genetic diversity in the maremmano horse and its relationship with other european horse breeds
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Genetic diversity in the Maremmano horse and its relationship withother European horse breeds
M. Felicetti*,1, M. S. Lopes1, A. Verini-Supplizi*, A. da Camara Machado, M. Silvestrelli*,
D. Mendonca and O. Distl
*Department of Pathology, Diagnostic and Veterinary Clinic, University of Perugia, Via San Costanzo 4, 06126 Perugia, Italy.Biotechnology Centre of Azores, Department of Agriculture, University of Azores, Terra-Cha, 9701-851 Angra do Herosmo, Portugal.Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bunteweg 17p, 30559 Hannover, Germany
Summary The Maremmano is an Italian warmblood horse breed from central Italy. We characterizedthe genetic diversity and the degree of admixture in Maremmano in comparison to 14 other
European horse breeds using 30 microsatellites. Between-breed diversity explained about 9
per cent of the total genetic diversity. Cluster analysis, genetic distances and genetic dif-
ferentiation coefficients showed a close relationship of Maremmano with Hanoverian and
Lusitano in accordance with breed history.
Keywords clustering analysis, Equus caballus, Maremmano.
The Maremmano is an Italian warmblood horse mostly bred
in the provinces of Grosseto and Viterbo (Central Italy). It is
believed that the origin of this horse breed goes back to local
Etruscan horse populations which were crossbred with
modern breeds in the last centuries. Pedigree analysis of the
Maremmano horses revealed four male lines contributing
11.1% to the genetic diversity of all Maremmano stallions
(Silvestrelli 1991). In 1980, Maremmano breeders estab-
lished a stud book for Maremmano.
The objective of this study was to investigate the genetic
structure and the degree of admixture of Maremmano to-
gether with the Italian nucleus of Lipizzan and Lusitano as
well as twelve further horse breeds previously characterized
by Aberle et al. (2004). This data set consisted of Hanove-
rian, Arabian, Exmoor, Icelandic, Przewalski, Sorraia and
all German coldblood horse breeds. We used the same
marker set of 30 autosomal microsatellites and the same
PCR conditions and reference samples as described else-
where (Aberle et al. 2004).
A total of 146 animals that were not closely related were
genotyped in this study: Maremmano (n = 50), the Italian
nucleus of Lipizzan (n = 49) and Lusitano (n = 47) from
Portugal. DNA was extracted from EDTA-blood using
Genetic variability of the breeds genotyped in this study
was determined by the mean number of alleles, observed
and expected heterozygosities, breed-specific alleles and the
molecular variance (AMOVA) using GENALEX 6 (Peakall &
Smouse 2006). Excess and deficiency of heterozygotes,
which are deviations from Hardy-Weinberg equilibrium,
were estimated using GENEPOP (Raymond & Rousset
1995). Molecular genetic relationships among populations
were estimated using Wrights FST and Neis standardgenetic distance (GST; Nei 1973) by bootstrapping 1000
replicates using MICROSAT (Minch 1997). Phenograms
based on Neis GST and genetic distances among all 549animals were drawn using the unweighted pair group
method with arithmetic mean algorithm (UPGMA) by
PHYLIP (Felsenstein 1989) and displayed by TREEVIEW
(Page 1996). The Bayesian clustering procedure of
STRUCTURE was employed to investigate the genetic
structure and the degree of admixture of the 15 horse breeds
(Pritchard et al. 2000). A 20 000 initial burn-in was used,
followed by 100 000 MCMC iterations as recommended by
Falush et al. (2007) with 10 independent replicates each.
All runs used an admixture model with correlated fre-
quencies and the parameter of individual admixture alpha.
The mean number of alleles was 4.7 for the Lipizzan, 6.7
for the Lusitano and 7.3 for the Maremmano. For Marem-
mano and Lipizzan breeds the total observed heterozygosity
was higher than the expected, whereas for Lusitano breed
Address for correspondence
Prof. Dr O. Distl, Institute for Animal Breeding and Genetics, University
of Veterinary Medicine Hannover, Bunteweg 17p, 30559 Hannover,
1These authors have contributed equally to this work.
Accepted for publication 20 September 2010
2010 The Authors, Journal compilation 2010 Stichting International Foundation for Animal Genetics, 41 (Suppl. 2), 5355 53
19 out of the 30 loci showed observed heterozygosity values
lower than the expected ones (Table S1).
The AMOVA indicated that for Maremmano, Lipizzan and
Lusitano 9% of the total genetic variability is attributed to
significant differences between the horse breeds, whereas
91% of the observed variation was from within the breeds.
The AMOVA performed for the five riding horse breeds
(Maremmano, Lipizzan, Lusitano, Arabian, Hanoverian),
considered separately from the others, showed 11% of the
genetic variability due to breed differences. When the Ger-
man coldblood breeds were included, the variation among
breeds increased only slightly to 12%; with all 15 breeds the
total genetic variability rose to 14.6%.
Maremmano showed the lowest genetic differentiation
with the Hanoverian (5.5%); in addition, the Lipizzan and
Lusitano, when matched with the Maremmano, showed the
lowest FST values (11.4% and 6%, respectively). Among the
three breeds genotyped here, the Maremmano showed
the least differentiation when compared to the coldblood
breeds (Table S2). Similar results were obtained when the
genetic differentiation based on Neis GST among breed pairs
was used. The only exception was for the Maremmano,
which showed lowest genetic differentiation (14.7%) with
The phylogenetic tree based on Neis GST (Fig. 1) and thedendrogram based on the proportion of shared alleles
(Fig. S1) displayed three main clusters representing riding
horses, the Exmoor, Przewalski and Sorraia group and the
German draught horses. The Icelandic horses did not cluster
with any of the other breeds.
Clustering using STRUCTURE separated for K = 3 horses
into riding horse breeds, ancient and isolated breeds, and
German draught horses. For K = 9, Maremmano clustered
together with Lusitano, but Hanoverian and Arabian were
separate clusters. When K = 14, Maremmano and Lusitano
also clustered in their own pre-defined populations (Fig. S2;
From the three breeds genotyped in this study, both the
Maremmano and the Lusitano showed a high level of
genetic variability and similar to that observed for the same
breeds in other studies (Lus et al. 2007; Zuccaro et al.
2008). The high level of genetic differentiation observed for
the Maremmano may partly reflect contributions from
several breeds, although a significant proportion of the
Maremmano is descending from a reduced number of male
lines. The Italian nucleus of Lipizzan horses showed low
levels of variation similar to those observed by Achmann
et al. (2004). The small number of founders resulting in a
small effective population size and the traditional pure-
breeding system within a nucleus without any crossbreed-
ing may explain the reduced variability within the Lipizzan.
In agreement with the results of Lus et al. (2007) in the
Lusitano, levels of observed heterozygosity were lower than
their expected counterpart.
Pairwise FST values among Maremmano, Lipizzan, Lu-
sitano and Hanoverian were in a similar range as those
among closely related coldblood breeds (Aberle et al. 2004;
Druml et al. 2007). Inclusion of Hanoverian and German
coldblood increased the proportion of variance among
breeds only slightly, indicating a close relationship of these
breeds with Maremmano, Lipizzan and Lusitano in contrast
to Arabian, pony and primitive horse breeds. The two
genetic differentiation measures and the two model-based
clustering approaches also revealed a genetic proximity of
Maremmano with the Hanoverian and the Lusitano. Two
male lines founded by thoroughbred stallions (Aiace and
Ingres) and a Trakehner stallion might have created the
relationship between Maremmano and Hanoverian, as
thoroughbred and Trakehner stallions have been intensely
used in the Hanoverian warmblood (Hamann & Distl 2008).
Regarding the close genetic proximity between Maremmano
and Lusitano, it is believed that Iberian horses, the ancestors
of the Lusitano and the Andalusian, were in the Stato dei
Reali Presidi di Spagna (15571800), located between Stato
Pontificio and Granducato di Toscana, and therefore might
have influenced the founder lines of the Maremmano breed.
Figure 1 Dendrogram showing the genetic relationships among the 15
horse breeds inferred from microsatellite data. The tree is based on Neisstandard genetic distance (GST). Bootstrap values higher than 50 are
shown in the tree.
2010 The Authors, Journal compilation 2010 Stichting International Foundation for Animal Genetics, 41 (Suppl. 2), 5355
Felicetti et al.54
In conclusion, the Maremmano retained a high genetic
diversity and the results reported here can be used to pre-
vent genetic erosion of the Maremmano breed.