Inter-primer Binding Site Research Articles

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

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. 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. 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.

Inter-primer binding site (iPBS) retrotransposon markers provide insights into thegenetic diversity and population structure of carrots (Daucus, Apiaceae)

Studies of genetic diversity and population structure are essential as an initial step in conservation and breeding programs for modern crops. Carrot (Daucus spp.) is among the ten most important vegetables worldwide, however, its genetic structure and phylogenetic relationships are not totally deciphered. Here, we explored the utility of 21 inter-primer binding site (iPBS) retrotransposon markers to determine the genetic diversity and population structure of 38 accessions of Daucus and one accession of a related genus. The manual scoring revealed 309 bands based on their presence/absence. The dendrogram based on the UPGMA clustering algorithm and a principal coordinate analysis (PCoA) indicated the presence of four clusters. The Daucus species with 2n = 18 chromosome (subclade A´) separated from the other two species D. pusillus and D. muricatus, which were positioned into two individual clusters. The other clade includes the Daucus from the B group. It was also noticed that few accessions were intermixed amongst clusters. Different genetic diversity parameters were estimated based on the four clusters (populations) defined by STRUCTURE software, demonstrating that clusters 3 and 4 possessed the lowest and highest diversity values, respectively. AMOVA showed variation between and within clusters of 41.85% and 58.15%, respectively. The highest population divergence (Fst) was observed between clusters 2 and 3 (0.579), on the other hand, clusters 1 and 4 depicted the lowest Fst with 0.160. Our research highlighted that iPBS markers were successful and effective to study Daucus genetic diversity. These results will contribute to the genetic improvement of carrots and sustainable management of its diversity.

Genetic relationship of the winter barley varieties assessed by the inter-Primer Binding Site (iPBS) DNA profiling method

ABSTRACT Winter barley (Hordeum vulgare L.) is an important crop with great impact on the nutrition of the world population. Many of the barley varieties used in industry are obtained from private and public seed collection organizations. Understanding the relationships among barley genotypes within these collections is crucial for breeding and maintenance. The objective of this study was to determine the genetic diversity in winter barley from the Kuban State Agrarian University collection by using DNA-profiling polymerase chain reaction (PCR) methods based on a single primer complementary to the primer binding site (PBS) region downstream of the long terminal repeat (LTR) for retrotransposons. In total, 1293 PCR amplicons were identified, of which 646 were polymorphic. Barley genotypes from this collection were mainly divided into two populations based on their geographical origin. Analysis of Molecular Variance (AMOVA) results indicated a high level of genetic diversity among the barley genotypes (83%). Relationships of the studied barley genotypes and their ancestry were established using clustering and Principal coordinate analysis (PCoA) analysis. The results of PCoA and clustering were similar and close genetic relationships of selected accessions were confirmed. Surprising results were obtained for Timofei and Vasya genotypes that are not supposed to be closely related, but were positioned together. Similar unexpected results were shown for Samson and Iosif varieties. Nevertheless, obtained data can be useful for planning of future crossing by breeders using distantly related crossing pairs.

Genetic variation and population differentiation of Dorystaechas hastata endemic to Turkey: Implications for conservation management

Dorystaechas hastata is a relict endemic medicinal and aromatic plant under risk of extinction. Assessment of genetic diversity and association between geographic distribution and genetic variation could benefit to the conservation efforts. The objectives of this study were to assess genetic diversity and population differentiation of the species in its natural habitat. The genetic structure of 56 accessions sampled from 15 populations encompassing whole natural distribution of the species were analyzed with both sequencerelated amplified polymorphism (SRAP) and inter-primer binding site (iPBS) molecular markers. The 146 polymorphic markers were generated by 13 SRAP and 11 iPBS primers. The similarity of the accessions ranged from 53% to 91%, with a mean similarity value of 72%. The populations formed two main groups. Principle component analyses (PCoA) and cluster analysis explained 64.9% and 9.4% of the variations with the first and second eigen vectors. The overall genetic diversity of D. hastata was relatively high at the species level, while it is relatively low at the population level. The pairwise allelic differences between populations (FST), an indication of population differentiation, ranged from 0.15 (Tünektepe -Hacısekiler) to 0.76 (Altınyaka - Sivridağ). The AMOVA results indicated that 51% and 49% of the total variation resided among and within populations, respectively. Geographic distance and/or isolation seem to have strong effect on the genetic differentiation among populations. Results indicate that a representative core collection can be established without significant compromise on genetic diversity. Having the highest genetic diversity Tahtalı, Güllük (Termessos), Beldibi, and Beycik populations must be conserved immediately in nature and might be used to initiate domestication/breeding programs.

POPULATION STRUCTURE AND LINKAGE DISEQUILIBRIUM ANALYSES OF MODEL PLANT Brachypodium distachion THROUGH iPBS MARKERS

In the current study, a total of 235 genotypes of Brachypodium distachyon (BD) were studied and targeted to evaluate the genetic diversity, population structure and relative kinship of these genotypes using inter-primer binding site (iPBS) markers. Twenty-eight iPBS markers were used, and 184 clear and sharp polymorphic bands were produced. The polymorphism information content and diversity parameters indicated the existence of an appropriate amount of genetic diversity in the analysed genotypes. The results of the dendrogram with heat map and principal component analysis (PCoA) revealed that 235 BD genotypes were grouped into two separate clusters. The population structure was calculated using the STRUCTURE software, and two major sub-groups (K = 2) were established. A total of 95.1% of the relative kinship estimates being less than 0.4 for all genotypes. The findings of this study concluded that iPBS markers are highly polymorphic and are very efficient in the evaluation of the genetic diversity of B. distachyon.

Retrotransposon-based molecular markers as a tool in delimiting species in section Ryncholotus, a recent radiation group of Macaronesian Lotus

Island species are particularly vulnerable to extinction and decline due to a range of factors, including isolation, small population sizes, climate change, or the introduction of alien species. Given their high levels of biodiversity, the preservation and protection of island endemic species are fundamental to reducing the loss of global diversity. Therefore, species delimitation, description and identification are among the most important tasks in conservation biology. However, determining species boundaries in some cases can be challenging, especially in groups that have radiated recently, where frequently used molecular markers do not have enough discrimination power. Thus, it is important to look for new approaches with higher resolution. In the present study, we test the use of retrotransposon-based molecular markers to investigate the taxonomic status of five endemic and endangered Lotus species in the Canary Islands. Our analysis revealed that the five species conform different entities, in concordance with their morphological differences, and shown that the technique named inter-Primer Binding Site (iPBS) is a reliable molecular marker system that allows to discriminate among Lotus and has a potential value for taxonomy and conservation.

Retrotransposon-based genetic variation and population structure of Impatiens macrovexilla Y. L. Chen in natural habitats and the implications for breeding

Impatiens macrovexilla Y. L. Chen is a potential ornamental herbaceous plant originated from a relatively small area of subtropical Karst region in southern China, and natural populations of this species presented strong adaptability to a range of environmental conditions, with strong horticultural characteristics including bright colour, large flowers, early flowering, long flowering, and floriferousness. Overall, these adventive traits are significant for future selective breeding. Using a ‘jumping gene’ based or retrotransposon-based Inter-Primer Binding Site (iPBS) marker system to evaluate the genetic variation of nine I. macrovexilla populations collected from contrasting environments. The electrophoretic patterns of the PCR products produced for all the accessions were applied to evaluate the genetic diversity and differentiation between populations. The genotyping results distinguished all the 91 individual accessions in nine I. macrovexilla populations from varied microclimates, and population G was separated as a particular variety (I. macrovexilla var. yaoshannensis) which agrees with former botanical classifications about this species. Results from the AMOVA analysis showed that the greatest genetic variation (77 %) was observed within populations whilst 23 % was recorded among populations, indicating a wide range of biotypes at each location where germplasm collected. Results from our PCoA analysis, the phylogenetic dendrogram, and the Bayesian clustering analysis showed obvious subdivision among populations analyzed. The genetic relationships for population G (Jinxiu) and population H (Gantang) were the most different from each other and showed the highest genetic distance of 0.254. Characteristics of specific traits for each population and the genetic relationships of these populations could be utilized in optimizing potential parental candidate for cross combinations. Variations of the genetic pattern and the relationships among and within the populations will impact profoundly on our selective breeding of the species in the future.

Inter-primer binding site (iPBS) markers reveal the population genetic diversity and structure of tropical climbing Cissampelopsis (Asteraceae) in Thailand

Abstract. Vanijajiva O, Pornpongrungrueng P. 2020. Inter-primer binding site (iPBS) markers reveal the population genetic diversity and structure of tropical climbing Cissampelopsis (Asteraceae) in Thailand. Biodiversitas 21: 3919-3928. Cissampelopsis is a small climbing tropical Asian genus of Asteraceae-Senecioneae. In Thailand, the genus is represented by two species, C. corifolia and C. Volubilis, distributed through the mountain evergreen forest. Study on the genetic diversity and structure of populations of both Cissampelopsis species provide better understanding of the biology and pattern of species diversification in the genus. To identify the genetic diversity, we used the inter-primer binding site (iPBS) retrotransposon system, in 96 accessions of Cissampelopsis species collected from different regions in Thailand. A total of 120 iPBS bands were scored as presence⁄ absence characters. Results from UPGMA and PCoA analyses indicated that C. corifolia and C. volubilis are different species. Genetic diversity and genetic differentiation among and within populations of C. volubilis is higher than C. corifolia. Molecular Variance (AMOVA) analysis of both species indicated that the genetic variance value within populations is higher than among populations of each species. Bayesian model-based STRUCTURE analysis detected two gene pools for both Cissampelopsis and showed admixture within individuals. Differences among the two Cissampelopsis species, in total diversities and levels of population differentiation, indicated that the genetic structure of Cissampelopsis populations are congruent with long-lived perennial habit with regional distribution, even for congeneric species, may vary considerably. This study suggests the effectiveness of the iPBS marker system to estimate the population genetic diversity and structure of Cissampelopsis genotypes.

Genetic diversity of pinto and fresh bean (Phaseolusvulgaris L.) germplasm collected from Erzincan province of Turkey by inter-primer binding site (iPBS) retrotransposon markers

In this study, genetic diversity among 71 bean genotypes (41 pinto beans and 30 fresh bean genotypes) and 4 commercial cultivars (Aleyna, Gina, Perolar, and Serra) commonly grown at Erzincan province in Turkey were investigated. The iPBS (inter-primer binding site) markers were used to investigate the genetic diversity of the genotypes. Polymorphism ratio was identified as 100% in all primers. Twenty-seven primers were studied and number of alleles in primers varied between 13?69 (with an average value of 37.14). According to cluster analysis based on iPBS data, genotypes were divided into 2 groups. Polymorphic information content (PIC) values varied between 0.19 (iPBS 2077) and 0.42 (iPBS 2381, iPBS 2385, and iPBS 2389) with an average PIC value of 0.33. According to genetic structure analysis, genotypes were divided into 2 subpopulations. Majority of the genotypes of the regions from where beans were collected were placed in the same groups.

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.

Genetic diversity of Alternaria species associated with black point in wheat grains.

The genus Alternaria is a widely distributed major plant pathogen that can act as a saprophyte in plant debris. Fungi of this genus frequently infect cereal crops and cause such diseases as black point and wheat leaf blight, which decrease the yield and quality of cereal products. A total of 25 Alternaria sp. isolates were collected from germ grains of various wheat cultivars from different geographic regions in Kazakhstan. We investigated the genetic relationships of the main Alternaria species related to black point disease of wheat in Kazakhstan, using the inter-primer binding site (iPBS) DNA profiling technique. We used 25 retrotransposon-based iPBS primers to identify the differences among and within Alternaria species populations, and analyzed the variation using clustering (UPGMA) and statistical approaches (AMOVA). Isolates of Alternaria species clustered into two main genetic groups, with species of A.alternata and A.tennuissima forming one cluster, and isolates of A. infectoria forming another. The genetic diversity found using retrotransposon profiles was strongly correlated with geographic data. Overall, the iPBS fingerprinting technique is highly informative and useful for the evaluation of genetic diversity and relationships of Alternaria species.

Molecular and pathogenic characterization of Cochliobolus anamorphs associated with common root rot of wheat in Azerbaijan

Genetic variation among the Azerbaijani isolates of anamorphs of Cochliobolus spp., the causal agents of common root rot of wheat, was evaluated using pathogenicity assessments, sequence analyses of the internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene, as well as inter-simple sequence repeat (ISSR) and inter-primer binding site (iPBS) markers. Twenty-eight isolates used in this study were obtained from diseased wheat plants in cereal growing regions of Azerbaijan in 2017. Bipolaris sorokiniana, Curvularia spicifera, and Curvularia inaequalis were identified. Bipolaris sorokiniana isolates were the most virulent on wheat seedlings, followed by isolates of C. spicifera and C. inaequalis. Phylogenetic analyses of a combined dataset of the ITS and GPDH regions grouped the isolates into three clusters, each of which contained isolates of one species. The dendrogram derived from the unweighted pair-grouped method by arithmetic average (UPGMA) cluster analyses based on the data of ISSR and iPBS markers divided the isolates into three clusters in concordance to their taxonomic grouping at species level, but without correlation to their geographic origins. Population structure of isolates was estimated based on Bayesian modelling, and this showed three populations (K = 3) supporting the separation of isolates in the dendrogram with the greatest mean value of Ln likelihood (-893,8). Utilization of the markers either separately or together produced a high level of polymorphism at interspecies level, which allowed for the separation of species. Although both marker systems had similar discrimination power to reveal genetic differences among the species, ISSR markers were more informative for eliciting intraspecies polymorphisms within B. sorokiniana and C. spicifera isolates. This is the first study on genetic diversity and population structure of anamorphic stages of Cochliobolus associated with common root rot of wheat using iPBS markers.

Genetic diversity analysis in the Turkish pepper germplasm using iPBS retrotransposon- based markers

Capsicum is one of the most important and diverse plant taxa, widely used as a spice and vegetable worldwide, including Turkey. Germplasm characterization is an essential step for crop breeding. In the present study, we characterized the genetic diversity and population structure of a collection of 94 pepper accessions using inter-primer binding site (iPBS) retrotransposon-based markers. A total of 20 iPBS primers were used that generated 172 bands (mean = 8.6 bands/primer), of which ~92% were polymorphic in the entire germplasm collection, whereas 83%, 69%, and 80% of the bands were polymorphic within the C. annuum, C. chinense, and C. frutescens subsets, respectively. All of the taxa analyzed were clearly differentiated by the iPBS markers. The polymorphism information content of the markers ranged between 0.15 and 0.99, with an average of 0.66. Cluster analyses by different methods (UPGMA, STRUCTURE, and principal coordinate analysis) revealed a clear separation of all of the C. annuum accessions from the other pepper species, with a few subclusters observed among the latter, including groups with accessions of both C. frutescens and C. chinense. At the interspecies level, the 3 clustering methods clearly discriminated C. annuum from C. frutescens and C. chinense. No clear association was found between the iPBS-based clustering and geographical origin or fruit characteristics of the accessions. This is the first report characterizing the genetic diversity and population structure in the Turkish pepper germplasm using iPBS markers. It is expected that these data will serve as a foundation for the development of new and improved pepper varieties.

Exploring the Genetic Diversity and Population Structure of Turkish Laurel Germplasm by the iPBS-Retrotransposon Marker System

Laurel is a medicinally important plant and is known to the world for its essential oil. Turkey is the main market in the laurel leaf trade by sharing about 90% of the world trade. Here we made an effort to elucidate genetic diversity and population structure of 94 Turkish laurel genotypes collected from 26 provinces and four geographical regions using inter-primer binding site (iPBS) retrotransposon markers. A total of 13 most polymorphic primers were selected which yielded 195 total bands, of which 84.10% were found polymorphic. Mean polymorphism information content (PIC) was (0.361) and diversity indices including mean effective number of alleles (1.36), mean Shannon’s information index (0.35) and overall gene diversity (0.22) revealed the existence of sufficient amount of genetic diversity in the studied plant material. Most diversity was found in genotypes collected from the Mediterranean region. Analysis of molecular variance (AMOVA) revealed that most of the variation (85%) in Turkish laurel germplasm is due to differences within populations. Model-based structure, principal coordinate analysis (PCoA) and neighbor-joining algorithms were found in agreement and clustered the studied germplasm according to their collection provinces and regions. This is a very first study exploring the genetic diversity and population structure of laurel germplasm using iPBS-retrotransposon marker system. We believe that information provided in this work will be helpful for the scientific community to take more interest in this forgotten but the medicinally important plant.

Utility of iPBS retrotransposons markers for molecular characterization of African Gnetum species

Species of African Gnetum are lianas used as vegetables, medicines and for generating income. Despite the taxonomic confusion, identification of new species and diverse morphological characters in African Gnetum, molecular markers on these plants are lacking. However, the inter-primer binding site (iPBS) retrotransposons markers could be simple and excellent molecular markers for African Gnetum. The objective of this study was to determine the efficiency of iPBS markers in detecting genetic differentiation in African Gnetum species. A set of 21 iPBS markers were analysed on 14 accessions including G. africanum Welw., G. buchholzianum Engl. and the recently identified species G. latispicum. Six best selected primers generated 103 bands in G. africanum, 95 in G. buchholzianum and 24 in G. latispicum. Cluster analysis divided the accessions into two major groups. The first group contained all the accessions of G. africanum, whereas the second group was further divided in two subgroups representing accessions of G. buchholzianum and G. latispicum. Additionally, the Jaccard similarity coefficient indicated a close relationship between accessions of G. buchholzianum and G. latispicum. The iPBS marker system revealed genetic differentiation within African Gnetum and could be useful for evaluating genetic diversity, conservation, taxonomy and evolution studies.

Genetic structure and diversity of Adonis L. (Ranunculaceae) populations collected from Turkey by inter-primer binding site (iPBS) retrotransposon markers

Genetic structure and diversity of Adonis L. (Ranunculaceae) populations collected from Turkey by inter-primer binding site (iPBS) retrotransposon markers

On the diversity and origin of the barley complex agriocrithon inferred by iPBS transposon markers

The six-rowed barley with brittle rachis Hordeum agriocrithon A.E. Aberg is a diverse taxon connected with the evolution and domestication processes in the genus Hordeum. However, the origin and patterns of taxonomic divergence of this barley type is still hotly disputed. Here we utilized inter-primer binding site (iPBS) retrotransposon marker analysis to assess the genetic diversity of the complex taxonomic group H. agriocrithon in reference to the other two main barley taxa H. spontaneum K. Koch and H. vulgare L.. Based on selected iPBS primers, we demonstrated that the long-term process of natural evolution and human-driven domestication have influenced the dynamics of the TE fraction of the barley genome. iPBS markers reliably differentiated at molecular level the three main Hordeum taxa but also revealed genetic and phenotypic diversity patterns at within taxon level. Although H. agriocrithon can be considered as an autochthonous species due to its genetic divergence from H. spontaneum and H. vulgare, our results have shown that most of representatives of this taxon are of hybrid origin and hybridization events have shaped its highly heterogeneous genetic structure. Furthermore, our results strongly support the notion that H. spontaneum accessions are ancestors of the H. agriocrithon subgroup paradoxon and that the Caspian sea region is the likely place of initial cultivation and domestication of the six-rowed barley.

Retrotransposon-based genetic variation of Poa annua populations from contrasting climate conditions.

BackgroundPoa annua L. is an example of a plant characterized by abundant, worldwide distribution from polar to equatorial regions. Due to its high plasticity and extraordinary expansiveness, P. annua is considered an invasive species capable of occupying and surviving in a wide range of habitats including pioneer zones, areas intensively transformed by human activities, remote subarctic meadows and even the Antarctic Peninsula region.MethodsIn the present study, we evaluated the utility of inter-primer binding site (iPBS) markers for assessing the genetic variation of P. annua populations representing contrasting environments from the worldwide range of this species. The electrophoretic patterns of polymerase chain reaction products obtained for each individual were used to estimate the genetic diversity and differentiation between populations.ResultsiPBS genotyping revealed a pattern of genetic variation differentiating the six studied P. annua populations characterized by their different climatic conditions. According to the analysis of molecular variance, the greatest genetic variation was recorded among populations, whereas 41.75% was observed between individuals within populations. The results of principal coordinates analysis (PCoA) and model-based clustering analysis showed a clear subdivision of analyzed populations. According to PCoA, populations from Siberia and the Kola Peninsula were the most different from each other and showed the lowest genetic variability. The application of STRUCTURE software confirmed the unique character of the population from the Kola Peninsula.DiscussionThe lowest variability of the Siberia population suggested that it was subjected to genetic drift. However, although demographic expansion was indicated by negative values of Fu’s FS statistic and analysis of mismatch distribution, it was not followed by significant traces of a bottleneck or a founder effect. For the Antarctic population, the observed level of genetic variation was surprisingly high, despite the observed significant traces of bottleneck/founder effect following demographic expansion, and was similar to that observed in populations from Poland and the Balkans. For the Antarctic population, the multiple introduction events from different sources are considered to be responsible for such an observation. Moreover, the results of STRUCTURE and PCoA showed that the P. annua from Antarctica has the highest genetic similarity to populations from Europe.ConclusionsThe observed polymorphism should be considered as a consequence of the joint influence of external abiotic stress and the selection process. Environmental changes, due to their ability to induce transposon activation, lead to the acceleration of evolutionary processes through the production of genetic variability.

Estimation of genomic instability and DNA methylation due to aluminum (Al) stress in wheat ( Triticum aestivum L.) using iPBS and CRED-iPBS analyses

Aluminum (Al) toxicity is a serious factor restricting crop productivity in acid soil, and Al is the major cause of phytotoxicity. However, the role of Al toxicity in interprimer binding site (iPBS) polymorphism, genomic instability, and DNA methylation has not been fully investigated. In the current study, the effects of different Al concentrations on iPBS polymorphism, genomic instability, and DNA methylation were investigated in seedlings of three wheat cultivars: Haymana 79, Kilciksiz, and Bezostaja 1. A higher aluminum concentration increased the polymorphism rate of the iPBS profile, but decreased genomic template stability in all cultivars . A higher Al concentration was found to cause DNA methylation. Furthermore, the coupled restriction enzyme digestion-iPBS technique was used to detect DNA cytosine methylation level, which could help in understanding the epigenetic mechanism. The occurrence of hypermethylation and hypomethylation was observed with respect to Al stress treatment, and Al was found to cause DNA methylation. Polymorphism in the CRED-iPBS profile and DNA methylation can be correlated to evaluate epigenetic changes under stress.

Molecular characterization of Fagaceae species using inter-primer binding site (iPBS) markers.

Retrotransposons (RTNs) contribute for genome evolution, influencing its size and structure. We investigated the utility of the RTN-based markers inter-primer binding site (iPBS) for the molecular characterization of 25 Fagaceae species from genera Castanea, Fagus and Quercus. The assessment of genetic diversity, relationships and structure, as well as taxonomic classification of Fagaceae based on molecular data is important for definition of conservation, forestry management strategies and discrimination among natural hybrids and their parents since natural hybridization may increase with the climate changes. Here, iPBS primers designed by other authors were tested alone and combined. Some of them were discriminative, revealed polymorphism within and among taxa allowing the production of a total of 150 iPBS markers. In addition, several monomorphic iPBS markers were also amplified in each taxon. The UPGMA dendrogram based on the pooled iPBS data revealed 27% of genetic similarity among species. The individuals were clustered per genus and most of the oaks per infrageneric group corroborating the adopted taxonomy. Globally, the iPBS markers demonstrated suitability for DNA fingerprinting, determination of phylogenies and taxonomic discrimination in Fagaceae, and could constitute a useful and alternative tool for germplasm characterization, and for definition of conservation strategies and forestry management. Moreover, these markers would be useful for fingerprinting natural hybrids that share morphological similarities with their parents. Since iPBS markers could also enable insights about RTNs evolution, an eventual correlation among iPBS polymorphism, variability of RTN insertions and/or genome size in Fagaceae is discussed.