A fragment of 466 base pairs from a highly variable peripheral region of the mitochondrial D-loop of horses was amplified and analyzed by single stranded conformational polymorphism (SSCP). Fourteen distinct SSCP variants were detected in 100 horses belonging to four breeds (Arabian, ARB; Thoroughbred, TB; Argentinian Creole, ARC; and Peruvian Paso from Argentina, PPA). Each breed showed four to eight SSCP variants, many of which were shared between two or three of the studied breeds. Arabian horses were the most variable (eight variants), with three variants unique to the breed. PPA and ARC showed two and one characteristic SSCP variants, respectively, while TB shared all its variants with at least one of the other breeds. An analysis based on the presence/absence of the variants revealed a closer relationship between PPA and TB, which was not completely unexpected considering the mixed ancestry of the PPA mares. The results also confirm the efficiency of SSCP to detect variability in horse mitochondrial DNA.Instituto de Genética Veterinari

Dulout, Fernando Noel

Mirol, Patricia Mónica

Peral García, Pilar

Genetics and Molecular Biology

English

SEDICI - Repositorio de la UNLP

Genetics and Molecular Biology, 25, 1, 25-28 (2002)
Copyright by the Brazilian Society of Genetics. Printed in Brazil 
www.sbg.org.br
Mitochondrial variability in the D-loop of four equine breeds shown by 
PCR-SSCP analysis
Patricia M. Mirol, P. Peral García and F.N. Dulout
Centro de Investigaciones en Genética Básica y Aplicada, Facultad de Ciencias Veterinarias, 
Universidad Nacional de La Plata, La Plata, Argentina
Abstract
A fragment of 466 base pairs from a highly variable peripheral region of the mitochondrial D-loop of horses was 
amplified and analyzed by single stranded conformational polymorphism (SSCP). Fourteen distinct SSCP variants 
were detected in 100 horses belonging to four breeds (Arabian, ARB; Thoroughbred, TB; Argentinian Creole, ARC; 
and Peruvian Paso from Argentina, PPA). Each breed showed four to eight SSCP variants, many of which were 
shared between two or three of the studied breeds. Arabian horses were the most variable (eigth variants), with three 
variants unique to the breed. PPA and ARC showed two and one characteristic SSCP variants, respectively, while 
TB shared all its variants with at least one of the other breeds. An analysis based on the presence/absence of the 
variants revealed a closer relationship between PPA and TB, which was not completely unexpected considering the 
mixed ancestry of the PPA mares. The results also confirm the efficiency of SSCP to detect variability in horse 
mitochondrial DNA.
Key words: MTDNA, equine, PCR.
Received: March 24, 2000; accepted: March 15, 2002.
Introduction
Mammalian mitochondrial DNA (mtDNA) is mater­
nally inherited, although rare events of paternal leakage 
have been reported (Kondo et al., 1990; Gyllensten et al., 
1991; Avise, 1991;Kaneda et al., 1995). The rate of nucle­
otide substitution is 5 to 10 times higher in mtDNA com­
pared to nuclear DNA (Brown et al., 1979). In humans, the 
substitution rate of the non-coding control region (D-loop) 
has been estimated to be 2.8 to 5 times higher than the rate 
for the rest of the mitochondrial genome (Aquadro and 
Greenberg, 1982); and within this region, the two periph­
eral domains (hypervariable region) evolve even faster 
(Pesóle et al., 1999). Given the maternal transmission, the 
reported absence of recombination (Brown, 1985; 
Hagelberg et al., 1999) and the high substitution rate, 
mtDNA has proven to be a powerful tool in the analysis of 
intra- and interspecific variation, population structure and 
phylogeny. Recent reports have described mitochondrial 
D-loop variation in horses (Ishida et al., 1994a,b; 1995; 
Marklund et al., 1994; Dove et al., 1996; Bowling et al., 
1998; Kavar et al., 1999). In this study we used SSCP
Send correspondence to P.M. Mirol, Centro de Investigaciones en 
Genética Basica y Aplicada, facultad de Ciencias veterinarias, 
universidad Nacional de La Plata, CC 296 (B1900AVW) La Plata, 
Argentina. E-mail: pmirol@fcv.medvet.unlp.edu.ar. 
analysis (Orita et al., 1989) of a peripheral region of the 
D-loop to estimate the variability and relationship among 
four horse breeds, Argentine Creole (ARC), Peruvian Paso 
from Argentina (PPA), Arabian (ARB) and Thoroughbred 
(TB). The Arabian horse is an ancient breed which origi­
nated on the Arabian Peninsula (Bailey and Lear, 1994; 
Andrade, 1954). The Thoroughbred breed originated in the 
United Kingdom from the crossbreeding of English mares 
of Tarpan ancestry with Arabian, Spanish, Turkish and 
Barb horses (Bailey and Lear, 1994). The origin of the Pe­
ruvian Paso can be traced to the 16th century, from Barb 
and Andalusian horses brought to Peru by Spanish con­
querors. The Argentinean Creole horse constitutes a direct 
descendent of the Iberian horses of the 1500s, especially 
Andalusian, Spanish Pure Breed, Barb and Arabian 
(Cabrera, 1945).
In this report we analyze the variability of a fragment 
of the mitochondrial D-loop from four horse breeds to es­
tablish a preliminary pattern of variability prior to investi­
gating sequence variation.
Material and Methods
The ARB, TB and ARC horses used in this study were 
traced back three maternal generations, and the PPA two 
generations. Total horse DNA was extracted from 500 pL 
of blood with DNAzol (Gibco BRL) following the
26 Mirol et al.
manufacturer’s recommendations. The PCR primers used 
were 5 ’-AGGACTATC AAAGGAGAAGCTCTA-3 ’
(DI 4991, Ishida et al., 1994a) and 
5’-CCTGAAGTAGGAACCAGATG-3’ (H16498, Meyer 
et al., 1990), which amplify a 466 bp region situated be­
tween the tRNATI" (position 15397, Xu and Amason, 1994) 
and the central domain of the D-loop (position 15863, Xu 
and Amason, 1994). The horse D-loop contains four con­
served blocks (CSB) and a region of direct eight base pair 
repeats (Xu and Amason, 1994; Ishida et al., 1994a). The 
fragment amplified lies outside these blocks, and includes 
the most variable region of the D-loop (Ishida et al., 1994a). 
The 50 pL reaction mix contained 100 ng total horse DNA, 
0.5 pM of each primer, 0.1 mM dNTPs and 2 U Taq poly­
merase (Gibco BRL) in 20 mM Tris-HCl (pH 8.4), 50 mM 
KC1 and 2mM MgCL, under mineral oil. The PCR con­
sisted of a first denaturation step at 96 °C for two minutes 
followed by 35 one-minute cycles at 94 °C, 30 s at 55 °C 
and one minute at 72 °C, with an elongation step of 5 min at 
72 °C in the last cycle. The size of the products was esti­
mated by 1.5% agarose gel electrophoresis with pBR322 
Msp I Digest as size marker.
Fifteen microliters of each PCR product were added 
to 40 pL of LIS dye (10% sucrose, 0.01% bromophenol 
blue and 0.01% xylene cyanol FF; Maruya et al., 1996). 
The samples were then heated at 96 °C for 10 min, cooled 
on ice for at least 5 min and loaded onto a 10% polya­
crylamide gel (491 acrylamide biacrylamide). Electropho­
resis was carried out at 4 °C, 200V in 0.5 x TBE buffer for 
18 h. The gels were subsequently silver-stained, fixed in 
ethanol 5%, stained with AgNO3 0.2% and revealed with 
2% CaCO3.
A total of 100 horses were examined (45 ARC, 18 
ARB, 30 PPA and 7 TB). The PCR products were of the 
same length, approximately 460 base pairs, which is in 
agreement with the published horse sequences (Ishida et 
al., 1994a).
Results and Discussion
SSCP analysis revealed 14 variants which were con­
sistently obtained in different runs and in different SSCP 
conditions (Figure 1A). Heteroplasmy was not detected. 
Table I shows the number of variants per breed, and indi­
cates those patterns unique to each breed. ARC and PPA 
horses had five distinct SSCP patterns, TB had four and 
ARB horses had eight patterns. The high variability sug­
gested by the existence of 14 different SSCP variants is also 
in agreement with the comparison of three sequences corre­
sponding to the entire D-loop published by Ishida et al. 
(1994a), who found most of the substitutions (11 out of 18) 
within the fragment amplified in the present work. Our re­
sults are also similar to those of Marklund et al. (1994), 
who found 15 SSCP variants in a similar fragment of the 
D-loop corresponding to 78 horses from five Scandinavian 
breeds, each one of these showing between 5 and 10 vari­
ants. In a study of the same portion of the D-loop, Kavar et 
al. (1999) found three SSCP variants in 16 maternal lines of
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Figure 1 - A. Silver stained polyacrylamide gel showing the 14 SSCP 
variants detected in this study. B. Tree obtained with the Wagner parsi­
mony method. ARC, Argentinean Creole; TB, Thoroughbred; PPA, Peru­
vian Paso from Argentina; ARB, Arabian.
Table I - Number and type of SSCP variants detected in each horse breed.
Breed n nV SSCP variant
1 2 3 4 5 6 7 8 9 10 11 12 13 14
ARC 45 5 - 2 26 - - - - - 10 - 1 6 - -
TB 7 4 - - 2 - - - - 1 - - - 3 - 1
PPA 30 5 - 5 - - 12 - - 6 - 1 - 6 - -
ARB 18 8 1 3 - 1 - 6 3 - 1 - - - 1 3
ARC, Argentinean Creole; TB, Thoroughbred; PPA, Peruvian Paso from Argentina; ARB, Arabian. In bold, those variants that are unique for each breed, 
n = number of individuals.
nV = number of SSCP variants.
S SCP variation in horse mitochondrial DNA 27
the Lipizzan horse breed, which included 13 haplotypes. 
Furthermore, Bowling etal. (1998) found28 base substitu­
tions (corresponding to 22 haplotypes) in a study based on 
398 bp of the D-loop from 43 Arabian horses. The results 
indicate a considerable degree of variation within the Ara­
bian breed, which was found to be the most variable in our 
sample.
As it can be observed from Table I, Arabian and Peru­
vian Paso presented five and two characteristic patterns, re­
spectively, not seen in the other breeds. Thoroughbred 
horses showed no characteristic pattern, and only one SSCP 
variant was found exclusively in Argentine Creole horses. 
Six SSCP variants were shared by the studied breeds, al­
though none by the four breeds simultaneously. Each breed 
shared at least one variant with each one of the others. The 
most common variants in PPA (variant 5) or ARB (variant 
6) were unique to those breeds, while the most common 
variants in ARC (variant 3) and TB (variant 12) were 
shared by TB and ARC and PPA, respectively.
A phenogram based on a discrete two-state character 
data matrix (0/1, 1/0; presence/absence ofthe SSCP variant 
or allele) using the Wagner parsimony method was con­
structed with the MIX program of the PHYLIP software 
package (Felsenstein, 1993). The tree (Figure IB) shows a 
close relationship between PPA and TB with ARB as a 
more basal taxa followed by the Argentine Creole breed.
The interpretation of the results obtained is not 
straightforward. Arabian and TB are considered the foun­
dation stock for many modem horse breeds (Bowling, 
1994). They have probably contributed in varying degrees 
to the formation of American breeds. Arabian and Barbs 
were brought to America by the Spanish conquerors along 
with horses such as the Spanish Pure Breed and the Anda­
lusian. Their ancestry to South American breeds has been 
suggested by blood group and polymorphic protein loci 
analyses (De Andrés Cara, 1982; Rodríguez-Gallardo et 
al., 1992). A correlation between some of these races based 
on five polymorphic protein loci (Peral-García et al., 1996) 
resulted in similar values between the ARC, Andalusian 
and PP from Pem. However, the Argentinean Pemvian 
Paso is a breed in formation and, therefore, even when stal­
lions are brought from Pemvian Paso in Pem, the mares are 
frequently not pure PP. Their position on the tree in Figure 
IB may be explained by the fact that the maternal history is 
followed through the mtDNA, which is probably of mixed 
ancestry. On the other hand, the position of the ARC breed, 
a well established breed in Argentina, in Figure IB is not 
clear, although it is well differentiated from the other 
breeds examined. We must note here that this unclear posi­
tion could be related to the missing information about the 
ancestral breeds of the analyzed samples.
The reliability of these results lies fundamentally on 
the assumption that each SSCP variant corresponds to a dif­
ferent haplotype. It is generally accepted that SSCP can de­
tect even single base substitutions (Takeda et al., 1995; 
Ostellari etal., 1996). The resolution power of this method 
is crucially dependent on the fragment examined and on the 
conditions of the experiment (temperature, gel concentra­
tion, ran length). Under different experimental conditions, 
we have always obtained the same patterns. If some point 
mutations did not produce a new variant, the variability 
would be underestimated and the results would represent a 
first approximation to the relationship and variability of the 
analyzed breeds. In conclusion, SSCP allowed the detec­
tion of extensive variability in PPA, ARC, TB and ARB 
horse breeds, which makes this technique a valuable first 
step in future phylogenetic studies, including sequencing 
techniques.
Acknowledgements
This work was supported by National Research 
Council (CONICET) and Universidad de la Plata grants to 
P.P.G. and F.N.D., and SECyT grant to P.M.M. The au­
thors wish also thank Mariana Kienast for collecting the 
samples and David Bilton for useful comments.
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Mitochondrial variability in the D-loop of four equine breeds shown by PCR-SSCP analysis

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Mitochondrial variability in the D-loop of four equine breeds shown by PCR-SSCP analysis

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