Sibel Yilmaz

Variations in BARE-1 insertion patterns in barley callus cultures.

The stability of aging barley calli was investigated with the barley retroelement 1 (BARE-1) retrotransposon specific inter-retrotransposon amplified polymorphism (IRAP) technique. Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were cultured on callus induction MS medium supplemented with 3 mg/L 2,4-D and maintained on the same medium for 60 days. Ten IRAP primers were used in 25 different combinations. The similarity index between 30-day-old and 45-day-old calli was 84%; however, the similarity index between mature embryos and 45-day-old calli was 75%. These culture conditions caused BARE-1 retrotransposon alterations to appear as different band profiles. This is the first report of the use of the IRAP technique in barley in an investigation of callus development.

BARE1 AND BAGY2 RETROTRANSPOSON MOVEMENTS AND EXPRESSION ANALYSES IN DEVELOPING BARLEY SEEDLINGS

ABSTRACT Retrotransposons are genetic elements that can move within the genome. They can cause mutations by inserting themselves near or within genes. They may also have an important role in the regulation of the development. Barley is an important model plant in addition to its commercial importance. Retrotransposons constitute more than 50% of the barley (Hordeum vulgare L.) genome. In this study, we used mature embryo, leaf and root tissues grown from the same barley plant, to investigate BARE1 and BAGY2 retrotransposon movements, and to analyze the expression of inner domains of BARE1-gag, BAGY2-env (envelope) and rt (reverse transcriptase), using IRAP (Inter-Retrotransposon Amplified Polymorphism) and reverse transcriptase PCR (RT-PCR) techniques, respectively. Barley seeds germinated in Petri dishes under sterile conditions for 16 h were used for mature embryo dissection and genomic DNA isolation. Genomic DNA was also isolated from the leaves and roots of 5 individual seedlings which were harvested on the 10th day of germination. IRAP-PCR was performed with each DNA template for BARE1 and BAGY2 retrotransposons. BAGY2 was found to be more stable, while BARE1 polymorphisms were observed among embryos, 10-day-old roots and 10-day-old leaves. We found 50 % similarity between the roots and the leaves, 55 % between the embryo and the roots, and 66 % between the embryo and the leaves. Different PCR products of cDNA samples from embryos, roots and leaves demonstrated that the expression profile might change among individuals. The obtained findings are expected to contribute to our understanding of the effects of epigenetic changes during barley development.

Effects of epirubicin on barley seedlings

Epirubicin (EPI) is one of the anthracycline antibiotics, which is used in cancer chemotherapy. It inhibits DNA and RNA synthesis and causes cell death by DNA cleavage and production of free radicals. In this study, phytotoxicity of EPI was investigated on root and shoot growth, antioxidant enzymes and retrotransposons’ movements in 10- and 20-day-old barley seedlings. Mature embryos of barley were germinated on Murashige and Skoog medium supplemented with 250 and 500 μg/ml EPI. Our results showed that EPI treatment significantly inhibited shoot and root growth when compared with control group. Treatment with 250 and 500 μg/ml of EPI reduced shoot length in the 10-day-old plants by approximately 1.5- and 2-fold, respectively; the same treatments reduced total root length by 2- and 4-folds, respectively. However, the shoot and root lengths of 20-day-old plants were observed to be more affected by EPI-treatment. A 500-μg/ml concentration decreased total protein levels and peroxidase (EC 1.11.1.11) activity and increased superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) activities. To investigate the effect of EPI on the movements of BARE-1, SUKKULA and BAGY2 retrotransposons, inter-retrotransposon amplified polymorphism technique was performed. While some polymorphic polymerase chain reaction bands were observed for BARE-1, no polymorphism was identified in SUKKULA and BAGY2 movements. To our knowledge, this is the first report showing phytotoxic effects of EPI on plant germination and retrotransposons’ movements.

Analysis of Hopi/Osr27 and Houba/Tos5/Osr13 retrotransposons in rice

ABSTRACT We investigated Hopi/Osr27 (gypsy) and Houba/Tos5/Osr13 (copy) retrotransposon movements in 10-day-old roots and leaves of Oryza sativa cvs. Ipsala, Beser and Osmancik-97. Seeds from these three cultivars were germinated between filter papers in Petri dishes for 10 days. Three biologically independent (nonrelated) seeds were germinated for each cultivar. Then, roots and leaves grown from the same rice plant were harvested and used for genomic DNA isolation. Inter-retrotransposon amplified polymorphism–polymerase chain reaction with suitable primers was performed with each DNA template to analyze the movements of Hopi/Osr27 and Houba/Tos5/Osr13 retrotransposons. Polymorphism ratios were evaluated both among cultivars and among roots and leaves from the same cultivar. The polymorphism ratios ranged from 0% to 17% for Hopi/Osr27 and from 10% to 87% for Houba/Tos5/Osr13. The obtained results at retrotransposon and varietal levels indicated that the retrotransposon type and genotype dependence are responsible for the occurrence of different variations. Transposable elements are very important for understanding the relationship between cultivars and evolution. Our findings are expected to contribute to the understanding of spontaneous genomic insertion events and their effects on the genetic and epigenetic changes during rice development.

Human endogenous retrovirus-H insertion screening

Endogenous retroviruses (ERVs) and ERV-like sequences comprise 8% of the human genome. We aimed to analyze genome integration polymorphisms of human endogenous retrovirus (HERV)-H by the inter-retrotransposon amplified polymorphism (IRAP) technique using the sequences of LTR7A (450 bp), LTR7B (445 bp) and LTR7C (471 bp). Blood samples from 20 individuals (10 females and 10 males) of diverse ethnic origins were used for the determination of integration variations at the genomic level. Isolated genomic DNA was screened using 3 pairs of primers corresponding to LTR regions of the HERV-H gene. We observed insertion polymorphism patterns between 0-87% in all subjects. The findings obtained contribute to our understanding of the effects of HERV-H on variations within the human genome.

Sukkula retrotransposon insertion polymorphisms in barley

Tissue culture could be considered as one of the stress factors that affect the activation of retrotransposons. In this study, mature barley embryos (Hordeum vulgare L., cv. Tokak 157/37) were cultured for callus formation. Sukkula (a non-autonomous retrotransposon) polymorphism was investigated in calli with different culturing time (40- and 80-day-old), which was derived from the same embryo in MS medium supplemented with 3 mg/L of Dicamba. Polymorphism was detected using Inter-Retrotransposon Amplified Polymorphism (IRAP) and Inter-Primer Binding Site Amplification (iPBS) techniques with two different Sukkula-specific primers. Noncultured five mature embryos were used as a control. While up to 14% polymorphism rate was observed in the case of noncultured embryos, 61 and 0% polymorphism rates were observed with IRAP analyses for 40- and 80-day-old calli, respectively. iPBS analysis revealed the polymorphism ratio up to 29% in noncultured material, and up to 58 and 70% in 40- and 80-day-old callus materials, respectively. In addition, there were 50 and 74% polymorphism rates between 40- and 80-day-old calli by IRAP and iPBS analyses, respectively. The results obtained showed that tissue culture conditions and callus age affected Sukkula retrotransposon movements, and all individuals did not present the same effect.

Transposon Activity in Plant Genomes

Transposable elements (TEs) were first discovered in maize plants. However, they exist in all plant species investigated so far. Although plants with small genomes have smaller transposon percentages, plants with large genomes have high transposon percentages. For example,Arabidopsis thaliana has a genome size of 125 Mb, which comprises 14 % transposons, and theHordeum vulgare genome (5300 Mb) has 80 %. TEs are classified into two major groups based on their transposition mechanism. Class I elements are characterized by DNA sequences with homology to reverse transcriptase, and they are often referred to as retroelements, retrotransposons, or retrovirus-like elements. Retrotransposons function by a copy-and-paste transposition mechanism. Class II TEs (DNA transposons) move by a cut-and-paste mechanism. TEs affect the genome dynamics of plants by regulation of gene expression and chromosomal mutations (such as duplications, insertions/deletions, and structural variations). Transposition rates among generations are about 10−3 to 10−4, which is a higher rate than spontaneous mutations. All TEs in a cell are named as transposomes, and transposomics is a new area to work with transposomes. Although some bioinformatics software has recently been developed for the annotation of TEs in sequenced genomes, there are very few computational tools strictly dedicated to the identification of active TEs using genome-wide approaches. In this review article, after a brief introduction and review of the transposable elements, we discuss the effects of TEs in plant gene expression and evolution, and also present our recent research data on barley retrotransposons.

Short-term mutagenicity test by using IRAP molecular marker in rice grown under herbicide treatment

ABSTRACT Rice is an economically important plant as well as a model organism. The rice genome consists of 35% retrotransposons. Although most of the retrotransposons are inactivated through evolutionary processes, they can be activated under various biotic and abiotic stress conditions. The main objective of this study was to explore the effects of herbicides on retrotransposon activities and the usage of retrotransposons in short-term mutagenicity tests. In this study, bentazone and an MCPA (2-methyl-4-chlorophenoxyacetic acid)-containing herbicide was used. Plant samples were grouped into three classes: control (untreated), 1% and 2% herbicide treatment. Retrotransposon activities were investigated by using the inter-retrotransposon amplified polymorphism (IRAP) marker technique. IRAP analyses were performed for Houba (Tos5/Osr13) retrotransposon. Polymorphism ratios were calculated with the Jaccard similarity index, and the significance of polymorphism was evaluated by one-way analysis of variance (ANOVA). We observed that the polymorphism ratios ranged from 8%–90% for Houba among plant samples. ANOVA showed that these variable ratios were statistically significant. Bentazone and the MCPA-containing herbicide increased the retrotransposon activities, and they might be responsible for DNA mutations. This study indicated valuable data for establishing retrotransposon-based short-term mutagenicity test in rice with suitable retrotransposons such as Houba.