Agricultural and Food Science in Finland 233 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 233–237. phisms, i.e. blood groups and serum proteins. Particularly, microsatellites (Weber and May 1989, Litt and Luty 1989) have proved suitable due to their unique polymorphism and Mendeli- an codominant inheritance (e.g. Litt and Luty 1989). In addition, these short tandem repeats (STR’s) are readily typed and scored with auto- mated procedures, which makes them amenable for routine laboratory practice (Goor et al. 1998). In an ideal situation all laboratories would © Agricultural and Food Science in Finland Manuscript received January 1999 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 233–237. Research Note Microsatellite panels suggested for parentage testing in cattle: informativeness revealed in Finnish Ayrshire and Holstein-Friesian populations Peter Bredbacka Finnish Animal Breeding Association, FABALAB, PO Box 40, FIN-01301 Vantaa, Finland, e-mail: pete@faba.fi Mikko T. Koskinen Finnish Animal Breeding Association, FABALAB, PO Box 40, FIN-01301 Vantaa, Finland. Current address: Department of Ecology and Systematics, Division of Population Biology, PO Box 17, FIN-00014 University of Helsinki, Finland Informativeness of eleven microsatellite markers suggested for parentage control in cattle by the International Society for Animal Genetics (ISAG) was studied in Finnish Ayrshire and Holstein- Friesian populations. Calculations were based on a sample of 100 non-sib artificial insemination bulls. Assuming one known parent the nine loci suggested for routine testing exhibited exclusion probabilities of 99.84% in the Ayrshires and 99.91% in the Holstein-Friesians. The addition of mark- ers INRA23 and TGLA53, recommended for further investigations, increased the attained values to 99.94% in Ayrshires and to 99.98% in Holstein-Friesians. The recommended core set of six microsat- ellites provided a combined exclusion probability of 98.25% in Ayrshires and 99.32% in Holstein- Friesians. Although the combined values were high in general, a relatively low level of polymor- phism was detected in some instances. Key words: animal identification, bovine, exclusion probability, microsatellite Introduction Correct assignment of livestock pedigrees is par- ticularly important when artificial insemination is widely used, as for example in cattle: errors have a direct effect to the genetic response (Aren- donk et al. 1998). In recent years, nuclear DNA loci have become the markers of choice for par- entage verification over the traditional polymor- mailto:pete@faba.fi 234 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Bredbacka, P. & Koskinen, M. T. Microsatellite panels for parentage testing in cattle be using the same markers and recognising the same nomenclature for allele scoring. Therefore, in 1996 ISAG’s (International Society for Ani- mal Genetics) cattle standing committee suggest- ed a set of nine microsatellites combined in three multiplex-PCR (Polymerase Chain Reaction) reactions as a standard protocol for parentage testing in bovines. Four additional markers were proposed for further investigations. In 1998 the committee recommended a core set of six loci as a minimum assay for all typed animals (http:/ / w w w . i m m g e n . c o m / h t m l / p r i m e r s e - quences.html). We are routinely using eleven of these mic- rosatellites to ensure correct pedigrees for cattle in Finland. Since 1996 over 2500 cattle, repre- senting six different breeds (mainly Finnish Ayr- shire and Holstein-Friesian), have been typed in FABALAB (laboratory of the Finnish Animal Breeding Association). The purpose of this work was to evaluate the efficiency of the three pan- els consisting of 11, 9 and 6 markers (suggested by ISAG), in detecting false parentage assign- ments in Finnish Ayrshire and Holstein-Friesian cattle. Material and methods Our sample consists of 100 bulls representing two of Finland’s most common breeds, the Finn- ish Ayrshire (50) and the Holstein-Friesian (50). These individuals are all being used for artifi- cial insemination purposes, thus having a high number of offspring, and representing the Finn- ish populations well. DNA was extracted from hair bulbs using PCR buffer containing 8 µg of proteinase K / 40 µl reaction. Alternatively, extraction was per- formed from semen or blood using standard pro- tocols. Microsatellites were amplified in three reactions: Multiplex 1, Multiplex 2 and Multi- plex 3 (Table 1). Prior to electrophoresis, 0.5 µl of the PCR products were pooled, 12 µl forma- mide and 0.5 µl TAMRA 350 internal lane stand- ard (Applied Biosystems) were added and the mixture was kept in 95°C for 3 minutes for de- naturation and quickly cooled on ice. Fragment separation and allele size scoring were performed using the ABI 310 Genetic Analyser and the GeneScan v. 2.1 software (Applied Biosystems). Locus specific probabilities of excluding a falsely assigned sire (or dam) were calculated using formulae adapted from Jamieson (1994): P En = Σp i (1-p i )2- Σ (p i p j )2 [4-3(p i + p j )], where P En is the probability of exclusion in a given locus with n alleles and p i and p j refer to the frequencies of alleles. Panel specific combined exclusion probabil- ities were calculated as: P Ec = 1-[(1-P E1 )(1-P E2 )…(1-P En )], where P Ec is the combined exclusion proba- bility over all loci in the panel and P E1 , P E2 …P En are the exclusion probabilities in the individual loci. This approach for calculating probabilities of exclusion is appropriate in situations where the Table 1. Multiplexes, microsatellites and their PCR (Polymerase Chain Reaction) parameters. Multiplex Locus and its forward and PCR reverse primer amounts parameters 1 BM1824 15pmol 94°C 3min BM2113 3pmol 27x 94°C 30s SPS115 7pmol 58°C 1min 72°C 1min 72°C 5min 2 ETH3 5pmol 94°C 3min ETH10 5pmol 27x 94°C 30s ETH225 5pmol 66°C 1min 75°C 30s 75°C 5min 3 TGLA227 5pmol 94°C 3min TGLA126 11pmol 29x 94°C 30s TGLA122 3pmol 55°C 1min TGLA53 5pmol * 75°C 30s INRA023 2pmol * 75°C 5min * Markers recommended for further investigations includ- ed to Multiplex 3 235 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 233–237. the other parent’s genotype is known. Thus, it can be used for farm animal populations (Jamie- son and Taylor 1997). In theory, the formula of Jamieson (1994) assumes that investigated marker alleles are in- herited in Hardy-Weinberg (H-W) proportions. H-W exact probability tests of Guo and Thomp- son (1992), implemented by the computer pro- gram GENEPOP v.3.1b (Raymond and Rousset 1995), were therefore conducted for the eleven loci of Finnish Ayrshire and Holstein-Friesian populations. Corrections for multiple signifi- cance tests were performed by applying a se- quential Bonferroni correction (Rice 1989). Results Twenty-two exact tests (2 populations, 11 loci) revealed no highly significant (P<0.01) devia- tions from H-W equilibrium. Genotypes of the locus BM1824 expressed marginal departures (0.0199.9%, 93.7% to >99.9% and 79.1% to 99.2% in the Ayrshires and 97.3% to >99.9%, 95.4% to 99.7% and 88.1% to 99.3% in the Holstein-Friesians for the eleven, nine and six loci, respectively. These re- sults emphasize the assay’s efficiency but also point out the problems the minimum number of markers might give rise to. Having an international agreement on mic- rosatellites and fragment size calling serves when sperm or embryos are imported: the bull’s read- ily available genotype greatly reduces problems with uncertain pedigrees. To avoid retyping it would be beneficial to have a high number of commonly investigated markers. With efficient multiplexing and stable assays the additional effort and cost is marginal. For instance, the set of eleven cattle microsatellites can be analysed in a single multiplex (Goor et al. 1998). In setting up guidelines for testing cattle par- entage it is important to survey as many breeds as possible as bovine microsatellite allele fre- quencies and exclusion probabilities tend to vary across breeds (e.g. Moazami-Goudarzi et al. 1997, Usha et al. 1995, Glowatzki-Mullis et al. 1995). Herein we report, to our knowledge, the first results of the informativeness of the com- bined sets of markers suggested by ISAG for cattle parentage testing. With this survey we con- clude that the set of nine STR-loci provides high probabilities of exclusion in the Finnish Ayrshire and Holstein-Friesian populations. Addition of INRA23 and TGLA53 increases the attained values most likely enough for solving even the most troublesome cases. However, in the two studied breeds SPS115 exhibits considerably lower polymorphism than the other loci. If sim- ilar conclusions are made with other populations a possible exchange of the marker should per- haps be considered. Furthermore, at least in the Finnish Ayrshires, the minimum panel of six markers seems relatively inefficient for solving the most difficult disputes of parentage. Acknowledgements. The authors thank Kaarina Pirhonen and Raili Toivanen for their assistance. References Arendonk, J.A.M. van, Spelman, R.S., Waaij, E.H. van der, Bijma, P. & Bovenhuis, H. 1998. Livestock breed- ing schemes: challenges and opportunities. 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Comparison of three proba- bility formulae for parentage exclusion. Animal Ge- netics 28: 397–400. Litt, M. & Luty, J.A. 1989. A hypervariable microsatellite 237 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 233–237. revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Ameri- can Journal of Human Genetics 44: 397–401. Moazami-Goudarzi, K., Laloe, D., Furet, J.P. & Gro- sclaude, F. 1997. Analysis of genetic relationships between 10 cattle breeds with 17 microsatellites. Animal Genetics 28: 338–345. Raymond, M. & Rousset, F. 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. Journal of Heredity 86: 248–249. Rice, W.R. 1989. Analysing tables of statistical tests. Evolution 43: 223–225. Usha, A.P., Simpson, S.P. & Williams, J.L. 1995. Proba- bility of random sire exclusion using microsatellite markers for parentage verification. Animal Genetics 26: 155–161. Weber, J.L. & May, P.E. 1989. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. American Journal of Hu- man Genetics 44: 388–396. SELOSTUS Suomalaisten ayrshire- ja holstein-friisiläisrotuisten nautojen mikrosatelliitti-DNA:han perustuvan polveutumismäärityksen tehokkuus Peter Bredbacka ja Mikko T. Koskinen Kotieläinjalostuskeskus-FABA ja Helsingin yliopisto Kansainvälisen eläingenetiikan järjestö (ISAG) on ehdottanut mikrosatelliitti-DNA:han perustuvaa me- netelmää yleisesti käytettäväksi nautojen polveutu- misten varmistamisessa. Eri rotujen välillä esiintyväs- tä mikrosatelliittien polymorfian vaihtelusta johtuen ISAGin ehdottaman menetelmän tehokkuus vaihtelee rodun mukaan. Tämän tutkimuksen tarkoituksena oli selvittää ehdotettujen vaihtoehtojen (11, 9 tai 6 lo- kusta) tehokkuus suomalaisten ayrshire- ja holstein- friisiläisrotujen väärien isyyksien paljastajana. Tulok- set osoittavat yhdeksän rutiinitestaukseen tarkoitetun mikrosatelliitin paljastavan väärät isyydet ayrshirel- lä 99,84 %:n ja holstein-friisiläisellä 99,91 %:n var- muudella. Kahden lisätutkimuksiin tarkoitetun mik- rosatelliitin lisäys nostaa vastaavat arvot 99,94 %:iin ayrshirellä ja 99,98 %:iin holstein-friisiläisellä. Kuu- den mikrosatelliitin muodostaman menetelmän avulla voidaan väärät isyydet paljastaa 98,25 %:n todennä- köisyydellä ayrshirellä ja 99,32 %:n todennäköisyy- dellä holstein-friisiläisellä. Vaikka edellä mainitut to- dennäköisyydet osoittavat, että väärät isyydet voidaan sulkea pois tehokkaasti, havaitsimme tietyissä yksit- täisissä tapauksissa (esim. lokus SPS115) suhteelli- sen vähäistä polymorfiaa. Jos vastaavia tuloksia saa- daan jatkossa myös muilla roduilla, tulisi kyseisten lokusten mahdollista vaihtoa harkita. Ongelmat ko- rostuvat esim. tilanteissa, joissa emästä ei ole saata- vissa näytettä. 238 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Bredbacka, P. & Koskinen, M. T. Microsatellite panels for parentage testing in cattle Title Introduction Material and methods Results Discussion References Selostus