Molecular genetic polymorphism at the K-casein and (3-lactoglobulin loci in Finnish dairy hulls Riikka Velmala, Esa A. Mäntysaari and Asko Mäki-Tanila Velmala, R., Mäntysaari, E. A. & Mäki-Tanila, A. 1993. Molecular genetic poly- morphism at the K-casein and li-lactoglobulin loci in Finnish dairy bulls. Agric. Sci. Finl. 2: 431-435. (Agric. Res, Centre of Finland, Inst. Animal Prod., FIN-31600 Jokioinen, Finland.) Dairy bulls have been genotyped for K-casein and P-lactoglobulin from semen samples by methodology based on a polymerase chain reaction (PCR), In this study, a previously described method for K-casein A and B variants was extended to cover also the detection of the E variant. For P-lactoglobulin the variants A and B were genotyped by another PCR-based method. The frequencies of the K-casein and P-lactoglobulin alleles were determined from 308 and 291 Finnish Ayrshire and 42 and 44 Finnish Friesian bulls, respectively. The bulls had been bom between 1973 and 1988. There was no noticeable trend in the differences between allele frequencies over the years, the overall frequen- cies of K-casein A, B and E being 0.62,0.09 and 0.29 in the Finnish Ayrshires and 0.85, 0.14 and 0.01 in Finnish Friesians. The overall frequencies ofP-lactoglobulin A and B alleles were 0.25 and 0.75 in Ayrshires and 0.56 and 0.44 in Friesians. Key words: dairy cattle, PCR, K-casein, P-lactoglobulin, genetic variation Introduction The genetic polymorphism of the four major casein proteins (asi, ctS2, P and k) and two whey proteins (a-lactalbumin and P-lactoglobulin) in cattle is well documented (Grosclaude 1988). It derives both from substitutions and deletions in the amino acid sequences of these proteins (Eigel et al. 1984). Protein electrophoresis has been used to identify the different protein variants of bovine milk. This kind of methodology for the determination of bull genotypes requires the analysis of multiple dam/daughter pairs, thereby considerably delaying the results. To overcome these restrictions, DNA analysis techniques have been applied to type an- imals for milk protein loci (LEVÉZIEL et al. 1988, ROGNE et al. 1989). Among DNA amplification techniques, several PCR-based methods for geno- typing milk protein loci have been published (Me- drano and Aguilar-Cordova 1990a, 1990b, David and Deutch 1992,Lien et al. 1992). We have recently initiated a study to analyze the associations between production traits and milk protein genotypes in Finnish dairy cattle. The pre- sent paper is the first phase of the work. Here we describe some modifications to existing PCR-based methodology and present the allele frequencies of K-casein (CASK) and P-lactoglobulin (LGB) for the major Finnish dairy breeds from a sample of artificial insemination (AI) bulls. Material and methods Frozen semen pellets from altogether 324 Finnish Ayrshire and 53 Finnish Friesian bulls were ob- 431 Agric. Sei. Finl. 2 (1993) tained from five Finnish AI societies. The birth years of the bulls ranged from 1973 to 1988. Ayr- shire bulls were divided into three age groups ac- cording to their birth year: 1973-81, 1982-85 and 1986-88. The method described by Zadworny and Kuhnlein (1990) was applied to isolate DNA from the semen. The DNA concentration was determined by fluorescence (Hoefer TKO 100 Fluorometer). The yield was in the range of 10-30 |ig DNA per semen pellet. The isolation of DNA from whole blood was done with a rapid method described by Kawasaki (1990). The PCR reactions, restriction enzyme diges- tions and gel runs for CASK A and B variants and for LGB A and B variants were performed under the conditions described by Medrano and Agui- lar-Cordova (1990a, 1990b), with following modifications: The amount of DNA in the reaction was 100 ng; the reaction volume was 25 pi; the reaction buffer did not contain gelatin; and 1.25 units of Taq DNA polymerase (Promega) were used per reaction. The oligonucleotides used for the amplification ofCASK were K346A (5 ’-C ATTTATGGCCATTCCACCAA AG-3 ’) and K3468 (5’-C ATTTCGCCTTCTCTGTAACAG-3 ’). These primers are otherwise the same as JKSOI and JK3O2 (Medrano and Aguilar-Cordova 1990a) but they are 2 and 4 bases shorter. Also, K 3468 is an inversion of the JK3O2 orientated erroneously, as could be deduced from the K-casein sequence (Alexander et al. 1988). For CASK the PCR programme consisted of denaturation for 3 min at 94°C followed by 1 min at 94°C, 50 sec at 60°C, 30 sec at 72°C for 10 cycles, 1 min at 94°C, 50 sec at 60°C, 45 sec at 72°C for 20 cycles, 1 min at 94°C, 50 sec at 60°C, 1 min at 72°C for 10 cycles. A final extension of primers for 5 min at 72°C was included for both PCR programmes. To improve specificity and yield of the reaction, the ’hot start’ technique was applied to start the reactions: the nucleotides needed for amplification were added during the first denaturation step of the PCR pro- gramme. The amplifications were performed with a Hybaid Thermal Reactor (Hybaid Limited, Middle- sex, UK). CASK E variant has been genotyped at DNA level using the restriction enzyme Hae 111 (SCHLIE- ben et al. 1991).We found that with the PCR-prod- uct obtained by the method of Medrano and Aguilar-Cordova (1990a), also a specific band- ing pattern for E variant could be observed after digestion with Hae 111. Thus, the method for CASK was extended to cover also the detection of the E variant. An aliquot of 10 p.l of the PCR product was used for all restriction enzyme digestions. Prior to this study, 20 cows of Finnish Ayrshire breed with known CASK and LGB genotypes had been tested with the above genotyping assays from blood samples. The results deduced from the DNA analysis were identical to those obtained at protein level. Results and discussion In addition to CASK A and B variants we also detected the E variant. The mutation in codon 155 (Ser to Gly) is known to be responsible for this variant (Erhardt 1989). The digestion of the CASK PCRproduct (346 bp) with Hae 111 resulted in fragments 337 bp (and 9 bp) for non-E and 192 bp, 145 bp (and 9 bp) for the E variant. Bands were easily distinguishable since a strong amplification product was obtained in most cases (Figure 1). The polymorphism at codon 148 was detected using the procedure of Medrano and Aguilar-Cordova (1990a) for CASK A and B variants. The polymor- phism at codon 118 was utilized for LGB (Me- drano and Aguilar-Gordova 1990b). A successful amplification was obtained from 308 and 291 Ayrshire samples and from 42 and 44 Friesian samples in CASK and LGB, respectively. The allele distributions calculated from these res- ults are presented in Table 1. For the Finnish Ayr- shire breed, the allelic frequencies of A, B and E at the CASK locus were 0.61, 0.10 and 0.29, and the frequencies ofA and B at the LGB locus were 0.25 and 0.75. For the Finnish Friesian, the allelic fre- quencies of A, B and E at CASK were 0.85, 0.14 432 Agric. Sei. Finl. 2 (1993) and 0.01 and those of A and B at LGB were 0.56 and 0.44. Our results for the overall allele frequencies of CASK for Ayrshires and Friesians are in agreement with previous results obtained for these breeds by protein electrophoresis (Piironen et al. 1992). In Ayrshires, there was some variation in the fre- quency of A and E allele between the age groups. These differences, however, did not represent any trend in time. There had been no drastic alterations in selection criteria over the studied period, so that the changes could not be explained as being result- ing from a correlated response to selection for pro- duction. The frequency fluctuations are most likely due to genetic sampling involving only a small number of parents sireing the bulls analyzed. In LGB, there were no noticeable differences in allele frequencies between the age groups. The fre- quency of LGB B allele, however, was somewhat higher in Ayrshires and lower in Friesians com- pared to previous results for these breeds (Aal- tonen and Antila 1987, Atroshi et al. 1982, Piironen et al. 1992, Tervala et ai. 1983). The differences here, too, may be due to genetic sampl- ing. On the other hand, the present study was made with bulls whereas the previous ones have dealt with cows. Another factor is that in the 1980‘s there were substantial semen contributions by Holstein bulls into the Friesian population, which may have changed the allele distribution in Friesian bulls. Several studies have investigated the associa- tion between milk protein variants and various economically important milk traits, including manufacturing properties (e.g. AALTONEN and AN- TILA 1987, Ng-Kwai-Hang et al. 1990). Current molecular genetic techniques allow a thorough screening of genetic variation among Al-bulls for milk protein loci. The analyses presented here clearly indicate that a routine and inexpensive test- ing for CASK and LGB is feasible. However, a more direct and detailed analysis of the association between the economic traits and milk protein vari- ants is required. In that context, the CASK locus should be analyzed together with other closely linked casein loci (Lien et al. 1993). Acknowledgements. We wish to thank the Al societies for providing the required semen samples. We are grateful to Peter Bredbacka, Juha Kantanen and Johanna Viikki for technical support and useful discussions, and to the Finnish Ministry of Agriculture for part of the funding. Table 1. Number of samples and allele frequencies of K-casein and B-lactoglobulin loci in Al bulls by breed and age group. K-casein P-lactoglobulin ~~Ä B E Ä B Finnish Ayrshire No. No. 1973-81 82 0.71 0.07 0.23 75 0.26 0.74 1982-85 85 0.52 0.11 0.36 81 0.28 0.72 1986-88 141 0.62 0.10 0.27 135 0.22 0.78 total 308 0.62 0.10 0.29 291 0.25 0.75 Finnish Friesian 1975-88 42 0.85 0.14 0.01 44 0.56 0.44 No-number of animals analyzed. Fig. 1. Discrimination of K-casein genotypes as detected by agarose gel electrophoresis of Hinfl (lanes 1,3,5) and Hae 111 digested (lanes 2,4, 6) PCR products. The three samples presented here were typed to be DNA genotypes EE (lanes 1 and 2), AE (lanes 3 and 4) and AB (lanes 5 and 6) of the CASK gene. (Photo: R. Velmala). 433 Agric. Sei. Fint. 2 (1993) References Aaltonen, M.-L. & Antila, V. 1987. Milk renneting proper- ties and the genetic variants of proteins. Milchwissen- schaft 42: 490-492. Alexander,L.J., Stewart, A.F., Mackinlay, A.G., Kapelin skaya, T.V., Tkach, T.M. & Gorodetsky, S.I. 1988. Isolation and characterization of bovine K-casein gene. J. Biochem. 178: 395-401. Atroshi, F., Kangasniemi, R„ Honkanenßuzalski, T. & Sandholm, M. 1982. p-lactoglobulin phenotypes in Finn - ish Ayrshires and Friesian cattle with special reference to mastitis incidence. Acta Vet. Scand. 23: 135-143. David, V.A. & Deutch, A.H. 1992. Detection of bovine a s i -casein genomic variants using the allele-specific po- lymerase chain reaction. Anim. Genet. 23: 425-429, Eioel, W.N., Butler, J.E., Ernstrom, C.A., Farrell, H.M., Harwalkar, V.R., Jennes, R. & Whitney, R. MCE 1984. Nomenclature of proteins of cow’s milk. J. Dairy Sci. 67: 1599-1631. Erhardt, G. 1989. K-Kaseine in Rindermilch-Nachweis eines weiteren Allels (K-CnE) in verschiedenen Rassen. J Anim. Breed. Genet. 106: 225-231. Grosclaude, F. 1988. Le polymorphisme génétique des principales lactoprotéines bovines. INRA Prod. Anim. 1: 5-17. Kawasaki, E, S. 1990. Sample preparation from blood, cells and other fluids. In: Innis et al, (eds,). PCR Protocols, A guide to methods and applications. Academic Press, INC., USA, p. 148, Levéziel, H., Méténier, l„ mahé, M.-F., Choplain, J.,Furet, J.-P., Paboeuf, G., Mercier, J.C. & Grosclaude, F. 1988. Identification of the two common alleles of the bovine K-casein locus by the RFLP technique using the enzyme Hind BL Génét. Sél. Evol. 20: 247-254. Lien, S., ALESTRÖM, P., Klungland, H. & Roone, S. 1992. Detection of multiple (3-casein (CASE) alleles by amplification created restriction sites (ACRS). Anim. Genet. 23: 333-338. & Rogne, S. 1993. Bovine casein haplotypes: Number, frequencies and applicability as genetic markers, Anim Genet. 24: 373. Medrano, J.F. & Aouilar-Cordova, E. 1990a. Genotyping of bovine kappa-casein loci following DNA sequence amplification. Bio/technology 8: 144-146. & Aouilar-Cordova, E. 1990b. Polymerase chain reac- tion amplification of bovine {J-lactoglobulin genomic se- quences and identification of genetic variants by RFLP. Anim. Biotechn. 1: 73-77. Ng-Kwai-Hang, K.F., Monardes, H.G. & Hayes, J.F. 1990. Association between genetic polymorphism of milk pro- teins and production traits during three lactations. J. Dairy Sci. 73: 3414. Piironen, T., Ojala, M., Niini, T., Syväoja, E.-L. & Setälä, J. 1992. Effects of milk protein variants and lactation stage on renneting properties of bovine milk, 43rd An- nual Meeting of the EAAP, Madrid. Abstracts, Vol 2:46. Rogne, S., Lien, S., Vegarud, G., Steine, T., Langsrud, T. & Aleström, P. 1989. A method for K-casein genotyping of bulls. Anim. Genet. 20; 317-321. Schlieben, S., Erhardt, G. & Senft, B. 1991 . Genotyping of bovine K-casein (k-CNA, k-CNB, k-CNC, k-CNE) fol- lowing DNA sequence amplification and direct sequenc- ing of k-CNE PCR product. Anim. Genet. 22: 333-342. Tervala, H.-L., Antila, V., Syväjärvi, J. & Lindström, U.B. 1983. Variations in the renneting properties of milk. Meijeritieteellinen Aikakauskitja 41: 24-33. Zadworny, D. & Kuhnlein, U. 1990. The identification of the kappa-casein genotype in Holstein dairy cattle using the polymerase chain reaction. Theor. Appi. Genet. 80: 631-634. Manuscript received May 1993 Riikka Velmala Esa A. Mäntysaari Asko Mäki-Tanila Agricultural Research Centre of Finland Institute of Animal Production Section of Animal Breeding FIN-31600 Jokioinen, Finland 434 Agric. Sei. Fint. 2 (1993) SELOSTUS K-kaseiinin ja (3-laktoglobuliinin varianttien molekyyligeneettinen tunnistaminen ja muuntelu suomalaisissa lypsykarjasonneissa Riikka Velmala, Esa A. Mäntysaari ja Asko Mäki-Tanila Maatalouden tutkimuskeskus Kotieläintuotannon tutkimuslaitoksen eläinjalostusyksikössä on aloitettu tutkimus maitoproteiinivarianttien yhteyksistä eläinten tuotanto-ominaisuuksiin. Tässä julkaisussa esitetään työn ensimmäinen vaihe, jossa molekyyligeneettistä menetel- mää sovellettiin keinosiemennyssonnien K-kaseiinin ja |l-lak- toglohuliinin genotyypitykseen. Tuloksista laskettiin näiden lokusten alleelifrekvenssit ayrshire- ja friisiläisroduille. DNA-monimuotoisuutta analysoitiin entsymaattisen mo- nistamisen (PCR) ja DNA:ta spesifisesti pilkkovien entsyy- mien avulla. K-kaseiinin variantit A, B ja E sekä fJ-laktoglobu- liinin variantit A ja B määritettiin spermanäytteistä. Yhteensä 308/291 ayrshire-ja 42/44 friisiläisnäytettä analysoitiin K-ka- seiinin/p-laktoglobuliinin suhteen. K-lokuksen A-, B- ja E-al- leelien frekvenssit olivat ayrshire-sonneilla 0,61,0,10 ja 0,29 ja friisiläissonneilla 0,85, 0,14 ja 0,01. p-laktoglobuliinilo- kuksessa olivat A- ja B-alleelien frekvenssit ayrshirella 0,25 ja 0,75 ja friisiläisillä vastaavasti 0,56 ja 0,44. Allee- lifrekvenssejä tutkittiin ayrshirellä myös ikäryhmittäin sonni- en syntymävuosien (1973-88) mukaan. Frekvensseissä ei ha- vaittu systemaattista muutosta tutkittuna ajanjaksona. Näiden rotujen K-lokuksen alleelifrekvenssit vastasivat hyvin jo ai- emmin maidosta proteiinielektroforeesilla määritettyjä frek- venssejä. (f-lakloglobuliinin alleelifrekvensseissä havaittiin vähäisiä eroja aikaisempiin tutkimuksiin verrattuna. Erot saat- tavat johtua analysoidun aineiston koosta. Lisäksi aikaisem- mat tutkimukset on tehty lehmillä kun taas tässä tutkimukses- sa käytettiin sonneja. Maitoproteiinialleelien ja taloudellisten ominaisuuksien välisistä yhteyksistä tarvitaan yksityiskohtaisempaa analyy- siä. Tällaisessa analyysissä K-kaseiinilokus pitäisi analysoida yhdessämuiden geneettisesti kytkeytyneiden kaseiinilokusten (etsi-, «S2- ja fi-kaseiinin> kanssa. 435 Agric. Sei. Fint. 2 (1993)