Random Amplified Polymorphic DNA Markers Research Articles

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Reactive oxygen species (ROS) are produced in both stressed and unstressed cells. Superoxide dismutase (SOD) and phenylalanine ammonia lyase (PAL) play an important role in the defense against ROS. Eight different turf grass species were used in order to detect their ability to withstand environmental stress through investigating SOD and PAL gene expression and also the genetic relationship among them using random amplified polymorphic DNA (RAPD) molecular markers. The levels of expression of PAL , SOD genes and mRNA varied with the type of turf; both PAL and SOD gene expressions were low in cold season turf grasses (kentucky blue grass and fine fescue), moderate for bermuda hybrids (tifgreen and tifway) and high in Paspalum vaginatum . Primer 3 (UBC-245) can be used to distinguish between Paspalum species, also between common burmuda ( Cynodon dactylon ) and burmuda hybrids. It was concluded that hot season genera can withstand environmental stress more than cold season ones since they have more SOD and PAL gene expressions. Also, DNA markers can be used to differentiate between different turf genera which are hard to be differentiated morphologically. Keywords: Turf, superoxide dismutase, phenylalanine ammonia lyase, RAPD markers, oligonucleotide primers, burmuda hybrids. African Journal of Biotechnology , Vol 13(24) 2394-2399

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The random amplified polymorphic DNA (RAPD) technique has been widely applied to identify different varieties of plants for molecular breeding. However, application of RAPD markers to identify parthenogenesis in plants has not been reported. In this investigation, we used pedigree and RAPD markers to differentiate different Taxus chinensis var. mairei cultivars that were associated with parthenogenesis. Among 180 and eighty RAPD primers used, 108 primers generated RAPD bands from genomic DNA of T. chinensis var. mairei cultivars (“Jingzhou” and “Baokang”). Six hundred and thirty-three RAPD loci bands were produced and used to identify eight cultivars with unique banding patterns. Pedigree and RAPD data demonstrated that T. chinensis var. mairei cultivars with parthenogenesis were separated from others in both “Jingzhou” and “Baokang” cultivars. These results provide evidence for identification of parthenogenesis and confirm that the RAPD technique is especially suitable for identification of T. chinensis var. mairei cultivars for agricultural purposes.   Key words: Cultivar identification, DNA extraction, parthenogenesis, pedigree, random amplified polymorphic DNA (RAPD), Taxus chinensis var. mairei.

Miscanthus: genetic diversity and genotype identification using ISSR and RAPD markers.

Due to the limited number of molecular studies focused on European gene pool investigation, it is necessary to perform plant material recognition. Eighteen accessions of three Miscanthus species, namely, M. × giganteus, M. sinensis, M. sacchariflorus were evaluated with the use of molecular marker systems such as: inter simple sequence repeats (ISSRs), random amplified polymorphic DNA (RAPD), and by estimation of ploidy level based on flow cytometry. As a result, only one ISSR primer (ISSR1) and three RAPD primers (RAPD1, RAPD2, RAPD4) were required to identify all genotypes. Moreover, the use of the above mentioned molecular markers enable the proper species recognition of the interspecific hybrid M. × giganteus “Floridulus,” which has been previously mislabeled as M. floridulus. The highest genetic similarity coefficient (0.94) was observed between M. × giganteus clones, which indicates that the genetic diversity within this species was very low. Whereas M. sinensis genotypes represented a relatively wide diversity with similarity coefficient of 0.58. Cluster analysis using UPGMA grouped the 18 accessions in three clusters according to species affiliation including relabeled M. × giganteus “Floridulus,” which proved to be closely related to M. × giganteus. Similar groupings were evident in the PCoA analysis.

Analysis of genetic and virulence variability of Stemphylium lycopersici associated with leaf spot of vegetable crops

Stemphylium lycopersici (Enjoji) W. Yamam was initially described from tomato and has been reported to infect different hosts worldwide. Sequence analyses of the internal transcribed spacer (ITS) regions 1 and 2, including 5.8S rDNA (ITS-5.8S rDNA) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), as well as virulence studies were conducted to analyze 46 S. lycopersici isolates. Stemphylium lycopersici isolates used in this study were obtained from diseased tomato (Solanum lycopersicum L.), eggplant (Solanum melongena L.), pepper (Capsicum annuum L.) and lettuce (Lactuca sativa L.) from major vegetable growing regions of Malaysia, including the three states of Pahang, Johor and Selangor between 2011 and 2012. Phylogenetic analysis of a combined dataset of the ITS-5.8S rDNA and gpd regions indicated that all isolates were clustered in the sub-cluster that comprised S. lycopersici, and were distinguished from other Stemphylium species. Cluster analyses using the UPGMA method for both RAPD and ISSR markers grouped S. lycopersici isolates into three main clusters with similarity index values of 67 and 68 %. The genetic diversity data confirmed that isolates of S. lycopersici are in concordance to host plants, and not geographical origin of the isolates. All S. lycopersici isolates were pathogenic on their original host plants and showed leaf spot symptoms; however, virulence variability was observed among the isolates. In cross-inoculation assays, the representative isolates were able to cause leaf spot symptoms on eggplant, pepper, lettuce and tomato, but not on cabbage.

Appraisal of RAPD and ISSR markers for genetic diversity analysis among cowpea (Vigna unguiculata L.) genotypes

The genetic variability and relationships among 11 cowpea genotypes representing two cultivars and nine elite genotypes were analyzed using 22 random amplified polymorphic DNA (RAPD) and nine inter-simple sequence repeat (ISSR) markers. ISSR markers were more efficient than RAPD assay with regards to polymorphism detection. But the average numbers of polymorphic loci per primer and resolution power were found to be higher for RAPD than for ISSR. Also, the total number of genotype specific marker loci, Nei’s genetic diversity, Shannon’s information index, total heterozygosity, and average heterozygosity were prominent in RAPD as compared to ISSR markers. The regression test between the two Nei’s genetic diversity indices showed low regression (0.3733) between ISSR and RAPD + ISSR-based similarities but maximum (0.9823) for RAPD and RAPD + ISSR-based similarities. The RAPD- and ISSR-generated cultivar- or genotype-specific unique DNA fingerprints able to identify the most diverse genotypes. A dendrogram constructed based on RAPD and ISSR combined data indicated a very clear pattern of clustering according to the groups (cultivars and elite genotypes). The results of principal coordinate analysis were comparable to the cluster analysis. Cluster analysis showed that most diverse genotypes (GP-125 — small size with good seed quality; GP-129, GP-90L — big size with poor seed quality) were separated from moderately diverse cultivars and genotypes. The genetic closeness among GP-129 and GP-90L, JCPL-42, and JCPL-107 could be explained by the high degree of commonness in these genotypes.

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The species of Asparagus are very important as they are used for ornamental, vegetable and medicinal purposes since ancient time. In the present study, random amplified polymorphic DNA (RAPD) markers were used to evaluate genetic diversity among nine species of Asparagus and six cultivars of Asparagus officinalis L. RAPD analysis using seven random oligonucleotide primers yielded a total amplification of 245 bands, among which 220 (89.80%) were polymorphic with an average of 31.4 bands per primers. Highest number of 39 (97.50%) polymorphic bands were obtained with primer OPC-07, while minimum polymorphic bands were 18 (69.23%) with primers OPA-01. Genetic similarity coefficient ranged from 0.75 to 0.96 with an average of 0.85. Phenogram clustered all Asparagus species and A. officinalis L. cultivars into two clear clusters. One cluster comprised of all cultivars of A. officinalis L. while the second cluster comprised of all the Asparagus species. The present study reveals that RAPD markers were more convincing for analyzing genetic diversity among Asparagus species and cultivars of A. officinalis L. Key words: Asparagus officinalis L., genetic diversity, random amplified polymorphic DNA (RAPD), Phenogram.

English

Panicum maximum Jacq. is an important forage grass of African origin largely used in the tropics. The genetic breeding of this species is based on the hybridization of sexual and apomictic genotypes and selection of apomictic F1 hybrids. The objective of this work was to identify molecular markers linked to apomixis in P. maximum to determine easily and at an early stage, the reproductive mode of F1 hybrids, so to assist the breeding program. A bulked segregant analysis was performed using 184 random amplified polymorphic DNA (RAPD) primers in an F1 population of P. maximum segregating for reproductive mode. Four RAPD markers linked to apomixis were identified and mapped in this population. These markers showed good selection efficiency, ranging from 77.3 to 88%, with 81.3% when analyzed together. Two of these markers were easily transferred to another F1 population of P. maximum. In this population, the selection efficiency was also high for both markers; 84.8 and 90%, when analyzed together. These markers may be used in the assisted selection for reproductive mode in both F1 progenies of P. maximum studied here and in other populations to which they can be transferred.   Key words: Guinea grass, apospory, reproduction mode, random amplified polymorphic DNA (RAPD), selection efficiency.

Molecular characterization of Dendrobium nobile Lindl., an endangered medicinal orchid, based on randomly amplified polymorphic DNA

Dendrobium nobile Lindl., is an orchid species of immense biopharmaceutical and horticultural importance. The knowledge of its existent genetic variations within wild accessions is crucial for formulation of sustainable utilization strategies. It is distributed in a large landscape area and can grow at altitudes ranging from 800 to 2,000 m. Given that Orchidaceae is one of the largest families of flowering plants, reports on their existing genetic diversity are extremely limited. Molecular study on D. nobile can provide valuable information about the genetic diversity level and genetic relatedness of this important medicinal orchid species. Knowledge of genetic structure of a species also provides resources of a species with its current feature and future evolutionary potential. The genetic structure of D. nobile from Northeast India was investigated using randomly amplified polymorphic DNA (RAPD).The PIC value of RAPD primers was 0.74 and Rp values ranged between 6.80 and 13.23. Overall the Shannon Index, Global coefficient of genetic differentiation (G ST), showed that relative genetic diversity of the populations was fairly high. However, the low value of gene flow (Nm = 0.27) was revealed by the RAPD marker. The results of analysis of molecular variance (AMOVA) revealed that variation amongst the populations was significantly higher than within the populations. A combination of UPGMA and STRUCTURE analysis was employed to estimate the genetic relationships of D. nobile germplasm; interestingly, both the methods presented similar grouping pattern with few differences. Results revealed that 60 individuals belonging to six natural populations in Northeast India were clustered into two major groups. The data represented in this study suggested that the RAPD method was a valuable tool for estimation of genetic diversity and genetic relatedness of the D. nobile germplasm. The present findings are useful outcomes for germplasm conservation and formulation of new breeding strategies for this extremely important medicinal orchid species.

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Knowledge of genetic distances between genotypes is important for efficient organization and conservation of plant genetic resources for crop improvement programs. In this study genetic distances between genotype pairs (complements of Jaccard's similarity coefficient) were estimated from Random Amplified Polymorphic DNA (RAPD) data collected from 48 Zimbabwean sorghum landraces. These varieties showed variation in their seed proanthocyanidin (PAs) levels with 16 and 29 of them having detectable and non-detectable PA levels respectively. RAPDs revealed considerable genetic variation between the varieties used and 2.7 polymorphisms per primer were obtained.  Ninety nine polymorphic RAPD bands were used to calculate genetic distances and the mean genetic distance between the genotypes was 0.494 (± 0.113) with a range of 0.051 to 0.761. A multidimensional scaling (MDS) plot of the distance matrix revealed two distinct clusters of cultivated and wild sorghums. No clustering of genotypes according to their seed proanthocyanidin levels was revealed by MDS analysis; also the mean genetic distances of genotypes in the low, medium and high PA categories were not different from each other and none of them was significantly different from the mean genetic distances between all the groups. The RAPD markers used in the present study could not distinguish between sorghums with different PA levels in their seeds; however, the protocol established could be useful in further analysis of this trait in near isogenic lines.   Key words:  Genetic distances, multidimensional scaling, proanthocyanidins, Random Amplified Polymorphic DNA (RAPD), Sorghum bicolor, Zimbabwe.

Fertility restoration in three CMS systems of eggplant by the Rf genes of each other's systems and their SCAR marker

Three pollen non-formation type cytoplasmic male sterility (CMS) systems of eggplant (Solanum melongena L.) were developed in our previous study, through repeated backcrossing, by using wild Solanum species Solanum aethiopicum L. Aculeatum Group, Solanum anguivi Lam. and Solanum grandifolium C. V. Morton as the cytoplasm donor and eggplant ‘Uttara’ as the recurrent pollen parent. Each CMS system has male fertile (MF) line which carries the fertility restorer (Rf) genes for the pollen non-formation type sterility. In the present study, fertility restoration ability in three CMS systems of eggplant by the Rf genes of each other's systems was evaluated. The male sterile (MS) line of each CMS system was pollinated with the pollen of MF lines of other CMS systems as well as its own CMS system. Among the nine cross combinations, six groups were obtained from crossing MS lines with MF lines of other CMS systems and three groups were obtained from crossing MS lines with MF lines of their own CMS system. The segregation of MF and MS plants was fitted well with a 3:1 ratio in almost all cross combinations. The segregation patterns revealed that two independent dominant Rf genes restore fertility in each CMS system. Pollen stainability and in vitro pollen germination rate of the progenies obtained from different cross combinations were varied from 61.0 to 89.8% and 0.7 to 8.0%, respectively. Each Rf gene of three CMS systems restores fertility of other CMS systems as well as its own CMS system. Moreover, fertility recovery action in these three kinds of CMS is similar. DNA markers linked to Rf genes were screened by bulked segregant analysis (BSA) using random amplified polymorphic DNA (RAPD). A total of 420 primers were used, and more than 65% of the primers yielded a considerable number of RAPD fragments in both MF and MS bulks. Only one Operon primer OPAB10 gave a reproducible polymorphic band of 1.9kbp (OPAB101900) between bulks and was identified as linked to the Rf genes. Additionally, the RAPD marker OPAB101900 was converted to sequenced characterized amplified region (SCAR) marker designated as SCAB101900 by nucleotide sequencing. Findings of the present study may give us a potential way to explore CMS and the corresponding Rf genes from wild Solanum resources for the breeding program of eggplant.

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Ocimum spp, popularly known as Tulsi, has great commercial (food and perfumery industries) and medicinal importance due to antispasmodic, stomachicum, carminative, antimalarial and febrifuge properties. The aroma and flavor in Ocimum spp. is distinct due to the predominant aroma compound eugenol, camphor, citral etc. We used molecular techniques to assess the genetic variability and relatedness of 20 accessions of three germplasm of Ocimum spp. (Ocimum gratissimum, Ocimum sanctum and Ocimum basilicum) collected from different places of India. DNA was isolated by fixing a sample in alcohol without using liquid nitrogen. 20 accessions were analyzed through random amplified polymorphic DNA (RAPD) profiling for similarity and genetic distance, using 18 primers. The binary (1/0) data was analyzed with REEPLOT to infer the genetic distance and to construct the unweighted pair group method with arithmetic mean (UPGMA) based dendrogram. High degree of polymorphism (82.78%) was shown by RAPD markers. There was total 122 bands generated, 101 bands were polymorphic. Highest similarity was measured at approximately 0.97% and least was 0.46%. The present work showed interesting finding and proved to be a bidirectional evolution in Ocimum species. Therefore, RAPD markers can be used in the systematic study of wild plants and new crop. The present study would provide suitable keys for further studies. Key words: Ocimum, Tulsi, genetic diversity, degree of divergence, R marker.

Seed-specific identification of Larix gmelinii, Larix olgensis, and Larix principis-rupprechtii using sequence-characterised amplified region markers

Larix gmelinii, Larix olgensis, and Larix principis-rupprechtii are the three native and sympatric larch species in North China, and each of these species has a distinctive ecological niche. It is difficult to identify them based only on certain morphological characters, particularly the seed appearance. In this study, the seed endosperms of these three larch species were analysed using the random amplified polymorphic DNA (RAPD) technique to screen for interspecific differences. The following three RAPD markers linked to species-specific segments were observed in the different species: 1475-bp (Larix gmelinii and L. olgensis), 505-bp (Larix principis-rupprechtii), and 1121-bp (Larix gmelinii) markers. The three seed-specific fragments amplified by the RAPD markers were sequenced, and the sequences were used to design and synthesise species-specific SCAR markers. The size of the SCAR fragments was concordant with that of the RAPD species-specific fragments. Therefore, these SCAR markers can be used to identify the seeds of different larch species, thereby providing a new molecular tool for the identification of larch seeds that leads to considerable savings in terms of time and economic resources.

Typha glauca Godron and its parental plants in Poland: taxonomic characteristics

Natural interspecific hybridization between Typha latifolia L. and Typha angustifolia L. was analyzed by morpho-anatomical and molecular methods to determine whether the hybrid Typha glauca Godr. is present in Poland and to identify the best diagnostic traits for its identification. Eighty-three samples of the Typha species were collected. Nine random amplified polymorphic DNA (RAPD) primers provided 12 fragments specific for T. angustifolia and eight fragments specific for T. latifolia. DNA of all sampled individuals was analyzed with 20 diagnostic RAPD markers. The morpho-anatomical variability of T. glauca F1 was found to be quite similar to that observed in parental plants. All of the 41 traits examined in the hybrids overlapped with those observed in the parents, however, the hybrids were visibly closer to T. angustifolia than to T. latifolia. The most discriminate characteristics were the length and pedicel width, as well as the epidermal cell thickness located above vascular bundles in leaf blades. Moreover, preliminary observations of seed sculpture showed that the length of testa cells could also be used to identify T. glauca. Clusters and the hybrid index (for molecular and morphological data) were highly coincident and support the hybridization hypothesis.

Genetic diversity of Vicia faba L. based on random amplified polymorphic DNA and simple sequence repeat markers

The genetic diversity of 13 Tunisian faba bean (Vicia faba L.) accessions, and the commercial variety “Aguadulce”, were studied using simple sequence repeats (SSRs) and random amplified polymorphic DNA (RAPD) markers. In comparison to RAPD markers, SSRs showed a higher level of polymorphism and greater information content, as assessed by the expected heterozygosity. One hundred and fourteen bands were amplified using six SSR primer combinations and 376 bands were observed using four RAPD primers. The percentages of polymorphic fragments were 100% and 60.63% for SSR and RAPD markers, respectively. The polymorphism information content, gene diversity and population differentiation test were 0.370, 0.490 and 0.746, respectively, for SSR markers and 0.319, 0.406 and 0.775 for RAPD markers. The correlation coefficients of similarity based on the Mantel test were statistically significant for the both marker systems used, but were higher for SSR data than for RAPD. Dendrogram topologies, conducted separately on polymorphic RAPD and SSR markers, showed minor differences in the grouping of accessions. The dendrogram based on combined RAPD and SSR data sets identified clustering of accessions according to their geographic diffusion. Collectively, these results will aid Tunisian faba bean germplasm management, conservation and breeding.

KARAKTERISASI BEBERAPA STRAIN GURAMI Osphronemus gouramy Lac. MENGGUNAKAN MARKA RAPD

Giant gouramy, Osphronemus gouramy Lac. is a popular fish species in Indonesia, especially in Java and Sumatera as this freshwater fish species has a high economic value of stable price. Fish farmers in Bogor divide giant gouramy into six strains based on egg productivity, growth rate, and maximum weight of the adult. They are soang, jepang, blue saphire, paris, bastar, and porselin. These various strains lead to the need of study on the genetic relationship among them, which can be performed by the use of RAPD (Random Amplified Polymorphic DNA) marker. This study aims to determine primers producing consistent and polymorphic RAPD markers, determine specific RAPD markers, and know the genetic relationship among several giant gouramy strains. The strains used in this study are soang, jepang, and blue saphire. Survey method was applied employing purposive random sampling technique. Total genomic DNA was isolated using Genjettm genomic DNA purification kit (Fermentas), which was then used as template to amplify RAPD markers with primers OPA-07, OPA-09, OPA-11, OPA-20, OPAH-01, OPAH-08, OPAH-09, and OPAC-14. The variables examined were patterns and numbers of specific DNA fragments as the PCR amplification products. Selected primers were determined descriptively on the basis of specific DNA bands appearing on the agarose gel. Genetic diversity was predetermined by changing qualitative band pattern into quantitative binnary data. Genetic relationship was analyzed using cladistic method with PAUP software. The results showed that only five of the eight primers produce consistent and polymorphic RAPD markers, i.e. OPA-11, OPA-20, OPAH-1, OPAH-8, and OPAH-9. Specific RAPD markers which can be used to distinguish several gouramy strains are those amplified with OPA-20 of 786 bp, OPA-20 of 1,176 bp, OPAH-8 of 1,000 bp and OPAC-14 of 1,607 bp. Nervertheless, it was found that RAPD markers cannot be used to clearly determine genetic relationship among gouramy strains.

Morpho-molecular analysis as a prognostic model for repulsive feedback of the medicinal plant “Andrographis paniculata” to allogamy

Andrographis paniculata Nees. (AP) is a self-pollinated medicinal herb with a wide range of pharmaceutical properties, facing a low diversity in Malaysia. Cross-pollination of AP accessions leads to considerable rates of heterosis in the agro-morphological characteristics and anticancer phytochemicals of this eminent medicinal herb. However, the poor crossability of the plant at the interpopulation or intraspecific levels is an obstacle from the evolutionary and breeding points of view as an average of 4.56% crossability was recorded for AP in this study. Hence, this research aimed to elicit the impact of parental genetic distances (GDs) on the rate of crossability of AP using seven accessions in 21 possible cross combinations. To this end, a set of 55 randomly amplified polymorphic DNA (RAPD) primers and a total of 13 agro-morphological markers were employed to test the hypothesis. Twenty-two out of the 55 RAPD primers amplified a total of 257 bands of which 107 bands were found to be polymorphic. The principal component analysis (PCA) based on the RAPD markers revealed that the studied AP accessions were distributed to three distinct groups. Furthermore, it was noticed that even a minor increase in GD between two parents can cause a decline in their crossability. Unlike, the morphological-based GDs acted neutrally to crossability. This finding suggests that, despite the low genetic diversity among the Malaysian APs, a population prescreening using RAPD markers would be useful to enhance the rate of fruit set through selecting the genetically adjacent parents.

Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus

Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis) techniques yields inaccurate results. The objective of this study was to develop species-specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L. pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria.

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Citrus is an economically important fruit crop with a long history of cultivation worldwide. A great number of varieties of citrus are extensively grown in the Middle East including Iraq for domestic consumption and exports. However, the genetic diversity of this genus in Iraq has not been reported. Therefore, the objective of this study was to evaluate genetic relationships of Iragi citrus genotypes to provide useful information for germplasm conservation and planning of breeding strategies. Twenty decamer primers were used to generate RAPD markers to evaluate genetic relationship among 16 genotypes (14 species and hybrids) of cultivated Citrus in Iraq. Based on RAPD polymorphisms, the citrus genotypes were classified into two main groups; the first consisted of citron (Citrus medica) and its hybrids (lime and lemon). The second group contained the remaining genotypes including three sub-groups; the first being formed of sour orange (Citrus aurantium), sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi), the second the mandarins (Citrus reticulata) and the third the pummelo (Citrus grandis). The RAPD-based classification was consistent with previous studies based on other types of molecular markers.   Key words: Citrus, genetic diversity, Iraq, oranges, random amplified polymorphic DNA (RAPD) markers.&nbsp

Use of Tradescantia clone 4430 for direct long-term soil mutagenicity studies

Soil mutagens permanently and directly affect terrestrial plants, soil microorganisms and invertebrates and indirectly impact vertebrate and human populations. However, the dynamic response to soil mutagens under conditions of long-term exposure has been studied insufficiently. The clonal nature of Tradescantia #4430 and the well-developed associated test systems for evaluation of genotoxicity allow investigation of the permanent effects of direct exposure to the soil not only on plants of the same genotype, but even with the same individual plants, as was done in the present study. Twenty soil samples of different origin were studied in Tradescantia after 0.5, 1 and 2 years of direct exposure to the soil, in order to examine the induction of pink cells (PC), colorless cells (CC) and branched hairs (BH). Additionally, after 0.5 years, micronucleus (MN) formation was assessed, and after 1 year, the variation of random amplified polymorphic DNA (RAPD) markers was evaluated. Nine soil samples were of urban origin, and the others came from different soils from former military grounds. The results of the 0.5-year exposure were compared with the effects of aqueous/DMSO extracts tested in previous work. The long-term exposure period allowed not only a better differentiation of the soils within territories, but also permitted detection of differences between soils of urban and military origin. The results also depended on the test used to evaluate genotoxicity. The ‘burst’ phenomenon, consisting of a sudden increase of BH variation and, especially, of CC, was observed only in tests of samples from several military sites, mainly after a 2-year exposure. MN formation was less frequent after 0.5 years of exposure compared with the results from aqueous extracts, and this difference was highly significant. The variation of the RAPD markers in a significant portion of the soil samples coincided with a higher level of variation according to other tests (PC, CC, BH), but predicting the genotoxicity of the soils based on their elemental soil composition at the end of the experiment was impossible. At several points where a ‘burst’ was observed, the concentration of a number of elements was lower. A cumulative effect is hypothesized to explain these observations. Tradescantia clone #4430 is the most suitable system for such studies.

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Genetic diversity within Gerbera jamesonii Bolus is the key to genetic improvement of this important ornamental species. In the present study, genetic diversity of 12 accessions of gerbera was assessed through random amplified polymorphic DNA (RAPD) markers. A total of 40 RAPD primers belonging to OPD, OPE, OPF and OPG series of universal primers set were used out of which, only 10 primers produced clear, reproducible and scorable bands. Ten (10) decamer RAPD primers produced a total of 49 scorable bands from 12 genotypes of G. jamesonii Bolus, out of which 42 were polymorphic and seven were monomorphic. The percentage of polymorphism ranged from a maximum of 100.00% by OPE-02, OPE-14, OPF-18, OPG-18, OPG-16 and OPG-17 to a minimum of 50.00% by OPE-08. The RAPD profiles generated were further evaluated for studying the Jaccard’s similarity coefficient. The average genetic similarity coefficient for the 12 accessions evaluated by Jaccard index was 0.66 ranging from 0.35 to 0.86. The RAPD amplification data were used to obtain similarity matrix and for generation of dendrogram using unweighted pair group method with arithmetic averages (UPGMA) method. Based on the dendrogram, all 12 genotypes could be distinctly divided into two clusters and the accession CF Orange was found most dissimilar from other accessions. This study shows that DNA based molecular marker RAPD is a powerful, less time consuming and cost effective molecular technique for assessment of genetic diversity among different genotypes of G. jamesonii Bolus. The availability of these gerbera RAPD markers would facilitate the use of molecular markers in gerbera breeding and genetic studies.   Key words: Gerbera jamesonii Bolus, molecular markers, genetic diversity, RAPD.