genetic diversity in the maremmano horse and its relationship with other european horse breeds

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  • doi:10.1111/j.1365-2052.2010.02102.x

    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

    standard methods.

    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 Lusitano.

    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;

    Table S3).

    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.



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