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Microsatellite DNA Variation within and among European Cattle Breeds Author(s): David E. Machugh, Ronan T. Loftus, Daniel G. Bradley, Paul M. Sharp and Patrick Cunningham Source: Proceedings: Biological Sciences, Vol. 256, No. 1345 (Apr. 22, 1994), pp. 25-31Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/49589Accessed: 17-09-2015 11:21 UTC

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Microsatellite DNA variation within and among European cattle breeds

DAVID E. MACHUGH, RONAN T. LOFTUS, DANIEL G. BRADLEY, PAUL M. SHARP AND PATRICK CUNNINGHAM

Department of Genetics, Trinity College, Dublin 2, Ireland

SUMMARY

Microsatellite markers offer great potential for genetic comparisons within and between populations. We report the analysis of 12 microsatellite loci in six breeds of European cattle. This yielded a wide spectrum of variability with observed heterozygosities ranging from 0.00 to 0.91. Deviations from Hardy-Weinberg equilibrium were noted for some locus-population combinations, particularly at a microsatellite located within the prolactin gene. Also, significant linkage disequilibrium was detected between two microsatellite loci located within the bovine major histocompatibility complex, and this association was maintained across breeds, providing evidence for marker stability during short-term evolution. The mode of mutation was investigated by comparing the observed data with that expected under the infinite alleles model of neutral mutation, and six of the microsatellite loci were found to deviate significantly, suggesting that a stepwise mutation model may be more appropriate. One indication of marker utility is that, when genetic distance estimates were computed, the resultant dendrogram showed concordance with known breed histories.

1. INTRODUCTION

Cattle breeds have existed as discrete socioeconomic entities for some hundreds of years at most, although undoubtedly many have been separated as landraces for much longer. Because of their cultural importance, the study of the genetic relatlionships between cattle breeds has been historically a very active area of biological systematics. Previous blood group and protein studies have illustrated the close genetic relationships among breeds of European Bos taurus cattle, and their marked distinction from the humped zebu or Bos indicus cattle breeds of Asia and Africa (for review see Baker & Manwell (1991)). Also, we have recently shown that, whereas a matriarchal phylogeny derived from mitochondrial sequence data provides sufficient data to support a deep dichotomy between B. taurus and B. indicus, there is no evidence of mito- chondrial substructuring within European cattle (Loftus et al. 1994).

Recently the microsatellite, a new class of genetic marker, has been described which is based on length variation within tandem arrays of di-, tri- or tetra- nucleotide motifs (Litt & Lutty 1989; Weber & May 1989). These regions are widely dispersed in mam- malian genomes and can be typed by using the polymerase chain reaction (PCR). The underlying mutational dynamics of microsatellites are still poorly understood, and there have been few systematic studies cataloguing the extent of polymorphism within and between populations. Slipped-strand mispairing dur- ing DNA replication is accepted as the predominant

mutational event at these loci (Levinson & Gutman 1987), but it is unclear whether the allelic distributions are in accordance with the infinite alleles model (IAM)

of mutation (Kimura & Crow 1964) or with a stepwise mutation model (sMM) (Kimura & Ohta 1978). The former model assumes that new mutations occurring are always different from the extant alleles in the population. Conversely, the incremental stepwise model implies the possibility of length convergence among alleles which are not identical by descent (homoplasy), a phenomenon which may complicate comparisons between populations (Valdes et al. 1993).

Currently, there is a concerted effort to construct a high-density map of the bovine genome (Fries et al. 1993) and, as a result, many microsatellite markers are becoming available. If these markers prove amenable to population-level comparisons, they will become valuable tools for evolutionary studies, for guiding genetic conservation of endangered breeds, and for historical studies of cattle domestication and migration.

In this paper we present results from a survey of 12 microsatellite loci in six geographically distinct breeds of European Bos taurus cattle. We describe the population genetics of these loci by using standard genetic parameters, and estimate the relationships among the breeds.

2. MATERIALS AND METHODS

(a) Sample collection

Blood samples were collected from bulls in Artificial Insemination stations throughout Ireland, and pedigree

Proc. R. Soc. Lond. B (1994) 256, 25-31 25 C) 1994 The Royal Society Printed in Great Britain

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Table 1. Microsatellite loci used in the study

(Locus code and map position or synteny group from the bovine gene map (Fries et al. 1993). Number of alleles detected and most common allele were derived from the pooled European sample.) r

may- number common gene-clone locus code synteny repeat type PCR primers (5'-3') of alleles allele b.p. source or reference

,B-globin HBB U19 (TA)n(CA). GAT ATA AAA AAG AAG ACC CAG TAG 9 112 Moore (1992) TAC CTG AGT CAT ATG TAA TAT TCC

MHC class II DR /3 pseudogene BOLA-DRBP1 23 (CA)n(CT)n ATG GTG CAG CAG CAA GGT GAG CA 11 120 Creighton et al. (1992) GGG ACT CAG TCT CTC TAT CTC TTT G

MHC class II DR ,62 gene BOLA-DR2B 23 (CA).(CT). AGG CAG CGC CGA GGT GAG CGA 2 152 Fries et al. (1993) TCC AAC ACT CAC CTG GAC GTA GC

RAS p2l protein activator RASA 7 (CA). CCC TTC CGC TTT AGT GCA GCC AG 5 188 Eggen et al. (1992) GGG CCA CAG CCC AGG ATC GGG AGC

Retinol-binding protein 3 RBP3 U29 (CA) (TA)n TGT ATG ATC ACC TTC TAT GCT TC 4 141 GenBank (Borst et al. GCT TTA GGT AAT CAT CAG ATA GC 1989) M20748

Opioid binding molecule OCAM U7 (CT), CCT GAC TAT AAT GTA CAG ATC CCT C 4 184 Moore et al. (1991) GCA GAA TGA CTA GGA AGG AAG GAT GGC A

Prolactin PRL 23 (CA). GGA AAG TGA ACA TGA CTG TCT AG 3 162 Creighton et al. (1992) GCC CTC TCT TCT ACA ATG AAC AC

MISATA not assigned not assigned (CA). AGT CCA TGG GAT TGA AAG AGT TGG 7 113 GenBank (Kaukinen & CTT TTA TTC AAC AGC TAT TTA ACA AGG Varvio 1992) X65202

ETH152 D5S1 5 (CA). TAC TCG TAG GGC AGG CTG CCT G 7 199 Steffen et al. (1993) GAG ACC TCA GGG TTG GTG ATC AG

ETH225 DU2S1 U2 (CA). GAT CAC CTT GCC ACT ATT TCC T 6 150 Steffen et al. (1993) ACA TGA CAG CCA GCT GCT ACT

ILSTS001 not assigned not assigned (CA). GGT GCT GTT ATC TAG AAT TTG G 7 93 Brezinzky et al. (1992a) GGA GTC ATA CAC AAC TGA GC

ILSTS014 not assigned not assigned (CA). CTG ACT ATG GTG ATA ATC CC 3 130 Brezinzky et al. (1992b) TCT TTT CCC TTT CCT TCC CC

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Microsatellite variation in cattle D. E. MacHugh and others 27

0.5 05 (a) (b)

0.4

0.3

0.2- - __

0.1

0.0

0.5 (c) (d)

0.4 -___._

0.3 -

0.2 -

0.1

0.0

0.5 (e) (f)

0.4

0.3__ _

0.2-

0.1

0.0 _ 140 142 144 146 148 150 152 140 142 144 146 148 150 152

allele length / b.p. allele length / b.p.

Figure 1. Allelic distributions for the ETH225 microsatellite in the six breeds in the study: (a) Aberdeen Angus (n = 66); (b) Jersey (n = 68); (c) Hereford (n = 68); (d) Friesian (n = 80); (e) Simmental (n = 72); (f) Charolais (n = 72). The sample size (n) is the number of chromosomes tested. PCR product lengths (allele classes) are indicated.

records were consulted to ensure the animals were unrelated. Supplementary semen straw samples (20 for each breed) were obtained from the British Milk Marketing Board (Genus). The breeds included are: Aberdeen Angus (33), northeast Scotland; Charolais (36), central France; Friesian (40), The Netherlands; Hereford (34), west England; Jersey (34), Channel Islands; Simmental (36), west Switzerland.

DNA from blood samples was extracted by using standard procedures (Sambrook et al. 1989). DNA from semen was extracted by using a mercaptoethanol-proteinase K-based procedure according to Andersson et al. (1986).

(b) Genetic loci studied

The 12 microsatellite loci studied and their sources are detailed in table 1. Ten of the markers were obtained from published reports, and two markers were characterized by searching the GenBank/EMBL/DDBJ DNA sequence data library for bovine sequence entries containing dinucleotide repeat regions.

(c) Genotype determinattions

Polymerase chain reactions were done on 30 ng template DNA in 10 p1 reaction volumes using 0.5 units of Taq

polymerase with reaction buffer comprising (in millimoles per litre): 500 KCl, 100 Tris-HCl, pH 9.0; 15 MgCl2, 1 % Triton X-100, 200 ZtM dATP, dGTP, dTTP, 10 ZtM dCTP; 0.3 ZtM each primer was added, as was 0.5 iCit [x_32P] dCTP at 3000 Ci mmol-1 per reaction. These were overlaid with oil, and amplifications were done in a Hybaid OmniGene thermal cycler using 96-well microtitre plates. A 4 min denaturation step at 94 ?C was followed by 35 cycles of 45 s denaturation at 93 ?C, 45 s annealing at 55, 60 or 65 ?C (depending on the primer pair), 45 s extension at 72 ?C, and a 4 min final extension step. Samples were mixed with 1 volume of formamide dye solution, denatured at 95 ?C for 3 min and loaded onto a standard 60% polyacrylamide denaturing sequencing gel (Sambrook et al. 1989); pUC19 sequencing templates or previously amplified samples were also run to allow determination of allele lengths.

(d) Computations and statistics

Allele frequencies were determined by gene counting, and standard errors were computed as the square root of the variance of a binomial distribution. Unbiased estimates of gene diversity or expected heterozygosity (H) and X2 tests of

t I Ci = 3.7x 1010Bq.

Proc. R Soc. Lond. B (I1994)

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28 D. E. MacHugh and others Microsatellite variation in cattle

Table 2. For each locus-population combination, gene diversity estimates, observed heterozygosity, and x2 valuesfor deviationsfrom Hardy- Weinberg equilibrium are shown in vertical sequence

(The x2 values were calculated from expected genotype frequencies against observed genotype frequencies. Degrees of freedom are enclosed in parentheses after each x2 value.)

A. Angus Charolais Friesian Hereford Jersey Simmental pooled locus (n = 33) (n = 36) (n = 40) (n = 34) (n = 34) (n = 36) (n = 213)

HBB 0.56 0.72 0.74 0.64 0.67 0.72 0.77 0.82 0.61 0.78 0.76 0.62 0.78 0.73 11.0 (3) 39.0 (21)** 26.3 (28) 7.1 (6) 10.6 (10) 10.6 (21) 86.2 (36)***

BOLA-DRBP1 0.78 0.83 0.80 0.81 0.82 0.85 0.84 0.73 0.81 0.90 0.91 0.82 0.81 0.83

22.0 (21) 29.5 (45) 28.7 (28) 14.0 (21) 27.1 (21) 26.5 (36) 50.2 (45) BOLA-DR2B 0.34 0.33 0.43 0.09 0.16 0.32 0.29

0.36 0.42 0.45 0.09 0.18 0.28 0.30 0.3 (1) 2.5 (1) 0.2 (1) 0.1 (1) 0.3 (1) 0.5 (1) 0.1 (1)

RASA 0.50 0.57 0.56 0.47 0.36 0.52 0.54 0.55 0.53 0.55 0.38 0.44 0.61 0.51 0.4 (1) 0.6 (3) 10.7 (6) 1.1 (1) 0.7 (1) 3.9 (6) 56.0 (10)***

RBP3 0.03 0.18 0.17 0.06 0.26 0.41 0.20 0.03 0.19 0.18 0.06 0.18 0.47 0.19 0.0 (1) 0.4 (6) 0.4 (6) 0.0 (1) 6.5 (3) 1.8 (3) 3.5 (6)

OCAM 0.12 0.03 0.05 0.06 0.00 0.06 0.05 0.12 0.03 0.05 0.09 0.00 0.06 0.06 0.1 (1) 0.0 (1) 0.0 (1) 0.0 (3) 0.0 (1) 0.2 (6)

MISATA 0.50 0.71 0.66 0.58 0.63 0.64 0.68 0.55 0.67 0.55 0.65 0.59 0.67 0.61 3.1 (3) 12.6 (6) 6.9 (15) 1.4 (3) 3.2 (6) 4.4 (10) 26.1 (21)

PRL 0.58 0.13 0.18 0.16 0.29 0.06 0.26 0.27 0.08 0.20 0.06 0.29 0.06 0.16

20.7 (3)*** 4.5 (1)* 0.5 (3) 13.7 (1)*** 0.0 (1) 0.0 (1) 47.8 (3)*** ETH152 0.59 0.74 0.72 0.67 0.62 0.55 0.72

0.61 0.75 0.73 0.68 0.68 0.47 0.65 14.7 (15) 2.8 (6) 17.7 (15) 1.3 (6) 3.0 (6) 2.9 (10) 19.6 (21)

ETH225 0.75 0.65 0.80 0.79 0.68 0.74 0.79 0.85 0.58 0.80 0.79 0.82 0.83 0.78 17.0 (10) 15.6 (10) 15.7 (15) 12.1 (15) 7.2 (6) 7.4 (10) 17.6 (15)

ILSTSOO1 0.09 0.41 0.47 0.14 0.03 0.18 0.24 0.09 0.47 0.53 0.15 0.03 0.19 0.25 0.1 (1) 1.8 (3) 6.8 (15) 0.2 (6) 0.0 (1) 0.4 (3) 4.3 (21)

ILSTSO14 0.14 0.22 0.27 0.37 0.40 0.13 0.26 0.15 0.25 0.28 0.41 0.29 0.14 0.25 0.2 (1) 0.7 (1) 0.0 (1) 1.9 (3) 2.0 (1) 0.2 (1) 0.6 (3)

*p <0.05. **p<0.01. ***p<0.001.

genotype frequencies for deviations from Hardy-Weinberg equilibrium were calculated by using the GENET2 program (Quesada et al. 1992). Linkage disequilibrium arising from allelic association between markers known to be linked or on the same chromosome was tested by using the ASSOCIATE

computer program (Ott 1985). Expectations for the number of alleles under the infinite alleles neutral mutation model were derived as described by Ely et al. (1992). Genetic distance estimation and phylogenetic analyses were done using the DISPAN computer program (T. Ota, personal communication).

3 RESULTS

(a) Allele frequencies

Allele frequencies and their standard errors were calculated from the genotypes of individual animals for

the 72 combinations of 12 loci and six populations (data available from the authors on request). All loci were found to be polymorphic in the six breeds, with the single exception of OCAM in the sample of 34 Jersey animals. In this population an allele length of 184 base pairs (b.p.) appears to be fixed. Allelic frequencies for most loci were variable among breeds, and only six markers were found to have the same predominant allele in all breeds (BOLA-DR2B; RBP3; OCAM; PRL; ILSTSOO1; ILSTS014). Figure 1 shows the allelic distributions obtained for ETH225, which illustrates an average level of variability among populations. However, the number of alleles detected at some loci seems to vary quite dramatically between breeds (e.g. HBB: three alleles in A. Angus, eight in Friesian). Also, one consistent trend that emerges is that there is a reduction in the observed number of

Proc. R. Soc. Lond. B (1994)

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Microsatellite variation in cattle D. E. MacHugh and others 29

(a)

A. Angus * Charolais 0.123 ****

Friesian 0.102 0.041 **** Hereford 0.129 0.077 0.051 ****

Jersey 0.088 0.089 0.078 0.091 **** Simmental 0.118 0.056 0.041 0.066 0.095 **** N'Dama 0.248 0.142 0.183 0.228 0.235 0.178 ****

(b) 81 A.Angus

34 Jersey

41 - Hereford

72 Friesian

Simmental

Charolais 0 0.05

N'Dama Figure 2. Genetic distance values calculated from 11 microsatellite loci (excluding BOLA-DR2B; see text) and resultant dendrogram. (a) Matrix of Nei's (1978) standard genetic distance. (b) Dendrogram constructed by the neighbour-joining method. Numbering on the nodes indi- cated the number of times a particular branch was recovered per 100 bootstrap replications following 1000 replications. The scale bar represents a length of 0.05.

alleles across all loci in the British Isles breed group (A. Angus, Hereford and Jersey: mean = 3.3 alleles) as opposed to the mainland Europe group (Charolais, Friesian and Simmental: mean = 4.3 alleles).

(b) Tests for Hardy- Weinberg equilibrium

A X2 test for deviations from expected Hardy- Weinberg genotype frequencies was done for each locus-population combination and detailed in table 2. Three of the microsatellite loci (HBB, RASA and PRL) deviate from Hardy-Weinberg equilibrium (HWE) in the combined sample, and this is probably due to the pooling of data from reproductively isolated populations (the Wahlund effect). Only PRL shows consistent deviation across breed groups. The PRL microsatellite is a (TG)f(TA)1(TG). (n 6) repeat region with three alleles (158 b.p., 162 b.p. and 164 b.p.) located between a 5' distal regulatory element and the bovine prolactin coding sequence (Wolf et al. 1990). A similar allele distribution is seen in genetically distant Indian and African Bos indicus populations (unpublished data), suggesting it is probably of ancient origin. Prolactin is a major protein product of the anterior pituitary gland of mammals, it is essential for traits such as milk production, and the observed deviation from HWE at the PRL microsatellite may be due to linkage disequilibrium with functionally rel- evant sequence variants in this or neighbouring genes. European cattle have been exposed to centuries of

artificial selection for milk production, and this has intensified dramatically since the 1950s with the introduction of modern breeding programmes and artificial insemination. Interestingly, however, the populations displaying the genotypic imbalance are the three breeds with beef production as a primary selection goal in breeding programmes (Angus, Charolais and Hereford).

(c) Linkage disequilibrium on chromosome 23

In cattle, the prolactin gene and the major histo- compatibility complex (BoLA) have both been map- ped to chromosome 23 by in situ hybridization (Fries et al. 1986; Hallerman et al. 1988). Strong allelic association (P < 0.01), presumably due to linkage disequilibrium, was detected between BOLA-DRBPI and BOLA-DR2B in all breeds except the Hereford, and was maintained in the pooled sample (P < 0.00 1). The Hereford population is almost fixed for the 152 b.p. allele (P = 0.91), and this would make allelic association difficult to detect without a much larger sample size. Maintenance of linkage disequilibrium across breeds in the pooled sample suggests that few new length mutations have occurred since the breeds diverged from a common ancestor, an observation which contributes to the growing body of evidence that microsatellite polymorphisms are stable in a short-term evolutionary context (Sherrington et al. 1991; Richards et al. 1992). No linkage disequilibrium was detected between either of the BoLA microsatellites and the PRL microsatellite and, presumably, the reported recombination rate of 5 0o (Creighton et al. 1992) is sufficient to prevent allelic association between these two regions.

(d) Evaluation of mutation models

The expected number of alleles under the infinite alleles model (IAM) of neutral mutation was calculated and compared with the actual number of alleles observed in the populations. Means for the observed and expected values were also calculated under the assumption that they represent an average of in- dependent samples of six typical cattle populations. A paired t-test was calculated to determine if there were any significant differences between the observed number of alleles and the expectations under the IAM.

If the observed number of alleles is significantly lower than the expected number of alleles it suggests that the SMM is a better description of the allelic distribution at a particular locus (Deka et al. 1991). Under these criteria, six of the loci investigated in these populations (HBB; BOLA-DRBPI; RASA; MISATA; ETH152 and ETH225) are in closer accordance with a stepwise allelic distribution.

(e) Genetic distance values and dendrogram

An outgroup population was included for the genetic distance and phylogenetic analysis. This consisted of 63 animals of the taurine N'Dama breed sampled in Guinea, West Africa. Nei's (1978) standard genetic

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30 D. E. MacHugh and others Microsatellite variation in cattle

distances were estimated for the 21 pairwise breed comparisons using 11 loci, excluding the least in- formative of the two loci found to be in linkage disequilibrium (BOLA-DR2B), and are presented in figure 2. Also shown is a rooted neighbour-joining tree summarizing these values (Saitou & Nei 1987), including bootstrap values from 1000 replications of resampled loci. The most robust features of the topology are the Jersey-Angus grouping and the placement of the Charolais breed as an outgroup to the other breeds. Notably, the overall topology is consistent with that expected on biogeographical grounds, and short branch lengths and low bootstrap values between breeds suggest historical admixture between popula- tions.

4. DISCUSSION

Generally Hardy-Weinberg equilibrium was ob- served within the populations tested. However, signifi- cant deviations detected in a subset of loci in the pooled sample suggest some degree of genetic heterogeneity among European cattle populations. Similarly, Ed- wards et al. (1992) found significant deviations from HWE in their pooled sample of human populations, and few deviations within populations. Artificial selection is the major genetic influence on modern domestic cattle populations, and the only locus found to be deviating from HWE in more than one breed is a microsatellite within the gene encoding prolactin, a major deter- minant of milk production in mammals.

Linkage disequilibrium was detected between two very closely linked microsatellites on chromosome 23 and was maintained under testing of a pooled sample of all breeds. This would not be expected if the mutation rate at either locus were high enough to mimic the effects of recombination through convergent allele evolution (homoplasy). Indeed, one of the loci found to show strong linkage disequilibrium (BOLA- DRBP 1) was the most variable of all 12 loci examined with 11 allele length classes and represents one of the most polymorphic of all reported bovine microsatellites (Fries et al. 1993). This result suggests that micro- satellite markers are sufficiently stable for informative comparisons between even distantly related popula- tions. In addition, the utility of microsatellites for population-level discrimination may be enhanced by haplotypic analysis of several closely linked loci.

The estimated genetic distance values for the six breeds and groupings summarized in the dendrogram (figure 2) can be compared with the classification scheme discussed by Baker & Manwell (1991) based on protein polymorphisms. In their study, the Hereford and Aberdeen Angus breeds were clustered as expected on biogeographical grounds. However, contrary to our results, the Channel Island breeds formed a grouping distinct from the other British breeds. In our study, the Hereford breed does not show strong affinities with the other two British Isles breeds, and this may reflect historical genetic input from northern Europe, which is known to have occurred before the establishment of the Hereford herd in 1846 (Heath-Agnew 1983). As demonstrated by the short branch lengths and low

bootstrap values on two nodes of the dendrogram, there are no clear-cut breed clusters other than the Jersey-Angus pairing. Both of these breeds may have been isolated from the extensive cross-channel traffic of cattle in the 18th and 19th Centuries: Jersey cattle were isolated by post- 1763 laws forbidding importation of livestock to the Channel Islands, and the Angus breed by their remote geographical location in north- east Scotland. The lack of differentiation among the other European populations may be due to this historical admixture before the demarcation of various groups into reproductively isolated breeds in the 19th Century. The status of the Charolais breed as an outgroup is in accordance with a tradition tracing the breed's origins to cattle brought to France by the invading Roman legions.

Previously, the overriding concern about the use of microsatellite loci for population genetic studies was that noise due to slippage-based mutation and conse- quent allelic homoplasy would overwhelm any useful phylogenetic or biogeographical information. How- ever, observations here and estimates of mutation rates which are intermediate between protein and variable number of tandem repeat (VNTR) loci (Edwards et al. 1992; Dallas 1992) should allay this concern. Allelic homoplasy may only be a problem for comparisons at a more divergent, interspecific level. It is hoped that an accurate estimate of the degree of homoplasy at individual microsatellite loci will emerge from the examination of the unique sequence surrounding the repeat regions for different alleles in distantly related populations. For intraspecific comparisons, a panel of markers can be chosen which is appropriate to the problem in hand (e.g. for the study of a hybrid zone or admixture, markers showing alleles unique to one or other population would be favoured). Microsatellite loci can be typed far more rapidly than restriction fragment length polymorphism (RFLP) or protein- based techniques, particularly if a multiplex PCR

strategy is used. In conclusion, with the current intensive character-

ization of microsatellite polymorphisms in a wide array of species, the increased use of this class of highly variable marker may eventually prove as much a watershed for population and ecological genetics as it has for gene mapping and medical genetics.

We thank Andrew Lloyd and Ciarain Meghan for discussion and comments; the staff of various Artificial Insemination stations in Ireland for supplying blood and semen samples; Peter Merson of Genus for supplying supplementary semen samples; Gareth Tyrell for providing blood samples from his herd of Jersey cattle; Momadou Diallo of Boke, Guinea for the provision of N'Dama DNA samples; Rudi Fries for microsatellite primer sequences; and Masatoshi Nei for providing a copy of the DISPAN computer program. This work was supported by a grant from the EEC, contract no. TS2- 0165.

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Received 8 November 1993; accepted 6 January 1994

Proc. R. Soc. Lond. B (1994)

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