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Allele frequencies for SNPs in the αS1-casein gene (CSN1S1) 5′ flanking region in European cattle and association with economic traits in German Holstein
Livestock Production Science 98 (2005) 155 – 160 www.elsevier.com/locate/livprodsci
Allele frequencies for SNPs in the aS1-casein gene (CSN1S1) 5V flanking region in European cattle and association with economic traits in German Holstein Eva-Maria Prinzenberg a,*, Horst Brandt a, Joern Bennewitz b, Ernst Kalm b, Georg Erhardt a b
a Institute for Animal Breeding and Genetics, Justus-Liebig-University, Ludwigstr. 21B, 35390 Giessen, Germany Institute for Animal Breeding and Husbandry, Christian-Albrechts-University, Olshausenstr. 40, 24098 Kiel, Germany
Abstract Allele frequencies of a polymorphism in the a S1 -casein 5V flanking region (CSN1S1-5V) were determined in 14 Central and Eastern European cattle breeds by single strand conformation polymorphism analysis. Allele frequencies ranged from 0.00 to 0.35 (allele 1), 0.08 to 0.91 (allele 2), 0.04 to 0.41 (allele 3) and from 0.00 to 0.16 (allele 4). Allele 5 was not found. Presence of allele 2 in all breeds highlights its probable wild type status. A loss of alleles and genotypes occurred in some breeds, but differentiation within the breed groups milk, beef and dual purpose was higher than between these groups. An analysis of the variance in a German Holstein granddaughter design with family and CSN1S1-5V genotype as fixed effects revealed significant effects ( P b 0.05 each) of the CSN1S1-5V genotype on breeding values for dairy character, fore udder attachment, length of productive life and highly significant effects on somatic cell score ( P b 0.001). Genotype LS-means assigned favourable values for udder health and longevity to genotype 24. Past selection for milk yield or beef production does not seem to have favoured indirectly specific promoter alleles. No negative effects of genotype 24 were evident in Holstein, but selection for 23 requires careful inspection of unfavourable side effects. D 2005 Elsevier B.V. All rights reserved. Keywords: Alpha-S1-casein; Promoter polymorphism; Allele frequency; Economic traits
1. Introduction A four allele polymorphism in the bovine a S1 casein gene promoter (CSN1S1-5V) has previously * Corresponding author. Tel.: +49 641 9937626; fax: +49 641 9937629. E-mail address: [email protected] (E.-M. Prinzenberg). 0301-6226/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.livprodsci.2005.10.015
been used for linkage mapping of the gene and analysis of effects on milk production traits in German Holsteins. This study indicated significant and for genotype 24 positive effects of CSN1S1-5V on protein content and possibly linked effects on yield traits in a granddaughter design. The effect on milk protein content was hypothesized to be a direct effect of polymorphisms in regulatory elements at the 5V end of
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the casein locus (Prinzenberg et al., 2003). This makes CSN1S1-5V an interesting candidate for marker or gene assisted selection programmes. A fifth allele was recently reported in West African Bos indicus cattle (Ibeagha-Awemu et al., 2004). QTL studies identified several putative or significant QTL within a region spanning about 60 cM on BTA 6. These include two major regions affecting milk production traits (Khatkar et al., 2004), but also other traits contributing to an economical production as milking speed, size characteristics (Schrooten et al., 2000; Boichard et al., 2003; Hiendleder et al., 2003), stillbirth/direct effect (Ku¨hn et al., 2003), foot angle, general quality of feet and legs and udder conformation (Hiendleder et al., 2003). Especially the last study, based on the same half-sib families as ours, identified a QTL cluster for 8 body conformation traits at positions 85–89 cM on BTA 6, close to the map position of CSN1S1-5V at 93.9 cM (Prinzenberg et al., 2003). Klungland et al. (2001) reported a QTL for clinical mastitis on BTA 6, which is located around position 37 cM (near BM143), but the same QTL position was not supported in analysis of somatic cell counts. A recent analysis within an enlarged granddaughter design revealed a QTL for somatic cell score at position 99 (of 135 cM) on BTA 6 (Bennewitz et al., 2004). Knowledge on allele frequencies for different breeds and evaluation of associations between potentially favourable alleles or genotypes and unfavourable side effects on traits influenced by linked QTL will be a prerequisite for implementation of marker assisted breeding strategies. Therefore a range of breeds including beef and milk selected cattle were chosen to estimate allele frequencies. Based on the present status of QTL detection on BTA 6 in the German granddaughter design, associations of CSN1S1-5V genotypes with potentially linked QTL were also examined.
2. Materials and methods 2.1. Samples and genotyping Cattle DNA samples from 14 breeds from Central and Eastern Europe (Angler, Ayrshire, Bohemian Red, British Frisian, Charolais, German Angus, German
Holstein, German Simmental, German Yellow, German Black Pied (resource population), Hereford, Jersey, Pinzgauer, Polish Red) collected at our institute during past years and projects were used for genotyping the CSN1S1-5V polymorphism by single strand conformation polymorphism (SSCP) analysis as described in Prinzenberg et al. (2003) or using a 348 bp fragment and a shortened electrophoresis run (650 V, 5 h, 5 8C, 12% PA, 37 : 1). Numbers of samples per breed and the main use of the current population in Europe (milk, beef or dual purpose) are given in Table 1. A second sample set, a total of 678 Holstein bulls representing great-grandsires, grandsires and the offspring of the 7 heterozygous grandsires of the German resource families were used to evaluate associations between CSN1S1-5V genotype and economic traits. One big family with 316 half-siblings from a genotype 22 homozygous grandsire was also included. 2.2. Allele frequencies and population differentiation in milk, beef and dual purpose breeds Allele and genotype frequencies for different breeds, Hardy Weinberg tests and tests for population differentiation were computed using CONVERT (Glaubitz, 2004), GENEPOP version 3.4 (Raymond and Rousset, 1995) and FSTAT version 2.9.3.2 (Goudet, 2002) softwares. In addition to counting alleles per population, FSTAT also applies a correction for the use of different sample sizes, which gives the so called ballelic richnessQ in the output file. Pair wise F ST values and tests for population differentiation were calculated for all breeds separately and also within and between sample sets for the three groups bmilkQ (Angler, Ayrshire, Bohemian Red, British Frisian, German Holstein, German Black Pied (resource population), Jersey, Polish Red), bbeefQ (Charolais, German Angus, Hereford) and bdual purposeQ (German Simmental, German Yellow, Pinzgauer) against each other. A Bonferroni correction for multiple tests is included in FSTAT. 2.3. Data collection and statistical analyses of association with economic traits Estimated breeding values (EBVs) of German Holstein bulls from the granddaughter design were
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Table 1 Allele and genotype frequencies of CSN1S1-5V in 14 cattle breeds of Central and Eastern Europe Breed1 n
Main Allelic Allele frequency use2 richness 1 2 3
4
11
12
13
14
22
23
24
33
34
44
ANG AYR BRE BRF CHA GAN GBP GHO GSI GYE HER JER PIN PRE Total
M M M M B B GR M DP DP B M DP M
0.112 – 0.022 0.050 0.094 0.038 0.024 0.038 0.076 – 0.146 0.163 – 0.049
– – – – – 0.113 – – 0.017 – – 0.115 – 0.024
– 0.042 0.200 0.060 0.245 0.189 0.071 0.055 0.153 0.194 0.063 0.058 0.143 0.073
– – 0.089 – – 0.113 – 0.010 – 0.056 – 0.269 – 0.073
– – – – 0.019 0.075 – – – – – 0.135 – –
0.673 0.833 0.244 0.720 0.377 0.340 0.667 0.549 0.424 0.639 0.604 0.038 0.743 0.268
0.122 0.104 0.333 0.120 0.189 0.151 0.143 0.267 0.220 0.111 0.042 0.019 0.114 0.341
0.122 – 0.044 0.080 0.151 – 0.048 0.057 0.119 – 0.250 – – 0.073
0.020 0.021 0.089 – – 0.019 0.071 0.043 0.034 – – 0.192 – 0.122
0.020 – – 0.020 0.019 – – 0.018 0.034 – 0.042 0.154 – 0.024
0.041 – – – – – – – – – – 0.019 – –
49 48 45 50 53 53 42 490 59 36 48 52 35 41 1145
3.00 2.85 3.88 3.92 4.00 3.96 3.88 3.76 4.00 3.00 3.92 4.00 3.00 3.99
– 0.021 0.144 0.030 0.132 0.302 0.036 0.033 0.093 0.125 0.031 0.346 0.071 0.098
0.796 0.906 0.533 0.850 0.670 0.509 0.798 0.738 0.669 0.792 0.781 0.077 0.871 0.512
0.092 0.073 0.300 0.070 0.104 0.151 0.143 0.191 0.161 0.083 0.042 0.413 0.057 0.341
Genotype frequency
1
ANG = Angler, AYR = Ayrshire, BRE = Bohemian Red, BRF = British Frisian, CHA = Charolais, GSI = German Simmental, GAN = German Angus, GBP = German Black Pied (genetic resource population), GHO = German Holstein, GYE = German Yellow, HER = Hereford, JER = Jersey, PIN = Pinzgauer, PRE = Polish Red. 2 M = milk production, B = beef production, DP = dual purpose (milk and beef), GR = genetic resource.
available for 11 linear body conformation traits (dairy character, height at sacrum/stature, strength, body depth, rear leg set side and rear view, hocks, foot angle, rump width and angle, angularity), 6 udder conformation traits (teat length and placement, fore udder attachment, rear udder height, suspensory ligament, udder depth) and for milking speed and temperament. These traits and the scoring process are described by Hiendleder et al. (2003) in detail. Additionally, relative breeding values (RBVs) for somatic cell score (given as normalized RZS), length of productive life (normalized as RZN) and a composite relative breeding value for body conformation (RZE) were analysed. All data were from the national routine sire evaluation in November 1999. A description of the genetic evaluation procedures is available at www.vit.de. Bulls with rare genotypes (less than 15 records, present in less than 4 of the 8 families) were excluded from the statistical analyses. Thus the final data set encompassed 635 bulls from 8 families ranging from 11 to 316 sons (average 84) and the four CSN1S1-5V genotypes 12 (75 records, 5 families), 22 (372 records, 7 families), 23 (114 records, 8 families) and 24 (74 records, 5 families) (for details see Prinzenberg et al., 2003). Due to missing breeding values for some traits, number of bulls in the analysis ranged from 140
to 635 depending on the trait examined. Reliabilities of the breeding values varied considerably depending on the individual trait and bull with a minimum of 54% (foot angle) and a maximum of 99% (all traits) and means ranging from 75% (foot angle) to 93% (somatic cell score). All EBVs and RBVs were analysed separately using the GLM procedure of the SAS program package (SAS, 1989) and the model Yijk = l + CSgi + F j + e ijk (with Yijk = EBV/RBV for the trait examined of individual bull k with genotype i of family j, l = overall overall mean, CSgi = CSN1S1-5V genotype i (12, 22, 23, 24), F j = family j (1–8), e ijk = random residual effect). The individual reliability per trait and bull was included as weighting factor. Pair wise tests for genotype differences were calculated with the contrast option of the GLM procedure.
3. Results and discussion 3.1. Allele frequencies and population differentiation in milk, beef and dual purpose breeds Genotyping of CSN1S1-5V revealed 4 alleles and all 10 resulting genotypes in the complete sample set. The number of alleles and genotypes in individual
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breeds ranged from 3 to 4 alleles and 3 to 9 genotypes (Table 1). No CSN1S1-5V*5, was found. Allelic richness ranged from 2.85 in Ayrshire to 4.00 in Charolais, German Simmental and Jersey. All breeds were in Hardy Weinberg Equilibrium (significance level P b 0.01). Alleles 2 and 3 and the genotypes 22 and 23 were present in all breeds investigated and allele 2 was predominant over all breeds, except in Jersey, with allele 3 and genotype 13 found most frequently. This supports the possible wild type status of allele 2, which was already suggested by the most probable sequence based evolution presented by IbeaghaAwemu et al. (2004). Pair wise F ST values ranged from 0.001 for German Holstein/German Black Pied to 0.513 for Jersey/Ayrshire in the whole sample set. In general, Jersey showed high pair wise F ST values with all other breeds (0.163 vs. Polish Red to 0.513 vs. Ayrshire), and also were identified as highly significant different from all other breeds in tests for pair wise population differentiation. In these pair wise tests, Bonferroni correction lowered the 5% level to 0.0005. German Simmental and German Black Pied each differed from two breeds only: Jersey and Ayrshire and Jersey and German Angus, respectively. The remaining breeds differed significantly from four to eight breeds. Pair wise F ST within the breed groups ranged from 0.001 to 0.513 (milk), 0.024 to 0.136 (beef) and 0.003 to 0.056 (dual purpose). Excluding the Jersey breed from the milk group narrowed the range to 0.001–0.230. Pair wise F ST between the groups milk, beef and dual purpose ranged from 0.011 (milk/dual purpose) to 0.021 (milk/beef). Excluding Jersey from the milk group results in a lower F ST for milk/dual purpose (0.008) and a higher F ST for milk/beef (0.033). Therefore, the genetic differentiation calculated from CSN1S1-5V allele frequencies is higher within the groups milk/beef/dual purpose than between these three groups. Beef breeds, represented by Charolais, German Angus and Hereford, did not display an overall trend in allele distribution nor any marked differences compared to milk breeds (Table 1). Therefore selection for beef production did not indirectly favour different CSN1S1 promoter alleles than in milk breeds. Allele frequencies in German Holstein, which have been strongly selected for milk production over
the past 40 years, and the original German Black Pied cattle from the resource population were nearly the same (Table 1) and very low differentiation (expressed as F ST) between these two breeds was found. This underlines, that selection schemes applied to German Holstein, which focused only on milk yield only over a long time, did not favour certain allelic variants in the promoter region of CSN1S1 or cause any shift in allele frequencies. Breeds like Polish and Bohemian Red showed similar allele frequencies and low pair wise F ST values (0.007) also, reflecting their relationship and introgression of Polish into Bohemian Red cattle. A general decrease in allelic richness was found in two milk and dual purpose breeds each (Table 1). 3.2. Association of CSN1S1-5V genotypes with economic traits in Holstein In German Holstein bulls, analysis of associations between CSN1S1-5V genotype and body conformation traits revealed significant effects for EBV for fore udder attachment (FUA) and dairy character (DAICHA). No significant effect was detected for the composite relative breeding value body conformation, RZE. Regarding functional traits, significant CSN1S15V effects ( P b 0.05) were found for the relative breeding value for functional life (RZN). Highly significant effects ( P b 0.001) of CSN1S1-5V genotypes were found for somatic cell score (RZS) (Table 2). No significant effects were found for milking speed and temperament, even if a suggestive QTL for milking speed was reported on BTA 6 (Boichard et al., 2003). Our analysis did not identify significant effects of CSN1S1-5Von traits with previously reported QTL for body conformation close the casein cluster, but indicated associations with udder shape. For fore udder attachment, udder depth and suspensory ligament markedly lower LS means were associated with genotype 23. The same genotype was also associated with highest somatic cell scores, reflecting the influence of udder shape on udder health (Table 2). This underlines presence of udder conformation QTL in the same chromosome area as the caseins, but on the other hand considerable recombination seems to be present between these loci and CSN1S1-5V. The high significant effects of CSN1S1-5V on somatic cell score are in line with the QTL report of
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Table 2 Effect of CSN1S1-5V genotypes on estimated breeding values for body conformation and functional traits Variable1
n
Overall mean
SD
P for effect of CSN1S1
LS-means in CSN1S1-5V genotype groups 12
22
23
24
12 vs. 22
23 vs. 22
24 vs. 22
RZE RZN RZS ANGULAR BODYDE DAICHA FANGLE FUA HOCKS RLSRV RLSSV RUH RUMPA RUMPW STATURE STRE SUSP TEATL TEATPL UDDERD MSPEED TEMPER
620 635 634 620 620 617 620 614 140 143 620 620 620 620 620 614 620 620 620 620 212 212
100.03 102.13 100.33 102.24 100.53 102.67 97.03 100.62 106.69 98.97 101.08 97.93 101.10 97.03 97.00 97.07 99.51 99.42 99.54 99.90 99.53 101.73
11.98 12.37 11.14 11.99 12.37 12.07 11.79 12.40 10.55 13.12 10.78 11.21 11.65 11.39 11.51 12.10 13.11 11.60 12.58 11.65 13.73 14.82
0.795 0.011* b0.001*** 0.064 0.831 0.021* 0.307 0.023* 0.718 0.833 0.878 0.491 0.209 0.146 0.154 0.419 0.336 0.722 0.190 0.125 0.573 0.888
95.19 97.93 96.60 99.30 99.22 99.62 96.23 98.05 102.00 97.86 99.52 97.37 100.93 95.40 94.67 96.68 92.56 100.38 97.04 96.94 102.76 102.08
94.55 101.90 99.03 97.74 98.24 98.06 95.92 97.23 104.21 98.79 100.89 96.08 98.19 97.07 95.25 97.28 95.44 101.42 95.63 98.06 102.31 103.16
95.02 98.51 96.30 101.17 97.78 101.85 96.16 93.78 102.42 95.78 100.73 95.67 97.25 99.09 97.58 95.57 94.97 100.72 94.32 95.19 98.95 104.70
95.90 102.91 101.38 98.89 98.82 99.06 98.94 95.52 106.54 99.18 100.62 94.44 97.68 98.33 97.65 98.16 95.04 101.93 97.83 98.03 99.32 104.95
0.364 0.024* 0.104 0.364 0.547 0.302 0.866 0.553 0.427 0.965 0.417 0.317 0.090 0.289 0.753 0.688 0.068 0.403 0.309 0.523 0.809 0.946
0.008 0.008** 0.011* 0.008 0.747 0.002** 0.948 0.006** 0.563 0.373 0.899 0.792 0.434 0.100 0.075 0.159 0.699 0.610 0.349 0.018 0.229 0.495
0.408 0.594 0.011* 0.493 0.581 0.528 0.059 0.340 0.526 0.913 0.827 0.316 0.659 0.397 0.152 0.499 0.984 0.573 0.139 0.966 0.452 0.637
P for contrast
Significant P values are in bold letters. Traits are grouped in the following order: relative breeding values, linear body and udder conformation traits, traits contributing to milking ability. *p b 0.05; **p b 0.01, ***p b 0.001. 1 RZE = composite relative breeding value body conformation; RZN = relative breeding value productive life; RZS = relative breeding value somatic cells; ANGULAR = angularity, BODYDE = body depth, DAICHA = dairy character; FANGLE = foot angle; FUA = fore udder attachment; HOCKS = hocks; RLSRV = rear leg set rear view; RLSSV = rear leg set side view; RUH = rear udder height; RUMPA = rump angle; RUMPW = rump width; STATURE = height at sacrum; STRE = strength; SUSP = suspensory ligament; TEATL = teat length; TEATPL = teat placement; UDDERD = udder depth; MSPEED = milking speed; TEMPER = temperament.
Bennewitz et al. (2004), who identified the adjacent marker interval CSN3-BP7 as location of the respective QTL. According to Klungland et al. (2001), QTL for milk production and mastitis would be expected to be close to each other, because of the known negative genetic correlations for milk yield and udder health. A fact contributing to both, udder health and longevity might be the total milk yield. Reduced yields were earlier found with genotype 24 (Prinzenberg et al., 2003) and this genotype is found with most desirable values for somatic cell score and length of productive life in comparison to the remaining genotype groups now. On the other hand genotype 23, and less obvious also genotype 12, seem to be associated with less desirable udder shape and breeding values for somatic cells in German Holstein. This is far from explaining completely the
effects found in this study, but is in accordance with present knowledge.
4. Conclusion Allele frequencies over all breeds show a high variability and also a minimum of 3 alleles per breed. Past selection schemes in specialised milk breeds, which focused only on milk yield for decades and very recently included content traits, did not indirectly select for certain alleles in the CSN1S1 promoter, which is shown by the unchanged allele frequencies comparing German Holstein and German Black Pied cattle as well as no clear allelic or genotypic differentiation between milk and beef breeds. Differences in allele frequen-
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cies detected in the 14 European breeds are more likely a result of breed historical aspects and geographic factors than a consequence of selection for milk or meat production. Based on our present results, genotype 24 (or allele 4) is unlikely to have negative side effects on body and udder conformation in German Holstein. Nevertheless, associations of CSN1S1-5V with economically important traits like longevity and somatic cell scores were found. These are positive for genotype 24 in the bulls studied here, but will require monitoring if any gene assisted selection is to be implemented. For all other breeds, these associations need to be evaluated, before considering CSN1S1-5V as marker in selection programmes. Acknowledgements We thank Sandra Stein and Renate Latzke-Reinhardt for skilful technical support in genotyping and Christina Peter for discussions and advice regarding statistical evaluation of population differentiation. DNA samples were kindly provided by J. Citek, J.L. Williams, R. Zieminski and by the German cattle breeders association ADR. Estimated breeding values were kindly provided by Vereinigte Informationssysteme Tierhaltung (VIT). A part of this project was financially supported by the Federal Ministry of Education and Research (Project No. 031 1020A). References Bennewitz, J., Reinsch, N., Guiard, V., Fritz, S., Thomsen, H., Looft, C., Ku¨hn, C., Schwerin, M., Weimann, C., Erhardt, G., Reinhardt, F., Reents, R., Boichard, D., Kalm, E., 2004. Multiple Quantitative Trait Loci Mapping with Cofactors and Application of Alternative Variants of the False Discovery Rate in an Enlarged Granddaughter Design. Genetics 168, 1019–1027. Boichard, D., Grohs, C., Bourgeois, F., Cerqueira, F., Faugeras, R., Neau, A., Rupp, R., Amigues, Y., Boscher, M.Y., Leveziel, H.,
2003. Detection of genes influencing economic traits in three French dairy cattle breeds. Genet. Sel. Evol. 35, 77 – 101. Glaubitz, J., 2004. CONVERT: a user-friendly program to reformat diploid genotypic data for commonly used population genetic software packages. Mol. Ecol. Notes 4, 309 – 310. Goudet, J. 2002. FSTAT A software developed by Je´roˆme Goudet. available online at: http://www2.unil.ch/izea/softwares/fstat. html. Hiendleder, S., Thomsen, H., Reinsch, N., Bennewitz, J., LeyheHorn, B., Looft, C., Xu, N., Medjugorac, I., Russ, I., Kuhn, C., Brockmann, G.A., Blumel, J., Brenig, B., Reinhardt, F., Reents, R., Averdunk, G., Schwerin, M., Forster, M., Kalm, E., Erhardt, G., 2003. Mapping of QTL for body conformation and behavior in cattle. J. Heredity 94, 496 – 506. Ibeagha-Awemu, E.M., Prinzenberg, E.-M., Erhardt, G., 2004. High variability of milk protein genes in Bos indicus cattle breeds of Cameroon and Nigeria and characterization of a new as1-casein promoter allele. J. Dairy Res. 71. Khatkar, M.S., Thomson, P.C., Tammen, I., Raadsma, H.W., 2004. Quantitative trait loci mapping in dairy cattle: review and metaanalysis. Genet. Sel. Evol. 36, 163 – 190. Klungland, H., Sabry, A., Heringstad, B., Olsen, H.G., GomezRaya, L., Vage, D.I., Olsaker, I., Odegard, J., Klemetsdal, G., Schulman, N., Vilkki, J., Ruane, J., Aasland, M., Ronningen, K., Lien, S., 2001. Quantitative trait loci affecting clinical mastitis and somatic cell count in dairy cattle. Mamm. Genome 12, 837 – 842. Ku¨hn, C., Bennewitz, J., Reinsch, N., Xu, N., Thomsen, H., Looft, C., Brockmann, G.A., Schwerin, M., Weimann, C., Hiendleder, S., Erhardt, G., Medjugorac, I., Fo¨rster, M., Brenig, B., Reinhardt, F., Reents, R., Russ, I., Averdunk, G., Blu¨mel, J., Kalm, E., 2003. Quantitative trait loci mapping of functional traits in the German Holstein cattle population. J. Dairy Sci. 86, 360 – 368. Prinzenberg, E.M., Weimann, C., Brandt, H., Bennewitz, J., Kalm, E., Schwerin, M., Erhardt, G., 2003. Polymorphism of the bovine CSN1S1 promoter: linkage mapping, intragenic haplotypes, and effects on milk production traits. J. Dairy Sci. 86, 2696 – 2705. Raymond, M., Rousset, F., 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Heredity 86, 248 – 249. SAS, 1989. SAS/STAT User’s Guide, Version 6. SAS Institute Inc., Cary, NC. Schrooten, C., Bovenhuis, H., Coppieters, W., Van Arendonk, J.A., 2000. Whole genome scan to detect quantitative trait loci for conformation and functional traits in dairy cattle. J. Dairy Sci. 83, 795 – 806.
Allele frequencies for SNPs in the aS1-casein gene (CSN1S1) 5V flanking region in European cattle and association with economic traits in German Holstein Eva-Maria Prinzenberg a,*, Horst Brandt a, Joern Bennewitz b, Ernst Kalm b, Georg Erhardt a b
a Institute for Animal Breeding and Genetics, Justus-Liebig-University, Ludwigstr. 21B, 35390 Giessen, Germany Institute for Animal Breeding and Husbandry, Christian-Albrechts-University, Olshausenstr. 40, 24098 Kiel, Germany
Abstract Allele frequencies of a polymorphism in the a S1 -casein 5V flanking region (CSN1S1-5V) were determined in 14 Central and Eastern European cattle breeds by single strand conformation polymorphism analysis. Allele frequencies ranged from 0.00 to 0.35 (allele 1), 0.08 to 0.91 (allele 2), 0.04 to 0.41 (allele 3) and from 0.00 to 0.16 (allele 4). Allele 5 was not found. Presence of allele 2 in all breeds highlights its probable wild type status. A loss of alleles and genotypes occurred in some breeds, but differentiation within the breed groups milk, beef and dual purpose was higher than between these groups. An analysis of the variance in a German Holstein granddaughter design with family and CSN1S1-5V genotype as fixed effects revealed significant effects ( P b 0.05 each) of the CSN1S1-5V genotype on breeding values for dairy character, fore udder attachment, length of productive life and highly significant effects on somatic cell score ( P b 0.001). Genotype LS-means assigned favourable values for udder health and longevity to genotype 24. Past selection for milk yield or beef production does not seem to have favoured indirectly specific promoter alleles. No negative effects of genotype 24 were evident in Holstein, but selection for 23 requires careful inspection of unfavourable side effects. D 2005 Elsevier B.V. All rights reserved. Keywords: Alpha-S1-casein; Promoter polymorphism; Allele frequency; Economic traits
1. Introduction A four allele polymorphism in the bovine a S1 casein gene promoter (CSN1S1-5V) has previously * Corresponding author. Tel.: +49 641 9937626; fax: +49 641 9937629. E-mail address: [email protected] (E.-M. Prinzenberg). 0301-6226/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.livprodsci.2005.10.015
been used for linkage mapping of the gene and analysis of effects on milk production traits in German Holsteins. This study indicated significant and for genotype 24 positive effects of CSN1S1-5V on protein content and possibly linked effects on yield traits in a granddaughter design. The effect on milk protein content was hypothesized to be a direct effect of polymorphisms in regulatory elements at the 5V end of
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the casein locus (Prinzenberg et al., 2003). This makes CSN1S1-5V an interesting candidate for marker or gene assisted selection programmes. A fifth allele was recently reported in West African Bos indicus cattle (Ibeagha-Awemu et al., 2004). QTL studies identified several putative or significant QTL within a region spanning about 60 cM on BTA 6. These include two major regions affecting milk production traits (Khatkar et al., 2004), but also other traits contributing to an economical production as milking speed, size characteristics (Schrooten et al., 2000; Boichard et al., 2003; Hiendleder et al., 2003), stillbirth/direct effect (Ku¨hn et al., 2003), foot angle, general quality of feet and legs and udder conformation (Hiendleder et al., 2003). Especially the last study, based on the same half-sib families as ours, identified a QTL cluster for 8 body conformation traits at positions 85–89 cM on BTA 6, close to the map position of CSN1S1-5V at 93.9 cM (Prinzenberg et al., 2003). Klungland et al. (2001) reported a QTL for clinical mastitis on BTA 6, which is located around position 37 cM (near BM143), but the same QTL position was not supported in analysis of somatic cell counts. A recent analysis within an enlarged granddaughter design revealed a QTL for somatic cell score at position 99 (of 135 cM) on BTA 6 (Bennewitz et al., 2004). Knowledge on allele frequencies for different breeds and evaluation of associations between potentially favourable alleles or genotypes and unfavourable side effects on traits influenced by linked QTL will be a prerequisite for implementation of marker assisted breeding strategies. Therefore a range of breeds including beef and milk selected cattle were chosen to estimate allele frequencies. Based on the present status of QTL detection on BTA 6 in the German granddaughter design, associations of CSN1S1-5V genotypes with potentially linked QTL were also examined.
2. Materials and methods 2.1. Samples and genotyping Cattle DNA samples from 14 breeds from Central and Eastern Europe (Angler, Ayrshire, Bohemian Red, British Frisian, Charolais, German Angus, German
Holstein, German Simmental, German Yellow, German Black Pied (resource population), Hereford, Jersey, Pinzgauer, Polish Red) collected at our institute during past years and projects were used for genotyping the CSN1S1-5V polymorphism by single strand conformation polymorphism (SSCP) analysis as described in Prinzenberg et al. (2003) or using a 348 bp fragment and a shortened electrophoresis run (650 V, 5 h, 5 8C, 12% PA, 37 : 1). Numbers of samples per breed and the main use of the current population in Europe (milk, beef or dual purpose) are given in Table 1. A second sample set, a total of 678 Holstein bulls representing great-grandsires, grandsires and the offspring of the 7 heterozygous grandsires of the German resource families were used to evaluate associations between CSN1S1-5V genotype and economic traits. One big family with 316 half-siblings from a genotype 22 homozygous grandsire was also included. 2.2. Allele frequencies and population differentiation in milk, beef and dual purpose breeds Allele and genotype frequencies for different breeds, Hardy Weinberg tests and tests for population differentiation were computed using CONVERT (Glaubitz, 2004), GENEPOP version 3.4 (Raymond and Rousset, 1995) and FSTAT version 2.9.3.2 (Goudet, 2002) softwares. In addition to counting alleles per population, FSTAT also applies a correction for the use of different sample sizes, which gives the so called ballelic richnessQ in the output file. Pair wise F ST values and tests for population differentiation were calculated for all breeds separately and also within and between sample sets for the three groups bmilkQ (Angler, Ayrshire, Bohemian Red, British Frisian, German Holstein, German Black Pied (resource population), Jersey, Polish Red), bbeefQ (Charolais, German Angus, Hereford) and bdual purposeQ (German Simmental, German Yellow, Pinzgauer) against each other. A Bonferroni correction for multiple tests is included in FSTAT. 2.3. Data collection and statistical analyses of association with economic traits Estimated breeding values (EBVs) of German Holstein bulls from the granddaughter design were
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Table 1 Allele and genotype frequencies of CSN1S1-5V in 14 cattle breeds of Central and Eastern Europe Breed1 n
Main Allelic Allele frequency use2 richness 1 2 3
4
11
12
13
14
22
23
24
33
34
44
ANG AYR BRE BRF CHA GAN GBP GHO GSI GYE HER JER PIN PRE Total
M M M M B B GR M DP DP B M DP M
0.112 – 0.022 0.050 0.094 0.038 0.024 0.038 0.076 – 0.146 0.163 – 0.049
– – – – – 0.113 – – 0.017 – – 0.115 – 0.024
– 0.042 0.200 0.060 0.245 0.189 0.071 0.055 0.153 0.194 0.063 0.058 0.143 0.073
– – 0.089 – – 0.113 – 0.010 – 0.056 – 0.269 – 0.073
– – – – 0.019 0.075 – – – – – 0.135 – –
0.673 0.833 0.244 0.720 0.377 0.340 0.667 0.549 0.424 0.639 0.604 0.038 0.743 0.268
0.122 0.104 0.333 0.120 0.189 0.151 0.143 0.267 0.220 0.111 0.042 0.019 0.114 0.341
0.122 – 0.044 0.080 0.151 – 0.048 0.057 0.119 – 0.250 – – 0.073
0.020 0.021 0.089 – – 0.019 0.071 0.043 0.034 – – 0.192 – 0.122
0.020 – – 0.020 0.019 – – 0.018 0.034 – 0.042 0.154 – 0.024
0.041 – – – – – – – – – – 0.019 – –
49 48 45 50 53 53 42 490 59 36 48 52 35 41 1145
3.00 2.85 3.88 3.92 4.00 3.96 3.88 3.76 4.00 3.00 3.92 4.00 3.00 3.99
– 0.021 0.144 0.030 0.132 0.302 0.036 0.033 0.093 0.125 0.031 0.346 0.071 0.098
0.796 0.906 0.533 0.850 0.670 0.509 0.798 0.738 0.669 0.792 0.781 0.077 0.871 0.512
0.092 0.073 0.300 0.070 0.104 0.151 0.143 0.191 0.161 0.083 0.042 0.413 0.057 0.341
Genotype frequency
1
ANG = Angler, AYR = Ayrshire, BRE = Bohemian Red, BRF = British Frisian, CHA = Charolais, GSI = German Simmental, GAN = German Angus, GBP = German Black Pied (genetic resource population), GHO = German Holstein, GYE = German Yellow, HER = Hereford, JER = Jersey, PIN = Pinzgauer, PRE = Polish Red. 2 M = milk production, B = beef production, DP = dual purpose (milk and beef), GR = genetic resource.
available for 11 linear body conformation traits (dairy character, height at sacrum/stature, strength, body depth, rear leg set side and rear view, hocks, foot angle, rump width and angle, angularity), 6 udder conformation traits (teat length and placement, fore udder attachment, rear udder height, suspensory ligament, udder depth) and for milking speed and temperament. These traits and the scoring process are described by Hiendleder et al. (2003) in detail. Additionally, relative breeding values (RBVs) for somatic cell score (given as normalized RZS), length of productive life (normalized as RZN) and a composite relative breeding value for body conformation (RZE) were analysed. All data were from the national routine sire evaluation in November 1999. A description of the genetic evaluation procedures is available at www.vit.de. Bulls with rare genotypes (less than 15 records, present in less than 4 of the 8 families) were excluded from the statistical analyses. Thus the final data set encompassed 635 bulls from 8 families ranging from 11 to 316 sons (average 84) and the four CSN1S1-5V genotypes 12 (75 records, 5 families), 22 (372 records, 7 families), 23 (114 records, 8 families) and 24 (74 records, 5 families) (for details see Prinzenberg et al., 2003). Due to missing breeding values for some traits, number of bulls in the analysis ranged from 140
to 635 depending on the trait examined. Reliabilities of the breeding values varied considerably depending on the individual trait and bull with a minimum of 54% (foot angle) and a maximum of 99% (all traits) and means ranging from 75% (foot angle) to 93% (somatic cell score). All EBVs and RBVs were analysed separately using the GLM procedure of the SAS program package (SAS, 1989) and the model Yijk = l + CSgi + F j + e ijk (with Yijk = EBV/RBV for the trait examined of individual bull k with genotype i of family j, l = overall overall mean, CSgi = CSN1S1-5V genotype i (12, 22, 23, 24), F j = family j (1–8), e ijk = random residual effect). The individual reliability per trait and bull was included as weighting factor. Pair wise tests for genotype differences were calculated with the contrast option of the GLM procedure.
3. Results and discussion 3.1. Allele frequencies and population differentiation in milk, beef and dual purpose breeds Genotyping of CSN1S1-5V revealed 4 alleles and all 10 resulting genotypes in the complete sample set. The number of alleles and genotypes in individual
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breeds ranged from 3 to 4 alleles and 3 to 9 genotypes (Table 1). No CSN1S1-5V*5, was found. Allelic richness ranged from 2.85 in Ayrshire to 4.00 in Charolais, German Simmental and Jersey. All breeds were in Hardy Weinberg Equilibrium (significance level P b 0.01). Alleles 2 and 3 and the genotypes 22 and 23 were present in all breeds investigated and allele 2 was predominant over all breeds, except in Jersey, with allele 3 and genotype 13 found most frequently. This supports the possible wild type status of allele 2, which was already suggested by the most probable sequence based evolution presented by IbeaghaAwemu et al. (2004). Pair wise F ST values ranged from 0.001 for German Holstein/German Black Pied to 0.513 for Jersey/Ayrshire in the whole sample set. In general, Jersey showed high pair wise F ST values with all other breeds (0.163 vs. Polish Red to 0.513 vs. Ayrshire), and also were identified as highly significant different from all other breeds in tests for pair wise population differentiation. In these pair wise tests, Bonferroni correction lowered the 5% level to 0.0005. German Simmental and German Black Pied each differed from two breeds only: Jersey and Ayrshire and Jersey and German Angus, respectively. The remaining breeds differed significantly from four to eight breeds. Pair wise F ST within the breed groups ranged from 0.001 to 0.513 (milk), 0.024 to 0.136 (beef) and 0.003 to 0.056 (dual purpose). Excluding the Jersey breed from the milk group narrowed the range to 0.001–0.230. Pair wise F ST between the groups milk, beef and dual purpose ranged from 0.011 (milk/dual purpose) to 0.021 (milk/beef). Excluding Jersey from the milk group results in a lower F ST for milk/dual purpose (0.008) and a higher F ST for milk/beef (0.033). Therefore, the genetic differentiation calculated from CSN1S1-5V allele frequencies is higher within the groups milk/beef/dual purpose than between these three groups. Beef breeds, represented by Charolais, German Angus and Hereford, did not display an overall trend in allele distribution nor any marked differences compared to milk breeds (Table 1). Therefore selection for beef production did not indirectly favour different CSN1S1 promoter alleles than in milk breeds. Allele frequencies in German Holstein, which have been strongly selected for milk production over
the past 40 years, and the original German Black Pied cattle from the resource population were nearly the same (Table 1) and very low differentiation (expressed as F ST) between these two breeds was found. This underlines, that selection schemes applied to German Holstein, which focused only on milk yield only over a long time, did not favour certain allelic variants in the promoter region of CSN1S1 or cause any shift in allele frequencies. Breeds like Polish and Bohemian Red showed similar allele frequencies and low pair wise F ST values (0.007) also, reflecting their relationship and introgression of Polish into Bohemian Red cattle. A general decrease in allelic richness was found in two milk and dual purpose breeds each (Table 1). 3.2. Association of CSN1S1-5V genotypes with economic traits in Holstein In German Holstein bulls, analysis of associations between CSN1S1-5V genotype and body conformation traits revealed significant effects for EBV for fore udder attachment (FUA) and dairy character (DAICHA). No significant effect was detected for the composite relative breeding value body conformation, RZE. Regarding functional traits, significant CSN1S15V effects ( P b 0.05) were found for the relative breeding value for functional life (RZN). Highly significant effects ( P b 0.001) of CSN1S1-5V genotypes were found for somatic cell score (RZS) (Table 2). No significant effects were found for milking speed and temperament, even if a suggestive QTL for milking speed was reported on BTA 6 (Boichard et al., 2003). Our analysis did not identify significant effects of CSN1S1-5Von traits with previously reported QTL for body conformation close the casein cluster, but indicated associations with udder shape. For fore udder attachment, udder depth and suspensory ligament markedly lower LS means were associated with genotype 23. The same genotype was also associated with highest somatic cell scores, reflecting the influence of udder shape on udder health (Table 2). This underlines presence of udder conformation QTL in the same chromosome area as the caseins, but on the other hand considerable recombination seems to be present between these loci and CSN1S1-5V. The high significant effects of CSN1S1-5V on somatic cell score are in line with the QTL report of
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159
Table 2 Effect of CSN1S1-5V genotypes on estimated breeding values for body conformation and functional traits Variable1
n
Overall mean
SD
P for effect of CSN1S1
LS-means in CSN1S1-5V genotype groups 12
22
23
24
12 vs. 22
23 vs. 22
24 vs. 22
RZE RZN RZS ANGULAR BODYDE DAICHA FANGLE FUA HOCKS RLSRV RLSSV RUH RUMPA RUMPW STATURE STRE SUSP TEATL TEATPL UDDERD MSPEED TEMPER
620 635 634 620 620 617 620 614 140 143 620 620 620 620 620 614 620 620 620 620 212 212
100.03 102.13 100.33 102.24 100.53 102.67 97.03 100.62 106.69 98.97 101.08 97.93 101.10 97.03 97.00 97.07 99.51 99.42 99.54 99.90 99.53 101.73
11.98 12.37 11.14 11.99 12.37 12.07 11.79 12.40 10.55 13.12 10.78 11.21 11.65 11.39 11.51 12.10 13.11 11.60 12.58 11.65 13.73 14.82
0.795 0.011* b0.001*** 0.064 0.831 0.021* 0.307 0.023* 0.718 0.833 0.878 0.491 0.209 0.146 0.154 0.419 0.336 0.722 0.190 0.125 0.573 0.888
95.19 97.93 96.60 99.30 99.22 99.62 96.23 98.05 102.00 97.86 99.52 97.37 100.93 95.40 94.67 96.68 92.56 100.38 97.04 96.94 102.76 102.08
94.55 101.90 99.03 97.74 98.24 98.06 95.92 97.23 104.21 98.79 100.89 96.08 98.19 97.07 95.25 97.28 95.44 101.42 95.63 98.06 102.31 103.16
95.02 98.51 96.30 101.17 97.78 101.85 96.16 93.78 102.42 95.78 100.73 95.67 97.25 99.09 97.58 95.57 94.97 100.72 94.32 95.19 98.95 104.70
95.90 102.91 101.38 98.89 98.82 99.06 98.94 95.52 106.54 99.18 100.62 94.44 97.68 98.33 97.65 98.16 95.04 101.93 97.83 98.03 99.32 104.95
0.364 0.024* 0.104 0.364 0.547 0.302 0.866 0.553 0.427 0.965 0.417 0.317 0.090 0.289 0.753 0.688 0.068 0.403 0.309 0.523 0.809 0.946
0.008 0.008** 0.011* 0.008 0.747 0.002** 0.948 0.006** 0.563 0.373 0.899 0.792 0.434 0.100 0.075 0.159 0.699 0.610 0.349 0.018 0.229 0.495
0.408 0.594 0.011* 0.493 0.581 0.528 0.059 0.340 0.526 0.913 0.827 0.316 0.659 0.397 0.152 0.499 0.984 0.573 0.139 0.966 0.452 0.637
P for contrast
Significant P values are in bold letters. Traits are grouped in the following order: relative breeding values, linear body and udder conformation traits, traits contributing to milking ability. *p b 0.05; **p b 0.01, ***p b 0.001. 1 RZE = composite relative breeding value body conformation; RZN = relative breeding value productive life; RZS = relative breeding value somatic cells; ANGULAR = angularity, BODYDE = body depth, DAICHA = dairy character; FANGLE = foot angle; FUA = fore udder attachment; HOCKS = hocks; RLSRV = rear leg set rear view; RLSSV = rear leg set side view; RUH = rear udder height; RUMPA = rump angle; RUMPW = rump width; STATURE = height at sacrum; STRE = strength; SUSP = suspensory ligament; TEATL = teat length; TEATPL = teat placement; UDDERD = udder depth; MSPEED = milking speed; TEMPER = temperament.
Bennewitz et al. (2004), who identified the adjacent marker interval CSN3-BP7 as location of the respective QTL. According to Klungland et al. (2001), QTL for milk production and mastitis would be expected to be close to each other, because of the known negative genetic correlations for milk yield and udder health. A fact contributing to both, udder health and longevity might be the total milk yield. Reduced yields were earlier found with genotype 24 (Prinzenberg et al., 2003) and this genotype is found with most desirable values for somatic cell score and length of productive life in comparison to the remaining genotype groups now. On the other hand genotype 23, and less obvious also genotype 12, seem to be associated with less desirable udder shape and breeding values for somatic cells in German Holstein. This is far from explaining completely the
effects found in this study, but is in accordance with present knowledge.
4. Conclusion Allele frequencies over all breeds show a high variability and also a minimum of 3 alleles per breed. Past selection schemes in specialised milk breeds, which focused only on milk yield for decades and very recently included content traits, did not indirectly select for certain alleles in the CSN1S1 promoter, which is shown by the unchanged allele frequencies comparing German Holstein and German Black Pied cattle as well as no clear allelic or genotypic differentiation between milk and beef breeds. Differences in allele frequen-
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cies detected in the 14 European breeds are more likely a result of breed historical aspects and geographic factors than a consequence of selection for milk or meat production. Based on our present results, genotype 24 (or allele 4) is unlikely to have negative side effects on body and udder conformation in German Holstein. Nevertheless, associations of CSN1S1-5V with economically important traits like longevity and somatic cell scores were found. These are positive for genotype 24 in the bulls studied here, but will require monitoring if any gene assisted selection is to be implemented. For all other breeds, these associations need to be evaluated, before considering CSN1S1-5V as marker in selection programmes. Acknowledgements We thank Sandra Stein and Renate Latzke-Reinhardt for skilful technical support in genotyping and Christina Peter for discussions and advice regarding statistical evaluation of population differentiation. DNA samples were kindly provided by J. Citek, J.L. Williams, R. Zieminski and by the German cattle breeders association ADR. Estimated breeding values were kindly provided by Vereinigte Informationssysteme Tierhaltung (VIT). A part of this project was financially supported by the Federal Ministry of Education and Research (Project No. 031 1020A). References Bennewitz, J., Reinsch, N., Guiard, V., Fritz, S., Thomsen, H., Looft, C., Ku¨hn, C., Schwerin, M., Weimann, C., Erhardt, G., Reinhardt, F., Reents, R., Boichard, D., Kalm, E., 2004. Multiple Quantitative Trait Loci Mapping with Cofactors and Application of Alternative Variants of the False Discovery Rate in an Enlarged Granddaughter Design. Genetics 168, 1019–1027. Boichard, D., Grohs, C., Bourgeois, F., Cerqueira, F., Faugeras, R., Neau, A., Rupp, R., Amigues, Y., Boscher, M.Y., Leveziel, H.,
2003. Detection of genes influencing economic traits in three French dairy cattle breeds. Genet. Sel. Evol. 35, 77 – 101. Glaubitz, J., 2004. CONVERT: a user-friendly program to reformat diploid genotypic data for commonly used population genetic software packages. Mol. Ecol. Notes 4, 309 – 310. Goudet, J. 2002. FSTAT A software developed by Je´roˆme Goudet. available online at: http://www2.unil.ch/izea/softwares/fstat. html. Hiendleder, S., Thomsen, H., Reinsch, N., Bennewitz, J., LeyheHorn, B., Looft, C., Xu, N., Medjugorac, I., Russ, I., Kuhn, C., Brockmann, G.A., Blumel, J., Brenig, B., Reinhardt, F., Reents, R., Averdunk, G., Schwerin, M., Forster, M., Kalm, E., Erhardt, G., 2003. Mapping of QTL for body conformation and behavior in cattle. J. Heredity 94, 496 – 506. Ibeagha-Awemu, E.M., Prinzenberg, E.-M., Erhardt, G., 2004. High variability of milk protein genes in Bos indicus cattle breeds of Cameroon and Nigeria and characterization of a new as1-casein promoter allele. J. Dairy Res. 71. Khatkar, M.S., Thomson, P.C., Tammen, I., Raadsma, H.W., 2004. Quantitative trait loci mapping in dairy cattle: review and metaanalysis. Genet. Sel. Evol. 36, 163 – 190. Klungland, H., Sabry, A., Heringstad, B., Olsen, H.G., GomezRaya, L., Vage, D.I., Olsaker, I., Odegard, J., Klemetsdal, G., Schulman, N., Vilkki, J., Ruane, J., Aasland, M., Ronningen, K., Lien, S., 2001. Quantitative trait loci affecting clinical mastitis and somatic cell count in dairy cattle. Mamm. Genome 12, 837 – 842. Ku¨hn, C., Bennewitz, J., Reinsch, N., Xu, N., Thomsen, H., Looft, C., Brockmann, G.A., Schwerin, M., Weimann, C., Hiendleder, S., Erhardt, G., Medjugorac, I., Fo¨rster, M., Brenig, B., Reinhardt, F., Reents, R., Russ, I., Averdunk, G., Blu¨mel, J., Kalm, E., 2003. Quantitative trait loci mapping of functional traits in the German Holstein cattle population. J. Dairy Sci. 86, 360 – 368. Prinzenberg, E.M., Weimann, C., Brandt, H., Bennewitz, J., Kalm, E., Schwerin, M., Erhardt, G., 2003. Polymorphism of the bovine CSN1S1 promoter: linkage mapping, intragenic haplotypes, and effects on milk production traits. J. Dairy Sci. 86, 2696 – 2705. Raymond, M., Rousset, F., 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Heredity 86, 248 – 249. SAS, 1989. SAS/STAT User’s Guide, Version 6. SAS Institute Inc., Cary, NC. Schrooten, C., Bovenhuis, H., Coppieters, W., Van Arendonk, J.A., 2000. Whole genome scan to detect quantitative trait loci for conformation and functional traits in dairy cattle. J. Dairy Sci. 83, 795 – 806.