع در ژن‎های کاندیدا BMPR1B و GDF9 میش‎های مغانی، افشاری و بلوچی

نوع مقاله : مقاله پژوهشی

نویسندگان

1 استادیار، گروه علوم دامی، دانشکده کشاورزی و دامپروری، مجتمع آموزش عالی تربت جام

2 دانشیار، گروه علوم دامی، دانشکده کشاورزی، دانشگاه فردوسی مشهد

3 دانشیار بخش تحقیقات علوم زراعی و باغی، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی خراسان رضوی، مشهد،

چکیده

گوسفند، به عنوان یک مدل ژنتیکی برای مطالعه ارتباط بین تنوع ژنتیکی و میزان تخمک‎گذاری در تحقیقات پیشین مورد استفاده قرار گرفته است. مطالعات اخیر نشان داده است که تنوع در میزان تخمک‎گذاری و صفت چندقلوزایی می‌توانند توسط مجموعه ژن‎هایی به نام ژن‎های کنترل کننده باروری کنترل شوند. در این راستا، سه ژن عملکرد باروری به نام‎های BMPR1B یا FecB، GDF9 یا FecG و BMP15 یا FecX در گوسفند شناسایی شده‎اند. روش مولکولی Tetra-ARMS PCR یک جایگزین مناسب برای روش‌های پرهزینه مانند توالی‎یابی و PCR-RFLP در شناسایی اسنیپ‎هایی است که توالی آنها شناخته شده هستند. در سالهای اخیر، ورود آلل برولا به نژاد گوسفند افشاری کشور موارد چند تخمک‎گذاری را افزایش داد. هدف از مطالعه حاضر، بررسی چند شکلی دو ژن باروری شامل FecB و FecG در گوسفندان نژاد مغانی، افشاری و بلوچی خراسان رضوی و ارتباط آن‎ها با صفت چندقلوزایی بود.
نمونه‎های خون از مجموع، تعداد ۹۵ رأس گوسفند افشاری، بلوچی و مغانی از طریق ورید وداجی اخذ شد. دو جفت آغازگر کنترل (خارجی) و اختصاصی (داخلی) برای تکثیر قطعات ژن‎های FecB و FecG در روش Tetra-ARMS PCR مورد استفاده قرار گرفتند. برنامه دمایی برای تکثیر قطعات ژن FecB به صورت واسرشت سازی اولیه در C 94 به مدت 4 دقیقه، سپس، 35 چرخه دمایی به ترتیب C 94 به مدت 25 ثانیه، دمای اتصال در C 54 به مدت 35 ثانیه، بسط در دمای C 72 به مدت 20 ثانیه و بسط نهایی در C 72 به مدت 5 دقیقه انجام شد. برای تکثیر قطعات ژن FecG از دمای اتصال C 54 به مدت 35 ثانیه، بسط در دمای C 70 به مدت 40 ثانیه و دمای بسط نهایی C 70 به مدت 5 دقیقه استفاده شد.
در بررسی نتایج بدست آمده، سه ژنوتیپ برای ژن FecB در گوسفند افشاری شامل هموزیگوت وحشی، هتروزیگوت و هموزیگوت جهش یافته مشاهده شدند. همه گوسفندان بلوچی و گوسفند مغانی برای این ژن هموزیگوت وحشی بودند. فراوانی ژنوتیپ هموزیگوت وحشی برای سه نژاد گوسفند فوق الذکر بالا بود. فراوانی آلل وحشی در این نژاد 39/0 و فراوانی آلل جهش یافته 61/0 بود، در مجموع، برای سه نژاد به ترتیب 8/0 و 2/0 اندازه گیری شد. نتیجه Tetra-ARMS PCR برای جهش نقطه‎ای G1 ‏درجایگاه GDF9 برای همه نژادها چندشکلی نشان داد. هرچند، فراوانی هموزیگوت وحشی بالا بود. برای نژاد گوسفند بلوچی فقط چهار درصد از حیوانات ژنوتیپ هتروزیگوت داشتند و ۹۶ درصد حیوانات ژنوتیپ وحشی را نشان دادند. برای نژاد گوسفند مغانی ۱۶ درصد ژنوتیپ هتروزیگوت را نشان دادند و فقط چهار درصد از حیوانات ژنوتیپ هموزیگوت جهش یافته داشتند. همچنین، تنها یک ژنوتیپ هتروزیگوت در نژاد افشاری مشاهده شد. مطالعات متعددی ارتباط بین ژن‎های FecB، FecG و FecX را با چند قلوزایی در گوسفند نشان داده است. به دلیل فراوانی بالای هموزیگوت وحشی و این حقیقت که میش‎های بلوچی و مغانی عمدتاً، تک قلوزا بودند، نقش جهش FecB در چند قلوزایی گوسفند افشار ی می‎تواند قابل توجه باشد. قوچ مورد استفاده در این گله هتروزیگوت بود. به طور کلی، می‌توان نتیجه گرفت که آلل جهش یافته FecB با چندقلوزایی گوسفندان ایرانی ارتیاط دارد. هرچند، تایید آماری این موضوع نیاز به یک مطالعه اختصاصی همرا با ثبت دقیق رکورد صفات تولید مثلی دارد.
نتایج این مطالعه چندشکلی آلل برولا در میش‎های افشاری که فرزندان یک قوچ ژنوتیپ هتروزیگوت بودند را نشان داد. همچنین، کل این میش‎ها در شکم اول و 50 درصد آن‎ها در شکم دوم چند قلوزا بودند. بنابراین، جهش برولا ممکن است باعث چند قلوزایی در میش‎ها ‎شود. هرچند، تایید ارتباط چندشکلی‎های ژنی مرتبط با افزایش باروری و چندقلوزایی نیاز به مطالعه جامع‌تری به همراه اندازه‎گیری شاخص‎هایی مانند غلظت هورمون‎های جنسی، میزان تخمک‎گذاری و اندازه فولیکول‎ها دارد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Investigation on single nucleotide polymorphism of BMPR1B and GDF9 genes in Mughani, Afshari and Baluchi ewes

نویسندگان [English]

  • Reza Tohidi 1
  • Ali Javadmanesh 2
  • Elias Ebrahimi Khoramabadi 1
  • Kamal Ghasemi Bezdi 3
1 Assistant Professor, Department of Animal Science, Faculty of Agriculture and Animal Husbandry, Torbat Jam Higher Education Complex
2 Associate Professor, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad
3 Associate Professor, Agricultural and Horticultural Science Research Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, Mashhad,
چکیده [English]

Sheep are used as a genetic model to study the relationship between genetic diversity and ovulation rate. Previous studies have shown that ovulation rate and litter size can be controlled by a set of genes called fecundity genes . Three fertility genes have been identified in sheep called BMPR1B or FecB, GDF9 or FecG and BMP15 or FecX. Tetra-ARMS-PCR is a suitable alternative method in the context of expensive methods such as sequencing and PCR-RFLP to identify SNPs whose sequences are known. The entry of the Booroola allele into the Afshari breed increased the frequency of litter size. The aim of this study was to investigate the polymorphism of two fertility genes including FecB and FecG in Mughani, Afshari and Baluchi sheep in Khorasan Razavi province.
Blood samples were taken from 95 Afshari, Baluchi and Mughani sheep. Two pairs of control (outer) and specific (inner) primers were used to amplify FecB and FecG gene fragments by the Tetra-ARMS PCR method. Temperature cycling of PCR amplification for FecB started at 94°C for 4 minutes, followed by 35 cycles consisting of 94°C for 25 seconds, annealing temperature at 54°C for 35 seconds, extension at 72°C for 20 seconds and a final extension at 72°C for 5 minutes. For amplification of the G1 point mutation on the FecG gene, the annealing temperature was 54°C for 35 s, extension was 70°C for 40 s, and final extension was 70°C for 5 minutes. The PCR products were electrophoresed on a 2% agarose gel.
Three genotypes have been observed for the FecB gene in Afshari sheep; wild homozygous (++), heterozygous (B+) and homozygous mutant (BB). All Mughani and Baluchi sheep were homozygous for this gene. A 108 bp band was detected for mutant homozygotes. A band of 213 bp was observed in wild homozygotes. The frequency of wild homozygotes was high in the three breeds,. The frequency of the wild allele in this breed was 0.39 and that of the mutant allele 0.61, the overall frequencies for the three breeds being 0.8 and 0.2, respectively. The result of Tetra-ARMS-PCR for the G1 point mutation of the GDF9 gene showed polymorphism in all three breeds, however, the frequency of wild homozygotes was high. In the Balochi breed, only two animals (4%) were heterozygous and 96% of the animals showed the wild homozygous genotype. In the Mughani breed, 16% were heterozygous and only one animal was homozygous mutant. In addition, only one heterozygote was observed in the Afshari breed. Several studies have shown the association of the FecB, FecG and FecX genes with litter size in sheep. Due to the high frequency of wild homozygotes and the fact that Mughani and Baluchi ewes mostly gave birth to single lambs, the role of FecB mutation in litter size of Afshari ewes was observed. The ram used in this herd was heterozygous for FecB. In general, it can be concluded that the mutant FecB allele has a correlation with litter size in Iranian sheep. However, the statistical confirmation of this issue requires a dedicated study with a detailed record of reproductive traits.
The results of this study demonstrated the presence of a Booroola mutation in the Afshari ewes that were offspring of heterozygous rams. In addition, all of these ewes had litter size in at least one of the two pregnancies. Hence, the Booroola mutation may potentially increase the litter size of ewes. However, a more comprehensive study measuring biological indicators such as sex hormone levels, ovulation rate and follicle size is needed to demonstrate the effect of these types of mutations on increasing fertility.

کلیدواژه‌ها [English]

  • Candidate gene
  • Fecundity
  • Polymorphism
  • Sheep
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