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Barcode DNA length polymorphisms vs fatty acid profiling for adulteration detection in olive oil
(Elsevier Sci Ltd, 2017) Uncu, Ali Tevfik; Uncu, Ayse Ozgur; Frary, Anne; Doganlar, Sami
The aim of this study was to compare the performance of a DNA-barcode assay with fatty acid profile analysis to authenticate the botanical origin of olive oil. To achieve this aim, we performed a PCR-capillary electrophoresis (PCR-CE) approach on olive oil: seed oil blends using the plastid trnL (UAA) intron barcode. In parallel to genomic analysis, we subjected the samples to gas chromatography analysis of fatty acid composition. While the PCR-CE assay proved equally efficient as gas chromatography analysis in detecting adulteration with soybean, palm, rapeseed, sunflower, sesame, cottonseed and peanut oils, it was superior to the widely utilized analytical chemistry approach in revealing the adulterant species and detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Thus, the present research has shown the feasibility of a PCR-CE barcode assay to detect adulteration in olive oil. (C) 2016 Elsevier Ltd. All rights reserved.
A barcode-DNA analysis method for the identification of plant oil adulteration in milk and dairy products
(Elsevier Sci Ltd, 2020) Uncu, Ayse Ozgur; Uncu, Ali Tevfik
In the present work, a barcode-DNA analysis method is described for the detection of plant oil adulteration in milk and dairy products. The method relies on the fact that plant DNA should not be present in readily detectable amounts in a dairy product unless it contains undeclared plant material. Thus, a universal plant barcode is chosen as the target to be amplified from dairy samples. Accordingly, barcode PCR-CE (PCR-capillary electrophoresis) assays are described, which do not require preliminary information on the species source of the adulterant oil type. Two PCR-CE assays, one operating on the plastid trnL (UAA) intron and the other targeting its inner P6 loop in nested format, were shown to detect corn, soybean, rapeseed and sunflower oils in clarified butter, milk and yogurt. Both barcodes are robustly amplified with extremely conserved primers. While the intron provides the species discrimination ability, the P6 loop provides superior detection sensitivity.
Construction and characterization of a de novo draft genome of garden cress (Lepidium sativum L.)
(Springer Heidelberg, 2022) Patat, Aysenur Soyturk; Sen, Fatima; Erdogdu, Behic Selman; Uncu, Ali Tevfik; Uncu, Ayse Ozgur
Garden cress (Lepidium sativum L.) is a Brassicaceae crop recognized as a healthy vegetable and a medicinal plant. Lepidium is one of the largest genera in Brassicaceae, yet, the genus has not been a focus of extensive genomic research. In the present work, garden cress genome was sequenced using the long read high-fidelity sequencing technology. A de novo, draft genome assembly that spans 336.5 Mb was produced, corresponding to 88.6% of the estimated genome size and representing 90% of the evolutionarily expected orthologous gene content. Protein coding gene content was structurally predicted and functionally annotated, resulting in the identification of 25,668 putative genes. A total of 599 candidate disease resistance genes were identified by predicting resistance gene domains in gene structures, and 37 genes were detected as orthologs of heavy metal associated protein coding genes. In addition, 4289 genes were assigned as transcription factor coding. Six different machine learning algorithms were trained and tested for their performance in classifying miRNA coding genomic sequences. Logistic regression proved the best performing trained algorithm, thus utilized for pre-miRNA coding loci identification in the assembly. Repetitive DNA analysis involved the characterization of transposable element and microsatellite contents. L. sativum chloroplast genome was also assembled and functionally annotated. Data produced in the present work is expected to constitute a foundation for genomic research in garden cress and contribute to genomics-assisted crop improvement and genome evolution studies in the Brassicaceae family.
A Cost-Efficient and Simple Plant Oil DNA Extraction Protocol Optimized for DNA-Based Assessment of Product Authenticity
(Springer, 2018) Uncu, Ayse Ozgur; Torlak, Emrah; Uncu, Ali Tevfik
DNA-based assays offer precision in ascertaining the species/cultivar origin of agro-food products. Yet, obtaining DNA of sufficient quality and quantity is the main challenge while performing DNA-based food authentication analyses. The aim of the present work was to standardize a cost-efficient, easy-to-apply, yet effective plant oil DNA isolation protocol that allows reliable downstream PCR-based analyses. Because capillary electrophoresis (CE) separation of species/cultivar discriminating genomic fragments is a widely adopted approach in food genomics, a CE system was utilized in order to assess the performance of the proposed cetyl trimethyl ammonium bromide (CTAB)-based protocol. A plastid intergenic spacer and a nuclear olive gene were used as targets in order to evaluate the amplificability of DNA extracted with the CTAB-based protocol. The plastid barcode not only allowed assessing the reproducibility of PCR amplifications from the extracted oil DNA samples (olive, hazelnut, corn, rapeseed, cottonseed, and soybean oils) but also proved successful in discriminating all tested oil crop species based on amplified fragment length polymorphisms. Moreover, the barcode assay proved successful in correctly identifying the tested olive oil: cottonseed oil blends as admixtures of the two oil species. Thus, it was also feasible to demonstrate the potential of the barcode sequence as a discriminatory analyte to detect adulteration in plant oils. In addition, application of a CAPS (cleaved amplified polymorphic sequence) assay designed to genotype a nuclear SNP (single nucleotide polymorphism) marker resulted in the successful identification of the two single-cultivar olive oils included in the study. As a result of the present work, it was feasible to standardize a reliable and cost-efficient DNA extraction protocol that works well with both unrefined (olive and hazelnut) and refined (corn, rapeseed, cottonseed, and soybean) oils.
Determination of Resistance Levels to Fusarium oxysporum f. sp. melonis and ZYMV and Homogeneity in Some Melon Genotypes
(Centivens Inst Innovative Research-Ciir, 2021) Cetin, Aye Nur; Uncu, Ali Tevfik; Sen, Fatima; Erdeger, Seyma Nur; Turkmen, Onder
ZYMV and Fusarium oxysporum f. sp. melonis are one of the main disease factors limiting melon cultivation. ZYMV (Zucchini Yellow Mosaic Virus) is one of the most important diseases caused by potyvirus and causing the most important yield losses in melons. Another important disease agent, Fusarium oxysporum f. sp. Melonis, is a soil- borne disease with four races FOM 0, 1, 2, 1-2. The most common races are 1 and 2 in Turkey. This study, 87 melon accessories ZYMV and Fom 1 and Fom 2 races resistance levels and homogeneity testing levels of genotypes were determined. Result of the study, when the resistance among the genotypes for two races was examined, Fom 1 and Fom 2 strains, 23 genotypes were found to have homozygous resistance alleles against to both races, while for Fom 1 there were 46 allele resistance lines in total, and 30 genotypes had homozygous resistance alleles, 16 of them had heterozygous resistance alleles. This situation for Fom 2, it was found that there were 75 allele resistant alleles, 69 genotypes had homozygous allele and 16 genotypes had heterozygous allele. All genotypes are sensitive against to ZYMV. Homogeneity levels,29 of the genotypes were observed to be between 85-95%.
Genes involved in mRNA surveillance are induced in Brachypodium distachyon under cadmium toxicity
(Springer, 2022) Aksoy, Emre; Uncu, Ali Tevfik; Filiz, Ertugrul; Orman, Sule; Cetin, Durmus; Akbudak, M. Aydin
Background Cd accumulation in plant cells results in dramatic problems including oxidative stress and inhibition of vital enzymes. It also affects mineral uptakes by disrupting membrane permeability. Interaction among Cd and other plant nutrient elements changes the nutritional contents of crops and reduces their yield. Methods and results In the present study, Cd stress in Brachypodium distachyon led to the upregulation of some heavy metal transport genes (influx or efflux) encoding cation-efflux proteins, heavy metal-associated proteins and NRAMP proteins. The Arabidopsis orthologs of the differentially expressed B. distachyon genes (DEGs) under Cd toxicity were identified, which exhibited Bradi4g26905 was an ortholog of AtALY1-2. Detailed co-expression network and gene ontology analyses found the potential involvement of the mRNA surveillance pathway in Cd tolerance in B. distachyon. These genes were shown to be downregulated by sulfur (S) deficiency. Conclusions This is the first transcriptomic study investigating the effect of Cd toxicity in B. distachyon, a model plant for genomic studies in Poaceae (Gramineae) species. The results are expected to provide valuable information for more comprehensive research related to heavy metal toxicity in plants.
Genome-wide SNP discovery and QTL mapping for fruit quality traits in inbred backcross lines (IBLs) of solanum pimpinellifolium using genotyping by sequencing
(Bmc, 2017) Celik, Ibrahim; Gurbuz, Nergiz; Uncu, Ali Tevfik; Frary, Anne; Doganlar, Sami
Background: Solanum pimpinellifolium has high breeding potential for fruit quality traits and has been used as a donor in tomato breeding programs. Unlocking the genetic potential of S. pimpinellifolium requires high-throughput polymorphism identification protocols for QTL mapping and introgression of favourable alleles into cultivated tomato by both positive and background selection. Results: In this study we identified SNP loci using a genotyping by sequencing (GBS) approach in an IBL mapping population derived from the cross between a high yielding fresh market tomato and S. pimpinellifolium (LA1589) as the recurrent and donor parents, respectively. A total of 120,983,088 reads were generated by the Illumina HiSeq next-generation sequencing platform. From these reads 448,539 sequence tags were generated. A majority of the sequence tags (84.4%) were uniquely aligned to the tomato genome. A total of 3.125 unique SNP loci were identified as a result of tag alignment to the genome assembly and were used in QTL analysis of 11 fruit quality traits. As a result, 37 QTLs were identified. S. pimpinellifolium contributed favourable alleles for 16 QTLs (43.2%), thus confirming the high breeding potential of this wild species. Conclusions: The present work introduced a set of SNPs at sufficiently high density for QTL mapping in populations derived from S. pimpinellifolium (LA1589). Moreover, this study demonstrated the high efficiency of the GBS approach for SNP identification, genotyping and QTL mapping in an interspecific tomato population.
High-Throughput Genomic Simple Sequence Repeat (SSR) Marker Development and Construction of a High Resolution Physical Map in Button Mushroom (Agaricus bisporus) Genome
(2018) Uncu, Ali Tevfik; Özgür Uncu, Ayşe
Button mushroom [Agaricus bisporus (J.E. Lange) Imbach], is both a model organism and a worldwide cultivated edible mushroom species. Despite its scientific and economic relevance, efforts to develop molecular markers in A. bisporus genome are scarce and relatively recent. The present research reports the development of 3943 novel simple sequence repeat (SSR) markers specific to the A. bisporus genome. The marker set is well-distributed among the 13 A. bisporus chromosomes, saturating the entire genome with sequence-specific markers. The large number of novel, A. bisporus specific DNA markers introduced in the present work constitute a valuable resource for molecular genetic research in button mushroom, including strain identification and protection, and gene/QTL (Quantitative Trait Locus) mapping.
High-throughput simple sequence repeat (SSR) mining saturates the carrot (Daucus carota L.) genome with chromosome-anchored markers
(Taylor & Francis Ltd, 2020) Uncu, Ayse Ozgur; Uncu, Ali Tevfik
Carrot (Daucus carota L.) is a versatile vegetable crop and the most economically important member of the Apiaceae family. While there are several important cultivated species in the family such as celery, parsley, cumin, fennel, coriander and parsnip, molecular genetic research in Apiaceae is relatively limited compared to other agriculturally important taxa. In the present work, an in silico approach was employed in order to develop chromosome-anchored simple sequence repeat (SSR) markers from the carrot genome assembly. A total of 55,386 markers were developed and marker loci that correspond to protein coding sequences were determined. In silico mapping analysis predicted that 51,160 of these were single-locus markers and 4,226 amplified more than one locus. Cross-species transferability of the markers was assessed using the fennel (Foeniculum vulgare Mill.) draft genome sequence, resulting in the identification of 578 low-copy transferable markers. These markers can serve for the purposes of interspecific genomic synteny studies and comparative gene identification/cloning. A subset of 50 markers was evaluated on DNA from 17 accessions of carrot. As a result, 46 (92%) produced amplicons from all genotypes, of which 28 (61%) displayed polymorphisms among the 17 carrot accessions, confirming the potential of the newly developed markers to reveal genotypic diversity in cultivated carrot. With the present work, carrot chromosomes were saturated with sequence-specific markers, which constitute a physical map of the carrot genome. The collection of markers will serve as practical molecular tools for germplasm characterization, gene tagging and molecular breeding studies in this important crop species.
A new molecular approach to the diagnosis of small ruminant morbillivirus with EvaGreen based assay
(Tubitak Scientific & Technological Research Council Turkey, 2021) Oz, Mustafa Emin; Torlak, Emrah; Avci, Oguzhan; Dogan, Muge; Uncu, Ali Tevfik
The present work describes a new method that is anticipated to contribute to the global control and eradication strategies of small ruminant morbillivirus (SRMV), which negatively affects sheep and goat breeding in many countries where outbreaks emerge. Described assay was developed to detect SRMV by targeting the fusion glycoprotein gene. In the study, the EvaGreen based real-time RT-PCR method is compared with real-time RT-PCR based on TaqMan probe, which is recommended as the diagnostic test by World Organisation for Animal Health (OIE). Detection limit, normalized fluorescence values, and sensitivity of the methods were evaluated in the analysis. The lowest limit of detection obtained was approximately 12 RNA copies per reaction corresponding to a C-T value of 36.79. According to the results, it was observed that the developed in-house method has a specific, sensitive, and powerful detection limit as much as the gold standard test. As a result, it was concluded that the proposed method, which was developed as a more cost-effective and easily optimized analysis, could provide an advantage for detecting SRMV in diagnostic laboratories.
De novo assembly and characterization of the first draft genome of quince (Cydonia oblonga Mill.)
(Nature Portfolio, 2021) Soyturk, Aysenur; Sen, Fatima; Uncu, Ali Tevfik; Celik, Ibrahim; Uncu, Ayse Ozgur
Quince (Cydonia oblonga Mill.) is the sole member of the genus Cydonia in the Rosacea family and closely related to the major pome fruits, apple (Malus domestica Borkh.) and pear (Pyrus communis L.). In the present work, whole genome shotgun paired-end sequencing was employed in order to assemble the first draft genome of quince. A genome assembly that spans 488.4 Mb of sequence corresponding to 71.2% of the estimated genome size (686 Mb) was produced in the study. Gene predictions via ab initio and homology-based sequence annotation strategies resulted in the identification of 25,428 and 30,684 unique putative protein coding genes, respectively. 97.4 and 95.6% of putative homologs of Arabidopsis and rice transcription factors were identified in the ab initio predicted genic sequences. Different machine learning algorithms were tested for classifying pre-miRNA (precursor microRNA) coding sequences, identifying Support Vector Machine (SVM) as the best performing classifier. SVM classification predicted 600 putative pre-miRNA coding loci. Repetitive DNA content of the assembly was also characterized. The first draft assembly of the quince genome produced in this work would constitute a foundation for functional genomic research in quince toward dissecting the genetic basis of important traits and performing genomics-assisted breeding.
Plastid trnH-psbA intergenic spacer serves as a PCR-based marker to detect common grain adulterants of coffee (Coffea arabica L.)
(Elsevier Sci Ltd, 2018) Uncu, Ali Tevfik; Uncu, Ayse Ozgur
The present work describes the utilization of interspecific length polymorphisms within the chloroplastic trnH-psbA intergenic spacer to standardize a DNA-based method that identifies adulteration in Arabica coffee (Coffea arabica L.). Adulteration of coffee with roasted cereal grains and soybean is a common fraudulent practice, yet, research to design sensitive DNA-based tests in order to establish traceability in coffee is extremely limited. In the present study, PCR-CE (PCR-capillary electrophoresis) analysis of the trnH-psbA intergenic spacer proved successful to discriminate Arabica coffee and its common adulterants, namely corn, soybean, rice, wheat and barley. When the barcode PCR-CE assay was tested on admixtures, adulteration as low as 1% was successfully detected simply through barcode amplification and capillary electrophoresis separation. Moreover, it is important to note that the proposed barcode assay not only detects adulteration but also reveals the identity of the adulterant species based on barcode amplification profiles obtained from reference sets of anticipated adulterants. (C) 2018 Elsevier Ltd. All rights reserved.
Transcriptomic analysis of asymptomatic and symptomatic severe Turkish patients in SARS-CoV-2 infection
(Kare Publ, 2022) Pence, Sadrettin; Caykara, Burcu; Pence, Halime Hanim; Tekin, Saban; Keskin, Birsen Cevher; Uncu, Ali Tevfik; Uncu, Ayse Ozgur
OBJECTIVE: Coronavirus disease 2019 (COVID-19), leading to mild infection (MI), acute respiratory distress syndrome or death in different persons. Although the basis of these variabilities has not been fully elucidated, some possible findings have been encountered. In the present study, we aimed to reveal genes with different expression profiles by next-generation sequencing of RNA isolated from blood taken from infected patients to reveal molecular causes of different response. METHODS: Two healthy, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-negative control individuals (NCI), two SARS-CoV-2-positive patients who have MI, and two patients who have critical infection (CI) were included in the study. Total RNA was extracted from blood samples and sequenced. Raw RNA-Seq data were analyzed on Galaxy platform for the identification of differentially expressed genes and their pathway involvements. RESULTS: We found that 199 and 521 genes were downregulated in whole blood of COVID-19-positive CI patients compared to NCI and MI patients, respectively. We identified 21 gene ontology pathways commonly downregulated in CI patients compared to both NCI and MI, mostly associated with innate and adaptive immune responses. Three hundred and fifty-four and 600 genes were found to be upregulated compared to NCI and MI, respectively. Upregulated six pathways included genes that function in inflammatory response and inflammatory cytokine release. CONCLUSION: The transcriptional profile of CI patients deviates more significantly from that of MI in terms of the number of differentially expressed genes, implying that genotypic differences may account for the severity of SARS-CoV-2 infection and inflammatory responses through differential regulation of gene expression. Therefore, further studies that involve whole genome analysis coupled with differential expression analysis are required in order to determine the dynamics of genotype - gene expression profile associations.
Transcriptomic analysis of asymptomatic and symptomatic severe Turkish patients in SARS-CoV-2 infection
(Kare Publ, 2022) Pence, Sadrettin; Caykara, Burcu; Pence, Halime Hanim; Tekin, Saban; Keskin, Birsen Cevher; Uncu, Ali Tevfik; Uncu, Ayse Ozgur
OBJECTIVE: Coronavirus disease 2019 (COVID-19), leading to mild infection (MI), acute respiratory distress syndrome or death in different persons. Although the basis of these variabilities has not been fully elucidated, some possible findings have been encountered. In the present study, we aimed to reveal genes with different expression profiles by next-generation sequencing of RNA isolated from blood taken from infected patients to reveal molecular causes of different response. METHODS: Two healthy, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-negative control individuals (NCI), two SARS-CoV-2-positive patients who have MI, and two patients who have critical infection (CI) were included in the study. Total RNA was extracted from blood samples and sequenced. Raw RNA-Seq data were analyzed on Galaxy platform for the identification of differentially expressed genes and their pathway involvements. RESULTS: We found that 199 and 521 genes were downregulated in whole blood of COVID-19-positive CI patients compared to NCI and MI patients, respectively. We identified 21 gene ontology pathways commonly downregulated in CI patients compared to both NCI and MI, mostly associated with innate and adaptive immune responses. Three hundred and fifty-four and 600 genes were found to be upregulated compared to NCI and MI, respectively. Upregulated six pathways included genes that function in inflammatory response and inflammatory cytokine release. CONCLUSION: The transcriptional profile of CI patients deviates more significantly from that of MI in terms of the number of differentially expressed genes, implying that genotypic differences may account for the severity of SARS-CoV-2 infection and inflammatory responses through differential regulation of gene expression. Therefore, further studies that involve whole genome analysis coupled with differential expression analysis are required in order to determine the dynamics of genotype - gene expression profile associations.
The trnL (UAA)-trnF (GAA) intergenic spacer is a robust marker of green pea (Pisum sativum L.) adulteration in economically valuable pistachio nuts (Pistacia vera L.)
(Wiley, 2020) Sen, Fatima; Uncu, Ayse Ozgur; Uncu, Ali Tevfik; Erdeger, Seyma Nur
BACKGROUND Pistachio (Pistacia vera L.) is an expensive culinary nut species; it is therefore susceptible to adulteration for economic profit. Green pea (Pisum sativum L.) kernels constitute the most common material used for adulterating chopped / ground pistachio nuts and pistachio paste. Food genomics enables the species composition of a food sample to be ascertained through DNA analysis. Accordingly, a barcode DNA genotyping approach was used to standardize a test method to identify green pea adulteration in pistachio nuts. RESULTS The trnL (UAA)-trnF (GAA) intergenic spacer in the plastid genome was the target analyte in the present study. The barcode locus displayed a significant, discriminatory size difference between pistachio and pea, with amplicon sizes of 449 and 179 bp, respectively. Polymerase chain reaction-capillary electrophoresis (PCR-CE) analysis of the intergenic spacer resulted in the successful identification of species composition in the in-house admixtures, which contained 5% to 30% of green pea. CONCLUSION The present work describes a fast and straightforward DNA test that identifies green pea adulteration in pistachio nuts without requiring a statistical data interpretation process. The plastid trnL (UAA)-trnF (GAA) intergenic spacer length widely varies among plant taxa, so the PCR-CE protocol that operates on the intergenic spacer holds the potential to reveal adulteration with a plethora of adulterants. The PCR-CE assay described in the present work can be adopted readily by food-quality laboratories in the public sector or the food industry as an easy and reliable method to analyze pistachio authenticity. (c) 2020 Society of Chemical Industry
Use of gaseous ozone to reduce aflatoxin B1 and microorganisms in poultry feed
(Pergamon-Elsevier Science Ltd, 2016) Torlak, Emrah; Akata, Ilgaz; Erci, Fatih; Uncu, Ali Tevfik
This study was undertaken to evaluate the efficacy of gaseous ozone for the degradation of aflatoxin B-1 (AFB(1)) and inactivation of indigenous microflora in poultry feed. Feed samples were treated with continuous stream of two different constant concentrations (2.8 and 5.3 mg/L) of ozone at room temperature up to 240 min. The initial AFB(1) level in artificially contaminated feed samples, determined as 32.8 mu g/kg, decreased by 74.3 and 86.4% after 240 min of exposure at 2.8 and 5.3 mg/L, respectively. At the both ozone concentrations, 240 min exposure was reduced the aerobic plate and yeast and mold counts below the detection limit (<10 CFU/g) with a reduction more than 3.2 and 2.7 log, respectively. The thiobarbituric acid reactive substances (TSARS) assay indicated that no significant (P >= 0.05) increase occurred in the level of lipid oxidation in feed samples during 120 min ozonation at 2.8 mg/L. At the end of the 240 min of exposure at 2.8 and 5.3 mg/L, initial TBARS concentration, determined as 2.4 mg/kg, reached to 4.4 and 5.3 mg/kg with a significant (P < 0.05) increases, respectively. The results presented in this study suggested that significant (P < 0.05) reductions in the AFB(1) level and microbial population can be achieved in poultry feed by ozonation with an acceptable changes in lipid oxidation. (C) 2016 Elsevier Ltd. All rights reserved.

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