Kilis, Halep ve Kıl Keçilerinde Beta-Kazein (CSN2) Genindeki Çeşitliliğin Allel Spesifik PCR, Real-Time PCR ve Sekans Analizi ile Araştırılması

Yıl 2019, , 70 - 76, 01.07.2019

Faruk Bozkaya , Akın Yiğin Mehmet Osman Atlı

https://doi.org/10.31196/huvfd.592558

Öz

Bu çalışmada Kilis, Halep ve Kıl keçilerinde
Beta-kazein geninin (CSN2) promotor bölgesi ile 7. eksonunun belirli
nükleotitlerindeki farklılıkların allel spesifik polimeraz zincir reaksiyonu
(AS-PCR), Real-Time PCR ve sekans analizi yöntemleriyle araştırılarak söz
konusu keçi populasyonlarının bu açıdan genetik çeşitliliğinin belirlenmesi
amaçlanmıştır. Bu amaçla toplam 246 DNA örneği kullanılmıştır. CSN2 geninin
promotor bölgesindeki g1311T>C mutasyonu AS-PCR ile, 7. eksonun 404.
pozisyonundaki C>T mutasyonunun (CSN2C alleli) varlığı ise Real-Time PCR ve
sekans analizi ile incelenmiştir. AS-PCR işlemi sonucunda CSN2 geni promotor
bölgesi 1311. nükleotitte T allelinin sıklığı bütün populasyonlarda yüksek bulunmakla
birlikte Kıl keçilerinde C allelinin sıklığı (0.0489) Halep (0.0190) ve Kilis
(0.0188) keçilerinden daha yüksek bulunmuştur. Bütün populasyonlar
değerlendirildiğinde 7. eksonunun 404. pozisyonundaki T nükleotitinin (CSN2C
alleli) sıklığının (0.824) C nükleotitinden (0.176) daha yüksek olduğu
gözlenmiştir. En yüksek CSN2C alleli sıklığı Kıl keçilerinde (0.8864) en düşük
ise Kilis keçilerinde (0.6364) gözlenmiştir. Sonuç olarak Kilis, Halep ve Kıl
keçilerinde CSN2 geninin promotor bölgesindeki g1311T>C mutasyonu ile 7.
eksonun 404. pozisyonundaki C>T mutasyonu açısından çeşitlilik gösterdiği
tespit edilmiştir. Bu çeşitliliğin keçi sütünün bileşimi ve beta kazein
içeriğini etkileyip etkilemediğinin belirlenmesi için daha fazla araştırmaya
ihtiyaç bulunmaktadır.

Anahtar Kelimeler

Keçi, Süt, CSN2, Polimorfizm

Destekleyen Kurum

Harran üniversitesi bilimsel araştırmalar projeleri koordinasyon birimi

Proje Numarası

15116/2017

Teşekkür

Bu çalışma Harran üniversitesi bilimsel araştırmalar projeleri koordinasyon birimi tarafından desteklenmiştir (Proje No.15116/2017).

Investigation on the polymorphism of Beta-Casein gene (CSN2) in Kilis, Aleppo and Hair Goats by using allele specific PCR, Real-Time PCR and sequence analysis

Öz

The objective of this study
was to investigate the genetic variability of Beta-casein gene (CSN2) with
respect to certain nucleotides at the promotor region and exon 7 in Kilis,
Aleppo and Hair goats by using allele specific polymerase chain reaction
(AS-PCR), real-time PCR and sequencing methods. A total of 246 DNA samples were
used. Presence of the mutation at the promotor region (g1311T>C) of CSN2
gene was searched by using AS-PCR, while the polymorphism at the 404th
nucleotide of the exon 7 was analysed using Real-Time PCR and sequence
analysis. By using AS-PCR frequency of the T allele at the 1311th
nucleotide of the promotor region was found to be higher than that of C allele,
while the frequency of the C allele was higher in Hair goats (0.0489) than that
in Aleppo (0.0190) and Kilis goats (0.0188). When all three populations were
considered, frequency of T nucleotide (CSN2C allele) at the 404th
nucleotide position of the exon 7 (0.824) was higher than that of C nucleotide
(0.176). The highest frequency of the CSN2C allele was found in Hair goats
(0.8864) while the lowest frequency of this allele was in Kilis goats (0.6364).
The results indicated that polymorphism was present with respect to g1311T>C
mutation at the promotor region and at the 404th nucleotide of the
exon 7 of the CSN2 gene among Kilis, Aleppo and Hair goats. Further studies are
required for determining the effect of these polymorphism on the composition
and beta-casein content the milk in these goat populations.

Anahtar Kelimeler

Goat, Milk, CSN2, Polymorphism

Proje Numarası

15116/2017

Kaynakça

• Balteanu VA, Pascal C, Vlaic A, 2015: Genetic polymorphisms of Αs1-Caseın (CSN1S1) and Β-casein (CSN2) genes in carpathian goat breed. Lucrări Științifice - Seria Zootehnie, 63, 193-198.
• Bozkaya F, Mundan D, Karabulut O, Yerturk M, Gurler S, Aral F, 2008: An investigation on the distribution of O and D alleles of the CSN1S2 gene in goat populations raised in south eastern region of Turkey. Small Ruminant Res, 78, 193-196.
• Bozkaya F, Gürler Ş, Yertürk M, 2013: Genetic variability of CSN1S1 gene in goat populations raised in Southeastern Region of Turkey. Kafkas Univ Vet Fak Derg, 19, 147-152.
• Caroli A, Jann O, Budelli E, Bolla P, Jager S, Erhardt G, 2001: Genetic polymorphism of k-casein (CSN3) in different breeds and characterization at DNA level. Anim Genet, 32, 226-230.
• Caroli A, Chiatti F, Chessa C, Rignanese D, Bolla P, Pagnacco G, 2006: Focusing on the goat casein complex. J Dairy Sci, 89, 3178-3187.
• Chessa S, Budelli E, Chiatti F, Cito AM, Bolla P, Caroli A, 2005: Predominance of β-Casein (CSN2) C allele in goat breeds reared in Italy. J Dairy Sci, 88, 1878-1881.
• Chianese L, Garro G, Nicolai MA, Mauriello R, Ferrani P, Pizzano R, Cappucio LP, Addeo F, Ramunno L, Rando A, Rubino R, 1993: The nature of β-casein heterogeneity in caprine milk. Lait, 73,533-547.
• Cosenza G, Pauciullo A, Gallo D, Di Berardino D, Ramunno L, 2005: A SspI PCR-RFLP detecting a silent allele at the goat CSN2 locus. J Dairy Res, 72, 456-459.
• Cosenza G, Paciullo A, Colimono L, Mancus A, Di Berardino D, Ramunno L, 2007: An SNP in the goat CSN2 promotor region is associated with the absence of β-casein in milk. Anim Genet, 38, 655-658.
• Cosenza G, Iannaccone M, Pico BA, Ramunno L, Capparelli R, 2016: The SNP g.1311T>C associated with the absence of β-casein in goat milk influences CSN2 promoter activity. Anim Genet, 47,615-617.
• Cunsolo V, Galliano F, Muccilli V, Saletti R, Marletta D, Bordonaro S, Foti S, 2005: Detection and characterization by high-performance liquid chromatography and mass spectrometry of a goat β-casein associated with a CSN2 nullallele. Rapid Commun Mass Sp,19, 2943-2949.
• Feligini M, Frati S, Cubric CV, Brambilla A, Parma P, Curik I, Enne G, 2005: Caprine αs1-casein polymorphism: characterization of A, B, E and F variants by means of various biochemical and molecular techniques. Food Technol Biotech, 43,123-132.
• Hall TA, 1999: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic acids symposium series, 41,95-98.
• Korkmaz AÖ, Çınar KB, Akyüz B, Elmaz Ö, Özçelik MM, Saatçi M, Ertuğrul O, 2012: Identification of β-lactoglobulin gene SacII polymorphism in Honamli, Hair and Saanen goat breeds reared in Burdur vicinity. Kafkas Univ Vet Fak Derg, 18, 385-388.
• Kusza S, Ilie DE, Sauer M, Nagy K, Patras I, Gavojdian D, 2016: Genetic polymorphism of CSN2 gene in Banat White and Carpat in a goats. Acta Biochim Pol, 63,577-580.
• Kyseľová J, Rychtářová J, Sztankóová Z, Czerneková V, 201: Simultaneous identification of CSN3 and LGB genotypes in cattle by high-resolution melting curve analysis. Livestock Sci, 145, 275-279.
• Liu J, Huang S, Sun M, Liu S, Liu Y, Wang W, Xiurong Z, Hanzhong W, Hua W, 2012: An improved allele-specific PCR primer design method for SNP marker analysis and its application. Plant Methods, 8, 34.
• Marletta D, Bordonaro A, Guastella AM, D'Urso G 2005: Genetic polymorphism of calcium sensitive caseins in Sicilian Girgentana and Argentatadell’Etna goat breeds. J Anim Breed Genet,121, 52-56.
• Marletta D, Criscione A, Bordonaro S, Guastella AM, D'Urso G, 2007: Casein polymorphism in goat's milk. Lait, 87, 491-504.
• Moatsou G, Samolada M, Panagiotou P, Anifantakis E, 2004: Casein fraction of bulk milks from different caprine breeds. Food Chem, 87, 75-81.
• Neveu C, Molle D, Moreno J, Martin P, Leonil J, 2002: Heterogeneity of caprine beta-casein elucidated by RP-HPLC/MS: Genetic variants and phosphorylations. J Protein Chem, 21, 557-567.
• Paksoy N, İriadam M, 2012: Kilis keçilerinde serum selenyum düzeylerinin araştırılması. Harran Univ Vet Fak Derg, 1, 6-8.
• Pazzola M, Dettori ML, Pira E, Noce A, Paschino P, Vacca GM, 2014: Effect of polymorphisms at the casein gene cluster on milk renneting properties of the Sarda goat. Small Ruminant Res, 117, 124-130.
• Persuy MA, Printz C, Medrano JF, Mercier JC, 1999: A single nucleotide deletion resulting in a premature stop codon is associated with marked reduction of transcripts from a goat β-casein null allele. Anim Genet, 30, 444-451.
• Prinzenberg EM, Gutscher K, Chessa S, Caroli A, Erhardt G, 2005: Caprine K-casein (CSN3) polymorphism: new developments in molecular knowledge. J Dairy Sci, 88, 1490-1498.
• Ramunno L, Cosenza G, Pappalardo M, Pastore N, Gallo D, Digregorio P, Masina P, 2000: Identification of the goat CSN1S1 F allele by means of PCR-RFLP method. Anim Genet, 31, 342-343.
• Ramunno L, Longobardi E, Pappalardo M, Rando A, Digregorio P, Cosenza G, Mariani P, Pastore N, Masina P, 2001: An allele associated with a non-detectable amount of αs2 casein in goat milk. Anim Genet, 32, 19-26.
• Rijnkels M, 2002: Multispecies comparison of the casein gene loci and evolution of casein gene family. J Mammary Gland Biol, 7, 327-345.
• Roberts B, Di Tullio P, Vitale J, Hehir K, Gordon K, 1992: Cloning of the goat β-casein-encoding gene and expression in transgenic mice. Genetics, 121, 255–62.
• Sambrook J, Fritsch EF, Maniatis T, 1989: Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York.
• Sacchi P, Chessa S, Budelli E, Bolla P, Ceriotti G, Soglia D, Caroli A 2005: Casein haplotype structure in five Italian goat breeds. J Dairy Sci, 88, 1561-1568.
• Singh LV, Jayakumar S, Sharma A, Gupta SK, Dixit SP, Gupta N, Gupta SC, 2015: Comparative screening of single nucleotide polymorphisms in β-casein and κ-casein gene in different live stock breeds of India. Metagene, 4, 85-91.
• Sztankóová Z, Kyseľová J, Kott T, Kottová, E, 2008: Technical Note: Detection of the C allele of β-Casein (CSN2) in Czech dairy goat breeds using LightCycler analysis. J Dairy Sci, 91, 4053–4057.
• Trujillo AJ, Casals I, Guamis B, 2000: Analysis of major caprine milk proteins by reverse-phase high-performance liquid chromatography and electro sprayionization-mass spectrometry. J Dairy Sci, 83, 11-19.
• TÜİK, 2019: Türkiye İstatistik Kurumu, Temel İstatistikler, Hayvansal Üretim. http://tuik.gov.tr/UstMenu.do?metod=temelist, Erişim Tarihi;04.02.2019.
• Vacca GM, Dettori ML, Piras G, Manca F, Paschino P, Pazzola M, 2014: Goat casein genotypes are associated with milk production traits in the Sarda breed. Anim Genet, 45, 723-731.