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Analysis of Physio-Biochemical Responses and Expressional Profiling Antioxidant-Related Genes in Some Neglected Aegilops Species under Salinity Stress
(Mdpi, 2023) Jamshidi, Bita; Pour-Aboughadareh, Alireza; Bocianowski, Jan; Shooshtari, Lia; Bujak, Henryk; Turkoglu, Aras; Nowosad, Kamila
Wild common wheat species represent a significant pool of resistance genes to various environmental stresses. In this study, we examined several physiological traits and the activity of three antioxidant enzymes-namely, catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX)-as well as the expression patterns of their encoding genes in three neglected Aegilops species with alien genomes (including Ae. triuncialis (UUCC-genome), Ae. neglecta (UUMM-genome) and Ae. umbellulata (UU-genome)) under two control (0 mM NaCl) and salinity (250 mM NaCl) conditions. The results of the analysis of variance (ANOVA) showed highly significant effects of salinity stress, accessions, and their interaction on most physio-biochemical traits, root and shoot dry biomasses, and antioxidant-related gene expression level. As a result of comparison between Aegilops species and a bread wheat cultivar (cv. Narin as a salt-tolerant reference variety), Ae. triuncialis responded well to salinity stress, maintaining both ionic homeostasis capability and biochemical ability. Moreover, transcriptional data revealed the prominence of Ae. triuncialis over other Aegilops species and salt-tolerant bread wheat [cv. Narin] in terms of the level of expression of antioxidant genes (APX, SOD, and CAT). This result was further supported by a biplot rendered based on principal component analysis (PCA), where this wild relative showed a positive association with most measured traits under salinity stress. Moreover, we speculate that this accession can be subjected to physiological and molecular studies, and that it can provide new insights into the use of the alien genomes in future wheat breeding programs.
Assessment of genetic diversity and population structure in local alfalfa genotypes using iPBS molecular markers
(Springer, 2023) Eren, Baris; Keskin, Bilal; Demirel, Fatih; Demirel, Serap; Turkoglu, Aras; Yilmaz, Abdurrahim; Haliloglu, Kamil
iPBS retrotransposon markers, in other words, inter-primer binding site markers based on retrotransposon, have been helpful for the determination of genetic diversity in several plants. The study was evaluated using 10 iPBS molecular markers on the level of genetic diversity and genetic structure of alfalfa genotypes. A total of 280 alleles in 50 alfalfa genotypes (48 local genotypes and 2 commercial varieties) were obtained by 267 polymorphic markers with an average of 28 per locus, ranging from 9 to 41 alleles. The rate of polymorphism of the markers ranged from 86.36 to 100% with an average of 93.71%. The average number of polymorphic bands per marker was detected as 26.7. The mean PIC value and Dice's similarity index were calculated as 0.14 and 0.50 respectively. The results of UPGMA analysis, principal coordinate analysis (PCoA), and STRUCTURE demonstrated that the 50 alfalfa genotypes could be classified into 4 subpopulations, namely the Q1, Q2, Q3, and Q4. The Nei's genetic distances ranged from 0.0121 to 0.0359. iPBS markers and alfalfa genotypes used in this study may be used in studies of alfalfa breeding and germplasm conservation.
Determining Genetic Diversity and Population Structure of Common Bean (Phaseolus vulgaris L.) Landraces from Turkiye Using SSR Markers
(Mdpi, 2022) Ozkan, Guller; Haliloglu, Kamil; Turkoglu, Aras; Ozturk, Halil Ibrahim; Elkoca, Erdal; Poczai, Peter
Assessment of genetic diversity among different varieties helps to improve desired characteristics of crops, including disease resistance, early maturity, high yield, and resistance to drought. Molecular markers are one of the most effective tools for discovering genetic diversity that can increase reproductive efficiency. Simple sequence repeats (SSRs), which are codominant markers, are preferred for the determination of genetic diversity because they are highly polymorphic, multi-allelic, highly reproducible, and have good genome coverage. This study aimed to determine the genetic diversity of 40 common bean (Phaseolus vulgaris L.) landraces collected from the Ispir district located in the Northeast Anatolia region of Turkiye and five commercial varieties using SSR markers. The Twenty-seven SSR markers produced a total of 142 polymorphic bands, ranging from 2 (GATS91 and PVTT001) to 12 (BM153) alleles per marker, with an average number of 5.26 alleles. The gene diversity per marker varied between 0.37 and 0.87 for BM053 and BM153 markers, respectively. When heterozygous individuals are calculated proportional to the population, the heterozygosity ranged from 0.00 to 1.00, with an average of 0.30. The expected heterozygosity of the SSR locus ranged from 0.37 (BM053) to 0.88 (BM153), with an average of 0.69. Nei's gene diversity scored an average of 0.69. The polymorphic information content (PIC) values of SSR markers varied from 0.33 (BM053) to 0.86 (BM153), with an average of 0.63 per locus. The greatest genetic distance (0.83) was between lines 49, 50, 53, and cultivar Karacasehir-90, while the shortest (0.08) was between lines 6 and 26. In cluster analysis using Nei's genetic distance, 45 common bean genotypes were divided into three groups and very little relationship was found between the genotypes and the geographical distances. In genetic structure analysis, three subgroups were formed, including local landraces and commercial varieties. The result confirmed that the rich diversity existing in Ispir bean landraces could be used as a genetic resource in designing breeding programs and may also contribute to Turkiye bean breeding programs.
The Effect of Mammalian Sex Hormones on Polymorphism and Genomic Instability in the Common Bean (Phaseolus vulgaris L.)
(Mdpi, 2022) Turkoglu, Aras; Haliloglu, Kamil; Balpinar, Ozge; Ozturk, Halil Ibrahim; Ozkan, Guller; Poczai, Peter
Mammalian sex hormones are steroid-structured compounds that support the growth and development of plants at low concentrations. Since they affect the physiological processes in plants, it has been thought that mammalian sex hormones may cause modifications to plant genomes and epigenetics. This study aims to determine whether different mammalian sex hormones (17 beta-estradiol, estrogen, progesterone, and testosterone) in several concentrations (0, 10(-4), 10(-6), and 10(-8) mM) affect genetic or epigenetic levels in bean plants, using in vitro tissue cultures from plumule explants. We investigated levels of DNA damage, changes in DNA methylation and DNA stability in common bean exposed to mammalian sex hormones (MSH) using inter-primer binding site (iPBS) and Coupled Restriction Enzyme Digestion-iPBS (CRED-iPBS) assays, respectively. The highest rate of polymorphism in iPBS profiles was observed when 10(-4) mM of estrogen (52.2%) hormone was administered. This finding indicates that genetic stability is reduced. In the CRED-iPBS profile, which reveals the methylation level associated with the DNA cytosine nucleotide, 10(-4) mM of estrogen hormone exhibited the highest hypermethylation value. Polymorphism was observed in all hormone administrations compared to the control (without hormone), and it was determined that genomic stability was decreased at high concentrations. Taken together, the results indicate that 17 beta-estradiol, estrogen, progesterone, and testosterone in bean plants affect genomic instability and cause epigenetic modifications, which is an important control mechanism in gene expression.
Effects of mammalian sex hormones on in vitro organogenesis of common bean (Phaseolus vulgaris L.)
(Nature Portfolio, 2023) Haliloglu, Kamil; Turkoglu, Aras; Balpinar, Ozge; Ozturk, Halil Ibrahim; Ozkan, Guller; Poczai, Peter
Beans are an important plant species and are one of the most consumed legumes in human nutrition, especially as a protein, vitamin, mineral, and fiber source. Common bean (Phaseolus vulgaris L.) is a plant that also has an important role in natural nitrogen fixation. Currently, in vitro regeneration and micropropagation applications are limited in relation to genetic factors in bean Accordingly, there is great need to optimize micropropagation and tissue culture methods of the bean plant. To date, the effect of mammalian sex hormones (MSH) on in vitro conditions in P. vulgaris L. is poorly understood. This study examined the effects of different types of explants (embryo, hypocotyl, plumule, and radicle), MSH type (progesterone, 17 beta-estradiol, estrone, and testosterone), and MSH concentration (10(-4), 10(-6), 10(-8) and 10(-10) mmol L-1) on the responding explants induction rate (REI), viability of plantlets rate (VPR), shoot proliferation rate (SPR), root proliferation rate (RPR), and callus induction rate (CIR). The effects of mammalian sex hormones, concentrations, explant type, and their interactions were statistically significant (p <= 0.01) in all examined parameters. The best explants were embryo and plumule. Our results showed that the highest REI rate (100%) was recorded when 10(-10) mmol L-1 of all MSH was applied to MS medium using the plumule explant. The highest VPR (100%) was obtained when 10(-10) mmol L-1 of all MSH was applied to MS medium using the plumule explant. The highest root proliferation rates (77.5%) were recorded in MS medium supplemented with 10(-8) mmol L-1 17 beta-estradiol using embryo explant. The highest percentage of shoot-forming explants (100%) generally was obtained from embryo and plumule cultured in the MS culture medium with low MSH concentration. In addition, the highest CIR (100%) was obtained from embryo and plumule explant cultured in MS medium containing 10(-10) mmol L-1 of all MSH types. In conclusion, we observed that mammalian sex hormones may be used in bean in vitro culture.
Effects of mammalian sex hormones on regeneration capacity, retrotransposon polymorphism and genomic instability in wheat (Triticum aestivum L.)
(Springer, 2023) Turkoglu, Aras
Mammalian steroid hormones (MSH) are also inherently synthesized by several plant species. However, external application of steroid hormones further stimulates cell division, pollen germination, plant growth and development. There is little known about the effects of MSH on polymorphism and DNA damage in Triticum aestivum L under in vitro conditions. In this study, inter-primer binding site (iPBS) retrotransposon and coupled restriction enzyme digestion-iPBS (CRED-iPBS) markers were used for detection of the variation in responded embryogenic callus (REC) that were obtained from endosperm-supported mature embryo of wheat in Murashige and Skoog (MS) medium containing different concentrations [0 (control), 10(-4), 10(-6) and 10(-8) m mol L-1] of MSH (progesterone, 17 beta-estradiol, estrone and testosterone). Responded embryogenic callus (REC) and regenerable callus (RE) induction from mature embryos varied with MSH type and concentrations. The highest level of genomic template stability (GTS) value (80.52%) was obtained from 10(-8) mM progesterone treatments to the lowest value (68.83%) from 10(-4) mM 17 beta-estradiol treatments. Epigenetic changes were more frequent and variable than the genetic changes. While DNA hypermethylation was observed at higher 17 beta-estradiol concentrations, DNA hypomethylation was observed in progesterone treatments. It was concluded based on iPBS and CRED-iPBS findings that high MSH concentrations caused DNA changes and methylation. Key message Variability among regenerates caused by MSH treatments in different concentration were observed. iPBS polymorphisms indicated the presence of genetic variation among the wheat regenerates. Application of MSH in high concentration had a clearly effective on polymorphism and genomic instabilityin the wheat mature embryo.
Effects of Zinc, Copper and Iron Oxide Nanoparticles on Induced DNA Methylation, Genomic Instability and LTR Retrotransposon Polymorphism in Wheat (Triticum aestivum L.)
(Mdpi, 2022) Haliloglu, Kamil; Turkoglu, Aras; Balpinar, Ozge; Nadaroglu, Hayrunnisa; Alayli, Azize; Poczai, Peter
Nanomaterials with unique and diverse physico-chemical properties are used in plant science since they improve plant growth and development and offer protection against biotic and abiotic stressors. Previous studies have explored the effects of such nanomaterials on different plant mechanisms, but information about the effects of nanomaterials on induced DNA methylation, genomic instability and LTR retrotransposon polymorphism in wheat is lacking. Therefore, the present study highlights the key role of nanoparticles in DNA methylation and polymorphism in wheat by investigating the effects of ZnO, CuO and gamma-Fe3O4 nanoparticles (NPs) on mature embryo cultures of wheat (Triticum aestivum L.). Nanoparticles were supplemented with Murashige and Skoog (MS) basal medium at normal (1X), double (2X) and triple (3X) concentrations. The findings revealed different responses to the polymorphism rate depending on the nanoparticle type and concentration. Genomic template stability (GTS) values were used to compare the changes encountered in iPBS profiles. ZnO, CuO and gamma-Fe3O4 NPs increased the polymorphism rate and cytosine methylation compared to the positive control while reducing GTS values. Moreover, non-gamma-Fe3O4 NPs treatments and 2X ZnO and CuO NP treatments yielded higher polymorphism percentages in both MspI-and HpaII-digested CRED-iPBS assays and were thus classified as hypermethylation when the average polymorphism percentage for MspI digestion was considered. On the other hand, the 3X concentrations of all nanoparticles decreased HpaII and MspI polymorphism percentages and were thus classified as hypomethylation. The findings revealed that MS medium supplemented with nanoparticles had epigenetic and genotoxic effects.
Ethyl Methanesulfonate (EMS) Mutagen Toxicity-Induced DNA Damage, Cytosine Methylation Alteration, and iPBS-Retrotransposon Polymorphisms in Wheat (Triticum aestivum L.)
(Mdpi, 2023) Turkoglu, Aras; Haliloglu, Kamil; Tosun, Metin; Bujak, Henryk; Eren, Baris; Demirel, Fatih; Szulc, Piotr
The use of mutagens in plant breeding is used to create new germplasm, increase agricultural yield, quality, and resistance to diseases and pests. Mutagens are physical or chemical factors that can alter the DNA or RNA structure of an organism, causing mutations above the expected level. One of the most common and potent chemical mutagens is EMS (ethyl-methane sulfonate), which produces point mutations in plants, but to a lesser degree can also cause the loss or deletion of a chromosomal region. This study used inter-primer binding site (iPBS) and coupled restriction enzyme digestion inter-primer binding site (CRED-iPBS) technique analysis to determine the effect of EMS mutagens on methylation rates in wheat genotypes at seedling growth stage. Treatments with five different EMS concentrations (0%; control, 0.1%, 0.2%, 0.3%, and 0.4%) at four different times (0; control, 3, 6, and 9 h) were used. Inter-primer binding site (iPBS) markers were employed to assess genomic instability and cytosine methylation in treated wheat. In seeds treated with EMS at different concentrations and times, the disappearance of regular bands and the formation of new bands due to the effects of the EMS mutagen revealed that genetic diversity exists. The CRED-iPBS analysis revealed that the 3 h + 0.1% EMS treatment produced the highest MspI polymorphism value (19.60%), while the 9 h + 0.1% EMS treatment produced the lowest value (10.90%). The mutagenic effects of EMS treatments had considerable polymorphism on a variety of impacts on the cytosine methylation and genomic instability of wheat. According to the current research, EMS mutagenesis may be a practical method for accelerating breeding programs to produce enough genetic diversity in wheat populations. Mutation-assisted breeding and the subsequent selection of desirable mutants using genetic markers may also be carried out in wheat utilizing an integrated strategy.
Evaluation of Sodium Azide as a Chemical Mutagen in Developing Cold-Tolerant Quinoa (Chenopodium quinoa Willd.) Lines
(Springer Int Publ Ag, 2023) Ilhan, Emre; Kasapoglu, Ayse Gul; Turkoglu, Aras; Aygoren, Ahmed Sidar; Muslu, Selman; Aydin, Murat; Aldaif, Muhammed
Quinoa (Chenopodium quinoa Willd.) is an allotetraploid grain crop belonging to the Amaranthaceae family. With a nutritionally well-balanced nature and rich oil, protein, vitamin, carbohydrate, and amino acid content, it has various health benefits. Hybridization programs to induce genetic variation are difficult processes in quinoa plants; however, the introduction of variations through mutation to obtain promising genotypes is much easier. In this research, quinoa cultivar Atlas seeds were exposed to different doses and application durations of sodium azide (NaN3) were cultivated in pots. The present findings revealed distinctive differences in all studied traits between native and mutant plants. NaN3 mutagenesis caused genetic variations that were categorized based on studied traits and RAPD data, which led to two distinct groups during M-2. It was noted that 2 mM + 3 h NaN3 treatments had the greatest effects on genetic variations and germination parameters. Present findings were also confirmed by molecular analysis. This research will provide new insights into molecular breeding programs to be conducted to improve present quinoa genotypes and produce better genotypes.
Exploring genetic diversity and Population structure of five Aegilops species with inter-primer binding site (iPBS) markers
(Springer, 2022) Kizilgeci, Ferhat; Bayhan, Bora; Turkoglu, Aras; Haliloglu, Kamil; Yildirim, Mehmet
Background Turkey is not only a center of origin for wheat, but also contains wild forms of various cereals. Turkey, located in the Fertile Crescent, has conserved its genetic richness to the present day. The aim of the study was to investigate the genetic diversity of 70 wild wheat species, to evaluate the structure of diversity in germplasm and to generate useful data for further breeding programs. Methods and results Genetic diversity and population structure of 70 wild wheat species (Ae. cylindrica, Ae. geniculata, Ae. triuncialis, T. dicocoides, Ae. columnaris) collected from Eastern and Southeastern Anatolia regions of Turkey were investigated in this study with the use of inter-primer binding site (iPBS) markers. Of 35 iPBS primers used, 11 yielded a total of 61 alleles. Number of alleles per marker varied between 2 (iPBS-2085) and 9 (iPBS-2394) with an average value of 5.55. Polymorphic information content (PIC) values varied between 0.22 and 0.47, with an average value of 0.35. Average number of effective alleles (Ne) was identified as 1.9488, Nei's genetic diversity (H) as 0,4861 and Shannon's information index (I) as 0.6791. Cluster analysis through unweighted pair-group mean average (UPGMA) method revealed that 70 wild wheats were divided into three main clusters. Genetic similarity between the genotypes, calculated with the use of NTSYS-pc software, varied between 19% (YB2 and YB70) and 98% (YB66 and YB67). Principal coordinate analysis (PCoA) revealed that three principal coordinates explained 62.33% of total variation. Moreover, population structure analysis showed that all genotypes formed three sub-populations. Expected heterozygosity values varied between 0.2666 (the first sub-population) and 0.2330 (third sub-population), with an average value of 0.2500. Average population differentiation measurement (Fst) was identified as 0.3716 for the first sub-population, 0.3930 for the second subpopulation and 0.4804 for the third sub-population. Conclusions Based on present findings population structure of 70 wild wheat genotypes collected from Eastern and Southeastern Anatolia regions of Turkey were successfully characterized with the use of iPBS markers. Present findings suggested that iPBS-retrotransposon markers could reliably be used to elucidate genetic diversity of Aegilops genotypes.
Exploring the genetic diversity and population structure of fenugreek (Trigonella foenum-graecum L.) genotypes through inter-primer binding site (iPBS)-retrotransposon marker system
(Springer, 2024) Haliloglu, Kamil; Ozer, Hakan; Melik, Seda; Coban, Furkan; Turkoglu, Aras
Fenugreek, a key medicinal-aromatic plant, offers rich bioactive compounds and nutritional value. Its diverse applications in cuisine and pharmaceuticals, coupled with health benefits like anti-diabetic and antioxidant properties, underscore its significance. Assessing genetic diversity becomes crucial for effective conservation and utilization. In this study, we examined the molecular diversity and population structure of 34 fenugreek genotypes collected from 18 countries worldwide using 24 inter-primer binding site (iPBS) markers. The iPBS primers produced 499 bands, with the total number of bands per primer ranging from 15 (iPBS-2224) to 26 (iPBS-2077), averaging 20.79. Polymorphism information content (PIC) ranged from 0.03 (iPBS-2374) to 0.34 (iPBS-2237), averaging 0.23. In the molecular analysis, the G1 genotype (Isfahan/Iran) exhibited the maximum effective number of alleles (Ne), Nie's gene diversity (He), and Shannon's information index (I) at 1.946, 0.486, and 0.679, respectively. Conversely, the G34 genotype (India/B) displayed the lowest values at 1.539, 0.350, and 0.535, respectively. Utilizing the unweighted pair-group means average (UPGMA) method, the iPBS-based tree revealed three distinct groups corresponding to the genomic constitution of fenugreek genotypes, a pattern partially corroborated by principal component analysis (PCA). Further model-based cluster analysis classified the 34 genotypes into four subpopulations, with expected heterozygosity (He) values of 0.428, 0.390, 0.426, and 0.007, respectively. The F-statistic (Fst) values for these subpopulations were 0.197, 0.210, 0.187, and 0.356, respectively. These findings underscored significant genetic variation among the tested fenugreek genotypes, thereby demonstrating the efficacy of iPBS markers in accurately assessing genetic diversity and phylogenetic relationships within fenugreek populations.
Genetic Diversity and Population Structure in Turkiye Bread Wheat Genotypes Revealed by Simple Sequence Repeats (SSR) Markers
(Mdpi, 2023) Turkoglu, Aras; Haliloglu, Kamil; Mohammadi, Seyyed Abolgahasem; Ozturk, Ali; Bolouri, Parisa; Ozkan, Guller; Bocianowski, Jan
Wheat genotypes should be improved through available germplasm genetic diversity to ensure food security. This study investigated the molecular diversity and population structure of a set of Turkiye bread wheat genotypes using 120 microsatellite markers. Based on the results, 651 polymorphic alleles were evaluated to determine genetic diversity and population structure. The number of alleles ranged from 2 to 19, with an average of 5.44 alleles per locus. Polymorphic information content (PIC) ranged from 0.031 to 0.915 with a mean of 0.43. In addition, the gene diversity index ranged from 0.03 to 0.92 with an average of 0.46. The expected heterozygosity ranged from 0.00 to 0.359 with a mean of 0.124. The unbiased expected heterozygosity ranged from 0.00 to 0.319 with an average of 0.112. The mean values of the number of effective alleles (Ne), genetic diversity of Nei (H) and Shannon's information index (I) were estimated at 1.190, 1.049 and 0.168, respectively. The highest genetic diversity (GD) was estimated between genotypes G1 and G27. In the UPGMA dendrogram, the 63 genotypes were grouped into three clusters. The three main coordinates were able to explain 12.64, 6.38 and 4.90% of genetic diversity, respectively. AMOVA revealed diversity within populations at 78% and between populations at 22%. The current populations were found to be highly structured. Model-based cluster analyses classified the 63 genotypes studied into three subpopulations. The values of F-statistic (Fst) for the identified subpopulations were 0.253, 0.330 and 0.244, respectively. In addition, the expected values of heterozygosity (He) for these sub-populations were recorded as 0.45, 0.46 and 0.44, respectively. Therefore, SSR markers can be useful not only in genetic diversity and association analysis of wheat but also in its germplasm for various agronomic traits or mechanisms of tolerance to environmental stresses.
Genotype-Trait (GT) Biplot Analysis for Yield and Quality Stability in Some Sweet Corn (Zea mays L. saccharata Sturt.) Genotypes
(Mdpi, 2023) Stansluos, Atom Atanasio Ladu; Ozturk, Ali; Niedbala, Gniewko; Turkoglu, Aras; Haliloglu, Kamil; Szulc, Piotr; Omrani, Ali
A strong statistical method for investigating the correlations between traits, assessing genotypes based on numerous traits, and finding individuals who excel in particular traits is genotype-trait (GT) biplot analysis. The current study was applied to evaluate 11 sweet corn (Zea mays L. saccharata) genotypes and correlate them based on genotype-trait (GT) biplot analysis for two cropping seasons in Erzurum, Turkiye using the RCBD experimental design with three reputations. The results showed that the genotypes were significantly different for the majority of the examined variables according to the combined analysis of variance findings at 0.01 probability level. An ecological analysis was performed to evaluate sweet corn varieties and environmental conditions and interactions between them (genotype x environmental conditions). Our results showed that the summation of the first two and second main components was responsible for 73.51% of the combined cropping years of the sweet corn growth and development variance, demonstrating the biplot graph's optimum relative validity, which was obtained. In this study, the Khan F1 (G6) genotype was found to be the stablest genotype, and the Kompozit Seker (G7) genotype was the non-stable genotype, moreover based on the first cropping year, second cropping year, and the average mean of the two cropping years. As a conclusion, the Khan F1 (G6) genotype is the highest-yielding genotype, and the Kompozit Seker (G7) is the lowest. Based on the heat map dendrogram, the context of the differential extent of trait association of all genotypes into two clusters is indicated. The highest genetic distance was shown between the BATEM Tatli (G3) and Febris (G5) genotypes. Our results provide helpful information about the sweet corn genotypes and environments for future breeding programs.
Identification of Novel QTLs Associated with Frost Tolerance in Winter Wheat (Triticum aestivum L.)
(Mdpi, 2023) Bolouri, Parisa; Haliloglu, Kamil; Mohammadi, Seyyed Abolghasem; Turkoglu, Aras; Ilhan, Emre; Niedbala, Gniewko; Szulc, Piotr
Low temperature (cold) and freezing stress is a major problem during winter wheat growth. Low temperature tolerance (LT) is an important agronomic trait in winter wheat and determines the plants' ability to cope with below-freezing temperatures; thus, the development of cold-tolerant cultivars has become a major goal of breeding in various regions of the world. In this study, we sought to identify quantitative trait loci (QTL) using molecular markers related to freezing tolerance in winter. Thirty-four polymorphic markers among 425 SSR markers were obtained for the population, including 180 inbred lines of F-12 generation wheat, derived from crosses (Norstar x Zagros) after testing with parents. LT50 is used as an effective selection criterion for identifying frost-tolerance genotypes. The progeny of individual F-12 plants were used to evaluate LT50. Several QTLs related to wheat yield, including heading time period, 1000-seed weight, and number of surviving plants after overwintering, were identified. Single-marker analysis illustrated that four SSR markers with a total of 25% phenotypic variance determination were linked to LT50. Related QTLs were located on chromosomes 4A, 2B, and 3B. Common QTLs identified in two cropping seasons based on agronomical traits were two QTLs for heading time period, one QTL for 1000-seed weight, and six QTLs for number of surviving plants after overwintering. The four markers identified linked to LT50 significantly affected both LT50 and yield-related traits simultaneously. This is the first report to identify a major-effect QTL related to frost tolerance on chromosome 4A by the marker XGWM160. It is possible that some QTLs are closely related to pleiotropic effects that control two or more traits simultaneously, and this feature can be used as a factor to select frost-resistant lines in plant breeding programs.
Improvement of Drought Tolerance by Exogenous Spermidine in Germinating Wheat (Triticum aestivum L.) Plants Is Accompanied with Changes in Metabolite Composition
(Mdpi, 2022) Gholizadeh, Fatemeh; Janda, Tibor; Gondor, Orsolya Kinga; Pal, Magda; Szalai, Gabriella; Sadeghi, Amirali; Turkoglu, Aras
Drought is one of the most important environmental factors reducing the yield and production of crops, including wheat. Polyamines are closely associated with plant stress tolerance. The present study investigated the mechanisms through seed germination with spermidine protecting wheat varieties from drought stress. In the first experiment, the effects of spermidine on the germination of wheat varieties, namely Rakhshan, Mihan, Sirvan and Pishgam, were investigated in three drought levels, namely 0, -2, and -4 MPa induced by polyethylene glycol 6000. Analysis of variance indicated that spermidine, drought stress and interaction between varieties and drought stress were significant for all traits, and with severity of stress, all traits significantly decreased. In the second experiment, detailed gene expression and non-targeted metabolomics analyses were carried out using the Rakhshan and Mihan varieties after germination, with or without spermidine treatment and/or drought stress. According to the biomass parameters, the Mihan variety showed relatively better growth compared to the other variety, but the Rakhshan one showed more pronounced responses at gene expression level to exogenous spermidine than the Mihan variety. Overall, these results showed that spermidine increased the drought tolerance of wheat at the germination stage, due to specific role of polyamine metabolism in the development of effective responses under drought stress.
Investigation of the Influence of Polyamines on Mature Embryo Culture and DNA Methylation of Wheat (Triticum aestivum L.) Using the Machine Learning Algorithm Method
(Mdpi, 2023) Eren, Baris; Turkoglu, Aras; Haliloglu, Kamil; Demirel, Fatih; Nowosad, Kamila; Ozkan, Guller; Niedbala, Gniewko
Numerous factors can impact the efficiency of callus formation and in vitro regeneration in wheat cultures through the introduction of exogenous polyamines (PAs). The present study aimed to investigate in vitro plant regeneration and DNA methylation patterns utilizing the inter-primer binding site (iPBS) retrotransposon and coupled restriction enzyme digestion-iPBS (CRED-iPBS) methods in wheat. This investigation involved the application of distinct types of PAs (Put: putrescine, Spd: spermidine, and Spm: spermine) at varying concentrations (0, 0.5, 1, and 1.5 mM). The subsequent outcomes were subjected to predictive modeling using diverse machine learning (ML) algorithms. Based on the specific polyamine type and concentration utilized, the results indicated that 1 mM Put and Spd were the most favorable PAs for supporting endosperm-associated mature embryos. Employing an epigenetic approach, Put at concentrations of 0.5 and 1.5 mM exhibited the highest levels of genomic template stability (GTS) (73.9%). Elevated Spd levels correlated with DNA hypermethylation while reduced Spm levels were linked to DNA hypomethylation. The in vitro and epigenetic characteristics were predicted using ML techniques such as the support vector machine (SVM), extreme gradient boosting (XGBoost), and random forest (RF) models. These models were employed to establish relationships between input variables (PAs, concentration, GTS rates, Msp I polymorphism, and Hpa II polymorphism) and output parameters (in vitro measurements). This comparative analysis aimed to evaluate the performance of the models and interpret the generated data. The outcomes demonstrated that the XGBoost method exhibited the highest performance scores for callus induction (CI%), regeneration efficiency (RE), and the number of plantlets (NP), with R-2 scores explaining 38.3%, 73.8%, and 85.3% of the variances, respectively. Additionally, the RF algorithm explained 41.5% of the total variance and showcased superior efficacy in terms of embryogenic callus induction (ECI%). Furthermore, the SVM model, which provided the most robust statistics for responding embryogenic calluses (RECs%), yielded an R-2 value of 84.1%, signifying its ability to account for a substantial portion of the total variance present in the data. In summary, this study exemplifies the application of diverse ML models to the cultivation of mature wheat embryos in the presence of various exogenous PAs and concentrations. Additionally, it explores the impact of polymorphic variations in the CRED-iPBS profile and DNA methylation on epigenetic changes, thereby contributing to a comprehensive understanding of these regulatory mechanisms.
iPBS-Retrotransposon Markers in the Analysis of Genetic Diversity among Common Bean (Phaseolus vulgaris L.) Germplasm from Turkiye
(Mdpi, 2022) Haliloglu, Kamil; Turkoglu, Aras; Ozturk, Halil Ibrahim; Ozkan, Guller; Elkoca, Erdal; Poczai, Peter
Beans are legumes that play extremely important roles in human nutrition, serving as good sources of protein, vitamins, minerals, and antioxidants. In this study, we tried to elucidate the genetic diversity and population structure of 40 Turkish bean (Phaseolus vulgaris L.) local varieties and 5 commercial cultivars collected from 8 different locations in Erzurum-Ispir by using inter-primary binding site (iPBS) retrotransposon markers. For molecular characterization, the 26 most polymorphic iPBS primers were used; 52 bands per primer and 1350 bands in total were recorded. The mean polymorphism information content was 0.331. Various diversity indices, such as the mean effective allele number (0.706), mean Shannon's information index (0.546), and gene diversity (0.361) revealed the presence of sufficient genetic diversity in the germplasm examined. Molecular analysis of variance (AMOVA) revealed that 67% of variation in bean germplasm was due to differences within populations. In addition, population structure analysis exposed all local and commercial bean varieties from five sub-populations. Expected heterozygosity values ranged between 0.1567 (the fourth sub-population) and 0.3210 (first sub-population), with an average value of 0.2103. In contrary, population differentiation measurement (Fst) was identified as 0.0062 for the first sub-population, 0.6372 for the fourth subpopulations. This is the first study to investigate the genetic diversity and population structure of bean germplasm in Erzurum-Ispir region using the iPBS-retrotransposon marker system. Overall, the current results showed that iPBS markers could be used consistently to elucidate the genetic diversity of local and commercial bean varieties and potentially be included in future studies examining diversity in a larger collection of local and commercial bean varieties from different regions.
Machine Learning Analysis of the Impact of Silver Nitrate and Silver Nanoparticles on Wheat (Triticum aestivum L.): Callus Induction, Plant Regeneration, and DNA Methylation
(Mdpi, 2023) Turkoglu, Aras; Haliloglu, Kamil; Demirel, Fatih; Aydin, Murat; Cicek, Semra; Yigider, Esma; Demirel, Serap
The objective of this study was to comprehend the efficiency of wheat regeneration, callus induction, and DNA methylation through the application of mathematical frameworks and artificial intelligence (AI)-based models. This research aimed to explore the impact of treatments with AgNO3 and Ag-NPs on various parameters. The study specifically concentrated on analyzing RAPD profiles and modeling regeneration parameters. The treatments and molecular findings served as input variables in the modeling process. It included the use of AgNO3 and Ag-NPs at different concentrations (0, 2, 4, 6, and 8 mg L-1). The in vitro and epigenetic characteristics were analyzed using several machine learning (ML) methods, including support vector machine (SVM), random forest (RF), extreme gradient boosting (XGBoost), k-nearest neighbor classifier (KNN), and Gaussian processes classifier (GP) methods. This study's results revealed that the highest values for callus induction (CI%) and embryogenic callus induction (EC%) occurred at a concentration of 2 mg L-1 of Ag-NPs. Additionally, the regeneration efficiency (RE) parameter reached its peak at a concentration of 8 mg L-1 of AgNO3. Taking an epigenetic approach, AgNO3 at a concentration of 2 mg L-1 demonstrated the highest levels of genomic template stability (GTS), at 79.3%. There was a positive correlation seen between increased levels of AgNO3 and DNA hypermethylation. Conversely, elevated levels of Ag-NPs were associated with DNA hypomethylation. The models were used to estimate the relationships between the input elements, including treatments, concentration, GTS rates, and Msp I and Hpa II polymorphism, and the in vitro output parameters. The findings suggested that the XGBoost model exhibited superior performance scores for callus induction (CI), as evidenced by an R-2 score of 51.5%, which explained the variances. Additionally, the RF model explained 71.9% of the total variance and showed superior efficacy in terms of EC%. Furthermore, the GP model, which provided the most robust statistics for RE, yielded an R-2 value of 52.5%, signifying its ability to account for a substantial portion of the total variance present in the data. This study exemplifies the application of various machine learning models in the cultivation of mature wheat embryos under the influence of treatments and concentrations involving AgNO3 and Ag-NPs.
Mammalian Sex Hormones as Steroid-Structured Compounds in Wheat Seedling: Template of the Cytosine Methylation Alteration and Retrotransposon Polymorphisms with iPBS and CRED-iBPS Techniques
(Mdpi, 2023) Demirel, Fatih; Turkoglu, Aras; Haliloglu, Kamil; Eren, Baris; Ozkan, Guller; Uysal, Pinar; Pour-Aboughadareh, Alireza
Phytohormones are chemical compounds found naturally in plants that have a significant effect on their growth and development. The increase in research on the occurrence of mammalian sex hormones (MSHs) in plants has prompted the need to investigate the functions performed by these hormones in plant biology. In the present study, we investigated the effects of MSHs on DNA damage and DNA methylation of wheat (Triticum aestivum L.) during the seedling growth stage, using the CRED-iPBS (coupled restriction enzyme digestion/inter primer binding site) assay and iPBS analysis to determine DNA methylation status. Exogenous treatment with four MSHs (17-& beta;-estradiol, estrogen, progesterone, and testosterone) was carried out at four different concentrations (0, 0.05, 0.5, and 5 & mu;M). The highest genomic template stability (GTS) value (80%) was observed for 5 & mu;M 17-& beta;-estradiol, 0.5 & mu;M testosterone, and 0.05 & mu;M estrogen, while the lowest value (70.7%) was observed for 5 & mu;M progesterone and 0.5 & mu;M estrogen. The results of the CRED-iPBS analysis conducted on MspI indicate that the 0.05 & mu;M estrogen-treated group had the highest polymorphism value of 40%, while the 5 & mu;M progesterone-treated group had the lowest value of 20%. For HpaII, treatment with 0.5 & mu;M 17-& beta;-estradiol had the highest polymorphism value of 33.3%, while the group treated with 0.05 & mu;M 17-& beta;-estradiol and 0.05 & mu;M progesterone had the lowest value of 19.4%. In conclusion, MSH treatments altered the stability of the genomic template of wheat plants and affected the cytosine methylation status at the seedling growth stage. Upon comprehensive examination of the results, it was seen that the employed methodology successfully detected alterations in cytosine methylation of genomic DNA (gDNA), as well as changes in the pattern of genomic instability.
Modeling Callus Induction and Regeneration in Hypocotyl Explant of Fodder Pea (Pisum sativum var. arvense L.) Using Machine Learning Algorithm Method
(Mdpi, 2023) Turkoglu, Aras; Bolouri, Parisa; Haliloglu, Kamil; Eren, Baris; Demirel, Fatih; Isik, Muhammet Islam; Piekutowska, Magdalena
A comprehensive understanding of genetic diversity and the categorization of germplasm is important to effectively identify appropriate parental candidates for the goal of breeding. It is necessary to have a technique of tissue culture that is both effective and reproducible to perform genetic engineering on fodder pea genotypes (Pisum sativum var. arvense L.). In this investigation, the genetic diversity of forty-two fodder pea genotypes was assessed based on their ability of callus induction (CI), the percentage of embryogenic callus by explant number (ECNEP), the percentage of responding embryogenic calluses by explant number (RECNEP), the number of somatic embryogenesis (NSE), the number of responding somatic embryogenesis (RSE), the regeneration efficiency (RE), and the number of regenerated plantlets (NRP). The findings of the ANOVA showed that there were significant differences (p < 0.001) between the genotypes for all in vitro parameters. The method of principal component analysis (PCA) was used to study the correlations that exist between the factors associated with tissue culture. While RE and NRP variables were most strongly associated with Do & gbreve;ruyol, Ova & ccedil;evirme-4, Do & scedil;eli-1, Yolge & ccedil;mez, and Incili-3 genotypes, RECNEP, NSE, RDE, and RECNEP variables were strongly associated with Avc & imath;lar, Ova & ccedil;evirme-3, and Ardahan Merkez-2 genotypes. The in vitro process is a complex multivariate process and more robust analyses are needed for linear and nonlinear parameters. Within the scope of this study, artificial neural network (ANN), random forest (RF), and multivariate adaptive regression spline (MARS) algorithms were used for RE estimation, and these algorithms were also compared. The results that we acquired from our research led us to the conclusion that the employed ANN-multilayer perceptron (ANN-MLP) model (R-2 = 0.941) performs better than the RF model (R-2 = 0.754) and the MARS model (R-2 = 0.214). Despite this, it has been shown that the RF model is capable of accurately predicting RE in the early stages of the in vitro process. The current work is an inquiry regarding the use of RF, MARS, and ANN models in plant tissue culture, and it indicates the possibilities of application in a variety of economically important fodder peas.

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