BOVINES GENOTYPING GENOTIPIZAREA LA BOVINE

Transcription

1 Lucrări ştiinţifice Zootehnie şi Biotehnologii, vol. XXXVIII (2005), Timişoara BOVINES GENOTYPING GEORGESCU S.E. *, CONDAC E. *, REBEDEA MARIANA **, DINISCHIOTU ANCA *, TESIO C. D. *, COSTACHE MARIETA * *University of Bucharest, Faculty of Biology, Molecular Biology Center, Bucharest **Institute of Animal Biology and Nutrition, Baloteşti GENOTIPIZAREA LA BOVINE During the last two decades major advances have been made in mammalian genetics. Recently, breeders have turned to molecular biology and they are using PCR (polymerase chain reaction) applied to the detection of short sequence repeats - microsatellites. Microsatellite markers are evenly distributed across genome and can be identified within DNA samples using PCR. In our experiment we analyzed Romanian Black Spotted cattle from IBNA Baloteşti with Stock Marks Kit. The number of allele peaks depends on whether the individual tested is a heterozygote or homozygote. Bovine identification and offspring determination by amplification of the STR loci was realized using modern technology of DNA extraction and automatic genotyping. The designation and number of microsatellites that should be used in parentage testing is yet a matter of discussion and depends on the characteristics of each locus and on the variability of the breed under study. Key Words: bovine, microsatellite, parentage testing, genotyping. Introduction During the last two decades major advances have been made in mammalian genetics. New methods have been developed and applied to investigating the genetics of the cattle and to improve its performance. Animals with superior traits, such as high milk production and lean carcasses in the case of cattle, or speed and strength in the case of horses, are used as breeding stock for subsequent generations. The recent developments in molecular genetics allowing the detection of genes responsible for economic traits have opened a new area in farm animal selection. The classical method for selection is based on physical observation of such traits in adult animals and the careful maintenance of lineage records by breeding organizations. Recently, breeders are using Polymerase Chain Reaction for detection of short sequence repeats (microsatellites). Eventually, DNA markers will be used to identify genetically superior animals more quickly, reliably, and inexpensively. The DNA profile test is more powerful than conventional methods because it detects DNA-sequence information that is highly variable. This technology 763

2 provides a sensitive method for parentage verification, individual identification and the estimation of relatedness. Materials and Methods Blood samples were obtained from Romanian Black Spotted, from IBNA, Baloteşti. The isolation of genomic DNA from the white blood cells was performed with Promega Genomic DNA Extraction Kit. Amplification of the STR loci was realized by multiplex PCR using StockMarks Paternity PCR Typing Kit (Applied Biosystems), according to the procedure recommended by the manufacturer. The reagents in the kit are used to amplify DNA samples using the fluorescent dye-labelled primers specific to the relevant loci. The kit contains all of the labelled and unlabeled primers, AmpliTaq Gold DNA Polymerase, control DNA, dntps mix and buffers. As the samples migrate past the fluorescence detector, the GeneScan Software collects the signal and assigns a base pair size for each sample. Allelic nomenclature is in order by size in base pairs, from smallest to largest. PCR amplifications were performed in 0.2 ml tubes, in AppliedBiosystem 2700 Thermocycler, using 31 cycles with denaturation at 94ºC (45 s), annealing at 61 C (45 s) and extension at 72 C (60 s). The first denaturation step was performed at 95 ºC (10 min) and the last extension was to 60 min at 72 C. Additionally, performed one cycle at 25 C for 2 hours. The PCR final volume was 15 µl. For each tested animal, the sample (10 µl) was diluted with 90µl deionised water for the PCR amplification reaction. The reaction mixture was composed of: 1,0 µl of the diluted eleven-plex reaction product, 11.5 µl Hi-Di Formamide and 0.5 µl GeneScan-500 ROX Size Standard. The mixture was spinned briefly in a microcentrifuge and heated at 95 C for 2 min before loading. Then, immediately, the tubes were put on ice for 3 min. Finally the samples were sequenced into the ABI PRISM 310 instrument. Results and Discussions The heritability estimation is a tool which breeders can use to help guide decisions of which cattle to mate based on superior performance for the trait in question, and also to predict the improvement of that trait in the offspring. Even though certain traits are affected primarily by environment, others are determined by genetics and still others are affected by both environment and genetics. The percentage that is due to genetics is an indication of the likelihood of a trait being passed from one generation to the next. In our experiment successful amplification yields allele peaks with the associated PCR stutter bands within a maximum range of eight base pairs from the allele peak. The number of allele peaks depends on whether the individual tested is a heterozygote or homozygote. For this StockMarks Kit, all eleven loci are dinucleotide repeats. Size calling is based on GeneScan-500 ROX Size Standard. 764

3 Figure 1: Genotyper software analysis of PCR amplification products using equine blue markers (ABI PRISM 310 Genetic Analyzer). Figure 2: Genotyper software analysis of PCR amplification products using equine green markers (ABI PRISM 310 Genetic Analyzer). 765

4 Figure 3: Genotyper software analysis of PCR amplification products using equine yellow markers (ABI PRISM 310 Genetic Analyzer). The GeneScan electrophoregram of a dinucleotide repeat marker from three cattle (one cow and two calf) of Romanian Black Spotted are shown in Figures 1, 2 and 3. In Table 1 are shown the allelic distribution for the three cattle on the specific loci. Table 1 Allelic distribution on the specific loci for three cattle Locus Cow (B1) Calf (B2) Calf (B3) TGLA , , ; BM ; ; ; TGLA ; ; ; 159 ETH ; ; ; SPS ; ; ; TGLA ; ; ; TGLA ; ; ; 150 INRA ; ; ; ETH ; ; ; ETH ; ; ; BM ; ; ;

5 Conclusions Cattle identification and offspring determination by amplification of the STR loci was realized using modern technology of DNA extraction and automatic genotyping with ABI PRISM 310 Genetic Analyzer. This technology is for the first time applied in Romania and provides a more efficiently and sensitive method for parentage and individual identification. The most efficient genetic markers for parentage testing are co-dominant and inherited by Mendelian principles. DNA testing techniques, now available and progressively adopted by most responsible laboratories, can greatly improve the success of parentage tests, providing an excellent alternative to traditional methods. Microsatellites are highly polymorphic genetic markers with co-dominantly inherited alleles that are relatively easy to score. A number of STR markers are now in use for parentage testing in several cattle breeds and other breed registries are now in the process of converting to a DNA based technology. The designation and number of microsatellites that should be used in parentage testing is yet a matter of discussion and depends on the characteristics of each locus and on the variability of the breed taken in study. Bibliography 1. Ashwell, M. S., Rexroad Jr., C.E., Miller, R.H., Van Raden, P. M. and Da, Y. Detection of loci affecting milk production and health traits in an elite US Holstein population using microsatellite markers, Animal Genetics 28: , [1997]. 2. Beever, J. E., George, P. D., Fernando, R. L., Stormont, C. J. and Lewin, H. A. - Associations between genetic markers and growth and carcass traits in a parental half sib family of Angus cattle, Journal of Animal Science 68:337, [1990]. 3. Beever J. E., Fisher S. R., Levin H. A. - Polymorphism identification in the ACADM, AT3, IL10, MYOG and TSHB genes of cattle. Animal Genetics 28, , [1997]. 4. Bishop, M. D., Hawkins, G. A. and Keeler, C. L. - Use of DNA markers in animal selection, Theriogenology 43: 61-70, [1995]. 5. Bovenhuis, H., van Arendonk, J. A. M., Davis, G., Elsen, J. M., Haley, C. S., Hill, W. G., Baret, P. V., Hetzel, D. J. S. and Nicholas, F. W. - Detection and mapping of quantitative trait loci in farm animals, Livestock Production Science 52: , [1997]. 6. Fries, R. and Ruvinsky, A. - The Genetics of Cattle, Ed. A.T. Bowling & A. Ruvinsky, ISBN , [1999]. 7. Georges, M., Nielsen, D., Mackinon, M., Mishra, A., Okimoto, R., Pasquino, A. T., Seargeant, L. S., Sorensen, A, Steele, M. R., Zhao, X., Womack, J. E. and Hoeschele, I. - Mapping quantitative trait loci 767

6 controlling milk production in dairy cattle by exploiting progeny testing, Genetics 139: , [1995]. 8. Georges, M., Lequarré, A-S., Castelli, M., Hanset, R. and Vassart, G. - DNA fingerprinting in domestic animals using four different minisatellite probes, Cytogenetics and Cell Genetics 47: , [1988]. 9. Hoeschele, I., Ulimari, P., Grignola, F. E., Zhang, Q. and Gage, K. M. - Advances in statistical methods to map quantitative trait loci in outbreed populations, Genetics 147: , [1997]. 10. Marklund, S., Ellegren, H., Eriksson, S., Sandberg, K. and Andersson, L. - Parentage testing and linkage analysis in horse using a set of highly polymorphic microsatellites, Animal Genetics 25:19-23, [1994]. 11. Spelman, R. and Garrick, D. - Utilisation of marker assisted selection in a commercial dairy cow population, Livestock Production Science 47: , [1997]. 12. Woodward, B. W., Du, F-X., Montaldo, H., Andersen, J. and DeNise, S. K. - Preliminary evidence for major genes controlling beef carcass traits in Limousine cattle. In: Proc. of the 6-th Word Congress on Genetics Applied to Livestock Production, Armidale, NSW, Vol. 25: , [1998]. GENOTIPIZAREA LA BOVINE GEORGESCU S.E. *, CONDAC E. *, REBEDEA MARIANA **, DINISCHIOTU ANCA *, TESIO C. D. *, COSTACHE MARIETA * *Facultatea de Biologie Bucureşti **Institutul de Biologie şi Nutriţie Animală, Baloteşti Rezumat: În ultimele două decenii, genetica mamiferelor a înregistrat progrese remarcabile. Recent, crescătorii de animale din întreaga lume s-au orientat către biologia moleculară şi folosesc tehnica PCR pentru a detecta anumite secvenţe repetitive scurte din genom - microsateliţii. Aceştia sunt distribuiţi în întreg genomul şi pot fi identificaţi din probe de ADN. În cadrul experimentelor realizate am analizat exemplare din rasa Bălţată cu negru românească provenite de la IBNA Baloteşti. Analizele le-am realizat cu ajutorul kitului StockMarks pentru bovine. Numărul de semnale care apar pe fiecare locus în parte depinde de genotipul individului pe acel locus: homozigot sau heterozigot. Identificarea bovinelor şi determinarea paternităţilor s-a realizat folosind o tehnologie modernă de extracţie şi amplificare a ADN şi o tehnică automată de genotipare. Desemnarea şi stabilirea numărului de microsateliţi care vor fi folosiţi în testele de paternitate este încă o problemă discutată şi acest lucru va depinde de caracteristicile fiecărui locus şi de variabilitatea raselor luate în studiu. Cuvinte cheie: bovine, microsateliţi, test de paternitate, genotipare. 768