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Very gracile dog bones dated to the 8–9 century AD were found at the Chotěbuz-Podobora archaeological site in the Czech part of Silesia near the town of Český Těšín. Osteological analysis indicated that the bones were from a greyhound. To confirm this hypothesis, genetic analysis of greyhound-specific Single nucleotide polymorphisms (SNP) in the control region (CD, also D-loop) of the mitochondrial DNA (mtDNA) was performed, together with analysis of buccal swabs from living greyhounds from the Czech Republic. Our genotyping results strongly support the hypothesis that bones found were from a greyhound which supports the second hypothesis that an elite order inhabited Chotěbuz-Podobora hillfort in the Middle Ages. ■ greyhound; mtDNA; Control region; Great Moravia; Middle Ages Received February 11, 2015 Issued October, 2015 the maximum length and the minimum width falls into the variation width of the ratio for greyhounds, mainly Polish greyhounds (Nývltová Fišáková 2007, 2010, fig. 8). The greyhound has been considered an exceptional animal since the very earliest times (Sullivan 1999, Morris 2001, Branigan 2004). During the Middle Ages these dogs could only have been owned by nobles and rulers (Kholová 1987, Stuchlý and Císařovský 1992, Sullivan 1999, Branigan 2004, Císařovský 2008). The greyhound was one of the heraldic symbols of kings and nobles in both England and France (Sullivan 1999, Branigan 2004), as well as in Czech lands (Mysliveček 1993, Buben 2003). This is the oldest evidence of a greyhound in the Czech Republic. Until this discovery, the oldest finding was the mummified English greyhound from the castle in Ždánice (Nývltová Fišáková, 2007). The main aim of this study was to analyze 17 excavated bones to test the hypothesis that these were the oldest greyhound remains within the Czech territory. We chose to analyse mitochondrial DNA (mtDNA) because of its special features: it is situated the outside nucleus in mitochondria, there are many copies while there are usually only two copies of each chromosome. Circular mtDNA is also more stable than linear nDNA, thus increasing the chance to obtain analyzable results even from degraded (ancient) DNA. The first published complete mitochondrial DNA (mtDNA) genome of the domestic dog (Canis familiaris) contained 16,727 bp (base pairs) with the control region (CR) spanning positions 15,458–16,727 (1,270 bp) (Kim et al. 1998). While the dog mtDNA genome closely resembles the mtDNA genome of other mammals, the dog (and other related canids) mtDNA CR differs due to the presence of a 10 bp repeating unit (5 ́-GTACACGT(A/G)C-3 ́) that begins at base 16,130 and varies in number and sequence both within and among individuals. The CR of dog mtDNA, like that of human mtDNA, has been the subject of a number of studies investigating variation among individuals (Vila et al. 1997, Kim et al. 1998, Webb and Allard 2009). Previous studies revealed more than 100 single nucleotide polymorphisms (SNPs) within the mtDNA CR of the domestic dog. Materials and methods The ancient material was found at Chotěbuz-Podobora site. These bones were osteologically very similar to greyhound-like dogs – long and generally slim in comparison with other dogs of a similar height (Text-fig. 1). For comparison with recent domestic dog, we took a buccal swab from a greyhound whose ancestors were born in 1840 in Great Britain. We also took swabs from 15 recent dogs (greyhound-like dogs) from the dog exhibition in Brno 2010 as suitably representative samples for verification of our results. Sample 1 is Romanov z Palatinu Morava (male), sample 2 is Body z Murky (male) (both are Russian barsoi). Sample 3 is Limia Lykon (female, afghan greyhound). Sample 4 is Glenoak Junah (female, Saluki). Samples 5 and 6 are Lajjina and Pussy Bohemia Genao (both Sloughi). Sample 7 is Arabian Sloughi Djawida EJJI Fatira of Love ARAB BEAUTY. MAYSUN Bohemia Genao is sample 8, whose grand-father was a Moroccan Sloughi. Sample 9 Takellaut N8FA BADAKA is a Sloughi-Azawakh, whose ancestor was from the Sahara. Sample 10 is a Polish greyhound Baldwin-Xanthusia (male). Uncle Teddy (male) and Hellega (female) are whippets, samples 11 and 12. Sample 13 is deerhound Greyrory ́s Draeura (female). Rendel (male) and Rosarka (female) are samples 14 and 15 are also barsois. Isolation of DNA from ancient bone Forensic DNA isolation methods were used which are a combination of the isolating methods described in the publications of Baron et al. (1996) and Yang et al. (1997). First we isolated DNA from the bone. The surfaces of both small bone fragments were mechanically cleaned with sandpaper and 96% ethanol was used to remove foreign substances, such as bacteria, fungi, or other foreign DNA. Usage of a more efficient bleaching liquid could be severely damaging for old bone. The next step of preparation for isolation of DNA was cutting the bone into thin slices with 18 Text-fig. 1. Bottom view of the spoke bone of the excavated greyhound-like dog (above) and the spoke bone of a dog of the same size category (below). The gracility of the spoke bone of the greyhound-like dog is clearly visible. Photo by D. Nývlt. an oscillatory handsaw (Eickemeyer). The slices were crushed with a lever press and then ground with a mill (Retsch MM 200) for 3 minutes. The grindings were collected into agate bowls. The final volume of bone powder was 3.73 g and 3.54 g in 50 ml tubes. The isolation process itself was done according to a protocol using phenol-chloroformisoamylalcohol. A total of 40 ml of EDTA (pH 8.0) was added to the bone powder and the solution was incubated for 24 hours at 4 °C (the process of decalcification) and then centrifuged at 4,000 rpm for 15 minutes. Forty ml of EDTA-Sarcosyl was then added and the solution centrifuged at 4,000 rpm for 15 minutes. The supernatant was then poured off and thus the residue of calcium was removed. Then 7 ml of EDTA-Sarcosyl (pH 8.0) and 200 μl Proteinase K (20 μg/ml) were added and the sample mixed thoroughly by vortexing. Incubation at 56°C followed and continued until dissolution of the solid particles was complete and then 7 ml of phenol:chlorophorm:isoamylalcohol, 25:24:1 was added. The sample was then gently shaken and centrifuged at 4,000 rpm for 15 minutes, the upper aqueous phase containing DNA was transferred to another clean tube and this organic extraction procedure was repeated twice more. To concentrate the DNA to a final volume of 150 μl, Centricon columns were used (Centricon YM, Centrifugal Filter Devices, MILLIPORE). Isolation of DNA from buccal swabs of recent greyhounds DNA from the buccal swabs of 15 recent dogs was isolated with a 5% suspension of Chelex (Walsh et al. 1991). Amplification of mtDNA from isolates of bone and buccal swabs Oligonucleotide primers used for amplification and sequencing of the canid mtDNA CR were created using a dog reference sequence (GenBank accession no. U96639). Convention nomenclature was used: the primer name indicates the position of the 5 ́ base. Forward primers were F15.719 (5 ́-GTAATGTCCCTCTTCTCGCT-3 ́) and F16.431 (5 ́-CACGCGCGTAAGACATTAAG-3 ́). Reverse primers were R16.114 (5 ́-CCTGAAACCATTGACTGAATAG-3 ́) and R42 (5 ́-GGCATTTTCAGTGCCTTGCTT-3 ́) (Gundry et al. 2007, see Text-fig. 2 for the position of primers). Lyophilized primers (Generi Biotech, Czech Republic) were suspended to produce a concentration of 10 mM in ddH2O. 5 μl of total genomic dog DNA was used to amplify the chosen regions in a 20 μl reaction volume containing the following reagents: 200 mM of each dNTP (Applied Biosystems), 0.6 mM each of primers F15,412; F16,431; R16,114 and R42, 5U AmpliTaq Gold DNA polymerase (Applied Biosystems) and 1 GeneAmpTM PCR Buffer containing 1.5 mM MgCl2 (Applied Biosystems). Amplifications were conducted in a GeneAmp 9700 PCR System thermal cycler (Applied Biosystems), and consisted of denaturation for 11 min. at 95 °C, followed by 30 cycles of 1 min. at 94 °C, 1 min. at 58.9 °C, and 2 min. at 72 °C, plus final incubation for 60 min. at 60 °C. Genomic DNA of recent dog was used as a positive control and sterile ddH2O as a negative control. A total of 2 μl of each sample was visualised on 2% Agarose gel, 5 V/cm/30 min. (Text-fig. 3). DNA isolation from the test objects were performed at an accredited workplace of OKTE (Department of Criminalistic Technique and Expertises), amplification and subsequent sequencing in accredited laboratories GeneProof, as and sequencing of mtDNA from bones was repeated 3 for each isolate with the same result. Isolation of mtDNA from the bones was repeated six months later in a laboratory which is normally working only with human DNA. Isolation of mtDNA from the Ulster greyhound was performed separately and after isolation of DNA from bone thus preventing any contamination.

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Genetic analysis of possibly the oldest greyhound remains within the territory of the Czech Republic as a proof of a local elite presence at Hotěbuz-Podobora hillfort in the 8th–9th century AD