The genetic variability of Hungarian Tsigai sheep

Microsatellite analysis was used to estimate the genetic origin, differences, relationship within 10 Hungarian Tsigai populations. The number of alleles was 262 at the 16 examined locus. Fifteen population specific alleles were detected. The mean number of alleles detected per locus ranged from 4.3 (OarAE119) to 11.9 (MAF70). Genetic distance values were calculated from Nei’s minimum genetic distance (DA) formula. Phylogenetic tree was constructed using UPGMA algorithm. The results indicated that the genetic difference was negligible between the following populations pair-wise: two Hungarian indigenous populations (Kardoskút1-IN and Kardoskút2-IN); the Hungarian indigenous population Soltszentimre-IN and the Milking Tsigai population Akasztó-ZO; the Hungarian indigenous population Csanádpalota-IN and the transitional type population Makó-Rákos-TR. Microsatellite genotyping prooved to be efficient tool for examing the genetic relationships among Hungarian Tsigai populations.


Introduction
In order to find out the possible methods of preservation for the Tsigai and other indigenous breeds of sheep in the in Central-, Eastern and South European regions the differences and relationships among the sheep populations of the various countries of the region were determined (DUCHEV andGROENEVELD 2006, DUCHEV et al. 2006).
There are different alternatives about history, origin of Tsigai sheep breed.There is a very strong opinion from DRAGANESCU (2003), that all members of this group originated from the Ruda sheep breed of Romania, while several others (e.g.KUKOVICS andJÁVOR 2002, KUSZA et al. 2008) are arguing this statement.
According to BREHM (1903) the Tsigai sheep got into Europe from Small Asia by the Bask people and took this breed along the North part of Mediterranean Sea up to Spain from where these sheep arrived to the British islands as well.An other resource stated (SCHANDL 1953) that there were two parts of the Tsigai migration to Europe, but the origin of the breed was the same as mentioned above.The first part came via North-West Caucasian region following the North coast of Black Sea, reached the Cream peninsula, the region of Azov Sea, South Ukraine, Bessarabia (Moldavia) and the ancient Romania, than to Transylvania, North and South Hungary.The second part came from the South coast of the Black Sea, and started to spread to North following the bed of Danube River, and reached the old Hungarian territories during the second half of the years 1700.
In Hungary, over the past two hundred years, the Tsigai breed, although in fluctuating ratio (1-10 %), has composed a constant part of sheep stock.Limited number of Tsigai sheep left in Hungary after the I. and II.World War, because the lands, breeding this breed, became abroad.
In order to improve the wool, milk and meat production traits different exotic breeds were used to cross the original Tsigai sheep over the last century.It was the background of the wide variability of the Tsigai sheep (KUKOVICS and JÁVOR 2002 a,b).
However, it could not say that Tsigai sheep is one breed the Hungarian Sheep Breeders' Association decided that these sheep are traditionally divided into two groups base on their phenotype: indigenous and milking (Zomborski) types.On the contrary to this only its colour could change between black and white, via light coffee colour.The adult body weight of the ewes is differing from 30 and 100 kg, and the body measurements also vary within wide range.Great variations could be recorded regarding milk production: The most Tsigai ewes are milked during a 50-200 days milking period (120-150 days in average).Daily milk production of lactating ewes varies between 0.470 l (MARGETIN et al. 1996) up to 1.25 l in Milking type Tsigai flocks (CAPISTRAK et al. 1997, KUKOVICS 2000, KUKOVICS et al. 2006).In milk composition also high differences were found.The fat-, protein-and lactose content varied between 4.6 to 10.1; 4.6 to 7.2 and 3.6 to 5.4 %, respectively (KUKOVICS and JÁVOR 2002 a,b).Great variations of wool productivity trait values could be observed in different Tsigai populations (greasy wool weight 2.0.-5.1 kg; clean wool weight 1.4-2.9kg; staple length 6.0-14.0cm; fibre diameter 23-40 microns) (KUKOVICS and JÁVOR 2002 a,b).Therefore Tsigai populations of the different regions could be hardly considered as a particular, single breed.
The purpose of this work was to carry out genetic characterisation of different Tsigai sheep population in Hungary in order to facilitate their rational development, utilization and conservation.

Animals
Hair and blood samples were taken from randomly selected individuals from different herds (Table 1).Pedigree information was available about selected individuals.3 to 5year-old animals with no shared common ancestor for at least two generations were selected.Numbers of animals studied per breed are presented in Table 4. Based on the studies of KUKOVICS et al. (2004KUKOVICS et al. ( , 2006) ) 10 different Tsigai sheep populations were sampled for this examination.Besides of the four indigenous-, one transitional and two Milking Tsigai flocks bred originally in Hungary, three imported (one from Romania; and two from Serbia) populations were included in the trial.The number of individuals per flock are presented in Table 1.Blood samples were taken from 252 individuals, belonging to three different (imported) flocks.Hair samples with bulbs were taken from the other seven flocks (altogether 317 individuals).

Statistical analysis
The number of alleles per locus and heterozygosity values (Hobs and Hexp) were estimated using GENEPOP program (RAYMOND-ROUSSET 1995).Populations (v.1.2.28) and ARLEQUIN (v.2.0) programmes were used for population datas (LANGELLA 1999, SCHNEIDER et al. 2000).Single locus F-statistics were calculated according to WEIR and COCKERHAM (1984).Genetic distances were estimated from microsatellite data by Population v. 1.2.28 (LANGELLA 1999) using the Nei standard genetic distance (DS) and minimum genetic distance formulas (DA) (NEI 1987).Phylogenetic tree of the populations were constructed by the Populations package using the Neighbor Joining algorithm with 1 000 bootstrap on individuals for DA.DRAWGRAM program of the PHYLIP package (v. 3.57c, FELSENSTEIN 1995) was used for the tree drawing.
Distribution of allele frequencies were very widespread.On a few locus there were alleles having high frequency in all examined population (eg.OarCP49 98 allele, MAF35 112 allele, except Cegléd-ZO).

Population analysis
The average number of alleles per locus for the populations varied between 6.63 (Makó-Rákos-TR) and 8.75 (Debrecen-CO).The observed and expected heterozigozities are presented in Table 4.The mean observed and expected heterozygosities per studied populations were between 0.500-0.629(Debrecen-ZO -Soltszentimre-IN) and 0.694-0.767(Cegléd-ZO -Csanádpalota-IN), respectively.All examined population was less heterozygous than it was expected.FIS value ranged between 0.1556 (indigenous Tsigai) and 0.3406 (Milking Tsigai).The heterozigosity deficit was the highest in Debrecen-ZO population and the smallest in Soltszentimre-IN among examined populations (Table 4).
DA distance was resulted from microsatellite data of population.Genetic distance between Cegléd-ZO and Kardoskút1-IN (0.922), Kardoskút2-IN (0.947) and between Debrecen-RU and Kardoskút1-IN (0.891), Kardoskút2-IN (0.911) were the largest among the studed Hungarian populations (Table 5, Figure).Makó-Rákos) was definitely indigenous Tsigai sheep, having no breeding relationship with the one kept and bred in Makó-Rákos.This latter one had refressing breeding stocks (rams) dominantly from the Milking Tsigai flock bred in Cegléd.At the same time (KUKOVICS et al. 2004(KUKOVICS et al. , 2006) ) even the body measurements showed a significant distance between these two populations.GÁSPÁRDY et al. (2004) used 8 microsatellites to determinate the genetic distance among 5 Tsigai populations in Hungary.They found that milking type of Tsigai from Cegléd was very much different from indigenous Tsigai from Jákotpuszta, Kardoskút, Akasztó, Makó.They stated that geographical localization of examined populations was in correlation with their genetic background.They made their own classification as Tsigai sheep from Jákotpuszta called as mountain type, while the others were so called plain type of this breed.This differentiation was not exactly correct because this population in Jákotpuszta was originated from mid Slovakia, having a different breeding background.
The results confirmed the previously known data about the genetic origin of these populations (KUKOVICS et al. 2004), however, some differences were found.In summary we have demonstrated that all sixteen microsatellite markers were able to be amplified in all examined populations and they could be used valuable for the present aim in Tsigai populations.

Figure
Figure Dendogramm showing the genetic relationship among Hungarian Tsigai populations using DA distances from 16 microsatellite loci Genetische Verhältnisse zwischen den Zackelschaf Beständen nach den DA-Werten der 16 Loci

Table 1
The number, type and label of examined Tsigai populations Typ, Anzahl und Bezeichnung der ungarischen Zackelschaf-Populationen (ZSOLNAI andtraction.Hair samples were taken by picking up and collected in paper bags.Genomic DNAs were extracted as previously described(ZSOLNAI and ORBÁN 1999,  FAO/IAEA 2004).Loci selection was based on their location in several chormosomes and recommendation of United States Department of Agriculture (USDA); Australian Gene Mapping Web Site; Food and Agricultural Organization (FAO); International Society for

Table 4
Mean number of allele.observed and expected heterozygosities and FIS values in the examined Hungarian populations Durchschnittliche Anzahl Allele, ermittelte und geschätzte Heterozygotie sowie FIS-Werte der Populationen

Table 5
Matrix of Nei's genetic distances (below diagonal) between the examined Hungarian populations Genetische Distanz (DA) der untersuchten Bestände Indigenous Tsigai, however, it was close to Csanádpalota-IN (0.175) and far from the other examined population.Kardoskút2-IN and Kardoskút1-IN were very close to each other and far from other Hungarian Tsigai groups.The tree suggest that other examined Hungarian Tsigai were genetically closer to each other than Kardoskút2-IN and Kardoskút1-IN populations.It was suprise that Debrecen-RU was close to Cegléd-ZO.