The Genetic Characterization of DGAT1 Gene in Donkey Populations Reared in Thrace Region of Turkey

AcylCoA: diacylglycerol acyltransferase (DGAT1) gene has a considerable effect on milk content and yield in cattle with a substitution of lysine by alanine in the exon 8 of the gene. Moreover there are many other researches comprising the DGAT1 gene on different farm animals, such as buffalo, sheep and goat but there is no information about the DGAT1 gene in donkeys. In this study, the polymorphism of DGAT1 gene in donkey populations reared in Thrace region of Turkey has been investigated by restriction fragment length polymorphism (RFLP) via EaeI (CfrI) restriction enzyme. EaeI restriction site was found in cattle breeds which resulted after K232A substitution, Lysine (AAG) to Alanine (GCG) variant but this restriction site was not found in donkey populations. A novel single-nucleotide polymorphism (G -> A substitution) in the DGAT1 gene at position 10,435 lacks this restriction site which results only Alanine variant (GCA) instead of Lysine variant. This novel single-nucleotide polymorphism in the DGAT1 gene was found in the studied donkey breeds

Genetic diversity of kappa-casein (CSN3) and lactoferrin (LTF) genes in the endangered Turkish donkey (Equus asinus) populations

In this study, the kappa-casein (CSN3) and lactoferrin (LTF) genes which were found in association with milk production traits in different animal species were studied firstly in Turkish donkey populations. A total of 108 donkeys from different regions of Turkey were used in order to reveal the different genotypes of CSN3 and LTF genes by using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing methods. To determine the genetic polymorphism, we attempted to digest a fragment of 235 bp of the CSN3 gene and a fragment of 751 bp of the LTF gene using Pstl, and DralI, EagI and MboI restriction enzymes, respectively. Neither the CSN3 gene nor the LTF gene had enzyme recognition sites with the PstI, DralI and MboI restriction enzymes in all of the studied samples. However, the LTF gene was only distinguished with the EagI restriction enzyme. Three genotypes were identified in the LTF gene with the EagI restriction enzyme: GG homozygotes (667, 84 bp), AG heterozygotes (751; 667, 84 bp) and AA homozygotes (751 bp). The transition from guanine to adenine in 89 bp of the LTF gene lacks the restriction site and different genotypes are obtained. This novel single nucleotide polymorphism (SNP) has been firstly detected in donkeys. According to the results, the G allele was predominant in the LTF-EagI gene in the studied Turkish donkey populations. In this study, all the genotype distributions of LTF-EagI were not found in Hardy-Weinberg equilibrium (P <0.05). The CSN3 and LTF genes have not been studied before in donkeys, so the results are the preliminary results of these gene regions in donkeys.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [TOVAG-215O555]This study was a part of the MSc thesis of Hasan Bulut in Tekirdag Namik Kemal University, Graduate School of Natural and Applied Sciences. Also, this study was supported by a grant from the Scientific and Technological Research Council of Turkey (TUBITAK TOVAG-215O555, project leader: Fulya Ozdil)

Evaluation of Variation on Myostatin (MSTN) Gene of Turkish Donkey Populations in Thrace Region of Turkey

The study aimed to determine the MSTN gene variation in 90 donkeys reared in the Thrace region of Turkey. Myostatin (MSTN), also named GDF-8 (growth differentiation factor 8) is a part of the transforming growth factor beta (TGF-beta) superfamily and it has a negative regulator role on muscle mass, growth and development in mammalian species. MSTN gene regulates the skeletal muscle growth in a negative way and has a significant role in homeostasis of skeletal muscles. Also, in muscle fibers balance of protein has been promoted by Myostatin factor. The total of 866 bp long partial intron 1 and 2, whole exon 2 regions of MSTN gene was amplified and PCR products analysed using DNA sequencing. In this study, a novel synonymous SNP was determined as g.4183919 G>A in the second exon region of the MSTN gene which does not cause an amino acid change in the protein. The G>A transition caused a silent mutation in leucine (leu) amino acid. Alterations in mRNA level and functionality of protein can occur due to synonymous mutations. Since leucine is an important amino acid that can avoid muscle mass loss and inhibits the expression of Myostatin, it can be said that silent mutation of Leu in donkeys may have altered the muscle mass and physical factor of donkeys in this study. Mutant leucine may have a lower efficient effect on preventing loss of muscles and causes more Myostatin protein expression. The identified SNP was firstly released and the DNA sequences of the MSTN gene in Turkish donkeys was revealed for the first time with recent study. Turkish donkeys lacked these mutations that were identified before in horses, which cause for the less might require for race ability of donkeys. The sequences of MSTN gene were submitted to the NCBI GenBank with the accession number: MW970078- MW970079. Further studies are needed to conduct, on protein and molecular levels, SNPs on the MSTN gene and their association with the morphological characters that may affect economic traits in donkey breeds

Morphometric and Genetic Characterization of Honey Bees (Apis mellifera L.) From Thrace Region of Turkey

A detailed morphological and genetic characterization of honey bees from the Thrace and west Anatolian regions of Turkey was surveyed. A total of 1650 worker bee samples (110 colonies) were evaluated with the forty-one morphological characters and 217 honey bee samples were analyzed via DNA sequencing of the tRNA(leu)-cox2 region. In this study, three different populations, Thrace (Tekirdag, Kirklareli and Edirne provinces), Island Gokceada, and western Anatolia were formed based on morphometrics, since the Marmara Sea has taken a very strong barrier role in this formation. The morphological similarity of the Thrace population was supported by the genetic analysis. The sequencing of the tRNA(leu)-cox2 region revealed twenty-two different haplotypes, sixteen of which are novel. The C2d, macedonica-like haplotype, was the most widely found haplotype (48%) all around the Thrace region. Along with the C2d haplotype, previously published C2s, C2v, C2i, C2j, and C2h haplotypes, and the newly found haplotypes were also observed but less frequently. In this study, Thrace honey bees were found to more similar to A. m. macedonica through the mtDNA sequence analysis, whereas carnica-like honey bees were only found near the Istranca mountain ridges, Kirklareli province and macedonica-like honey bees all around the Thrace region. According to our results, some of the Thrace honey bee populations may be both A. m. carnica and A. m. macedonica but the assignment to the latter subspecies seems more likely due to its geographic range.Scientific and Technological Research Council of Turkey-TUBITAK [3001-TOVAG 114O883]The authors are deeply indebted to numerous people that have contributed to this study for providing honey bee reference samples; Ljubia Z. Stanisavljevi for providing A. m. carnica samples from Serbia and Leonidas Charistos for providing A. m. macedonica samples from Greece. Financial support for this research was provided by The Scientific and Technological Research Council of Turkey-TUBITAK through the Project 3001-TOVAG 114O883, Project Coordinator Fulya ozdil

Relocation Facilitates the Acquisition of Short Cis-Regulatory Regions that Drive the Expression of Retrogenes during Spermatogenesis in Drosophila

Retrogenes are functional processed copies of genes that originate via the retrotranscription of an mRNA intermediate and often exhibit testis-specific expression. Although this expression pattern appears to be favored by selection, the origin of such expression bias remains unexplained. Here, we study the regulation of two young testis-specific Drosophila retrogenes, Dntf-2r and Pros28.1A, using genetic transformation and the enhanced green fluorescent protein reporter gene in Drosophila melanogaster. We show that two different short (< 24 bp) regions upstream of the transcription start sites (TSSs) act as testis-specific regulatory motifs in these genes. The Dntf-2r regulatory region is similar to the known beta 2 tubulin 14-bp testis motif (beta 2-tubulin gene upstream element 1 [beta 2-UE1]). Comparative sequence analyses reveal that this motif was already present before the Dntf-2r insertion and was likely driving the transcription of a noncoding RNA. We also show that the beta 2-UE1 occurs in the regulatory regions of other testis-specific retrogenes, and is functional in either orientation. In contrast, the Pros28.1A testes regulatory region in D. melanogaster appears to be novel. Only Pros28.1B, an older paralog of the Pros28.1 gene family, seems to carry a similar regulatory sequence. It is unclear how the Pros28.1A regulatory region was acquired in D. melanogaster, but it might have evolved de novo from within a region that may have been preprimed for testes expression. We conclude that relocation is critical for the evolutionary origin of male germline-specific cis-regulatory regions of retrogenes because expression depends on either the site of the retrogene insertion or the sequence changes close to the TSS thereafter. As a consequence we infer that positive selection will play a role in the evolution of these regulatory regions and can often act from the moment of the retrocopy insertion.National Institute of General Medical Sciences of the National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [R01GM071813]; ARRA; UT; NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCESUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [R01GM071813] Funding Source: NIH RePORTERThe authors thank J. Coyne, P. Gibert, F. Lemeunier, M. Long, M.-L. Wu, C.-I. Wu, and the UC San Diego Drosophila Stock Center for providing wild-type Drosophila strains used in this work and Genetic Services, Inc. and the Bloomington Drosophila Stock Center at Indiana University for providing the mutant stocks used in this study. They also thank H. White-Cooper for providing them with her in situ hybridization protocol before publication and for all her advice on its implementation in their lab. They want to thank four anonymous reviewers for comments on this work. M. G. wants to thank Arndt von Haeseler for his support when finalizing the manuscript. This work was supported by, the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM071813, an ARRA Supplement and UT Arlington startup funds to E. B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Phylogenetic relationships of Turkish indigenous donkey populations determined by mitochondrial DNA D-loop region

In this study, to analyze the mtDNA D-loop region and the origin of the maternal lineages of 16 different donkey populations, and to assess the domestication of Turkish indigenous donkeys in seven geographical regions, we investigated the DNA sequences of the D-loop region of 315 indigenous donkeys from Turkey. A total of 54 haplotypes, resulting from 35 polymorphic regions (27 parsimoniously informative and 6 singleton sites), were defined. Twenty-eight of these haplotypes are unique (51.85%), and 26 are shared among different Turkish indigenous donkey populations. The most frequent haplotype was Hap 1 (45.71%), followed by two haplotypes (Hap 4, 15.55% and Hap 7, 5.39%). The breed genetic diversity, evaluated by the haplotype diversity (HD ) and nucleotide diversity (?D ), for the Turkish donkey populations ranged from 0.533 ± 0.180 (Tekirdağ–Malkara, MAL) to 0.933 ± 0.122 (Aydin, AYD), and from 0.01196 ± 0.0026 (Antalya, ANT) to 0.02101 ± 0.0041 (Aydin, AYD), respectively. We observed moderate-to-high levels of haplotype diversity and moderate nucleotide diversity, indicating plentiful genetic diversity in all of the Turkish indigenous donkey populations. Phylogenetic analysis (NJT) and median-joining network analysis established that all haplotypes were distinctly grouped into two major haplogroups. The results of AMOVA analyses, based on geographic structuring of Turkish native donkey populations, highlighted that the majority of the observed variance is due to differences among samples within populations. The observed differences between groups were found to be statistically significant. Comparison among Turkish indigenous donkey mtDNA D-loop regions and haplotypes, and different countries’ donkey breeds and wild asses, identified two clades and which is named Somali (Clade IV) and Nubian (Clade V) lineages. The results can be used to understand the origin of Turkish donkey populations clearly, and to resolve the phylogenetic relationship among all of the different regions. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.Türkiye Bilimsel ve Teknolojik Araştirma Kurumu, TÜBITAK: 215O555This research was funded by TUBITAK (The Scientific and Technological Research Council of Turkey), grant number 215O555, project leader Fulya ?zdil. The authors would like to kindly thank to Selen Yatkin who provide help during the sample collection. We are also grateful to all farmers and pastoralists for allowing us to use their animal to collect blood samples for free. Many thanks to our lab team ?eref M?cahit Topalo?lu and Ayla Fidan for helping laboratory experiment. The authors want to thank four anonymous reviewers for comments on this work

Genetic characterization of native donkey (Equus asinus) populations of Turkey using microsatellite markers

This study presents the first insights to the genetic diversity and structure of the Turkish donkey populations. The primary objectives were to detect the main structural features of Turkish donkeys by microsatellite markers. A panel of 17 microsatellite markers was applied for genotyping 314 donkeys from 16 locations of Turkey. One hundred and forty‐two alleles were identified and the number of alleles per locus ranged from 4 to 12. The highest number of alleles was observed in AHT05 (12) and the lowest in ASB02 and HTG06 (4), while ASB17 was monomorphic. The mean HO in the Turkish donkey was estimated to be 0.677, while mean HE was 0.675. The polymorphic information content (PIC) was calculated for each locus and ranged from 0.36 (locus ASB02) to 0.98 (locus AHT05), which has the highest number of alleles per locus in the present study. The average PIC in our populations was 0.696. The average coefficient of gene differentiation (GST) over the 17 loci was 0.020 ± 0.037 (p < 0.01). The GST values for single loci ranged from −0.004 for LEX54 to 0.162 for COR082. Nei’s gene diversity index (Ht) for loci ranged from 0.445 (ASB02) to 0.890 (AHT05), with an average of 0.696. A Bayesian clustering method, the Structure software, was used for clustering algorithms of multi‐locus genotypes to identify the population structure and the pattern of admixture within the populations. When the number of ancestral populations varied from K = 1 to 20, the largest change in the log of the likelihood function (ΔK) was when K = 2. The results for K = 2 indicate a clear separation between Clade I (KIR, CAT, KAR, MAR, SAN) and Clade II (MAL, MER, TOK, KAS, KUT, KON, ISP, ANT, MUG, AYD and KAH) populations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Determination of the morphometric characteristics of donkey (Equus asinus) populations reared in Turkey

This research aims to determine the morphometric characteristics of the donkey (Equus asinus) populations reared in Turkey. For this purpose, live weights and body measurements were collected from 500 donkeys. The donkeys were grouped according to the factors of color, age, sex, and province and the live weights (LW) (kg), withers heights (WH), rump heights (RH), body lengths (BL), chest circumferences (CC), chest depths (CD), front shank circumferences (FSC), head lengths (HL) and ear lengths (EL) (cm) of the donkeys were measured. In the study, the males were found to have higher values of live weight, withers height, rump height, and chest depth than the females (p < 0.05). Significant differences in the live weights of the donkeys were seen by province, age, color, and sex (p < 0.01 and p < 0.05). Significant differences were found among the age groups as well (p < 0.01). Accordingly, the least squares means of the animals aged 1???3 years, 4???5 years, 6???8 years, and 9 years and over for live weight were measured as 112.10 ?? 3.11 kg, 141.54 ?? 2.76 kg, 153.98 ?? 2.42 kg, and 152.95 ?? 2.34 kg, respectively. The least squares mean of live weights were also determined as significant between the female and male animals (138.08 ?? 1.96 kg) and (142.21 ?? 2.25 kg), respectively (p 0.05). The highest correlation coefficient was calculated between live weight and body length among the donkeys (r = 0.83). Furthermore, the classical method (CM) and the fixed object photo (FOP) method were compared for photographed animals in the study. No difference in WH, RH, CD or HL was seen between the two methods (p 0.05). In conclusion, the morphometric characteristics of the donkeys were determined and it was shown that the populations were not distinguished clearly from each other and that this was fundamentally due to the transitions among the donkey populations for long years

Identification of factor XI deficiency in Holstein cattle in Turkey

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Determination of DGAT1 K232A Polymorphism in Donkey Populations Reared in Thrace Region of Turkey by DNA Sequencing

Diacylglycerol acyltransferase 1 (DGAT1) gene has become a potential candidate gene for milk composition and yield in cattle. A nonconservative lysine to alanine substitution (K232A) at position 10.433 and 10.434 in the eighth exon of DGAT1 gene has widely studied in cattle, goat, and sheep. This substitution conducted a considerable effect on milk fat, which was previously mapped to the centromeric end of bovine chromosome 14. Yet, no information has been found about DGAT1 gene in donkeys. In this study, the genetic variation of DGAT1 gene has been analyzed by CfrI (EaeI) restriction and DNA sequencing in 85 donkeys reared in Thrace region of Turkey. No digestion is found in the DGAT1 gene via CfrI restriction enzyme in the studied donkey populations. The DNA sequence of 437 bp of the DGAT1 gene in donkeys revealed only Alanine allele (K232A), which was reported in cattle with high milk fat content. But we found a novel polymorphism at position 10.435, from G to A, which also revealed Alanine allele. As a conclusion, CfrI digestion is not a diagnostic site in DGAT1 gene in donkeys. The DNA sequence of DGAT1 gene in donkeys is reported for the first time in this study, and this sequence is deposited to National Center for Biotechnology Information GenBank database with the accession number MK086162. © 2018 Elsevier Inc.NKUBAP.03Financial disclosure: The Scientific Research Projects Coordination Unit of Namık Kemal University, Turkey (Project No: NKUBAP.03.DS.17.129, Project Leader: Raziye IŞIK)