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  • Journal of Heredity 2011:102(6):753758doi:10.1093/jhered/esr091Advance Access publication September 13, 2011

    The American Genetic Association. 2011. All rights reserved.For permissions, please email: journals.permissions@oup.com.

    Genetic Analysis of SicilianAutochthonous Horse Breeds UsingNuclear and Mitochondrial DNAMarkersANNA MARIA GUASTELLA, ANTONIO ZUCCARO, ANDREA CRISCIONE, DONATA MARLETTA, ANDSALVATORE BORDONARO

    From the DISPA Sezione di Scienze delle Produzioni Animali, Universita degli Studi di Catania, via Valdisavoia, 5, 95123Catania, Italy.

    Address correspondence to D. Marletta at the address above, or e-mail: d.marletta@unict.it.

    Genetic diversity and relationship among 3 Sicilian horsebreeds were investigated using 16 microsatellite markersand a 397-bp length mitochondrial D-loop sequence. Theanalysis of autosomal DNA was performed on 191 horses(80 Siciliano [SIC], 61 Sanfratellano [SAN], and 50 SicilianOriental Purebred [SOP]). SIC and SAN breeds werenotably higher in genetic variability than the SOP. Geneticdistances and cluster analysis showed a close relationshipbetween SIC and SAN breeds, as expected according to thebreeds history. Sequencing of hypervariable mitochondrialDNA region was performed on a subset of 60 mares (20 foreach breed). Overall, 20 haplotypes with 31 polymorphicsites were identified: A higher haplotype diversity wasdetected in SIC and SAN breeds, with 13 and 11 haplotypesrespectively, whereas only one haplotype was found in SOP.These were compared with 118 sequences from GenBank.BLAST showed that 17 of the 20 haplotypes had beenreported previously in other breeds. One haplotype, found inSIC, traces back to a Bronze Age archaeological site (InnerMongolia). The 3 Sicilian breeds are now rare, and 2 of themare officially endangered. Our results represent a valuabletool for management strategies as well as for conservationpurposes.

    Key words: conservation, D-loop, genetic diversity,microsatellites, mitochondrial DNA, Sicilian horses

    Sicily detains an ancient tradition of horse breeding whichgoes back to Greek domination (600 BC). Until 16th century,2 different types of horses were mainly reared in Sicily: theAsiatic and the North African. The crossbreed between these2 genetic types gave origin to the Sicilian breed (Chiari1901). Thanks to its beauty and harmony of shapes, it wasconsidered an admirable example, thus at the end of 15thCentury, Leonardo da Vinci chose a noble Sicilian breed

    horse (the Ciciliano horse of Galeazzo Sanseverino) as amodel for his studies on horse body proportions. At the

    beginning of 20th century, Sicilian breed still maintained its

    excellent reputation and was one of the few recognized Italian

    horse breeds (Diffloth 1923).Nowadays, about 30 000 horses are counted in the island

    but only the 8% is represented by autochthonous horses,

    named Siciliano (SIC), Sanfratellano (SAN), and Sicilian

    Oriental Purebred (SOP).SIC (400 heads) is an heterogeneous and largely unmanaged

    population, reared in extensive system. This population, not yet

    officially recognized, represents one of the most authentic

    Sicilian equine genetic heritage. SAN (1600 heads) and SOP

    (150 heads) are considered at risk of extinction (for more

    details, see Zuccaro et al. 2008).Genetic characterization of the endangered breeds is a

    compelling prerequisite for preservation and management

    strategies. Mitochondrial DNA (mtDNA) was used to

    investigate the origins and the genetic relationships of

    several horse breeds (Royo et al. 2005; Aberle et al. 2007;

    Glazewska 2010); combined with historical information,

    mtDNA was able to evaluate maternal inheritance and to

    identify founder mares in some breeds (Bowling et al. 2000;

    Hill et al. 2002).Microsatellite markers (short tandem repeats) have been

    widely used in horse studies, also associated with mtDNA

    information (Kakoi et al. 2007; Perez-Gutierrez et al. 2008), in

    order to quantify genetic variation and to address conserva-

    tion programmes (Marletta et al. 2006; Thirstrup et al. 2008).

    In this study, genetic variability has been assessed in 3 Sicilian

    indigenous horse breeds. The use of molecular information

    supplied by nuclear and mtDNA markers was aimed to

    provide suitable strategies for management and conservation.

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  • Materials and Methods

    Sampling and DNA Extraction

    DNA was extracted from blood samples (10 ml in K3-EDTAtubes) collected all over Sicily from 191 minimally relatedSicilian horses: 80 SIC, 61 SAN, and 50 SOP (Figure 1). Asubset of 60 maternally unrelated mares (20 per each breed)was submitted to the mtDNA analysis.

    Microsatellite Amplification and Analysis

    A set of 16 STRs (HTG6, HTG10, VHL20, HTG7, HTG4,AHT5, AHT4, HMS3, HMS6, HMS7, HMS2, ASB2, HMS8,HTG15, HMS1, and HMS5) was used for genotyping by anABI PRISM 377 genetic analyzer (Applied Biosystems, FosterCity, CA). The main parameters of genetic variability wereobtained by running the software Molkin v.3.0 (Gutierrezet al. 2005), which was also implemented to assess thecontribution of each breed to the total genetic diversityaccording to the methods proposed by Caballero and Toro(2002) and Petit et al. (1998).

    In order to assess the distribution of genetic variabilitywithin and among breeds, FSTAT ver.2.9.3 software (Goudet2001) was employed to estimate F-statistics (FIT, FIS, andFST), including population pair-wise FST; statistical signifi-cance was inferred by randomization-based methods andcorrected by Bonferroni method to account for multiplecomparisons. The model-based approach proposed by Falushet al. (2003) in the software STRUCTURE 2.2 was used toassess the genomic clustering of the sample. The admixturemodel was implemented to infer the population structureusing no prior information (500 000 burn-ins, 500 000iterations). The range of possible clusters (K) tested was from1 to 10 (10 runs for each K).

    MtDNA Amplification and Sequences Analysis

    Mitochondrial control region (D-loop) was amplified accord-ing to Cozzi et al. (2004) using primers designed according tothe horse sequence (Acc Num X79547). The amplicons, of397-bp length, were purified and sequenced using the BigDyeTerminator v1.1 Kit, on an ABI PRISM 310 DNA Sequencer(Applied Biosystems).

    The aligned sequences were edited in MEGA 4 (Tamuraet al. 2007) in order to identify the polymorphic sites andaccomplish the BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

    Arlequin ver.3.1 (Excoffier et al. 2005) was used toachieve the analysis of molecular variance (AMOVA) withinthe Sicilian breeds (Slatkin linearized indexes VST calcula-tion) as well as to evaluate haplotype diversity (h), nucleotidediversity (pn), and the mean number of pair-wise differences(p) of Sicilian sample and 118 sequences (397-bp length)acquired from the literature.

    Maximum parsimony network (Polzin and Daneschmand2003) was constructed using the median-joining method(Bandelt et al. 1999). The analysis, performed by NETWORKver.4.5.0.1 (http//:www.flexus-engineering.com), includedsequences belonging to several European and geographicallydistant breeds.

    Results

    Microsatellites

    Notable autosomal polymorphism (Table 1) and a relativelymoderate gene flow among breeds (FST index 5.7%; P ,0.001) were observed. SIC was the most heterogeneouspopulation in our sampling, SOP showed the lowestvariability. FIS inferred per breed was always close to zero.A total of 26 breed-specific alleles (14 in SOP, 9 in SIC, and 3in SAN) were detected, always at a frequency lower than0.010 (data not shown). The pair-wise FST values weresignificant (P , 0.01), and a notably higher differentiationwas observed between SAN and SOP (10.3%), followed bythe between SIC and SOP FST (5.9%), whereas SIC andSAN accounted for the least genotypic diversity (2.6%).Reynolds distance (DR) between SAN and SOP (0.112) wasthe highest detected, whereas the closest breeds resulted SICand SAN (0.031).

    Clustering analysis inferred 3 ancestral subdivisions inthe whole sample; genome structure of SAN and SOP areclearly referable to distinct clusters with a probability of83.6% and 95.1% each; SIC grouped mainly in a thirdcluster with an estimated membership of 71.4%.

    According to a conservation program, aimed at pre-serving the maximum amount of total gene diversity (GDT)and the maximum amount of allele diversity (ART), eachbreed showed favorable contribution to the variability of thewhole set, except for SAN when we took into account themethod based on the rarefacted number of alleles proposedby Petit et al. (1998).

    Table 1 Sample size, mean number of alleles per locus (An), allelic richness (Ar), observed and expected heterozygosity (Ho, He), FISvalues; within breed (GDW), between breed (GDB), and total contribution to the gene diversity (GDT) according to Caballero and Toro(2002), within breed (ARW), between breed (ARB), and total contribution to the allelic richness (ART) according to Petit et al. (1998) in 3Sicilian autochthonous horse breeds

    Breeds Size An Ar Ho He FIS GDW/GDB GDT ARW/ARB ART

    SIC 80 7.00 6.77 0.725 0.740 0.027 2.975/1.384 1.591 4.061/0.255 4.316SAN 61 6.25 6.16 0.710 0.702 0.003 0.497/1.072 0.575 0.864/0.313 1.177SOP 50 5.88 5.88 0.696 0.670 0.029 1.884/2.698 0.814 3.197/8.187 4.989

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    http://blast.ncbi.nlm.nih.gov/Blast.cgihttp://blast.ncbi.nlm.nih.gov/Blast.cgihttp//:www.flexus-engineering.com

  • Mitochondrial Sequences

    Twenty ha