Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis The Use of Crispr Cas9 Technology To Reduce Polyphenol Oxidase (PPO) Enzyme Activity in Eggplant(01. Izmir Institute of Technology, 2024) Doğanlar, Sami; Dalgıç, Fatma Hacımalak; Doğanlar, SamiPatlıcan (Solanum melongena), Solanaceae ailesinin önemli bir üyesidir ve ekonomik potansiyeli ve zengin besin değeriyle öne çıkmaktadır. Patlıcan, dünyada en fazla üretimi yapılan sebzelerden biridir. Ayrıca, patlıcanın benzersiz genetik yapısı ve zorlu çevre koşullarına uyum sağlayabilme potansiyeli, ürün dayanıklılığı, verim ve besin içeriğini yükseltmeyi amaçlayan çalışmalar için önemli bir hedef haline getirmektedir. Genom düzenleme teknolojilerindeki son gelişmeler, özellikle CRISPR/Cas9 teknolojisi, patlıcanda spesifik genlerin düzenlemesi için umut vadetmektedir. Patlıcanın hasat edildikten sonra işlenmesinde önemli bir sorun olan enzimatik kararma, fenolik bileşiklerin oksidasyon reaksiyonundan kaynaklanmaktadır. Polifenol oksidazlar, kısaca PPO'lar bu reaksiyonu katalize etmektedir. Patlıcanda enzimatik esmerleşme yalnızca görünümde olumsuz etkilere neden olmamakla birlikte, raf ömrünü ve tüketici talebini de negatif şekilde etkilemektedir. Çalışmada, bir Türk patlıcan çeşidi olan Aydın Siyahı çeşidinin enzimatik esmerleşme sorununa hızlı ve kalıcı bir çözüm sağlamak amacıyla CRISPR/Cas9 teknolojisi kullanılmıştır. PPO genlerinde mutasyonlar oluşturularak, ilgili genlerin ifadesinde azalma ve esmerleşmenin azalması hedeflenmektedir. Geleneksel yöntemlerle esmerleşme probleminin çözümü zaman alıcı olacaktır ve ayrıca tüketicilerin tercih ettiği özelliklerin kaybolmasına neden olabilir. Sonuç olarak, bu çalışma ile Türk tipi patlıcanda birden fazla PPO genini hedefleyebilen CRISPR/Cas9 protokolü başarıyla optimize edilmiş ve uygulanmıştır. Bir Türk patlıcan çeşidi olan Kemer tipi Aydın Siyahı için, maksimum üç rehber RNA içerecek şekilde hazırlanan vektörler ve doku kültürü rejenerasyon protokolleri bu çalışmanın önemli başarılarıdır.Master Thesis Development and Characterization of Solanum Linnaeanum Hairy Root Lines(01. Izmir Institute of Technology, 2024) Kaya, Aysu; Doğanlar, SamiSolanum lınnaeanum, birçok Afrika ülkesinde geleneksel olarak tedavi edici özellikleri nedeniyle sıkça kullanılır ve Solanum melongena'nın yabani bir akrabasıdır. Fenolik bileşikler ve glikoalkaloitler de dahil olmak üzere bu bitkinin ürettiği birçok ikincil metabolit, antibakteriyel, antifungal ve antiviral özellikler gösterir. Tüylü kök kültürleri ikincil metabolitlerin üretimi ve bitkinin metabolik yolaklarının çalışılması için sıkça kullanılan bir bitki doku kültürü yöntemidir. Bu çalışmada, S. l?nnaeanum ve S. melongena eksplantlarından tüylü kök hatları oluşturmak için Agrobacter?um rh?zogenes'in agropin suşlarından biri olan ATCC 43057 kullanıldı. İstatistiksel olarak anlamlı olmamasına rağmen, transformasyon verimliliği iki tür için de hipokotil eksplantlarında kotiledon eksplantlarına göre daha yüksek olarak bulundu. T-DNA bölgesinin bitki genomuna entegrasyonu PCR ile doğrulandıktan sonra, transforme edilen tüylü kökler sıvı MS besi yerinde büyütüldü ve biyokimyasal analizler yapıldı. Analizler sonucunda, S. melongena tüylü kök hatlarıyla karşılaştırıldığında, S. l?nnaeanum tüylü kök hatlarında total fenolik (5,75 mg/g kuru ağırlık) ve total flavonoid (14,85 mg/g kuru ağırlık) içeriğinin ve total antioksidan kapasitesinin (11,45 mg/g kuru ağırlık) daha yüksek olduğu gözlemlendi. Ayrıca, myb1, HQT ve Game9 genlerinin ekspresyon analizi S. l?nnaeanum tüylü kök kültürlerinde daha fazla mRNA seviyeleri gösterdi. Bu sonuçlar, ikincil metabolit üretimi için S. l?nnaeanum tüylü kök kültürlerinin, elisitörler veya biyoreaktörler kullanılarak daha da geliştirilebilecek potansiyel bir kaynak olabileceğini göstermektedir.Master Thesis Induction of Hairy Root Culture in Eggplant With Rhizobium Rhizogenes(Izmir Institute of Technology, 2021) Öztürk, Tuba; Doğanlar, SamiIn this study, It has been used two agropine strains (ATCC 15834 and ATCC 43057) of R. rhizogenes to compare hairy root induction in hypocotyl and cotyledon explants of eggplant. Transformation frequency was found to be the highest in hypocotyl explants (49.4%) infected with ATCC 15834. Thirty nine hairy root clones were selected based on their hairy root phenotype. The transgenic status of 15 of these clones was tested by PCR using rol gene primers. Hairy roots infected with ATCC 15834 were grown for 8 weeks in MS liquid medium. At the end of this growth period, the growth ratio of the hairy roots was measured. The root biomass was 6.52 g/DW for hypocotyl and 9.83 g/DW for cotyledon-derived clones. Biochemical analyses of clones were performed. Total phenolic contents were evaluated for hypocotyl and cotyledon-derived clones yielding 8.56 mg/g DW and 8.81 mg/g DW, respectively. The average total flavonoid content was 2.12 mg/g DW in hypocotyl-derived clones, and 1.90 mg/g DW in cotyledon-derived clones. On average, these amounts of both phenolic and flavonoid contents were significantly higher than those obtained for untransformed eggplant roots. Overall the results indicated that eggplant hairy roots are a useful source of secondary metabolites. In the future, the production of metabolites of interest such as steroidal glycoalkaloids or phenolics can be improved using elicitors in eggplant.Master Thesis Development of Single Nucleotide Polymorphism Markers Using Genotyping by Sequencing Technique for Determination of Genetic Diversity and Population Structure in Hazelnut(01. Izmir Institute of Technology, 2021) Yanar, Ertuğrul Gazi; Doğanlar, SamiHazelnut (Corylus avellana L.) is a critical commodity for Turkey due to its economic and nutritional value. Turkey ranks first in world hazelnut production with 65-75% of the market. Due to the signifacance of this crop, it is crucial to preserve Turkish hazelnut genetic diversity. In the current study, a panel representing the entire national collection of 430 accessions was used. Genetic characterization of the panel revealed 7609 high-quality SNPs, 5567 of which were physically mapped to the Tombul reference genome. Fingerprint analysis indicated that all individuals could be distinguished with only seven SNP markers. Population structure analysis of the dataset indicated that the panel's genetic relationships were explained by three clusters containing 8, 17, and 25 accessions, respectively. Nearly half of the accessions had admixed ancestry. The admixed material contained 8 cultivars, 22 landraces, and 12 wild accessions indicating that nearly 50% of each type of material had admixed ancestry. An unweighted neighbor-joining dendrogram was constructed using a distance matrix computed with the identity by state distance measure. The calculated dissimilarity values ranged from 0.15 to 0.30 with a mean of 0.26. This study is the first time that the Gras-Di sequencing approach was used on a nut tree and provides a new perspective on hazelnut genetics. In addition, the panel will serve as a wellcharacterized genetic resource for future work on this economically important tree nut crop.Master Thesis Association Mapping for Oil and Protein Content in Hazelnut (corylus Avellana L.)(Izmir Institute of Technology, 2020) Söylemez, Fatmanur; Doğanlar, SamiEuropean hazelnut has an important place in terms of nutritional and economic value among tree nut species. Because of its nutritional content, the consumption of hazelnuts promotes human health in many ways. These nutritional components are controlled by multiple genes and affected by the environment; therefore, they are quantitative traits. The vast majority of world hazelnut production is provided by Turkey. So it is important to develop hazelnuts with higher nutritional quality for our country. In this work, we aimed to associate genetic diversity data and oil and protein content of hazelnuts to identify QTL. For this purpose oil and protein content were measured in kernels of 96 accessions. Genotypic data were obtained with 30 SSR markers and resulted in 407 polymorphic alleles. According to allelic data, the mean dissimilarity value was 0.52 (52%) for the 96 accessions. Population structure analysis resulted in three clusters with 30, 30, and 16 accessions. Twenty accessions could not be assigned to any cluster and were considered admixed. Association mapping between allelic and phenotypic data indicated that five loci were significantly associated with oil content. The most significant result for oil content belonged to B628-307 loci (p=0.0002, r2=0.145). Three loci were detected for protein content. Among them, A613-153 had the most significant effect (p=0.003, r2= 0.088). We hope that our survey of germplasm and the identified loci associated with oil and protein amount can accelerate hazelnut breeding. In the future this study can contribute to develop new genotypes with enhanced nutritional value.Master Thesis Metabolic and Genomic Profiling for Taste and Aroma Traits in Tomato (solanum Lycopersicum)(Izmir Institute of Technology, 2017) Çakıroğlu, Mehtap; Doğanlar, SamiTomato is one of the most important crops for humans both in our country and the world because of its economic importance and high nutritional quality. Nowadays many people complain about the flavor of tomato. In previous work, researchers have mainly focused on traits such as yield, color, biotic and abiotic stress tolerance which resulted in the loss of flavor. Volatile metabolites are one of the most important flavor contributors in tomato along with organic acids and sugars. More than 400 volatile molecules are found in tomato, but only 15 to 20 are significant in composing tomato’s characteristic flavor. Integration of metabolomics, transcriptomics and proteomics work in recent years has accelerated the speed of genomics studies. Despite this increased interest, there is a limited number of studies that identified quantitative trait loci (QTL) for flavor and aroma in tomato. The aim of this study was to identify QTL using an advanced backcross population of 85 interspecific inbred lines generated by crossing S. lycopersicum cv TA209 x S. pimpinellifolium wild LA1589. Identification of QTL for sugars, organic acids and volatile metabolites is essential to identify candidate genes involved in tomato flavor. Identification of QTLs was undertaken by screening advanced backcross lines using GC and HPLC. A total of 51 COSII and 10 COS markers were positioned on the IBL map. In this way, 25 QTLs were identified. Of these QTLs, one locus was identified for lactic acid and the other 24 loci were identified for volatile compounds. For some of the QTLs identified for taste and aroma characters, alleles from the S. pimpinellifolium parent were favorable. These results showed that S. pimpinellifolium can be used as a source of flavor traits in order to improve elite tomato lines.Master Thesis Development of High-Yielding and Disease Resistant Processing Tomato Lines Using Molecular Marker Technology(Izmir Institute of Technology, 2015) Özbek, Ayten Gizem; Doğanlar, SamiFresh market and processing tomatoes are an important part of the daily diet. Processing tomatoes are used for tomato paste, ketchup, soup and drying. Processing tomatoes are grown under field and/or greenhouse conditions where abiotic (cold, drought, salt) and biotic (bacteria, fungi, viruses and nematodes) stress factors can affect yield and quality. Breeding programs aim to provide disease resistant lines with high quality fruits to farmers and the processing industry. Classical breeding and markerassisted selection (MAS) are two important ways for development of novel inbred lines and tomato cultivars. Classical breeding is long term and based on phenotypic results while MAS provides genotypic information more easily and quickly. In this project, tomato lines were assessed for improved quality of various economic and agronomic features: fruit weight, external color, firmness, flowering, stem scar, fruit shape, wall thickness, yield, brix, internal color, number of flowers and number of fruits. F2 and F3 populations derived from high yielding tomato F1 hybrid MS1453 were scored for these phenotypic features. CAPs and SSR markers were used to genotype the plants and to determine QTL regions controlling the phenotypic features in the F2 population. In addition, disease resistance for Fusarium Crown and Root Rot, Tomato Mosaic Virus, Root Knot Nematode, Verticillium Wilt, and Tomato Yellow Leaf Curl Virus diseases were determined for 261 individuals to identify new sources of candidate disease resistance.Master Thesis Development of Subnils for Fine Mapping of Salt Tolerance in Tomato (solanum Lycopersicum)(Izmir Institute of Technology, 2014) Top, Oğuz; Doğanlar, SamiSalt tolerance is a complex trait that is not easily bred into plants. Salt tolerance can be manifested in several ways such as the ability to increase shoot and/or root mass despite salt stress, a greater than normal accumulation of antioxidants like phenolic compounds and flavonoids, and an improved ability to exclude sodium ions. In previous work, S. lycopersicum M82, S. pennellii LA716 and a S. pennellii IL population were evaluated for growth and their levels of antioxidant activity and content under both control and salt stress conditions (150 mM NaCl) (Frary et al., 2010). These data were used to identify QTLs responsible for controlling antioxidant parameters under both control and stress conditions. Salt tolerance characteristics were observed in IL11-1, IL6-1 and IL7-4-1. The aim of this project was the development of subNILs for fine mapping of salt tolerance related traits within these introgression regions. To this end, each Solanum pennellii IL was crossed with Solanum lycopersicum M82 to produce F2 populations of approximately 1600 individuals for IL6-1, 1600 individuals for IL7-4-1 and 3000 individuals for IL11-1. These individuals were screened with molecular markers that delimit the 30-40 cM introgressions contained in each line. Because of low number of recombinants in IL6-1 and IL7-4-1 populations, they were not selfed to produce subNILs for future analysis. Recombinant F2 plants in IL11-1 population were self-pollinated to generate F3 recombinant families. Each F3 recombinant plant was characterized with several codominant molecular markers in the introgression region. 228 homozygous recombinant F3 plants and 620 heterozygous recombinant plants were identified. At the end of this work, fine mapping populations were developed and in the future they will be grown hydroponically under both control and salt conditions and will be screened for physiological, mineral and biochemical parameters. By statistical comparison between control and salt-treated plants, it will then be possible to identify which recombinants carry regions with significant effects on the various salt tolerance responses. Thus, each gene will be narrowed down to a particular chromosome region.Master Thesis Molecular Mapping of Quantitative Trait Loci Conferring Resistance To Verticillium Wilt in Cotton(Izmir Institute of Technology, 2014) Akköse, Asena; Doğanlar, Sami; Doğanlar, SamiCotton with its valuable natural fiber is an economically important field plant. Resistance against stress conditions and diseases are desired traits in cotton production. Verticillium wilt is one of the major factors resulting in huge cotton yield losses. Control of Verticillium disease has not been successful because of low genetic diversity and a complex disease mechanism. Moreover resistance against Verticillium wilt is a quantitative trait that is regulated by more than one gene and under environmental effects. Due to this complex inheritance, conventional breeding methods have not resulted in efficient resistance. Therefore, it is necessary to develop cotton lines resistant to Verticillium wilt using molecular breeding techniques. In this study we aimed to identify the quantitative trait loci which are responsible for Verticillium wilt resistance. For this purpose, we carried out a Verticillium wilt disease test in a natural population composed of 118 individuals. We inoculated the population with Verticillium dahliae in five replicates. After one month, the symptoms were examined and phenotypic variation was detected and scored. We then used 100 SSR markers for genotyping of the population. Population structure was determined with STRUCTURE 2.2.3. According to results, we determined two clusters. There were 44 and 34 individuals in the first and second clusters, respectively. A total of 40 individuals remained intermixed. Association analysis between phenotypic and allelic data was carried out with TASSEL 2.1. In this way we identified 30 SSR markers associated with Verticillium wilt resistance at a significance level of p<0.05. The most significant SSR locus was DPL080-238 (p=0.0014). Moreover, SSRs DPL188-130 and DPL223-251 showed the highest values for dominant and recessive allelic effects, respectively. We hope that our study will be helpful for the development of marker-assisted strategies for breeding of Verticillium wilt resistant cotton cultivars.Master Thesis Molecular Genetic Analysis in Sesame (sesamum Indicum L.)(Izmir Institute of Technology, 2010) Seyitalioğlu, Ümmü; Doğanlar, SamiIn this study, 156 Turkish sesame (Sesamum indicum L.) accessions were characterized morphologically and 161 accessions were characterized genotypically. A total of 15 criteria were used for morphological characterization. Simple Sequence Repeats (SSRs, microsatellites) were used to characterize genetic variability among Turkish accessions. A total of 318 new EST based SSRs were developed for genotypic analysis. Also, these microsatellites were screened in Korean-Japan and African sesame accessions (parents) for future map construction studies. According to the morphological analysis, some characters showed variation whereas some characters had no variation among accessions. For example, height of first capsule, capsule number per plant, plant height, number of branches, number seeds/capsule, days to 1st flower and days to %50 flower characters showed variation among accessions. However, stem hair, leaf hairs, axil flower number, number of carpels/capsule and capsule hairs showed variation for only a few accessions. Also, growth habit, branching and capsule splitting characters had no variation. In accordance with genetic characterization, survey results showed only five polymorphic markers among 318 EST based SSR markers. Also, in this study, genetic distance of Turkish sesame accessions was calculated using DICE matrix and UPGMA (Unweighted Pair Group Method) arithmetical averages via 19 EST based SSR fragments. Genetic analysis showed that Turkish sesame accessions have fairly low genetic diversity. As a result, genetic diversity among Turkish sesame accessions were defined and the genetic relationships of Turkish sesame accessions were established.
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