Analysis of GH 1 , GHR and PRL gene polymorphisms for estimation of the genetic diversity of Buryat and Altai cattle breeds

Small and unique Buryat and Altai cattle breeds of Turano-Mongolian origin are well adapted to harsh con ditions of the continental climate to be their ha bitat. However, the population-genetic structure of the breeds has been poorly studied. This paper pre sents the re sults of analysis of polymorphisms GH1 (AC_000176.1: BTA 19, exon 5, rs41923484, g.2141C>G, L127V), GHR (AC_000177.1: BTA 20, exon 10, rs109300983, g.257A>G, S555G) and PRL (AC_000180.1: BTA 23, exon 3, g.35108342A>G) in the samples of Buryat cattle breed of Russia, China and Mongolia, and indigenous Altai cattle breed (Russia) that belong to Turano-Mongolian cattle. The Russian sample of Buryat breed was differentiated from the Mongolian sample based on pairwise G-test and FST values for the PRL-RsaI polymorphism and from the Chinese sample – based on pairwise G-test values for the GH1-AluI polymorphism. All the three samples of Buryat breed clearly differed from the sample of Altai breed based on pairwise G-test and FST values for the GHR-AluI polymorphism as well as on the base of FST values for the joint polymorphism of the three genes. Nei’s genetic distances calculated from the three gene polymorphisms also confirmed the difference between the two breeds. The results of AMOVA demonstrated that GHR gene variability (16 %) gave the largest contribution to the differentiation that was confirmed by FST values (0.12–0.27). The STRUCTURE software enabled us to reveal four clusters, with a specific ratio for each sample, in the Chinese and Mongolian samples of Buryat breed, and in the sample of Altai breed, while the Russian sample of Buryat breed had only three clusters. The differences within the breed level were determined based on the GH1-AluI and PRL-RsaI polymorphisms, while at the inter-breed level – based on the GHR-AluI polymorphism. Linkage Малочисленные уникальные бурятская и алтайская породы крупного рогатого скота турано-монгольского корня хорошо адаптированы к суровым условиям континентального климата региона обитания. Информация о популяционно-генетической структуре этих пород практически отсутствует. В настоящей работе выполнен анализ генетической изменчивости следующих генов-кандидатов: GH1 (AC_000176.1: хромосома 19, экзон 5, rs41923484, g.2141C>G, L127V), GHR (AC_000177.1: хромосома 20, экзон 10, rs109300983, g.257A>G, S555G), PRL (AC_000180.1: хромосома 23, экзон 3, g.35108342A>G) в выборках бурятской породы из трех сопредельных государств – России, Китая и Монголии, а также аборигенной алтайской породы (Россия), относящихся в соответствии с происхождением к турано-монгольскому корню. Российская выборка бурятского скота дифференцируется от монгольской выборки этой породы на основе попарных значений G-теста и FST по изменчивости RsaI-локуса гена PRL и от китайской выборки – на основе значений G-теста для AluI-локуса гена GH1. При этом все выборки бурятского скота ведут себя согласованно в отношении алтайской породы, четко отличаясь от нее по данным G-теста и FST для локуса гена GHR и по значениям FST для комплекса локусов генов PRL, GH1 и GHR. Генетические расстояния Нея на основе комплекса генов также отделяют бурятскую и алтайскую породу. Наибольший вклад в межпородную дифференциацию двух пород вносит изменчивость гена GHR, что зафиксировано в результатах AMOVA (16 %) и FST (0.12–0.27). Кластерный анализ, выполненный в программе STRUCTURE, выявил четыре кластера в алтайской породе крупного рогатого скота, китайской и монгольской выборках бурятского скота, которые представлены специфично в каждой выборке. У российского бурятского скота отсутствует один кластер, его частота в других выборках достигает 15 %. Различия на внутрипородном уровне у бурятской породы определяют локусы генов PRL и GH1, на межпородном с алтайской породой – исследованный локус гена GHR, что отражает неодинаковый вклад разных локусов. Анализ на неравновесие по сцеплению генов доказал достоверное сцепление следующих пар генов у бурятской породы: GH1-GHR, GH1-PRL, GHR-PRL.

As of today, Kalmykian and Yakutian cattle have been preserved in Russia, along with Buryat and Altai cattle.These breeds are well adapted to harsh conditions of the continental climate and specific habitat features and are characterized with high physical endurance, minimum human involvement in their management, year-round free grazing regime and abilities to maintain viability under low-caloric and deteriorating forage base in certain times of year, digest coarse forage and find it under the snow.These features are facilitated by physiological and morphological adaptations from specific features of digestive system structure and metabolism to developing longer and thicker fur in winter (Lazebnaya et al., 2010).
Lactation yield in Buryat and Altai cattle is not specifically high.According to M. N. Balkov (1962), milk yield in 114 cows in a 305-day lactation period, the number of calvings being four or above, was on average 911 kg with a fat percentage of 4.58.However, the simplicity and low cost of management for these breeds, as well as their high stability against diseases, make it possible to consider them not only unique, but irreplaceable for conventional breeding regions in terms of solving problems of providing environmentally healthy nourishment, preserving historical traditions of the local population, including technologies of making traditional dishes and clothing, and developing agro-and ecotourism.In addition, cattle breeds of such an ancient origin with aforementioned features are rare even on global scale, which makes it possible to consider them an important object for scientific investigation with an opportunity for finding possible uses for their genetic potential in present and future (Dmitriev, Ernest, 1989;Ukhanov et al., 1993;Moiseeva et al., 2006;Mwai et al., 2015;Shabtay, 2015).Despite the facts above, the genetic features of breeds of Turano-Mongolian origin are rather understudied.Individual candidate genes are investigated in Yakutian and Kalmykian breeds, whose SNPs are associated in cattle with productivity attributes of selection value (Lazebnaya et al., 2010(Lazebnaya et al., , 2013;;Gorlov et al., 2014).Polylocus (ISSR) and monolocus (SSR) DNA markers were used to study a variety of breeds, including Yakutian, Kalmykian, and Mongolian ones (Tapio et al., 2010;Stolpovsky et al., 2011).Whole genome SNP-analysis has been performed recently for a series of Russian breeds, which also included Buryat breed, to investigate their origin, phylogeny, and differentiation (Yurchenko et al., 2017).
Diversity in somatotropic axis genes, which are candidate genes to be associated with milk and meat yield traits, is well researched in individual commercial cattle breeds.Correlation is established between gene polymorphism in growth hormone (GH1), its receptor (GHR), and prolactin (PRL) and growth and development, weight gain rate, meat quality, and protein and fat content in milk (Mitra et al., 1995;Chung et al., 1996;Chrenek et al., 1999;Dybus et al., 2004;Di Stasio et al., 2005).The best-known SNPs for the listed genes are as follows: the ones of GH1 AluI (L127V, exon 5) (Dybus et al., 2004) and GHR AluI genes (AF140284: g.257A > G, exon 10, Ser/Gly) (Di Stasio et al., 2005), and the ones of PRL RsaI gene (A > G, 103 codon, exon 3) (Mitra et al., 1995).Comparative data on the diversity of the listed genes in Buryat and Altai cattle are not available.
The objective of the presented study was to assess the genetic structure in samples from two breeds of Turano-Mongolian origin, specifically Buryat breed from the neighboring territories of China, Mongolia, and Russia to discover diversity patterns within the same breed, and indigenous Russian Altai cattle based on polymorphism analysis for PRL, GH1, and GHR genes.
The data were statistically processed using the GenAlEx 6.503 (http://biology-assets.anu.edu.au/GenAlEx/Download_files/GenAlEx %206.503%20Download.zip),and STRUCTURE 2.3.4.software (https://web.stanford.edu/group/pritchardlab/structure_software/release_versions/v2.3.4/html/structure.html).Benjamini-Hochberg multiple comparison correction was introduced (Benjamini, Hochberg, 1995) to estimate the confidence of p Fst , p Gst , and p χ2 values.Gene linkage was assessed using linkage (D) and correlation (r) coefficients.The confidence of linkage disequilibrium was estimated by the χ 2 test using the Benjamini-Hochberg multiple comparison correction.Cluster analysis of genetic structure based on the complex of studied genes was performed using the STRUCTURE 2.3.4 software.Here, the basic set of models was analyzed with genetic admixture and allele frequency correlation both taken and not taken into account.
Models were analyzed in two modes, i. e. LOCPRIOR and without considering the general matrix division into studied samples.Nine hypotheses on population structure were tested within each model with K varying from 1 to 9. For each K, the simulation was repeated 10 times to collect the required statistical data on likelihood function logarithm values and its average variance.The quantity of possible populations was tested by 1 000 000 burn-in iterations with the first 100 000 iterations being discarded.

Results
Allele frequency distributions for the studied GH1, GHR, and PRL gene loci are presented in Table 1, along with chi-square test results with probability values for each of three candidate genes studied in the cattle samples of Buryat and Altai breeds considered.According to Table 1, allele frequency L of GH1 gene prevails in all samples and varies within the range of 0.654 in Buryat breed from China and up to 0.863 in the Russian sample of the breed.Allele A of PRL gene is also represented with high frequency in all samples of both breeds, frequency interval is 0.620 for Buryat breed from Mongolia and up to 0.814 in Buryat breed from Russia.Allele frequency distribution for GHR gene differs from the ones described above for GH1 and PRL genes, i. e. allele frequency A, 0.88, prevails in the Altai breed sample and allele frequencies G, 0.62 and 0.559, in Buryat breed samples from Mongolia and Russia respectively.It should be noted that the genotype frequency distribution observed showed no deviation from the one expected based on Hardy-Weinberg equilibrium for all GH1, GHR, and PRL gene loci in all samples.
Pairwise G-test values for the studied samples with the corresponding probability values are presented in Table 2.According to the Benjamini-Hochberg multiple comparison correction, probability values that fit condition p < 0.025 are significant.According to Table 2, differences between the Russian breed sample and Chinese and Mongolian samples were discovered in AluI locus of GH1 gene and RsaI locus of PRL gene, respectively.The difference between the Altai breed and Buryat breed samples was found in AluI locus of GHR gene regardless of origin.
Differentiation between the considered Buryat and Altai cattle breed samples was estimated using pairwise values of Wright's fixation index (F ST ) for individual genes and their complexes (Table 3).Confident F ST values given the Benjamini-Hochberg multiple comparison correction (p < 0.025) were identified by the considered GHR gene locus for Altai breed sample paired with Buryat breed samples from China (F ST = 0.117), Mongolia (F ST = 0.269), and Russia (F ST = 0.216).This regularity stands for this parameter in case of gene complexes as well.In addition, Russian Buryat breed sample is differentiated from the Mongolian sample (F ST = 0.046) by the studied PRL gene locus.
The analysis of Table 3 also shows that significant  Breeding • 2018 • 22 • 6 differentiation of Altai breed from Buryat breed samples from various regions is primarily determined by GHR gene diversity, which is further confirmed by AMOVA results.Thus, diversity between populations by three genes studied was rather high at 8 %, however for GHR gene it was 16 % and for GH1 and PRL genes -2 %.Ordination of the studied samples in the first two principal coordinates based on pairwise Nei's genetic distance (Nei, 1978) for the gene complex is shown in Fig. 1.

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The amount of diversity in the first principal component is 92.33 %, and in the second -7.67 %.All the studied samples of both breeds are differentiated from each other.The distance from Altai breed to Buryat breeds by samples increases as follows: China, Russia, and Mongolia.Here, the calculations show that average pairwise Nei's genetic distance inside the group formed by Buryat breed samples from various regions (D Nei = 0.071) is over three times as small as average pairwise Nei's genetic distance between Altai breed and each Buryat breed sample from China, Mongolia, and Russia (D Nei = 0.222).
Cluster analysis of genetic structure on the dataset, including the complex genotypes of GH1, GHR, and PRL genes in the Buryat breed samples from three regions, and Altai breed from Russia, was performed using the STRUCTURE software.After various models had been tested taking posterior probability into account, a model with genetic material admixture but with no allele frequency correlation was selected.Based on average likelihood function logarithm values and variances of its estimates obtained in 10 simulation runs with a specified set of respective parameters, the optimum number of clusters (K) turned out to be four.
It follows from Fig. 2 and Table 4 that each sample has its own cluster distribution.Most samples show all types of clusters, apart from Buryat breed from Russia, in which one cluster shown in blue in Fig. 2 and present in Table 4 under no. 3 is lacking, whereas its frequency in other samples does not exceed 0.154.In Buryat cattle from China, the presence of cluster 2 shown in green is high at 0.403, and it is only higher in Altai breed, in which it is vastly prevailing (0.820).Buryat breed sample from China is characterized by the most uniform distribution of various clusters.Buryat and Altai breeds from Russia show the significant prevalence of one cluster, namely clusters 1 and 2 (see Table 4) highlighted in red (0.800) and green respectively.
GH1, GHR, and PRL genes in B. taurus specimens are localized on different chromosomes, 19, 20, and 23, respectively, however linkages between individual loci remain a possibility.The performed analysis of pairwise linkage in studied loci of three genes in accordance with Benjamini-Hochberg correction showed confident probability values (p < 0.025) for loci of the following gene pairs: GH1-GHR in Buryat breed from Russia (D = 0.051, r = 0.297, p = 0.003) and Mongolia (D = 0.076, r = 0.391, p = 0.006), GH1-PRL genes in all Buryat breed sample pairs (D = 0.033-0.099,r = 0.248-0.470,p = 0.012-0.020),and Fig. 1.Principal coordinates analysis (PCoA) of pairwise Nei's genetic distances (Nei, 1978) from the three polymorphisms typed in four samples.Анализ полиморфизма генов GH1, GHR и PRL у бурятской и алтайской пород крупного рогатого скота GHR-PRL genes in Buryat breed from Russia (D = 0.063, r = 0.324, p = 0.001).Complex genotypes based on two and three genes for samples, in which disequilibrium gene linkage is established, are presented in Table 5.According to the provided complex genotype frequencies for GH1-PRL genes, the linkage between L-allele of GH1 gene with A-allele of PRL gene may be assumed in all Buryat breed samples.In addition, according to maximum complex genotype frequencies for GH1-GHR genes, L-allele of GH1 gene is also linked to A-and G-alleles of GHR gene in Buryat breeds from Russia and Mongolia.These regularities are also reflected in high complex genotype frequencies for three genes, in which L-allele of GH1 Differences in the genetic structure of studied samples were demonstrated by cluster analysis performed using the STRUCTURE software.Four clusters identified in Altai breed, as well as Chinese and Mongolian Buryat breed samples are present in a specific fashion in each sample.However, one cluster with relatively low frequencies in other samples is lacking in Russian Buryat breed.Presence of the same clusters in Buryat and Altai breeds may indicate the unity of origin expressed by belonging to Turano-Mongolian root breed, however their lengthy independent existence affected their genetic structure in the form of prevalence of one of clusters in Altai breed sample.In addition, differentiation within the breed may be observed in Buryat cattle.Note that differences within the breed in Buryat cattle are determined by PRL and GH1 gene loci and differences from Altai breed by the studied GHR gene locus, which reflects unequal contributions of various loci to differentiation depending on the level of comparison.To clarify whether the identified diversity pattern is maintained, when the dataset is expanded, larger-scale research needs to be carried out.However, the results obtained show the necessity of taking diversity in subpopulations into account, while preserving small breeds.Disequilibrium linkage analysis of the genes considered performed in the paper confidently showed its presence for the following gene pairs in Buryat breed: GH1-GHR, GH1-PRL, GHR-PRL.The use of primarily the same servicing bulls could be considered a possible explanation for the discovered similarity in disequilibrium linkage between alleles of the mentioned genes in all studied cattle samples, however our samples represent independent herds, which makes their mixing impossible.Thus, the discovered similarity in disequilibrium linkage between alleles of the mentioned genes is most likely to result from direct or indirect natural and-or artificial selection.Disequilibrium linkage of GH1 and PRL genes with GHR gene does not contradict with the available data on possible competition between prolactin and growth hormone genes for linkage with the latter's receptor (Inoue et al., 2001).
Maintaining genetic diversity and preventing inbreeding are especially important for small indigenous breeds in a critical state.Allele variety of genes subjected to strong selection pressure in commercial breeds may lead to significant allele frequency deviation in one direction or another up to their fixation, which may have a negative effect on their viability under changing climatic and agrobiocenotic conditions.Moreover, genetic potential of indigenous breeds well adapted to harsh ecological and geographical conditions, which also preserve stability against diseases most common for their habitat, may be desired in both conventional livestock breeding and developing new breeds.Given the fact that artificial insemination is rarely practiced in free grazing breeds, it is necessary to develop in situ and ex situ preservation strategies taking into account DNA marker monitoring.The data obtained are the first population-genetic characteristics of Buryat and Altai breeds based on candidate gene diversity.

Fig. 2 .
Fig. 2. Bayesian genotypic cluster analysis of the four samples in Buryat and Altai breeds based on the three polymorphisms (GH1, GHR, and PRL) at various values of K.X-axis: 1 -Altai cattle breed, 2 -Buryat cattle breed from China, 3 -Buryat cattle breed from Mongolia, 4 -Buryat cattle breed from Russia; each cluster designated with particular color (description in the text).

table 2 .
The pairwise G-test and corresponding probability values for each genetic marker Notes: pairwise G-test values for the considered samples are given below the diagonal, the corresponding probability values are above the diagonal.table 3. Pairwise genetic differentiation of Buryat and Altai breed samples on the basis of Wright's F-statistics (F ST ) and corresponding probability values given separately for the studied loci of GH1, GHR and PRL genes and for a complex of the genes Notes: the pairwise values of the F ST coefficient are given below the diagonal, the corresponding probability values are above the diagonal.
gene, A-and G-alleles of GHR gene, and A-allele of PRL gene are present in Russian Buryat breed sample.Mongolia, and Russia and Altai breed from Russia have been compared for the first time in the present paper.In none of these samples the observed genotype frequency distribution deviates from the one expected based on Hardy-Weinberg equilibrium.Russian Buryat breed sample is differentiated from the Mongolian sample of the same breed based on pairwise G-test and F ST values regarding diversity in RsaI locus of PRL gene and from Chinese sample -based on G-test values for AluI locus of GH1 gene.Here, all Buryat breed samples match in their behavior with respect to Altai breed, i. e. they clearly differ from it based on G-test and F ST data for GHR gene locus and F ST values for locus complexes of PRL, GH1, and GHR genes.Nei's genetic distances based on gene complexes also isolate the group of Buryat breed samples from Altai breed, which is indicated by the relationship between average pairwise Nei's genetic distance inside the Buryat breed group and average pairwise Nei's genetic distance for Buryat breed samples from China, Mongolia, and Russia and Altai breed.The largest contribution to differentiation between the two breeds is given by GHR gene diversity, which is reflected by AMOVA (16 %) and F ST results (0.12-0.27).

table 4 .
Percentage of the clusters identified in samples of Altai and Buryat cattle breeds

table 5 .
Complex genotypes of GH1, GHR and PRL genes with linkage disequilibrium in Buryat cattle breed Note. q is the frequency of the complex genotype.