Identification of Single Nucleotide Polymorphism (SNP) in β-Lactoglobulin Gene and Its Association with Milk Composition in Iranian Indigenous Khalkhali Goats

Document Type : Research Articles


1 Department of Animal Science, Faculty of Agricultural Science, University of Mohaghegh Ardabili, Ardabil, Iran

2 Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

3 Department of Animal Science, Faculty of Agriculture, Ilam University, Ilam, Iran


β-lactoglobulin is one of the important milk proteins in several ruminants like goats, sheep and cattle. This study was performed to investigate the genetic polymorphism of exon 7 and 3’ UTR region of β-lactoglobulin (BLG) gene and its association with milk composition in 120 unrelated individuals of Iranian indigenous Khalkhali goats. Purified polymerase chain reaction (PCR) products (427 bp) were sequenced under standard conditions using Sanger sequencing. Alignment of sequenced fragments against reference sequence leads to identification of one single nucleotide polymorphism (SNP), substitution A (frequency equal to 0.44) to C (frequency equal to 0.56) in the 3’ UTR region of BLG gene. Observed frequencies of AA, AC and CC genotypes were 0.12, 0.64 and 0.24, respectively. The effects of identified genotypes on milk composition were analysed using general linear model. We found that BLG gene genotypes have a significant effect on milk parameters except for lactose percentage (p < 0.05). The milk of goats with AA and AC genotypes had higher protein and fat percentages, respectively, in compaired with other genotypes (p < 0.05). Obtained results revealed that, identified genotypes in the BLG gene of Khalkhali goats is not in Hardy-Weinberg equilibrium.



Iran is one of the most likely locations for goat domestication about 10000 years ago (Haenlein, 2007; Naderi et al. 2008). Goats are considered an important domestic species in Iran due to regional and cultural resaons. Based on FAOSTAT (2017), Iran with almost 23.2 million goats (eighth in the world), is one of the important locations for goat rearing in the world. Due to different climates and breeding manners, several divers goat breeds have raise up in Iran, including Najdi, Tali, Raini, Markhaz, Bluchi, Mamasani, Adani and Khalkhali. Khalkhali goats are mainly dispersed in khalkhal, Dasht-e-Moghan (Ardabil province) and in some part of Azarbaijan-Sharghi province of Iran. This breed has a small body size (Figure 1) and mostly is breed for meat and milk production. Goat milk in compared with cattle milk, has favorable chemical, physical, organoleptic and nutritional characteristic that makes it a good nutritional source for infants and chidren and also a medicinal food (Silanikove et al. 2010). Most of milk proteins in ruminants are encoded by six genes including four casein genes as cluster of CSN1S1, CSN2, CSN1S2 and CSN3 (Rijnkels, 2002) and also two lactoglobulin genes including α- and β-lactoglobulin (α-LA and β-LG) (Martin et al. 2002). BLG gene contains 7 exons and is located on goat’s chromosome 11. This gene encodes a protein with 180 amino acid and mass of 19976 Da in goats (Uniprot accession: P02756) and considered as a important allergens component of milk in cow, sheep and goat (Kapila et al. 2013). It is worth to mention that this protein is absent in milk from camels, human and rabbits (Selvaggi et al. 2015).


Figure 1 A female Khalkhali goat



Several studies on genetic polymorphisms in the BLG gene and their association with the milk-related traits were performed in ruminants such as cattle (Ganai et al. 2009; Karimi et al. 2009; Vidovic et al. 2014; Ozdemir et al. 2018) and sheep (Çelik and Özdemir, 2006; Staiger et al. 2010; Kawecka and Radko, 2011). Also, previousley studies about milk-related traits in goats (Caravaca et al. 2011; Dagnachew et al. 2011; Palmeri et al. 2014; El Hanafy et al. 2015) confirmed the impact of genetic variants on some traites like protein content of milk that is a important attribute for produts like the cheese. An extensive number of genetic diversity have been reported in different parts of BLG gene in different species (Sardina et al. 2012; Yang et al. 2012; Özmen and Kul, 2016). Among them, the polymorphisms at exon 7 define one of important variation effect on milk composition in goat (Kumar et al. 2006; El Hanafy et al. 2015). Despite the investigation of genetic variants of BLG gene and their effects on milk composition in some Iranian goat breeds (Gharedaghi et al. 2016), but no similar information about BLG gene variation has been reported in the Khalkhali goat breed. Therefore the current study was design to investigate about genetic polymorphism of BLG gene and its impact on milk composition in Khalkhali goat.



Samples and DNA extraction

Based on survey information, a total of 120 unrelated individuals were randomly selected from four different herds of Khalkhali goat in the Northwest of Iran according to guidelines of animal care. The sampled animals were completely dependent on the same pastures; they were grazing in the pasture during the day and kept in a roofed place at night. All procedures used in this experiment approved by research council of university of Mohaghegh Ardabili. Milk records were obtained from all 120 Khalkhali goats, so that, milk of some goats were sampled in the first lactation and others in the second lactation. Individual milk samples were collected manually from morning milking at the end of each month of lactation and were stored at -20 ˚C until further use. For more accuracy, each sample was divided into two fractions for analysis of milk composition including protein, fat, lactose percentage and dry matter content using Milko-Scan FT 6000 (Foss Electric, Hillerød, Denmark). About 4 mL of blood was collected through the jugular vein of 120 sampled goats using venoject tubes containing lyophilized ethylenediaminetetraacetic acid (EDTA) (EDTA). Total genomic DNA was purified from whole blood using Exgene Cell SV kit (GENEALL Biotechnology co, LTD, Republic of Korea) and the quality of extracted DNA was assessed using 0.8% Agarose gel electrophoresis and Thermo Scientific™ NanoDrop spectrophotometers.


PCR amplification, sequencing, and statistical analysis

A 427 bp fragment of exon 7 and 3’ UTR of the caprine BLG gene was amplified (Figure 2) using following primers set: Forward 5-CGGGAGCCTTGGCCCCTCTGG-3 and Reverse 5-CCTTTGTCGAGTTTGGGTGT-3 (Kumar et al. 2006). Amplification of desired DNA fragment, was carried out in 25 µL volume reaction mixture containing 0.2 mM dNTP, 1.2 mM MgCl2, 1.5U Taq DNA polymerase (Ampliqon), 15 pMol of forward and reverse primer (Invitrogen) and 100 ng genomic DNA. The thermal profile consisted of denaturation at 95˚C for 5 min, followed by 33 cycles of 94 ˚C for 30s, annealing at 63 ˚C for 30 s and extension at 72 ˚C for 60 s, with a final extension step at 72 ˚C for 10 min. The PCR products were purfied using ExpinTM PCR SV kit from GENEALL company. Then, purified products were sent to Macrogen company (Seoul, South Korea) for sequencing under standard condition using sanger sequencing technology (Sanger et al. 1977). Chromas 2.33 ( was used to edit the sequenced fragnments. Then, the obtained sequences were analyzed and compared using MEGA 6.0 (Tamura et al. 2013).


Figure 2 PCR amplification of BLG gene (A fragment of 427 bp) in Khalkhali goat



The genotype frequency of single nucleotide polymorphisms (SNPs) was determined by direct counting methods. Finally, a fixed model was used to perform the milk composition association with BLG gene polymorphisms using generalize linear model (GLM) procedure of SAS software 9.2 (SAS, 2004). The used model includes herd, SNP genotype and lactation stage as fixed effects:

Yijkl= µ + Hi + Lj + GK + eijkl


Yijkl: milk traits (Fat, protein, lactose, solid material percentage).

μ: general mean for the certain trait.

Hi: fixed effect of ith herd (i=1, 2, 3 and 4).

Lj: fixed effect of jth lactation (j=1, 2).

Gk: fixed effect of kth genotype (k=AA, AC and CC).

eijkl: residual effect.

It should be noted that, the significance of deviations was verified with the Tukey-Kramer test. Also, we used chi-square test manually to test the population for Hardy-Weinberg equilibrium:

χ2= Ʃ (Oi–Ei)2 / Ei


Oi and Ei: observed and expected number of individuls for the ith genotype, respectively.



The means for milk composition of Khalkhali goats are present in Table 1. Milk samples were obtained from Khalkhali goats living in natural pastures belonging to Ardabil province during spring and first month of summer. The fat and protein percentage of Khalkhali breed milk in compared with goat breeds like Girgentana (Todaro et al. 2005), greek indigenous breed (Kondyli et al. 2012), Malaysian local breeds (Jamnapari, Shami and Toggenburg) (Mohsin et al. 2019), Italian local goats (Jonica, Mediterranean Red and Garganica), Saanen (Currò et al. 2019) and Alpine breeds (Da Costa et al. 2014) were low. Alignment of sequences against reference sequence (Accession number in NCBI: XM_018054689.1) showed a transversion polymorphic site in 3’ UTR that leads to substitution of A nucleotide to C nucleotide in position 362 bp (Figure 3), based on the reference sequence, the mutation was placed in 798 position. Based on detected variant, three different genotypes were detected in present study that called AA, AC and CC with frequency 0.12, 0.64 and 0.24 respectively (Table 2). Statistical analysis revealed that herd as a fixed effect has a significant impact on all investigated milk-related traits. Identified genotypes of BLG gene significantly influenced the milk composition parameters, i.e. fat, protein and solid material percent (P<0.05). However, observed genotypes had no significant effect on lactose percentage of obtained milk samples. While, milk from goats with AC genotype had significantly higher fat than milk of goats with CC genotypes. Also, AA genotype showed significantly higher protein and solid material in compared with CC genotype (Table 3). Obtained results based on chi-square test (P<0.01) showed that identified genotypes in the BLG gene of Khalkhali goats is not in Hardy-Weinberg equilibrium (Table 2). As mentioned earlier, the milk of goats with AC genotype has higher fat percentage and proper composition, therefore, it seems that this genotype may be a convenient option for breeders and ranchers. The results also revealed that the selection was probably against CC genotype due to lower fat and protein percentage in its milk in compared with the other two genotypes. The results are consistent with this fact that the percentage of fat and protein in goat's milk is considered a key factor among ranchers of area, in order to produce more butter and cheese. Kumar et al. (2006), amplified the BLG gene from exon 7 to the 3’ flanking region (426 bp) as we did, and genotyping of this region of gene revealed the presence of two alleles and three genotypes.


Table 1 Means of milk composition traits (Means±Standard deviation) in Khalkhali goat


The means within the same row with at least one common letter, do not have significant difference (P>0.05).


Figure 3 DNA sequencing chromatograms of CC, AA and AC genotypes of BLG gene in Khalkhali goat



Table 2 Allelic and genotypic frequencies of the BLG gene (n=120) in Khalkhali goats


HWE: Hardy-Weinberg equilibrium.


Table 3 Results of association analysis of BLG genotypes with milk composition in Khalkhali goat breed


The means within the same row with at least one common letter, do not have significant difference (P>0.05).


In addition to mentioned study, identification of variants in this region in our work and other studies (Pena et al. 2000; El Hanafy et al. 2015) revealed that this part of BLG gene considered as a variable region which affects milk composition. Although, variation detected in this part of caprine BLG gene in present study, dose not generate any changes in the amino acids, but it may effect on the regulation of gene or mRNA splicing or linked to other polymorphisms in the coding area, which may have effect on gene expression level. Evidence suggests that variants in nonexonic regions can have major effects on the phenotypes (Van Laere et al. 2003). In accordance with the current study, the results of investigation in sheep, goat and cattle breeds have shown that BLG polymorphisms significantly effects on milk composition (Dettori et al. 2015; Selvaggi et al. 2015; Cardona et al. 2016; Gras et al. 2016). However, there are some studies that failed to detect significant effect of variants on milk composition (Karimi et al. 2009).



This study has been investigated the association between the BLG genotypes and milk composition traits in Khalkhali goat breed. The obtained results indicate the relationship between identified genotypes and fat, protein percentage and solid content of Khalkhali goats milk. We found that the milk of goats with BLG AC genotype had higher fat and lactose percentage and also proper protein and solid content. Based on obtained results, we suggest that AC genotype is the best option for selection, as the breeders.



The authors thank owners of Khalkhali goat herds for the collaboration on collecting blood and milk samples from the goats.

Caravaca F., Ares J.L., Carrizosa J., Urrutia B., Baena F., Jordana J., Badaoui B., Sànchez A., Angiolillo A., Amills M. and Serradilla, J.M. (2011). Effects of α s1-casein (CSN1S1) and κ-casein (CSN3) genotypes on milk coagulation properties in Murciano-Granadina goats. J. Dairy Res. 78, 32-37.
Cardona S.J.C., Cadavid H.C., Corrales J.D., Munilla S., Cantet R.J. and Rogberg-Muñoz A. (2016). Longitudinal data analysis of polymorphisms in the κ-casein and β-lactoglobulin genes shows differential effects along the trajectory of the lactation curve in tropical dairy goats. J. Dairy Sci. 99, 7299-7307.
Çelik Ş. and Özdemir S. (2006). β-lactoglobulin variants in Awassi and Morkaraman sheep and their association with the composition and rennet clotting time of the milk. Turkish J. Vet. Anim. Sci. 30, 539-544.
Currò S., De Marchi M., Claps S., Salzano A., De Palo P., Manuelian C.L. and Neglia G. (2019). Differences in the detailed milk mineral composition of Italian local and Saanen goat breeds. Animals. 9, 412-420.
Da Costa W.K.A., de Souza E.L., Beltrao-Filho E.M., Vasconcelos G.K.V., Santi-Gadelha T., de Almeida Gadelha C.A., Franco O.L., do Egypto R.D.C.R. and Magnani M. (2014). Comparative protein composition analysis of goat milk produced by the Alpine and Saanen breeds in northeastern Brazil and related antibacterial activities. PLoS One. 9, e93361.
Dagnachew B.S., Thaller G., Lien S. and Ådnøy T. (2011). Casein SNP in Norwegian goats: Additive and dominance effects on milk composition and quality. Genet. Sel. Evol. 43, 31-38.
Dettori M.L., Pazzola M., Paschino P., Pira M.G. and Vacca G.M. (2015). Variability of the caprine whey protein genes and their association with milk yield, composition and renneting properties in the Sarda breed: 2. The LALBA gene. J. Dairy Res. 82, 434-441.
El Hanafy A.A.M., Qureshi M.I., Sabir J., Mutawakil M., Ahmed M.M.M., El Ashmaoui H., Ramadan H.A.M.I., Abou-Alsoud M. and Sadek M.A. (2015). Nucleotide sequencing and DNA polymorphism studies of β-lactoglobulin gene in native Saudi goat breeds in relation to milk yield. Czech J. Anim. Sci. 60, 132-138.
FAOSTAT. (2017). Database of the Food and Agricultural Organization (FAO) of the United Nations. Availabe at:
Ganai N.A., Bovenhuis H., Van Arendonk J.A.M. and Visker M.H.P.W. (2009). Novel polymorphisms in the bovine β‐lactoglobulin gene and their effects on β‐lactoglobulin protein concentration in milk. Anim. Genet. 40, 127-133.
Gharedaghi L., Shahrbabak H.M. and Sadeghi M. (2016). Identification of novel SNP in caprine β-lactoglobulin gene. J. Genet. 95, 485-490.
Gras M.A., Pistol G.C., Pelmus R.S., Lazar C., Grosu H. and Ghita E. (2016). Relationship between gene polymorphism and milk production traits in Teleorman Black Head sheep breed. Rev. MVZ Córdoba. 21, 5124-5136.
Haenlein G.F.W. (2007). About the evolution of goat and sheep milk production. Small Rumin. Res. 68, 3-6.
Kapila R., Kavadi P.K. and Kapila S. (2013). Comparative evaluation of allergic sensitization to milk proteins of cow, buffalo and goat. Small Rumin. Res. 112, 191-198.
Karimi K., Beigi Nassiri M.T., Mirzadeh K., Ashayerizadeh A., Roushanfekr H. and Fayyazi J. (2009). Polymorphism of the b-lactoglobulin gene and its association with milk production traits in Iranian Najdi cattle. Iranian J. Biotechnol. 7, 82-85.
Kawecka A. and Radko A. (2011). Genetic polymorphism of β-lactoglobulin in sheep raised for milk production. J. Appl. Anim. Res. 39, 68-71.
Kondyli E., Svarnas C., Samelis J. and Katsiari M.C. (2012). Chemical composition and microbiological quality of ewe and goat milk of native Greek breeds. Small Rumin. Res. 103, 194-199.
Kumar A., Rout P.K. and Roy R. (2006). Polymorphism of β-lacto globulin gene in Indian goats and its effect on milk yield. J. Appl. Genet. 47, 49-53.
Martin P., Szymanowska M., Zwierzchowski L. and Leroux C. (2002) The impact of genetic polymorphisms on the protein composition of ruminant milks. Reprod. Nutr. Dev. 42, 433-459.
Mohsin A.Z., Sukor R., Selamat J., Hussin A.S.M. and Ismail I.H. (2019). Chemical and mineral composition of raw goat milk as affected by breed varieties available in Malaysia. Int. J. Food Prop. 22, 815-824.
Naderi S., Rezaei H.R., Pompanon F., Blum M.G., Negrini R., Naghash H.R., Balkız Ö., Mashkour M., Gaggiotti O.E., Ajmone-Marsan P. and Kence A. (2008). The goat domestication process inferred from large-scale mitochondrial DNA analysis of wild and domestic individuals. Proc. Natl. Acad. Sci. 105, 17659-17664.
Ozdemir M., Kopuzlu S., Topal M. and Bilgin O.C. (2018). Relationships between milk protein polymorphisms and production traits in cattle: a systematic review and meta-analysis. Arch. Anim. Breed. 61, 197-206.
Özmen Ö. and Kul S. (2016). Investigating the genetic polymorphism in the exon 2 region of ovine β-lactoglobulin gene and its association with some milk traits. Ankara Univ. Vet. Fak. Derg. 63, 323-328.
Palmeri M., Mastrangelo S., Sardina M.T. and Portolano B. (2014). Genetic variability at α s 2-casein gene in Girgentana dairy goat breed. Italian J. Anim. Sci. 13, 2997-3001.
Pena R.N., Sanchez A. and Folch J.M. (2000). Characterization of genetic polymorphism in the goat β-lactoglobulin gene. J. Dairy Res. 67, 217-224.
Rijnkels M. (2002). Multispecies comparison of the casein gene loci and evolution of casein gene family. J. Mamm. Gland Biol. Neoplas. 7, 327-345.
Sanger F., Nicklen S. and Coulson A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. 74, 5463-5467.
Sardina M.T., Rosa A.J.M., Davoli R., Braglia S. and Portolano B. (2012). Polymorphisms of β-lactoglobulin promoter region in three Sicilian goat breeds. Mol. Biol. Rep. 39, 3203-3210.
SAS Institute. (2004). SAS®/STAT Software, Release 9.2. SAS Institute, Inc., Cary, NC. USA.
Selvaggi M., Laudadio V., Dario C. and Tufarelli V. (2015). β-lactoglobulin gene polymorphisms in sheep and effects on milk production traits: A review. Adv. Anim. Vet. Sci. 3, 478-484.
Silanikove N., Leitner G., Merin U. and Prosser C.G. (2010). Recent advances in exploiting goat's milk: Quality, safety and production aspects. Small Rumin. Res. 89, 110-124.
Staiger E.A., Thonney M.L., Buchanan J.W., Rogers E.R., Oltenacu P.A. and Mateescu R.G. (2010). Effect of prolactin, β-lactoglobulin, and κ-casein genotype on milk yield in East Friesian sheep. J. Dairy Sci. 93, 1736-1742.
Tamura K., Stecher G., Peterson D., Filipski A. and Kumar S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729.
Todaro M., Scatassa M.L. and Giaccone P. (2005). Multivariate factor analysis of Girgentana goat milk composition. Italian J. Anim. Sci. 4, 403-410.
Van Laere A.S., Nguyen M., Braunschweig M., Nezer C., Collette C., Moreau L., Archibald A.L., Haley C.S., Buys N., Tally M. and Andersson G. (2003). A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig. Nature. 425, 832-841.
Vidovic V., Lukac D., Nemes Z. and Trivunovic S. (2014). β-lactoglobulin genetic variants in Serbian Holstein-Friesian dairy cattle and their association with yield and quality of milk. Anim. Sci. Pap. Rep. 32(2), 179-182.
Yang F., Li L., Liu H., Cai Y. and Wang G. (2012). Polymorphism in the exon 4 of β-lactoglobulin variant B precursor gene and its association with milk traits and protein structure in Chinese Holstein. Mol. Biol. Rep. 39, 3957-3964.