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Blood Antigen, Serum Protein, and Milk Protein Gene Frequencies and Genetic Interrelationships in Holstein Cattle1
Blood Antigen, Serum Protein, and Milk Protein Gene Frequencies and Genetic Interrelationships in Holstein Cattle 1 H. C. HINES =, G. F. W. H A E N L E I N 3, J. P. Z l K A K I S s, and H. C. D I C K E Y 4 The Ohio State University and The Ohio Agricultural Research and Development Center Columbus 43210 3 Delaware Agricultural Experiment Station Newark 19711 4 University of Maine Orono 04473 ABSTRACT
Gene frequencies at ten blood group loci, one serum protein locus, and four milk protein loci were determined for the Holstein-Friesian breed in the United States. The sample consisted of 8630 cows in 51 herds from 10 states. Because of the close linkage among casein subloci and the concomitant rarity of crossoverrecombinant groups, casein gene combination or haplotype frequencies were determined also. As one means of comparison of systems, indices of homozygosity and number of effective alleles were calculated. These indices were proposed also to be useful tools for monitoring changes in genetic variability of breeds. Genotypes within codominant systems and phenotypes associated with paired-system combinations generally were not randomly occurring. Paired system phenotypes within the sire sample corresponded much more closely to expectations of randomness. INTRODUCTION
Frequencies of genes and genotypes are useful in establishing breed structures and
Received December 2, 1976. 1Data from the NE-62 Regional Research Project, Relationship Between Genetic Markers and Performance in Dairy Cattle. Members of the NE-62 Technical Committee: C. W. Arave, UT; H. C. Dickey, ME; S. N.
Gaunt, MA; G. F. W. Haenlein, DE; G. L. Hargrove, PA; H. C. Hines, OH; A. C. Hunter, MN; C. A. Kiddy, ARS-USDA; R. E. Mather, NJ; P. R. Shellenberger, PA; G. W. Trimberger, NY; C. W. Young, MN; J. P. Zikakis, DE.
interrelationships. As additional genetic marker loci are discovered in cattle, it becomes easier to infer about the development and relationships of breeds and to establish benchmarks from which subsequent changes in breed structure can be measured. The information reported here differs from that of previous studies by being more comprehensive in two respects: 1) it results from the typing of more than 8,000 Holstein-Friesian cattle, and 2) it consists of simultaneously determined genotypes at 15 loci. Thus, it probably provides the best basis to date for thorough assessment of several aspects of the genetic composition of the Holstein breed in particular and the bovine species in general. The 15 loci consisted of 10 red cell antigen loci (bloodgroups A, B, C, FV, J, L, M, S, Z, and R'S'), serum transferrin (Tf), and milk proteins beta-lactoglohulin (Lg), alphasl-casein (%l-Cn), beta-casein (/3-Cn), and kappa-casein (K-Cn). Many of the sires also were typed for hemoglobin (Hb), but no variation was detected. The blood group systems have been reviewed by Stormont (23), the hemoglobin and transferrin systems by Kiddy (10), and the milk protein systems by Aschaffenburg (2). Subsequent to the transferrin review, additional genetic variation at this locus was detected by improved electrophoretic techniques (14). Three linkage groups have been detected among the loci; otherwise the loci appear to be independent. Linkage groups are: a) the Hb and A blood group loci (7, 16), b) the Lg and J blood group loci (8, 17, 24), and c) the %1-,/3-, and •-Cn loci (6, 8, 18). Larsen and T h y m a n n (18) consider the casein linkage so close as to be virtually a single locus. In this respect it may behave as a kind of superlocus and as such m a y bear similarities to the more complex cattle
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blood group systems. As for these latter systems, it also may be desirable to represent the combined casein locus variants as phenogroups or haplotypes. This investigation formed an integral part of the Northeast Regional Research Project NE62, Relationships between genetic markers and performance in dairy cattle. The design of the project specified that a large and representative sample of cattle from a single breed simultaneously would be typed for genetic markers and evaluated for performance traits. The project provided for the development of the following objectives for this investigation: 1) determination of frequencies for the genetic marker systems which would be representative of frequencies in the Holstein-Friesian breed in the United States, 2) determination of comparative measures of variation at different genetic marker loci, 3) ascertainment of intra- and interlocus relationships within the sample as exemplified by Hardy-Weinberg equilibrium and inter-locus phenotype independence, and 4) construction of Holstein casein haplotypes and calculation of their frequencies within the breed.
red ceils, and .025 ml of normal rabbit serum (complement source) which was added after a 5-min red cell-reagent incubation period. The plates were covered with a plastic adhesive, shaken, and incubated at 30 C. Except for these modifications, the test was essentially the same as that described by Ferguson (5). In projects encompassing several years, changes in typing commonly occur, particularly with a battery of biological reagents in finite supply. The primary changes of this type were those associated with additions to the reagent bank over time. Reagents in this category included anti-A1, which enabled subdivision of the A system factor A into A1 and A2 subtypes; anti-U'2, which enabled us to differentiate the S system L?2 factor from the silent allele product (designated s); and anti-R' and anti-S', which allowed determination of genotypes at a separate new locus (R'S'). Most of the bulls siring females in this study were typed by the Serology Laboratory of the University of California (Davis). Typing techniques were similar to those of the Ohio laboratory. The participation of both laboratories in biennial comparison-standardization tests helped to standardize results (12).
EXPERIMENTAL PROCEDURE Source of Samples
Blood and milk samples were obtained from 8630 Holstein cows and heifers from 51 herds in 10 states: Delaware, Maine, Maryland (USDA), Massachusetts, Minnesota, New Jersey, New York, Ohio, Pennsylvania, and Utah. Private and institutional herds were represented. The animals typed were from 3 mo to 3 yr of age. Samples were collected from 1964 to 1974. Blood Group Typing
All typing of blood groups on cows and heifers was by the Cattle Blood Typing Laboratory of The Ohio State University. Blood samples were collected in isotonic sodium citrate, cooled to 4 C, and shipped to the Ohio laboratory for typing by hemolytic test procedures. The typing was in disposable plastic microtiter plates (Cooke Engineering Co., Alexandria, VA). Each test well contained .05 ml of specific hemolytic reagent (monospecific antiserum), .025 ml of a 2.5% suspension of washed Journal of Dairy Science Vo|. 60, No. 7
Elect rophoret ic Typing
Serum transferrin typing was by the Minnesota, Ohio, USDA, and Utah stations. The typing procedures were those described by Bailey and Kiddy (4), Jamieson (9), Kristjansson and Hickman (14), and Rausch et al. (21). In the early years of the project, the typing was for the undifferentiated D allele, but later TfD1 and D z were subtyped. Additionally, some of the samples typed earlier and still in frozen storage were retyped for DI and D2. Electrophoretic typing of milk protein was at Delaware, Massachusetts, Minnesota, Ohio, Pennsylvania, USDA, and Utah laboratories. Beta-lactoglobulin was typed according to the procedures described by Aschaffenburg and Thymann (3), Kiddy et al. (15), and Wake and Baldwin (26). Alphasl-casein and K-casein variants were typed by the methods outlined by Aschaffenburg and Thymann (3), Kiddy (11), Thompson et al. (25), and Woychik (27). The procedures for fi-Cn typing have been described by Aschaffenburg (1), Kiddy (11), and Peterson and Kopfler (20).
HOLSTEIN GENE FREQUENCIES
1145
TABLE 1. Genetic marker codominant and simple dominant system gene frequencies. System
Allele
Frequencya,b
A
.239
A
No. typed
8452
~
1
AH
.049 2H
D DH
.675 .037
F V
.874 .126
j js jcs
.589 .205 .206
1 L
.800 .200
m M2M'
.966 .034
s SH' UH' U' 1 U' 2 H'
.323 .114 .061 .026 .013 .463
z Z
.683 .317
R' S'
.024 .976
A D
.431 .540
.215 .024
1094-q 1094]
o45
109
.004
1094 I
8464
FV
8426
8086
M
8399
639 8468
1144
R'S'
8068
Tf
.249 .291 E
.029
A B
.526 .474
A B C
.003 .957 .040
A1 A2 A3 B
.415 .532 .028 .025
427~ 427~ 6465
Lg
6874
~sl-Cn
6575
13-Cn
Journal of Dairy Science Vol. 60, No. 7
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HINES ET AL.
TABLE 1. Continued. System
Allele
Frequencya,b
A B
.800 .200
•-Cn
No. typed 6531
aFrequencies for the A, J, and S systems were determined by the maximum likelihood method. Frequencies for the FV, Tf, Lg, ~slq2n, 3-Cn, and ~-Cn systems were determined by gene counting. Frequencies for the L, M, Z, and R'S' systems were determined by the square root method. bstandard deviations of frequency estimates, determined by the formula, S.D. = [p(1--p)] -5/n, ranged from .0004 to .0049. Although a variety of electrophoretic methods was utilized by the cooperating laboratories, standardization of results was verified by periodic comparisons. Similarly, although many of the bulls were typed electrophoretically by the California Serology Laboratory, standardization of results was corroborated likewise by regular participation in international comparison-standardization tests. Gene Frequency Determination
Gene frequencies were determined from blood and milk types of the 8630 females. Seven-hundred-seventy of the 900 sires of these females were bloodtyped, and these formed a sample which also was subjected to analyses of gene frequency and genetic interrelationship. Milk protein types of sires were determined by inference from their daughters' types. The results of the sire sample determinations are not reported in detail but only as they provide information related to aspects of the female sample findings. Four methods of calculation of gene frequency were utilized in computing of these estimates. Frequencies of alleles in codominant systems (FV, Tf, Lg, C~sl-Cn, /3-Cn, and K-Cn), where genotypes always can be determined from phenotypes, were calculated by gene counting. Simple dominant systems containing two alleles (L, M, and Z) were analyzed by extracting the square root of the frequency of the homozygous recessive genotype. Although the RtS ' system is codominant, the absence of anti-S' reagents from the typing battery of the Ohio laboratory for most of the typing period precluded analysis for codominant system. Instead, the system also was analyzed by the square root method as a simple dominant locus. Journal of Dairy Science Vol. 60, No. '7
More complex dominant systems with from three to ten alleles (A, J and S) were analyzed by a maximum likelihood method described and programmed for computer by Kurczynski and Steinberg (15). Complex genetic systems of more than ten alleles (B and C) were analyzed by a genotype allocation procedure essentially as described by Neimann-Sorensen (19) and by computer program developed at The Ohio State University. For cases in which improved typing techniques became available during the project resulting in subsequent recognition of additional alleles, the frequencies for such have been demonstrated only over that portion of the sample for which they were typed. Thus, their frequencies are neither biased nor do they bias other estimations; however, because of fewer numbers their frequencies are estimated with less precision than for alleles typed for the entire sample. Because of the apparently close linkage among the ~sl-, /3-, and K-casein loci and the concomitant rarity of crossover-recombinant groups, casein gene combination or haplotype frequencies also were determined. In the female sample, haplotypes were determined, when possible, directly from phenotypes, secondarily from pedigree information, and finally, as necessary, by allocation methods (19) to complete the frequency estimation. In the sire sample, haplotypes were determined solely from progeny information. Genetic Interrelationships
Genotype distribution within codominant systems was examined for Hardy-Weinberg equilibrium. Frequencies of paired system phenotypes were compared with those ex-
HOLSTEIN GENE FREQUENCIES TABLE 2. System C phenogroup frequencies (8219 animals typed).
1 147
RESULTS AND DISCUSSION Gene Frequencies
Allele code number
o 32 2 22 33 27 31 1 21 23 29 15 26 13 3 30 16 14 25 11 54 50 34 45 9 52 5 10 8 4 20 28 7 6 19 39
Phenogroup longhand designation
c
X2 CtE E X 2 L'
W Xt Cl CzW EW WX:~ C2 ER RWX 2 C2 C 1 EW WX 2 L' C 2 EW C2E EL' C1 X1 WL' L' C X2
CW C EL' X L' C ER C R C EX 1 C EWX 2 C R WX1 C 1EWX 1 Ct RW C 2 ERX 2 C2 EWX 2
a
Frequencya,b
.205 .168 .153 .134 .073 .065 .048 .032 .029 .019 .015 .010 .010 .010 .008 .005 .004 .003 .003 .001 .001 .0007 .0007 .0005 .0004 .0004 .0002 .0002 .0002 .00012 .00012 .00012 .00007 .00006 .00006 .00006
G e n e f r e q u e n c i e s in t h e c o d o m i n a n t a n d simple d o m i n a n t s y s t e m s are in T a b l e 1. Information on a portion of the sample pertinent to t h e alleic subdivision w h i c h r e s u l t e d f r o m improved typing techniques during the project is b r a c k e t e d . T h e r e d u c e d n u m b e r o f a n i m a l s so s u b t y p e d as well as t h e smaller n u m b e r t y p e d for U'e is i n d i c a t e d . In all o t h e r cases t h e n u m b e r of a n i m a l s t y p e d for a particular s y s t e m is a p p l i c a b l e to all of t h e alleles in t h a t system. P h e n o g r o u p (allele) f r e q u e n c i e s for t h e m o r e c o m p l e x C a n d B s y s t e m s are in T a b l e s 2 a n d 3. T h e B allele G 2 Y 2 E ' I Q ' o c c u r r e d m u c h m o r e frequently than any other B phenogroup. At t h e o p p o s i t e e x t r e m e , t h e 22 alleles t h a t were d e t e c t e d each o n l y o n c e m a y b e a t y p i c a l o f t h e Holstein breed. T h e i r d e t e c t i o n , as well as t h e d e t e c t i o n of a n y o t h e r alleles n o t specifically listed in T a b l e 3, in a b r e e d p u r i t y analysis o f a r e p o r t e d l y p u r e b r e d Holstein a n i m a l raises suspicion of foreign b l o o d a n d s h o u l d be f o l l o w e d b y e x h a u s t i v e pedigree checking. Casein Haplotypes
T h e d e t e c t e d casein h a p l o t y p e s a n d t h e i r c o r r e s p o n d i n g f r e q u e n c i e s are in T a b l e 4. T h e o b s e r v e d a n d e x p e c t e d f r e q u e n c i e s differ considerably in s o m e cases, m o s t n o t a b l y for h a p l o t y p e s BBB a n d C A 3 A , w h i c h o c c u r m u c h m o r e o f t e n t h a n w o u l d be e x p e c t e d f r o m a r a n d o m c o m b i n a t i o n of t h e i r c o m p o n e n t s . T h e overall d i s t r i b u t i o n of n u m b e r s o f alleles differed f r o m e x p e c t a t i o n ( P < . 0 0 1 ) . A similar d i s t r i b u t i o n a n d significant d e v i a t i o n f r o m exp e c t a n c y was n o t e d for the sire sample.
Frequencies determined by genotype allocation.
bstandard deviations of frequency estimates, determlned by the formula, S.D. = [p(1-p)] .5/n, ranged from .0001 to .0031.
pected from random combination of contributing individual s y s t e m p h e n o t y p e s . C o d o m i n a n t gene f r e q u e n c i e s in male a n d f e m a l e samples were c o m p a r e d . All of t h e s e e x a m i n a t i o n s involved chi-square tests a n d analyses of cont i n g e n c y tables as o u t l i n e d by Steel a n d T o r r i e (22).
Comparative Variation of Polymorphic Systems
If o n e o b t a i n s individual allele f r e q u e n c i e s , squares t h e m , a n d sums t h e r e s u l t a n t squares, he derives a value w h i c h is t h e e s t i m a t e d h o m o z y g o s i t y at t h a t locus in t h e p o p u l a t i o n . Such a figure is useful in evaluating s o m e effects of b r e e d i n g p r a c t i c e s in a p o p u l a t i o n . A n u m b e r o f such values, r e p r e s e n t a t i o n s of variat i o n at d i f f e r e n t loci, also m a y provide o n e i n d e x for c o m p a r i s o n of genetic variability a m o n g breeds. T h e reciprocal of t h e h o m o z y g o s i t y value of Journal of Dairy Science Vol. 60, No. 7
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HINES ET AL.
TABLE 3. System B phenogroup frequencies (8416 animals typed). Allele code number
Phenogroup longhand designation
Frequencya, b
39 307 3 202 46 232 62 84 22 0 38 89 338 93 30 85 101 312 5 23 79 337 247 2 35 231 49 9 76 17 61 50 12 41 78 87 176 218 88 37 68 94 63 65 25 174 82
G2 Y2 E't Q' Q' BO 1 I" 12 O 1A ' I " OxE'a G" O3J'K'O' OxD'Et3 F'G'O t BO2 Y: A'E'3 G'P'Q 'Gt'I't b B2GY 1 D t I'Q' I t' BOa ¥2 D'I" OxA'O'ptl t' B: G a I~ Ett OxY 2 A t OxE' 3 GtG t' BO l B' BGI 10 a T~ A' OxY t E' a G ' Y ' G " Q'I . . E'a FtI'I '' BI t G2 O1 Y2 I" OxE'a F'G'OtG t' O 1 Y2 A' 2 I" BO 1D' OxY a At Y' BO2 A'Et2 G'P'Q'G" OxK' Ot YI E'a G'G't BG2 KOxE' 2 +-F ' O ' I " B2 GOt Y2 D'Eta I~ Y2 E'x Y' OxE'a BO 2 QAtp'Q'I '' OxQE' a Q'I" E' a G'ItG"I 't BO, Y21" PI t l~' OxO' O3 Y2 J'K'O t PI" BG~ KOxA'E' a OtG '' BG2 KOxY2 A'E'~ I'O'Gttl '' Y:
.204 .065 .063 .058 .052 .048 .041 .040 .035 .033 .026 .024 .021 .020 .020 .018 .018 .018 .016 .015 .014 .011 . .010 .010 .010 .010 .007 .006 .005 .004 .004 .004 .004 .004 .004 .004 .004 .003 .003 .003 .002 .002 .002 .002 .002 .002 .002
Allele code number
Phenogroup longhand designation
Frequency
31 103 24 295 133 233 112 1 99 276 92 97 331 10 132 330 44 90 29 04 121 334 . 161 156 230 246 120 100 45 107 102 901 164 108 34 42 306 157 205 249 250 288 309 326 333
B: G 1 O 1 BG 2 KOxA' BG 2 KOxY2 A'Otl t' B~GO 1 Y. D t O~ A'D'G ~ OxE' 3 Gt'O'I '' BO~ E' 3 G " I " BOxI" QEt't E' 3 I'Q' OxAtp t IOxQA' E t 1 Kt' O 1 I tt B(O 2 )A'Q' O 1 D'(G t) OxA' 11 QE t OxEt3 O' B 2 G. O' BI'Q~I'' 110 a J'K'O t BGOx Y2 E t ( A ) Y2 y, E 2 GI" OxEt3 FtO ' DtEt 3 BO 3 J' K'O t OxY 1 E' 3 G ' G " O ~Q' BG:~ KOxY~ E' 3 0 ' G " B2 Y2 Q' B BG2 KOxA'E'3 BaG l O 1 Y~ It I' B 2 GOxO' 12 D'G t BG 2 KY~ E',zO' PQE' 1 I' BG 2 KOxY~ DtO ' O 1Q EG~ KOxA'E'~ G ' G " I t' E' 10*G"
.002 .002 .002 .002 .002 .002 .001 .001 .0009 .0007 .0007 .0007 .0006 .0006 .0006 .0005 .0005 .0004 .0004 .0004 .0004 .0003 .0003 .0003 .0003 .0003 .0002 .0002 .0002 .0002 .0002 .0002 .0001 .0001 .0001 .0001 .0001 .0001 .0001 ,0001 .0001 .0001 .0001 .0001 .0001
Other phenogroups c
.0013
aFrequencies determined by genotype allocation. bstandard deviations of frequency estimates, determined by the formula, S.D. = [p(1--p)] .5/n, ranged from .0001 to .0031. CTwenty-two other phenogroups were observed once each (individual frequency = .00006). These groups and their code numbers were: BG 2 KOxE' 3 (13), BG~ K O x A ' B ' K ' O ' I " (19), BG 2 K O x Y 1 A ' B ' E ' 3 0 ' (26), BG 2 KO 2 t ~ t t t t s tt tF I i t s t t i t t t Y1ABE3GKOYG I (28) B2GD (33) G 2 0 1 E a (36), G 3 O t T E 3 F K ( 5 4 ) , O 3 Q J K O (58), O 3 Y 1 D J K•O , (64), B G 2 K O x E,z O , (116) , G 2 O I Y 2 D,E ~, Q , ( 1 3 9 ) , O ~ E t 3 ( 1 4 5 ) , B | 2 O x Q ' (172),12 Y 2E1' (204), Y 1 I" (239), YI Y' (240), Y~ E' 1 (243), BOxO'I" (293), BY~ A'E' 3 G'P' (299), Y~ E' a Y' (310), O2A'E' 3 G'G" (327), OxY 1 E' a G ' O ' G " (328). Journal of Dairy Science Vol. 60, No. 7
HOLSTEIN GENE FREQUENCIES
1149
TABLE 4. Casein haplotype frequencies (6094 animals completely casein typed)•
Haplotype code
Longhand designation %1-, t3-, K-Cn haplotype components
Observed frequency a,b
Expected frequency c
221 211 222 212 242 331 321 231 111 241 311 322 332 232 312 121 112
BA:A BAIA BA2B BAIB BBB CAaA CA2A BA3A AA1A BBA CAIA CA~B CAaB BA3B CA~B AA2A AAaB
.382 .346 .132 .068 .024 ,024 .012 .003 .002 .002 •002 .0008 .0007 .0004 •0003 .0003 .0002
.391 •310 .114 .091 •006 .001 •016 •020 .001 .020 .013 .0048 .0002 .0059 .0038 .0011 •0003
122 131 132 141 142 341 342
AA2B AAaA AAaB ABA ABB CBA CBB
a
.0003 .00006 .00002 .00006 .00002 .0008 .0002
•
Frequenmes determined by genotype allocation•
bstandard deviations of frequency estimates, determined by the formula, S.D. = [p(1-p)] .5/n, ranged from .0001 to .0044• CFrequency resulting from random combination of individual %1-, j3-, and K-casein components of the haplotype.
a locus expresses the a m o u n t of genetic variation d e t e c t e d at that locus in terms of the n u m b e r of effective alleles. It is a useful way of transforming f r e q u e n c y i n f o r m a t i o n into terms which are m o r e meaningful for certain comparisons. These values and the corresponding h o m o zygosity values for t h e loci are in Table 5. The casein systems have been handled in a combined fashion. With respect to the a m o u n t of detectable genetic variability, the B system is by far the m o s t useful, f o l l o w e d by the C and the casein c o m p l e x while at the o p p o s i t e end of the s p e c t r u m the M and R ' S ' systems have little variability in this breed. Other factors enter into most evaluations of the utility of i n f o r m a t i o n on gene frequency. C o d o m i n a n t systems are for a n u m b e r of
purposes d i s p r o p o r t i o n a t e l y useful because of the absolute c o r r e s p o n d e n c e between g e n o t y p e and p h e n o t y p e . Conversely, usefulness of the C system is depreciated by the lack of correspondence between g e n o t y p e and p h e n o t y p e , and certainly the usefulness of the milk systems is affected adversely by their sex-limited nature•
Genotype and Phenotype Distribution
One of the most surprising observations concerned the e x t e n t to which g e n o t y p e s and p h e n o t y p e s deviated f r o m e x p e c t e d numbers b o t h within and b e t w e e n systems. Of the six c o d o m i n a n t systems in the female sample, three exhibited highly significant deviations f r o m e x p e c t e d g e n o t y p e distributions (P<.001), one Journal of Dairy Science Vol. 60, No. 7
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HINES ET AL.
TABLE 5. Comparative variation of polymorphic systems. Homozygosity
No. of effective alleles
n
System B
.069
C c~#K-Cn Tf S J Lg A Z L FV M R'S'
.126 .289 .333 .336 .431 .501 .506 .567 .681 .780 .935 .952
14.5 7.9 3.5 3.0 3.0 2.3 2.0 2.0 1.8 1.5 1.3 1.1 1.0
aPi = frequency of the ith allele in the designated system.
deviated at .05 probability and the remaining two had chi-square values which fell within the 5 to 20% range. The sire sample was not examined similarly because of biases from pedigree supplementations. These procedures invariably favor supplementation of heterozygous classes. A comparison of allelic frequencies for the six codominant systems revealed moderate differences between the male and female samples. One system differed at .0I probability, and three differed at .05. Treated in a combined fashion, the differences were real (P<.O01). At least as striking as the deviation from expectations within system was the degree of intersystem nonrandom phenotype occurrence. In the female sample 14 of 45 combinations of paired systems exhibited highly significant nonrandom association of phenotypes (P<.001), and four more had deviations differing from expectancy at the .01 probability. In contrast to this the sire sample had only four paired systems which deviated at .001 probability and an additional one at .01. Furthermore, three of the highly significant deviations in the sire sample were associated with known linkage groups and were most likely a result of linkage disequilibrium in the sample. The fourth example, involving FV and M system associations, Journal of Dairy Science Vol. 60, No. 7
is probably not a linkage phenomenon since close or even moderate linkage of the loci has been excluded (7, 8). Neither does it seem probable that the excess of M positive, V positive animals can be explained adequately by disproportionate representation of a few M positive, V positive sires of the tested bulls. A good explanation is not offered, but further study is urged. The extensive interaction of paired systems in the female sample is probably primarily a result of nonrandom breeding and extremely variable representation of sires in progeny numbers. A few sires with large numbers of progeny easily could have forced associations. It may be argued that the sample is not truly reflective of breed structure, particularly as a result of geographical and institutional bias. While it is true that areas of the United States were not represented in the sample, the geographical base was fairly broad, including regions of the midwest (Ohio and Minnesota) and west (Utah) in addition to the northeast. Similarly, while institutional herds formed a larger part of the sample than of the entire breed and by virtue of their different objectives probably followed differing breeding practices those of private herds, similar results were reported by Zikakis et al. (28) in predominantly private herds. Therefore, while this sample may not be strictly representative of the general population, it is typical enough to support the conclusion that breed structure is today considerably different from that associated with expectations of random breeding. That there are in the sample a few sires with large numbers of daughters is relatively typical of the general situation. Nevertheless, the detected polymorphic differences indicate extensive genetic variability. However, it would be wise to monitor genetic changes as they may be revealed by changes at genetic marker loci. Such monitoring would be easy with the blood typing programs by breed organizations. ACKNOWLEDGMENTS
We gratefully acknowledge the assistance provided by the Holstein-Friesian Association of America in supplying sires' blood types. Recognition also is given to the Serology Laboratory, University of California, Davis, for much of the blood typing of sires.
HOLSTEIN GENE FREQUENCIES REFERENCES
1 Aschaffenburg, R. 1966. Modified procedure of starch gel electrophoresis for 3-casein phenotyping. J. Dairy Sci. 49:1284. 2 Aschaffenburg, R. 1968. Reviews on the progress of dairy science. Section G. Genetics. Genetic variants of milk proteins: their breed distribution. J. Dairy Res. 35:447. 3 Aschaffenburg, R., and M. T h y m a n n . 1965. Simult a n e o u s p h e n o t y p i n g procedure for t h e principal proteins of cow's milk. J. Dairy Sci. 4 8 : 1 5 2 4 . 4 Bailey, L. F., and C. A. Kiddy. 1972. Resolution of cattle transferrin in starch-gel at pH 6.3. Anim. Blood Grps. Biochern. Genet. 3:245. 5 Ferguson, L. C. 1941. Heritable antigens in the erythrocytes o f cattle. J. Immunol. 40:213. 6 Grosclaude, F., J. Pujolle, J. Garnier, and B. Ribadeau-Dumas. 1965. Determinisme genetique des caseines k du lait de vache; etroite laison du locus ~-Cn avec tes loci %-Cn et 3-Cn. Compt. Rend. Acad. Sci. (Paris) 261 : 5229. 7 Hines, H. C., G. F. W. Haenlein, J. P. Zikakis, and C. A. Kiddy. 1976. Linkage relationships a m o n g bovine and milk polymorphisms. Program 71st Ann. Meeting Amer. Dairy Sci. Ass., p. 132. (Abstr.) 8 Hines, H. C., C. A. Kiddy, E. W. Bruin, and C. W. Arave. 1969. Linkage a m o n g cattle blood and milk polymorphisrns. Genetics 62:401. 9 Jamieson, A. 1965. The genetics of transferrin in cattle. Heredity 20:419. 10 Kiddy, C. A. 1964. Inherited differences in specific blood and milk proteins in cattle. A review. J. Dairy Sci. 47: 510. 11 Kiddy, C. A. 1975. Gel electrophoresis in vertical polyacrylamide beds. Procedure I, II, and Ill, pp. 14-19. In Methods of Gel Electrophoresis of Milk Proteins. H. Swaisgood, ed. 12 Kiddy, C. A., and N. W. Hooven, Jr. 1961. Results of repeatability and standardization tests in cattle blood typing. A R S 44-94. 13 Kiddy, C. A., R. E. Rollins, and J. P. Zikakis. 1972. Discontinuous polyacrylamide electrophoresis for 34actoglobulin typing of cow's milk. J. Dairy Sci. 55:1506. 14 Kristjansson, F. K., and C. G. Hickman. 1965. Subdivision of the allele Tf D for transferrins in
1151
Holstein and Ayrshire cattle. Genetics 52:627. 15 Kurczynski, T. W., and A. G. Steinberg. 1967. A general program for m a x i m u m likelihood estimation of gene frequencies. Amer. J. H u m a n Genet. 19:178. 16 Larsen, B. 1966. Test for linkage of the genes controlling haemoglobin, transferrin, and blood types in cattle. Yearb., 1966, Royal Vet and Agr. Coil., Copenhagen, Denmark, 41. 17 Larsen, B. 1970. Linkage relations of blood group and polymorphic protein systems in cattle. Aarsberem. Inst. Sterilitetsforsk, p. 165. 18 Larsen, B., and M. T h y m a n n . 1966. Studies on milk protein p o l y m o r p h i s m in Danish cattle and the interaction of the controlling genes. Acta Vet. Scand. 7:189. 19 Neimann-Sorensen, A. 1956. Blood groups and breed structure as exemplified by three Danish breeds. Acta Agr. Scand. 6:115. 20 Peterson, R. F., and F. C. Kopfler. 1966. Detection of new types of 3 casein by polyacrylamide gel electrophoresis at acid pH: A proposed n o m e n clature. Biochem. Biophys. Res. C o m m . 22: 388. 21 Rausch, W. H., T. M. Ludwick, and D. F. Weseli. 1 965. Determination o f bovine transferrin types by disc electrophoresis. J. Dairy Sci. 48:720. 22 Steel, R. G. D., and T. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill Book Company, Inc., New York. 23 Stormont, C. 1962. Current status o f blood groups in cattle. Ann. N. Y. Acad. Sci. 97:251. 24 Thatcher, W. W., and C. A. Kiddy. 1965. Associations a m o n g blood and milk p o l y m o r p h i s m s in dairy cattle. J. Dairy Sci. 48:1558. (Abstr.) 25 T h o m p s o n , M. P., C. A. Kiddy, J. O. J o h n s t o n , and R. M. Weinberg. 1964. Genetic p o l y m o r p h i s m in caseins of cow's milk. II. Confirmation of the genetic control of B-casein variation. J. Dairy Sci. 47:378. 26 Wake, R. G., and R. L. Baldwin. 1961. Analysis of casein fractions by zone electrophoresis in concentrated urea. Biochim. Biophys. Acta 47:225. 27 Woychik, J. H. 1964. Polymorphism in K-casein o f cow's milk. Biochem. Biophys. Res. C o m m . 16:267.
28 Zikakis, J. P., G. F. W. Haenlein, H. C. Hines, R. E. Mather, and S. Tung. 1974. Gene frequencies of electrophoretically determined p o l y m o r p h i s m s in Guernsey blood a n d milk. J. Dairy Sci. 57:405.
Journal of Dairy Science Vol. 60, No. 7
Gene frequencies at ten blood group loci, one serum protein locus, and four milk protein loci were determined for the Holstein-Friesian breed in the United States. The sample consisted of 8630 cows in 51 herds from 10 states. Because of the close linkage among casein subloci and the concomitant rarity of crossoverrecombinant groups, casein gene combination or haplotype frequencies were determined also. As one means of comparison of systems, indices of homozygosity and number of effective alleles were calculated. These indices were proposed also to be useful tools for monitoring changes in genetic variability of breeds. Genotypes within codominant systems and phenotypes associated with paired-system combinations generally were not randomly occurring. Paired system phenotypes within the sire sample corresponded much more closely to expectations of randomness. INTRODUCTION
Frequencies of genes and genotypes are useful in establishing breed structures and
Received December 2, 1976. 1Data from the NE-62 Regional Research Project, Relationship Between Genetic Markers and Performance in Dairy Cattle. Members of the NE-62 Technical Committee: C. W. Arave, UT; H. C. Dickey, ME; S. N.
Gaunt, MA; G. F. W. Haenlein, DE; G. L. Hargrove, PA; H. C. Hines, OH; A. C. Hunter, MN; C. A. Kiddy, ARS-USDA; R. E. Mather, NJ; P. R. Shellenberger, PA; G. W. Trimberger, NY; C. W. Young, MN; J. P. Zikakis, DE.
interrelationships. As additional genetic marker loci are discovered in cattle, it becomes easier to infer about the development and relationships of breeds and to establish benchmarks from which subsequent changes in breed structure can be measured. The information reported here differs from that of previous studies by being more comprehensive in two respects: 1) it results from the typing of more than 8,000 Holstein-Friesian cattle, and 2) it consists of simultaneously determined genotypes at 15 loci. Thus, it probably provides the best basis to date for thorough assessment of several aspects of the genetic composition of the Holstein breed in particular and the bovine species in general. The 15 loci consisted of 10 red cell antigen loci (bloodgroups A, B, C, FV, J, L, M, S, Z, and R'S'), serum transferrin (Tf), and milk proteins beta-lactoglohulin (Lg), alphasl-casein (%l-Cn), beta-casein (/3-Cn), and kappa-casein (K-Cn). Many of the sires also were typed for hemoglobin (Hb), but no variation was detected. The blood group systems have been reviewed by Stormont (23), the hemoglobin and transferrin systems by Kiddy (10), and the milk protein systems by Aschaffenburg (2). Subsequent to the transferrin review, additional genetic variation at this locus was detected by improved electrophoretic techniques (14). Three linkage groups have been detected among the loci; otherwise the loci appear to be independent. Linkage groups are: a) the Hb and A blood group loci (7, 16), b) the Lg and J blood group loci (8, 17, 24), and c) the %1-,/3-, and •-Cn loci (6, 8, 18). Larsen and T h y m a n n (18) consider the casein linkage so close as to be virtually a single locus. In this respect it may behave as a kind of superlocus and as such m a y bear similarities to the more complex cattle
1143
1144
HINES ET AL.
blood group systems. As for these latter systems, it also may be desirable to represent the combined casein locus variants as phenogroups or haplotypes. This investigation formed an integral part of the Northeast Regional Research Project NE62, Relationships between genetic markers and performance in dairy cattle. The design of the project specified that a large and representative sample of cattle from a single breed simultaneously would be typed for genetic markers and evaluated for performance traits. The project provided for the development of the following objectives for this investigation: 1) determination of frequencies for the genetic marker systems which would be representative of frequencies in the Holstein-Friesian breed in the United States, 2) determination of comparative measures of variation at different genetic marker loci, 3) ascertainment of intra- and interlocus relationships within the sample as exemplified by Hardy-Weinberg equilibrium and inter-locus phenotype independence, and 4) construction of Holstein casein haplotypes and calculation of their frequencies within the breed.
red ceils, and .025 ml of normal rabbit serum (complement source) which was added after a 5-min red cell-reagent incubation period. The plates were covered with a plastic adhesive, shaken, and incubated at 30 C. Except for these modifications, the test was essentially the same as that described by Ferguson (5). In projects encompassing several years, changes in typing commonly occur, particularly with a battery of biological reagents in finite supply. The primary changes of this type were those associated with additions to the reagent bank over time. Reagents in this category included anti-A1, which enabled subdivision of the A system factor A into A1 and A2 subtypes; anti-U'2, which enabled us to differentiate the S system L?2 factor from the silent allele product (designated s); and anti-R' and anti-S', which allowed determination of genotypes at a separate new locus (R'S'). Most of the bulls siring females in this study were typed by the Serology Laboratory of the University of California (Davis). Typing techniques were similar to those of the Ohio laboratory. The participation of both laboratories in biennial comparison-standardization tests helped to standardize results (12).
EXPERIMENTAL PROCEDURE Source of Samples
Blood and milk samples were obtained from 8630 Holstein cows and heifers from 51 herds in 10 states: Delaware, Maine, Maryland (USDA), Massachusetts, Minnesota, New Jersey, New York, Ohio, Pennsylvania, and Utah. Private and institutional herds were represented. The animals typed were from 3 mo to 3 yr of age. Samples were collected from 1964 to 1974. Blood Group Typing
All typing of blood groups on cows and heifers was by the Cattle Blood Typing Laboratory of The Ohio State University. Blood samples were collected in isotonic sodium citrate, cooled to 4 C, and shipped to the Ohio laboratory for typing by hemolytic test procedures. The typing was in disposable plastic microtiter plates (Cooke Engineering Co., Alexandria, VA). Each test well contained .05 ml of specific hemolytic reagent (monospecific antiserum), .025 ml of a 2.5% suspension of washed Journal of Dairy Science Vo|. 60, No. 7
Elect rophoret ic Typing
Serum transferrin typing was by the Minnesota, Ohio, USDA, and Utah stations. The typing procedures were those described by Bailey and Kiddy (4), Jamieson (9), Kristjansson and Hickman (14), and Rausch et al. (21). In the early years of the project, the typing was for the undifferentiated D allele, but later TfD1 and D z were subtyped. Additionally, some of the samples typed earlier and still in frozen storage were retyped for DI and D2. Electrophoretic typing of milk protein was at Delaware, Massachusetts, Minnesota, Ohio, Pennsylvania, USDA, and Utah laboratories. Beta-lactoglobulin was typed according to the procedures described by Aschaffenburg and Thymann (3), Kiddy et al. (15), and Wake and Baldwin (26). Alphasl-casein and K-casein variants were typed by the methods outlined by Aschaffenburg and Thymann (3), Kiddy (11), Thompson et al. (25), and Woychik (27). The procedures for fi-Cn typing have been described by Aschaffenburg (1), Kiddy (11), and Peterson and Kopfler (20).
HOLSTEIN GENE FREQUENCIES
1145
TABLE 1. Genetic marker codominant and simple dominant system gene frequencies. System
Allele
Frequencya,b
A
.239
A
No. typed
8452
~
1
AH
.049 2H
D DH
.675 .037
F V
.874 .126
j js jcs
.589 .205 .206
1 L
.800 .200
m M2M'
.966 .034
s SH' UH' U' 1 U' 2 H'
.323 .114 .061 .026 .013 .463
z Z
.683 .317
R' S'
.024 .976
A D
.431 .540
.215 .024
1094-q 1094]
o45
109
.004
1094 I
8464
FV
8426
8086
M
8399
639 8468
1144
R'S'
8068
Tf
.249 .291 E
.029
A B
.526 .474
A B C
.003 .957 .040
A1 A2 A3 B
.415 .532 .028 .025
427~ 427~ 6465
Lg
6874
~sl-Cn
6575
13-Cn
Journal of Dairy Science Vol. 60, No. 7
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HINES ET AL.
TABLE 1. Continued. System
Allele
Frequencya,b
A B
.800 .200
•-Cn
No. typed 6531
aFrequencies for the A, J, and S systems were determined by the maximum likelihood method. Frequencies for the FV, Tf, Lg, ~slq2n, 3-Cn, and ~-Cn systems were determined by gene counting. Frequencies for the L, M, Z, and R'S' systems were determined by the square root method. bstandard deviations of frequency estimates, determined by the formula, S.D. = [p(1--p)] -5/n, ranged from .0004 to .0049. Although a variety of electrophoretic methods was utilized by the cooperating laboratories, standardization of results was verified by periodic comparisons. Similarly, although many of the bulls were typed electrophoretically by the California Serology Laboratory, standardization of results was corroborated likewise by regular participation in international comparison-standardization tests. Gene Frequency Determination
Gene frequencies were determined from blood and milk types of the 8630 females. Seven-hundred-seventy of the 900 sires of these females were bloodtyped, and these formed a sample which also was subjected to analyses of gene frequency and genetic interrelationship. Milk protein types of sires were determined by inference from their daughters' types. The results of the sire sample determinations are not reported in detail but only as they provide information related to aspects of the female sample findings. Four methods of calculation of gene frequency were utilized in computing of these estimates. Frequencies of alleles in codominant systems (FV, Tf, Lg, C~sl-Cn, /3-Cn, and K-Cn), where genotypes always can be determined from phenotypes, were calculated by gene counting. Simple dominant systems containing two alleles (L, M, and Z) were analyzed by extracting the square root of the frequency of the homozygous recessive genotype. Although the RtS ' system is codominant, the absence of anti-S' reagents from the typing battery of the Ohio laboratory for most of the typing period precluded analysis for codominant system. Instead, the system also was analyzed by the square root method as a simple dominant locus. Journal of Dairy Science Vol. 60, No. '7
More complex dominant systems with from three to ten alleles (A, J and S) were analyzed by a maximum likelihood method described and programmed for computer by Kurczynski and Steinberg (15). Complex genetic systems of more than ten alleles (B and C) were analyzed by a genotype allocation procedure essentially as described by Neimann-Sorensen (19) and by computer program developed at The Ohio State University. For cases in which improved typing techniques became available during the project resulting in subsequent recognition of additional alleles, the frequencies for such have been demonstrated only over that portion of the sample for which they were typed. Thus, their frequencies are neither biased nor do they bias other estimations; however, because of fewer numbers their frequencies are estimated with less precision than for alleles typed for the entire sample. Because of the apparently close linkage among the ~sl-, /3-, and K-casein loci and the concomitant rarity of crossover-recombinant groups, casein gene combination or haplotype frequencies also were determined. In the female sample, haplotypes were determined, when possible, directly from phenotypes, secondarily from pedigree information, and finally, as necessary, by allocation methods (19) to complete the frequency estimation. In the sire sample, haplotypes were determined solely from progeny information. Genetic Interrelationships
Genotype distribution within codominant systems was examined for Hardy-Weinberg equilibrium. Frequencies of paired system phenotypes were compared with those ex-
HOLSTEIN GENE FREQUENCIES TABLE 2. System C phenogroup frequencies (8219 animals typed).
1 147
RESULTS AND DISCUSSION Gene Frequencies
Allele code number
o 32 2 22 33 27 31 1 21 23 29 15 26 13 3 30 16 14 25 11 54 50 34 45 9 52 5 10 8 4 20 28 7 6 19 39
Phenogroup longhand designation
c
X2 CtE E X 2 L'
W Xt Cl CzW EW WX:~ C2 ER RWX 2 C2 C 1 EW WX 2 L' C 2 EW C2E EL' C1 X1 WL' L' C X2
CW C EL' X L' C ER C R C EX 1 C EWX 2 C R WX1 C 1EWX 1 Ct RW C 2 ERX 2 C2 EWX 2
a
Frequencya,b
.205 .168 .153 .134 .073 .065 .048 .032 .029 .019 .015 .010 .010 .010 .008 .005 .004 .003 .003 .001 .001 .0007 .0007 .0005 .0004 .0004 .0002 .0002 .0002 .00012 .00012 .00012 .00007 .00006 .00006 .00006
G e n e f r e q u e n c i e s in t h e c o d o m i n a n t a n d simple d o m i n a n t s y s t e m s are in T a b l e 1. Information on a portion of the sample pertinent to t h e alleic subdivision w h i c h r e s u l t e d f r o m improved typing techniques during the project is b r a c k e t e d . T h e r e d u c e d n u m b e r o f a n i m a l s so s u b t y p e d as well as t h e smaller n u m b e r t y p e d for U'e is i n d i c a t e d . In all o t h e r cases t h e n u m b e r of a n i m a l s t y p e d for a particular s y s t e m is a p p l i c a b l e to all of t h e alleles in t h a t system. P h e n o g r o u p (allele) f r e q u e n c i e s for t h e m o r e c o m p l e x C a n d B s y s t e m s are in T a b l e s 2 a n d 3. T h e B allele G 2 Y 2 E ' I Q ' o c c u r r e d m u c h m o r e frequently than any other B phenogroup. At t h e o p p o s i t e e x t r e m e , t h e 22 alleles t h a t were d e t e c t e d each o n l y o n c e m a y b e a t y p i c a l o f t h e Holstein breed. T h e i r d e t e c t i o n , as well as t h e d e t e c t i o n of a n y o t h e r alleles n o t specifically listed in T a b l e 3, in a b r e e d p u r i t y analysis o f a r e p o r t e d l y p u r e b r e d Holstein a n i m a l raises suspicion of foreign b l o o d a n d s h o u l d be f o l l o w e d b y e x h a u s t i v e pedigree checking. Casein Haplotypes
T h e d e t e c t e d casein h a p l o t y p e s a n d t h e i r c o r r e s p o n d i n g f r e q u e n c i e s are in T a b l e 4. T h e o b s e r v e d a n d e x p e c t e d f r e q u e n c i e s differ considerably in s o m e cases, m o s t n o t a b l y for h a p l o t y p e s BBB a n d C A 3 A , w h i c h o c c u r m u c h m o r e o f t e n t h a n w o u l d be e x p e c t e d f r o m a r a n d o m c o m b i n a t i o n of t h e i r c o m p o n e n t s . T h e overall d i s t r i b u t i o n of n u m b e r s o f alleles differed f r o m e x p e c t a t i o n ( P < . 0 0 1 ) . A similar d i s t r i b u t i o n a n d significant d e v i a t i o n f r o m exp e c t a n c y was n o t e d for the sire sample.
Frequencies determined by genotype allocation.
bstandard deviations of frequency estimates, determlned by the formula, S.D. = [p(1-p)] .5/n, ranged from .0001 to .0031.
pected from random combination of contributing individual s y s t e m p h e n o t y p e s . C o d o m i n a n t gene f r e q u e n c i e s in male a n d f e m a l e samples were c o m p a r e d . All of t h e s e e x a m i n a t i o n s involved chi-square tests a n d analyses of cont i n g e n c y tables as o u t l i n e d by Steel a n d T o r r i e (22).
Comparative Variation of Polymorphic Systems
If o n e o b t a i n s individual allele f r e q u e n c i e s , squares t h e m , a n d sums t h e r e s u l t a n t squares, he derives a value w h i c h is t h e e s t i m a t e d h o m o z y g o s i t y at t h a t locus in t h e p o p u l a t i o n . Such a figure is useful in evaluating s o m e effects of b r e e d i n g p r a c t i c e s in a p o p u l a t i o n . A n u m b e r o f such values, r e p r e s e n t a t i o n s of variat i o n at d i f f e r e n t loci, also m a y provide o n e i n d e x for c o m p a r i s o n of genetic variability a m o n g breeds. T h e reciprocal of t h e h o m o z y g o s i t y value of Journal of Dairy Science Vol. 60, No. 7
1148
HINES ET AL.
TABLE 3. System B phenogroup frequencies (8416 animals typed). Allele code number
Phenogroup longhand designation
Frequencya, b
39 307 3 202 46 232 62 84 22 0 38 89 338 93 30 85 101 312 5 23 79 337 247 2 35 231 49 9 76 17 61 50 12 41 78 87 176 218 88 37 68 94 63 65 25 174 82
G2 Y2 E't Q' Q' BO 1 I" 12 O 1A ' I " OxE'a G" O3J'K'O' OxD'Et3 F'G'O t BO2 Y: A'E'3 G'P'Q 'Gt'I't b B2GY 1 D t I'Q' I t' BOa ¥2 D'I" OxA'O'ptl t' B: G a I~ Ett OxY 2 A t OxE' 3 GtG t' BO l B' BGI 10 a T~ A' OxY t E' a G ' Y ' G " Q'I . . E'a FtI'I '' BI t G2 O1 Y2 I" OxE'a F'G'OtG t' O 1 Y2 A' 2 I" BO 1D' OxY a At Y' BO2 A'Et2 G'P'Q'G" OxK' Ot YI E'a G'G't BG2 KOxE' 2 +-F ' O ' I " B2 GOt Y2 D'Eta I~ Y2 E'x Y' OxE'a BO 2 QAtp'Q'I '' OxQE' a Q'I" E' a G'ItG"I 't BO, Y21" PI t l~' OxO' O3 Y2 J'K'O t PI" BG~ KOxA'E' a OtG '' BG2 KOxY2 A'E'~ I'O'Gttl '' Y:
.204 .065 .063 .058 .052 .048 .041 .040 .035 .033 .026 .024 .021 .020 .020 .018 .018 .018 .016 .015 .014 .011 . .010 .010 .010 .010 .007 .006 .005 .004 .004 .004 .004 .004 .004 .004 .004 .003 .003 .003 .002 .002 .002 .002 .002 .002 .002
Allele code number
Phenogroup longhand designation
Frequency
31 103 24 295 133 233 112 1 99 276 92 97 331 10 132 330 44 90 29 04 121 334 . 161 156 230 246 120 100 45 107 102 901 164 108 34 42 306 157 205 249 250 288 309 326 333
B: G 1 O 1 BG 2 KOxA' BG 2 KOxY2 A'Otl t' B~GO 1 Y. D t O~ A'D'G ~ OxE' 3 Gt'O'I '' BO~ E' 3 G " I " BOxI" QEt't E' 3 I'Q' OxAtp t IOxQA' E t 1 Kt' O 1 I tt B(O 2 )A'Q' O 1 D'(G t) OxA' 11 QE t OxEt3 O' B 2 G. O' BI'Q~I'' 110 a J'K'O t BGOx Y2 E t ( A ) Y2 y, E 2 GI" OxEt3 FtO ' DtEt 3 BO 3 J' K'O t OxY 1 E' 3 G ' G " O ~Q' BG:~ KOxY~ E' 3 0 ' G " B2 Y2 Q' B BG2 KOxA'E'3 BaG l O 1 Y~ It I' B 2 GOxO' 12 D'G t BG 2 KY~ E',zO' PQE' 1 I' BG 2 KOxY~ DtO ' O 1Q EG~ KOxA'E'~ G ' G " I t' E' 10*G"
.002 .002 .002 .002 .002 .002 .001 .001 .0009 .0007 .0007 .0007 .0006 .0006 .0006 .0005 .0005 .0004 .0004 .0004 .0004 .0003 .0003 .0003 .0003 .0003 .0002 .0002 .0002 .0002 .0002 .0002 .0001 .0001 .0001 .0001 .0001 .0001 .0001 ,0001 .0001 .0001 .0001 .0001 .0001
Other phenogroups c
.0013
aFrequencies determined by genotype allocation. bstandard deviations of frequency estimates, determined by the formula, S.D. = [p(1--p)] .5/n, ranged from .0001 to .0031. CTwenty-two other phenogroups were observed once each (individual frequency = .00006). These groups and their code numbers were: BG 2 KOxE' 3 (13), BG~ K O x A ' B ' K ' O ' I " (19), BG 2 K O x Y 1 A ' B ' E ' 3 0 ' (26), BG 2 KO 2 t ~ t t t t s tt tF I i t s t t i t t t Y1ABE3GKOYG I (28) B2GD (33) G 2 0 1 E a (36), G 3 O t T E 3 F K ( 5 4 ) , O 3 Q J K O (58), O 3 Y 1 D J K•O , (64), B G 2 K O x E,z O , (116) , G 2 O I Y 2 D,E ~, Q , ( 1 3 9 ) , O ~ E t 3 ( 1 4 5 ) , B | 2 O x Q ' (172),12 Y 2E1' (204), Y 1 I" (239), YI Y' (240), Y~ E' 1 (243), BOxO'I" (293), BY~ A'E' 3 G'P' (299), Y~ E' a Y' (310), O2A'E' 3 G'G" (327), OxY 1 E' a G ' O ' G " (328). Journal of Dairy Science Vol. 60, No. 7
HOLSTEIN GENE FREQUENCIES
1149
TABLE 4. Casein haplotype frequencies (6094 animals completely casein typed)•
Haplotype code
Longhand designation %1-, t3-, K-Cn haplotype components
Observed frequency a,b
Expected frequency c
221 211 222 212 242 331 321 231 111 241 311 322 332 232 312 121 112
BA:A BAIA BA2B BAIB BBB CAaA CA2A BA3A AA1A BBA CAIA CA~B CAaB BA3B CA~B AA2A AAaB
.382 .346 .132 .068 .024 ,024 .012 .003 .002 .002 •002 .0008 .0007 .0004 •0003 .0003 .0002
.391 •310 .114 .091 •006 .001 •016 •020 .001 .020 .013 .0048 .0002 .0059 .0038 .0011 •0003
122 131 132 141 142 341 342
AA2B AAaA AAaB ABA ABB CBA CBB
a
.0003 .00006 .00002 .00006 .00002 .0008 .0002
•
Frequenmes determined by genotype allocation•
bstandard deviations of frequency estimates, determined by the formula, S.D. = [p(1-p)] .5/n, ranged from .0001 to .0044• CFrequency resulting from random combination of individual %1-, j3-, and K-casein components of the haplotype.
a locus expresses the a m o u n t of genetic variation d e t e c t e d at that locus in terms of the n u m b e r of effective alleles. It is a useful way of transforming f r e q u e n c y i n f o r m a t i o n into terms which are m o r e meaningful for certain comparisons. These values and the corresponding h o m o zygosity values for t h e loci are in Table 5. The casein systems have been handled in a combined fashion. With respect to the a m o u n t of detectable genetic variability, the B system is by far the m o s t useful, f o l l o w e d by the C and the casein c o m p l e x while at the o p p o s i t e end of the s p e c t r u m the M and R ' S ' systems have little variability in this breed. Other factors enter into most evaluations of the utility of i n f o r m a t i o n on gene frequency. C o d o m i n a n t systems are for a n u m b e r of
purposes d i s p r o p o r t i o n a t e l y useful because of the absolute c o r r e s p o n d e n c e between g e n o t y p e and p h e n o t y p e . Conversely, usefulness of the C system is depreciated by the lack of correspondence between g e n o t y p e and p h e n o t y p e , and certainly the usefulness of the milk systems is affected adversely by their sex-limited nature•
Genotype and Phenotype Distribution
One of the most surprising observations concerned the e x t e n t to which g e n o t y p e s and p h e n o t y p e s deviated f r o m e x p e c t e d numbers b o t h within and b e t w e e n systems. Of the six c o d o m i n a n t systems in the female sample, three exhibited highly significant deviations f r o m e x p e c t e d g e n o t y p e distributions (P<.001), one Journal of Dairy Science Vol. 60, No. 7
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HINES ET AL.
TABLE 5. Comparative variation of polymorphic systems. Homozygosity
No. of effective alleles
n
System B
.069
C c~#K-Cn Tf S J Lg A Z L FV M R'S'
.126 .289 .333 .336 .431 .501 .506 .567 .681 .780 .935 .952
14.5 7.9 3.5 3.0 3.0 2.3 2.0 2.0 1.8 1.5 1.3 1.1 1.0
aPi = frequency of the ith allele in the designated system.
deviated at .05 probability and the remaining two had chi-square values which fell within the 5 to 20% range. The sire sample was not examined similarly because of biases from pedigree supplementations. These procedures invariably favor supplementation of heterozygous classes. A comparison of allelic frequencies for the six codominant systems revealed moderate differences between the male and female samples. One system differed at .0I probability, and three differed at .05. Treated in a combined fashion, the differences were real (P<.O01). At least as striking as the deviation from expectations within system was the degree of intersystem nonrandom phenotype occurrence. In the female sample 14 of 45 combinations of paired systems exhibited highly significant nonrandom association of phenotypes (P<.001), and four more had deviations differing from expectancy at the .01 probability. In contrast to this the sire sample had only four paired systems which deviated at .001 probability and an additional one at .01. Furthermore, three of the highly significant deviations in the sire sample were associated with known linkage groups and were most likely a result of linkage disequilibrium in the sample. The fourth example, involving FV and M system associations, Journal of Dairy Science Vol. 60, No. 7
is probably not a linkage phenomenon since close or even moderate linkage of the loci has been excluded (7, 8). Neither does it seem probable that the excess of M positive, V positive animals can be explained adequately by disproportionate representation of a few M positive, V positive sires of the tested bulls. A good explanation is not offered, but further study is urged. The extensive interaction of paired systems in the female sample is probably primarily a result of nonrandom breeding and extremely variable representation of sires in progeny numbers. A few sires with large numbers of progeny easily could have forced associations. It may be argued that the sample is not truly reflective of breed structure, particularly as a result of geographical and institutional bias. While it is true that areas of the United States were not represented in the sample, the geographical base was fairly broad, including regions of the midwest (Ohio and Minnesota) and west (Utah) in addition to the northeast. Similarly, while institutional herds formed a larger part of the sample than of the entire breed and by virtue of their different objectives probably followed differing breeding practices those of private herds, similar results were reported by Zikakis et al. (28) in predominantly private herds. Therefore, while this sample may not be strictly representative of the general population, it is typical enough to support the conclusion that breed structure is today considerably different from that associated with expectations of random breeding. That there are in the sample a few sires with large numbers of daughters is relatively typical of the general situation. Nevertheless, the detected polymorphic differences indicate extensive genetic variability. However, it would be wise to monitor genetic changes as they may be revealed by changes at genetic marker loci. Such monitoring would be easy with the blood typing programs by breed organizations. ACKNOWLEDGMENTS
We gratefully acknowledge the assistance provided by the Holstein-Friesian Association of America in supplying sires' blood types. Recognition also is given to the Serology Laboratory, University of California, Davis, for much of the blood typing of sires.
HOLSTEIN GENE FREQUENCIES REFERENCES
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