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Assessment of the genetic structure of Central European cattle breeds based on functional gene polymorphism
Global Ecology and Conservation 17 (2018) e00525
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Original Research Article
Assessment of the genetic structure of Central European cattle breeds based on functional gene polymorphism czuk a, Karolina Kasprzak-Filipek a, Wioletta Sawicka-Zugaj a, *, Zygmunt Litwin a b c ta Sveistien e_ , Josef Bulla Witold Chabuz , Ru a
University of Life Sciences in Lublin, Institute of Animal Breeding and Biodiversity Conservation, Sub-department of Cattle Breeding and Genetic Resources Conservation, Poland b Lithuanian University of Health Sciences, Animal Science Institute, Lithuania c Slovak University of Agriculture in Nitra, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2018 Received in revised form 3 January 2019 Accepted 3 January 2019
As many European cattle breeds are considered to be threatened with extinction, and existing genetic variability is increasingly at risk of being irretrievably lost, its assessment is crucial. The aim of the study was to assess the genetic variability of seven breeds of cattle in Central Europe on the basis of polymorphism at the loci of functional genes, encoding blactoglobulin (LGB), leptin (LEP), prolactin (PRL), oestrogen receptor alpha (ERa) and growth hormone receptor (GHR). The research was carried out on 290 individuals e 50 Polish White-backed (PW), 50 Lithuanian White-backed (LWB), 50 Polish Red (PR), 50 Lithuanian Red (LR), 22 Carpathian Brown (CB), 18 Ukrainian Grey (UG) and 50 Slovak Pinzgauer (PG). Gene polymorphism was determined by PCR-RFLP. The statistical indicators estimated, i.e. the frequency of alleles and genotypes, observed and expected heterozygosity (HO and HE), F-statistics, gene flow (Nm), and genetic distances, were used to characterize the genetic structure of these cattle breeds. The research demonstrated that the populations analysed have undergone a bottleneck process as a consequence of the rapid decline in the size of individual populations. Owing to the introduction of genetic resources conservation programmes, endangered populations can slowly be restored to a state of genetic balance. © 2019 University of Life Sciences in Lublin. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
Keywords: Genetic variability Functional genes Central European cattle breeds Gene polymorphism Population genetic structure
1. Introduction The diversity within and between cattle breeds is closely linked to their origin, history and evolution (Bradley et al., 1996; MacHugh et al., 1997; Troy et al., 2001). A crucial element of biodiversity conservation is ‘in situ’ protection of indigenous, native breeds of cattle constituting a reservoir of unique combinations of genes and alleles, which in the future may prove highly valuable, e.g. for the creation of new livestock genotypes (Bulla et al., 2013). Although the diversity between the world's cattle breeds does not allow the species to be classified as endangered, the loss of local breeds is an irreversible and irreplaceable erosion of genetic resources (FAO, 2011; Taberlet et al., 2008). Genetic structure can be characterized using a variety
* Corresponding author. E-mail address: [email protected] (W. Sawicka-Zugaj). https://doi.org/10.1016/j.gecco.2019.e00525 2351-9894/© 2019 University of Life Sciences in Lublin. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
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of molecular markers, depending on the research problem (Groeneveld et al., 2010). In recent years, analysis of single nucleotide polymorphisms (SNP) has begun to dominate this research. The literature shows that the data obtained from SNP analysis explain the history of European cattle more accurately than analysis of microsatellite sequences, and for this reason they are increasingly used in biodiversity conservation (Gautier et al., 2007; McKay et al., 2008; Socol et al., 2015; Svensson et al., 2007; The Bovine HapMap Consortium, 2009). Particular importance is attributed to SNPs in the search for linkages between a marker with a specific location in the genome and an unknown gene locus. The search for such associations is important because they allow a phenotypic effect to be assessed by identifying its genetic basis (e.g. in the diagnosis of genetic ski, 2008). According to Socol et al. (2015), in the near diseases or analysis of the variability of a quantitative trait) (Swito n future characterization of genes located on different chromosomes will probably become an integral part of animal genetic resources (AnGR) conservation programmes. Knowledge based on SNP polymorphisms at loci of functional genes can be an important source of information for conservation decisions (FAO, 2012). New technologies using SNP polymorphism or whole genome scanning may revolutionize previous achievements in biodiversity assessment and genetic characterization of breeds, providing a more complete understanding of the molecular basis of functional diversity (Groeneveld et al., 2010). Research by Lasagna et al. (2015) suggests that functional markers are more suitable than neutral markers for evaluating the diversity of morphologically similar breeds. In addition, that study showed that the level of genetic variability analysed on the basis of functional markers is higher than for analysis based on neutral markers. The aim of the study was to characterize the genetic structure of seven Central European cattle breeds (Lithuanian Whitebacked, Polish White-backed, Polish Red, Lithuanian Red, Carpathian Brown, Ukrainian Grey and Slovak Pinzgauer) on the basis of polymorphism at loci of functional genes encoding b-lactoglobulin, leptin, prolactin, oestrogen receptor alpha, and growth hormone receptor. 2. Methods The research was carried out on 290 individuals of seven Central European cattle breeds e 50 Polish White-backed (PW), 50 Lithuanian White-backed (LWB), 50 Polish Red (PR), 50 Lithuanian Red (LR), 22 Carpathian Brown (CB), 18 Ukrainian Grey (UG) and 50 Slovak Pinzgauer (PG). The animals were from farms located in Poland, Lithuania, Ukraine and Slovakia. Hair bulbs were the biological material for analysis. DNA was isolated using ready-made commercial kits for isolating nucleic acids from biological traces (Sherlock AX A & A Biotechnology) according to the procedure given by the manufacturer. Genetic variability was evaluated by the PCR-RFLP method on the basis of polymorphism of loci of the functional genes encoding b-lactoglobulin (LGB), leptin (LEP), prolactin (PRL), oestrogen receptor alpha (ERa) and growth hormone receptor (GHR) (Table 1). The PCR reaction was carried out in an MJ Research thermal cycler (PTCe200 PELTIER THERMAL CYCLER). The fragments amplified by PCR were digested with FastDigest™ restriction enzymes (Thermo Scientific). The following endonucleases were used: for the b-lactoglobulin gene (LGB) e the enzyme HaeIII (Medrano and Aguilar-Cordova 1990), for the leptin gene (LEP) e Sau3AI (Liefers et al., 2002), for the prolactin gene (PRL) e RsaI (Lewin et al., 1992), for the growth hormone receptor gene (GHR) e AluI (Heap et al., 1995) and for the oestrogen receptor gene (ERa) e BglI (Szreder and Zwierzchowski, 2004). The digestion time and the composition of the reaction mixtures were in accordance with the procedure given by the manufacturer of the restriction enzymes. Fragments obtained by PCR-RFLP were electrophoresed on 2% agarose gels stained with 0.01% ethidium bromide (EtBr). Molecular-weight size markers (Thermo Scientific GeneRuler 100bp Plus DNA Ladder) were used to track the electrophoresis process and to assess the length of the fragments obtained. To visualize the genetic structure of the analysed cattle breeds, the results were subjected to statistical analysis in POPGENE v. 3.2. and CERVUS v. 3.0.7 software. The following indicators were estimated: frequency of alleles and genotypes at individual loci; observed heterozygosity (HO); expected heterozygosity (HE) according to Nei (1973); F-statistics estimated for groups and between groups, i.e. the Table 1 Primers used in this study. Gene
Primer sequences
Reference
b-lactoglobulin (LGB)
50 -GTCCTTGTGCTGGACACCGACTACA-30 50 -CAGGACACCGGCTCCCGGTATATGA-30 50 -TGGAGTGGCTTGTTATTTTCTTCT-30 50 -GTCCCCGCTTCTGGCTACCTAACT-30 50 -CGAGTCCTTATGAGCTTGATTCTT-30 50 -GCCTTCCAGAAGTCGTTTGTTTTC-30 50 -TGCGTGCACAGCAGCTCAACC-30 50 -AGCAACCCCACTGCTGGGCAT-30 50 -TTTGGTTAACGAGGTGGAG-30 50 -TGTGACACAGGTGGTTTTTC-30
(Medrano and Aquilar-Cordova, 1990)
Leptin (LEP) Prolactin (PRL) Growth hormone receptor (GHR) Oestrogen receptor alpha (ERa)
Liefers et al. (2002) Lewin et al. (1992) Heap et al. (1995) Szreder and Zwierzchowski (2004)
The reaction mixtures (with a final reagent volume of 12.5 ml) contained 100 ng DNA, primers at a final concentration of 10 mM, REDTaq®DNA Polymerase (1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs and 0.06 unit/mL of Taq DNA polymerase) and nuclease-free water (Sigma-Aldrich). The temperature programmes consisted of the following cycles: 1) initial denaturation (94-95 C/3e5 min), 2) cyclically repeating steps (30e36): DNA denaturation (94-95 C/30 s - 1 min), primer annealing and primer elongation (72 /1 min) and 3) final extension (72 C/5e10 min). The primer annealing temperatures ranged from 56.5 C to 66.8 C, i.e. 61 C for LGB, 65 C for LEP, 56.5 C for PRL, 66.8 C for GHR, and 56.9 C for ERa (Table 2).
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Table 2 PCR conditions for analysed genes. PCR cycles
Step
Temperature
Time
1 30e36
Initial denaturation Denaturation Annealing Extension Final extension
95 C 94 C 56.5 Ce66.8 C 72 C 72 C 4 C
5 min 40 s 1 min 1 min 7 min Indefinitely
1
inbreeding coefficient FIS (Wright, 1978), the fixation index FST according to Hartl and Clark (1997) and Weir (1990), and FIT; and gene flow (Nm) according to Slatkin and Barton (1989). Genetic distances were determined according to Nei (1972). On the basis of the estimated genetic distances, a phylogenetic tree was created to illustrate the diversity of the cattle breeds. Statistical analysis of Hardy-Weinberg equilibrium for the populations was based on the chi square test (c2). 3. Results The research carried out among individuals belonging to seven Central European cattle breeds identified three genotypes at each locus of the genes for b-lactoglobulin (LGB) e AA, AB and BB; leptin (LEP) e CC, CT and TT; prolactin (PRL) e AA, AB and BB; oestrogen receptor alpha (ERa) e AA, AG and GG, and growth hormone receptor (GHR) e AA, AB and BB. Restriction analysis of a fragment of the gene encoding b-lactoglobulin (LGB) identified three genotypes: AA, AB and BB. The highest frequency of the B allele (0.94) was recorded in individuals of the Ukrainian Grey breed, and the lowest (0.56) for the Polish White-backed breed. Individuals homozygous for the A allele at the LGB gene locus were observed only in the Polish White-backed breed (genotype frequency e 0.14; frequency of A allele e 0.44). At the locus of the leptin gene (LEP) in the breeds Polish White-backed (PWB), Lithuanian White-backed (LWB), Polish Red (PR), Lithuanian Red (LR) and Pinzgauer (PG), three genotypes were identified e CC, CT and TT, determined by two alleles, C and T, with average frequencies of 0.76 and 0.24, respectively, for all breeds. Among individuals of the Carpathian Brown (CB) and Ukrainian Grey (UG) breeds, the homozygous TT genotype was not found. The highest frequency of the T allele (0.36) was found in PR individuals and the lowest (0.17) in the UG breed. Restriction digestion of a 156 bp fragment of the prolactin gene (PRL) with the endonuclease RsaI identified three genotypes, AA, AB and BB, among individuals of the breeds LWB, PWB, PR and PG. In the Ukrainian Grey population, all individuals were homozygous for the A allele, while individuals with the BB genotype were not identified among the breeds LR and CB. The highest frequency of the B allele (0.47) was estimated for individuals of the Lithuanian White-backed breed. The highest frequency was found for heterozygous individuals (on average 0.6 for all breeds). Three genotypes were found at the locus of the oestrogen receptor gene: AA, AG and GG. Individuals of the UG and CB breeds were homozygous at the ERa locus for the G allele. AA homozygotes were identified only among Polish Red individuals (with a genotype frequency of 0.06). For all animals, the highest frequency was observed among homozygous GG individuals (with a frequency of 0.6). Three genotypes, AA, AB and BB, were identified at the locus of the growth hormone receptor gene (GHR) among individuals of the breeds LWB, PWB, PR, LR and PG. The highest average frequency was observed for the A allele (0.9). Individuals with the BB genotype were observed only in the PWB breed, in which the frequency of the A and B alleles was 0.77 and 0.23, respectively. Only individuals with AA genotypes were noted among the UG and CB breeds. The estimated values of the chi-square test (c2) in the Polish Red population showed that the frequency distribution of genotypes was consistent with Hardy-Weinberg equilibrium. The estimated frequencies of genotypes observed in the other breeds analysed, which differ statistically from the expected frequencies (in accordance with the HWE principle), indicate a lack of genetic equilibrium in these populations. The heterozygosity index (Table 3) indicates the highest average observed heterozygosity (HO ¼ 0.51) at the locus of the prolactin gene (PRL) and the lowest at the locus of the growth hormone receptor gene (GHR) (with a mean HO of 0.15 for all breeds). The estimated inbreeding coefficients (FIS) provided information regarding the proportions between observed (HO) and expected (HE) heterozygosity, making it possible to assess the processes that were likely to have influenced the current genetic structure of the populations. The negative values obtained for the FIS coefficient indicate an excess of heterozygotes in the populations. The F-statistics provided information on how genetic variability was distributed within and between the analysed populations (Table 4). A negative average value for FIT was noted for the populations. To interpret the values of the fixation index (FST), the following scale was adopted: FST <0.05 e very low or no variation between populations; 0.05e0.15 e low genetic variation; 0.15e0.25 e an intermediate level of variation, and >0.25 e high variation between populations (Weir and Cockerham, 1984). The FST values obtained for all breeds analysed, ranging from 0.0244 to 0.1605, indicate low diversity and a small degree of genetic isolation between them. The gene flow (Nm) between the populations was estimated based on the values of the FST fixation index. It was 2.74 for LGB, 10.00 for LEP, 1.31 for PRL, 2.06 for ERa, and 2.91 for GHR. The mean gene flow rate of 2.40 for all loci indicates about two
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Table 3 Genetic diversity of the seven cattle breeds. Observed heterozygosity (HO), expected heterozygosity (HE) and inbreeding coefficient (FIS). Coefficients
LGB
LEP
PRL
ERa
GHR
Breed
HO HE FIS HO HE FIS HO HE FIS HO HE FIS H HE FIS
Mean HO Mean HE Mean FIS
Mean
BGL
BGP
PC
LC
BU
SU
PG
0,72 0,46 0,56 0,28 0,3 0,05 0,9 0,5 0,81 0,24 0,21 0,14 0,3 0,26 0,18 0,49 0,35 0,33
0,6 0,49 0,22 0,34 0,39 0,14 0,66 0,46 0,45 0,54 0,39 0,37 0,34 0,35 0,04 0,50 0,42 0,02
0,52 0,38 0,35 0,48 0,46 0,04 0,42 0,35 0,19 0,5 0,43 0,17 0,12 0,11 0,06 0,41 0,35 0,16
0,3 0,26 0,18 0,4 0,42 0,05 0,74 0,47 0,59 0,18 0,16 0,10 0,16 0,15 0,09 0,36 0,29 0,18
0,59 0,42 0,42 0,5 0,38 0,33 0,05 0,04 0,02 0 0 e 0 0 e 0,23 0,17 0,26
0,11 0,1 0,06 0,33 0,28 0,20 0 0 e 0 0 e 0 0 e 0,09 0,08 0,13
0,3 0,26 0,18 0,28 0,3 0,05 0,8 0,49 0,62 0,42 0,33 0,27 0,1 0,1 0,05 0,38 0,30 0,21
0,45 0,34 0,28 0,37 0,36 0,04 0,51 0,33 0,45 0,27 0,22 0,06 0,15 0,14 0,07 0,35 0,28 0,19
Table 4 Wright's F-statistics for analysed genes. LOCUS
FIT
FST
LGB LEP PRL ERa GHR Mean
0.2152 0.0125 0.2953 0.0967 0.0259 0.1442
0.0835 0.0244 0.1605 0.1082 0.0792 0.0941
migrants per generation. The highest value, indicating the highest rate of gene flow, was noted at the leptin gene (LEP) locus and the lowest at the prolactin gene (PRL) locus. To determine the genetic differences and similarities between the populations of Central European cattle breeds included in the study, the genetic distances were estimated. The genetic distance and similarity values according to Nei (1972) made it possible to determine the phylogenetic relationships between the populations (Table 5). Based on the genetic distances obtained, the pair group method (UPGMA) was used to create a phylogenetic tree (Fig. 1) illustrating the genetic structure and genetic similarity between the Central European cattle breeds. 4. Discussion Despite the increasing number of studies assessing the genetic variability of various cattle breeds around the world, there are still few that focus on characterizing the genetic diversity between native Central European cattle breeds and analysing their genetic structure (Gutierrez et al., 2003; Petrakova et al., 2012). Analyses aimed at determining the phylogenetic affiliation of various breeds of cattle on the basis of functional gene polymorphism are also uncommon. As a rule, such research focuses on assessment of the genetic variability of various breeds of cattle from northern Europe, or determination of the phylogenetic affiliation of European and African cattle breeds based on haplotype variation (Gautier et al., 2007). As demonstrated by numerous studies (Brym et al., 2005; Buchanan et al., 2003; Curi et al., 2006; Dobicki et al., 2002; Ghasemi ski and Zabolewicz, 2000; Karimi et al., 2009; Komisarek and Antkowiak, 2007; Liefers et al., 2002; Rahbar et al., 2009; Kamin
Table 5 Genetic differences and similarities between the populations of seven Central European cattle breeds.
LWB PWB PR LR CB UG PG
LWB
PWB
PR
LR
CB
UG
PG
**** 0.0171 0.0451 0.0212 0.0613 0.0784 0.0180
0.9831 **** 0.0265 0.0395 0.0586 0.0880 0.0386
0.9559 0.9739 **** 0.0241 0.0355 0.0456 0.0287
0.9790 0.9613 0.9762 **** 0.0388 0.0360 0.0098
0.9406 0.9431 0.9652 0.9619 **** 0.0136 0.0629
0.9246 0.9158 0.9555 0.9647 0.9865 **** 0.0542
0.9821 0.9621 0.9717 0.9902 0.9391 0.9473 ****
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Fig. 1. Phylogenetic tree of genetic distance for assessed cattle breeds.
et al., 2010; Signorelli et al., 2009), functional gene polymorphism is closely linked to specific production traits in cattle, and analysis of variability at gene loci makes it possible to assess the predisposition of animals to a specific type of production. Based on the allele frequencies obtained for the b-lactoglobulin (LGB), leptin (LEP), prolactin (PRL), oestrogen (ERa) and growth hormone (GHR) genes, the observed (HO) and expected (HE) heterozygosity were estimated for the analysed cattle ndez et al. (2004), the higher the level of heterozygosity and the closer the frequency of the breeds. As reported by Ferna alleles, the greater the variability of the breed. For all breeds the mean observed heterozygosity (HO) was higher than the expected heterozygosity (HE). The highest mean observed heterozygosity (HO) was observed in the population of Polish White-backed cattle, and the lowest in the population of Ukrainian Grey cattle. The estimated observed heterozygosity for the seven Central European cattle breeds, ranging from 0.09 to 0.50, indicates a low level of genetic variation within the genes analysed. The observed heterozygosity values reported by Canon et al. (2001) for local breeds raised in Spain, Portugal and France, based on analysis of microsatellite markers, were significantly higher and ranged between 0.54 and 0.72. The FIS inbreeding coefficients were estimated on the basis of the HO and HE values. In most of the loci analysed, negative values were obtained for the inbreeding coefficient, indicating an excess of heterozygotes in the populations (Sawicka-Zugaj et al., 2018). The values obtained for the inbreeding coefficient are presumably indicative of a bottleneck effect in the populations, where the current population structure is the result of a sudden decline in the size of the population and its subsequent rebuilding from the group of survivors. The average FST value (0.0941) obtained in the study indicates that only 9% of the total variability results from inter-breed differences. The FST value of 0.07 (p < 0.01) obtained by Canon et al. (2001) for local breeds raised in Spain, Portugal and France demonstrates a similar (7%) level of variation between these breeds. The genetic distances were parameters describing the genetic diversity between the Central European cattle breeds. Toro et al. (2009) report that in recent years estimation of genetic distances between different populations of livestock animals has been based on information on microsatellite polymorphism. In the present study, we estimated genetic distances and similarities on the basis of polymorphism of functional genes. The highest genetic distance value (0.0880), indicating the least genetic similarity, was noted for the Polish White-backed and Ukrainian Grey breeds, while the greatest genetic similarity (with the lowest genetic distance value ¼ 0.0098) was observed between the Lithuanian Red and Pinzgauer breeds. The genetic similarity values (ranging from 0.9158 to 0.9902) indicate high genetic similarity between the Central European cattle breeds tested. In comparison with the results of research carried out by Martínez et al. (2012) for European breeds (including Spanish, Portuguese and British populations) and zebu cattle, where the distance values ranged from 0.01 to 0.33, much greater similarity was observed among the breeds investigated in our research. According to Barker (1999), phylogenetic diversity can be the best objective criterion in making conservation decisions within a given breed, and the most taxonomically distinct breeds should be specifically protected by a genetic resources conservation programme. An analysis of microsatellite polymorphism conducted by Grzybowski and Prusak (2004) indicates low, comparable genetic distances between nine European cattle breeds, testifying to a common ancestor. According to the rooted dendrogram created in our study and the low genetic distance values, all breeds included in the research are derived from a common ancestor and form two separate clades, which emerged at different times. The first clade consists of two further clades, of which the first comprises the Lithuanian White-backed and Polish White-backed breeds and the second the Polish Red breed with a separate group formed by Lithuanian Red and Pinzgauer cattle. The third, separate clade consists of the breeds Carpathian Brown and Ukrainian Grey. Assessment of genetic variation based on functional gene polymorphism can be used both to characterize the genetic structure of different breeds of cattle and to supplement analysis of genetic diversity based on polymorphisms of neutral
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markers. In our study on seven central European cattle breeds, little variation and genetic isolation were noted between them, which may be due to their close geographic proximity and gene flow between these breeds in the past. It should be noted that all the analysed populations represent local breeds which in the past were the most popular breeds in East-Central Europe, before intensification and globalization of agriculture led to their marginalization. The negative FIS coefficient estimated for all breeds indicates that they were subject to a bottleneck effect. Current genetic resource conservation programmes are contributing to an increase in their numbers and to preservation of the valuable gene reservoir. References Barker, J.S.F., 1999. Conservation of livestock breed diversity. Agri 25, 33e43. Bradley, D.G., MacHugh, D.E., Cunningham, P., Loftus, R.T., 1996. Mitochondrial diversity and the origins of African and European cattle. Proc. Natl. Acad. Sci. 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Identification of polymorphism in promoter region of growth hormone receptor (GHR) gene and its association with milk related traits in Holstein cows. Afr. J. Biotechnol. 9, 5460e5464. czuk, Z., Kasprzak-Filipek, K., 2018. Evaluation of reproductive performance and genetic variation in bulls of the Polish Sawicka-Zugaj, W., Chabuz, W., Litwin White-Backed breed. Reprod. Domest. Anim. 53, 157e162. , M.C., Catillo, G., Marchitelli, C., Moioli, B., 2009. Exploring polymorphisms and effects on milk Signorelli, F., Orrù, L., Napolitano, F., De Matteis, G., Scata traits of the DGAT1, SCD1 and GHR genes in four cattle breeds. Livest. Sci. 125, 74e79. Slatkin, M., Barton, N.H., 1989. A comparison of three indirect methods for estimating average levels of gene flow. Evolution 43, 1349e1368. Socol, C.T., Iacob, L., Mihalca, I., Criste, F.L., 2015. Molecular and population genetics tools for farm animal genetic resources conservation : brief overview. Anim. Sci. Biotechnol. 48, 95e102. Svensson, E., Anderung, C., Baubliene, J., 2007. Tracing genetic change over time using nuclear SNPs in ancient and modern cattle. Anim. Genet. 38, 378e383. ski, M., 2008. Advances in Domestic Animal Genomics, Nauka, vol. 1, pp. 27e43. Polish). Swito n Szreder, T., Zwierzchowski, L., 2004. Polymorphism within the bovine estrogen receptor-alpha gene 5’-region. J. App. Genet. 45, 225e236. Taberlet, P., Ajmone-Marsan, P., Valentini, A., Rezaei, H.R., Naderi, S., Pompanon, F., Negrini, R., Ajmone-Marsan, P., 2008. Are cattle, sheep, and goats endangered species? Mol. Ecol. 17, 275e284. The Bovine HapMap Consortium, 2009. Genome-wide survey of SNP variation uncovers the genetic structure of cattle breeds. Science 324, 528e532. ndez, J., Caballero, A., 2009. Molecular characterization of breeds and its use in conservation. Livest. Sci. 120, 174e195. Toro, M.A., Ferna
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Troy, C.S., MacHugh, D.E., Bailey, J.F., Magee, D.A., Loftus, R.T., Cunningham, P., Chamberlain, A.T., Sykes, B.C., Bradley, D.G., 2001. Genetic evidence for NearEastern origins of European cattle. Nature 410, 1088e1109. Weir, B., 1990. Genetic Data Analysis. Methods for Discrete Population Genetic Data. Sinauer Associates, Inc. Publishers, Sunderland, Massachusetts. Weir, B.S., Cockerham, C.C., 1984. Estimating F-statistics for the analysis of population structure. Evolution 38, 1358e1370. Wright, S., 1978. Evolution and the Genetics of Populations: 4. Variability in and Among Natural Populations. University of Chicago Press, Chicago, IL USA.
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Global Ecology and Conservation journal homepage: http://www.elsevier.com/locate/gecco
Original Research Article
Assessment of the genetic structure of Central European cattle breeds based on functional gene polymorphism czuk a, Karolina Kasprzak-Filipek a, Wioletta Sawicka-Zugaj a, *, Zygmunt Litwin a b c ta Sveistien e_ , Josef Bulla Witold Chabuz , Ru a
University of Life Sciences in Lublin, Institute of Animal Breeding and Biodiversity Conservation, Sub-department of Cattle Breeding and Genetic Resources Conservation, Poland b Lithuanian University of Health Sciences, Animal Science Institute, Lithuania c Slovak University of Agriculture in Nitra, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2018 Received in revised form 3 January 2019 Accepted 3 January 2019
As many European cattle breeds are considered to be threatened with extinction, and existing genetic variability is increasingly at risk of being irretrievably lost, its assessment is crucial. The aim of the study was to assess the genetic variability of seven breeds of cattle in Central Europe on the basis of polymorphism at the loci of functional genes, encoding blactoglobulin (LGB), leptin (LEP), prolactin (PRL), oestrogen receptor alpha (ERa) and growth hormone receptor (GHR). The research was carried out on 290 individuals e 50 Polish White-backed (PW), 50 Lithuanian White-backed (LWB), 50 Polish Red (PR), 50 Lithuanian Red (LR), 22 Carpathian Brown (CB), 18 Ukrainian Grey (UG) and 50 Slovak Pinzgauer (PG). Gene polymorphism was determined by PCR-RFLP. The statistical indicators estimated, i.e. the frequency of alleles and genotypes, observed and expected heterozygosity (HO and HE), F-statistics, gene flow (Nm), and genetic distances, were used to characterize the genetic structure of these cattle breeds. The research demonstrated that the populations analysed have undergone a bottleneck process as a consequence of the rapid decline in the size of individual populations. Owing to the introduction of genetic resources conservation programmes, endangered populations can slowly be restored to a state of genetic balance. © 2019 University of Life Sciences in Lublin. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
Keywords: Genetic variability Functional genes Central European cattle breeds Gene polymorphism Population genetic structure
1. Introduction The diversity within and between cattle breeds is closely linked to their origin, history and evolution (Bradley et al., 1996; MacHugh et al., 1997; Troy et al., 2001). A crucial element of biodiversity conservation is ‘in situ’ protection of indigenous, native breeds of cattle constituting a reservoir of unique combinations of genes and alleles, which in the future may prove highly valuable, e.g. for the creation of new livestock genotypes (Bulla et al., 2013). Although the diversity between the world's cattle breeds does not allow the species to be classified as endangered, the loss of local breeds is an irreversible and irreplaceable erosion of genetic resources (FAO, 2011; Taberlet et al., 2008). Genetic structure can be characterized using a variety
* Corresponding author. E-mail address: [email protected] (W. Sawicka-Zugaj). https://doi.org/10.1016/j.gecco.2019.e00525 2351-9894/© 2019 University of Life Sciences in Lublin. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
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of molecular markers, depending on the research problem (Groeneveld et al., 2010). In recent years, analysis of single nucleotide polymorphisms (SNP) has begun to dominate this research. The literature shows that the data obtained from SNP analysis explain the history of European cattle more accurately than analysis of microsatellite sequences, and for this reason they are increasingly used in biodiversity conservation (Gautier et al., 2007; McKay et al., 2008; Socol et al., 2015; Svensson et al., 2007; The Bovine HapMap Consortium, 2009). Particular importance is attributed to SNPs in the search for linkages between a marker with a specific location in the genome and an unknown gene locus. The search for such associations is important because they allow a phenotypic effect to be assessed by identifying its genetic basis (e.g. in the diagnosis of genetic ski, 2008). According to Socol et al. (2015), in the near diseases or analysis of the variability of a quantitative trait) (Swito n future characterization of genes located on different chromosomes will probably become an integral part of animal genetic resources (AnGR) conservation programmes. Knowledge based on SNP polymorphisms at loci of functional genes can be an important source of information for conservation decisions (FAO, 2012). New technologies using SNP polymorphism or whole genome scanning may revolutionize previous achievements in biodiversity assessment and genetic characterization of breeds, providing a more complete understanding of the molecular basis of functional diversity (Groeneveld et al., 2010). Research by Lasagna et al. (2015) suggests that functional markers are more suitable than neutral markers for evaluating the diversity of morphologically similar breeds. In addition, that study showed that the level of genetic variability analysed on the basis of functional markers is higher than for analysis based on neutral markers. The aim of the study was to characterize the genetic structure of seven Central European cattle breeds (Lithuanian Whitebacked, Polish White-backed, Polish Red, Lithuanian Red, Carpathian Brown, Ukrainian Grey and Slovak Pinzgauer) on the basis of polymorphism at loci of functional genes encoding b-lactoglobulin, leptin, prolactin, oestrogen receptor alpha, and growth hormone receptor. 2. Methods The research was carried out on 290 individuals of seven Central European cattle breeds e 50 Polish White-backed (PW), 50 Lithuanian White-backed (LWB), 50 Polish Red (PR), 50 Lithuanian Red (LR), 22 Carpathian Brown (CB), 18 Ukrainian Grey (UG) and 50 Slovak Pinzgauer (PG). The animals were from farms located in Poland, Lithuania, Ukraine and Slovakia. Hair bulbs were the biological material for analysis. DNA was isolated using ready-made commercial kits for isolating nucleic acids from biological traces (Sherlock AX A & A Biotechnology) according to the procedure given by the manufacturer. Genetic variability was evaluated by the PCR-RFLP method on the basis of polymorphism of loci of the functional genes encoding b-lactoglobulin (LGB), leptin (LEP), prolactin (PRL), oestrogen receptor alpha (ERa) and growth hormone receptor (GHR) (Table 1). The PCR reaction was carried out in an MJ Research thermal cycler (PTCe200 PELTIER THERMAL CYCLER). The fragments amplified by PCR were digested with FastDigest™ restriction enzymes (Thermo Scientific). The following endonucleases were used: for the b-lactoglobulin gene (LGB) e the enzyme HaeIII (Medrano and Aguilar-Cordova 1990), for the leptin gene (LEP) e Sau3AI (Liefers et al., 2002), for the prolactin gene (PRL) e RsaI (Lewin et al., 1992), for the growth hormone receptor gene (GHR) e AluI (Heap et al., 1995) and for the oestrogen receptor gene (ERa) e BglI (Szreder and Zwierzchowski, 2004). The digestion time and the composition of the reaction mixtures were in accordance with the procedure given by the manufacturer of the restriction enzymes. Fragments obtained by PCR-RFLP were electrophoresed on 2% agarose gels stained with 0.01% ethidium bromide (EtBr). Molecular-weight size markers (Thermo Scientific GeneRuler 100bp Plus DNA Ladder) were used to track the electrophoresis process and to assess the length of the fragments obtained. To visualize the genetic structure of the analysed cattle breeds, the results were subjected to statistical analysis in POPGENE v. 3.2. and CERVUS v. 3.0.7 software. The following indicators were estimated: frequency of alleles and genotypes at individual loci; observed heterozygosity (HO); expected heterozygosity (HE) according to Nei (1973); F-statistics estimated for groups and between groups, i.e. the Table 1 Primers used in this study. Gene
Primer sequences
Reference
b-lactoglobulin (LGB)
50 -GTCCTTGTGCTGGACACCGACTACA-30 50 -CAGGACACCGGCTCCCGGTATATGA-30 50 -TGGAGTGGCTTGTTATTTTCTTCT-30 50 -GTCCCCGCTTCTGGCTACCTAACT-30 50 -CGAGTCCTTATGAGCTTGATTCTT-30 50 -GCCTTCCAGAAGTCGTTTGTTTTC-30 50 -TGCGTGCACAGCAGCTCAACC-30 50 -AGCAACCCCACTGCTGGGCAT-30 50 -TTTGGTTAACGAGGTGGAG-30 50 -TGTGACACAGGTGGTTTTTC-30
(Medrano and Aquilar-Cordova, 1990)
Leptin (LEP) Prolactin (PRL) Growth hormone receptor (GHR) Oestrogen receptor alpha (ERa)
Liefers et al. (2002) Lewin et al. (1992) Heap et al. (1995) Szreder and Zwierzchowski (2004)
The reaction mixtures (with a final reagent volume of 12.5 ml) contained 100 ng DNA, primers at a final concentration of 10 mM, REDTaq®DNA Polymerase (1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs and 0.06 unit/mL of Taq DNA polymerase) and nuclease-free water (Sigma-Aldrich). The temperature programmes consisted of the following cycles: 1) initial denaturation (94-95 C/3e5 min), 2) cyclically repeating steps (30e36): DNA denaturation (94-95 C/30 s - 1 min), primer annealing and primer elongation (72 /1 min) and 3) final extension (72 C/5e10 min). The primer annealing temperatures ranged from 56.5 C to 66.8 C, i.e. 61 C for LGB, 65 C for LEP, 56.5 C for PRL, 66.8 C for GHR, and 56.9 C for ERa (Table 2).
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Table 2 PCR conditions for analysed genes. PCR cycles
Step
Temperature
Time
1 30e36
Initial denaturation Denaturation Annealing Extension Final extension
95 C 94 C 56.5 Ce66.8 C 72 C 72 C 4 C
5 min 40 s 1 min 1 min 7 min Indefinitely
1
inbreeding coefficient FIS (Wright, 1978), the fixation index FST according to Hartl and Clark (1997) and Weir (1990), and FIT; and gene flow (Nm) according to Slatkin and Barton (1989). Genetic distances were determined according to Nei (1972). On the basis of the estimated genetic distances, a phylogenetic tree was created to illustrate the diversity of the cattle breeds. Statistical analysis of Hardy-Weinberg equilibrium for the populations was based on the chi square test (c2). 3. Results The research carried out among individuals belonging to seven Central European cattle breeds identified three genotypes at each locus of the genes for b-lactoglobulin (LGB) e AA, AB and BB; leptin (LEP) e CC, CT and TT; prolactin (PRL) e AA, AB and BB; oestrogen receptor alpha (ERa) e AA, AG and GG, and growth hormone receptor (GHR) e AA, AB and BB. Restriction analysis of a fragment of the gene encoding b-lactoglobulin (LGB) identified three genotypes: AA, AB and BB. The highest frequency of the B allele (0.94) was recorded in individuals of the Ukrainian Grey breed, and the lowest (0.56) for the Polish White-backed breed. Individuals homozygous for the A allele at the LGB gene locus were observed only in the Polish White-backed breed (genotype frequency e 0.14; frequency of A allele e 0.44). At the locus of the leptin gene (LEP) in the breeds Polish White-backed (PWB), Lithuanian White-backed (LWB), Polish Red (PR), Lithuanian Red (LR) and Pinzgauer (PG), three genotypes were identified e CC, CT and TT, determined by two alleles, C and T, with average frequencies of 0.76 and 0.24, respectively, for all breeds. Among individuals of the Carpathian Brown (CB) and Ukrainian Grey (UG) breeds, the homozygous TT genotype was not found. The highest frequency of the T allele (0.36) was found in PR individuals and the lowest (0.17) in the UG breed. Restriction digestion of a 156 bp fragment of the prolactin gene (PRL) with the endonuclease RsaI identified three genotypes, AA, AB and BB, among individuals of the breeds LWB, PWB, PR and PG. In the Ukrainian Grey population, all individuals were homozygous for the A allele, while individuals with the BB genotype were not identified among the breeds LR and CB. The highest frequency of the B allele (0.47) was estimated for individuals of the Lithuanian White-backed breed. The highest frequency was found for heterozygous individuals (on average 0.6 for all breeds). Three genotypes were found at the locus of the oestrogen receptor gene: AA, AG and GG. Individuals of the UG and CB breeds were homozygous at the ERa locus for the G allele. AA homozygotes were identified only among Polish Red individuals (with a genotype frequency of 0.06). For all animals, the highest frequency was observed among homozygous GG individuals (with a frequency of 0.6). Three genotypes, AA, AB and BB, were identified at the locus of the growth hormone receptor gene (GHR) among individuals of the breeds LWB, PWB, PR, LR and PG. The highest average frequency was observed for the A allele (0.9). Individuals with the BB genotype were observed only in the PWB breed, in which the frequency of the A and B alleles was 0.77 and 0.23, respectively. Only individuals with AA genotypes were noted among the UG and CB breeds. The estimated values of the chi-square test (c2) in the Polish Red population showed that the frequency distribution of genotypes was consistent with Hardy-Weinberg equilibrium. The estimated frequencies of genotypes observed in the other breeds analysed, which differ statistically from the expected frequencies (in accordance with the HWE principle), indicate a lack of genetic equilibrium in these populations. The heterozygosity index (Table 3) indicates the highest average observed heterozygosity (HO ¼ 0.51) at the locus of the prolactin gene (PRL) and the lowest at the locus of the growth hormone receptor gene (GHR) (with a mean HO of 0.15 for all breeds). The estimated inbreeding coefficients (FIS) provided information regarding the proportions between observed (HO) and expected (HE) heterozygosity, making it possible to assess the processes that were likely to have influenced the current genetic structure of the populations. The negative values obtained for the FIS coefficient indicate an excess of heterozygotes in the populations. The F-statistics provided information on how genetic variability was distributed within and between the analysed populations (Table 4). A negative average value for FIT was noted for the populations. To interpret the values of the fixation index (FST), the following scale was adopted: FST <0.05 e very low or no variation between populations; 0.05e0.15 e low genetic variation; 0.15e0.25 e an intermediate level of variation, and >0.25 e high variation between populations (Weir and Cockerham, 1984). The FST values obtained for all breeds analysed, ranging from 0.0244 to 0.1605, indicate low diversity and a small degree of genetic isolation between them. The gene flow (Nm) between the populations was estimated based on the values of the FST fixation index. It was 2.74 for LGB, 10.00 for LEP, 1.31 for PRL, 2.06 for ERa, and 2.91 for GHR. The mean gene flow rate of 2.40 for all loci indicates about two
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Table 3 Genetic diversity of the seven cattle breeds. Observed heterozygosity (HO), expected heterozygosity (HE) and inbreeding coefficient (FIS). Coefficients
LGB
LEP
PRL
ERa
GHR
Breed
HO HE FIS HO HE FIS HO HE FIS HO HE FIS H HE FIS
Mean HO Mean HE Mean FIS
Mean
BGL
BGP
PC
LC
BU
SU
PG
0,72 0,46 0,56 0,28 0,3 0,05 0,9 0,5 0,81 0,24 0,21 0,14 0,3 0,26 0,18 0,49 0,35 0,33
0,6 0,49 0,22 0,34 0,39 0,14 0,66 0,46 0,45 0,54 0,39 0,37 0,34 0,35 0,04 0,50 0,42 0,02
0,52 0,38 0,35 0,48 0,46 0,04 0,42 0,35 0,19 0,5 0,43 0,17 0,12 0,11 0,06 0,41 0,35 0,16
0,3 0,26 0,18 0,4 0,42 0,05 0,74 0,47 0,59 0,18 0,16 0,10 0,16 0,15 0,09 0,36 0,29 0,18
0,59 0,42 0,42 0,5 0,38 0,33 0,05 0,04 0,02 0 0 e 0 0 e 0,23 0,17 0,26
0,11 0,1 0,06 0,33 0,28 0,20 0 0 e 0 0 e 0 0 e 0,09 0,08 0,13
0,3 0,26 0,18 0,28 0,3 0,05 0,8 0,49 0,62 0,42 0,33 0,27 0,1 0,1 0,05 0,38 0,30 0,21
0,45 0,34 0,28 0,37 0,36 0,04 0,51 0,33 0,45 0,27 0,22 0,06 0,15 0,14 0,07 0,35 0,28 0,19
Table 4 Wright's F-statistics for analysed genes. LOCUS
FIT
FST
LGB LEP PRL ERa GHR Mean
0.2152 0.0125 0.2953 0.0967 0.0259 0.1442
0.0835 0.0244 0.1605 0.1082 0.0792 0.0941
migrants per generation. The highest value, indicating the highest rate of gene flow, was noted at the leptin gene (LEP) locus and the lowest at the prolactin gene (PRL) locus. To determine the genetic differences and similarities between the populations of Central European cattle breeds included in the study, the genetic distances were estimated. The genetic distance and similarity values according to Nei (1972) made it possible to determine the phylogenetic relationships between the populations (Table 5). Based on the genetic distances obtained, the pair group method (UPGMA) was used to create a phylogenetic tree (Fig. 1) illustrating the genetic structure and genetic similarity between the Central European cattle breeds. 4. Discussion Despite the increasing number of studies assessing the genetic variability of various cattle breeds around the world, there are still few that focus on characterizing the genetic diversity between native Central European cattle breeds and analysing their genetic structure (Gutierrez et al., 2003; Petrakova et al., 2012). Analyses aimed at determining the phylogenetic affiliation of various breeds of cattle on the basis of functional gene polymorphism are also uncommon. As a rule, such research focuses on assessment of the genetic variability of various breeds of cattle from northern Europe, or determination of the phylogenetic affiliation of European and African cattle breeds based on haplotype variation (Gautier et al., 2007). As demonstrated by numerous studies (Brym et al., 2005; Buchanan et al., 2003; Curi et al., 2006; Dobicki et al., 2002; Ghasemi ski and Zabolewicz, 2000; Karimi et al., 2009; Komisarek and Antkowiak, 2007; Liefers et al., 2002; Rahbar et al., 2009; Kamin
Table 5 Genetic differences and similarities between the populations of seven Central European cattle breeds.
LWB PWB PR LR CB UG PG
LWB
PWB
PR
LR
CB
UG
PG
**** 0.0171 0.0451 0.0212 0.0613 0.0784 0.0180
0.9831 **** 0.0265 0.0395 0.0586 0.0880 0.0386
0.9559 0.9739 **** 0.0241 0.0355 0.0456 0.0287
0.9790 0.9613 0.9762 **** 0.0388 0.0360 0.0098
0.9406 0.9431 0.9652 0.9619 **** 0.0136 0.0629
0.9246 0.9158 0.9555 0.9647 0.9865 **** 0.0542
0.9821 0.9621 0.9717 0.9902 0.9391 0.9473 ****
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Fig. 1. Phylogenetic tree of genetic distance for assessed cattle breeds.
et al., 2010; Signorelli et al., 2009), functional gene polymorphism is closely linked to specific production traits in cattle, and analysis of variability at gene loci makes it possible to assess the predisposition of animals to a specific type of production. Based on the allele frequencies obtained for the b-lactoglobulin (LGB), leptin (LEP), prolactin (PRL), oestrogen (ERa) and growth hormone (GHR) genes, the observed (HO) and expected (HE) heterozygosity were estimated for the analysed cattle ndez et al. (2004), the higher the level of heterozygosity and the closer the frequency of the breeds. As reported by Ferna alleles, the greater the variability of the breed. For all breeds the mean observed heterozygosity (HO) was higher than the expected heterozygosity (HE). The highest mean observed heterozygosity (HO) was observed in the population of Polish White-backed cattle, and the lowest in the population of Ukrainian Grey cattle. The estimated observed heterozygosity for the seven Central European cattle breeds, ranging from 0.09 to 0.50, indicates a low level of genetic variation within the genes analysed. The observed heterozygosity values reported by Canon et al. (2001) for local breeds raised in Spain, Portugal and France, based on analysis of microsatellite markers, were significantly higher and ranged between 0.54 and 0.72. The FIS inbreeding coefficients were estimated on the basis of the HO and HE values. In most of the loci analysed, negative values were obtained for the inbreeding coefficient, indicating an excess of heterozygotes in the populations (Sawicka-Zugaj et al., 2018). The values obtained for the inbreeding coefficient are presumably indicative of a bottleneck effect in the populations, where the current population structure is the result of a sudden decline in the size of the population and its subsequent rebuilding from the group of survivors. The average FST value (0.0941) obtained in the study indicates that only 9% of the total variability results from inter-breed differences. The FST value of 0.07 (p < 0.01) obtained by Canon et al. (2001) for local breeds raised in Spain, Portugal and France demonstrates a similar (7%) level of variation between these breeds. The genetic distances were parameters describing the genetic diversity between the Central European cattle breeds. Toro et al. (2009) report that in recent years estimation of genetic distances between different populations of livestock animals has been based on information on microsatellite polymorphism. In the present study, we estimated genetic distances and similarities on the basis of polymorphism of functional genes. The highest genetic distance value (0.0880), indicating the least genetic similarity, was noted for the Polish White-backed and Ukrainian Grey breeds, while the greatest genetic similarity (with the lowest genetic distance value ¼ 0.0098) was observed between the Lithuanian Red and Pinzgauer breeds. The genetic similarity values (ranging from 0.9158 to 0.9902) indicate high genetic similarity between the Central European cattle breeds tested. In comparison with the results of research carried out by Martínez et al. (2012) for European breeds (including Spanish, Portuguese and British populations) and zebu cattle, where the distance values ranged from 0.01 to 0.33, much greater similarity was observed among the breeds investigated in our research. According to Barker (1999), phylogenetic diversity can be the best objective criterion in making conservation decisions within a given breed, and the most taxonomically distinct breeds should be specifically protected by a genetic resources conservation programme. An analysis of microsatellite polymorphism conducted by Grzybowski and Prusak (2004) indicates low, comparable genetic distances between nine European cattle breeds, testifying to a common ancestor. According to the rooted dendrogram created in our study and the low genetic distance values, all breeds included in the research are derived from a common ancestor and form two separate clades, which emerged at different times. The first clade consists of two further clades, of which the first comprises the Lithuanian White-backed and Polish White-backed breeds and the second the Polish Red breed with a separate group formed by Lithuanian Red and Pinzgauer cattle. The third, separate clade consists of the breeds Carpathian Brown and Ukrainian Grey. Assessment of genetic variation based on functional gene polymorphism can be used both to characterize the genetic structure of different breeds of cattle and to supplement analysis of genetic diversity based on polymorphisms of neutral
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markers. In our study on seven central European cattle breeds, little variation and genetic isolation were noted between them, which may be due to their close geographic proximity and gene flow between these breeds in the past. It should be noted that all the analysed populations represent local breeds which in the past were the most popular breeds in East-Central Europe, before intensification and globalization of agriculture led to their marginalization. The negative FIS coefficient estimated for all breeds indicates that they were subject to a bottleneck effect. Current genetic resource conservation programmes are contributing to an increase in their numbers and to preservation of the valuable gene reservoir. References Barker, J.S.F., 1999. Conservation of livestock breed diversity. Agri 25, 33e43. Bradley, D.G., MacHugh, D.E., Cunningham, P., Loftus, R.T., 1996. Mitochondrial diversity and the origins of African and European cattle. Proc. Natl. Acad. Sci. 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