PSXII-1 Tracing the historical genetic components in turano-mongolian cattle breeds based on the microsatellite analysis of modern and museum samples

In their centuries-long history, the nomads of the south of Russia developed the local cattle breeds, which are well adapted to the harsh steppe conditions. In 20th century, the population size of such cattle breeds was dramatically decreased. At the same time, intensive cross breeding with high-producing transboundary breeds had been practiced on the small remaining part of population. The aim of our study was to trace the historical genetic components in modern populations of local cattle breeds of the turano-mongolian origin. The historical specimens of Kalmyk (n = 10), Kyrgyz (n = 11) and Kazakh (n = 3) cattle dated by the first quarter of the 20th century were derived from the craniological collection of the E.F. Liskun Museum for Animal Husbandry and subjected to the study. The modern representatives of Kalmyk (n = 28), Kyrgyz (n = 20), Kazakh White-headed (n = 30), Hereford (n = 26), and Mongolian (n = 41) cattle were used for comparison. All works with historical samples were performed in dedicated facility of the L.K. Ernst research Center for Animal Husbandry. The samples were genotyped for 11 microsatellite loci (BM1818, BM2113, BM1824, ETH10, ETH225, INRA023, SPS115, TGLA53, TGLA122, TGLA126, and TGLA227). For historical samples, the PCR reactions were carried out in five replicates to determine the consensus genotypes. In total, 132 alleles were identified, including 97 alleles in historical samples and 124 alleles in modern samples. The values of unbiased expected heterozygosity were 0.767–0.776 and 0.653–0.778 for the historical and modern samples, respectively. The STRUCTURE clustering showed the visible differences in genetic structure between the historical and modern populations of Kalmyk, Kyrgyz and Kazakh cattle breeds; however, the historical genetic components were still maintained in modern representatives of all of studied breeds. The research results will be useful for the sustainable breeding and conservation of valuable local genetic resources. The study was funded by the RSF No. 19-76-20012.

Validation of methodology for efficient genotyping of CYP2D6 and CYP2C19

There are some data associating variants in the CYP2D6 and/or CYP2C19 genes with concentration-to-dose ratios, efficacy, and retention in treatments. However, much of the above arises from relatively small studies or large datasets with limited genotyping methodologies. Our aim was to develop and validate comprehensive and accurate genotyping methodology for these two genes to facilitate regenotyping in large datasets and hence the generation of more accurate clinical associations. TaqMan copy number variant (CNV) assays for CYP2D6 were used to identify samples from a relevant large dataset (GENDEP study, N = 853) with particularly challenging genotypes to call. These and those representing as broad a range of CYP2D6 and CYP2C19 genotypes as possible by prior available data (AmpliChip CYP450 and TaqMan CYP2C19*17) were chosen for further analysis (N = 96). Genotyping techniques employed were: Luminex CYP2D6 xTAGv3 and Luminex CYP2C19 xTAGv3, PharmacoScan, the Ion S5 AmpliSeq Pharmacogenomics Panel, TaqMan single nucleotide variant (SNV) assays, and, for the CYP2D6 hybrids, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing. Agena was also used for CYP2C19. The TaqMan SNV assays were able to assist with identifying which gene was duplicated or in tandem for multiple copy variants. A multiplex assay was adaptable for analysis of CYP2D6 hybrid genes, with Sanger sequencing data being consistent with the data arising; we provide these data for efficient genotyping of such CYP2D6 hybrid genes with adaptable multiplex methods. Consensus genotypes generated to date resulted in revision of assigned enzyme activity score for 28/96(29%) and 2/93 samples (2.2%) for CYP2D6 and CYP2C19, respectively.

Comparative Study of the Genetic Diversity of Local Steppe Cattle Breeds from Russia, Kazakhstan and Kyrgyzstan by Microsatellite Analysis of Museum and Modern Samples

The comparative molecular genetic study of museum and modern representatives of cattle breeds can help to elucidate the origin and maintenance of historical genetic components in modern populations. We generated the consensus genotypes for 11 microsatellite loci for 24 museum samples of Kalmyk, Kyrgyz, and Kazakh cattle, dated from the first quarter of the 20th century, and compared them with those of modern Kalmyk, Kyrgyz, and Kazakh white-headed breeds. The level of genetic diversity of the modern Kalmyk and Kyrgyz cattle (uHe = 0.771–0.778) was similar to those observed in the museum samples (uHe = 0.772–0.776), while a visible decrease in genetic variability in the modern Kazakh white-headed breed compared to museum Kazakh cattle was detected (uHe = 0.726 and 0.767, respectively). The PCA plot, FST- and Jost’s D-based networks, and STRUCTURE clustering provided strong evidence of the maintenance of the historical genetic background in modern populations of Kalmyk and Kyrgyz cattle. In spite of the allele pool of Kazakh white-headed cattle having undergone great changes compared to the museum Kazakh cattle, several animals still carry the visible aspect of the historical genetic components. Our results can be used for the selection of individuals for the creation of gene banks and may significantly improve the efficiency of conservation programs aimed at preserving genetic diversity in the national genetic resources of cattle.

Genetic Diversity of Historical and Modern Populations of Russian Cattle Breeds Revealed by Microsatellite Analysis

Analysis of ancient and historical DNA has great potential to trace the genetic diversity of local cattle populations during their centuries-long development. Forty-nine specimens representing five cattle breeds (Kholmogor, Yaroslavl, Great Russian, Novgorod, and Holland), dated from the end of the 19th century to the first half of the 20th century, were genotyped for nine polymorphic microsatellite loci. Using a multiple-tube approach, we determined the consensus genotypes of all samples/loci analysed. Amplification errors, including allelic drop-out (ADO) and false alleles (FA), occurred with an average frequency of 2.35% and 0.79%, respectively. A significant effect of allelic length on ADO rate (r2 = 0.620, p = 0.05) was shown. We did not observe significant differences in genetic diversity among historical samples and modern representatives of Kholmogor and Yaroslavl breeds. The unbiased expected heterozygosity values were 0.726–0.774 and 0.708–0.739; the allelic richness values were 2.716–2.893 and 2.661–2.758 for the historical and modern samples, respectively. Analyses of FST and Jost’s D genetic distances, and the results of STRUCTURE clustering, showed the maintenance of a part of historical components in the modern populations of Kholmogor and Yaroslavl cattle. Our study contributes to the conservation of biodiversity in the local Russian genetic resources of cattle.

Validating the use of non-invasively sourced DNA for population genetic studies using pedigree data

Non-invasive genetic sampling has provided valuable ecological data for many species – data which may have been unobtainable using invasive sampling methods. However, DNA obtained non-invasively may be prone to increased levels of amplification failure and genotyping error. Utilizing genotype data from 32 pedigreed koalas, this study aimed to validate the reliability of final consensus genotypes obtained using DNA isolated from koala scats. Pedigree analysis, duplicate genotyping, analysis of mismatched loci and tests for null alleles were used to look for evidence of errors. All genetically confirmed parent–offspring relationships were found to follow Mendelian rules of inheritance. Duplicate genotypes matched in all cases and there was no evidence of null alleles. Related individuals always had different 12-marker genotypes having a minimum of three unique loci (in one full sibling pair), a mode of seven unique loci and a maximum of 11 unique loci. This study demonstrates the capacity of DNA recovered from koala scats to provide reliable genotypes that can unequivocally discriminate individuals and infer parentage, provided data are missing from no more than two loci. Validating data obtained using non-invasive sampling is an important step, allowing potential problems to be identified at an early stage.

Non-invasive genetic approaches for estimation of ungulate population size: a study on roe deer (Capreolus capreolus) based on faeces

Estimating population size is particularly difficult for animal species living in concealing habitats with dense vegetation. This is the case for roe deer as for many other ungulates. Our objective was to develop a non–invasive genetic capture–mark–recapture approach based on roe deer faeces collected along transects. In a pilot study, we collected 1,790 roe deer faeces during five sampling days in a forested study area in south western Germany. We extracted DNA from 410 of these samples and carried out microsatellite analysis using seven dinucleotide markers. The analyses resulted in 328 useable consensus genotypes which were assigned to 174 individuals. The population size estimated using a Bayesian approach was 94 (82–111) male and 136 (121–156) female roe deer. Our study shows that non–invasive genetic methods are a valuable management tool for roe deer.