24th Int. Symp. “Animal Science Days”, Ptuj, Slovenia, Sept. 21st−23rd, 2016. Acta argiculturae Slovenica, Supplement 5, 37–40, Ljubljana 2016 COBISS: 1.08 Agris category code: L10 GENETIC DIFFERENTIATION OF SLOVAK PINZGAU, SIMMENTAL, CHAROLAIS AND HOLSTEIN CATTLE BASED ON THE LINKAGE DISEQUILIBRIUM, PERSISTENCE OF PHASE AND EFFECTIVE POPULATION SIZE Veronika KUKUČKOVÁ 1, Nina MORAVČÍKOVÁ 2, Anna TRAKOVICKÁ 3, Ondrej KADLEČÍK 4, Radovan KASARDA 5 Genetic differentiation of Slovak Pinzgau, Simmental, Charolais and Holstein cattle based on the linkage disequilibrium, persis- tence of phase and effective population size 1 Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, Department of Animal Genetics and Breeding Biology, Tr. A. Hlinku 2, 949 76, Nitra, Slovak Republic, e-mail: veron.sidlova@gmail.com 2 Same address as 1, e-mail: nina.moravcikova@uniag.sk 3 Same address as 1, e-mail: anna.trakovicka@uniag.sk 4 Same address as 1, e-mail: ondrej.kadlecik@uniag.sk 5 Same address as 1, e-mail: radovan.kasarda@uniag.sk ABSTRACT The aim of this study was to estimate the linkage disequilibrium extent (LD), effective population size (Ne) and the persistence of phase (R) among cattle breeds for syntenic marker pairs using genome-wide single-nucleotide polymor- phisms genotyped in Slovak Pinzgau, Simmental, Charolais and Holstein cattle. The average LD ranged from 0.29 ± 0.23 (Charolais) to 0.36 ± 0.29 (Simmental) for markers in the 0 to 10 kb bins. The recent Ne was from 58 (Holstein) to 85 (Charolais). The persistence of phase as a measure of the prediction reliability for markers in one breed compare to an- other breeds markers was estimated. Markers in the 0 to 10 kb bin could be predicted with a maximal accuracy of 0.65 between Simmental and Charolais. The lowest correlation was observed between Slovak Pinzgau and Charolais (0.46 in the 0 to 10 kb bins) what indicated that phase was not strongly preserved between breeds. Results of this study could be the basis for the implementation of genomic selection programs in the Slovak Pinzgau breed and the differentiation of cattle populations during the selection process. Key words: endangered breed, selection criteria, BovineSNP50 BeadChip, linkage disequilibrium, effective popu- lation size, persistence of phase et al., 2007). Moreover, observed LD is related to historical effective population sizes (Ne), and can provide insights into the genetic diversity history of populations (Biegelmeyer et al., 2016). The square correlation of alleles at two loci (r2) is considered the most robust measure of LD (Badke et al., 2012). Persistence of phase (R) is a measure of the degree of agreement of LD phase for pairs of SNP between two populations (Badke et al., 2012). Persistence of allele phase between breeds can be used to identify the history and the relationship among breeds within a species or to determine the marker density to conduct multi-breed genomic selec- tion (Makina et al., 2016). If r2 between two markers is equal in two populations, but their corresponding r has the opposite sign, the gametic 1 INTRODUCTION Knowledge about the extent and the pattern of linkage disequilibrium (LD) in livestock populations is essential to determine the density of single nucleotide polymorphisms (SNPs) required for accurate genome-wide association and genomic selection studies (Biegelmeyer et al., 2016). The observed correlation between alleles at different loci in the genome is a determinant parameter in many applications of molecular genetics. With the wider use of genomic technol- ogies in animal breeding and genetics, it is worthwhile revis- ing and improving the current knowledge and understand- ing of cattle LD (Pérez O’Brien et al., 2014). LD is common between alleles at neighbouring loci that tend to be inher- ited together and associated in a segregating population (Du Acta agriculturae Slovenica, Supplement 5 – 201638 V. KUKUČKOVÁ et al. ers and individuals with call rates less than 90 % were ex- cluded, as well as markers with highly significant deviations (p < 10−5) from Hardy-Weinberg Equilibrium were also re- moved using PLINK v1.9 (Purcell et al., 2007). Pérez O’Brien et al. (2014) observed no evidence of a minor allele frequency (MAF) influence on r2 estimates and concluded that unbiased estimates of LD were obtained pro- vided MAF > 0.05 unless low density SNP coverage assays were used. Consequently, the MAF threshold in this study was set to 0.05. Genome-wide LD as r2 for different genetic distances between SNPs were calculated for PIN (dual purpose and dairy breed), SIM (dual purpose breed), CHA (beef breed) and HOL (dairy breed). The persistence phase of LD (R) be- tween alleles on the same chromosome was calculated as the Pearson correlation coefficient for marker pairs that were in common within four breeds. The correlations among r val- ues (the root of LD estimator r2) between breeds were esti- mated for 10 kb intervals (from 0 to 500 kb) using PROC CORR command in SAS 9.3 programme (SAS Institute, 2011). 3 RESULTS AND DISCUSSION Genome-wide LD as r2 for different distances between SNPs was calculated for PIN, SIM, CHA and HOL. Average LD for all breeds is presented in Figure 1. The average r2 was from 0.29 ± 0.23 (CHA) to 0.36 ± 0.29 (SIM) and the aver- age r was from 0.49 ± 0.23 (CHA) to 0.54 ± 0.26 (SIM) for markers in 0 to 10 kb bin. All studied breeds showed an in- verse relationship between LD and inter-marker distances. The decline of LD as a distance function agreed with other studies based on the r2 estimates in cattle (Du et al., 2007; phase is reversed. High positive values are a result of high r2 and equal phase in both breeds (Uimari and Tapio, 2011). The Slovak Pinzgau breed is a dual purpose endan- gered breed due to a significant decline of the population in the recent years (Kasarda et al., 2014). Thanks to its unique traits as longevity, fertility, health and grazing ability it is bred in the mountain regions of northern Slovakia. The population faced the bottleneck effect and the loss of diver- sity due to unequal use of founders. Further population size decreasing can lead to serious problems. Therefore, it is nec- essary to preserve the recent population in both types (dairy and beef; Pavlík et al., 2013). A better monitoring system is needed to increase the population size without a genetic di- versity reduction (Kadlečík et al. 2011). The objective of this study was to estimate LD levels, persistence of phase and effective population size in Slovak Pinzgau breed in comparison with Simmental, Charolais and Holstein cattle. 2 MATERIAL AND METHODS All Pinzgau sires (n = 19) in Slovakia (PIN) used in insemination were analysed. Genomic DNA was isolated from the semen samples and genotyped at a commercial lab using the Illumina BovineSNP50v2 BeadChip (Illu- mina Inc., San Diego, CA). The genotyping array contained 54,609 SNPs. Genotyping information (BovineSNP50v2 BeadChip) for Simmental (SIM; n = 84), Charolais (CHA; n = 55) and Holstein (HOL; n = 100) breeds were retrieved from the public digital repository (McTavish et al., 2013b). For the retrieved dataset the quality control has already been performed according to McTavish et al. (2013a). The same criteria were used in Slovak Pinzgau genotyping data. Mark- Figure 1: Decrease of linkage disequilibrium (r2) by physical distance in studied breeds Acta agriculturae Slovenica, Supplement 5 – 2016 39 GENETIC DIFFERENTIATION OF SLOVAK PINZGAU, SIMMENTAL ... PHASE AND EFFECTIVE POPULATION SIZE lowest R between PIN and HOL was obtained. The corre- lations between alleles at adjacent loci indicated that phase was not strongly preserved within the breed pairs and it decreased with the increasing of physical distance among breeds (Table 1). Correlation of phase as well as Ne declined among breeds from about 2000 generations to 100 generations ago (Fig. 2). As reported Garcia-Ruiz et al. (2015) the per- sistence of LD phase was decreasing when the distance between markers was increasing. The value R refers to the correlation of LD of SNP pairs in one breed with LD of the same pairs in the other breed. The persistence of phase (cor- relation of r values) among populations showed the genetic relationship among them (Badke et al., 2012) and thus it was used as a measure among the breeds in this study. Observed correlations of allele phases among breeds in this study were much lower than previously reported between European cattle breeds at a short range (<10 kb; Biegelmeyer et al., 2016), but higher than among South Af- rican breeds (Makina et al., 2016). The difference between the persistence of phase among Badke et al., 2012; Pérez O’Brien et al. 2014; Garcia-Ruiz et al., 2015; Biegelmeyer et al., 2016; Makina et al., 2016) where was shown that LD is a population specific. There- fore, it could be heterogeneous among populations depend- ing on the demographic history of the populations. Past and recent effective population sizes were esti- mated from the average r2 for markers separated by various genomic distances. The extent of LD over distances approxi- mately 6000 kb indicated more recent Ne (Ne ≈ 10 genera- tions ago) while that over shorter distances (60 kb) provided historical Ne (Ne ≈ 1000). Historical Ne were from 1713 (HOL) to 2304 (PIN), while more recent Ne were only from 58 (HOL) to 85 (CHA). The most rapid decline of Ne was found in PIN in the last five to six generations, confirming PIN considered as endangered breed from the year 1993. The persistence of phase between PIN, SIM, CHA and HOL populations was calculated at the interval distances of 10 kb up to 500 kb. The largest R was observed between SIM and CHA pair (0.65 in 0 to 10 kb bin), while the lowest R was observed between PIN and CHA (0.46 in 0 to 10 kb bin). However, for the distance of bins 50 kb and more, the Breed pairs bins distances (kb) 10 20 50 100 200 500 Correlation of phase (R) Slovak Pinzgau - Simmental 0.5899 0.4415 0.4315 0.3582 0.1885 0.0666 Slovak Pinzgau - Charolais 0.4561 0.4020 0.4483 0.3541 0.1868 0.0585 Slovak Pinzgau - Holstein 0.5687 0.4318 0.3574 0.2860 0.1676 0.0111 Simmental - Charolais 0.6497 0.5524 0.5486 0.4871 0.3088 0.0904 Simmental - Holstein 0.6018 0.4184 0.3842 0.3224 0.2521 0.0268 Charolais - Holstein 0.5191 0.4570 0.4460 0.3373 0.2501 0.0427 Table 1: Correlations of allele phase among all evaluated pairs of breeds by physical distance Figure 2: Correlations of allele phases between all evaluated breeds pairs by physical distance represented as generations in the past Acta agriculturae Slovenica, Supplement 5 – 201640 V. KUKUČKOVÁ et al. the breeds in this study at the shortest distances ranged from 0.02 to 0.19, what was much higher than among Holstein at the shortest distance (0.01–0.04; Garcia-Ruiz et al., 2015). Similar results were obtained among Angus, Holstein and South African breeds (Makina et al., 2016) or different spe- cie like pigs (Badke et al., 2012). Correlation between alleles at adjacent loci indicat- ed that phase was not strongly preserved among breeds, whereas R reached moderately positive values in all popula- tion pairs in the distance < 500 kb. The statistically signifi- cant slightly negative values of R between PIN and HOL in 720 to 730 kb bin as well as between PIN and CHA in 740 to 750 kb bin were observed. According to Uimari and Tapio (2012) the only high positive values are the result of a strong r2 and an equal phase in both breeds, while negative values reveals the reversed gametic phase. 4 CONCLUSIONS Previous studies have shown that LD is a population specific and could be heterogeneous among populations depending on the demographic history of the populations. In this study, average LD was from 0.29 (Charolais) to 0.36 (Simmental) for markers in 0 to 10 kb bin. The correlation of allele phase referred to the correlation of LD values of SNP pairs in one breed with LD values of the same pairs in the other breed. The largest persistence of phase (R) was observed between Simmental and Charolais (0.65 in 0 to 10 kb bin), while the lowest one between Slovak Pinzgau and Charolais (0.46 in 0 to 10 kb bin). 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