genetic diversity in swiss cattle breeds

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  • 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

    U.S. Copyright Clearance Center Code Statement: 09312668/99/16010001 $14.00/0

  • 2 M. Schmid et al.

    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 topologie