توالي يابي و آناليز بيوانفورماتيكي ژن كاپا كازئين در جمعيت بزهاي خلخالي ايران و ساير دام هاي اهلي

Detection of Polymorphism of Kappa Casein Gene in Iranian Khalkhali Goats

در سال هاي اخير به طور عمده چند شكلي هاي ژنتيك پروتئين شير مورد توجه و تحقيق بيشتري بوده است. زيرا بين پروتئين شير و صفات اقتصادي مهم در دام رابطه مستقيم وجود دارد. كاپا كازئين به عنوان عمده ترين پروتئين موجود در شير نقش مهمي در شكل گيري ميسل هاي موجود شير و كمك به افزايش حلاليت كلسيم و فسفر ايفا مي كند، از اين رو اين مطالعه با هدف و رويكرد بررسي ژنتيكي و فيلوژنتيكي ناحيه اگزون 4 ژن كاپا كازئين با روش توالي يابي در جمعيت بز خلخالي و مقايسه آن با ساير گونه ها انجام گرفت. نتايج حاصل از توالي هاي منجر به شناسايي يك جهش در نمونه هاي توالي يابي شده گرديد كه باعث تغيير نوكلئوتيد C به T در جايگاه اگزون چهار ژن كاپا كازئين گرديد كه منجر به تغيير كدون CCT به TCT مي شود و باعث جايگزيني اسيد آمينه پرولين به جاي سرين مي شود. آناليز تنوع ژنتيكي در جمعيت بز خلخالي 0/524 به دست آمدكه نشان دهنده تنوع ژنتيكي نسبتا مناسب مي باشد. مقايسه توالي هاي جمعيت خلخالي با گونه هاي مختلف مانند بز، گاو ، گوسفند و گاو ميش منجر به شناسايي 41 جهش شد كه ايجاد 29 هاپلوتايپ مختلف با تنوع هاپلوتايپي 0/833 در گونه هاي مورد مطالعه مي كند. تنوع نوكلئوتيدي و ميانگين تفاوت نوكلئوتيدي (k) در بين گونه ها نيز به ترتيب برابر 0/04469 و 75436/ 11 به دست آمد.

Introduction: In compare with other dairy animal، the most important components of goat milk are protein and fat. Due to association between milk proteins and economic traits، there are a lot of studies about genetic structure of milk proteins. Nowadays، the effect of genetic polymorphisms in goats and other species and association with production traits and health widely was investigated. One of the most important topics for the genetic characterization of dairy breeds is investigation of the casein genes. The goat casein genes including CNS1S1، CSN2، CSN1S2 and CSN3 are clustered on chromosome 6. The Ks1-casein protein encoded by theCSN1S1 gene and known as candidate gene. The funding of different studies indicates the effect of ks1-casein gene on milk traits in goat. Khalkhali goat is one of the native dairy goats in Iran that is distributed in North West at Ardabil province. Due to less economically viable، the population of this breed have a declining trend. Therefore consider and attention to this breed is very important. K-casein gene is a useful genetic marker to improve animal breeding programs. The polymorphisms of k-casein gene have significant effect on milk composition and production. To the best of our knowledge، no study has yet considered the about casein gene in Khalkhali goats. This study aimed to assess the variation and polymorphisms at CSN3 loci on Khalkhali goats. Materials and Methods: In this study for analyzing of polymorphisms in Kappa casein gene، 100 blood samples were collected from Khalkhali goats. Genomic DNA was extracted using DNA isolation kit for mammalian blood and the exon 4 of Kappa casein gene amplified using specific primers including: Forward primer F: 5’-GGT ATC CTA GTT ATG GAC TCA AT-3’ and revers primer R:5’-GTT GAA GTA ACT TGG GCT GTG T -3’. Genotyping were determined using PCR-SSCP and were sequenced through detection of different pattern. The sequences were analyzed using bioinformatics software’s. Estimates of evolutionary divergence between the sequences were conducted using the maximum composite likelihood method by using MEGA version 6.10 software. Phylogenetic tree was constructed using the Neighbor-Joining method by using the same software. Haplotype analysis and the network analysis of haplotypes was constructed using DnaSp 5.10 and Network 4.613 respectively. Results and Discussion: The results of sequencing lead to identification of one polymorphic site in kappa casein gene sequences، that nucleotide C substituted by T in exon 4 of kappa casein gene which caused changing CCT codon to TCT، subsequently، based on IUPAC standard genetic code lead to conversion of Proline with Serine amino acid. Genetic diversity of Khalkhali goat was 0.524 which evidences intermediate genetic diversity in this breed. Comparison between species identified 41 mutations that cause 29 haplotype with 0.833 haplotype diversity. Nucleotide diversity and average nucleotide differences (K) among species were 0.04469 and 11.75436 respectively. The analyzing of 129 sequences of farm animal showed that the biggest haplotype is belong to Hap 1 and Hap 2 with 35 and 36 frequency respectively in capra hircus and Khalkhali goat population. Haplotype diversity، haplotype diversity variance، nucleotide diversity in each loci، ، average of the number of nucleotide diversity (k) and tajima D were 0.833، 0.0004، 0.0447، 11.7544 and 1.5899 respectively. Tajima D was non-significant at p>0.1 that could be due to use of less samples in this analysis. enetic differentiate (Fst) and Genetic distance (DXY) between farm animal based on CSN3 gene were analyzed and the most genetic differentiate were observed between sheep and buffalo and also we estimated less and negative genetic differentiate between Khalkhali goat and capra hircus. That could be because of allele frequencies distribution in marker loci is not normal. Conclusion: Results illustrated in this study could be useful in characterization of specific dairy products linked to the Khalkhali breed، which are certainly a suitable tool to conserve local breeds and biodiversity. based on mutation identified in k-casein gene and this substation caused changing in Amino acids and based on reports about this gene as good marker genetics in animal breeding programs، recommended that association of this mutation with milk traits would be investigated and if that is significant should be used in Khalkhali goat breeding program. These results could be preferred in future planning of breeding programs and selection schemes for enhancing economic traits.