Polymorphism of selected microsatellite DNA sequences in
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Polymorphism of selected microsatellite DNA sequences in
Animal Science Papers and Reports vol. 24 (2006) Supplement 2, 71-77 Institute of Genetics and Animal Breeding, Jastrzębiec, Poland Presented at the III Conference “Genetic and environmental possibilities of adjusting the slaughter value and meat quality of animals to consumers’ requirements” 7-8 September 2006, Lublin-Krasnobród, Poland Polymorphism of selected microsatellite DNA sequences in Simmental cattle chosen for identification of QTLs for meat traits Bogumiła Choroszy1, Andrzej Janik2, Zenon Choroszy1, Tomasz Ząbek3 1 Department of Animal Genetics and Breeding, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland 2 Department of Animal Nutrition and Feed Science, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland 3 Department of Animal Immuno- and Cytogenetics, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland The polymorphism of microsatellite DNA markers (TGLA227, BM2113, TGLA53, ETH10, SPS115, TGLA126, TGLA122, INRA23, ETH3, ETH225, BM1824) was determined in Simmental bulls chosen for identification of QTLs of meat traits. Genomic DNA isolated from blood was amplified in a Gene Amp PCR System 9600 thermocycler in a multiplex PCR, using starter sequences from the commercial Stock Marks for Cattle Parentage Verification Kit, Bovine II version 2”. Automated DNA sizing technology was applied. The results showed that the 11 DNA microsatellite markers considered were highly polymorphic and the selected population of Simmental bulls was highly heterozygous. The microsatellite DNA sequences analysed could be useful for the identification of QTLs for meat traits in Simmental cattle. KEY Words: cattle / beef / meat traits / microsatellite DNA sequences / QTL / Simmental 71 Bogumiła Choroszy et al. Many cattle production traits of economic importance, such as milk yield, milk composition, milk technological value, growth rate, carcass quality and meat quality, are quantitative traits. The values of these traits result from overlapping of genes known as quantitative traits loci (QTL) and environmental factors [Kurył 2000]. These genes can be identified and mapped by analysing their linkages with genetic markers [Soller 1991]. The first studies on the linkages between QTL and genetic markers were conducted using markers such as blood groups, polymorphic proteins or histocompatibility antigens. The discovery of molecular markers such as microsatellite DNA sequences and the development of methods for their analysis (identification) opened up new possibilities for such studies. Microsatellite markers are highly polymorphic and occur within the whole genome, which makes them a very useful tool for the identification and mapping of QTL. The relationship or linkage between a marker and the level of a quantitative trait can indicate the probable localization of the gene affecting this trait on the same chromosome and in the area of the marker’s locus [Kurył et al. 1997]. In most countries, and in Poland, Simmental cattle, one of the most popular breeds in the world, represent the dual-purpose type, with good dairy traits and welldeveloped beef traits [Choroszy and Choroszy 2004a]. Because of selective breeding to improve meat performance, a considerable increase in growth intensity has been achieved in the European population of cattle. The standard of slaughtered cattle has been raised as a result of breeding for increased carcass muscling without excessive fatness. Simmental cattle play a significant role in beef production. Their bulls’ semen accounts for approximately 30% of inseminations in commercial crossbreeding. High slaughter performance is characteristic not only of purebred Simmentals, but also of their crosses with dairy breeds [Choroszy and Choroszy 2004b]. The good beef performance of the breed in question and the great interest from breeders result from consistent breeding work, in which the appropriate selection of tested bulls for reproduction plays a significant role. A potentially important element of breeding work is the preliminary selection of breeding bulls as early as possible, by working out the genome of Simmental cattle and, if possible, by identifying QTL(s) for meat traits [Janik et al. 2001, Sőlkner et al. 2005]. The identification of QTL for meat traits in Simmental cattle is organized into three stages: - the analysis of DNA polymorphism in Simmental bulls tested for meat traits and chosen for the identification of QTL(s) for meat traits; - the evaluation of meat traits in bulls from the population studied; - the analysis of linkages between microsatellite loci and meat traits. The aim of the present study, which is the first stage in research on the identification of QTL for meat traits in Simmental cattle, was to determine the polymorphism of 11 DNA microsatellite sequences in a selected population and to evaluate their usefulness for studies on the identification of QTL for meat traits. 72 Adjusting the slaughter value and meat quality of animals to consumers’ requirements Material and methods A total of 92 Simmental bulls were used, station-evaluated for live and post mortem meat traits. The meat traits to be analysed for linkages included net daily live weight gain, carcass weight, meat content of carcass, and the area of the longissimus dorsi muscle. The polymorphism of 11 microsatellite DNA markers (TGLA227, BM2113, TGLA53, ETH10, SPS115, TGLA126, TGLA122, INRA23, ETH3, ETH225 and BM1824) of known localization in the genome was determined in blood samples. Genomic DNA isolated from blood samples was amplified in a Gene Amp PCR System 9600 thermocycler in a multiplex PCR, using starter sequences from the commercial Stock Marks for Cattle Parentage Verification Kit, Bovine II version 2. Automated DNA sizing technology was applied in an ABI PRISM 377 automatic DNA sequencer. The size of the DNA fragments analysed and the genotype of the animals studied were determined using specialist software (GeneScan v.2.1 and Genotyper 2.0). The results were evaluated including (i) calculation of the frequency of the alleles identified at particular loci after Lubieniecka et al. [1999], (ii) the degree of heterozygosity (H) after Ott [1992] and (iii) the polymorphism information content (PIC) after Botstein et al. [1980]. Results and discussion Among the basic indicators most often used for analysing the polymorphism of microsatellite DNA sequences in different breeds or populations of animals are the number and frequency of alleles identified at individual loci, the effective number of alleles per locus, the degree of hetero/homozygosity and the PIC. In the Simmental population considered, a total of 80 alleles were identified at 11 loci of DNA microsatellites, their number ranging from four (loci ETH3 and BM1824) to 16 (locus TGLA53). The mean number of alleles per locus was 7.27 (Tab. 1). In 10 of the 11 loci analysed, considerable differences were found in the frequency of the identified alleles. High frequency was characteristic of all four alleles with 178 bp (0.1703), 180 bp (0.3297), 182 bp (0.1593) and 188 bp (0.3407) detected at the BM1824 locus. Janik et al. [2001] also observed a high allele frequency (180 bp, 182 bp, 184 bp, 190 bp) at this locus in Simmentals. However, on the basis of earlier research it is impossible to give conclusive reasons for the distribution of alleles at this locus. Most of the microsatellite loci analysed presented high polymorphism, as indicated by the number of alleles detected at particular loci and by the PIC value, which was above 0.5. The most polymorphic of the 11 loci were: TGLA53 (16 alleles with sizes ranging from 146 to 190 bp, PIC=0.851), TGLA227 (10 alleles ranging from 77 to 97 bp, PIC=0.824), BM2113 (8 alleles ranging from 125 to 139 bp, PIC=0.744), SPS115 (8 alleles ranging from 248 to 262 bp, PIC=0.615) and INRA23 (8 alleles ranging from 198 to 218 bp, PIC=0.665). The high polymorphism at these loci in the Simmental breed has already been described by Janik et al. [2001]. Very 73 Bogumiła Choroszy et al. Table 1. Polymorphism at the 11 microsatellite loci in Simmental cattle raised in Poland 74 Locus Number of alleles Alleles Allele frequency TGLA227 10 77 79 81 83 87 89 91 93 95 97 BM2113 8 TGLA53 ETCH10 PIC* H** 0.0110 0.0934 0.3022 0.0879 0.0879 0.0989 0.0879 0.0495 0.0275 0.1538 0.824 0.890 125 127 129 131 133 135 137 139 0.0220 0.1264 0.0055 0.3736 0.0824 0.0769 0.0879 0.2253 0.744 0.758 16 146 154 160 162 164 166 168 170 172 174 176 182 184 186 188 190 0.0055 0.1703 0.0275 0.0275 0.0824 0.0549 0.2033 0.1538 0.1648 0.0055 0.0220 0.0165 0.0330 0.0220 0.0055 0.0055 0.851 0.747 6 213 215 217 219 221 223 0.0055 0.0165 0.7418 0.1813 0.0165 0.0385 0.376 0.418 Adjusting the slaughter value and meat quality of animals to consumers’ requirements 75 Bogumiła Choroszy et al. high polymorphism at the TGLA53 locus was also reported by Heyen et al. [1997] in American Simmentals. The high degree of heterozygosity observed in 10 of the 11 DNA microsatellite loci analysed (from 0.593 at the ETH3 locus to 0.890 at the locus TGLA227) shows considerable variation within the Simmental population studied. A high heterozygosity at the loci of the same set of microsatellites (from 0.580 at the TGLA126 locus to 0.880 at the locus TGLA227) in Simmental cattle was previously reported by Janik et al. [2001]. For most of the loci considered, high PIC and H values (above 0.5) were reported by Usha et al. [1995] who studied the polymorphism of other microsatellite markers in British Simmental cattle, by Glowatzki-Mullis et al. [1995] in Swiss Simmentals, and by Heyen et al. [1997] in American Simmentals. The results of the present study confirm the high polymorphism of the 11 microsatellite DNA markers considered and the high heterozygosity of the analysed Simmental population with reference to this group of markers. 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Bogumiła Choroszy, Andrzej Janik, Zenon Choroszy, Tomasz Ząbek Polimorfzm wybranych sekwencji mikrosatelitarnych bydła simentalskiego wytypowanego do badań nad identyfikacją QTLs cech mięsnych Streszczenie W badaniach nad identyfikacją QTL cech mięsności buhajków simentalskich ocenianych pod względem cech mięsnych określono polimorfizm wybranych sekwencji mikrosatelitarnych DNA. Wykazano rozległy polimorfizm 11 użytych do badań markerów mikrosatelitarnych DNA oraz wysoką heterozygotyczność analizowanej populacji bydła simentalskiego. Badane sekwencje mikrosatelitarne DNA mogą być przydatne w identyfikacji QTLs cech mięsnych tego bydła. 77