DNA MICROSATELLITE ANALYSIS OF HORSE AND CATTLE BREEDS

THESIS OF DOCTORAL (PhD) DISSERTATION PANNON UNIVERSITY GEORGIKON FACULTY OF AGRICULTURAL SCIENCES Department of Animal Science and Animal Breeding Head of Department Dr. Ferenc Husvéth (D.Sc.) Animal Sciences Doctoral School (assignee: Animal and Agrarian Environment Sciences Doctoral School) Former Head of Programme Dr. Ferenc Szabó (D.Sc.) Head of Programme Dr. Angéla Anda (D.Sc.) Supervisor Dr. Ferenc Husvéth (D.Sc.) Co-Supervisor Dr. Hedvig Fébel (C.Sc.) Consultant Beáta Bán DNA MICROSATELLITE ANALYSIS OF HORSE AND CATTLE BREEDS Written by Csilla Józsa KESZTHELY 2008

1. INTRODUCTION AND OBJECTIVES OF THE STUDY By means of selection, men have been taking hand in the genetic structure of the cultured populations since the beginning of domestication. The diversity of the known characteristics of the currently cultured species has been developed as a result of this work. Nevertheless, new procedures (such as microsatellite analysis) and other molecular genetic methods always accompany conventional selection methods, providing more reliable results both for researchers and animal breeders. In my doctoral work genetic characteristics of cattle and horse species commonly bred in Hungary were investigated using DNA microsatellites. The analyses were performed in the Central Animal Breeding Laboratory, Animal Breeding Directorate, Central Agricultural Office, Budapest, Hungary. The blood samples used were samples obligatory submitted for routine laboratory examination between 2004 and 2006 for origin control (by DNA microsatellite analysis), selected in a randomized manner in both species (horse, cattle). The studied population included both female and male animals. The main objectives of the examinations carried out within the framework of the thesis are summarized as follows: Survey of the genetic diversity of the horse (Thoroughbred, American Trotter, Lipizzaner, Hungarian Draught Horse) and cattle (Hungarian Simmental, Limousin, Hereford, Charolais, Aberdeen Angus) breeds bred in Hungary using microsatellite DNA markers. Defining the number and frequency of the alleles on the microsatellite loci, searching for individual alleles in the above mentioned horse and cattle breeds. 2

Evaluation of the genetic distance between the particular horse and cattle breeds in Hungary using DNA microsatellite analysis. Evaluation of the degree of inbreeding using DNA microsatellite analysis in the populations and breeds involved in the study. Estimation of the applicability of microsatellite DNA markers supporting advance in Hungarian animal breeding. 2. MATERIALS AND METHODS Within the framework of the examination of horse breeds, randomly selected animals of the Lipizzaner (n=630), the Thoroughbred (n=200), the American Trotter (n=198) and the Hungarian Draught Horse (n=58) were investigated. Population analysis was performed with Thoroughbred blood samples received from two studs: Dióspuszta (n=72) and Kerteskő (n=39), both submitted in 2005. In the case of beef cattle species, individual blood samples of five breeds, namely: the Hungarian Simmental (n=252), Limousin (n=127), Hereford (n=144), Charolais (n=254) and Aberdeen Angus (n=337) were studied. The animals involved in the study were selected in a randomized manner. Within the Hungarian Simmental breed the DNA microsatellite analysis was also conducted from samples collected from populations of the Petőfi Agricultural Ltd. of Derecske (Derecske Petőfi Mezőgazdasági Kft.; n=40) and the Béke Agricultural Society in Hajdúböszörmény (Béke Agrárszövetkezet, n=55) in 2004 and 2006. In addition to the 9 microsatellites set out by ISAG (International Society for Animal Genetics) that are to be analysed obligatorily when controlling the origin of horses, in my work 8 more microsatellites (VHL20, HTG4, AHT4, HMS7, HTG6, AHT5, HMS6, ASB23, ASB2, HTG10, HTG7, HMS3, HMS2, ASB17, LEX3, HMS1, CA425) in the horse breeds, while 11 microsatellites (BM1824, BM2113, 3

ETH10, ETH225, INRA23, SPS115, TGLA122, TGLA126, TGLA227, ETH3, TGLA53) in the cattle breeds were analyzed. In the first phase of the studies genomic DNA from lymphocytes was isolated and after obtaining the purified DNA the template was amplified. For the amplification the multiplex kits of Applied Biosystems StockMarks developed for horse and cattle species (Applied Biosystems, 2001) were used. The separation of the fragments amplified in a multiplex form was performed with capillary electrophoresis using ABI PRISM TM 310 genetic analyzer (Applied Biosystems, USA). 3. RESULTS The analysis of the 11 cattle and 17 horse loci revealed rather polymorph results regarding the observed (n a ) and effective number of alleles (n e ) and the observed (H o ) and expected heterozygosity (H e ). The observed microsatellite alleles and allele frequencies also showed great variability. Concerning the number and frequency of observed alleles the Hungarian Simmental proved to be the most diverse cattle breed (mean n a =10.1818) and the Lipizzaner was the most diverse horse breed (mean n a =10.6471). The majority of the loci did not differ significantly (p>0.05) from the Hardy- Weinberg equilibrium in the studied breeds (except for the Lipizzaner horse and Charolais cattle breeds). A significant change at 10 loci in the Lipizzaner breed (p<0.001; p<0.01; p<0.05) and at 6 out of 11 microsatellites in the Hungarian Charolais population (p<0.001; p<0.05) was detected. For the observed heterozygosity neither value reached the maximum level (i.e. H o =1) in the breeds/populations studied. This indicates that the studied breeds/populations originated from the appropriate number of parent generations. F IS values for 3 out of the 4 horse breeds studied (Thoroughbred, Hungarian Draught Horse, Lipizzaner) and for all cattle breeds studied indicated inbreeding (F IS >0). Mean expected and observed heterozygosity values indicated inbreeding for all studied horse and cattle breeds since mean 4

H e values exceeded mean H o values (H e >H o ) in all cases. The breeds studied showed great allele variability. As a conclusion, the genetic backgrounds of the studied horse and cattle breeds commonly bred in Hungary were successfully analyzed in this work. The Lipizzaner horse showed the highest number of breed-specific alleles (e.g. HMS7-P, HMS7-Q, HTG10-S), and so was found the Limousin cattle, as the allele 177 of the TGLA122 microsatellite had a frequency of 3.15%, while this allele could not be detected in the other breeds. According to our results, the applied analytic procedures based on multiple factors and subsystems proved to adequately distinguish between the individual animals and breeds and to determine their degree of relatedness. With these methods the gene flow and the degree of inbreeding could also be analyzed. According to these results, remarkable differences can be detected with DNA microsatellite analysis between the horse and cattle breeds, respectively. With this method the origin of an individual animal can be unambiguously determined (with 97.85-99.46% confidence in cattle breeds or 99.80-99.98% confidence in horse breeds) and monitoring of genetic changes and the degrees of relatedness within breeds is also possible. 4. DISCUSSION The DNA microsatellite marker analysis of horse and cattle species bred in Hungary revealed that the total Hungarian animal stock was genetically rather heterogeneous and had considerable genetic resources. This especially applies to the Lipizzaner breed within horses and to almost all cattle breeds. In addition, the studied breeds show signs of inbreeding, as it was confirmed with various genetic parameters. The Hungarian horse and cattle breeds are genetically closely related, respectively. Values reflecting genetic distance were relatively small. The F ST values of the breed pairs also support this conclusion: F ST <0.05 reflects small degree of differentiation, while a value higher than 0.25 indicates significant degree of isolation and fragmentation 5

within the species. In the present study, F ST values for all studied horse and cattle breeds were below 0.25, and in some cases it was even below 0.05. Taking this into account, horse and cattle breeds can be well differentiated with 17 and 11 DNA microsatellite markers, respectively. Total Hungarian horse and cattle stock studied did not differ significantly from the Hardy- Weinberg equilibrium (except for the Lipizzaner and Charolais breeds). This result supports the notion that there is no inbreeding among the animals in these breeds. However, a few genetic parameters (comparison of the expected and observed heterozygosity, positivity and negativity of the F IS value) indicated some degree of inbreeding among both beef cattle and horse breeds studied, respectively. In order to maintain breeding of horses and cattle for the future in our country, an important issue would be to refresh the bloodlines of these inbred populations by involving animals from the same breeds (from abroad or from Hungary) into the breeding process. Prior to this, performing a molecular genetic analysis of the hereditable alleles in the newly involved animals is inevitable in order to ensure genetic variability of the breeds. An issue for the future could be the assessment of the larger Hungarian horse and cattle breeding stocks and the evaluation of the impact of the individual male animals on the genetics of the animal population. Molecular genetic methods and utilization of the results obtained in our investigations are recommended to be used in the breeding programs of horse and cattle. Genetic variability of Hungarian breeds has to be preserved and molecular genetic procedures are excellent methods for this purpose. Importing breeding animals could be carried out in order to establish genetic variability in cases where it seems to be necessary according to the results of this analysis. Molecular genetic analysis of individual animals are recommended prior to their inclusion into the breeding process in order to make sure that only animals with truly diverse, perhaps even with special alleles refresh the Hungarian animal stock and therefore improve the genetic structure of the breeds. 6

For increasing the efficiency of the parentage control the 17 (horse) and 11 (cattle) microsatellites tested in this study are suggested to be applied in the routine parentage analysis of horse and cattle stocks in our country. 5. NEW SCIENTIFIC RESULTS 1. The 17 DNA microsatellite markers examined in the present study proved to accurately distinguish the horse breeds bred in Hungary according to their genetic markers. The analyzed loci showed high genetic variability. Extremely high degree of microsatellite polymorphism was found in some cases (e.g. VHL20, ASB2, CA425, ASB17, and LEX3) among the Hungarian horse breeds. A total of 193 alleles were identified at the analyzed 17 loci. The allele number per locus varied between 7 (HTG7) and 16 (ASB2, ASB23) in the four horse breeds analyzed. 2. The 11 microsatellites analyzed were adequate (and sufficient) to reveal the differences between the Hungarian cattle breeds studied. The TGLA loci (122, 126, 227, and 53) and the INRA23 and SPS115 from the analyzed microsatellites showed especially high variability and a high allele number. A total of 136 alleles at the analyzed 11 loci were identified in the cattle breeds studied. The allele number per locus varied between 6 (BM1824) and 21 (TGLA122). 3. According to my molecular genetic study, the ratio of the positive and negative F IS values and the observed mean heterozygosity values show signs of inbreeding among the Hungarian horse and cattle breeds, respectively. The mean expected heterozygosity value exceeded the observed heterozygosity value in every breed in this study, which is also regarded as a sign of inbreeding. In the case of the breeds examined (except for the Lipizzaner horse breed and Charolais cattle) the expected genotype distribution calculated 7

according to the Hardy-Weinberg equilibrium does not differ significantly at the majority of loci. 4. My results suggest that the horse breeds studied are genetically closely related and that the differentiation among breeds is low (genetic distance: 0.2803-0.4869; F ST value: 0.0502-0.0717). Furthermore, the results of our study confirm that the Lipizzaner and Hungarian Draught Horse and the Thoroughbred and American Trotter are the two most closely related breed pairs, respectively. 5. Our investigations proved that the Hungarian cattle breed studied are genetically closely related and that the differentiation among these breeds is low (genetic distance: 0.1052-0.4284; F ST value: 0.0212-0.0716). The closest relationships are showed between the Hungarian Simmental and Charolais breeds, but the Charolais and Limousin breeds also showed only small genetic distance. This small genetic distance between the Charolais and Limousin breeds indicates that both French cattle breeds originate form a genetically very similar animal population. Despite the fact that both breeds belong to the British cattle breeds, a larger genetic distance was detected between the Aberdeen Angus and Hereford bred in Hungary. 6. Regarding the TGLA122 microsatellite alleles, there was a high allele frequency of allele 177 in the Limousin, of allele 179 in Charolais and of allele 185 in Hereford breeds, while they could not be detected in other breeds. Therefore these alleles might be regarded as breed-specific alleles. 7. The high allele frequency observed for some of the analyzed alleles in the horse breeds studied (Thoroughbred: AHT4-G, HTG6-R; American Trotter: HMS1-Q; Hungarian Draught Horse: ASB17-T, -K; Lipizzaner: ASB23-Q, HTG10-S, CA425-P, HMS7-P, -Q,) also indicates the possibility that they might be breed-specific. 8

8. Regarding to the efficiency of the parentage testing it was demonstrated that the microsatellites applied on the inland routine parentage control could be used by the checking of the issue pedigree (horse: 17 locus; cattle: 11 locus). This study appointed that the microsatellites mentioned above can be applied in the parentage control of cattle s tested with the efficiency of 97.85%- 99.46%, and with the efficiency of 99.80-99.98% in the horse breeds studied. 6. LIST OF PUBLICATIONS Papers published in peer-reviewed journals/ books in English: A. Szontagh, B. Bán, I. Bodó, E. G. Cothran, W. Hecker, Cs. Józsa, Á. Major (2005): Genetic diversity of the Akhal-Teke horse breed in Turkmenistan based on microsatellite analysis, Convervation genetics of endangered horse breeds, Wageningen Academic Publishers, Wageningen, Netherlands, 123-129. S. Mihók, B. Bán, Cs. Józsa, I. Bodó (2005): Estimation of genetic distance between traditional horse breeds in Hungary, Convervation genetics of endangered horse breeds, Wageningen Academic Publishers, Wageningen, Netherlands, 111-123. Cs. Józsa, F. Husvéth, B. Bán, E. Takács (2006): DNA microsatellite test of Thoroughbred and Trotter horses in Hungary. Állattenyésztés és takarmányozás. 55. (4) 313-322. 9

Papers published in peer-reviewed journals/ books in Hungarian: Józsa Cs., Husvéth F., Bán B., Takács E. (2006): A telivér és ügető fajták D- vércsoport és biokémiai polimorf rendszerek vizsgálata. Állattenyésztés és takarmányozás. 55. (2) 117-125. Bán B., Bodó I., Józsa Cs., Mihók S. (2006): A mezőhegyesen kitenyésztett lófajták vércsoport, biokémiai polimorfizmus és DNS mikroszatellit vizsgálata, összehasonlításuk a hucul és az angol telivér fajtákkal - könyvrészlet, Génmegőrzés, Hagyományos háziállatfajták genetikai és gazdasági értékének tudományos feltárása, 44-55., Debreceni Egyetem Agrárátudományi Centrum, Debrecen Publications in conference proceedings in English: Beáta Bán, Alice Gyurmán, Csilla Józsa (2002): Thoroughbred and Trotter blood group and DNA allele frequency in Hungary (poster) D003, XXVIII. International Conference on Animal Genetics, ISAG, Germany, Göttingen Csilla Józsa, Ferenc Husvéth, Beáta Bán, Alice Gyurmán, Ferenc Szabó (2004): Microsatellite DNA polymorphism of four beef cattles in Hungary (poster) - E021, XXIX. International Conference on Animal Genetics, ISAG, Japan, Tokyo Cs. Józsa, B. Bán, S. Mihók, I. Bodó, F. Husvéth (2005): DNA microsatellite test of Hutsul horses in Hungary, Systems of identification in horses, Book of Abstracts of the 56th Annual Meeting of the European Association for Animal Production, H1-22 (poster), Uppsala, Sweden 10

Csilla Józsa, Beáta Bán, Erzsébet Takács, Ferenc Szabó, Ferenc Husvéth (2006): Microsatellite DNA polymorphism of Lipizzan horses in Hungary (poster) A573, XXX. International Conference on Animal Genetics, ISAG, Brazília, Porto Seguro Publications in conference proceedings in Hungarian: Józsa Cs., Bán B., Gyurmán A., Husvéth F., Németh K. (2003): A sportlovak származásellenőrzésének vizsgálata vér polimorfizmusok és DNS mikroszatellitek segítségével (előadás), Akadémiai Beszámolók, Genetika, MTA Állatorvos-Tudományi Bizottsága, Budapest Németh K., Balogh K., Ribiczeyné Sz. P., Mézes M., Gaál T., Husvéth F., Józsa Cs. (2003): Az E-vitamin- és a metionin-kiegészítés hatása a növendék baromfi antioxidáns rendszerére (előadás). Akadémiai Beszámolók. Élettan, Biokémia, Morfológia. MTA Állatorvos-Tudományi Bizottsága, Budapest Józsa Cs., Bán B., Gyurmán A., Mátrai R., Husvéth F. (2005): A lipicai fajta származásellenőrzésének vizsgálata genetikai markerek segítségével (előadás), MTA Állatorvos Tudományi Bizottsága, Akadémiai Beszámolók, Genetika, Budapest Józsa Cs., Bán B., Gyurmán A., Szabó F., Husvéth F. (2005): DNS mikroszatellit vizsgálatok húsmarha fajtákban (előadás), MTA Állatorvos Tudományi Bizottsága, Akadémiai Beszámolók, Genetika, Budapest Józsa Csilla, Bán Beáta, Péntek István, Husvéth Ferenc (2006): A lipicai fajta származásellenõrzésének vizsgálata DNS mikroszatellitek segítségével (előadás), Lótenyésztési Tudományos Napok, Debreceni Egyetem Agrártudományi Centrum, Debrecen 11

Lectures in conferences in English: S. Mihók, B. Bán, Cs. Józsa, E. Takács, I. Bodó (2004): Estimation of genetic distance between traditional horse breeds in Hungary, Free communications Horse Production, Book of Abstracts of the 55th Annual Meeting of the European Association for Animal Production, EAAP-Bled, Slovenia, H4-2. A. Szontagh, B. Bán, I. Bodó, E. G. Cothran, W. Hecker, Cs. Józsa (2004): Genetic diversity of the Akhal-Teke horse breed in Turkmenistan based on microsatellite analysis, Rare breeds: global genetic distance, Book of Abstracts of the 55th Annual Meeting of the European Association for Animal Production, EAAP-Bled, Slovenia, H3-5. Others: Jenny Cahill, Bokor Árpád, Józsa Csilla (2007): Lovak származás-ellenőrzése Új-Zélandon - Lovas Nemzet, XIII. évf., 3. szám, 40-41. Józsa Csilla, Bán Beáta, Bokor Árpád (2007): Lovak származás-ellenőrzése Magyarországon - Lovas Nemzet, XIII. évf., 4. szám, 48-50. 12