ISSN 1313 - 8820Volume 5, Number 4

December 2013

2013

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English Editor

Yanka Ivanova (Bulgaria)

2013

ISSN 1313 - 8820 Volume 5, Number 4December 2013

Genetic diversity and distance between two Bulgarian local sheep breeds assessed by microsatellite markers

1 2 1 3 4 1S. Georgieva , E. Todorovska , D. Hristova , I. Dimitrova , N. Stancheva , Ts. Yablanski

1Department of Genetics, Breeding and Reproduction, Faculty of Agriculture, Trakia University, 6000 Stara Zagora, Bulgaria2AgroBioInstitute, 8 Dragan Tsankov, 1164 Sofia, Bulgaria3University of Forestry, 10 Kliment Ohridski, 1756 Sofia, Bulgaria4Agricultural Institute, 3 Simeon Veliki, 9700 Shumen, Bulgaria

Abstract. In the present study 37 blood samples from Copper-Red Shumen and 38 blood samples from Karakachan sheep breeds were used to extract DNA followed by polymerase chain reaction (PCR). Based on the set of six microsatellite markers the genetic diversity was investigated in the examined breeds. Allele diversity, observed heterozygosity, expected heterozygosity, average gene diversity over loci, coefficient of inbreeding and chi-square test for Hardy-Weinberg equilibrium were calculated. Within breeds, the mean number of alleles varied from 9.5 in Karakachan to 10.5 in Copper-Red Shumen. The mean observed heterozygosity (Ho) ranged from 0.543 in Karakachan to 0.621 in Copper-Red Shumen, while the mean expected heterozygosity (He) from 0.774 in Karakachan to 0.812 in Copper-Red Shumen.The coefficient of inbreeding (Fis) was positive, ranging from 0.298 in Karakachan to 0.235 in Copper-Red shumen, indicating a risk of inbreeding. The low distance value (0.083) was found between the populations Karakachan and Copper-Red Shumen. The data obtained in the present study can contribute towards development of an effective conservation strategies for these traditional sheep breeds in Bulgaria.

Keywords: local sheep breeds, microsatellite markers, diversity, genetic structure, distances

Abbreviation: CRSH – Copper-Red Shumen; KKCH – Karakachan sheep

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No 4, pp , 2013367 - 370

Introduction level of polymorphism, co-dominant inheritance and easy to analyze (Cañon et al., 2006). Microsatellites have been wide by used for assessment of genetic diversity in sheep. (Diez-Tascon et al., 2000; Sheep play an important role in maintenance of rural Hassan et al., 2003; Arora and Bhatia, 2004; Gutiérrez-Gil et al. populations and also have a particular cultural importance due to its 2006; El-Nahas et al., 2008).traditional use in rites and celebrations. They are also used for meat,

In the present study, a set of six microsatellite markers were milk and wool production. Copper-Red Shumen (CRSH) and used to evaluate the genetic diversity within and between two Karakachan (KKCH) sheep breeds are important local sheep breeds Bulgarian local sheep breeds and to measure the distance among in Bulgaria and they are the object of this study. the breeds.CRSH is distributed mainly in the North-Eastern part of

Bulgariain regions. Representatives of this breed are medium-to-large with average body weight of 47kg with compact body. Fleece

Material and methods colour is rusty-red, while one can find individuals with black fleeces. Ewes are hornless, but rams have twisted well-developed horns. They usually have well covered body and legs free of wool A total of 75 individuals representing two local Bulgarian sheep (EASRAB, 2011). The average body weight varyed from 49.12 to breeds (KKCH and CRSH) were analyzed. For each breed unrelated 55.46kg (Staykova, 2005). The results of polymorphic analyses of individuals were selected (37 blood samples for CRSH and 38 for blood serum proteins showed the presence of some rare alleles KKCH). Blood samples of 5–10 ml were collected in EDTA tubes. such as Tf G, Alb F, Alb S, Alb W in this breed (Makaveev and Nakev, DNA was extracted from the whole blood with Illustra Blood 1987; Baulov and Alexieva, 1991). GenomicPrep DNA Purification Kit (GE Healthcare, UK) according

KKCH is spread throughout mountain areas in Bulgaria.In the to the manufacturer's instructions and stored at -20ºC until the past the breed was one of the most widespread on the Balkans. analysis was performed.Animals are small, vigorous, strong-boned with short legs, resistant A set of six microsatellite markers (Table 1) was chosen based to unfavourable climatic and feeding conditions, and diseases. The of their level of polymorphism location on different chromosomes, animals have a vivid temperament, they are energetic and mobile preferably unlinked following the recommendation of the Food and and can endure long-distance marches. Their face, ears, legs and Agriculture Organization (FAO) and the International Society for nose are black. Most of the population has pigmented grey to black Animal Genetics (ISAG). wool, sometimes brown, predominantly coarse. The average body PCR amplifications were carried out in total 10µl volumes, weight is 44.04–54.10kg (Staykova, 2005). containing 50ng DNA template, 20pM primers and 1x AmpliTaq Gold

Microsatellites have often been used for genetic diversity PCR Master mix (Applied Biosystems, USA). Forward primers were studies, because of their distribution throughout the genome, high Cy5 fluorescently labelled. PCR amplification was performed in a

367

* e-mail: sgeorg@af.uni-sz.bg

368

thermocycler GeneAmp 9700 (Applied Biosystems) using the Yong, 1999; Labate, 2000). This program was used also to establish following conditions: for loci OarFCB20, OarJMP58 and MAF65 an the genetic distances (D ) between populations according to the A

initial denaturation step at 95ºC for 5 min, followed by 35 cycles of method of Nei (1978). The ARLEQUIN software, version 3.5.1.3 was 95ºC for 30s, annealing at 55ºC for 35s, extension at 72ºC for 1 min used for population data analysis (Excoffier and Lischer, 2010). The and final extension at 72ºC for 10 min; for marker MAF70 the number of alleles of each locus, heterozygosities values were annealing temperature was increased to 60 ºC. For loci ILSTS11 and calculated for two investigated breed. The same software was used OarCP20 a two-step cycling procedure was used: denaturation at to evaluate the coefficient of inbreeding (Fis) and to check deviation 95ºC for 5 min, followed by 9 cycles of 95ºC for 30s, annealing at from Hardy-Weinberg equilibrium (HWE). 58ºC or 62ºC for 35s, 72ºC for 1 min, then 25 cycles at 95ºC for 30s, annealing at 55ºC or 60ºC for 35s, 72ºC for 1 min and final extension at 72ºC for 10 min. PCR amplified products were separated on 6% Results denaturing polyacrylamide gel (ReproGel High Resolution) using automated laser sequencer (ALF Express II Amersham All markers were highly polymorphic. In Table 2 are displayed Biosciences). The electrophoresis was performed in 1x TBE buffer the variability parameters the investigated loci. The number of (0.09M Tris-borate, pH 8.3; 2mM EDTA) under the following running alleles per locus observed in two breeds ranged from 5 to 21. Based parameters: 1500V, 60mA, 30W, 55ºC, sample interval 2s, run time on the mean number of alleles per population, more diverse 450 min. Fragment sizes were determined using the computer population was CRSH (10.5) in comparison to KKCH (9.5).program Allele Locator, version 1.03 (Amersham Pharmacia In the examined populations the Ho levels were lower than the Biotech, 1998) with internal size standarts and data were imported expected heterozygosities (He). The mean Ho and He per studied and converted to pseudogel images. populations were 0.621–0.812 for CRSH and 0.543–0.774 for KKCH

The allele diversity, number of alleles per locus and their (Table 3). The coefficient of inbreeding (Fis) was positive, ranging richness, observed and expected heterozygosity values (H and H ) o e from 0.235 (CRSH) to 0.298 (KKCH), indicating a risk of inbreeding. were calculated using POPGENE software, version 1.31, (Yeh and The values of chi-square test (11.80 and 9.02) point that both studied

Table 1. SSR locus information

Marker name Origin Chromosome location Primer sequences F / R

2

4

9

15

21

26

Ovine

Ovine

Bovine

Ovine

Ovine

Ovine

OarFCB20

MAF70

ILSTS11

MAF65

OarCP20

OarJMP58

F:AAATGTGTTTAAGATTCCATACAGTG

R:GGAAAACCCCCATATATACCTATAC

F:GCAGGACTCTACGGGCCTTTGC

R:CACGGAGTCACAAAGAGTCAGACC

F:GCTTGCTACATGGAAAGTGC

R:CTAAAATGCAGAGCCCTACC

F:AAAGGCCAGAGTATGCAATTAGGAG

R:CCACTCCTCCTGAGAATATAACATG

F:GATCCCCTGGAGGAGGAAACGG

R:GGCATTTCATGGCTTTAGCAGG

F:GAAGTCATTGAGGGGTCGCTAACC

R:CTTCATGTTCACAGGACTTTCTCTG

sheep breeds are in accordance with HWE, even though a deficiency of heterozygocity was indicated (Table 3).

Five population-specific alleles were detected in the two breeds CRSH and KKCH in three microsatellite loci (Table 4).The relationships among the examined populations were determined using minimum genetic distance formula (D ). The distance value A

between CRSH and KKCH was low (0.083).

Table 2. Number of alleles per locus

Number of alleles

SSR locusPopulation

OarFCB20 MAF70 ILSTS11 MAF65 OarCP20 OarJMP58

Mean

CRSH

KKCH

8

9

18

21

7

7

8

6

10

5

12

9

10.500

9.500

Table 3. Mean observed (Ho) and expected (He)heterozygosities, average gene diversity over loci (Gdv),coefficient of inbreeding (Fis) and chi-square test for HWE in the examined populations

Population chi-squareFisGdvHeHo

CRSH

KKCH

0.621

0.543

0.812

0.774

0.812

0.774

0.235

0.298

11.80

9.02

369

current status of the genetic diversity and relationships among two Discussionlocal sheep breeds in Bulgaria. Using a panel of 6 polymorphic SSR markers high level of genetic diversity was established in the studied The genetic characterization of a breed is very important for the sheep populations. Based on their cultural, historical and evaluation of genetic variability, which is an substantial element in environmental importance the data obtained here could be considered as an initial guide for the development of rational breeding strategies for preservation and utilization of the autochthonous sheep breeds in Bulgaria.

Acknowledgment

This research was a part of the project 501/22, 05.12.2012. (2012-2014) "Development of DNA markers (CAST, MSTN) for fattening ability and meat quality in Synthetic population Bulgarian Milk, Karakachanian and Copper Red Shumen sheep breeds" financed by the Ministry of Education, Youth and Science, Republic of Bulgaria.conservation of genetic resources and for breeding strategies. In

present study the allele diversity and genetic relationship of 2 indigenous sheep breeds in Bulgaria were examined using a set of 6 polymorphic microsatellites. The relatively high mean number of Referencesalleles per locus (10.5 and 9.5 respectively) indicates the usefulness of the selected markers for studying the genetic diversity in

Alvarez I, Royo L, Fernandez I, Gutierrez J, Gomez E and Bulgarian sheep populations. This observation is in accordance with

Goyache F, 2004. Genetic relationships and admixture among other studies (Alvarez et al. 2004; Arranz et al., 2001) and confirms

sheep breeds from Northern Spain assessed using microsatellites. the suitability of this marker in sheep genetic diversity.

Journal of Animal Science, 82, 2246-2252.The lack of clear differentiation between Bulgarian sheep

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54, 3, 227-230.present gene flow among them. As the breeds considered in this

Arranz J, Bayoan Y and San Primitivo F, 2001. Genetic variation at study have never been genetically characterized before it was not

microsatellite loci in Spanish sheep. Small Ruminant Research, 39, always possible to compare our data with that reported in the

3-10.literature. Previous studies (Kusza et al., 2010) on the genetic

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In this study the genetic distances among the examined Mahfouz Eman R, Bibars Mona A, Oraby Hanaa AS and de Hondt populations were relatively low, but higher than 0.05, which may HA, 2008. Analysis of genetic variation in different sheep breeds indicate certain differences in their genetic structure. The closed using microsatellites, African Journal of Biotechnology, 7, 8, 1060-genetic relatedness (0.083) found between CRSH and KKCH is in 1068.accordance with their similar phenotypic traits. Both breeds are short Excoffier L and Lischer H, 2010. Arlequin suite ver 3.5 (09.2011). thin-tailed type with black-brown colour of the wool, predominantly A new series of programs to perform population genetics analyses coarse. Panaiotov et al. (2003) recorded similar average live weight under Linux and Windows. Molecular Ecology Resources 10, 564-of animals at 18 months for CRSH and KKCH (35.8 and 32.6 kg, 567. http://cmpg.unibe.ch/software/arlequin3. respectively). The ratio body height/length is also similar (59/65sm Gutiérrez-Gil B, Uzun M, Arranz J, Primitivo F S, Yildiz S, for CRSH and 57/65.5sm for KKCH (EASRAB, 2011). Cenesiz M and Bayon Y, 2006. Genetic diversity in Turkish sheep,

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Table 4. Population-specific alleles

Locus Frequency of specificalleles (%)

Breed

KKCHMAF70

ILSTS11

OarJMP58 CRSH

Allele size(bp)

149

171

286

133

173

15.789

15.789

5.263

2.703

8.108

370

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Genetics and Breeding

Nutrition and Physiology

Investigation on the possibility to efficiently use Ukrainian cultivars for developing of early winter wheat linesI. Grain productivityN. Tsenov, T. Petrova, E. Tsenova

Combinig ability for grain yield of late maize linesN. Petrovska

Use of recurrent selection in middle late synthetic maize populationI. Results of the first cycle in synthetic “1/2005”N. Petrovska, V. Valkova

Genetic diversity and distance between two Bulgarian local sheep breeds assessed by microsatellite markersS. Georgieva, E. Todorovska, D. Hristova, I. Dimitrova, N. Stancheva, Ts. Yablanski

Testing of new Bulgarian sunflower hybrids under the conditions of North-East BulgariaI. Productivity and traits related to productivityG. Georgiev, P. Peevska, E. Penchev

Comparative morphological study of new Burley tobacco linesT. Radoukova, Y.Dyulgerski

Effect of genotypic and environmental factors on the inheritance of the main characters in chickpea and relationships between themR. Sturzu, T. Nistot, Cr. Melucă, Fl. Bodescu, A. Stoilova

Evaluation of double haploid lines of winter malting barley using selection indicesB. Dyulgerova, D. Valcheva

Evaluation of the combining ability of grain yield of mutant maize lines M. Ilchovska

Comparative study of some biochemical indicators in Karakachan and Copper-Red Shumen sheep breedsG. Angelov, I.Dimitrova, T. Mehmedov, P. Stamberov, N. Stancheva, S. Georgieva, Zh. Nakev

Impaired pancreatic function in mulard ducks with experimental aflatoxicosis I. Valchev, N. Grozeva, D. Kanakov, Ts. Hristov, L. Lazarov, R. Binev, Y. Nikolov

Comparative investigations on feeding efficiency in growing and fattening DanBred and Topigs hybrid pigsG. Ganchev, A. Ilchev

Blood parameters in yearling sheep fed Paulownia (Paulownia spp.) leavesI. Varlyakov, V. Radev, T. Slavov, G. Ganchev

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Changes in some blood parameters in yearling rams fed diets with different protein and lipid levelsV. Radev, T. Slavov, I. Varlyakov

Effect of the sowing norm and nitrogen fertilization on the yield from dry bean (Phaseolus vulgaris L.) cultivar Beslet G. Milev

Evapotranspiration of corn crop for silageR. Bazitov, A. Stoyanova

Productivity and economic traits of winter oilseed rape (Brassica napus var. biennis) under the conditions of DobrudzhaG. Georgiev, G. Georgiev, P. Chamurliyski

Feasibility of the use of heat energy from alternative sources for air conditioning in sows facilityK. Peichev, R. Georgiev

Productivity of green beans, irrigated at different pre-irrigation soil moistureR. Petrova, A. Matev, K. Koumanov, B. Harizanova-Petrova

Comparative assessment of plant resources as substrates for bioshlam productionZ. Shindarska, V. Kirov, G. Kostadinova, B. Baykov

The influence of organic carbon on bioremediation process of wastewater originate from aquaculture with use of microalgae from genera Botryococcus and Scenedesmus I. Sirakov, K. Velichkova, G. Beev, Y. Staykov

Sanitary hygienic assessment of drinking water from underground source at a pig farmG. Kostadinova

Study of bee honey by spectral analysis in the near infrared spectrumI. Zhelyazkova, S. Atanasova , K. Elencheva – Karaneycheva

Comparative GC/MS analysis of lavender (Lavandula angustifolia Mill.) inflorescence and essential oil volatilesT. Zagorcheva, S. Stanev, K. Rusanov, I. Atanassov

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thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, Sept.11 – 14, Berlin, Germany, p. 302 (Abstr.).Thesis:Penkov D, 2008. Estimation of metabolic energy and true digestibility of amino acids of some feeds in experiments with muscus duck (Carina moshata, L). Thesis for DSc. Agrarian University, Plovdiv, 314 pp.

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