J. Anim. Breed. Genet. 116 (1999), 18 Ms. received: 6.4.1998 1999 Blackwell Wissenschafts-Verlag, BerlinISSN 09312668

Institute of Animal Breeding, University of Berne, 3012 Berne, Switzerland

Genetic diversity in Swiss cattle breeds

By M. SCHMID, N. SAITBEKOVA, C. GAILLARD and G. DOLF

Introduction

First attempts at establishing the genetic relationships among cattle populations relied onarcheological evidence (EPSTEIN 1971; EPSTEIN and MASON 1984) and protein polymorphisms(BAKER and MANWELL 1980, 1991). LOFTUS et al. (1994) examined mitochondrial DNA todetermine the divergence time between Bos taurus and Bos indicus. Today most studies ongenetic diversity are based on microsatellite analysis (LITT and LUTY 1989; TAUTZ 1989;WEBER and MAY 1989). Microsatellites were used in, e.g. man (BOWCOCK et al. 1994), canids(ROY et al. 1994; FREDHOLM and WINTERO 1995) and sheep (BUCHANAN et al. 1994). Recentstudies in cattle are also microsatellite based (e.g. MACHUGH et al. 1994; CIAMPOLINI et al.1995; MOAZAMI-GOUDARZI et al. 1997) and aim at facilitating the development of man-agement programs for endangered breeds (FAO 1981).

Our microsatellite-based investigation on the genetic diversity between and within Swisscattle breeds included Original Swiss Brown, purebred Simmental, Holstein, Herens andEvole`nard. Previous studies in Swiss breeds made use of blood group systems (REUSE1969), serum transferrin and hemoglobin (KRUMMEN 1964), amylase (BUSER 1970) andcarboanhydrases (KASTLI et al. 1980).

The Herens breed is endemic to the canton of Wallis. The Evole`nard, which are very fewin numbers and restricted to a single valley in the canton of Wallis, are phenotypically verysimilar to the Herens with the exception of the coat colour. In the Aosta valley (Italy) whichborders the canton of Wallis these two breeds find their counterparts. The phenotype ofthe Aosta Chestnut fits the Herens and the Aosta Black Pied fits the Evole`nard. TheHolstein breed replaced the Fribourg breed which was a colour variant of the purebredSimmental breed (ENGELER et al. 1961) and is now extinct. The Original Swiss Brown andthe purebred Simmental are endemic to Switzerland.

Materials and methods

Animals entering this study were randomly chosen among primiparous cows and bullsbetween 1 and 2 years of age, without checking their relatedness. Blood samples werecollected from 44 females and six males each in Holstein, purebred Simmental, OriginalSwiss Brown and Herens, and 15 cows in Evole`nard. DNA was extracted using theChelex method (Chelex 100 Resin, BIO-RAD, Hercules, CA, USA) (WALSH et al. 1991).

The 30 microsatellites analysed (Table 2) correspond to the loci on a list currently inuse in an European project focusing on genetic diversity in European cattle breeds. Thelist of loci together with the relevant references is available at http://www.ri.bbsrc.ac.uk/cdivwww/homepage.htm.

ILSTS005 was analysed using radioactive labelling as previously described (DOLF et al.1996). All other loci were analysed using nonradioactive labelling. The polymerase chain

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Table 1. PCR reaction mixture for the microsatellites investigated by infrared labelling (LI-COR)

Quantity of DNA 2 ml Chelex extraction of 50 ml blooddNTP 52 08 pmol/mlTris-HCl 2 01 nmol/mlMgCl2 156 25 pmol/mlKCl 5 2 nmol/mlPrimers (infrared labelled) 0 2 pmol/mlTaq polymerase (Appligene) 0 029 units/mlEnd volume 12 ml

reaction (PCR) mixture for these microsatellites is given in Table 1. The PCR productswere run on a 18 cm, 8% denaturing polyacrylamide gel on a LI-COR DNA sequencer(model 4000 L; Li-COR, Lincoln, NE, USA) according to the manufacturers rec-ommendations. In addition to an infrared-labelled marker (IRD 400044, MWG), a cattlefamily including sire, mother and one offspring was used to verify the correct range of theobserved alleles.

Allele frequencies and HardyWeinberg equilibrium were calculated using the GENE-POP package version 3.1 (RAYMOND and ROUSSET 1995). Genetic diversity (H) can bemeasured as the amount of actual or potential heterozygosity as H = 1 S pi2 (pi is thefrequency of the ith allele). This corresponds to the probability that two randomly chosenalleles are different. If the population is in HardyWeinberg equilibrium, then H is ameasure of the actual heterozygosity. The level of subdivision in large populations can bemeasured by comparing the diversity of alleles within a subpopulation (HS) to that of thetotal population (HT) which measures the diversity of the total population if it wereconverted into a single randomly mating unit. The coefficient of gene differentiation, GST(NEI, 1973), measures the relative extent of gene differentiation among subpopulations andis defined as GST = (HT HS)/HT (HS is the average diversity of the subpopulations). It wascomputed by the program DISPAN (OTA, 1993).

Genetic distances between populations were measured with Cavalli-Sforza and Edwardschord distance DC (CAVALLI-SFORZA and EDWARDS 1967). This distance was calculated withthe program PHYLIP (FELSENSTEIN 1995). In addition, phylogenies were also estimated bythe restricted maximum likelihood method (REML) (FELSENSTEIN 1981) using the programPHYLIP. Neighbour-joining trees (SAITOU and NEI 1987) were constructed with the pro-gram PHYLIP. The reliability of the trees was obtained by a bootstrap test with 1000 re-samplings per locus. The principal component analysis was computed using the SAS pro-gram PRINCOMP (SAS 1990) according to the recommendation of CAVALLI-SFORZA et al.(1994).

Results

Table 2 summarizes the data characterizing the 30 microsatellites investigated. All loci werepolymorphic in the five breeds with total allele numbers ranging from 2 in ILSTS005 to 16in TGLA122. Great variations were found among breeds with respect to the total numberof alleles at a given locus, e.g. no variation at ILSTS005 and INRA005 but a difference ofsix alleles at HEL9 and TGLA53. The Holstein displayed the largest total number of alleles(n = 196) and the Evole`nard showed the lowest total number of alleles (n = 141).

The overall heterozygosity of the whole population investigated amounted to 0.70 (Table2). All values for the single loci but two ranged from 0.55 to 0.89. The two values mentionedbelonging to loci ILSTS005 and INRA035 were below 0.4. The average coefficient of gene

3Genetic diversity in Swiss cattle breeds

Table 2. Number of alleles in each breed, average heterozygosity HT and coefficient of dif-ferentiation GST of each microsatellite (30 loci)

Total no. Number of alleles in each breed Total population 95Marker alleles breeds)

SI HOL OB HER EVO HT GSTBM1818 8 6 6 6 6 5 0 66 0 07BM1824 6 4 5 5 5 4 0 72 0 08BM2113 8 7 7 8 7 6 0 86 0 10CSRM60 7 6 6 6 5 4 0 71 0 04CSSM66 13 9 10 9 7 5 0 72 0 11ETH3 8 6 6 4 6 5 0 68 0 04ETH10 7 3 7 4 4 3 0 64 0 16ETH152 6 4 6 4 5 4 0 67 0 05ETH185 11 4 9 7 6 5 0 74 0 06ETH225 8 6 7 6 6 5 0 72 0 06HAUT24 9 6 6 7 6 3 0 80 0 09HAUT27 10 6 8 7 5 5 0 79 0 09HEL1 8 6 7 6 6 4 0 69 0 03HEL5 8 7 6 7 6 3 0 71 0 15HEL9 13 4 9 5 10 5 0 80 0 13HEL13 4 3 3 4 4 3 0 55 0 02ILSTS005 2 2 2 2 2 2 0 39 0 21ILSTS006 7 6 5 5 6 4 0 71 0 15INRA005 3 3 3 3 3 3 0 65 0 03INRA023 11 7 7 7 10 9 0 76 0 10INRA032 7 5 6 4 5 5 0 65 0 08INRA035 5 3 3 4 2 2 0 35 0 03INRA037 12 10 7 6 9 8 0 76 0 06INRA063 7 6 4 5 3 2 0 58 0 12MM12 9 6 5 7 6 4 0 71 0 05SPS115 9 4 7 6 8 8 0 67 0 06TGLA53 15 10 13 10 8 7 0 89 0 09TGLA122 16 8 12 9 8 7 0 83 0 13TGLA126 6 4 5 4 6 4 0 69 0 09TGLA227 12 7 9 9 11 7 0 83 0 06

Total 255 168 196 176 181 141 0 70 0 09

SI, Simmental; HOL, Holstein; OB, Original Brown Swiss; HER, Herens; EVO, Evole`nard

differentiation (GST) over all 30 loci was 0.09. The GST values for single loci ranged from0.02 for HEL13 to 0.21 for ILSTS005.

The average expected heterozygosity within breeds (HS) ranged from 0.60 in Simmentalto 0.69 in Holstein (Table 3). The number of private alleles (Table 3) found among the fivebreeds varied considerably, from 1 in Evole`nard to 26 in Holstein. However, only very fewof the private alleles showed a frequency greater than 0.1.

The HardyWeinberg equilibrium was tested for all breedlocus combinations (n = 150)and combined by breed using Fishers method (FISHER 1958). With the exception of Herensall breeds were in HardyWeinberg equilibrium. In Herens the loci ETH152 and TGLA227showed a highly significant departure from HWE (p 0.001).

The matrix of DC values given in Table 4 compared each pair of breeds. The low value of0.029 between Evole`nard and Herens indicated a close relationship between these twobreeds. All the other distances were two to three times larger. The values involving Holsteinwere generally the largest ranging from 0.070 to 0.084. Based on the matrix of DC values a

4 M. Schmid et al.

Table 3. Average heterozygosity HS and and private alleles for each breed

Standard Number of private allelesHS error Total Frequency 0.1

Simmental 0 60 2 0 03 6 1Holstein 0 69 2 0 02 26 3Original Swiss Brown 0 67 2 0 02 14 3Herens 0 63 2 0 03 8 0Evole`nard 0 65 2 0 03 1 1

Table 4. Matrix of Cavalli-Sforza and Edwards chord distance DC calculated with 30 micro-satellite loci

SI HOL OB HERSimmental (SI)Holstein (HOL) 0 070Original Swiss Brown (OB) 0 058 0 070Herens (HER) 0 066 0 078 0 068Evole`nard (EVO) 0 071 0 084 0 070 0 029

phylogenetic tree was constructed using Holstein as an outgroup (Fig. 1). Herens andEvole`nard clearly formed a tight cluster supported by a bootstrap value of 100%. Thecluster formed by Simmental and Original Brown Swiss was weakly supported with abootstrap value of 35%. The topologies of the phylogenetic trees based on the REMLmethod as well as the corresponding bootstrap values were very similar to the data presentedin Figure 1 and therefore omitted.

Principal component analysis including all 30 microsatellite loci confirmed the phylo-genetic trees (Figure 2). The first three components explained 86% of the total variation,that is the first component 35%, the second 29% and the third 22%. The first componentclearly separates Holstein from Herens and Evole`nard but also from Simmental and OriginalSwiss Brown. The second component further separates Simmental and Original SwissBrown from Holstein but also from Herens and Evole`nard. In addition, Original SwissBrown is slightly set apart from Simmental. The third component differentiates Simmentalfrom the other breeds, notably from Original Swiss Brown.

Discussion

In this survey we used the Cavalli-Sforza and Edwards chord distance DC and the REMLmethod to reconstruct the phylogenies because the primary determinant of evolutionarydifferentiation is the genetic drift. In a simulation study TAKEZAKI and NEI (1996) were ableto show, that the probability to obtain a correct tree topology with DC is better than otherdistance measures irrespective of population size changes.

The most striking result is the very close kinship between the Herens and the Evole`narddocumented by the very low value of DC, the clustering of those breeds with a bootstrapvalue of 100% and the very close position nearly in the same spot in the principal componentanalysis. This is in accordance with historical evidence for the close kinship and ancientorigin of Herens and Evole`nard (ANONYMOUS, 1997). In the 19th century these breeds were

5Genetic diversity in Swiss cattle breeds

Fig. 1. Phylogenic tree based on the matrix of Cavalli-Sforza and Edwards chord distance DC

Fig. 2. Principal components analysis based on allele frequencies of 30 loci. SI, Simmental; OB, OriginalBrown Swiss; HOL, Holstein; HER, Herens; EVO, Evole`nard

recognized based on their coat colour. Although solid coloured animals only have beenaccepted in the Herens herd book since 1920, animals showing white markings still do

6 M. Schmid et al.

occur. The coat colour is the only means to distinguish the pied Evole`nard from the solidcoloured Herens.

The weak clustering of Simmental with Original Swiss Brown may hint at the possibilityof limited admixture. Although very distinct in phenotype and history these two breedsshared the alpine region for many centuries whereas Holstein joined the endemic Swissbreeds only very recently.

The deviation from Hardy-Weinberg equilibrium for ETH152 and TGLA227 was causedby an excess of homozygous animals in Herens. Assortative mating, population subdivisionsand occurrence of null alleles (CHAKRABORTY et al. 1992; CALLEN et al. 1993) are possibleexplanations for such a situation. In this context assortative mating offers the best expla-nation since Herens are bred for aggressive behavior to be displayed in cow fights, a selectioncriteria unique to this breed. Other markers, such as blood group systems or serum proteins,were investigated several times over the years (KRUMMEN 1964; REUSE 1969; BUSER 1970,KASTLI et al. 1980, our own unpublished results) but only the F/V blood group system wasnot in HWE in Herens (our own unpublished results).

The relative low number of alleles found in Evole`nard can be attributed to the smallpopulation size where loss of alleles are expected due to fixation. The large number ofprivate alleles in Holstein may reflect their distance to the other breeds.

Conclusions

The present investigation confirms the expected close relationship between Herens andEvole`nard and shows a tentative relationship between Simmental and Original Swiss Brown.The evaluation of our data is a first step in the European-wide effort (http://www.ri.bbsrc.ac.uk/cdivwww/homepage.htm) to establish genetic relationships among the Europeancattle breeds. Only the joint analysis of all the data generated within this European col-laboration will make full use of the data supplied by the single laboratories.

Acknowledgements

We thank the Swiss Association for Artificial Insemination for financial support and the SchweizerischeFleckviehzuchtverband, Schweizerische Braunviehzuchtverband, Federation Suisse delevage Holsteinand the Federation delevage de la race dHerens for the sampling of their animals. Also we wantto thank all who helped collecting blood samples, notably J. CARRUZZO, A. FLUCKIGER and F-J.SCHAWALDER. We thank B. COLOMB and U. SATTLER for their technical support.

Summary

The estimation of the genetic diversity in Swiss cattle breeds was based on the analysis of 30 micro-satellites. DNA from a total of 215 randomly chosen animals belonging to five breeds (Holstein,Original Swiss Brown, Simmental, Herens and Evole`nard) was investigated. The overall heterozygosityof the whole population amounted to 0.70. The average expected heterozygosity within breeds rangedfrom 0.60 in Simmental to 0.69 in Holstein. All breeds except the Herens were in HardyWeinbergequilibrium. The loci ETH152 and TGLA227 in Herens deviated significantly from HardyWeinbergequilibrium. A very close kinship between the Herens and the Evole`nard was made evident by the verylow value of the genetic distance DC, the phylogenetic trees and the very close position in the principalcomponent analysis. No other significant clustering was found. The Holstein showed the largest valuesof genetic distances to the other breeds.

Zusammenfassung

Genetische Diversitat zwischen und innerhalb der Schweizer Rinderrassen

Die Schatzung der genetischen Diversitat zwischen und innerhalb der Schweizer Rinderrassen basierteauf der Analyse von 30 Mikrosatelliten. DNA von 215 zufallig ausgewahlten Tieren, welche funf Rassenangehorten (Holsteiner, Original Braunvieh, Simmentaler, Eringer und Evole`ne), wurde untersucht.Der Heterozygotiegrad uber alle Loci der Gesamtpopulation belief sich auf 0.70. Der durchschnittlicheHeterozygotiegrad innerhalb der Rassen variierte zwischen 0.60 bei den Simmentalern und 0.69 bei

7Genetic diversity in Swiss cattle breeds

den Holsteinern. Alle Rassen mit Ausnahme der Eringer waren im HardyWeinberg-Gleichgewicht.Bei den Eringern zeigten die Loci ETH152 und TGLA227 eine signifikante Abweichung vom HardyWeinberg-Gleichgewicht. Die sehr enge Verwandtschaft zwischen den Eringern und den Evole`ne kamdeutlich zum Ausdruck bei den phylogenetischen Baumen und dem tiefen Wert der genetischen DistanzDC. Zudem liegen diese beiden Rassen in der Hauptkomponentenanlyse sehr nahe beieinander. Eswurden keine weiteren signifikanten Kladen gefunden. Die Holsteiner wiesen die hochsten Werte dergenetischen Distanz zu den anderen Rassen auf.

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Authors address: Gaudenz DOLF, Institute of Animal Breeding, University of Berne, Bremgar-tenstrasse 109a, 3012 Berne, Switzerland. E-mail: dolf.gaudenz@itz.unibe.ch