Investigation of the Influence of Polyamines on Mature Embryo Culture and DNA Methylation of Wheat (Triticum aestivum L.) Using the Machine Learning Algorithm Method.

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 R2 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 R2 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.

Use of retrotransposon based iPBS markers for determination of genetic relationship among some Chestnut Cultivars (Castanea sativa Mill.) in Türkiye.

BACKGROUND: The aim of this study was to reveal the genetic relationships among some economically important chestnut cultivars for Türkiye by using retrotransposon-based inter primer binding site (iPBS) markers. METHODS AND RESULTS: In this study, a total of 19 iPBS markers were used to determine the genetic relationships among 11 chestnut cultivars (Castanea sativa Mill.). In the study, chestnut cultivars named Haciömer, Osmanoglu, Sariaslama, Erfelek, Kemer, Isiklar, Sekerci, Siyah Bursa, Tülü, Bouche De Betizac and Marigoule were the preferred cultivars utilised. Using the online marker efficiency calculator (iMEC), some indices of polymorphism, such as the mean heterozygosity, polymorphism information content, marker index and discriminating power, were determined. In addition, the size ranges of alleles, number of average alleles, number of total alleles, number of polymorphic alleles, and polymorphism rate were determined at a successful level. The chestnut cultivars of Haciömer and Sekerci were determined to be the most similar cultivars with a similarity coefficient value of 0.924, and they formed a subgroup together with the chestnut cultivars Osmanoglu and Erfelek, showing close similarity with these two cultivars. CONCLUSIONS: The use of iPBS markers in chestnuts in Türkiye was carried out for the first time in this study. The power of iPBS markers to evaluate the genetic relationship for our preferred chestnut cultivars was revealed. For this reason, it has emerged that it will be useful in the molecular characterization of both genotypes in natural chestnut populations and chestnut breeding materials such as varieties and cultivars in chestnut breeding programs.

Exploring the genetic diversity and population structure of upland cotton germplasm by iPBS-retrotransposons markers.

BACKGROUND: Upland cotton is one of the utmost significant strategic fiber crops, and play a vital role in the global textile industry. METHODS AND RESULTS: A total of 128 genotypes comprised Gossypium hirsutum L, Gossypium barbadense L., and pure lines were used to examine genetic diversity using iPBS-retrotransposon markers system. Eleven highly polymorphic primers yielded 287 bands and 99.65% polymorphism was recorded. The mean polymorphism information content was estimated at 0.297 and the average diversity indices for the effective number of alleles, Shannon's information index, and overall gene diversity were 1.481, 0.443, and 0.265, respectively. The analysis of molecular variance (AMOVA) revealed that 69% of the genetic variation was within the population. A model-based STRUCTURE algorithm divided the entire germplasm into four populations and one un-classified population, the genotypes G42 (originating in Egypt) and G128 (originating in the United States), showed the highest genetic distance (0.996) so these genotypes could be suggested for breeding programs as parental lines. CONCLUSIONS: This is the first investigation using an iPBS-retrotransposon marker system to examine the genetic diversity and population structure of upland cotton germplasm. The rich diversity found in upland cotton germplasm could be exploited as a genetic resource when developing breeding programs and could also help with efforts to breed cotton around the world. These findings also show the applicability and effectiveness of iPBS-retrotransposons for the molecular characterization of cotton germplasm.

The comparison of polymorphism among Avena species revealed by retrotransposon-based DNA markers and soluble carbohydrates in seeds.

Here, we compared the polymorphism among 13 Avena species revealed by the iPBS markers and soluble carbohydrate profiles in seeds. The application of seven iPBS markers generated 83 bands, out of which 20.5% were polymorphic. No species-specific bands were scored. Shannon's information index (I) and expected heterozygosity (He) revealed low genetic diversity, with the highest values observed for A. nuda (I = 0.099; He = 0.068). UPGMA clustering of studied Avena accessions and PCoA results showed that the polyploidy level is the main grouping criterion. High-resolution gas chromatography revealed that the studied Avena accessions share the same composition of soluble carbohydrates, but significant differences in the content of total (5.30-22.38 mg g-1 of dry weight) and particular sugars among studied samples were observed. Sucrose appeared as the most abundant sugar (mean 61.52% of total soluble carbohydrates), followed by raffinose family oligosaccharides (31.23%), myo-inositol and its galactosides (6.16%), and monosaccharides (1.09%). The pattern of interspecific variation in soluble carbohydrates, showed by PCA, was convergent to that revealed by iPBS markers. Thus, both methods appeared as a source of valuable data useful in the characterization of Avena resources or in the discussion on the evolution of this genus.

Genetic variation of Cerastium alpinum L. from Babia Góra, a critically endangered species in Poland.

Babia Góra massif is the only site of occurrence of the Cerastium alpinum L. in Poland, an arctic-alpine perennial plant with a wide distribution in North America, northwestern Asia, and Europe. To determine whether the isolated Polish populations are genetically distinct, we have performed an evaluation of C. alpinum from Babia Góra with the use of iPBS markers. A total number of 133 individuals of C. alpinum from seven populations representing four localizations of the species were analyzed, i.e., from Babia Góra (Poland), Alps (Switzerland), Nuolja massif (Sweden), and Kaffiøyra (Svalbard, Norway). Genetic analysis of all C. alpinum samples using eight PBS primers identified 262 bands, 79.4% of which were polymorphic. iPBS markers revealed low genetic diversity (average He = 0.085) and high population differentiation (FST = 0.617). AMOVA results confirmed that the majority of the genetic variation (62%) was recorded among populations. The grouping revealed by PCoA showed that C. alpinum from Svalbard is the most diverged population, C. alpinum from Switzerland and Sweden form a pair of similar populations, whereas C. alpinum from the Babia Góra form a heterogeneous group of four populations. Results of isolation by distance analysis suggested that the spatial distance is the most probable cause of the observed differentiation among populations. Although significant traces of a bottleneck effect were noted for all populations of C. alpinum from Babia Góra, the populations still maintain a low but significant level of genetic polymorphism. These results are of great importance for developing conservation strategies for this species in Poland.

Genetic diversity and population structure of Saccharomyces cerevisiae isolated from Turkish sourdough by iPBS-retrotransposons markers.

Molecular DNA markers are valuable tools for analyzing genetic variation among yeast from different populations to reveal the genetically different autochthonous strains. In this study, we employed inter-primer binding site (iPBS) retrotransposon polymorphism to assess the genetic variation and population structure of 96 Saccharomyces cerevisiae isolates from four different regions in Turkey. The nine selected iPBS primers amplified 102 reproducible and scorable bands, of which 95.10% were polymorphic with an average of 10.78 polymorphic fragments per primer. The average polymorphism information content and the resolving power were 0.26-3.58, respectively. Analysis of molecular variance (AMOVA) revealed significant (P < 0.001) genetic differences within populations (88%) and between populations (12%). The unweighted pair group mean with arithmetic (UPGMA) dendrogram grouped 96 S. cerevisiae strains into two main clusters, where the highest probability of the data elucidating the population structure was obtained at ΔK = 2. There was not an obvious genetic discrimination of the populations according to geographical regions on UPGMA, supported by principal coordinate analysis. However, the individuals of the closer provinces in each population were more likely to group together or closely. The results indicate that iPBS polymorphism is a useful tool to reveal the genetically diverse autochthonous S. cerevisiae strains that may be important for the production of sourdough or baked goods.

Molecular Characterization of Native Entomopathogenic Fungi from Ambrosia Beetles in Hazelnut Orchards of Turkey and Evaluation of Their In Vitro Efficacy.

Ambrosia beetles, Anisandrus dispar Fabricius, Xylosandrus germanus Blandford, and Xyleborinus saxesenii Ratzeburg (Coleoptera: Curculionidae: Scolytinae) are among the most significant hazelnut pests in Turkey. The control of these pests is difficult and expensive due to their biology. The present study aimed to isolate entomopathogenic fungi (EPF) from A. dispar, X. germanus, and X. saxesenii individuals that were obtained from the main hazelnut production areas of Turkey, characterize the EPF isolates using internal transcribed spacer (ITS)-DNA sequencing and iPBS profiling, and determine the efficacy of the isolates against A. dispar, X. germanus, and X. saxesenii under laboratory conditions. Phylogenetic analyses based on ITS revealed that the 47 native isolates were Beauveria bassiana (11), B. pseudobassiana (8), Cordyceps fumosorosea (6), Cordyceps farinosa (1), Akanthomyces lecanii (13), Purpureocillium lilacinum (3), Clonostachys rosea (2) and Metarhizium anisopliae (3). For the first time, the primer binding site (PBS) marker system, based on retrotransposons, was used to discriminate successfully among the EPF species. Some isolates of B. bassiana, B. pseudobassiana, C. fumosorosea, A. lecanii, and M. anisopliae caused 100% mortality of the beetle species within 7 to 9 days. The findings of this study indicated that some isolated entomopathogenic fungi provide an essential basis for the development of bioproducts, as well as a promising alternative method for controlling these ambrosia beetles.

Mutagenic effects of sodium azide on in vitro mutagenesis, polymorphism and genomic instability in wheat (Triticum aestivum L.).

INTRODUCTION: Breeding studies are commonly conducted to develop new cultivars with high yield levels and improved quality traits. Chemically-induced mutations are used to create genetic variations in wheat genomes. Various physical and chemical mutagens are used to increase frequency of mutations and facilitate the selection processes. Sodium azide (SA) is largely employed to induce mutations of the genes regulating essential traits. Such mutations may also elucidate gene functions of the mutant phenotypes. Present experiments were conducted to investigate potential use of conventional chemical mutagenesis technique through SA for mature embryo culture in wheat. METHODS AND RESULTS: Sodium azide mutagenesis was experimented with 4 treatment durations (1, 2, 3 and 4 h) and 5 treatment concentrations (0, 1, 2, 3 and 4 mM). Mature embryos were subjected to experimental treatments to detect optimum doses of mutagenesis and to estimate polymorphism and genomic instability. Primarily, 50% reduction in number of regenerated plants as compared to the control (LD50) was adopted as the optimum dose. Based on LD50 criterion, the optimum value was achieved at 1 h duration of 4 mM SA concentration. Afterwards, inter-primer binding site markers were applied to investigate polymorphism and genomic instability in the regenerated plants. CONCLUSIONS: Present findings revealed that efficiency of chemical mutagenesis could be improved through the use of molecular technology and such mutations may assist plant breeders in developing high-yield cultivars.

The Effect of Mammalian Sex Hormones on Polymorphism and Genomic Instability in the Common Bean (Phaseolus vulgaris L.).

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 ß-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 ß-estradiol, estrogen, progesterone, and testosterone in bean plants affect genomic instability and cause epigenetic modifications, which is an important control mechanism in gene expression.

Exploring genetic diversity and Population structure of five Aegilops species with inter-primer binding site (iPBS) markers.

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.

Applicability of inter-primer binding site iPBS- retrotransposon marker system for the assessment of genetic diversity and population structure of Peruvian rosewood (Aniba rosaeodora Ducke) germplasm.

BACKGROUND: Rosewood (Aniba rosaeodora Ducke), which has a great demand due to its essential oil globally, is an evergreen tree of the Amazon forests. Rosewood natural stands have been depleted through deforestation and the destruction of habitat. Currently, rosewood is included in the ICUN red list of endangered species. METHODS AND RESULTS: The 11 highly polymorphic primers amplified total 305 bands of which 301 (98.69%) were polymorphic. The number of effective alleles (Ne), Shannon's information index (I), overall gene diversity (Ht), gene diversity (h), and polymorphism information content (PIC) were (1.562), (0.505), (0.330), (0.337) and (0.343), respectively. These diversity indices explored high genetic diversity in rosewood germplasm. Among studied germplasm, the Santa Marta population was found most diverse by reflecting higher values of diversity indices while the Zungarococha population was found least diverse. The analysis of molecular variance (AMOVA) revealed that 79% of the genetic variation was within the populations. The STRUCTURE algorithm, unweighted pair group with arithmetic mean (UPGMA), and principal coordinate's analysis (PCoA) separated all germplasms into different population groups according to their geographic locations. Santa Marta population was found more diverse by reflecting higher values of diversity indices. The maximum genetic distance (0.868) was found between the Huajoya-10 and Nanay-3. In this investigation, iPBS- retrotransposon marker system was used to explore the genetic diversity of Peruvian rosewood germplasm. CONCLUSIONS: The results in this study such as higher genetic diversity indices, AMOVA (79%) within population and PIC value (0.343) showed the utility and reproducibility of iPBS-retrotransposons in this species successfully. The STRUCTURE algorithm separated the germplasms into six population groups according to their geographic locations. These results have valuable information for the conservation, management strategies and future breeding activities of rosewood.

Genetic analysis and population structure of wild and cultivated wishbone flower (Torenia fournieri Lind.) lines related to specific floral color.

BACKGROUND: The wishbone flower or Torenia fournieri Lind., an annual from tropical Indochina and southern China, is a popular ornamental plant, and many interspecific (T. fournieri × T. concolor) hybrid lines have been bred for the international market. The cultivated lines show a pattern of genetic similarity that correlates with floral color which informs on future breeding strategies. This study aimed to perform genetic analysis and population structure of cultivated hybrid lines comparing with closely related T. concolor wild populations. METHODS: We applied the retrotransposon based iPBS marker system for genotyping of a total of 136 accessions from 17 lines/populations of Torenia. These included 15 cultivated lines of three series: Duchess (A, B, C); Kauai (D, E, F, G, H, I, J); Little Kiss (K, L, M, N, P) and two wild T. concolor populations (Q and R). PCR products from each individual were applied to estimate the genetic diversity and differentiation between lines/populations. RESULTS: Genotyping results showed a pattern of genetic variation differentiating the 17 lines/populations characterized by their specific floral colors. The final PCoA analysis, phylogenetic tree construction, and Bayesian population structural bar plot all showed a clear subdivision of lines/populations analysed. The 15 cultivated hybrid lines and the wild population Q that collected from a small area showed the lowest genetic variability while the other wild population R which sampled from a larger area had the highest genetic variability. DISCUSSION: The extremely low genetic variability of 15 cultivated lines indicated that individual line has similar reduction in diversity/heterozygosity from a bottleneck event, and each retained a similar (but different from each other) content of the wild genetic diversity. The genetic variance for the two wild T. concolor populations could be due to our varied sampling methods. The two wild populations (Q, R) and the cultivated hybrid lines (I, K, M, N, P) are genetically more closely related, but strong positive correlations presented in cultivated lines A, C, E, M, and N. These results could be used to guide future Torenia breeding. CONCLUSIONS: The genetic variation and population structure found in our study showed that cultivated hybrid lines had similar reduction in diversity/heterozygosity from a bottleneck event and each line retained a similar (but different from each other) content of the wild genetic diversity, especially when strong phenotypic selection of floral color overlaps. Generally, environmental factors could induce transposon activation and generate genetic variability which enabled the acceleration of the evolutionary process of wild Torenia species. Our study revealed that wild Torenia populations sampled from broad geographic region represent stronger species strength with outstanding genetic diversity, but selective breeding targeting a specific floral color decreased such genetic variability.

Genetic Diversity and Pathogenicity of Rhizoctonia spp. Isolates Associated with Red Cabbage in Samsun (Turkey).

A total of 132 Rhizoctonia isolates were recovered from red cabbage plants with root rot and wirestem symptoms in the province of Samsun (Turkey) between 2018 and 2019. Based on the sequence analysis of the internal transcribed spacer (ITS) region located between the 18S and 28S ribosomal RNA genes and including nuclear staining, these 124 isolates were assigned to multinucleate Rhizoctonia solani, and eight were binucleate Rhizoctonia. The most prevalent anastomosis group (AG) was AG 4 (84%), which was subdivided into AG 4 HG-I (81%) and AG 4 HG-III (3%), followed by AG 5 (10%) and AG-A (6%), respectively. The unweighted pair group method phylogenetic tree resulting from the data of 68 isolates with the inter-PBS amplification DNA profiling method based on interspersed retrotransposon element sequences confirmed the differentiation of AGs with a higher resolution. In the greenhouse experiment with representative isolates (n = 24) from AGs on red cabbage (cv. Rondale), the disease severity index was between 3.33 and 4.0 for multinucleate AG isolates and ranged from 2.5 to 3.17 for AG-A isolates. In the pathogenicity assay of six red cabbage cultivars, one isolate for each AG was tested using a similar method, and all cultivars were susceptible to AG 4 HG-I and AG 4 HG-III isolates. Redriver and Remale were moderately susceptible, while Rescue, Travero, Integro, and Rondale were susceptible to the AG 5 isolate. The results indicate that the most prevalent and aggressive AGs of Rhizoctonia are devastating pathogens to red cabbage, which means that rotation with nonhost-crops for these AGs may be the most effective control strategy. This is the first comprehensive study of Rhizoctonia isolates in red cabbage using a molecular approach to assess genetic diversity using iPBS-amplified DNA profiling.

Variability of Seed Germination and Dormancy Characteristics and Genetic Analysis of Latvian Avena fatua Populations.

Avena fatua is an economically detrimental weed found in cereal growing areas worldwide. Knowledge about the variation of dormancy and germination characteristics, as well as of the genetic diversity and structure can provide additional information about different populations within a region. Identification and development of potential indicators or markers of seed germination behavior, would be useful for modelling emergence and seed bank dynamics. This study aimed to describe the initial germination, dormancy, and morphological characteristics of seeds from different Latvian populations of A. fatua and to investigate the genetic structure of local populations. Seed samples from 26 to 41 locations in different regions of Latvia were collected over three years. Seed morphology, initial germination, and the effect of dormancy-breaking treatments were determined. Seeds from 18 Latvian populations with contrasting seed dormancy characteristics were genotyped. Although morphological differences between seeds from different regions were revealed, genetic analysis of the selected populations indicated that the population structure was not related to geographical location, which probably reflects the impact of anthropogenic dispersal of A. fatua seeds. The effect of dormancy-breaking treatments varied between years, between climatic zones and between populations, indicating environmental influences as well as inherited dormancy.

Influence of Environmental Factors on the Genetic and Chemical Diversity of Brickellia veronicifolia Populations Growing in Fragmented Shrublands from Mexico.

Brickellia veronicifolia is a native Asteraceae from Mexico that persists in fragmented habitats. This investigation reports the genetic and chemical diversity of B. veronicifolia. The diversity analysis based on iPBS markers showed an averaged Shannon index (S) of 0.3493, a Nei genetic diversity (h) of 0.2256, and a percentage of polymorphic loci average (P) of 80.7867%. The population structure obtained by AMOVA revealed that the highest variation found within populations was 94.58%. GC-MS profiling of six populations indicated that major volatiles were ß-caryophyllene (11.63%), spathulenol (12.85%), caryophyllene oxide (13.98%), α-cadinol (7.04%), cubedol (6.72%) and tau-muurolol (4.81%). Mantel tests suggested a statistically significant relationship between minor volatiles and geographical distance (r = 0.6163; p = 0.0470; p ˂ 0.05). Likewise, major volatiles showed a significant correlation with the soil pH (r = 0.6862; p = 0.0230) and maximum temperature (r = 0.4999; p = 0.0280). Our study suggests that the variation and genetic divergence of B. veronicifolia has no relationship with climatic parameters, whereas the volatiles are probably influenced by environmental factors and not by the genotype per se. Based on the characteristics of B. veronicifolia, this plant could be considered as a candidate for restoring fragmented shrublands in Mexico.

Retrotransposon-based genetic diversity of Deschampsia antarctica Desv. from King George Island (Maritime Antarctic).

Deschampsia antarctica Desv. can be found in diverse Antarctic habitats which may vary considerably in terms of environmental conditions and soil properties. As a result, the species is characterized by wide ecotypic variation in terms of both morphological and anatomical traits. The species is a unique example of an organism that can successfully colonize inhospitable regions due to its phenomenal ability to adapt to both the local mosaic of microhabitats and to general climatic fluctuations. For this reason, D. antarctica has been widely investigated in studies analyzing morphophysiological and biochemical responses to various abiotic stresses (frost, drought, salinity, increased UV radiation). However, there is little evidence to indicate whether the observed polymorphism is accompanied by the corresponding genetic variation. In the present study, retrotransposon-based iPBS markers were used to trace the genetic variation of D. antarctica collected in nine sites of the Arctowski oasis on King George Island (Western Antarctic). The genotyping of 165 individuals from nine populations with seven iPBS primers revealed 125 amplification products, 15 of which (12%) were polymorphic, with an average of 5.6% polymorphic fragments per population. Only one of the polymorphic fragments, observed in population 6, was represented as a private band. The analyzed specimens were characterized by low genetic diversity (uHe = 0.021, I = 0.030) and high population differentiation (F ST = 0.4874). An analysis of Fu's F S statistics and mismatch distribution in most populations (excluding population 2, 6 and 9) revealed demographic/spatial expansion, whereas significant traces of reduction in effective population size were found in three populations (1, 3 and 5). The iPBS markers revealed genetic polymorphism of D. antarctica, which could be attributed to the mobilization of random transposable elements, unique features of reproductive biology, and/or geographic location of the examined populations.

Primer Binding Site (PBS) Profiling of Genetic Diversity of Natural Populations of Endemic Species Allium ledebourianum Schult.

Endemic species are especially vulnerable to biodiversity loss caused by isolation or habitat specificity, small population size, and anthropogenic factors. Endemic species biodiversity analysis has a critically important global value for the development of conservation strategies. The rare onion Allium ledebourianum is a narrow-lined endemic species, with natural populations located in the extreme climatic conditions of the Kazakh Altai. A. ledebourianum populations are decreasing everywhere due to anthropogenic impact, and therefore, this species requires preservation and protection. Conservation of this rare species is associated with monitoring studies to investigate the genetic diversity of natural populations. Fundamental components of eukaryote genome include multiple classes of interspersed repeats. Various PCR-based DNA fingerprinting methods are used to detect chromosomal changes related to recombination processes of these interspersed elements. These methods are based on interspersed repeat sequences and are an effective approach for assessing the biological diversity of plants and their variability. We applied DNA profiling approaches based on conservative sequences of interspersed repeats to assess the genetic diversity of natural A. ledebourianum populations located in the territory of Kazakhstan Altai. The analysis of natural A. ledebourianum populations, carried out using the DNA profiling approach, allowed the effective differentiation of the populations and assessment of their genetic diversity. We used conservative sequences of tRNA primer binding sites (PBS) of the long-terminal repeat (LTR) retrotransposons as PCR primers. Amplification using the three most effective PBS primers generated 628 PCR amplicons, with an average of 209 amplicons. The average polymorphism level varied from 34% to 40% for all studied samples. Resolution analysis of the PBS primers showed all of them to have high or medium polymorphism levels, which varied from 0.763 to 0.965. Results of the molecular analysis of variance showed that the general biodiversity of A. ledebourianum populations is due to interpopulation (67%) and intrapopulation (33%) differences. The revealed genetic diversity was higher in the most distant population of A. ledebourianum LD64, located on the Sarymsakty ridge of Southern Altai. This is the first genetic diversity study of the endemic species A. ledebourianum using DNA profiling approaches. This work allowed us to collect new genetic data on the structure of A. ledebourianum populations in the Altai for subsequent development of preservation strategies to enhance the reproduction of this relict species. The results will be useful for the conservation and exploitation of this species, serving as the basis for further studies of its evolution and ecology.

Retrotransposable Elements: DNA Fingerprinting and the Assessment of Genetic Diversity.

Retrotransposable elements (RTEs) are highly common mobile genetic elements that are composed of several classes and make up the majority of eukaryotic genomes. The "copy-out and paste-in" life cycle of replicative transposition in these dispersive and ubiquitous RTEs leads to new genome insertions without excision of the original element. RTEs are important drivers of species diversity; they exhibit great variety in structure, size, and mechanisms of transposition, making them important putative components in genome evolution. Accordingly, various applications have been developed to explore the polymorphisms in RTE insertion patterns. These applications include conventional or anchored polymerase chain reaction (PCR) and quantitative or digital PCR with primers designed for the 5' or 3' junction. Marker systems exploiting these PCR methods can be easily developed and are inexpensively used in the absence of extensive genome sequence data. The main inter-repeat amplification polymorphism techniques include inter-retrotransposon amplified polymorphism (IRAP), retrotransposon microsatellite amplified polymorphism (REMAP), and Inter-Primer Binding Site (iPBS) for PCR amplification with a single or two primers.

The utility of iPBS retrotransposons markers to analyze genetic variation in yeast.

Yeasts are one of the main organisms in the food industry and effective components of many ecosystems. The method for identifying and detecting certain yeast species or strains is a crucial step for the food industry and should be simple, reliable, fast, and inexpensive. In our study, inter-priming binding sites (iPBS) retrotransposon marker system was employed to elucidate the genetic variability at intraspecies and interspecies levels among 112 yeast strains belonging to eight species previously obtained from fermented foods. The molecular identification of yeast strains was firstly confirmed by sequencing the D1/D2 domain of the 26S rRNA. The eight selected retrotransposon-based primers produced 278 bands, all of which were polymorphic with an average of 34.75 polymorphic fragments per primer. The averages of polymorphism information contents and the resolving power values for the iPBS marker system were 0.23 and 10.11, respectively. The genetic parameters within each yeast species obtained from iPBS markers were observed as; the percentage of polymorphic loci for each species ranging from 19.23% to 71.21%, Nei's gene diversity from 0.085 to 0.228, while Shannon's information index values ranging from 0.125 to 0.349. The value of gene flow (0.09) and genetic variation among the populations (0.85) showed higher genetic variation among the species. UPGMA analyses demonstrated considerable genetic variability in the yeast strains, clustered them according to their species, and revealed the intraspecific variation. Each of the selected iPBS primer provided enough species-discrimination. Present evaluations suggest the utility of iPBS marker system to estimate the genetic variation of yeast strains. This study is a preliminary point for further studies on the identification methodology, and population genetics of yeast species having importance in the food industry with iPBS markers.

Inter-retrotransposon amplified polymorphism markers revealed long terminal repeat retrotransposon insertion polymorphism in flax cultivated on the experimental fields around Chernobyl.

Ionizing radiation in environment comes from various natural and anthropogenic sources. The effect of radioactivity released after the CNPP (Chernobyl Nuclear Power Plant) on plant systems remains of great interest. Even now, more than three decades after the nuclear accident, the long-lived radionuclides represent a strong stress factor. Herein, the emphasis has been placed on analysis of genetic variability represented by activation of LTR (Long Terminal Repeat)-retrotransposons. Polymorphism in LTR-retrotransposon insertions has been investigated throughout the genome of two flax varieties, Kyivskyi and Bethune. For this purpose, two retrotransposon-based marker techniques, IRAP (Inter-Retrotransposon Amplified Polymorphism) and iPBS (inter-Primer Binding Site), have been employed. The hypothesis that chronic radioactive stress may induce mechanism of retransposition has been supported by the activation of FL9, FL11 and FL12 LTR-retrotransposons in flax seeds harvested from radioactive environment. Out of two retrotransposon-based approaches, IRAP appears to be more suitable for identification of LTR-retrotransposon polymorphism. Even though the LTR-retrotransposon polymorphism was identified, the results suggest the high level of plant adaptation in the radioactive Chernobyl area. However, it is not really surprising that plants developed an effective strategy to survive in radio-contaminated environment over the past 30 years.