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 (πD), for the Turkish donkey populations ranged from 0.533 ± 0.180 (Tekirdag-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.

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 (K) 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.

Leptin Receptor Gene Polymorphisms in Some Turkish Donkey Populations.

Leptin receptor is a fundamental regulator in physiological functions of the regulation of food intake, energy homeostasis, immune function, and reproduction as well as on ovarian follicular cells on the placenta and lactating mammary glands. The aim of this study was to investigate the LEPR gene polymorphism in 60 donkeys reared in Thrace region of Turkey. A 585 bp long partial intron 6, exon 7, intron 7, and exon 8 regions of LEPR gene were amplified, and polymerase chain reaction products analyzed via DNA sequencing. A novel single-nucleotide polymorphism (SNP) was identified as g.713668A>G in the seventh exon region of LEPR gene. This novel SNP was first identified, and the partial DNA sequence of LEPR gene in donkeys was reported for the first time in this study, and these sequences were deposited to NCBI Genbank database with the accession number: MK807114-MK807115. The A>G transition revealed a silent mutation (CAA-CAG) in glutamine amino acid. This nucleotide mutation could cause the changes of secondary structure of protein and expression level of LEPR hormone. For this reason, additional studies are needed to reveal new SNPs and in the LEPR gene that may affect economic traits and structure of protein in donkey breeds.

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

In this study, the κ -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 PstI, and DraII, EagI and MboI restriction enzymes, respectively. Neither the CSN3 gene nor the LTF gene had enzyme recognition sites with the PstI, DraII 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.

Genetic characterization of some Turkish sheep breeds based on the sequencing of the Ovar-DRB1 gene in the major histocompatibility complex (MHC) gene region.

In this research, Ovar-DRB1 gene in the major histocompatibility complex (MHC) gene region was surveyed by DNA sequencing in some of the native sheep breeds that are reared in Turkey. A total of 80 samples were collected from eight different Turkish native sheep breeds, and these samples were used for DNA sequencing. The exon 2 region of Ovar-DRB1 in the MHC gene region was polymerase chain reaction (PCR) amplified and sequenced. A total of 25 new alleles were revealed in the Ovar-DRB1 gene in Turkish native sheep breeds with 24 variable sites; only 13 sites were parsimony informative. The average pairwise genetic distance was 0.029 % for the Ovar-DRB1 gene exon 2 region. The sequence variations at eight different positions (7026, 7036, 7040, 7053, 7059, 7069, 7131 and 7214) are found in all of the studied samples. G → C transversion at position 7081 is only seen in Akkaraman sheep breed, whereas T → C transition at position 7097 is only seen in one sample from the Akkaraman breed. Overall, two main groups were detected among the 25 alleles from Turkish native sheep breeds. All Daǧliç and Kivircik alleles and one allele from Karayaka, Malya and Sakiz are grouped together while all the other breeds are grouped in the other branch.

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 ß2 tubulin 14-bp testis motif (ß2-tubulin gene upstream element 1 [ß2-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 ß2-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.

Diversity of Apis mellifera subspecies from Turkey revealed by sequence analysis of mitochondrial 16s rDNA region.

Mitochondrial DNA sequence variation can be used to infer honeybee evolutionary relationships. In this study, DNA sequence diversity of the mitochondrial 16s rDNA region was investigated in 112 honeybees from 15 populations in Turkey, which is mainly populated with Apis mellifera anatoliaca, A. m. caucasica, and A. m. meda. The study revealed 11 haplotypes for this segment, with 13 variable sites and nine parsimony informative sites. The haplotypes were not discriminated according to their geographical locations in a neighbor-joining dendrogram based on 16s rDNA sequences available in Genbank, but all the haplotypes obtained in this study are clustered with published haplotypes such as A. mellifera TAS (AF214666) and A. m. ligustica (EF116868) and with some unpublished Genbank records (HQ318928, HQ318934, and HQ318938). This study expands the knowledge of the mitochondrial 16s rDNA region, and it presents the first comprehensive sequence analysis of this region in Turkish honeybees.

Evaluation of ISSR markers for genetic diversity analysis in Anatolian water buffaloes.

BACKGROUND: The utilisation of molecular markers has increased in molecular research recently. Inter-simple sequence repeat (ISSR) markers allow the analysis of genomes without preliminary sequence information, since random primers are used. The purpose of the present study was to evaluate ISSR markers for assessing the genetic diversity of indigenous Anatolian Water Buffaloes reared in Afyon, Konya and Sivas provinces of Turkey, with a view to conservation of the gene resources. RESULTS: The 11 ISSR primers chosen for the analysis revealed a total of 110 bands, of which 76 (69.09%) were polymorphic. Also, genetic similarity, polymorphic information content (PIC), resolving power (R(p)) and mean resolving power (R(p)), heterozygosity (H) and Shannon index (I) were calculated as 0.9479-0.9562, 0.35 ± 0.20, 2.73, 0.27, 0.18 ± 0.07 and 0.28 ± 0.11, respectively. CONCLUSION: The ISSR markers were found to be promising for assessing the genetic diversity in buffalo populations. Potential genetic parameters such as PIC, R(p), R(p), H and I were effectively used in this study.

Identification of factor XI deficiency in Holstein cattle in Turkey.

BACKGROUND: Factor XI (FXI) is a plasma protein that participates in the formation of blood clots. Factor XI deficiency is autosomal recessive hereditary disorder that may be associated with excess bleeding in Holstein cattle. METHODS: In this study, 225 Holstein cows reared in Turkey were screened in order to identify FXI genotypes. DNA extractions were obtained from the fresh blood of the cows. Amplicons of FXI exon 12 were obtained by Polymerase Chain Reaction (PCR), and analyzed by 2% agarose gel electrophoresis stained with ethidium bromide. Additionally, all cows were confirmed by DNA sequencing to determine whether or not there was a mutant allele. RESULTS: Carriers of the FXI deficiency have two DNA fragments of 320 bp and 244 bp in size. The results of our study demonstrated that only four out of the 225 Holstein cows tested in Turkey carried the FXI deficiency. The frequency of the mutant FXI allele and the prevalence of heterozygous cows were found as 0.9% and 1.8%, respectively. CONCLUSION: The DNA-based test determines all genotypes, regardless of phenotype or FXI activity. The mutation responsible for the FXI deficiency had not been detected in Holstein cattle in Turkey before prior to this study. The frequency of the mutant FXI allele needs to be confirmed by carrying out further analyses on cattle in Turkey and the selection programs should be developed to eliminate this genetic disorder.