Showing papers on "RAPD published in 2016"

Genetic characterization of six parasitic protozoa: Parity between random-primer DNA typing and multilocus enzyme electrophoresis (phylogeny/clonal theory/Trypanosoma/Leishmania/Plasmodium)

Abstract: We have assayed genetic polymorphisms in several species of parasitic protozoa by means of random amplified polymorphic DNA (RAPD). One goal was to ascer- tain the suitability of RAPD markers for investigating genetic and evolutionary problems, particularly in organisms, such as the parasitic protozoa, unsuitable for traditional methods of genetic analysis. Another goal was to test certain hypotheses concerning Trypanosoma cruzi, and other protozoa, that have been established by multilocus enzyme electrophoresis. The RAPD results corroborate the hypothesis that the population structure of T. cruzi is clonal and yield a phylogeny of the clonal lineages in agreement with the one obtained by enzyme elec- trophoresis. This parity between the two sets of results confirms that RAPD markers are reliable genetic markers. The RAPD markers are also suitable for reconstructing species phyloge- nies and as diagnostic characters of species and subspecific lineages. The number of DNA polymorphisms that can be detected by the RAPD method seems virtually unlimited, since the number of primers can be increased effectively at will. The RAPD method is well suited for investigating genetic and evolutionary questions in certain organisms, because it is cost effective and demands no previous genetic knowledge about the organism.

Molecular typing and virulence analysis of multidrug resistant Klebsiella pneumoniae clinical isolates recovered from Egyptian hospitals.

Abstract: Klebsiella pneumonia infection rates have increased dramatically. Molecular typing and virulence analysis are powerful tools that can shed light on Klebsiella pneumonia infections. Whereas 77.7% (28/36) of clinical isolates indicated multidrug resistant (MDR) patterns, 50% (18/36) indicated carpabenem resistance. Gene prevalence for the AcrAB efflux pump (82.14%) was more than that of the mdtK efflux pump (32.14%) in the MDR isolates. FimH-1 and mrkD genes were prevalent in wound and blood isolates. FimH-1 gene was prevalent in sputum while mrkD gene was prevalent in urine. Serum resistance associated with outer membrane protein coding gene (traT) was found in all blood isolates. IucC, entB, and Irp-1 were detected in 32.14%, 78.5% and 10.7% of MDR isolates, respectively. We used two Polymerase Chain Reaction (PCR) analyses: Enterobacterial Repetitive Intergenic Consensus (ERIC) and Random Amplified Polymorphic DNA (RAPD). ERIC-PCR revealed 21 and RAPD-PCR revealed 18 distinct patterns of isolates with similarity ≥80%. ERIC genotyping significantly correlated with resistance patterns and virulence determinants. RAPD genotyping significantly correlated with resistance patterns but not with virulence determinants. Both RAPD and ERIC genotyping methods had no correlation with the capsule types. These findings can help up better predict MDR Klebsiella pneumoniae outbreaks associated with specific genotyping patterns.

Comparison of RAPD, ISSR, and AFLP Molecular Markers to Reveal and Classify Orchardgrass (Dactylis glomerata L.) Germplasm Variations

Abstract: Three different DNA-based techniques, Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) markers, were used for fingerprinting Dactylis glomerata genotypes and for detecting genetic variation between the three different subspecies. In this study, RAPD assays produced 97 bands, of which 40 were polymorphic (41.2%). The ISSR primers amplified 91 bands, and 54 showed polymorphism (59.3%). Finally, the AFLP showed 100 bands, of which 92 were polymorphic (92%). The fragments were scored as present (1) or absent (0), and those readings were entered in a computer file as a binary matrix (one for each marker). Three cluster analyses were performed to express–in the form of dendrograms–the relationships among the genotypes and the genetic variability detected. All DNA-based techniques used were able to amplify all of the genotypes. There were highly significant correlation coefficients between cophenetic matrices based on the genetic distance for the RAPD, ISSR, AFLP, and combined RAPD-ISSR-AFLP data (0.68, 0.78, 0.70, and 0.70, respectively). Two hypotheses were formulated to explain these results; both of them are in agreement with the results obtained using these three types of molecular markers. We conclude that when we study genotypes close related, the analysis of variability could require more than one DNA-based technique; in fact, the genetic variation present in different sources could interfere or combine with the more or less polymorphic ability, as our results showed for RAPD, ISSR and AFLP markers. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationship among genotypes of Dactylis glomerata.

Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant.

Abstract: Pittosporum eriocarpum Royle, a medicinally important taxon, is endemic to Uttarakhand region of Himalaya. It has become endangered due to over-collection and the loss of habitats. As raising plants through seeds in this plant is problematic, a reliable protocol for micropropagation using nodal explants has been developed. High shoot regeneration (95%) occurred in MS medium augmented with BA 0.4mg/l in combination IBA 0.6mg/l. In vitro regenerated shoots were rooted in MS medium supplemented with three auxins, of which 0.6 mg/l indole butyric acid proved to be the best for rooting (90%) with maximum number of roots per shoot. Thereafter, rooted plants were hardened and nearly 73% of rooted shoots were successfully acclimatized and established in the field. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity amongst nine in vitro raised plantlets with mother plant. DNA fingerprints of in vitro regenerated plantlets displayed monomorphic bands similar to mother plant, indicating homogeneity among the micropropagated plants with donor mother plant. The similarity values were calculated based on SCoT, ISSR and RAPD profiles which ranged from 0.89 to 1.00, 0.91 to 1.00 and 0.95 to 1.00 respectively. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis revealed 97% similarity amongst micropropagated plants with donor mother plant, thus confirming genetic homogeneity of micropropagated clones. This is the first report on micropropagation and genetic homogeneity assessment of P. eriocarpum. The protocol would be useful for the conservation and large scale production of P. eriocarpum to meet the demand for medicinal formulations and also for the re-introduction of in vitro grown plants in the suitable natural habitats to restore the populations.

Green Nanoparticles Engineering on Root-knot Nematode Infecting Eggplants and Their Effect on Plant DNA Modification.

Abstract: Background: Root-knot nematodes are known to cause significant damage to eggplants. New approaches by green silver nanoparticles (GSN) are used to control plant-parasitic nematode to avoid chemical nematicide hazards.Objectives: Analyses of the incorporation of different concentrations of nanoparticles on two different algae (Ulva lactuca and Turbinaria turbinata) were carried out. Furethermore, the effect of GSN on the eggplant DNA profile was studied using RAPD and EST molecular markers. Materials and Methods: Green Silver Nanoparticles (GSN) have been synthesized and characterized using the algal extract solution prepared from two algal genera. Nematicidal effect of the GSN was evaluated in greenhouse on eggplants (Solanum melongena cv. Login). Genomic DNA was extracted for use in molecular analysis. Both RAPD and EST molecular markers were used to study the GSN effect on eggplant DNA modification.Results: GSN (17 mg.mL-1) obtained from U. lactuca was more effective in reducing second-stage juveniles (J2s) of M. javanica (69.44%) population in soil. All treatments improved eggplants growth parameters. Change in DNA profile using of both RAPD and EST markers was noted. Conclusions: GSN (12.75 mg.100 mL-1) were effective on controlling the root-knot nematode (both T. turbinataand and U. lactuca algae), similar to chemical control in eggplants. GSN did not cause any phototoxicity in eggplants under treatment.

Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA , rpoB Gene Sequencing and RAPD-PCR

Abstract: Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

RAPD-SCAR Markers: An Interface Tool for Authentication of Traits

Abstract: The versatility of the PCR technique is that several kinds of primers can be explored for genome analysis depending on the purpose of study. The easy to access and low cost PCR-based markers include Random Amplified Polymorphic DNA (RAPD). The RAPD markers are easy to develop but lack of reproducibility makes it less reliable and obstacles to their further use in authentication of traits. In addition, other PCR and non PCR based markers like Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeat (SSR) and Restriction Fragment Length Polymorphism (RFLP) are also employed in authentication of traits with certain restrictions vis-a-vis use of radioactive materials, high cost and requirement of sequence information etc. Therefore, this problem can be overcome by converting RAPD markers to more robust sequence characterized amplified regions i.e. SCAR markers. SCARs are locus specific, co-dominant in nature and amplified by PCR using specific 15 - 30 bp DNA fragments. For developing SCAR markers, primers are designed from the nucleotide sequences of a cloned RAPD fragments linked to a trait of interest. SCAR markers are easy to develop and reliable tools for DNA fingerprinting. This mini review is an attempt to summarize efficacy of RAPD-SCAR interface in authentication of traits.

In vitro propagation and assessment of genetic stability of acclimated plantlets of Cornus alba L. using RAPD and ISSR markers.

Abstract: Cornus alba L. (white dogwood) is an important ornamental shrub having a wide range of applications such as reforestation programs and soil retention systems. The vegetative propagation of dogwood by cuttings may be slow, difficult, and cultivar dependent; therefore, an improved micropropagation method was developed. Nodal stem segments of C. alba cultivars ‘Aurea’ and ‘Elegantissima’ were cultured on media enriched with six different sources of macronutrients. Media were supplemented with either N 6-benzyladenine (BA) or thidiazuron (TDZ) in combination with 1-naphthaleneacetic acid (NAA). Regardless of the cultivar, the best shoot proliferation was observed on Lloyd and McCown medium (woody plant medium (WPM)) at pH 6.2, containing 1.0 mg L−1 BA, 0.1 mg L−1 NAA, and 20–30 g L−1 sucrose. Rooting of regenerated shoots was achieved by an in vitro method when different concentrations of NAA or indole-3-butyric acid (IBA) were tested. Microcuttings were rooted for 8 wk on medium enriched with 0.25 mg L−1 NAA and potted into P9 containers in the greenhouse. The final survival rate of the plants after 20 wk was 80% for ‘Aurea’ and 90% for ‘Elegantissima’. Genetic stability of the micropropagated plants was confirmed by using two DNA-based molecular marker techniques. A total of 30 random amplified polymorphic DNA (RAPD) and 20 inter-simple sequence repeat (ISSR) primers resulted in 197–199 and 184–187 distinct and reproducible band classes, respectively, in ‘Aurea’ and ‘Elegantissima’ plantlets. All of the RAPD and ISSR profiles were monomorphic and comparable with the mother plant.

Phenotypic and genotypic characterization of Pseudomonas spp. present in spoiled poultry fillets sold in retail settings.

Abstract: The objective of this study was to characterize and compare the phenotypic and genotypic diversity of most frequent Pseudomonas associated with spoilage at 4 °C in skinless marinated poultry breast fillets. We selected four or five prevalent Pseudomonas isolated 2 days after expiration date from 11 poultry samples with different production dates. Most Pseudomonas were Pseudomonas fragi (n = 24) and Pseudomonas fluorescens (n = 14). Phenotypical tests showed that P. fluorescens had proteolytic, lipolytic and lecithinase activities while P. fragi produced mainly proteolytic enzymes. Genotypic characterization showed a high number of RAPD types (≥85% similarity); nevertheless, some isolates from different samples had similar RAPD types. 14 P. fluorescens were grouped into 13 RAPD types, 3 Pseudomonas lundensis into 2 RAPD types and 24 P. fragi into 13 RAPD types. The findings revealed a high level of phenotypic and genotypic variability between and within Pseudomonas species isolated from poultry. In addition, some strains with similar RAPD types showed intra strain variability, despite not having the same phenotypic profile. This study has illustrated biological variability of Pseudomonas microbiota present in spoiled poultry fillets. Such variability may have strong importance across individual processors and spoilage and should be considered for risk assessments and when developing HACCP plans.

Molecular diversity of Annona species and proximate fruit composition of selected genotypes

Abstract: Understanding the genetic variation in germplasm is of utmost importance for crop improvement. Therefore, efforts were made to analyse the molecular marker based genetic diversity of 20 Annona genotypes from five different species of family Annonaceae. During analysis, a set of 11 RAPD primers yielded a total of 152 bands with 80.01 % polymorphism and PIC for RAPD ranged from 0.86 to 0.92 with a mean of 0.89. With 93.05 % polymorphism, 12 SSR primers produced 39 amplicons. The PIC for SSRs ranged from 0.169 to 0.694 with of average of 0.339. The dendrogram produced from pooled molecular data of 11 RAPD and 12 SSR primers showed seven clusters at a cutoff value of 0.78. The dendrogram discriminated all the Annona genotypes suggesting that significant genetic diversity was present among the genotypes. Proximate fruit composition study of nine fruiting genotypes of Annona revealed that A. squamosa possessed significantly higher amount of most of studies biochemical which gives an opportunity to fruit breeders to improve the other Annona species. Likewise, A. muricata being rich in seed oil content can be exploited in oil industries.

Direct shoot organogenesis from rhizomes of medicinal zingiber Alpinia calcarata Rosc. and evaluation of genetic stability by RAPD and ISSR markers

Abstract: A simple and efficient protocol for direct in vitro shoot multiplication and plant regeneration was established for an important aromatic medicinal plant, Alpinia calcarata. Preinduction of rhizome segments in medium containing 8.8 μM 6-benzylamino purine (BAP) rescued the buds from dormancy in 60% of the cultures. An average of 6.2 shoots were produced from rhizomatous bud explants on Murashige and Skoog (MS) medium supplemented with 5 μM BAP, 10 μM kinetin, and 2.5 μM α-Naphthalene acetic acid (NAA). The mother cultures retained their morphogenetic potential upto four subcultures and a maximum of 1.77-fold increase in shoot multiplication was recorded after the 3rd subculture. Rooting was simultaneously induced during subculture on shoot multiplication medium eliminating an additional step for rooting induction. Rooted plantlets were successfully acclimatized in pots in the greenhouse and subsequently established in the experimental garden without any visible symptoms of wilting and necrosis. The genetic fidelity of regenerated plants was evaluated by adapting to two PCR-based DNA marker techniques, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) which detected no variability in the in vitro multiplied plantlets of A. calcarata. This efficient method of clonal multiplication may be useful for commercial scale multiplication, and in situ and ex situ conservation of elite germplasm of A. calcarata.

Characterization of Grain Amaranth (Amaranthus spp.) Germplasm in South West Nigeria Using Morphological, Nutritional, and Random Amplified Polymorphic DNA (RAPD) Analysis

Abstract: Efficient utilization of plant genetic resources for nutrition and crop improvement requires systematic understanding of the important traits. Amaranthus species are distributed worldwide with an interesting diversity of landraces and cultivars whose leaves and seeds are consumed. Despite their potential to enhance food security and economic livelihoods, grain amaranth breeding to improve nutritional quality and adoption by farmers in sub-Saharan Africa is scanty. This study assessed the variation among 29 grain amaranth accessions using 27 phenotypic (10 morphological and 17 nutritional) characters and 16 random amplified polymorphic DNA (RAPD) primers. Multivariate analysis of phenotypic characters showed the first four principal components contributing 57.53% of observed variability, while cluster analysis yielded five groups at 87.5% similarity coefficient. RAPD primers generated a total of 193 amplicons with an average of 12.06 amplicons per primer, 81% of which were polymorphic. Genetic similarities based on Jaccard’s coefficient ranged from 0.61 to 0.88. The RAPD-based unweighted pair group method with arithmetic mean dendrogram grouped the accessions into nine clusters, with the same species clustering together. RAPD primers distinguished the accessions more effectively than phenotypic markers. Accessions in the different clusters as obtained can be exploited for heterotic gain in desired nutritional traits.

In vitro plant regeneration from cotyledonary explants of Cucumis melo L. var. cantalupensis and genetic stability evaluation using RAPD analysis

Abstract: An efficient plant regeneration protocol useful in genetic studies for important breeding lines of cantaloupe melons was developed. Three cultivars (Charentais-T, Vedrantais and Isabelle) and three doubled-haploid melon lines (NAD, DH-L2 and DH-L6) were tested for their potential to regenerate under in vitro conditions. Cotyledon explants from 6-day-old seedlings were cultured on three Murashige and Skoog based medium supplemented with different combinations of 6-benzylaminopurine (BAP) and indoleacetic acid (IAA). After 5 weeks of culture all the genotypes were able to regenerate on all media, however the regeneration frequency varied widely among the cultivars and the medium combinations used. The Charentais-T cultivar developed the highest number of shoots/explant on media containing BAP 1.0-IAA 1.0 × 10−2 mg/L (4.1) and BAP 1.2-IAA 1.2 × 10−2 mg/L (4.4) while on medium supplemented with BAP 1.3-IAA 1.3 × 10−2 mg/L the Vedrantais genotype gave the best result (2.4). The work first highlighted a good morphogenetic response of the DH lines; among all the genotypes and combination used, NAD (65.6 %) and DH-L6 (85.4 and 50.0 %) had the highest yield of regenerated plants. The results confirmed that the regeneration competence was strongly affected by genotype. Plantlets with well-developed shoot and root systems were successfully acclimatized and exhibited normal morphology and growth characteristics. In order to confirm genetic stability, a PCR-based RAPD analysis was carried out using 20 decamer-primers. No polymorphic bands were observed among all the plants revealing the genetic homogeneity of the regenerants. Furthermore, the ploidy level of regenerated plants was confirmed by flow cytometry.

Detection of genetic variation in sandalwood using various DNA markers

Abstract: In the present study, 20 sandalwood (Santalum album L.) genotypes were characterized using RAPD, ISSR and SSR markers. Twenty-five RAPD and twenty-one ISSR primers that generated clear and reproducible banding patterns amplified 225 and 208 bands, respectively, among 20 sandalwood genotypes. Out of 225, 181 (83.13 %) RAPD bands were polymorphic while out of 208, 156 (75.77 %) ISSR bands were polymorphic. The average polymorphism information content (PIC) for RAPD and ISSR was 0.84 and 0.86, respectively. A good correlation (0.96) was observed between the matrices produced by RAPD and ISSR primers. Though, there was high similarity among genotypes (0.79 for RAPD and 0.70 for ISSR), the observed genetic diversity was found good enough for the characterization of sandalwood genotypes. Cross-species transferability SSR markers developed in S. austrocaledonicum and S. insulare were found to be monomorphic. The results of the present investigation would provide valid guidelines for collection, conservation and characterization of sandalwood genetic resources.

Morphological and Molecular Genetic Characterization of Soft Date Palm (Phoenix dactylifera L.) Cultivars in Egypt

Abstract: The fruits of the date palm (Phoenix dactylifera L.) are sweet berries with a sugar content of more than 50% (El-Sharabasy, 2009). The origin of the date palm is supposed to be in Middle East (Zohary and Speigel-Roy, 1975; Zo- hary and Hopf, 1988; and Amer, 2000). In Arab countries and in the Middle East the date palm is a staple food that can be pro- duced easily under unfavorable natural and economic conditions (El-Sharabasy,2009). During the past decades, classical methods to evaluate genetic variation have been complemented by molecular techniques. The development of so-called "DNA markers" which are based on po- lymorphisms found in proteins or DNA has greatly facilitated research in a variety of many biological branches such as tax- onomy, phylogenetic relationships and genetics (Carlson et al., 1991; Halward et al., 1992 and Abdelsalam et al., 1998). There are a number of molecular techniques available for characterization of the variation at the DNA level, e.g. RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism) and ISSR (Inter Simple Sequence Repeats). Furthermore, these techniques are able to reveal a vir- tually unlimited number of markers. For genetic diversity studies, the RAPD tech- nique shows some important advantages (Williams et al., 1990). The ISSR strategy was therefore performed to access the DNA diversity among crop genotypes (Zehdi et al., 2004). Soliman et al. (2003) used RAPD markers to study three males and four females date accessions from Egypt. Soliman et al. (2006) identify the genetic polymorphism for semi-dry date palm in Egypt using RAPD and ISSR markers. The genetic improvement of a crop species depends on the ability to select promising plant material. To facilitate the selection process, molecular markers that are associated with important traits can be used as selection tools. The markers can then be used to establish genetic maps, which in turn are important tools for more refined marker-assisted selection in breed- ing programs as well as for in-depth ge- netic and systematic analyses (Soliman et al., 2006). Cleary, an integrated approach is needed incorporating morphological and genetic studies to improve the know- ledge of date palm taxonomy and diversi- ty. Proteins or/and DNA attributes can be used successfully for variety identifica- tion, source of information of date palm gene bank and for studying the genetic diversity of cultivars. So, date palm can be promoted best through better characte- rization and evaluation (Soliman et al., 2006). The objective of the present study was simplified over view framework on the identification, characterization, evalu- ation and documentation the genetic di- versity of soft date palm (Phoenix dactyli- fera L.) cultivars in Egypt. Genetic diver- sity of nine soft date palm cultivars grow- ing in Egypt was addressed to identify and describe DNA markers and morpho- logical important traits as well as molecu- lar genetic characterization relationships were examined. The achieved results have been utilized for establishing genetic markers in order to discriminate among the date palm cultivars in Egypt.

Potential of RAPD and ISSR markers for assessing genetic diversity among Stevia rebaudiana Bertoni accessions

Abstract: In the present study, genetic diversity among 16 collections of Stevia rebaudiana Bertoni was assessed to compare the efficiency of two-marker systems over one marker using RAPD and ISSR markers. How well these two marker systems discriminated the Stevia collections was compared. In fact, all the Stevia accessions included in the present study have been growing under different altitudes, ranging 250 to 2250 m at different geographical conditions. The 22 selected RAPD primers yielded 66 scorable bands, 54 of which were polymorphic (81.8%). While the 23 selected ISSR primers amplified 49 bands, 44 of which were polymorphic (89.8%). The genetic similarity among the accessions varied from 0.365 to 0.887 when pooled data from ISSR and RAPD were analyzed through UPGMA. The analysis of combined data set of both the techniques clustered the genotypes, based on their geographic locations. Both genotypes A & B collected from Solan (Himachal Pradesh) were known to be contrasting for different levels of rebaudioside-A and stevioside and have been singled out by all the dendrograms generated by the techniques separately. High polymorphism obtained indicates that both techniques are efficient for evaluating genetic diversity in S. rebaudiana, though ISSR yielded more polymorphism. Usually, a combination of diverse marker types is recommended to provide an accurate assessment by comparison of results.

Genetic Diversity Among Some Promising Indian Local Selections and Hybrids of Cashew Nut Based on Morphometric and Molecular Markers

Abstract: The present investigation reports an elucidation of genetic diversity among 12 promising Indian cashew nut cultivars, including 9 local selections and 3 hybrids on the basis of morphometric and yield parameters as well as random amplified polymorphic DNA (RAPD) markers. Dice dissimilarity coefficient was used to discriminate cultivars with respect to gross phenotypic characters such as plant growth, flowering, fruit, and nut characters. DNA from these cultivars were subjected to polymerase chain reaction (PCR) using 35 decamer oligonucleotide primers of which 15 primers were able to generate informative polymorphism (average 79.187%). The unweighted pair group method using arithmetic average (UPGMA) dendrogram segregated the cashew cultivars into two main clusters at 0.53 Jaccard’s similarity coefficient. Five different primers were identified with the ability to discriminate eight cultivars based on a unique band pattern. Although both morphometric descriptors and RAPD markers proved their effect...

Antimicrobial Potential, Identification and Phylogenetic Affiliation of Wild Mushrooms from Two Sub-Tropical Semi-Evergreen Indian Forest Ecosystems.

Abstract: The diversity of wild mushrooms was investigated from two protected forest areas in India and 231 mushroom specimens were morphologically identified. Among them, 76 isolates were screened for their antimicrobial potential against seven bacterial and fungal pathogens. Out of 76 isolates, 45 isolates which displayed significant antimicrobial activities were identified using ITS rRNA gene amplification and subsequently phylogenetically characterized using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Sequencing of the ITS rRNA region classified the isolates into 16 genera belonging to 11 families. In total, 11 RAPD and 10 ISSR primers were selected to evaluate genetic diversity based on their banding profile produced. In total 337 RAPD and 312 ISSR bands were detected, among which percentage of polymorphism ranges from 34.2% to 78.8% and 38.6% to 92.4% by using RAPD and ISSR primers respectively. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of selected two methods were structured similarly, grouping the 46 isolates into two clusters which clearly showed a significant genetic distance among the different strains of wild mushroom, with an similarity coefficient ranges from 0.58 to 1.00 and 0.59 to 1.00 with RAPD and ISSR analysis respectively. This reporthas highlighted both DTR and MNP forests provide a habitat for diverse macrofungal species, therefore having the potential to be used for the discovery of antimicrobials. The report has also demonstrated that both RAPD and ISSR could efficiently differentiate wild mushrooms and could thus be considered as efficient markers for surveying genetic diversity. Additionally, selected six wild edible mushroom strains (Schizophyllum commune BPSM01, Panusgiganteus BPSM27, Pleurotussp. BPSM34, Lentinussp. BPSM37, Pleurotusdjamor BPSM41 and Lentinula sp. BPSM45) were analysed for their nutritional (proteins, carbohydrates, fat and ash content), antioxidant potential. The present findings also suggested that the wild edible mushroom strains do not have only nutritional values but also can be used as an accessible source of natural antioxidants.

Varietal Discrimination and Genetic Variability Analysis of Cymbopogon Using RAPD and ISSR Markers Analysis

Abstract: Cymbopogon is an important genus of family Poaceae, cultivated mainly for its essential oils which possess high medicinal and economical value. Several cultivars of Cymbopogon species are available for commercial cultivation in India and identification of these cultivars was conceded by means of morphological markers and essential oil constitution. Since these parameters are highly influenced by environmental factors, in most of the cases, it is difficult to identify Cymbopogon cultivars. In the present study, Random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) markers were employed to discriminate nine leading varieties of Cymbopogon since prior genomic information is lacking or very little in the genus. Ninety RAPD and 70 ISSR primers were used which generated 63 and 69 % polymorphic amplicons, respectively. Similarity in the pattern of UPGMA-derived dendrogram of RAPD and ISSR analysis revealed the reliability of the markers chosen for the study. Varietal/cultivar-specific markers generated from the study could be utilised for varietal/cultivar authentication, thus monitoring the quality of the essential oil production in Cymbopogon. These markers can also be utilised for the IPR protection of the cultivars. Moreover, the study provides molecular marker tool kit in both random and simple sequence repeats for diverse molecular research in the same or related genera.

Development of a SCAR marker and a strain-specific genomic marker for the detection of the biocontrol agent strain CPA-8 Bacillus amyloliquefaciens (formerly B. subtilis)

Abstract: In this work, reliable tools were developed to detect and identify the biocontrol strain CPA-8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA-8 that was sequenced and used to design SCAR (sequence-characterised amplified regions) primer pairs. The SCAR-4 marker amplified a semi-specific fragment of 665 bp not only for the strain CPA-8 but also for other 12 strains whose morphology was completely different from CPA-8. Another approach was developed to obtain a strain-specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA-8. Our results revealed that these two molecular markers, SCAR-4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA-8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA-8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.