Inter-retrotransposon Amplified Polymorphism Research Articles

Calcium-Binding Protein and Polymorphism in Musa spp. Somaclones Resistant to Fusarium oxysporum

The fresh fruits of ‘Grande Naine’ (Cavendish AAA—Musa spp.) dominate the world market, especially in countries with a population in a situation of social vulnerability. However, Fusarium wilt, caused by the fungus Fusarium oxysporum f.sp. cubense race 4 Subtropical (Foc ST4), emerges as a serious threat to banana production, requiring the development of resistant cultivars based on biotechnological strategies, such as the induction of mutation in tissue culture. This study aimed to identify and characterize genetic variation in somaclones resistant to Fusarium oxysporum f.sp. cubense subtropical race 4 (Foc ST4), derived from ‘Grand Naine’ bananas, by molecular markers based on retrotransposons IRAP (Inter-retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism). Nine combinations of IRAP and six combinations of REMAP primers were used. The low number of polymorphic bands did not allow for genetic diversity studies; however, ten polymorphic bands between the somaclones and control were sequenced. Of these, three presented good base calling and were aligned, namely, 1AF, 2AF, and 3AF bands. Only the 1AF band presented function related to stress response with homology to a calcium-binding protein. These proteins act early in plant infection as secondary messengers activated by pathogen-associated molecular patterns (PAMPs), initiating the cascade of plant defense signals. The fact that this band is present in all somaclones reinforces previous assessments of their resistance to Foc ST4. The use of markers IRAP and REMAP produced polymorphic bands that can, through future primer design and field validations, accelerate the identification of resistant banana genotypes for use in banana genetic breeding programs.

Molecular characterization and evaluation of different irrigation regimens on yield and other agronomic traits of some Egyptian wheat cultivars

ABSTRACT The objective of this study was to determine the optimal irrigation needs of six popular Egyptian bread wheat (Triticum aestivum L.) cultivars and to establish baseline effect of reduced irrigation on yield. The varieties included were Sids1, Sids14, Sakha94, Sakha95, Gemmiza12, and Shandawee1. The experiment was conducted at two different locations, Nubaria and Sids Agricultural Research Stations, Egypt over two consecutive seasons, 2020–2021 and 2021–2022. There was a reduction in yield with the decrease in the number of irrigations. However, several cultivars performed better for yield and other traits with one less irrigation than the current irrigation practices in the region. Therefore, the study may help revise irrigation recommendations. Overall, the cultivars Sakha95, Sids1, and Sids14 were taller and had more kernels per spike, higher seed index, and greater grain yield under reduced irrigations and showed the best drought tolerance index. The tested bread wheat cultivars were genotyped using Inter-Retrotransposon Amplified Polymorphism (IRAP) and Start Codon Targeted (SCoT) markers which showed relation among the cultivars. Among the two types of markers, IRAP was better than SCoT markers for reliability and robustness. Among the six cultivars, 58% of the IRAP markers detected polymorphism as compared to 38% with SCoT markers. The two wheat cultivars Sids1 and Sids14, which performed better for important agronomic traits under water stress conditions, could be used as donors for the national wheat breeding program for drought tolerance.

ANALYSES of P-Tst-1, P-Tst-3 and P-Tst-6 RETROTRANSPOSONS IN CONVENTIONALLY AND ORGANICALLY PRODUCED TOMATOES

Tomato is one of the best-studied cultivated dicotyledonous plants in molecular studies. Mobile genetic elements constitute large parts of plant genomes. Retrotransposons are mobile genetic elements within the genome and constitute more than 60% of the tomato genome. Transposable elements (TE) or transposons are DNA sequences that can alter their position within a genome, cause mutations and change the genetic identity of the cells and genome size. We aimed to analyze potato specific-P-Tst-1, P-Tst-3 and P-Tst-6 retrotransposon movements in tomatoes at different developmental stages (mature seedling, flowering stage and fruiting stage) under different cultural conditions (organic and conventional) by IRAP (Inter-Retrotransposon Amplified Polymorphism) technique. We found polymorphism rates between 1-100% for P-Tst-1, P-Tst-3 and 0-86% for P-Tst-6. When compared to organically produced tomatoes, conventionally produced tomatoes showed high polymorphism. Moreover, polymorphism ratios were different at developmental stages. This is the first report to analyze potato-specific retrotransposon movements in tomatoes grown under different conditions. Obtaining findings are expected to understand the evolutionary relationships between tomato and potato, and even the effects of different growing conditions on tomato genome to increase yield in agriculture. Key Words: IRAP, Mobile genetic elements, Solanum lycopersicum

Phenotypic variability evaluation and genetic variation in F2 intraspecific hybrids of cucumber (Cucumis sativus L.) using retrotransposon-based markers

Abstract. Setiawan AB, Zahidah QA, Kaltsum DN, Purwantoro A. 2023. Phenotypic variability evaluation and genetic variation in F2 intraspecific hybrids of cucumber (Cucumis sativus L.) using retrotransposon-based markers. Biodiversitas 24: 2596-2604. Cucumber (Cucumis sativus L.) has a narrow genetic base compared to other Cucumis species. The genetic base of the cucumber can be improved by crossing two distantly related cultivars from different geographical locations. This study aimed to evaluate phenotypic variability and investigate genetic variation of F2 cucumber population using two retrotransposon-based markers, namely Inter Retrotransposon Amplified Polymorphism (IRAP)andInter-SINE Amplified Polymorphism(ISAP). Two parental lines and the selected 59 genotypes from F2 population were subjected to PCR analysis for DNA profiling. The phenotypic variation of F2 genotypes revealed significant diversity in fruit weight, fruit diameter, fruit radian, pedicel length, fruit length, and fruit ratio (length-diameter). The genetic variation of the F2 hybrids was successfully detected and discriminated by IRAP and ISAP markers. Both IRAP and ISAP markers showed high heterozygosity ranged from 0.4021 to 0.4997, with an average of 0.4649 and moderate polymorphic information content ranged from 0.3489 to 0.3929 with an average of 0.3648. Cluster analysis showed that the F2 population and parental lines were grouped into two major clusters with a similarity coefficient ranged from 0.67 to 0.96. These findings imply that the F2 population is highly segregated and can be utilized to create new cultivars of cucumber.

Assessment of genetic diversity of Prunus salicina 'Shazikongxinli' by morphological traits and molecular markers

Prunus salicina 'Shazikongxinli' is one of China’s most economically valuable and reputable Prunus salicina cultivars. Understanding the genetic diversity and population structure of 'Shazikongxinli' is imperative for excellent germplasm breeding and the preservation of genetic resources. This work used morphological markers, inter-retrotransposon amplified polymorphism (IRAP), and inter simple sequence repeat (ISSR) to assess the genetic variation status of 50 'Shazikongxinli', with 18 plum cultivars selected as the control outgroup. The results showed that the average Shannon–Weaver diversity index (H') of 32 qualitative traits in 50 tests 'Shazikongxinli' was 0.557, the average coefficient of variation of 11 quantitative traits was 15.57%, and a total of 232 and 105 polymorphic loci were obtained from 22 IRAP and 15 ISSR primers, respectively. All three marker systems showed relatively rich polymorphism, especially the IRAP markers. This might be related to the nature of the retrotransposons in the IRAP markers, which might be more suitable for intraspecific variability detection than ISSR. In addition, all three markers clustered the 68 tested germplasms into two groups. All of 'Shazikongxinli' clustered into one group, and most outgroup plum cultivars clustered into another group. This suggested a relatively narrow genetic base within the 'Shazikongxinli' population. These results would be useful in understanding the genetic diversity of the germplasm resources of 'Shazikongxinli' and provide a basic theoretical reference for breeding superior germplasm.

IRAPs in Combination with Highly Informative ISSRs Confer Effective Potentials for Genetic Diversity and Fidelity Assessment in Rhododendron.

The species belonging to the Rhododendron genus are well-known for their colorful corolla. Molecular marker systems have the potential to elucidate genetic diversity as well as to assess genetic fidelity in rhododendrons. In the present study, the reverse transcription domains of long terminal repeat retrotransposons were cloned from rhododendrons and used to develop an inter-retrotransposon amplified polymorphism (IRAP) marker system. Subsequently, 198 polymorphic loci were generated from the IRAP and inter-simple sequence repeat (ISSR) markers, of which 119 were derived from the IRAP markers. It was shown that in rhododendrons, IRAP markers were superior to the ISSRs in some polymorphic parameters, such as the average number of polymorphic loci (14.88 versus 13.17). The combination of the IRAP and ISSR systems was more discriminative for detecting 46 rhododendron accessions than each of the systems on their own. Furthermore, IRAP markers demonstrated more efficiency in genetic fidelity detection of in-vitro-grown R. bailiense Y.P.Ma, C.Q.Zhang and D.F.Chamb, an endangered species recently recorded in Guizhzhou Province, China. The available evidence revealed the distinct properties of IRAP and ISSR markers in the rhododendron-associated applications, and highlighted the availability of highly informative ISSR and IRAP markers in the evaluation of genetic diversity and genetic fidelity of rhododendrons, which may facilitate preservation and genetic breeding of rhododendron plants.

Food Dye Amaranth-caused Retrotransposon Polymorphism and Salicilic Acid Protection on Triticum aestivum L.

In the present study, amaranth, which is a very important azo dye used in food, drug, paper, cosmetic and textile industries, was evaluated for its genotoxic potential by the plant model organism Triticum aestivum (wheat). The impacts of salicylic acid on genotoxic damage, and the alterations in DNA methylation due to amaranth stress in Triticum aestivum L. were also investigated. The marker techniques of Inter-retrotransposon amplified polymorphisms (IRAP) and Inter-Simple Sequence Repeats (ISSR) were used to identify genotoxic damage. Amaranth at four different doses (50, 100, 250, 500 mg/l) and salicylic acid at two different doses (0.50 mM and 0.75 mM) were used. Amaranth at all doses caused genotoxic damage and reduced Genomic template stability (GTS %). It was found that there was an increase in genomic stability due to doses of salicylic acid in combination with Amaranth.

Identification of genetic regions associated with sex determination in date palm: A computational approach

Sex determination of date palm seedlings is the challengeable effort for breeders. Different studies based on molecular markers and genome sequencing have provided some insight in to the genetic regions related to sex determination in date palms in general. But due to differences in cultivar population structure and also cost of whole genome sequencing, we may need a more suitable approach in developing countries for this task. Therefore, we suggest using a combination of different available molecular markers and a computational approach to identify the genetic regions involved in sex differentiation in date palm cultivars. In this study we used twenty-three cultivars including 7 male and 16 female cultivars that were examined by 30 different dominant and co-dominant molecular markers which deal with different genomic regions. Grouping of the tree samples based on 178 loci resulted in genetic differentiation of the studied male and female palm trees. Multiple correspondence analysis (MCA) bi-plot also showed genetic difference within male and female trees. Heatmap plot specified those markers which differentiate date palm trees. SSR (simple sequence repeats) and IRAP (inter retrotransposon amplified polymorphism) markers provided sex linked markers for male cultivars. In present study, we introduced sex specific alleles for Iranian male date palm cultivars as a fast track in seedlings. Different association studies performed identified the candidate genetic regions which are significantly associated with sex differentiation in date palm cultivars.

Evaluation of genetic diversity and Gene-Pool of Pistacia khinjuk Stocks Based On Retrotransposon-Based Markers

Pistachio genetic variety includes a wide range of female variations and male genotypes, and Iran is regarded as one of the critical sites for this diversity in the world. The genus Pistacia consists of eleven species that only have edible nuts and are commercially important. Four important species of pistachios include Pistacia vera, P. khinjuk Stocks, P. eurycarpa Yalt. (P. atlantica subsp. Kurdica Zoh.), and P. atlantica Dsef are found in Iran. Genetic diversity is one aspect of biological diversity that is extremely important for conservation strategies, especially in rare and narrowly endemic species. In Iran, there is no knowledge concerning the genomic organization of the population, genetic diversity, or phenotypic variations of the species. Pistacia khinjuk has eight distinct regional populations, all of which were studied for genetic variation and demographic organization because of the species’ therapeutic value. For this reason, we employed six inter-retrotransposon amplified polymorphism (IRAP) indicators and 15 mixed IRAP indicators to highlight genomic variation in this plant both within and across populations in this study. It was discovered that 73% of overall genomic variability was related to within-population variety and 27% was attributable to inter-population genomic divergence using the AMOVA test among the examined populations (PhiPT = 0.49, P = 0.010). It was discovered by the Mantel analysis that there was a substantial positive association between genomic isolation and geographic distance among the tested populations. STRUCTURE analyses and population assignment tests revealed some degree of gene flow among these populations. There was consistency between the MDS plots of communities and the NJ grouping of molecular information. Based on (IRAP) indicators, these findings demonstrated that regional communities of the plant Pistacia khinjuk are well distinct.

Genetic diversity and gene-pool of Medicago polymorpha L. based on retrotransposon-based markers

The genus Medicago L. (Fabaceae) comprises approximately 87 different species of herbs and shrubs widespread from the Mediterranean to central Asia. Medicago polymorpha is a herbaceous legume that can be a useful pasture plant, in particular, in regions with a Mediterranean climate. It had aroused great interest due to high nutritious quality, highly palatability and N-fixing plan in neutral soil. There is no information on its population genetic structure, genetic diversity, and morphological variability in Iran. Due to the medicinal importance of this species, a genetic variability and populations’ structure study is performed studying 15 geographical populations of Medicago polymorpha. Therefore, we used six inter-retrotransposon amplified polymorphism (IRAP) markers and 15 combined IRAP markers to reveal within and among population genetic diversity in this plant. AMOVA test produced significant genetic difference (PhiPT = 0.46, P = 0.010) among the studied populations and also revealed that, 66% of total genetic variability was due to within population diversity while, 34% was due to among population genetic differentiation. Mantel test showed positive significant correlation between genetic distance and geographical distance of the studied populations. STRUCTURE analyses and population assignment test revealed some degree of gene flow among these populations. PCoA plot of populations was in agreement with UPGMA clustering of molecular data. These results indicated that geographical populations of Medicago polymorpha are well differentiated based on (IRAP) markers.

Development of Retrotransposon-Based Molecular Markers for Characterization of Persea americana (Avocado) Cultivars and Horticultural Races

Persea americana (avocado) represents one of the most demanded food products worldwide, with an important impact in several agronomy-based economies. The avocado is one of the most salt-sensitive and valuable crops. It is therefore necessary to use salt-tolerant varieties, such as the West Indian, for cultivation in locations with soil salinity problems, such as the Canary Islands. Therefore, characterization of avocado cultivars is in demand, as well as development of molecular tools able to easily identify the main avocado cultivars and horticultural races. In the present work, inter-Primer Binding Site (iPBS) and Inter-Retrotransposon Amplified Polymorphism (IRAP) techniques, which are based on retrotransposon with Long Terminal Repeats (LTR), have been implemented for the first time in P. americana, allowing the characterization of genetic variation among cultivars from the three main horticultural races and the identification of potential P. americana LTR sequences. The iPBS approach showed clear advantages over its technical implementation, and allowed a better delimitation of horticultural races, especially when focused on West Indian cultivars. However, both techniques generated reproducible genetic fingerprints that not only allowed genetic characterization of each cultivar analyzed, but also revealed potential molecular markers for the identification of avocado cultivars and horticultural races.

High genetic diversity in Aegilops tauschii Coss. accessions from North Iran as revealed by IRAP and REMAP markers

BackgroundAegilops tauschii Coss. as a donor of wheat D genome has an important role in wheat breeding programs. Genetic and phylogeographic diversity of 79 Ae. tauschii accessions collected from north and northwest of Iran were analyzed based on retroelement insertional polymorphisms using inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers. ResultsIn total, 306 and 151 polymorphic bands were amplified in IRAP and REMAP analyses, respectively. As a result, a high level of polymorphism was observed among the studied accessions as revealed by an average of 25.5 bands per primer/primer combination and mean PIC value of 0.47 in IRAP and an average of 25.16 bands per primer combination and mean PIC value of 0.47 in REMAP. Genetic relationships of the accessions were analyzed using distance- and model-based cluster analyses. ConclusionThe result showed that genetic distance did not seem to be related to geographic distribution, and the accessions could be divided into three groups, which was further supported by principal coordinate analysis. These results on genetic diversity and population structure of Ae. tauschii in Iran should provide important knowledge on genetic resources and their applications in wheat breeding programs.

Characterisation of LTR-Retrotransposons of Stevia rebaudiana and Their Use for the Analysis of Genetic Variability

Stevia rebaudiana is one of the most important crops belonging to the Asteraceae family. Stevia is cultivated all over the world as it represents a valid natural alternative to artificial sweeteners thanks to its leaves, which produce steviol glycosides that have high sweetening power and reduced caloric value. In this work, the stevia genome sequence was used to isolate and characterise full-length long-terminal repeat retrotransposons (LTR-REs), which account for more than half of the genome. The Gypsy retrotransposons were twice as abundant as the Copia ones. A disproportionate abundance of elements belonging to the Chromovirus/Tekay lineage was observed among the Gypsy elements. Only the SIRE and Angela lineages represented significant portions of the genome among the Copia elements. The dynamics with which LTR-REs colonised the stevia genome were also estimated; all isolated full-length elements turned out to be relatively young, with a proliferation peak around 1–2 million years ago. However, a different analysis conducted by comparing sequences encoding retrotranscriptase showed the occurrence of an older period in which there was a lot of LTR-RE proliferation. Finally, a group of isolated full-length elements belonging to the lineage Angela was used to analyse the genetic variability in 25 accessions of S. rebaudiana using the Inter-Retrotransposon Amplified Polymorphism (IRAP) protocol. The obtained fingerprints highlighted a high degree of genetic variability and were used to study the genomic structures of the different accessions. It was hypothesised that there are four ancestral subpopulations at the root of the analysed accessions, which all turned out to be admixed. Overall, these data may be useful for genome sequence annotations and for evaluating genetic variability in this species, which may be useful in stevia breeding.

Comparative retrotransposon analysis of mutant and non-mutant rice varieties grown at different salt concentrations

This study investigated the effects of retrotransposon movements on the regeneration of 15- and 30-day-old leaves and roots of four different rice varieties grown in tissue culture medium at different salt concentrations (0, 50, 100, and 200 mmol/L) by Inter Retrotransposon Amplified Polymorphism (IRAP) and Retrotransposon Microsatellite Amplified Polymorphism (REMAP) polymerase chain reaction (PCR)-based marker methods. The study used Hopi, Houba, Osr30, Tos17 primers for IRAP analysis. The (GA)9C ISSR primer was used with four IRAP primers in REMAP analyses. There were different band profiles and polymorphism results between different salts concentrations of the same variety and various rice varieties. We observed that the polymorphism ratios for Houba ranged from 0 to 98% in IRAP and REMAP analyzes in the calculation made with the Jaccard similarity index in mutant samples. We report that mutant varieties give more valuable IRAP and REMAP results than non-mutant varieties. The results reported in this study contribute to understanding the effect of salinity stress on rice in terms of retrotransposon-based molecular markers. These results suggest that all tested retrotransposons are still active, causing genomic polymorphism among rice plantlets.

Iron toxicity-induced DNA damage, DNA methylation changes, and LTR retrotransposon polymorphisms in Zea mays

The impact of Fe2+ (iron)toxicity on genomic instability, DNA methylation status, and Long Terminal Repeat Retrotransposons (LTR RTs) polymorphisms on Zea mays is unknown. We investigated the toxicity of Fe2+ using Random Amplified Polymorphic DNA (RAPD), Coupled Restriction Enzyme Digestion-Random Amplification (CRED-RA) and Inter Retrotransposon Amplified Polymorphism (IRAP) assays in Zea mays seedlings, respectively. The results indicated that each dose of FeSO4 (50, 100, 200, and 300 mg/L) had a reducing effect on Genomic Template Stability (GTS) and increasing in RAPD pattern changes (DNA damage). The value of DNA methylation rised gradually depending on FeSO4 doses. Moreover, five LTR RTs (Wltr2105, Nikita-N57, Sukkula, Nikita-E2647, and Stowaway) of the maize genome revealed polymorphism in all FeSO4 doses. Furthermore, the present study indicated that there is a relationship between DNA methylation alterations and LTR RTs mobilization. It was concluded that iron caused DNA methylation changes as well as genotoxic damage in the maize genome. Also, considering the increase in some LTR RTs polymorphism we can say that it may be a part of the defense mechanism of the plant during stress

Retrotransposon-based markers revealed a repartition depending on geographical origin and breeding status of Tunisian pistachio species

Abstract Retrotransposon movements are considered to be an important factor in evolutionary processes and speciation as well as a source of genetic variation. In order to analyze genetic diversity and population structure in Tunisian pistachio species, nine inter-retrotransposon amplified polymorphism (IRAP) markers were used. As a result, eighty-six amplicons were produced among which 98.15 % were polymorphic. Mean numbers of the effective number of alleles (Ne), Shannon’s information index (I) and Nei’s genetic diversity (H) were respectively 1.529, 0.478, and 0.310. The average within-population genetic diversity (Hs) was 0.24 and the total diversity (Ht) was 0.3. The Tunisian pistachio populations exhibited high genetic differentiation (Gst =0.275) and gene flow (Nm = 1.888). The Analysis of Molecular Variance (AMOVA) indicated that variation was very high within populations (83 %). Phylogenetic tree using neighbor- joining (NJ) method and Principal Coordinates Analysis (PCoA) depicted that groupings of Tunisian varieties were made independently of the sex of the trees, but depending on their geographical origin and their breeding status. The modelbased Bayesian clustering (STRUCTURE) confirmed these observations. The inter-retrotransposons amplification polymorphism markers were significantly informative at the interspecific level. Findings reported in our study will be essential toward breeding for new pistachio genotypes with developed chemical and horticultural features.

Analysis of genetic variability in F2 interspecific hybrids of mung bean (Vigna radiata) using inter-retrotransposon amplified polymorphism marker system

Abstract. Fatmawati Y, Setiawan AB, Purwantoro A, Respatie DW, Teo CH. 2021. Analysis of genetic variability in F2 interspecific hybrids of mung bean (Vigna radiata) using inter-retrotransposon amplified polymorphism marker system. Biodiversitas 22: 4880-4889. Mung bean (Vigna radiata L. Wilczek) categorized as one of the pivotal annual crops of Vigna genera is commonly cultivated in rotation with the cereal crops during the drought season. Conversely, to ameliorate its stunted productivity, the interspecific hybridization technique has been introduced between the mung bean and the common bean to promote genetic improvement with the breeding projects in Indonesia. However, since mung bean is a self-pollinated crop and has a narrow genetic base, the selection and improvement of a specific trait using marker-assisted selection is more challenging. Hence, a precautionary investigation is imperative to evaluate the progenies resulting from interspecific hybridization using an ideal marker. This study aimed to investigate the genetic variability of the F2 population of the interspecific mung bean hybrids using retrotransposon-based markers, particularly Inter-Retrotransposon Amplified Polymorphism (IRAP) markers. In this study, we identified retrotransposon from the mung bean genome and determined the Long Terminal Repeat (LTR) sequence using the LTR Finder. The IRAP primers were designed from a conserved region of the LTR sequence. One hundred of the F2 interspecific hybrids generated from the crossing between mung bean and common bean were successfully discriminated by IRAP markers. The IRAP marker showed high heterozygosity and moderate Polymorphic Information Content (PIC) values. The IRAP markers were able to detect genetic variability in the F2 progenies resulting from the interspecific hybridization. Cluster analysis showed that 100 of the F2 progenies were grouped into three clusters. This study demonstrated that retrotransposon-based markers can offer an effective approach for evaluating the segregation in the F2 population of intercross hybrids in the mung bean.

Retrotransposons and multilocus sequence analysis reveals diversity and genetic variability in endophytic fungi-associated with Serjania laruotteana Cambess.

The composition of endophytic communities is dynamic and demonstrates host specificity; besides, they have great intra- and interspecific genetic variability. In this work, we isolated leaf endophytic fungi from Serjania laruotteana, identify them using multilocus analysis, and evaluate the genetic variability using IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microssatellite amplified polymorphism). A total of 261 fungi were isolated and 58 were identified. Multilocus phylogenetic analysis using the partial sequences from the ITS1-5.8S-ITS2 regions, elongation factor 1-alpha, β-tubulin, actin, glyceraldehyde-3-phosphate dehydrogenase, and calmodulin genes identify that most strains belonged to the Colletotrichum and Diaporthe genera, other isolated genera were Xylaria, Phyllosticta, Muyocopron, Fusarium, Nemania, Plectosphaerella, Corynespora, Bipolaris, and Curvularia. The IRAP and REMAP analyzes were performed with Colletotrichum and Diaporthe genera and showed 100% of polymorphism and high intra- and interspecific variability. This is the first report of the diversity of endophytic fungi from S. laruotteana. In addition, it demonstrated that the IRAP and REMAP can be used to distinguish morphologically similar lineages, revealing differences even strains of the same species.

Molecular marker based assessment of genetic homogeneity within the in vitro regenerated plants of Crocus sativus L. – a globally important high value spice crop

The global market for spice is steadily increasing and saffron is one of the costliest spices in the arena. However, there is a huge gap in the availability of quality planting materials (QPM) of saffron which can only be effectively redressed by appropriate application of plant tissue culture protocols. Nevertheless, one of the major embargoes in the sustainable utilization of in vitro technologies is occurrence of cryptic clonal variabilities defined as somaclonal variations. In the present research, we report a reproducible, cost effective and simple molecular marker mediated assessment of genetic fidelity within the in-vitro propagated shoots of saffron in order to ensure genetic uniformity during cormlet production. The in vitro propagated shoots of Crocus sativus were raised from field grown corms using an earlier reported method. The genetic stability of in vitro propagated shoots was then assessed using molecular markers namely, Start Codon Targeted Polymorphism (SCoT), Amplification of Minisatellite DNA (DAMD) and Inter Retro Transposon Amplified Polymorphism (IRAP). The isolated genomic DNA samples (mother and regenerated plants) were subjected to PCR amplification with 22 best reproducible primers (14 SCoT, 4 DAMD and 4 IRAP). The combined assay of molecular markers revealed a clonal variability of 7.81% thereby, indicating largely stable clonal lines. The present research documents a fast, reproducible and cost effective methodology for assessment of genetic variability amongst the regenerants of saffron and also ensures the true-to-type nature of the micropropagated plants with a target goal of sustainable propagation.

α-Thujone exhibits an antifungal activity against F. graminearum by inducing oxidative stress, apoptosis, epigenetics alterations and reduced toxin synthesis

Specific mechanisms contributing to the antifungal activity of α-thujone, one of the main compounds of essential oils in herbs, against Fusarium graminearum, which causes various diseases of cereals, were studied for the first time. Minimum inhibitory concentration (MIC) value of α-thujone was 3.2 μg μL−1 for F. graminearum. Its toxigenic effect was confirmed by WST-1-based cytotoxicity assay. Due to α-thujone treatment, changes in genomic template stability (GTS), type II and III methylation profiles were detected. GTS rates were calculated as 89.33 and 94% according to random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP) assays, respectively. Mean values of polymorphisms were 28.92 and 36.42% by evaluating digestion profiles. Gene expression analysis showed α-thujone caused upregulation of mgv1, mst20 and cat genes but downregulation of tri5. Fold change values were 1.86 ± 0.14, 3.10 ± 0.03, 2.56 ± 0.078 and 0.43 ± 0.15 respectively. The acridine orange-ethidium bromide (Ao/Eb) and 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) staining assays revealed that α-thujone treatment triggers apoptosis and oxidative stress in F. graminearum at cellular level. Additionally, 6.51-fold increase in catalase activity in comparison with the control was detected. There were no statistically significant changes in water loss rates (WLRs) when Hordeum vulgare L. cv. Akhisar seedlings were treated with α-thujone. According to the water loss rate values, α-thujone is not a potential abiotic stress factor that negatively affects the physiological development of barley plantlets. Outcomes showed that α-thujone leads to serious morphological, genetic, epigenetic, cellular alterations on F. graminearum, so could be a candidate as an alternative herbal antifungal compound.