Evaluating genetic diversity of geographically diverse populations of Embelia ribes Burm f., a highly medicinal woody liana from the Western Ghats of India, using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats (ISSR) markers.

BACKGROUND: Embelia ribes Burm f. (Primulaceae) is a medicinal and vulnerable woody liana distributed throughout India. Embelin, a well-recognized active phytoconstituents in berries, is commonly used in ayurvedic formulations. Due to over-exploitation, the status of the plant is vulnerable. Previous studies on this species mainly focused on its phytochemical analysis, which led to overexploitation and loss of the germplasm. METHODS AND RESULTS: In the present study, 20 RAPD and 18 ISSR markers were employed to assess genetic divergence in 40 genotypes of E. ribes collected from different parts of the Western Ghats of India. In RAPD analysis, all 40 accessions with 20 RAPD primers amplified 282 fragments, with 83.91% average polymorphism and with an average of 14.10 bands per primer. The size of amplicons varied from 200 to 2500 bp. While, ISSR primers produced 203 fragments of which 161 were polymorphic with an average of 11.28 bands per primer with 73.25% average polymorphism. The size of amplicons ranges from 200 to 2500 bp. RAPD and ISSR markers were also assessed by calculating polymorphic information content (PIC) to discriminate the genotypes; the average PIC value for RAPD, ISSR, and combined RAPD + ISSR markers obtained was more than 0.50 suggesting the informativeness of markers. UPGMA analysis based on Jaccard's similarity coefficient for RAPD, ISSR, and RAPD + ISSR data reveals that 40 accessions of E. ribes were depicted in four clusters. The clustering pattern of all individuals in PCoA analysis agreed with the UPGMA dendrograms, which further confirms the genetic relationships explained by cluster analysis. AMOVA analysis of RAPD, ISSR, and combined marker system revealed variation within the population, ranging from 41 to 44%, and among the population, it ranged from 56 to 59%. CONCLUSION: The present study provides an optimized method for evaluating the genetic diversity of Embelia ribes using RAPD and ISSR markers which are useful for further sustainable utilization and conservation of natural populations in the Western Ghats of India.

Random amplified polymorphic DNA and inter simple sequence repeat markers reveals genetic diversity between micro propagated, wild and field cultivated genotypes of Gloriosa superba: an endangered medicinal plant.

Gloriosa superba L., an endangered medicinal plant with global interest due to presence of colchicine, an important alkaloid used in formulations of Indian and Traditional medicine. The plant has become endangered due to its unscientifically exploitation and high medicinal values. In the Present study 10 randomly amplified polymorphic DNA (RAPD) and 6 ISSR markers were employed to assess genetic divergence among micro propagated, wild and field cultivated plants of Gloriosa superba collected from different parts of India. In RAPD analysis, all the 10 accession with 10 RAPD primers amplified 466 fragments, with 96.43 % polymorphism and with an average of 46.6 bands per primer. The size of amplicons varied from 1656 to 100 bp. While, ISSR primers produced 328 fragments of which 298 were polymorphic with an average of 49.7 bands per primer with 91.83% polymorphism. The size of amplicons ranges from 2395 to 181 bp. RAPD, ISSR markers were also assessed by calculating polymorphic information content (PIC) to discriminate the genotypes, Average PIC value for RAPD, ISSR and combined RAPD + ISSR markers obtained was ≤ 0.50 suggesting the informativeness of markers. Jaccard's coefficient ranges from 0.18 to 0.75 (RAPD) and 0.17 to 0.61 (ISSR) and 0.21-0.52 for pooled ISSR and RAPD markers. The clustering pattern based on UPGMA analysis of the genotypes in the combined analysis revealed that the majority of the genotypes remained similar to the ISSR dendrogram, while the RAPD-based dendrogram showed some variation in the clustering of genotypes. The result of PCA scattered plot obtained were in agreement with the UPGMA dendrogram, which further confirms the genetic relationships explain by cluster analysis. Results confirmed that the genotype studied had good genetic diversity and can be used for identification, conservation, and future breeding program of Gloriosa species and consequently for the benefit of the pharmaceutical industries.

Characterization of Streptococcus lutetiensis isolated from clinical mastitis of dairy cows.

Streptococcus lutetiensis, previously termed Streptococcus bovis type II/1, has rarely been associated with bovine mastitis. The objectives of this work were to characterize the molecular diversity, antimicrobial resistance profiles, virulence genes of Strep. lutetiensis (n = 37) isolated from bovine clinical mastitis, as well as its pathogenic effects in a murine mastitis model. Genetic relationships of isolates were determined by random amplified polymorphic DNA (RAPD)-PCR, virulence genes were detected by PCR. Antimicrobial susceptibility testing was carried out by broth microdilution technique. The pathogenic effects of Strep. lutetiensis were studied with 2 infection models: bovine mammary epithelial cells cultured in vitro and murine mammary infection in vivo. Streptococcus lutetiensis isolates were clustered into 5 RAPD-types (A-E), with a dominant type A representing 84% of isolates. Eighteen (49%), 16 (43%), and 9 (24%) isolates were resistant to ceftiofur, tetracycline, and erythromycin, respectively. Prevalence of multidrug resistance (resistant to ≥3 classes of antimicrobials) was 24% (9/37). The most prevalent virulence genes were bca (100%), speG (100%), hly (97%), scpB (95%), and ssa (95%). There was no difference between isolates from mild and moderate cases of bovine mastitis in prevalence of virulence genes. Streptococcus lutetiensis rapidly adhered to and subsequently invaded (1 and 3 h after infection, respectively) bovine mammary epithelial cells, resulting in elevated lactate dehydrogenase release (4 h after infection). Edema and hyperemia were observed in challenged mammary glands and bacteria were consistently isolated at 12, 24, and 48 h after infection. In addition, numerous neutrophils migrated into gland alveoli and interstitium of infected mammary tissue. We concluded that Strep. lutetiensis had potential to spread within a dairy herd and good adaptive ability in bovine mammary cells or tissue, which are generally characteristics of a contagious mastitis pathogen.

Characterisation of Thai strawberry (Fragaria × ananassa Duch.) cultivars with RAPD markers and metabolite profiling techniques.

Strawberries (Fragaria × ananassa Duch.) are one of the most economically important fruit crops worldwide, several commercially viable cultivars are cultivated in the northern region of Thailand. The morphological characters at the young vegetative seedling stage can be very similar, which has hindered breeding efforts. The present study assesses the ability of random amplification of polymorphic DNA (RAPD) markers and metabolomics techniques to distinguish six strawberry cultivars. Both techniques showed congruent results for the leaf tissue and classified the cultivars into three major clusters. For the most different cultivars, Akihime and Praratchatan No.80, fruits were analysed at eight fruit ripening stages. The data highlighted a broad biological variation at the early ripening stages and less biological variation at the mature stages. Key metabolic differences included the polyphenol profile in Praratchatan No.80 and fatty acid synthesis/oxidation in Akihime. In summary, the RAPD and metabolite data can be used to distinguish strawberry cultivars and elucidate the metabolite composition of each phenotype. This approach to the characterisation of genotypes will benefit future breeding programmes.

Phenotypic and genetic characterisation revealed the existence of several biotypes within the Neorautanenia brachypus (Harms) C.A. wild accessions in South East Lowveld, Zimbabwe.

BACKGROUND: Local communities in the South Eastern Lowveld of Zimbabwe have adopted the feeding of livestock with Neorautanenia brachypus (Harms) C.A. tuber to mitigate against climate change. Differences within Neorautanenia brachypus (Harms) tuber flesh colour and preferences by cattle have been observed, suggesting possible diversity within the N. brachypus plant community. This study aimed at distinguishing the N. brachypus wild plant species through phenotypic and genetic characterization using morphological descriptors and random amplified polymorphic (RAPD) markers respectively. Leaf samples were selected using judgmental sampling techniques from wards 11-15 in Sengwe (Chiredzi district) for leaf morphology and molecular characterization. RAPD-PCR analysis was done using 18-screened random decamer primers to confirm the diversity in the plant population. The similarity of the biotypes was evaluated using binary coding on the basis of the presence or absence of a morphological indicator as well as distinct DNA amplicon fragments. Primer 7.0.13 was used to estimate morphological and genetic similarities using the unweighted pair group method with arithmetic average (UPGMA). The cluster number was estimated using the Elbow method part of the R package. RESULTS: Initially, 14 biotype groups were identified from 96 accessions visually characterized basing of leaf characteristics. All the leaf biotypes displayed arcuate venation with differences observed for leaf shape, tip shape and leaf margins. The 14 biotypes clustered into six groups based on the binary data of the morphological characteristics. RAPD primers generated three hundred and sixty eight distinct amplicons with 77.5% being polymorphic from the 14 biotypes. The number of bands produced per primer ranged from four (OPF-02) to 44 (UBC-746). The PIC value ranged from 0.1327 to 0.1873 for the RAPD primers. Use of molecular markers collapsed the biotypes into five clusters. Both the leaf descriptors and RAPD markers showed the existence of genetic diversity within the wild accessions of N. brachypus. CONCLUSIONS: A combination of morphological and RAPD markers effectively refined the resolution of the genetic diversity within the N. brachypus wild accessions to nine biotypes. These findings have indicated to the existence of more than one biotype of N. brachypus with potentially different properties. The favorable biotypes can further be promoted through incorporation in pastures as alternative feed or complementary feed to livestock. As such the output of this study will serve as a guide for N. brachypus germplasm management and improvement.

Ecological Distribution and Genetic Variations of Some Aloe Species in Taif, KSA.

BACKGROUND AND OBJECTIVES: Aloe is a medicinally and economically important genus. Many Aloes seem an endangered species because of over-collection, destruction of plants and destroyed of natural habitats. The objectives of current study was to survey, collect and identification of some Aloe species and to analyze genetic variations between the collected Aloe species. MATERIALS AND METHODS: Four Aloe species (A. armatissima, A. edentata, A. parvicoma and A. pseudorubroviolacea) and Agave americana (Asperagaceae) were used as plant materials for ecological and genetic studies. In RAPD and ISSR analysis 23 and 16 primers, respectively were screened. RESULTS: Ecological study showed that the 4 species are endemic: 2 are endangered (A. edentata and A. parvicoma) and the others are not-endangered (A. armatissima and A. pseudorubroviolacea), while A. americana was introduced as ornamental species. Concerning RAPD, a total of 134 reproducible bands of them 131 bands are polymorphic ~ 97.65% polymorphism were produced, which ranged from 9 bands (primer OPC-04) to 18 (primer OPA-03) bands, with an average 13.4 bands/ primer, ranging from ~300-2500 bp. According to ISSR, 113 reproducible bands were totally yielded with an average 12.6 bands/primer, from ~180-1500 bp, of which 107 poly-morphic bands number (PBN) ~94.96% polymorphism ranged from 10 bands (primer UBC-818 and primer UBC-819) to 14 (primer UBC-814) with an average of 11.9 PB/primer. CONCLUSION: The results revealed high genetic variations between 4 bands Aloe species and A. americana species, which will be in concern for improvement, breeding and conservation programs.

Determining Phylogenetic Relationships Among Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) Markers.

Investigation of genetic variation and phylogenetic relationships among date palm (Phoenix dactylifera L.) cultivars is useful for their conservation and genetic improvement. Various molecular markers such as restriction fragment length polymorphisms (RFLPs), simple sequence repeat (SSR), representational difference analysis (RDA), and amplified fragment length polymorphism (AFLP) have been developed to molecularly characterize date palm cultivars. PCR-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are powerful tools to determine the relatedness of date palm cultivars that are difficult to distinguish morphologically. In this chapter, the principles, materials, and methods of RAPD and ISSR techniques are presented. Analysis of data generated from these two techniques and the use of these data to reveal phylogenetic relationships among date palm cultivars are also discussed.

Development of diagnostic SCAR markers for genomic DNA amplifications in breast carcinoma by DNA cloning of high-GC RAMP-PCR fragments.

Cancer is genetically heterogeneous regarding to molecular genetic characteristics and pathogenic pathways. A wide spectrum of biomarkers, including DNA markers, is used in determining genomic instability, molecular subtype determination and disease prognosis, and estimating sensitivity to different drugs in clinical practice. In a previous study, we developed highly effective DNA markers using improved random amplified polymorphic DNA (RAPD) with high-GC primers, which is a valuable approach for the genetic authentication of medicinal plants. In this study, we applied this effective DNA marker technique to generate genetic fingerprints that detect genomic alterations in human breast cancer tissues and then developed sequence-characterized amplified region (SCAR) markers. Three SCAR markers (BC10-1, BC13-4 and BC31-2) had high levels of genomic DNA amplification in breast cancer. The PHKG2 and RNF40 genes are either overlapping or close to the sequences of SCAR marker BC13-4, while SCAR marker BC10-1 is in the intron and overlap the DPEP1 gene, suggesting that alterations in the expression of these genes could contribute to cancer progression. Screening of breast cancer cell lines showed that the mRNA expression levels for the PHKG2 and DPEP1 were lower in non-tumorigenic mammary epithelial cell MCF10A, but elevated in other cell lines. The DPEP1 mRNA level in invasive ductal carcinoma specimens was significantly higher than that of the adjacent normal tissues in women. Taken together, high-GC RAMP-PCR provides greater efficacy in measuring genomic DNA amplifications, deletion or copy number variations. Furthermore, SCAR markers BC10-1 and BC13-4 might be useful diagnostic markers for breast cancer carcinomas.

Molecular and phenotypic characterization of methicillin-resistant Staphylococcus aureus isolates from a tertiary hospital in the Philippines.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to public health worldwide. There are relatively few studies addressing the molecular epidemiology of MRSA in the Philippines. METHODS: This study characterized MRSA isolates in terms of their antimicrobial susceptibility profile, the SCCmec type, and the presence of lukF-lukS genes for Panton-Valentine leukocidin (PVL) and determined the relatedness of the isolates by random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR). RESULTS: A total of 236 S. aureus were isolated from clinical specimens of the Makati Medical Center in Makati City, Philippines, between January 2013 and June 2013, and 108 or 45.76 % were found to be MRSA. Results showed that the MRSA strains were resistant to trimethoprim-sulfamethoxazole (20.37 %), azithromycin (10.19 %), gentamicin (5.56 %), and linezolid (4.63 %), while all were susceptible to vancomycin, nitrofurantoin, levofloxacin, minocycline, rifampin, and tetracycline. One isolate was found positive for inducible clindamycin resistance. All of the 108 MRSA strains were confirmed to carry the mecA and SCCmec genes, while the PVL genes were detected in 41 (38 %) of the isolates. Ninety-six isolates (89 %) carried SCCmec type IV, while the remaining isolates carried SCCmec type I (11 isolates) or type III (one isolate). CONCLUSION: This study is the first to present a comprehensive MRSA surveillance data with molecular characterization in a tertiary hospital in the Philippines.

Enumeration and rapid identification of yeasts during extraction processes of extra virgin olive oil in Tuscany.

The aim of this study was to evaluate the occurrence of yeast populations during different olive oil extraction processes, carried out in three consecutive years in Tuscany (Italy), by analysing crushed pastes, kneaded pastes, oil from decanter and pomaces. The results showed yeast concentrations ranging between 10(3) and 10(5) CFU/g or per mL. Seventeen dominant yeast species were identified by random amplified polymorphic DNA with primer M13 and their identification was confirmed by restriction fragments length polymorphism of ribosomal internal transcribed spacer and sequencing rRNA genes. The isolation frequencies of each species in the collected samples pointed out that the occurrence of the various yeast species in olive oil extraction process was dependent not only on the yeasts contaminating the olives but also on the yeasts colonizing the plant for oil extraction. In fact, eleven dominant yeast species were detected from the washed olives, but only three of them were also found in oil samples at significant isolation frequency. On the contrary, the most abundant species in oil samples, Yamadazyma terventina, did not occur in washed olive samples. These findings suggest a phenomenon of contamination of the plant for oil extraction that selects some yeast species that could affect the quality of olive oil.

Penicillium purpurogenum cultures under ethanol-induced stress and its correlation with fungal adhesion and biodegrading ability.

Fungi are known to be affected by external environmental stimuli, resulting in different stress response effects, which in turn could be used to enhance its biodegrading ability. In a previous study, ethanol was used to manipulate cell-cell and cell-surface interaction to prevent cell loss and maximize the usage of Penicillium purpurogenum cells in the media, a correlation was drawn between ethanol oxidative stress, surface-bound proteins and fungal adhesion. The present study focuses on a more detailed study of the effect of ethanol on the same fungus. The results show that the presence of Yap1p gene and the detection of an oxidized form of glutathione (GSSG) suggest that a stress response might be involved in the adhesion process. The process of adhesion could be described as a signaling process and it is affected by the germ tube formation as an initial step in adhesion. Protein profile showed polymorphism in surface-bound proteins for cultures amended with ethanol when compared to control cultures. Ethanol also affected the DNA polymorphic profile of DNA, rendering the fungus genetically variable. P. purpurogenum produced phenol oxidase enzyme and could be used to degrade total phenols in olive mill waste water without the formation of biofilm on the surface of the containers.

Identification of velvet antler by random amplified polymorphism DNA combined with non-gel sieving capillary electrophoresis.

Mitochondrial DNA of velvet antler was amplified with random amplified polymorphic DNA (RAPD) technique and the PCR products were detected with non-gel sieving capillary electrophoresis to establish a RAPD-HPCE method used for identifying the authenticity of velvet antler or it counterfeits. Factors that could affect the PCR amplification and capillary electrophoresis were optimized. Under the optimized conditions, namely, 20 mmol L(-1) NaH2PO4-Na2HPO4-2 mmol L(-1) EDTA buffer solution [0.8% (W/V) HPMC, 15 mmol L(-1) TBAP and pH 7.3], -10 kV injection voltage and -8 kV separation voltage, Cervus nippon Temminck antler, Cervus elaphus Linnaeus antler, Rangifer tarandus antler, Cervus canadensis antler and Elaphurus davidianus antler were analyzed. The analysis on the similarity of obtained elctrophoretograms showed that there were significant differences in similarities of different velvet antlers, which could be used for the quick identification of the authenticity of velvet antler samples. It can be found that the technique of RAPD combined with HPCE is advantageous in rich polymorphism, high detection rate, simple and convenient performance, high efficiency, rapidness and sensitivity, indicating that it should be suitable for the quick identification of the authenticity of velvet antler samples.

Modulation of mitochondrial membrane integrity and ROS formation by high temperature in Saccharomyces cerevisiae

Background Yeast strains are exposed to numerous environmental stresses during industrial alcoholic fermentation. High temperature accumulated acetic acid, enhanced the growth inhibition and decreased ethanol production. Results In this study the influence of high temperature on the cellular and mitochondrial membrane integrity of Saccharomyces cerevisiae as well as reactive oxygen species (ROS) formation was investigated to understand the mechanisms of the high temperature fermentation process. However, increasing the temperature to 42°C resulted in a clear decrease in the cytoplasmic and mitochondrial membrane potential and an increase in intracellular ROS formation. It was also determined that the different thermostability between YZ1 and YF31 strains had a clear correlation with the yeast's intracellular trehalose content of the cell. Finally, random amplified polymorphic DNA (RAPD) was used to explore the genome differences between the YZ1 and YF31 strains. Conclusions Thus, the stability of the mitochondrial membrane and subsequently, the clearance ROS ability could be important factors for the viability of S. cerevisiae at high temperatures.

Identification of structural DNA variations in human cell cultures after long-term passage.

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8-12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.

Late-onset neonatal group B streptococcal disease associated with breast milk transmission: molecular typing using RAPD-PCR.

Group B Streptococcus (GBS) is considered to be the major cause of neonatal sepsis and meningitis of bacterial origin. Late-onset GBS infection is infrequent and occurs between 1 week and 3 months of age. The transmission of GBS through the ingestion of breast milk is reported in the literature, but only a few of these cases have been confirmed by molecular techniques. In this article we report five cases of late-onset GBS disease: transmission through maternal milk was confirmed in four cases, using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) typing assay. In addition, the RAPD-PCR assay showed that each of the isolated clones belonged to a different RAPD genotype, thus revealing that the late-onset GBS infections were not epidemiologically related.

First evidence of fish genotoxicity induced by heavy metals from landfill leachates: the advantage of using the RAPD-PCR technique.

Municipal leachates are loaded with heavy metals that can contaminate surface water before discharge into a receiving body of water. The aim of this study is to evaluate the genotoxic effects of heavy metals generated by domestic waste on the common roach Rutilus rutilus in the last of the four interconnected ponds at the Etueffont landfill. We used random amplified polymorphic DNA (RAPD) since it has been shown to be a powerful means of detecting a broad range of DNA damage due to environmental contaminants. Our results show the ability of RAPD analysis to detect significant genetic alterations in roach DNA, after contamination with a set of metals contained in the landfill leachates in comparison to a roach from a non-polluted reference pond. Analysis of electrophoresis profiles indicates apparent changes such as the appearance of new bands or disappearance of bands as compared to the control. In fact, mixed smearing and laddering of DNA fragments in muscle samples support the genotoxic effects of metal deposits in the roach. This study is the first evidence found via the RAPD-PCR technique in the detection of pollutant impacts on fish exposed to landfill leachates.

Genoprotectivity of methanol and ethanol extracted leaf sap of Trigonella foenum-graecum in Allium cepa root assay.

Fenugreek (Trigonella foenum-graecum) of Fabacecae family is widely distributed throughout the world and used as an old medicinal plant and traditional food. The present study deals with the investigation of the anti-genotoxic potential of methanol (MTG) and ethanol (ETG) extracted leaf sap of fenugreek on Allium cepa root tip cells, which were treated with cadmium sulfate (CdSO(4)). Three types of treatments were applied. First, roots were treated with different concentrations of methanolic and ethanolic extracts (0.1%, 0.5% and 1%) separately for 3 h each, followed by CdSO(4) treatment (at 250 ppm, for 3 h). Second, roots were first treated with CdSO(4) followed by extracts treatment. Third, root tips were treated with CdSO(4) with extracts treatments at the same time. For controls, roots with CdSO(4) (250 ppm) and distilled water served as positive and negative control, respectively. The results showed that the methanol and ethanol extracts of fenugreek modulated the genotoxic and clastogenic aberrations, which were induced by CdSO(4). The protection activity of MTG (1%) was 50% in the first treatment, 70% in the second treatment and 82% in the third treatment and 61%, 68% and 88% of ETG (1%), respectively. DNA rearrangements were also observed by revealing new RAPD bands in the total DNS samples isolated from Allium roots after treatmenst.