Genetic polymorphism of β-lactoglobulin in sheep raised for milk production

This article was downloaded by: [Instytut Zootechniki] On: 29 August 2012, At: 23:45 Publisher: Taylor & Francis Informa Ltd Registered in England and...
Author: Stewart May
0 downloads 0 Views 278KB Size
This article was downloaded by: [Instytut Zootechniki] On: 29 August 2012, At: 23:45 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Applied Animal Research Publication details, including instructions for authors and subscription information:

Genetic polymorphism of β-lactoglobulin in sheep raised for milk production a

Aldona Kawecka & Anna Radko a


National Research Institute of Animal Production, 32-083, Kraków, Poland

Version of record first published: 01 Jun 2011

To cite this article: Aldona Kawecka & Anna Radko (2011): Genetic polymorphism of β-lactoglobulin in sheep raised for milk production, Journal of Applied Animal Research, 39:1, 68-71 To link to this article:

PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

Journal of Applied Animal Research, Vol. 39, No. 1, March 2011, 6871

Genetic polymorphism of b-lactoglobulin in sheep raised for milk production Aldona Kawecka* and Anna Radko National Research Institute of Animal Production, 32-083 Krako´w, Poland

Downloaded by [Instytut Zootechniki] at 23:45 29 August 2012

(Received 18 February 2010; final version received 22 November 2010) To investigate b-lactoglobulin polymorphism using polymerase chain reaction  restriction fragment length polymorphism in Polish Mountain, East Friesian, Polish Merino and Austrian Bergschaf sheep raised for milk production, three b-LGB genotypes (AA, AB and BB) were found in all the groups, AB being the most frequent genotype in all the groups. The frequency of b-LGB A genes was 0.66 in Friesian, 0.55 in Bergschaf, 0.5 in Polish Mountain and 0.48 in Polish Merino sheep with no deviations from genetic equilibrium according to HardyWeinberg law. The relationship between b-lactoglobulin genotype and milk yield and milk composition was investigated, but no statistical differences were found. The present findings provide no conclusive evidence about the effect of different b-LGB variants on milk production traits in sheep. Keywords: sheep; b-lactoglobulin; polymorphism; milk

Introduction In Poland, raising sheep for milk is of regional importance and, until recently, milk was only obtained from Polish Mountain sheep. This production type is now attracting growing interest in lowlands also, where milk is obtained from Polish Merino sheep and their crossbreds with East Friesian milk sheep. Attempts were also made to milk Bergschaf sheep imported from Austria. The b-lactoglobulin (b-LGB) is the main whey protein of ruminant milk and contains 162 amino acids. Its biological role is not completely understood, but it probably takes part in the transport of vitamin A and other low-molecule compounds. The b-lactoglobulin gene locus in sheep is localised on chromosome 3 and the genetic polymorphism of b-lactoglobulin is determined by three alleles  A, B and C. Milk protein (b-lactoglobulin and casein) polymorphism was described in considerable detail in dairy cattle. In many cases, the genetically determined diversity of these proteins was shown to be associated with the yield of milk and milk components (Litwin´czuk et al. 2006). Milk protein genes can, therefore, be viewed as quantitative trait loci (QTL) markers in terms of dairy traits and can thus be used in selection programmes. The issue of polymorphism has not been adequately studied among Polish breeds of sheep raised for milk production. Studies on Polish Merino sheep (Mroczkowski et al. 2004) have shown that dairy traits vary according to b-LGB genotype. Whereas, other studies have reported no effect of this genotype *Corresponding author. Email: [email protected] ISSN 0971-2119 print/ISSN 0974-1844 online # 2011 Taylor & Francis DOI: 10.1080/09712119.2011.565223

on milk production traits (Piwczyn´ski et al. 2002). The aim of the study was to characterise b-LgB polymorphism using PCR-RFLP in sheep raised for milk production in Poland and to determine the relationship between the occurrence of different genetic variants and milk production traits.

Materials and methods Samples of blood collected from 181 sheep of the Friesian, Polish Merino, Bergschaf and Polish Mountain sheep were studied. Genomic DNA was isolated from peripheral blood leukocytes using proteinase K, following the method described by Kawasaki (1990). The b-LGB genotype was determined using PCRRFLP. Isolated DNA was amplified using the polymerase chain reaction (PCR). The PCR was performed in 25 ml of reaction mixture containing 10  PCR Buffer, MgCl2 (15 mM), dNTP nucleotides (2 mM), AmpliTaq Gold polymerase (5 U/m) and LGB1 primer sequences (0.4 mM): 5?-CAA CTC AAG GTC CCT CTC CA-3? and LGB2: 5?-CTT CAG CTC CTC CAC GTA CA-3? (lit). The reaction mixture was subjected to a thermal programme of 15 min initial denaturation of genomic DNA at 958C, 31 cycles of denaturation at 948C for 45 sec, annealing at 648C for 45 sec, elongation of primers at 728C for 1 min, and final elongation at 728C for 60 min. the PCR reaction was performed using GeneAmp PCR System 9600 cycler (Applied Biosystems). Digestion of 10 ml amplification product was performed using RsaI restriction enzyme (2 U/ml) at 378C for 2.5 h. The digestion products obtained were


Journal of Applied Animal Research

Downloaded by [Instytut Zootechniki] at 23:45 29 August 2012

separated electrophoretically. The digested product was electrophoresed in 3% agarose gel: Agarose for Routine Use-NuSive GTG agarose (1:3). The results of electrophoretic separation were analysed in UV light using a transilluminator. The conformity of the actual distribution of genotypes in the analysed sheep groups with theoretical distribution was verified by chi-square test using Statistica ver.7.1 (StatSoft). Lactating ewes were recorded for milk yield using control milkings performed at 30-day intervals. Samples were collected from the morning milk to determine basic composition of fat, protein, lactose, and solids not fat. Data on milk performance of the sheep were analysed by the least squares method with regard to the effect of genetic variants, using SAS software (SAS/STAT).

Results and discussion The PCR-RFLP method enabled b-LGB polymorphism to be accurately identified in the material analysed. A DNA product of 103 base pairs was obtained. Restriction fragment length polymorphism reflected differences in nucleotide sequences resulting from point mutations within a gene. This mutation created a restriction site for the RsaI enzyme, which cut DNA into two fragments of 66 and 37 base pairs. During electrophoresis, two bands of 66 and 37 base pairs were obtained for the AA genotype and a band of 103 base pairs for the BB genotype. All three bands were found in heterozygotes. Three genotypes (AA, AB and BB) were found to occur in all the groups of sheep (Table 1). There were no significant differences among their frequencies, which showed that the sheep populations studied were in genetic equilibrium according to Hardy-Weinberg law.

The present study demonstrated that in all breeds, animals with the b-LGB heterozygous genotype were predominant (Table 1). A similar relationship was observed by Piwczyn´ski et al. (2002) in Polish Merino and in crosses derived from Finn, Romanov and Booroola rams, and by Cubric-Curik et al. (2002) in Croatian Pag sheep. For native Turkish breeds, the frequency of AB genotypes was slightly lower than that of AA genotypes (Elmaci et al. 2006). In Indian Jalauni sheep the AB genotype was not found (Arora et al. 2010). In the sheep population studied, AA homozygotes were more frequent than BB homozygotes in Bergschaf and Friesian sheep. BB homozygotes predominated in Merino sheep, and both genotypes had the same frequency in Polish Mountain sheep. In the groups described, the frequency of b-LGB BB genotypes was low and did not exceed 15%. A less than 5% frequency of BB homozygotes was reported by Piwczynski et al. (2002) in Finn sheep, with no animals of this genotype observed among crossbreds derived from Finn rams and Merino sheep. The BB genotype was not seen in the Iranian, Russian (Mohammadi et al. 2006) and Romanian Karakul (Kevorkian et al. 2008). According to Arora et al. (2010), the B allele was more frequent in the majority of the Indian sheep populations. Similar observations were made for Moghani and Afshary Iranian sheep (Elyasi et al. 2004). This variant was dominant in native Spanish breeds Latxa, Manchega and Churra (Barillet et al. 2005). The A allele was dominant in the East Friesian breed mentioned above, as well as in Slovakian Valaska and Tsigai sheep (Michalcova and Krupova 2009) and in Lithuanian Native Coarsewooled and Blackface sheep (Kucˇinskiene et al. 2005).

Table 1. Distribution of b-lactoglobulin genotypes and allele frequencies. Allele frequency Breed Bergschaf N  68 Friesian N  31 Polish Merino N  48 Polish Mountain sheep N 34

Genotype Observed number Expected number AA AB BB AA AB BB AA AB BB AA AB BB

19 46 3 10 20 1 8 30 10 8 18 8

23.8 36.3 13.8 12.9 14.1 3.9 11.0 23.9 13.0 8.5 17 8.5

Genotype frequency 0.257 0.621 0.122 0.323 0.645 0.32 0.167 0.625 0.208 0.235 0.53 0.235

Value of x2 test
















A. Kawecka and A. Radko

Table 2. Effect of b-lactoglobulin genotype on milk production traits.

Downloaded by [Instytut Zootechniki] at 23:45 29 August 2012

blactoglobulin genotype Trait

AA N  19

AB N  46

BB N 9

Bergschaf sheep Mean daily milk yield (ml) Fat (%) Protein (%) Lactose (%) Solids not fat (%)

518.5 5.82 5.23 4.87 10.75

482.7 6.47 5.13 4.82 10.63

422.2 6.24 5.32 5.03 10.94

Friesian sheep Mean daily milk yield (ml) Fat (%) Protein (%) Lactose (%) Solids not fat (%)

784.6 6.84 5.59 4.73 10.96

658.3 6.94 5.48 4.68 10.79

730.0 6.63 5.92 4.75 11.42

Polish Mountain sheep Mean daily milk yield (ml) Fat (%) Protein (%) Lactose (%) Solids not fat (%)

320.77 5.52 5.04 4.9 10.37

337.25 6.06 5.44 4.9 11.2

267.69 5.75 5.62 4.6 11.13

Polish Merino sheep Mean daily milk yield (ml) Fat (%) Protein (%) Lactose (%) Solids not fat (%)

376.63 6.6 6.56 4.67 11.88

392.24 6.94 6.42 4.69 11.94

488.0 6.24 6.28 4.79 11.97

Note: Means denoted with different letters differ statistically: a, b, c: PB0.05.

In our study we found no statistically significant differences in milk yield according to genotype (Table 2). No association among b-LGB polymorphism and amount of milk was found in Fresian sheep (Staiger et al. 2010) and in Slovakian breeds (Michalcova and Krupova 2009). Ramos et al. (2002) observed higher milk yield in AB heterozygotes (significant differences) when investigating the effect of genotype on milk yield traits of Portuguese breeds of sheep raised for milk (Merino and Serra da Estrela). In the present experiment, the effect of b-LGB on the milk composition was not confirmed. Piwczynski et al. (2002), in studies on Polish Merino sheep and their crosses with prolific breeds, also found no close relationship between the genetic polymorphism of lactoglobulin and milk quality traits. The milk fat content was higher in Portuguese breeds with AB genotype of b-LGB than in the other groups. Higher fat content was observed in milk of the Italian Altamurana and Leccese sheep with AA and AB genotypes (Dario et al. 2005; Dario et al. 2008) and in Awassi sheep with BB genotype of lactoglobulin (Celik and Ozdemir 2006). When analysing the effect of the b-LGB genotype on milk production traits in Polish Merino sheep, Mroczkowski et al. (2004) found that BB homozygotes had an advantage

over heterozygotes in protein content. Also, Celik and Ozdemir (2006) showed these relationships in the native Turkish breed. Michalcova and Krupova (2009) did not find statistically significant differences for Slovakian breeds of sheep. The results obtained showed the distribution of genotypes and alleles in the population of sheep used for milk production. They provide no conclusive evidence (as in cattle) about the effect of different b-LGB variants on milk production traits in sheep. As in the studies by other authors, the results showed considerable variation. The present findings may increase our knowledge of the possibility of using milk protein genes as potential markers of milk yield traits, but a larger population would have to be studied. Under Polish conditions, this particularly refers to the Polish Mountain sheep whose milk is processed in the mountain region.

References Arora R, Bhatia S, Mishra BP, Sharma R, Pandey AK, Prakash B, Jain A. 2010. Genetic polymorphism of the b-lactoglobulin gene in native sheep from India. Biochemical Genetics 48(34):304311.

Downloaded by [Instytut Zootechniki] at 23:45 29 August 2012

Journal of Applied Animal Research Barillet F, Arranz J-J, Carta A. 2005. Mapping quantitative trait loci for milk production and genetic polymorphism of milk proteins in dairy sheep. Genetics Selection Evolution 37:109123. Celik S, Ozdemir S. 2006. b-lactoglobulin variants in Awassi and Morkaraman sheep and their association with the composition and rennet clotting time of the milk. Turkish Journal of Veterinary and Animal Sciences 30:539544. Cubric-Curik V, Feligni M, Lukac-Havranek J, Curik I, Enne G. 2002. Genetic polymorphism of b-lactoglobulin in native sheep from Island of Pag. Food Technology and Biotechnology 40(1):7578. Dario C, Carnicella D, Bufano G. 2005. Effect of blactoglobulin genotypes on ovine milk composition in Altamurana breed. Archivos de Zootecnia 54:105108. Dario C, Carnicella D, Dario M, Bufano G. 2008. Genetic polymorphism of b-lactoglobulin gene and effect on milk composition in Leccese sheep. Small Ruminant Research 74(13):270273. Elmaci C, Oner Y, Balcioglu MS. 2006. Genetic polymorphism of b-lactoglobulin gene in native Turkish sheep breeds. Biochemical Genetics 44:376381. Elyasi G, Shodja J, Nassiry M, Tahmasebi A, Pirhary O, Javanmard A. 2004. Polymorphism of b-lactoglobulin gene in Iranian sheep breeds using PCR-RFLP. The Joint Agriculture and Natural Resources Symposium; 2004 May; Tabriz-Ganja, Iran. p. 1416. Kawasaki ES. 1990. Sample preparation from blood, cell and other fluids. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. New York: Academic Press. p. 146152. Kevorkian S, Manea MA, Georgescu A, Dinischiotu A, Costache M. 2008. Genotyping of b-lactoglobulin gene in Karakul sheep breed. Zootehnie si Biotehnologii 41(1):112116. Kucˇinskiene J, Vagonis G, Malevicˇiu¯ te´ J, Maleviciute J, Tapio I. 2005. Genetic polymorphism of b-lactoglobu-


lin in Lithuanian blackface and Lithuanian native coarsewooled sheep. Veterinarija ir Zootechnika 51(29): 9092. Litwin´czuk A, Barłowska J, Kro´l J, Litwin´czuk Z. 2006. Białka polimorficzne mleka jako markery cech uz× ytkowych bydła mlecznego i mie¸snego [Milk protein polymorphism as markers of production traits in dairy and meat cattle]. Medycyna Weterynaryjna 62(1):610. Michalcova A, Krupova Z. 2009. Influence of b-lactoglobulin C genotypes on composition of milk and milk production traits of the Slovak ovine breeds. Acta fytotechnica et zootechnica  Mimoriadne cˇı´ slo, Slovaca Universitas Agriculturae Nitriae. p. 438446. Mohammadi A, Nassiry M, Elyasi G, Shodja J. 2006. Genetic polymorphism of b-lactoglobulin in certain Iranian and Russian sheep breeds. Iranian Journal of Biotechnology 4(4):265268. Mroczkowski S, Korman K, Erhardt G, Piwczyn´ski D, Borys B. 2004. Sheep milk protein polymorphism and its effect on milk performance of Polish Merino. Archiv fur Tierzucht 47:114121. Piwczyn´ski D, Borys B, Mroczkowski S, Erhardt G, Jarzynowska A. 2002. Charakterystyka polimorfizmu bialek mleka i cech mlecznos´ ci merynosa polskiego i jego mieszan´co´w z rasami plennymi [Milk protein polymorphism and milk production traits in Polish Merino and Polish Merino crosses with prolific breeds]. Prace i Materialy Zootechniczne 14:151161. Ramos AM, Matos C, Bettencourt C, Pinheiro C, RangelFigueiredo T. 2002. Influence of aS1-casein, b-lactoglobulin and prolactin genotypes on milk production traits in two Portuguese sheep breeds. 7th World Congress on Genetics Applied to Livestock Production; 2002 Aug 1923; Montpellier, France. Staiger EA, Thonney ML, Buchanan JW, Rogers ER, Oltenacu PA, Mateescu RG. 2010. Effect of prolactin, b-lactoglobulin, and k-casein genotype on milk yield in East Friesian sheep. Journal of Dairy Science 93(4):17361742.

Suggest Documents