Animal Genetics 1992,23, 133-142

Cloning of highly polymorphic microsatellites in the horse

H. ELLEGREN, M. JOHANSSON, K. SANDBERG & L. ANDERSSON

Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden

Summary, We have isolated equine microsatellites by screening a genomic library with (TG)n and (TC), probes. TG microsatellites were found to be more abundant than'TC repeats, with an estimated frequency of one per 100000bp. Sequence analysis of eight TG-positive clones revealed varying structures of the repeat regions; perfect stretches of TG repeats, imperfect stretches of TG repeats and compound regions of TG and TC repeats. Five loci were analysed by PCR and showed extensive polymorphism; three to seven alleles and heterozygosities of 0 . 4 0 4 7 6 were observed when screening 20-30 unrelated individuals. The high degree of polymor- phism, their abundance and the possibility of automating the typing procedure make these loci ideal for standardized paternity testing in the horse. Furthermore, we demonstrate that single hairs can be used as starting material for the PCR analysis. Keywords: microsatellites, PCR, polymorphism, horses, paternity testing

Introduction

The eukaryotic genome contains several types of repetitive DNA, from simple stretches of a single nucleotide to highly ordered hierarchical satellite DNA (Beridze 1986; Tautz et al. 1986). Some of these repetitive DNAs are highly polymorphic. One of the most common types of repetitive DNA is stretches of simple nucleotide motifs (Hamada et al. 1982, 1984; Tautz & Rentz 1984; Tautz et al. 1986; Epplen 1988). Typical motifs are mono-, di-, tri- and tetra-nucleotide blocks. The (TG)" motif seems to be the most widely distributed among species and is also the quantitatively most abundant dinucleotide repeat in mammals (Hamada et al. 1982; Epplen 1988).

Human simple repeat loci, generally termed microsatellites, have recently been shown to be highly polymorphic due to a VNTR-type of polymorphism (Litt & Luty 1989; Tautz 1989; Weber & May 1989). The analyses of microsatellite loci are carried out by in vitro amplification of the repeat region by the polymerase chain reaction (PCR; Saiki et al. 1988), using locus-specific primers complementary to sequences flanking the repeat region. The approach has already been utilized for gene mapping in man and mouse (e.g. Dracopoli & Meisler 1990; Love et al. 1990; Luty et al. 1990; Petersen et al. 1990; Wallis et al. 1990).

Correspondence: Hans Ellegren, Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Box 7055, S-750 07 Uppsala, Sweden. Accepted 28 May I991

133

134 H. Ellegren et al.

Besides the work in man and mouse, a few microsatellites have been analysed in some wildlife species (Tautz 1989). Analyses of one bovine and one ovine TG microsatellite have also been reported recently (Fries et al. 1990; Crawford et al. 1990). Here we report the cloning and analysis of several highly polymorphic TG and TC microsatellites in the horse.

Materials and methods

Construction of an equine genomic library A size-fractionated horse genomic library was constructed in the plasmid vector pBluescript SKI1 + (Strategene). Genomic DNA from five unrelated individuals (Swedish Trotters) was separately digested with AluI, HaeIII and HinfI, pooled and separated by agarose electrophoresis. Restriction fragments in the approximate size range of 200-400bp were cut out and purified from agarose by electroelution. After repair of protruding ends by T4 DNA polymerase (Amersham, Bucks, UK), fragments were blunt-end ligated into Sma I digested vector.

Screening of the library and DNA sequencing About loo00 clones were screened for the presence of microsatellites using the synthetic polynucleotides (TG), and (TC), (Pharmacia, Uppsala, Sweden) as probes. Plated bacteria were transferred to nitrocellulose filters (Millipore, Molsheim, France) according to Sambrook et al. (1989). Filters were prehybridized in 0 . 2 6 ~ Na2HP04, 7% SDS, 1% BSA and ImM EDTA for 2h at 65°C. Nick translated probe was added to the same solution and hybridization was carried out at 65°C overnight. The filters were washed in 0.7 x SSC, 2 x 30 min at 50°C.

DNA sequencing was performed with the dideoxy chain-termination method on alkali denatured plasmid DNA using T7 DNA polymerase (Pharmacia). Both strands were sequenced over the microsatellite region.

PCR analysis PCR primers complementary to sequences flanking the microsatellite were synthe- tized. For each PCR analysis, one primer was 5' end-labelled with [ Y ~ ~ P ] A T P (1 Curie per 10 pmol primer) using T4 polynucleotide kinase (Amersham). PCR was carried out on a thermal cycler (Coy Laboratory Products Inc., Michigan, USA) using AmpliTuq DNA polymerase (Perkin Elmer Cetus, Emeryville, USA). The standard PCR reaction contained 10 pmol each of the labelled and unlabelled primers, 1 . 5 m ~ MgCl, 5 0 m ~ KCI, 1OmM Tris, 200pM dNTP, 1.5U AmpliTaq and approximately 50ng genomic DNA in a reaction volume of 2 0 4 . Twenty-eight cycles were run, each comprising one min at 94"C, 30s at 55°C and one min at 72°C. After the last cycle a final extension of 10 min was added. The amplification reaction was mixed with formamide and dye, heated to 75°C and 1-1.5p1 was immediately separated on a 6% denaturing polyacryiamide gel.

Polymorphic equine microsatellites 135

The animals investigated were families and unrelated individuals of Swedish and North Swedish Trotters. For the purpose of this study, allele frequency estimates are only given for pooled data from the two breeds.

PCR analysis from single hairs Hair strands were pulled out from five unrelated horses. After carefully checking that the hair root was intact, a 1-cm portion from the root end of each hair was transferred to 150 ~ 1 5 % Chelex (BioRad, Richmond, USA) and incubated at 56°C overnight. DNA was extracted by vortexing for lOs, boiling for 8 min and vortexing again for 10s. After centrifugation, 1 5 ~ 1 of the DNA containing supernatant was taken as template for PCR amplification. PCR was carried out as described above with the exception that 30 cycles were run. Of the amplification reaction, 1 4 p1 was loaded on a polyacrylamide gel.

Results

Characterization of horse microsatellites Screening of 10000 recombinant clones yielded 30 positive clones with the (TG), probe and four with the (TC), probe. Eight TG clones (including one clone positive for both TG and TC) were sequenced and all were found to contain unique microsatellite loci (Table 1). Four clones included perfect stretches of TG, two clones had imperfect stretches of TG repeats including interrupting nucleotides at one or two sites, and two clones comprised compound repeats with a TC stretch following the TG repeat (one of the latter was the one positive for both TG and TC). One of the clones contained an (A)~o stretch situated 100bp from the TG-repeat.

Primer pairs, corresponding to the 5’ and 3’ flanking sequences, were synthetized for six of the eight sequenced clones (for the two remaining clones the microsatellite was situated immediately adjacent to the cloning site). Primer sequences are shown in Table 1; primers were designed following, as far as possible, the recommendations given by Luty et al. (1990).

Genetic polymorphism of equine microsatellite loci PCR analysis of 2@30 unrelated horses revealed extensive polymorphism for five (clones HTGZHTG6) out of the six loci. The sixth locus (clone HTG1) was also polymorphic (at least two alleles) but technical problems with the amplification of this clone prevented further evaluation. The sizes of alleles were determined by comparing the electrophoretic mobilities of a new allele and the cloned allele, with the aid of a sequence reaction as marker during electrophoresis. Allele size differences corresponding to 2, 4, 6, etc. nucleotides are most likely due to a variation in the copy number of the dinucleotide repeat.

The polymorphism data for clones HTGZHTG6 are summarized in Table 2. The number of alleles varied between three and seven, the heterozygosities and PIC

Tab

le 1

. R

epea

t com

posi

tion

and

prim

er s

eque

nces

for e

quin

e T

C m

icro

sate

llite

s

Clo

ne

nam

e P

rim

erA

(5’-3

’) R

epea

t st

ruct

ure

Prim

er B

(5’

-3’)

HT

G 1

HT

G2

HT

G3

HT

G4

HTG

S H

TG

6 H

115*

H

117*

TT

T A

AT

GA

TT

CT

T T

AC

T C

C A

GA

TT

T

GC

T A

AG

C A

AT

GG

AA

TG

AT

GG

GT

G

GA

TT

GG

CA

AC

AG

AT

GT

T A

AC

T C

GG

(T

G)4

GG

(GT

)l

CC

CC

AT

GA

GA

AC

T A

AC

AA

T G

TT

AG

T

AA

CC

TG

GG

TG

CA

AA

GC

CA

CC

CA

T

(TG

),,

TC

AG

GG

CC

AA

TC

TT

CC

TC

AC

CT A

TC

TC

AG

TC

TT

GA

TT

GC

AG

GA

C

(GT

)IIG

AT

(AG

)5

CT

CC

CT

CC

CT

CC

CT

CT

GT

TC

TC

T

GC

TA

AG

CC

TC

AG

CA

CA

TA

CA

(T

G)I

%

TG

GA

AA

TA

AG

GT

TA

GC

AG

GG

AT

GC

C

CT

GC

TT

GG

AG

GC

TG

TG

AT

AA

GA

T

(TG

)?,,

GT

TC

AC

T G

AA

T G

T C

AA

AT

TC

T G

CT

(G

ThG

C(G

T),

GC

(GT

),

(GT

)IY

(TG

)Iy(

AG

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* N

o pr

imer

s ha

ve b

een

desi

gned

for

H11

5 an

d H

117

sinc

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peat

reg

ions

wer

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o cl

ose

to th

e cl

onin

g si

te

Polymorphic equine rnicrosaiellites 137

values were in the range 0.40-4.76 and 0.35-0.72, respectively. Mendelian inheri- tance was confirmed for each locus by pedigree analyses. A representative picture of the polymorphic pattern is given for the clone HG3 in Fig. 1.

For all loci some faint extra bands surrounding each major band were observed (Fig. 1) as previously reported for microsatellite loci in other species (Litt & Luty 1989; W-eber & May 1989; Luty et al. 1990). These extra amplification products, which are likely to be PCR artifacts due to slippage in the DNA synthesis (e.g. Luty et al. 1990), were usually shorter than the major fragment and the sizes differed by multiples of two nucleotides. The intensity and relative position of the extra bands varied between loci. Although they did not seriously interfere with the interpretation and scoring of alleles, it was important to take into account the extra band pattern for a particular locus. By using only one labelled primer we avoided the problem caused by different mobilities of the two complementary DNA strands in the denaturing electrophoresis.

In order to evaluate the informativeness of the microsatellites in relation to their composition, have calculated the weighed average number of repeat units for each locus. This was done including all repeats in compound microsatellites as well as considering only uninterrupted stretches (see Table 2). At most, the number of TG repeats in the cloned allele differed by four repeats from the weighed mean value

Table 2. Summary ot the obsened poljmurphisin of five hoise TG niicrosatcllirr loci. For each locus the allele in italics represents the cloned allele

Clone designation

HTG2 HTG3 HTG4 HTG5 HTG6

Number of animals screened

21 20 32 36 20 .___

Allele sues (bp) 107:0.26 129:0,18 139:0@3 95:0.01 100t0.32 and allele 103:O-02 127:0.15 137:0.12 91:0.03 90:0.40 frequencies 99:0.71 125:Oa 135:0.08 89:0.04 88:0.02

121t060 133:0.38 85:0.76 84:0.25 119.0~02 131 :0. I6 83:O. 10

129.0.23 81:0.04 79:0.01

Heterozygosity* 0.42 0.58 0 76 0.40 0-67

PIC value** 0.35 0.54 0.72 0.39 0.60

Weighted average 10.1 17.2 15.8 15.0 15.9 number of repeats* * *

* Estimated heterozygosity calculated from observed allele frequencies. * * Polymorphism information content estimated according to Botstein et al. (1980) * * * Estimated according to Weber (1990).

138 H. Ellegren et al.

Figure 1. Polyacrylarnide (6%) gel electrophoresis of PCR amplifications of the equine microsatellite locus HTG5. Thirteen unrelated Swedish trotters (lanes A-M) and plasmid DNA from the cloned allele (lane N ) were analysed. Allele sizes (bp) are indicated to the left.

(HTG6, cf. Tables 1 and 2). Although our sample is limited, there was a significant positive correlation between the weighed average number of repeats at a locus (including all repeats in compound loci) and its PIC value (R, = 0.90, P < 0.05, Spearman rank correlation). The same trend was also evident when only the longest uninterrupted TG stretch was considered but this relationship did not reach statistical significance ( R , = 0.70, P > 0.05).

DNA typing of horse microsatellites f rom single huirs Amplification of DNA from single sperm cells or from single hairs has illustrated how powerful the PCR method is for analyses of small amounts of DNA (Higuchi et al. 1988; Li et al. 1988). In this study we were able to amplify microsatellites from single horse hairs (Fig. 2). The signals were generally weaker than that obtained with 50ng DNA prepared from blood as starting template material. However, detectable overnight signals from a 30-cycle PCR was obtained with as little as 5% of an amplification reaction using 5% of a DNA preparation from a single hair as template (data not shown).

Multiplex PCR We regularly analysed several microsatellite loci on the same polyacrylamide gel by multiple loadings in the same lanes. If the different loci showed a significant size divergence, amplifications were mixed and simultaneously loaded. If the locus sizes were similar, each amplification was loaded separately with intervals of 5-10 min.

We also used up to three different primer pairs in the same PCR reaction (i.e. multiplex PCR, see Chamberlain et al. 1990). This gave varying degree of non-specific amplification but the allelic fragments were always present and scoreable (Fig. 3).

Polymorphic equine microsatellites 139

Figure 2. Polyacrylamide (6%) gel electrophoresis of PCR amplifications of the equine microsatellite locus HTG6 in five unrelated horses. Genomic DNA used as template in PCR was prepared from blood (50ng) and from a single hair, respectively. In the latter case, 10% of the DNA preparation was taken to amplification and 20% of the amplification reaction was loaded on the gel. C+ is plasmid DNA from the cloned allele and C- is a negative control without template DNA. Allele sizes (bp) are indicated to the left.

Figure 3. Polyacrylamide (6%) gel electrophoresis of PCR amplifications of the equine microsatellite loci HTG2, HTG4 and HTGS in three unrelated North Swedish trotters. A is a multiplex amplification of HTG2, HTG4 and HTG5 (including plasmid DNA from the cloned alleles in the fourth popition), B is a multiplex amplification of HTG2 and HTG4, C, D and E are individual amplifications of HTGS, HTG2 and HTG4, respectively. Allele sizes (bp) are indicated to the left.

IXsrimion

High ly ,wriuhie eq 1.1 ine rii ic rosrrtelliies I'he present study demonstrates t h a t rnicrosatcllites with the simple T'G repeat motif are ( I ) highly abundant irl the equine genome and (2) display a considerable degree of polymorphism. I n the screening of approximately 10000 recombinant clones we obtained 30 TG positive clones. Assuming an average insert size of 300bp in the library. we may estimate the average frequericv of 7 G microsatellites in the horse to roughly one per 10000Obp. and thus a total copy number estimate of about 30000. Although o u r size-fractionated library may riot be completely representative for the entire genome, these estimates are comparable t o corresponding figures derived from other mammals (e.g. FIamada eta!. 1982). TC-positive clones were considera- bly less frequent than TG clones and the frequency of TC microsatellites in the cquine genome may be an order of magnitude lower than TG microsatellites. That 3.C repeats are as polymorphic as 7'G motifs remains to be elucidated. Given the variability and abundancy o f horse microsatellites indicated by our data. they will provide a rich source of markers for genetic studies in the horse.

Weber (1990) compiled data from 112 human 7'G microsatellites and concluded that the degree of polymorphism was positively correlated to the number of repeat units and that perfect repeats were more polymorphic than interrupted repeats. Our data support the former coiiclusion arid suggest that in the search for the most polymorphic microsatellites, loci with comparatively long stretches of 'TG repeats should be chosen. In practice, however. i t is important to be aware that the best predictor for the degree of polymorphism i s the size of the weighed average allele and the cloned allele i s only one representative for the locus. Moreover, Weber's data indicate that in spite of the overall relationship between repeat length and polymorphism, there is a considerable variation in the informativeness of different loci having similar mean repeat lengths.

Prospects f o r u stundurdized pnrertiit.v tcstitig system in the horre hused on PC'K-unulysed microsatellites In the light of the variability at some of the loci described herein, it is obvious that microsatellites could be powerful tools for paternity testing in the horse. LJsing a panel of highly polymorphic microsatellite loci, a discriminating power approaching that of DNA fingerprinting and exceeding that of conventional blood typing is feasible. We may illustrate this by considering several loci each having a variability similar to the most informative locus in our study (HTG4). Using the allele frequencies given in Table 2. the exclusion probability (Jamieson 1965) revealed by the HTG4 locus can be estimated at 0,54. Combining five such loci, an exclusion probability of 0.98 is reached and usirig 10 loci the probability would be as high as 0. Y 996.

Although several loci have to be screened. a PCK-based paternity testing system has several advantages. The procedure is easily automated (DNA preparation and amplificatiori can be made in automatically handled microtitre wells), and can be simplified either by loading several amplifications or! the same gel or by multiplex

Polymorphic equine microsatellites 141

PCR reactions. Moreover, the procedure is rapid and requires minute amounts of sample. Our data show that microsatellites can easily be amplified from single hairs, a fact that opens a new perspective to the sampling routines needed for standardized paternity testing in the horse. Perhaps most importantly, the genetic interpretations of microsatellite polymorphism, which is locus specific, are straightforward. This is a major advantage compared to DNA fingerprinting which gives a complex genetic polymorphism derived from multiple loci. Furthermore, microsatellite alleles are well defined by the length of the PCR products, allowing computerization and exchange of typing information between laboratories. A standardized paternity testing system based on PCR-analysed microsatellites is thus likely to be a future alternative to blood typing in the horse.

Acknowledgements

This work was financially supported by the Agria Foundation and the Swedish Trotting Associaxon.

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