Milk Protein Polymorphisms in California Dairy Cattle

ALISON VAN EENENNAAM ancl JUAN FERNANDO MEDRANo1Department of AnImal ScIence

University of CaUfomlaDavis 95616-8521

California's cheese production has increasedfrom 11.3 million kg in 1972 to almost 279million kg in 1989. Presently, almost 30% of

the state's milk production is utilized for themanufacture of cheese (5). It is important to thecheese industry that the milk it receives hashigh cheese-yielding potential and desirablemanufacturing properties.

Cheese-yielding capacity and coagulatingproperties of milk are influenced by geneticvariants of the milk proteins j>.lactoglobulin(LG), asl-casein (CN), i>-CN, and lC-CN. Ren­net clotting time, rate of curd formation, andcoagulum strength are all improved in milkcontaining the B variant of lC-CN (27, 28). Thissame variant and the j>.LG B variant are associ­ated with increased cheese yield (1, 18, 19,24).

A 9% increase in cheese DM yield wasfound using milk from ~-LG BB, /3-CN BB (orAB), and lC-CN BB cows compared with cowsof the all AA genotype (7). The reduced rennet­ing time and resultant decrease in manufactur­ing costs from this type of milk would be addedbenefits. Soft curd problems would be reducedby the increased curd tension of milk fromthese genotypes.

Part of the effect of genetic variants is theirinfluence on milk composition. The lC-CN Ballele has been associated with increased milkprotein content (25). AleanOO et al. (1) foundthat the differences in Parmesan cheese yieldbetween milk of lC-CN AA and lC-CN BB geno­type was greater than that which would havebeen predicted based on the difference in milkcomposition alone. Differences in the saltedcurd yield from milk of j>.LG genotype AA andBB was also greater than expected; the /3-LGB.B gen~ p~ced a greater than predictedYIeld This 'protem variant" effect on cheeseyield may be related to the associations be­tween the milk protein genotypes and caseincomposition. Both the /3-LG BB and lC-CN BBgenotypes are associated with higher caseinpercentage in milk. The lC-CN variants affectthe concentration and proportion of lC-CN (BB> AB > AA), the proportion of <Xsi-CN. and theconcentration of P-LG and a-lactalbumin (AA> AB > BB). Cows with the j>.LG AA geno-

1730

INTRODUCTION

Received August 29. 1990.l'cceptcd October 22, 1990.Reprint requests.

1991 J Dairy Sci 74:1730-1742

ABSTRACT

Milk samples from 1454 first lactationheifers were collected from five breeds ofcattle in a representative sampling of theCalifornia dairy cattle population. Sam­pies. were phenotype<! for Ctsl-casein, ~­casem, lC-casein, and ~-lactoglobulin us­ing PAGE. Lactation yields for milk, fat,and protein were available on 1166 heif­ers. Genetic linkage of the casein loci andthe relationship between milk proteingenotypes and production parameters(~ fat and protein yield, fat and pr0­tem percentage) was investigated. Genefrequencies were similar to those found inother studies of the American dairy cattlepopulation. Linkage was found between~-casein and lC-casein in four of the five~ .and between ~asein and <ls1­casem m the Jersey breed. Casein haplo­types BBB and CA3A (<lsI-, ~-, lC-caseinrespectively) occurred in the Holsteinpopulation more often than would be ex­pected from a random combination of thealleles. Least squares analysis of the pro­~ction data indicated that the only sig­nificant effect was that of lC-casein geno­type on protein yield; the highest yieldswere obtained for the BB genotype.(Key words: milk protein polymor­phisms, casein, California dairy)

Abbreviation key: CN = casein, and LG =lactoglobulin.

MllX PROTEIN POLYMORPHISMS IN CALIFORNIA 1731

type produce milk with a greater proportion ofprotein in the fonn of whey proteins (20).These differences collectively result in the as­sociation of specific alleles with increasedcheese yield.

The manufacturing properties of the futuremilk supply could be improved by implement­ing selective breeding for desired milk proteinalleles. This type of selection would differ fromtraditional selection for improved quantitativetraits. The recent development of moleculartechniques for genotyping animals allows forthe rapid genotypic classification of males andyoung animals (21, 22). Utilization of thesetechniques in a breeding program selecting forsingle genes would change genotypes relativelyrapidly.

The feasibility of a breeding plan that in­volves selection for specific alleles of singlegenes depends upon the frequencies of thosealleles in the population. If an allele is rare,then selection for it becomes restrictive; if it isubiquitous, little opportunity exists to increaseits frequency. Genetic progress will also beaffected by the degree of linkage between thealleles. Linkage between loci of bovine caseinvariants was first discovered by the noDin­dependent assortment that occurred between the€lsI- and ~-CN loci (8). On the basis of ex­pected allelic combinations, the alleles of €lsI­and ~-CN genes were not segregating indepen­dently. There was an excess of the combina­tions !lsI-CN B ~-CN B and €lsI-CN C 13-CNA, whereas €lsl-CN C ~-CN B was extremelyrare. Grosclaude (9) proposed a model thatconsiders the casein genes to be linked togetheras a cluster. In this cluster, the !ls1- and 13-CNgenes are considered to be situated very closelytogether on the chromosomal DNA. This hasrecently been confinned by the physical map­ping of the bovine casein genes (€lsl-' 13-, !ls2-,K-) to a 200-kb region on chromosome 6 (6,29).

The objective of this study was to determinethe frequencies and linkages of the geneticvariants of ~-LG, !ls1-CN, l3-CN, and K-CN andtheir effects on yield.

MATERIALS AND METHODS

Milk Sampling Plan

Milk samples were collected from herdsgeographically representative of the California

dairy cattle population (Figure 1). The samp­ling criteria for all breeds included herds thathad at least 10 first lactation heifers, wereenrolled in DIDA testing, and were on test formilk protein percentage. The Holstein herdswere further restricted to include only DIDregistry herds because these were thought to bethe most likely group to contribute gametes tothe next generation. As a result, they wereconsidered the best indicators of the futuretrends in the milk protein allelic frequencies.

Five breeds of cattle were phenotyped from21 commercial dairy farms. The sample consis­ted of 1152 Holstein, 172 Jersey, 50 BrownSwiss, 40 Guernsey, and 40 Milking Shorthornfirst lactation heifers. Milk samples were col­lected from DIDA laboratories throughout Cali­fornia in the period July through October 1989.All samples contained .1% Bronopol (D&FControl Systems, Inc., San Ramon, CA) as apreservative.

!J Holstein

-. EJ Milking Shorthorn

Ii Jersey

G' Brown Swiss

lI'Guemsey

Figure 1. Proportional distribution of cows sampled, bybreed, in 21 California dairy heros. (Symbols representapproximately 10 cows.)

Journal of Dairy Science Vol. 74, No.5, 1991

1732 VAN EENENNAAM AND MEDRANO

c-I~ _-= ,,-f3-CN

I~ _ =__-_- a-CN

12345678

Figure 2. Polyacrylamide alkaline (pH 95) urea geleleclrophoresis showing the classification of IXorcasein(eN) and ji-CN genotypes. Lanes I and 8 show IXoI-CNand ji-CN standards, respectively (Sigma Chemical Com­pany, St. Louis, MO). Genotypes for IXoI-CN and Ii-CNare, respectively: lane 2 AB, AA; lane 3 BB, BB; lane 4BB, AC; lane 5 AC, AA; lane 6 BC, AB; lane 7 CC, AA

Electrophoresis of Milk Proteins

Milk samples were precipitated at pH 4.6.,lyophilized, and stored at -20'C until analyzed.The variants of asl-CN, J3-CN, and J3-LG weretyped using alkaline, acid, and native polyacryl­amide gel systems (23). The lC-CN variantswere typed by running the lC-CN macropeptidefragment on alkaline polyacrylamide gels (30).A total of 1454 milk: samples were analyzed.Figures 2, 3, and 4 show the classification ofthe casein genetic variants by PAGE.

Milk Records

Milk records were obtained from DIDA rec­ord processing centers. Records initiated byabortion or of less than 240 d were discarded.Those greater than 240 and less than 305 dwere adjusted to 305-d values using DIDAcorrection factors. Yields were not adjusted tomature equivalent basis. A total of 1166 heifershad complete first lactation production records.

Analysis of Genotypes

Allelic frequencies were determined by genecounting. The genotype distribution withincodominant systems was examined for Hardy­Weinberg equilibrium. Comparison of allelicfrequencies for the four milk: protein gene locibetween breeds and testing of nonindependentsegregation between pairs of milk: protein geneswithin each breed was accomplished using two­way contingency tables. Linkage analysis of thecasein loci was done by comparing the ex­pected frequencies of casein haplotypes (asl-,

Journal of Dairy Science Vol. 74, No.5, 1991

~'~I/". p.'; •.•.... ].I.·.~ •.•.•..•. p.~. M.' It.,.,. /l-CN

1IIIilii1 2 3 4 5 6 7 8 9 10

FJgW'C 3. Polyacrylamide acid (pH 3.0) urea gel electro­phoresis showing the classification of ji-casein (eN) geno­types. Lane I Ii-CN standard (Sigma Chemical Company,St. Louis, MO). Genotypes at the B-CN locus are respec­tivelv: lane 2 A IA2; lane 3 AIAf; lane 4 AlA!; lane 5A2A"3; lane 6 A2c; lane 7 A2A2; lane 8 AlB; lane 9 A2B;lane 10 BB.

J3-, lC-CN) based on independent segregation ofthe alleles with their observed frequencies (1).Significant differences between the two valueswere considered to indicate linkage.

Statistical Analysis

Production data were analyzed with a fIXedlinear model consisting of the classificationfactors of breed, herd within breed, season, andgenotype of the four milk: proteins.

Yijklmnop = J.l. + Bi + Hj(BJ + Sk + asl-CNI+ ~-CNm + lC-CNn + ~-LGo

+ ~jklmnop

where:

Yijklmnop = the observation p on one of thefive dependent variables,

J.l. = the intercept,Bj = the breed (i = 1,2,... 5),

Hj(BJ = the herd within the breed (j =1,2,... 11),

Sk = the season (k = 1,2,... 7),asrCNI = the asrcasein genotype (1 =

1,2,....5),P-CNm = the p-casein genotype (m =

1,2,... 9),lC-CNn = the lC-casein genotype (n =

1,2,3),j3-LGo = the p-Iactoglobulin genotype (0

= 1,2,3), and~jklmnop = the residual error.

1733

Gene Frequencies

Table 1 summarizes the estimated gene fre­quencies of the ~LG, asl-CN, p-CN, and K­CN loci in the five breeds. Gene frequencieswere similar to those reported in the literaturefrom other studies of the American dairy cattlepopulation (Table 2). The asl-CN B variantwas most common in all of the breeds tested,and Milking Shorthorns were found to bemonomorphic for this allele. The frequency ofthe asl-CN C allele was highest in Jerseys, forwhich it was present in the homozygous formin 19 of the 172 animals. The as1-CN A allelehas not previously been associated with Jerseys.In this study, one Jersey cow had the genotypeCXsI-CN AC. A sample of this cow's milk. canbe seen in lane 5 of Figure 2. The asl-CN Aallele was also found in the heterozygous asl­CN AB form in 7 of the 1152 Holstein cows.

The rare /3-CN C allele was found exclu­sively in the heterozygous /3-CN AC form in asmall number of Guernsey cows. The p-CN Aallele was the most prevalent in all of thebreeds, and a small number of samples fromHolsteins carried the rare P-CN A3 variant.Acid gel electrophoresis revealed that all of the~ A alleles in Guernseys were the /3-CN A2variant. The other four breeds carried both the/3-CN Al and the ~CN A2 variants; the latter

K-CN

MILK PROTEIN POLYMORPHISMS IN CALIFORNIA

RESULTS AND DISCUSSION

~ Ii! 3415 B 7

Season consisted of seven 3-mo periods,commencing in winter 1988 (January throughMarch) and ending in summer 1989. All levelsof classification were required to have at leasttwo observations. The dependent variables weremilk (kilograms), fat (kilograms), and proteinyield (kilograms), and fat and protein percent­age. Fat and protein yields were derived frominfrared analysis of monthly milk. samples bythe DIDA laboratories. The data were analyzedwith the PROC GLM procedure of the SAS(26). Models evaluating interactions amonggenotypes were also considered, but no interac­tions were significant.

Figure 4. Polyacrylamide aIkaliDe (pH 9.5) urea gelelectrophoresis of K-casein (eN) macropeptide. Lane I K­CN standard (Sigma Chemical Company, Sl Louis, MO.).Genotypes at the IC-CN locus are, respectively: lane 2 Mlane 3 M lane 4 AB; lane 5 AB; lane 6 BB; lane 7 BB.

TABLE 1. Allelic frequencies (Freq.) and observed number of the ji-lactoglobulin, <lsI-casein, ji-casein, and IC-casein lociof five daily cattle breeds in California.

Breedl

H BS G MS J(n = 1152) (n = 50) (n =40) (n = 40) (n = 172)Freq. No. Freq. No. Freq. No. Freq. No. Freq. No.

Proteinji-LG A .43 996 .39 39 .21 17 .31 25 .37 129~LGB .57 1308 .61 61 .79 63 .69 55 .63 215

~AI .43 985 .18 18 .49 39 .17 58~A2 .55 1263 .66 66 .96 77 .49 39 .50 172~A3 .00 6~CN B .02 50 .16 16 .02 2 .33 114~NC .04 3

lXsI-CN A .00 7 .00 I<ls1-CN B .99 2281 .86 86 .88 70 1.0 80 .68 233lXsrCN C .01 16 .14 14 .12 10 .32 110

IC-CN A .82 1895 .33 33 .73 58 .89 71 .14 49IC-CN B .18 409 .67 67 .27 22 .11 9 .86 295

IH = Holstein, BS =Brown Swiss, G = Guernsey, MS =Milldng Shorthorn, and J = Jersey.

2LG =Lactoglobulin, CN = casein.

Journal of Dairy Science Vol. 74, No.5, 1991

I TABLE 2. Allelic frequencies of the ~lactoglobulio, lXsl-casein, lkasein, and lC-easein loci found over time in the US dairy cattle population. --...Il.Jo)

Holstein Jersey~

01965 to 19682 19673 1965 to 19704 1964 to 19745 19906 1965 to 19682 1965 to 19704 19906...

t1 Proteinl (n = 400t) (n = 754) (n = 494+) (n =6000+) (n = 1152) Protein (n =37 to 387) (n =270+) (n = 172)!.I3-LG A .46 .50 .53 .43 I3-LG A .41 .36 .37

~ I3-LG B .54 .50 .47 .57 ~LG B .59 .64 .63

H lXsI-CN A .04 .00 .00 .00 lXsI-CN A .00lXsI-CN B .91 .94 .96 .99 lXsI-CN B .77 .74 .68

~ !lsl-CN C .05 .06 .04 .01 lXsI-CN C .23 .26 .32

~I3-CN Al .40 .31 .49 .41 .43 ~CN Al .22 .09 .17I3-CN A2 .54 .62 .49 .53 .55 ~CN A2 .49 .54 .50

~ ~CN A3 .02 .05 .01 .03 .00 ~CN B .29 .37 .33I3-CN B .04 .02 .01 .03 .02 ~CN C .00

~.l'"lC-CN A .80 .75 .80 .82 lC-CN A .31 .12 .14

~ lC-CN B .20 .25 .20 .18 lC-CN B .69 .88 .86

I- Guernsey Brown Swiss

1965 to 19682 1965 to 19704 1969 to 19747 19896 1965 to 19682 1965 to 19704 19906

(n =200+) (n =260+) (n = 3861+) (n = 40) (n =21 to 203) (n =259+) (n =50)

I3-LG A .30 .38 .39 .21 ~LG A .27 .33 .39I3-LG B .70 .62 .61 .79 ~LG B .73 .67 .61 ~lXsI-CN B .65 .79 .74 .88 asl-CN B .97 .98 .86

~all-CN C .35 .21 .26 .12 lXsI-CN C .03 .02 .14

I3-CN Al .01 .06 .01 ~CN Al .14 .15 .18

~I3-CN A2 .97 .88 .96 .96 I3-CN A2 .66 .72 .66I3-CN A3 .01 ~CN B .18 .10 .16I3-CN B .01 .01 .02 I3-CN C .02 .03I3-CN C .05 .01 .04

lC-CN A .67 .59 .73 .73 lC-CN A .38 .41 .33lC-CN B .33 .41 .27 .27 lC-CN B .62 .59 .67

1LG =Lactoglobulio, CN =casein.

2Kiddy et aI. (12, 13, 14, 15).

3Arave et aI. (2).

4U et aI. (16).

5Hines et aI. (11).

6nns study.

7Haenlein et aI. (10).

MILK PROTEIN POLYMORPHISMS IN CALIFORNIA 1735

TABLE 3. Breed comparisons of allelic frequencies for the four milk protein gene loci.

Breedl df X2 P elf X2 P

!i-Lactoglobulin lXarCasein

BS VS. G 1 3.3 <.fJ1 1 0 <.84BS VS. H 1 .4 <.56 2 68.6 <.01BS VS. J 1 .0 <.85 2 6.4 <.04BS VS. MS 1 .6 <.45 1 6.1 <.01G VS. H 1 7.6 <.01 2 49.8 <.01G VS. J 1 3.8 <.05 2 6.3 <.04G VS. MS 1 l.0 <.31 1 5.3 <.02H VS. J 1 2.0 <.16 2 323.1 <.01H VS. MS 1 2.3 <.13 2 .4 <.82J VS. MS 1 .5 <.46 2 17.5 <.01

lJ-Casein x-Casein

BS VS. G 3 18.0 <.01 1 13.9 <.01BS VS. H 3 63.7 <.01 1 72.9 <.01BS VS. J 2 5.8 <.06 1 9.1 <.01BS VS. MS 2 11.8 <.01 1 28.3 <.01G VS. H 4 72.1 <.01 1 2.5 <.12G VB. J 3 38.3 <.01 1 58.5 <.01G VS. MS 3 282 <.01 1 3.4 <.07H VB. J 3 3062 <.01 1 354.1 <.01H VB. MS 3 1.1 <.77 1 1.1 <.29J VS. MS 2 25.5 <.01 1 89.0 <.01

IBS =Brown Swiss; G = Guernsey; H = Holstein; J =Jersey; MS =Milking Shorthorn.

TABLE 4. Detection of nonindependent assortment of genotypes at the !i-lactoglobulin, lXal-casein, li-casein, and x-caseinloci in five cattle breeds.

Loci I df X2 P df X2 P

Holstein Jersey

fi-LG VS. <XsI-CN 4 6.6 <.16 6 3.2 <.78fi-LG VS. li-CN 4 1.7 <.79 4 3.4 <.49fi-LG VS. x-CN 4 3.4 <.49 4 9.5 <.05(Xsl-CN VS. fi-CN 4 .5 <.97 6 56.1 <.001**<XsI-CN VS. x-CN 4 3.8 <.44 6 6.7 <.35fi-CN VS. x-CN 4 133 <.001** 4 17.8 <.001**

Brown Swiss Guernsey

fi-LG VS. lXal-CN 4 1.8 <.77 2 2.3 <.32fi-LG VS. fi-CN 4 4.1 <.40 2 .2 <.92fi-LG VS. x-CN 4 3.1 <.54 2 5.6 <.23Usl-CN VS. fi-CN 4 2.0 <.73 1 1.1 <.30<Xsl-CN VS. x-CN 4 4.9 <.30 2 1.1 <.57f3-CN VS. x-CN 4 3.6 <.46 2 16.9 <.001**

Milking Shorthorn

f3-LG VS. lXarCN 2 m2fi-LG VS. I3-CN 2 2.3 <.31I3-LG VS. x-CN 4.5 <.10UsI-CN vs. f3-CN NTUsI-CN VS. x-CN NTfi-CN VS. x-CN 1 7.3 <.007**

ILG =Lactoglobulin, CN = casein.

2NT = Not tested.

**p < .01.

Journal of Dairy Science Vol. 74, No.5, 1991

1736 VAN EENENNAAM AND MEDRANO

TABLE 5. Observed (Obs.) and expected (Exp.) fi:cqueDcies (Freq.) of the haplotypes for the <lsl-' fl-. and lC-casein loci,respectively. for the Holstein (n = 1152) and Jersey (n = 172) breeds.

Holstein

Haplo­type

BA2ABAlABA1BBA2BBB BBB ACA2ACA1ABA3AAA1AAA2ACA3AAA1BAA2BCA2BCBABA~CA1BCA~

Obs.freq}

.4639

.3393

.0847

.0792

.0124

.0091

.0037

.0016

.0014

.0013

.0011

.0010

.0004

.0002

.0002

.0002

.0001

.0001

.0001

.4460

.3483

.0754

.0966

.0038

.0175

.0032

.0025

.0020

.0011

.0014

ooסס.

.0002

.0003

.0007

.0001

.0004

.0005

ooסס.

Haplo­type

BB BCA2BBA2BBA1BBA2ACBBCA2ACA1BBB ABA1ACA1ACBAAAlAAAlBAA2AAA2BABAABB

Obs.freq.

.2689

.2129

.1817

.1265

.0698

.0422

.0342

.0240

.0174

.0131

.0036

.0029

.0007

.0007

.0007

.0007

ooסס.

ooסס.

Exp.freq.

.1923

.1373

.2904

.0982

.0481

.0909

.0227

.0464

.0318

.0162

.0077

.0150

.0001

.0004

.0002

.0013

.0001

.0009

Iprequencies determined on all possible combinations of alleles from the genotype of each animal.

2Prequencies determined assuming independent segregation of the alleles of the three casein gene loci.

predominated in most of the breeds. The high­est frequency of the I3-CN B allele was foundin Jerseys, which had a total of 20 homozygous~-CN BB samples. Interestingly, each of the 20samples had the genotype BB BB BB for the<ls1-, ~-, and K-CN loci.

The K-CN A genetic variant was most fre­quent in Guernseys, Milking Shorthorns, andHolsteins, whereas the lC-CN B allele had thehigher frequency in Jerseys and Brown Swiss.The observed genotypic frequencies for the

casein loci in the five breeds followed Hardy­Weinberg expectations.

The ~-LG B allele was the predominantallele in all five dairy breeds. Only J3-LG A and~-LG B alleles were found in Jerseys. Thisdiffers from Australian data in which a thirdallele, p-LG C, has been detected (20). The ~­

LG locus did not follow Hardy-Weinberg ex­pectations in three of the five breeds. In theHolstein, Jersey, and Brown Swiss breeds, agreater than expected number of animals were

TABLE 6. A comparison of observed (Obs.) and expected (Exp.) frequencies of selected casein haplotypes [<lsl-' fl-. andlC-casein (eN), respectively] in three separate studies involving the Holstein breed.

<lsi-, fl-. lC-CN

BBBBBACA3A

19901 19852 19773

(n = 1152) (n = 3063) (n = 6(94)

Obs. Exp. Obs. Exp. Obs. Exp.

.0124 .0038 .0143 .0068.024 .006

.0091 .0175 .0106 .0187.002 .020

.001 OO.0006סס. OO1.024סס. .001

Totaf(n = 10.3(9)

Obs. Exp.

204 6255 199

149 6

1Present study.

2Aleandri et aI. (1).

~ el aI. (11).

~ese values represent the sum of the total number of cows in the three experiments that were of the specifichaplotype compared with the expected number based on gene frequencies and independent segregation of the alleles.

Journal of Dairy Scieoce Vol. 74, No.5. 1991

MILK PROTEIN POLYMORPmSMS IN CALIFORNIA 1737

TABLE 7. Percentage frcqueucy at the common milk: casein genotype combiDalions for <lsl-' tl-, and lC-easein (eN) locifound in Holstein and Jersey cows from different countries.

Genotype

<lsI". tl-, x-Casein

BBAIA2AABBA2A2AABBAIA2ABBBAIA1AABBA1AIABBBA2A2ABBBA2B ABBBAIB AB

«Sl-' fl., x-Casein

BCA2B BBBBAIB BBBBB B BBCCA2A2BBBCA1A2BBBCA2A2ABBBA2B BBBCA2A2BBBBA2B AB

l'Ibis study.

2Aleandri el al. (I), Italy.

3McLean el al. (20), Anstrlllia.

"nata not reported for these values.

~ech el al. (4), Dc:nmark.

heterozygous (P < .05). This trend has not beenreported by other investigators. An excess ofheterozygotes indicates a difference in the se­lective value of one of the alleles at the locus(16). However, no obvious selection appears tohave been occurring for either the f3-CN A orf3-LG B, which can be seen by the apparentstability over time of the milk: protein allelicfrequencies in the American dairy cattle popu­lation (Table 2). No increasing trend in thefrequency of anyone allele is evident.

Significant differences among breeds werefound for most of the gene frequencies of thethree casein loci (Table 3). Only betweenGuernseys and Holsteins was a differencefound in the ~-LG allelic frequencies. Holsteinsand Milking Shorthorn breeds did not differ inthe gene frequencies found at any of the fourmilk protein loci.

(n = 260)1984Ansttalia3

22.14

14.718215.9

(n = 308)1984Austra1ia3

21.6

13.1

10.8

9.8

Linkage Between LOCi

Chi-square tests revealed there was no sig­nificant linkage between the ~-LG locus andthe three casein loci. However, nonindependentassortment ocCUIYed between the f3-CN and K­CN loci in Guernseys, Holsteins, and Jerseysand between the <XsI-CN and ~-CN loci inJerseys (Table 4). The small sample sizes in­volved in Guernseys, Brown Swiss, and Milk­ing Shorthorns could have influenced the segre­gation patterns observed here. These results arein agreement with a number of studies thathave shown nonindependent segregation of the€Xsi-CN, 13-CN, and K-CN variants (1, 8, 20).

Table 5 shows the observed and expectedfrequencies of the allelic combinations for <Xs1­CN, f3-CN, and K-CN (haplotypes) for Jerseysand Holsteins. The observed frequencies differfrom expected frequencies in both of the breeds

Joumal of DaiJy Science Vol. 74, No.5, 1991

1738 VAN BENENNAAM AND MEDRANO

TABLE 8. Allelic frequencies for the Ji-Iactoglobnlin (Ji­LG), <XsI-casein (<XsI-CN), Ji-casein (Ji-CN), and lC-casein(lC-CN) loci in Holstein and Jersey population of threecountries.

IThis study.

2Aleandri et aI. (I), Italy.

~Lean et aI. (20), Australia.

~ech et aI. (4), Denmark.

*Chi-square test significant P < .05.

study had the genotype BB BB BB, In accor­dance with Grosclaude's cluster hypothesis (9)<ls1-CN C and P-CN B combinations werefewer than expected.

The most prevalent genotypes occurring inthe Holstein and Jersey cattle populations froma number of countries are shown in Table 7,The gene frequencies of the same studies aregiven in Table 8. The California Holstein popu­lation has a low frequency of K-CN B allele,whereas in the Jersey population the frequencyis very high. Although comparisons may bebiased by sampling errors, they do serve to

(n = 260)1984Australia3

.39

.61

.63

.35

.00

.02

.00

.96

.04

.68

.32

.63

.37

.23

.77

(n = 308)1984Australia3

.33

.56

.11

.07

.57

.36

Holstein

(n = 1383)1985Itmy2

.70

.30

.31

.69

(n = 1152)1990us l

(n = 172)1990us l

.37

.63

.40

.60

.58

.40

.00

.02

.00

.98

.02

.73

.27

----- Jersey -----

(n = 157)1985Denmark4

.31

.68

.01

.07

.58

.35

.43

.57

.43

.55

.00

.02

.00

.99

.01

.82

.18

Ji-LG AJi-LG BJi-CN AI,·Ji-CN A2,·Ji-CN A3,·Ji-CN B·<XsI-CN A*<XsI-CN B*<XsI-CN c*lC-CN A*lC-CN B*

Ji-LG A*Ji-LG B*Ji-LG c*Ji-CN AI,· .17Jl-CN A2,. .50Ji-CN B* .33<XsI-CN A .00<XsI-CN B .68<XsI-CN C .32lC-CN A* .14lC-CN B* .86

(P < .(01). These results and the results ofothers (1, 4, 11, 20) indicate that the Usl-, ~,

and K-CN loci are situated on the same chro­mosome in close proximity to each other.

Three haplotypes markedly deviated fromexpected frequencies in three separate studies(Table 6). Casein haplotypes in the followingdiscussion will be expressed in the order Usl-,p-, and K-CN. There was clearly an excess ofthe BBB and CA3A haplotypes. Linkage ismaintaining the BBB haplotype at unexpectedlyhigh frequencies, thus preventing recombina­tion to form the BBA haplotype in both Hol­steins and Jerseys. The similarity between thebreeds with respect to these haplotype devia­tions suggests that they have descended fromthe same original casein gene cluster. Thiswould be expected, given that the formation ofbreeds is a relatively recent event in the historyof the species.

Grosclaude (9) hypothesized that the UsI-CNB and P-CN A2 alleles made up the ancestralcasein cluster and that successive mutations ateither the UsI-CN or P-CN locus led to theformation of the other gene complexes. Thismodel then implies that <ls1-CN C ~CN A2,<ls1-CN B P-CN B, or <ls1-CN B P-CN Cwould be mutant derivatives of the originaltype but that <ls1-CN C P-CN B would be arecombinant type. The rare occurrence of thiscombination is explained by the "very closelinkage between the <ls1-CN and P-CN cis­trons," which was recently confirmed (6, 29).

The original cluster in Holsteins likely wasBA2A, which is currently the most prevalenthaplotype in the Holstein breed (Table 5). Thehigh incidence of CA3A could be explained bya single recent mutation of the P-CN A2 alleleto the P-CN A3 in a CA2A haplotype followedby no further recombination. This would resultin the significant association of ~CN A3 allelewith the CA3A haplotype. Once the BBBhaplotype has formed, it is apparently transmit­ted with a very low probability of recombina­tion. This is of interest to breeding schemesthat are considering selection of animals carry­ing the B alleles.

Few studies have been done on the haplo­types of Jerseys. The most common haplotypefor Jerseys in this study was BBB with anobserved frequency of .27. As discussed previ­ously, all 20 of the ~CN BB samples in this

Journal of Dairy Science Vol. 74, No.5, 1991

MILK PROTEIN POLYMORPHISMS IN CALIFORNIA 1739

TABLE 9. Mean squares and signifIcance of !he sources of variation for total lactation milk, fat and protein yield, and fatand protein percentage.

Protein

19.56··2.35··

.29*

.10

.09

.01

.21

.13

Fat

(kg x 103)

120.87··36.19*·15.03··

.191.53.85

2.011.76

(kg x 103)

9631··23.49··7.67··1.43

.811.127.24··1.08

----- (%) -----3.26··1.03··.13t.04.01.OS.11 t.04

Milk

(kg x 104)15,052"·

2674··1162··

408736

304t114

Sourcel df

Breed 4Herd (breed) 16Season 6~LG 2~CN 8aal-eN 3,,-CN 2Error 1123

lLG =Lacloglobulin, CN = casein.

....p < .01.

"p < .05.

tp < .10.

Breed 4Herd (breed) 16Season 6~LG 2~CN 8aal-CN 3,,-CN 2Error 1123

demonstrate the areas in which frequencies of adesirable allele are high and how this affectsthe prevalent genotypes in the area. These vari­ations may be the result of breeding animalsfrom small fOlDldation stocks imported to thedifferent colDltries many years ago (3).

Effect 01 Protein Genotypeon Milk, Fat, and Protein Yield

Table 9 presents the analysis of variance forlactation milk, fat and protein yield. and fat andprotein percentage. The only significant effectof the milk protein genotypes was that of lC-CNon protein yield (P < .01). The lC-CN genotypealso approached significance for its effect onmilk yield and protein percentage (P < .10).

A comparison of least squares means (Table10) for the lC-CN AA and BB genotypes showsthat the lC-CN BB genotype increased first lac­tation yields by 296 kg of milk: and 16 kg ofprotein and increased protein percentage by.066%. The increase in milk yield is in agree­ment with Lin et al. (17), who found replace­ment of the lC-CN AA genotype with the lC-CNBB genotype also resulted in an increase in firstparity milk yield. The increased protein yield

associated with the lC-CN B allele has beenfOlDld in other studies, as has the increasedprotein percentage (1, 25).

A number of studies have found associationsbetween the other milk protein genotypes andmilk: yield or gross composition (1), but othershave found no effect of milk protein genotypeon yield traits (20). Overall, milk protein geno­types appear to have no consistently significanteffect on production traits. This idea is sup­ported by the absence of trends of allelic fre­quency change in the US dairy cattle popula­tion over time (Table 2).

The intensive selection for milk productionthat bas occurred in the last 30 yr would havefavored an increase in any allele positivelycorrelated with an increased milk yield. Be­cause the allelic frequencies have remained sta­ble, they likely are not highly correlated withmilk production. Correspondingly, selection forspecific milk protein alleles would not be ex­pected to affect the production potential of thedairy cattle population deleteriously.

CONCLUSIONS

Genetic studies of the manufacturing proper­ties of milk have suggested that selection for

Journal of Daily Scieoce Vol. 74, No.5. 1991

1740 VAN BENENNAAM AND MEDRANO

TABLE 10. Least squares meaDS of different genotypes and their standard errors for total lactation milk, fat and proteinyield, and fat and protein pc:rccntage.

Genotypel n Milk Pat Protein

(Irg)

X SE X SE X SE

~LG AA 175 7021 236 295 9.2 250 7.3~LG AB 624 6993 226 293 8.8 248 6.9~LG BB 366 6945 226 294 8.9 245 7.0

~CN AlAI 171 7042 201 298 7.9 248 6.2~CN A IA2 487 7100 182 297 7.1 251 5.6~CN A IA3 4 7152 575 293 23.0 259 17.7~CN AlB 40 6945 268 287 10.5 247 8.2~CN A2A2 363 7119 181 297 7.1 251 5.6~CN A2A3 2 7670 792 333 31.1 266 24.4~CN A2B 74 6811 230 286 9.0 243 7.1~CN A2C 2 6352 798 273 31.3 228 24.5~CN B B 22 6684 340 281 13.3 238 10.5

CXal-CN AB 6 7196 480 306 18.8 253 14.8CXarCN BB 1050 6853 184 287 7.2 240 5.7CXal-CN BC 92 6945 212 290 8.3 247 6.5CXal-CN CC 17 6952 347 293 13.6 251 10.7

K-CN AA 662 6839 236 290 9.3 240 7.3K-CN AB 346 6985 233 292 9.1 247 7.2K-CN BB 157 7135 239 299 9.4 256 7.4

(%)

~LGAA 175 4.276 .081 3.624 .046~LG AB 624 4.277 .077 3.602 .044~LG BB 366 4.306 .078 3.597 .044~ AlAI 171 4.295 .069 3.590 .039~ AIA2 487 4.252 .062 3.589 .035~ AIA3 4 4.212 .197 3.667 .112~ AlB 40 4.174 .092 3.598 .052~ A2A2 363 4.245 .062 3.597 .035~CN A2A3 2 4.437 .271 3.543 .154~CN A2B 74 4.278 .079 3.623 .045~ A2C 2 4.369 .274 3.630 .155~BB 22 4.314 .117 3.628 .066

CXaI-CN AB 6 4.323 .165 3.581 .093CXaI-CN BB 1050 4.287 .063 3.571 .036CXal-CN BC 92 4.261 .073 3.620 .041CXal-CN CC 17 4.274 .119 3.656 .067

K-CN AA 662 4.321 .081 3.578 .046K-CN AB 346 4.277 .080 3.600 .045K-CN BB 157 4.261 .082 3.644 .046

lLG = Lactoglobulin, CN = casein.

the combination of j3-LG B, P-CN B, and lC-CNB would improve the casein content, coagulat­ing properties, and cheese-yielding capacity ofmilk. The <Is1-CN genotype has not beenshown to have a significant effect on the manu­facturing properties of milk, so altering theallelic frequency of this gene should not beconsidered as a selection objective. Selection

Journal of Dairy Science Vol. 74, No.5, 1991

for the B alleles of p-LG and lC-CN would havethe greatest impact on improving milk for man­ufacturing purposes.

In the California Holstein cattle population,the frequency of the P-LG B allele is moderate,and the frequency of the lC-CN B allele is low.The P-LG and lC-CN genes are found on sepa­rate chromosomes, so linkage does not compli-

MILK PROTEIN POLYMORPHISMS IN CALIFORNIA 1741

cate their concurrent selection. Selection forhigh producing animals carrying these alleleswould be feasible given the current allelic fre­quency. Selection for the li-CN B allele. how­ever, would be too restrictive due to its scarci­ty. Analyses of the effects of milk proteingenotypes on production parameters indicatethat such a selection strategy would notdecrease milk yield and might have a beneficialeffect on protein yield, which would help toimprove the cheese yield of Holstein milk.

The Jersey population has a moderate levelof the j3-LG and li-CN B alleles and a highlevel of the x:-CN B allele. Selection for the 13­CN B allele must consider the linkage thatexists between the x:-CN and f3-CN genes.Selection for the j3-CN B allele could conceiva­bly be in conflict with selection for the x:-CN Ballele. The effects of the x:-CN B allele onimproving the manufacturing properties of milk.are more significant than those of the li-CN Ballele; consequently, lC-CN should be givenselection priority. Genetic progress will be ac­celerated if the j3-CN genotype is considered inselection decisions only after cows have al­ready been genotyped lC-CN BB. Fortunately,as a result of linkage, transmission of the j3-CNB lC-CN B haplotype to progeny intact is highlyprobable. Selection for the f3-CN BB cowsgenotyped in this trial would have also selectedfor x:-CN BB animals.

Current frequencies of milk. protein alleles inthe California dairy cattle population couldfeasibly be altered by appropriate single geneselection strategies, thus improving the manu­facturing properties and cheese yield capacityof the future milk. supply. Prior to adoptingsuch a breeding program, consideration must begiven to possible association of the geneticvariants with other factors of dairy productionand with the economic changes that might en­courage producers to select for specific milk.protein genes.

ACKNOWLEDGMENTS

The authors would like to thank Linda Shar­row for her laboratory expertise and helpfulnessin the development of this investigation and RC. Laben for his review of this manuscript.This work was supported in part with fundingfrom the California Milk Advisory Board.

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