Random Amplified Polymorphic DNA Analysis Research Articles

Molecular characterization and multifunctional evaluation of lactic acid bacteria isolated from traditional sourdough

The primary objective of this study was to characterize lactic acid bacteria (LAB) strains derived from sourdough for possible utilization as functional starters to produce sourdough and various cereal-based fermented foods. A total of 350 autochthonous LAB strains were isolated from 65 Type I sourdough samples and characterized using six random amplified polymorphic DNA (RAPD) primers at intra- and interspecific levels. Species identification of selected strains representing distinct clusters from RAPD analysis was performed based on the 16S rRNA region. The LAB strains were identified as Companilactobacillus crustorum (n = 135), Levilactobacillus brevis (n = 125), Latilactobacillus curvatus (n = 40), Companilactobacillus paralimentarius (n = 32), and Lactiplantibacillus plantarum (n = 18). A total of 66 LAB strains were selected for technological characterization along with two commercial strains. The characterization involved acidity development, EPS production potential, leavening activity, and growth abilities under harsh conditions. Principle component analysis (PCA) identified 2 Lp. plantarum and 14 Lev. brevis strains as the most relevant technologically. Among them, Lp. plantarum L35.1 and Lev. brevis L37.1 were resistant to tetracycline. Evaluation of probiotic characteristics (survival in pH 2.5 and bile presence, auto aggregation capacity, hydrophobic activity, antioxidant activity, antimicrobial activity) by PCA identified four strains with relevance to Lactobacillus rhamnosus GG (LGG), which were further selected for in vitro digestion assays. Lactiplantibacillus plantarum L7.8, Lev. brevis L55.1, and L62.2 demonstrated similar viability indices to LGG, along with increased auto aggregation capacity and antioxidant activity. These strains are promising as candidate starters for producing sourdough and sourdough-related fermented food products.

Genetic diversity of maize resources revealed by different molecular markers

Maize (Zea mays L.) is the third most important cereal crop in the world because of its nutritional value and industrial benefits. Molecular markers are used mainly by the breeders to study the genetic variability of genotypes and its application in the breeding process. Two types of molecular markers, 10 random amplified polymorphic DNA (RAPD) primers and 10 start codon target (SCoT) primers, were assayed to determine the genetic diversity of 25 Slovak maize lines and 25 maize cultivars. A high level of polymorphism was found with both RAPD and SCoT markers, which was confirmed by high average polymorphism information content (PIC) values using both techniques. The efficiency of individual marker techniques in the detection of genotype diversity can be compared by calculating the marker index (MI), detecting diversity index (DDI), discriminating power, resolving power (RP) and other indices. A higher MI (11.788), DDI (2.358) and RP (53.08) value was achieved by the SCoT technique compared to the RAPD method. Three joint dendrograms and PCoA plots constructed based on RAPD, SCoT and both methods combined confirmed the unambiguous separation of maize lines and cultivars from each other. The results obtained from the RAPD and SCoT analysis can be used for the selection of potentially suitable biological sources for further marker-assisted breeding.

Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an everincreasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg L-1 BAP, 0.3 mg L-1 GA3, and 0.3 mg L-1 ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity.

Screening of among seventeen sunflowers (Helianthus annuus L.) genotypes for oil content and the first flowering day using random amplified polymorphic DNA markers

Background Sunflower refers to the tribe Helianthus, subtribe Helianthinae, and family Asteraceae, which collectively contains 20 genera and 400 species. An important oilseed crop that yields edible oil is Helianthus annuus L. Objective The primary goal of the current study was to assess the genetic diversity of 17 genotypes of sunflower (Helianthus annuus L) To measure the oil content during the initial flowering period and to reach the highest percentage of oil can be obtained from the first flowering day. Materials and methods Five RAPD (random amplified polymorphic DNA) primers were used to detect the genetic diversity of the 17 sunflower hybrid genotypes obtained from Spain. Phylogenetic relationships of 17 sunflower genotypes were determined using three replications and 6 m lines on August 15, 2019, at the National Research Centre farm in Nubaria as part of a donation from the German corporation (strobe), Spain. To analyze the genetic diversity and phylogenetic linkages in sunflower germplasm, DNA fingerprinting and the Random Amplified Polymorphic DNA (RAPD) molecular marker approach were also used. Results and conclusion The oil content of 17 sunflower genotypes (Helianthus annuus L.) was assessed, with values ranging from 46 to 50%, with the highest values falling into five genotypes. However, the two genotypes were found to have the lowest oil percentage (46%). The early age and oil percentage differed among the varieties. In the Tornado and Elves genotypes, the longest and shortest days were 59 and 47, respectively. The means and standard errors for all statistical data are reported. Statistical significance was evaluated using the LSD. P values were considered statistically significant at P less than or equal to 0.05. According to the findings, RAPD primers generated 49 bands with a size range of 0.1–3 kb and an 87.75% polymorphism percentage. For RAPD, 43 polymorphic bands with distinct bands were observed. Morphological features and RAPD analysis separated the UPGMA Dendrogram into three groups. Jaccard’s coefficient was used to analyze the genetic similarity matrix, and a morphological study revealed that Tornado and Elvas, both from Spain, shared the most genetic similarity (0.970). RAPD analysis and morphological features are useful in identifying genetic variants. Conclusion, according to our findings, Helianthus annuus L. has a significant variation ratio. Indicating substantial diversity across the 17 sunflower genotypes, the genetic similarity index calculated using pooled data from RAPD markers showed an extensive range from 0.645 to 0.986. This study may be a reference for future research on Helianthus annuus L. and may support breeding initiatives and species concepts.

Genetic Diversity Analysis of Bitter Gourd (Momordica charantia L.) Germplasm using Molecular Markers

Bitter gourd [Momordica charantia L.] is an important cucurbitaceous crop of medicinal and nutraceutical value. The present study was conducted to compare the efficacy of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) molecular markers for genetic diversity analysis of bitter gourd. Twenty-one genotypes of bitter gourd, collected from different parts of India, were used for diversity analysis using two different types of markers. RAPD and ISSR markers yielded an average of 7.16 and 5.73 amplicon per primer, respectively. In the present study, RAPD and ISSR showed 89 and 63% polymorphic bands, respectively. Combined data analysis of RAPD and ISSR markers provided 73.66% polymorphism with 5.33 polymorphic amplicons per primer. The better discriminatory power of ISSR markers over RAPD markers may be due to comparatively higher values of average polymorphic information content (0.175) as well as the diverse nature of the genotypes. The information generated in this study would be helpful in designing breeding strategies for bitter gourd improvement.

Genotypic and antimicrobial susceptibility of Streptococcus agalactiae causing bovine mastitis in the central region of Thailand.

Streptococcus agalactiae is a highly contagious pathogen that causes bovine mastitis, leading to significant economic losses. This study aimed to (1) identify and characterize S. agalactiae strains responsible for bovine mastitis by examining their phenotypic and genotypic characteristics in Thai dairy-intensive farming areas and (2) determine their susceptibility profiles to antimicrobial agents. In total, 100 S. agalactiae isolates obtained from clinical and subclinical mastitis cases from 13 dairy herds located in the central region of Thailand were examined. To confirm the identity of the bacterial pathogens, conventional microbiological procedures recommended by the National Mastitis Council (NMC) and the VITEK® 2 system were employed. All 100 isolates were successfully identified as S. agalactiae using the NMC procedure, whereas 94 isolates were identified as S. agalactiae using the VITEK® 2 system. Finally, the S. agalactiae-specific gene dlt S was identified in all the examined isolates using polymerase chain reaction. Capsular polysaccharide (CPS) typing revealed that all strains belonged to CPS type Ia. Multilocus sequence typing identified 33 selected isolates as sequence type 103. Random amplified polymorphic DNA (RAPD) typing yielded 43 RAPD types, with 6 RAPD clusters identified. These results demonstrated a high level of genetic diversity among S. agalactiae within the studied herds. RAPD analysis suggested that specific S. agalactiae strains could persist in dairy farms for 2-12 months. Furthermore, antimicrobial susceptibility testing was performed using the broth microdilution method. Most strains demonstrated susceptibility to ampicillin, penicillin, penicillin/novobiocin, cephalothin, oxacillin, ceftiofur, and erythromycin. This study revealed the phenotypic and genotypic characteristics of S. agalactiae isolates responsible for bovine mastitis in the central region of Thailand. The rapid identification of S. agalactiae and application of molecular typing methods can provide valuable epidemiological information regarding S. agalactiae causing mastitis in dairy farms. The antimicrobial susceptibility of S. agalactiae indicates that antimicrobial treatment for control and eradication could be a successful protocol. Our findings revealed that a single clonal strain of S. agalactiae affected the 13 studied farms. Further research is needed to explore the feasibility of vaccine development and application.

Evaluation of Biological Control of Sorghum Strains Using Bacillus Thuringiensis and Pseudomonas Aeruginosa Under Drought Stress

Background: Sorghum is an economically significant staple food crop for more than half a billion people in developing nations, especially in arid and semi-arid locations where drought stress is a significant limiting factor. Despite usually being regarded as tolerant, sorghum suffers severely from drought stress, which lowers its productivity and nutritional quality throughout its principal cultivation areas. Objective: Improvements in DNA fingerprinting by ISSRs, SSRs, and RAPD markers have also been employed in sorghum genetic modification (GMOs) to enhance the economic characteristics of this crop. Materials and methods: To provide a natural defence against pests, the most tolerant plants among the seven varieties of sorghum bicolour were selected and planted in the second season of 2020–2021 under treatment with two microorganisms, B. thuringiensis and P. aeruginosa. This study considered seven varieties of sorghum bicolour planted under 50% water deficiency in 2019–2020. Genetic variability analysis of sorghum genotypes was performed using seven Inter-Simple Sequence Repeat (ISSR) primers, six Simple Sequence Repeat (SSR) primers, and five Random Amplified Polymorphic DNA (RAPD) primers. Seven Sorghum bicolour accessions were collected from various regions of Egypt and their phylogenetic relationships were evaluated. Additionally, DNA fingerprinting and analyses of the genetic diversity and evolutionary linkages in the sorghum germplasm employed the (ISSR) molecular marker technique. Results and conclusion: The Fisher Least Significant difference test (LSD) at P < 0.05, based on RAPD, ISSR, and SSR markers demonstrated a significant connection. The findings demonstrated that 51 bands with a size range of 100–1500 bp and polymorphism percentage of 72.5% were created using five RAPD primers. Seven ISSR primers generated 45 bands With a 57.8(%) polymorphism percentage, ranging in size from 100 to 3000 bp. six SSR primers generated 28 bands with (67.86%) polymorphism percentage of 67.86 %, ranging in size from 100 to 1500 bp. Morphological characteristics and ISSR, SSR, and RAPD analyses were used to group the UPGMA Dendrogram into groups. Jaccard's coefficient was used to analyse the genetic similarity matrix. The maximum similarity was observed for ISSR between Hybrid Sh1 and Hybrid Sh306 (0.984%), SSR between Hybrid Sh306 and Sudan grass (0.964%), and RAPD between Giza 15 and Indian Millet (0.706%). The classification of sorghum germplasm, breeding initiatives, and conservation efforts rely heavily on the determination of the genetic diversity among sorghum species. Identification of genetic variants, morphological features, and genetic analysis of ISSR, SSR, and RAPD are useful techniques. These findings demonstrate a large ratio of variation in sorghum. This work could serve as a guide for future research on sorghum and aid in the understanding of species and breeding initiatives.

Detected Genetic Markers for Three Varieties of Rice (Oryza sativa L.) under Nano- Particles

Despite the enormous advancements in breeding, fresh and accessible methods of plant development are preferred. Many research have been done to determine the impact of zinc oxide nanoparticles (ZnO NPs) and magnesium nanoparticles (MgONPs) on plant growth. ZnO NPs and MgONPs have been used extensively in agriculture. Few research have looked into the potential impact of ZnO NPs on grain Zn content or cereal yield formation to date. The goal of the study was to demonstrate how varied concentrations of nanoparticles affected root germination in three different rice types (Giza 177, Sakha super 300, and Sakha 108) and the fungus Fusariummoniliforme, which causes foot rot in rice. Here, we performed a pot experiment to assess the effects that ZnO nanoparticles and MgONPs had on the shoot length, root length, and germination of rice. The experiment involved three dosages of Zn and Mg nanoparticle solution at varying concentrations of 10, 20, and 100 ppm. The outcomes showed that, when compared to the control treatment, ZnO NPs and MgONPs increased the length of the shoot, the length of the roots, and the rate of germination. Using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers, the genetic makeup of plants treated with 10, 20, and 100 ppm AgNPs was also examined. In vitro rooted shoots were acclimated in a greenhouse before being tested for biochemical stability and phenotypic stability. In vitro, callogenesis and caulogenesis were both reduced by AgNPs at the greatest dose (100 ppm). In order to maximise genome coverage, three rice cultivars from distinct origins were chosen, and they were tested using three RAPD markers and seven selected ISSR markers. AgNPs-treated plants using ISSR markers produced 87 amplified bands in total, 45 polymorphic allelic variants, and 36 monomorphic allelic variations. The Pearson correlation coefficient was used in the Mantel test to determine whether there was a significant association between the Jaccard's dissimilarity matrices based on ISSR and RAPD markers (r = 0.69; P 0.05). Morphological characteristics, ISSR, and RAPD analysis were used to partition the UPGMA Dendrogram into two groupings. Jaccard's coefficient was used to analyse the genetic similarity matrix. Sakha Super 300 and Giza 177 had the most genetic similarity (95%) while Sakha 108 and Giza 177 had the lowest genetic similarity (90%) according to ISSR and RAPD study. The classification of rice germplasm, breeding initiatives, and conservation efforts all heavily rely on the determination of genetic diversity within the rice species. To find genetic variations, morphological characteristics, ISSR, and RAPD analysis are useful techniques. The findings demonstrated the large ratio of variation in rice. This work successfully established the possibility of using MgONPs and ZnO NPs as high-performing fertilisers to improve rice output and quality.This study could serve as a guide for future research on rice and could support efforts to understand the species and improve breeding stock.

Genetic diversity and some biological characteristics of antibacterial yeasts isolated from natural honey and beeswax in Son La province

Yeast living in honey, an environment with high sugar content (up to 70 %, w/v), normally shows good resistance to the high level of osmotic pressure; they are of high potential for application in many fields. There were not many studies on the genetic diversity and biological characteristics of yeast from honey in Vietnam. This study aims to (1) evaluate the genetic diversity of antibacterial yeast isolated from natural honey and beeswax in Son La province by RAPD (Random Amplified Polymorphic DNA) and (2) study some biological characteristics of them. The research results may contribute to the scientific basis for screening yeast strains applied in different fields such as bioethanol and probiotic production. Sixty-eight yeast strains were isolated from natural honey and beeswax collected in Son La. Among them, twenty-one strains showed antibacterial activity against at least a tested bacterium including Escherichia coli, Bacillus subtilis, and Serratia marcescens. These yeast strains were genetically distinct in the RAPD analysis using M13 and (GTG)5 primers. Evaluation of yeast growing in the medium containing high glucose concentration (30 - 40 %, w/v) or high ethanol concentration (5 - 10 %, v/v) has shown some yeast strains that can tolerate high osmotic pressure and high ethanol concentration for different applications. YC.8 and YC.61 strains exhibited relatively good survival rates in two phases of digestion and have a wide pH range (2 - 7). YC.8 strain expressed the most potential for human or animal probiotics.

Toxic effects of sodium dodecyl sulfate on planarian Dugesia japonica.

Sodium dodecyl sulfate (SDS) is an anionic surfactant, which is widely used in various fields in human life. However, SDS discharged into the water environment has a certain impact on aquatic organisms. In this study, planarian Dugesia japonica (D. japonica) was used to identify the toxic effects of SDS. A series of SDS solutions with different concentrations were used to treat planarians for the acute toxicity test , and the results showed that the semi-lethal concentration (LC50) of SDS to D. japonica at 24 h, 48 h, 72 h, and 96 h were 4.29 mg/L, 3.76 mg/L, 3.45 mg/L, and 3.20 mg/L respectively. After the planarians were exposed to 0.5 mg/L and 1.0 mg/L SDS solutions for 1, 3, and 5 days, the activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) content were measured to detect the oxidative stress and lipid peroxidation in planarians. Random amplified polymorphic DNA (RAPD) analysis was performed to detect the genotoxicity caused by SDS to planarians. The results showed that the activities of SOD, CAT, and MDA content increased after the treatment, indicating that SDS induced oxidative stress in planarians. RAPD analysis showed that the genomic template stability (GTS) values of planarians treated by 0.5 mg/L and 1.0 mg/L SDS for 1, 3, and 5 days were 67.86%, 64.29%, 58.93%, and 64.29%, 60.71%, 48.21%, respectively. GTS values decreased with the increasing of SDS concentration and exposure time, indicating that SDS had genotoxicity to planarians in a time and dose-related manner. Fluorescent quantitative PCR (qPCR) was used to investigate the effects of SDS on gene expression of planarians. After the planarians were exposed to 1.0 mg/L SDS solution for 1, 3, and 5 days, the expression of caspase3 was upregulated, and that of piwiA, piwiB, PCNA, cyclinB, and RAD51 were downregulated. These results suggested that SDS might induce apoptosis, affect cell proliferation, differentiation, and DNA repair ability of planarian cells and cause toxic effects on planarian D. japonica.

Dye decolorization of magnetically retrievable formulation of laccase with amine-functionalized microparticles

Laccase is an important enzyme used in many industries because of its multi-substrate catalyst. New immobilization agents are excellent tools for enhancing the abilities of this enzyme. In this study, immobilization of laccase on silica microparticles with NH2 (S-NH2) surface modification to use in dye removal applications was aimed. The yield of immobilization by this method was found to be 93.93 ± 2.86% under optimum conditions. In addition, this newly created immobilized enzyme was adapted to a decolorization application with 87.56 ± 1.60% efficiency. Silica microparticles with NH2 (S-NH2) surface modification were used for laccase immobilization and this immobilized laccase had quite good potential. Besides, Random Amplified Polymorphic DNA (RAPD) analysis in evaluating the toxicity of the decolorization process was utilized. After amplification with two RAPD primers, decreased toxicity of dye in this study was observed. This study showed that RAPD analysis in toxicity testing could be accepted as an alternative and practical method that this approach will contribute to the literature in terms of providing fast and reliable results. The use of amine-modified surface silica microparticles for laccase immobilization and RAPD for toxicity testing is a crucial aspect of our investigation.

Evaluation of Wild Almond Genotypes Grown as a Rain-fed Crop in Sulaimani Governorate using RAPD and ISSR Markers

Almond is considered the most common and essential nut grown in the rainfed area. It has many species wildly distributed in the Iraqi Kurdistan region, which are called Chaqala by local people. To understand and improve the characteristics of the cultivated almonds, a comprehensive study of wild almonds in terms of population structure and genetic diversity is needed to transfer new traits into the domesticated almonds. So, twelve different genotypes were collected and analyzed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. The results revealed polymorphic bands for both markers, the mean value of 5.8 for the RAPD marker and 7.8 for the ISSR marker was also documented. The polymorphic information content (PIC) values were obtained for RAPD primes that range between (0.64-0.85) and ISSR primers were also verified between (0.53-0.91), which shows the discriminatory power of these markers. “Jaccard similarity coefficients” were obtained between 0.34 (G11 vs. G12) to 0.77 (G1 vs. G10), and clustered into four groups with a mean similarity (0.65) for ten RADP markers. For ten ISSR markers, 0.35 (G8, G10) to 0.79 (G6 vs. G11) were also observed, which is clustered into a mean similarity (0.65). Structure analysis of RAPD and ISSR showed two major groups. Overall, these outcomes showed the diversity between the studied genotypes and among groups which are highly important for future almond breeding and conservation programs.

Analysis of the Genetic Distance of Several Generations of Barley (Hordeum valulgare L) by RAPD-PCR Technique

Random-amplified-polymorphic-DNA(RAPD) was assayed to detect the genetic variation of 6 barley generations from Iraq. Four primers generated a total of 17 scoreable bands in RAPD analysis) and resolving power, the three polymorphic primers differed (Rp). The use of RAPD marker systems to detect the genetic distance among barley generation was discovered to be beneficial. The RAPD dendrograms indicate a diverse grouping of 6 barely specimens, although we did see that certain groups were identical in several cases. As a result, the RAPD molecular markers reveal two genetic groups in the few specimens examined. Keywords. Barley, Genetic variation, RAPD-PCR.

Genetic Variation of Vanda foetida J. J. Sm. ; a Rare and Endemic Orchid in South Sumatra Based on RAPD Analysis

Vanda foetida J.J.Sm., which is an endemic orchid species in Sumatra has taxonomically disappeared for over 100 years since it was described in 1906. The distribution of V. foetida was limited due to over-exploitation of this orchid and changes to land-use. The study of genetic variation of V. foetida in its natural habitat, Mount Dempo and Padiampe, South Sumatra was conducted based on Random Amplified Polymorphic DNA (RAPD) analysis for genetic variation using 8 primers (OPU 3, OPU 5, OPU 6, OPU 7, OPU 12, OPU 13, OPU 14 and OPU 16). Genetic variation in each population was detected by h value (Nei’s genetic diversity) which was 0.1999 and 0.1778 for Mount Dempo and Padiampe, respectively. This value was higher compared to those of other rare orchid species even though it only has a small population. V. foetida originated from two populations forming two main clusters in dendrogram with 67 % (0.67) degree of similarity. The dendrogram indicated that the two populations are connected, and assumed as one large population in the past and were separated from larger population by habitat fragmentation. This species was not genetically in danger and will be able to survive if its natural habitats are remained.

Molecular Characterization of Different Soybean [Glycine max (L). Merril] genotypes by RAPD Markers

The present investigation was carried out on molecular characterization of different soybean genotypes by using RAPD markers. Genetic diversity of 10 soybean genotypes was studied using Random Amplified polymorphic DNA (RAPD). 12 RAPD primers were used for screening 10 soybean genotypes from which total 334 fragments were amplified. It was observed that 41.10% bands were polymorphic and 58.88% were monomorphic. The percent of polymorphic amplicons in banding pattern was calculated and it was highest in OPA-01 (67.85%) followed by OPA-11 (66.66%). Polymorphism information content (PIC) was calculated and highest PIC value was observed in OPA-01 (0.26) and lowest was observed in OPA-08 and OPA-05 (0.03). Maximum amplicons were produced in OPA-01 (56) and minimum in OPA-08 (11). Genetic relationship between soybean genotypes was determined on the basis of Jaccard’s pair wise similarity coefficient values and dendrogram was generated by UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cluster analysis using dice’s similarity matrix through NT-SYS pc software. The dendrogram of RAPD analysis shows 8 clusters and sub clusters were revealed. The value of similarity coefficient ranged from 0.19 to 0.71. The maximum similarity percentage i.e. 71% was found between KDS-726 and KDS-922 genotypes. The minimum similarity i.e. 19% was found between IVT-33 and IVT-13. According to this information it is concluded that the KDS-726 and KDS-922 genotypes showed minimum genetic diversity as compare to the IVT-33 and IVT-13 genotypes. Therefore, understanding the genetic diversity of soybean genotypes is essential to broaden the genetic base and to further utilize in the MAS breeding program.

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.

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. 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 2500bp. 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 2500bp. 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%. 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.

Antimicrobial resistance pattern, virulence determinants and molecular analysis of carbapenem-resistant Klebsiella pneumoniae isolated from clinical samples in Iran.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as an important global threat in recent years. The objective of the present study was to characterize the molecular characteristics, antibiotic resistance pattern and the distribution of virulence factors in CRKP isolated from different clinical specimens. A total of 60 clinical CRKP isolates were collected from clinical samples. Based on Clinical Laboratory Standards Institute guidelines, antimicrobial susceptibility testing was assessed by the disk diffusion method. Carbapenem and aminoglycoside resistance determinants in addition to virulence genes were inspected by PCR. Molecular characteristics of CRKP isolates were analyzed by random amplified polymorphic DNA (RAPD) PCR and enterobacterial repetitive intergenic consensus (ERIC) PCR. All isolates were resistant to imipenem, meropenem, cefoxitin, levofloxacin, cefotaxime, ceftazidime and ciprofloxacin. Resistance to tetracycline, gentamicin and kanamycin were detected in 53%, 75% and 72% of isolates, respectively. The most common carbapenem resistance genes were OXA-48 (28.5%) and NDM (22%). The most common aminoglycosides resistance genes were aac6´Ib (57%) and aac(3)-IVa (28%). The most prevalent virulence genes were mrkD (82%), entB (62%) and ybts (58%). ERIC and RAPD analyses revealed 55 and 53 different patterns of CRKP isolates, respectively. We conclude that CRKP infections have been associated with different genotypes and that the carbapenemase type (OXA-48) and AME gene (aac6´-Ib) were widely distributed in CRKP isolates in our hospital. Continued compliance with existing phenotypes and genotypes, and strict enforcement of infection control guidelines, are recommended approaches for the prevention and dissemination of these strains.

Genetic analysis of micro propagated plants of Tinospora cordifolia (Willd.) Miers by using RAPD–PCR markers

Random Amplified Polymorphic DNA (RAPD) is the simplest and cost-effective tool used for the analysis of the genetic fidelity of in vitro propagated plants of Tinospora cordifolia. A high-quality genomic DNA free from any contaminants, was isolated from five genotypes by the modified CTAB method. Out of 30 random decamer primers, OPA-02, OPA-03, OPA-09, OPA-10, OPB-02, OPB- 04, OPB-09 and OPK-06 showed fingerprints. Out of total 27 scored bands, 26 produced were monomorphic bands (96.2%) and one was polymorphic band (3.8%). The highest number of bands (6) were produced by OPA -03 and OPK-06 and minimum number of bands (1) was produced by OPB -04. The amplicon size varied from 200 bp to 1500 bp. A coefficient similarity value ranging from 0.87–0.93 was obtained. Maximum similarity value 0.93 was exhibited by both the genotypes namely, SB1 and SB3. From the present study, it has been concluded that the RAPD analysis can be used to check the genetic fidelity in the micro propagated plants of Tinospora cordifolia.

Genotoxicity Evaluation of Metformin in Freshwater Planarian Dugesia japonica by the Comet Assay and RAPD Analysis.

Objective To investigate the genotoxicity of metformin on planarian with different concentrations and exposure times. Methods The planarians were treated, respectively, with 10 mmol/L and 50 mmol/L metformin for 1, 3, and 5 days, and then, the comet assay and random amplified polymorphic DNA (RAPD) analysis were performed. 13 random primers were used for PCR amplification with the genomic DNAs as templates. Planarians cultured in clear water were used as the control. Genomic template stability (GTS) was calculated by comparing and analyzing the RAPD patterns of the control group and the treatment groups. Results In the comet assay, DNA damage of planarians treated with 10 mmol/L metformin for 1, 3, and 5 days was 10.2%, 25.4%, and 36.8%, respectively, and that of planarians treated with 50 mmol/L metformin was 40.6%, 62.8%, and 65.4%, respectively. GTS values of planarians exposed to 10 mmol/L metformin for 1, 3, and 5 days were 64.1%, 62.8%, and 52.6%, respectively, and those of planarians exposed to 50 mmol/L metformin for 1, 3, and 5 days were 52.6%, 51.3%, and 50%, respectively. DNA damage increased and GTS values decreased with the increasing metformin exposure concentration and exposure time. Conclusion Metformin has certain genotoxicity on planarian in a dose- and time-related manner. The comet assay and RAPD analysis are highly sensitive methods for detecting genotoxicity with drugs.

Genetic diversity and growth responses of Indonesian tomato (Solanum lycopersicum L.) genotypes under lead stress.

Tomato (Solanum lycopersicum L.) is cultivated and consumed worldwide, including in Indonesia. It is used in the food, cosmetic, and pharmaceutical industries, due to its high content of carotenoid (lycopene) compounds that have antioxidant and anticancer activities. In Indonesia, although several cultivars of tomato are cultivated, including Opal, Permata, Mutiara, and Rewako, studies on their genetic information are limited. Unpredicted climate change as well as heavy metal contamination, especially Pb pollution, has threatened Indonesian food security. Therefore, our study aimed to analyze the genetic diversity of the four local tomatoes using random amplified polymorphic DNA (RAPD) markers and to determine the growth responses of several local tomato genotypes under Pb stress. In this study, morphological responses to Pb, including plant height and root length were observed. The RAPD analysis showed that Rewako and Permata were distinct, whereas Opal and Mutiara were closely related, possessing 81.8% similarity. Pb stress influenced plant height and root length in the four tomato genotypes, with each genotype exhibiting different morphological responses than others. However, the closely related Mutiara and Opal genotypes demonstrated similar responses to Pb stress to Permata and Rewako. Our study demonstrates that RAPD are sensitive and efficient for elucidating the genomic profile of the tomato genotypes. In addition, our results suggest that genetic variation among tomato genotypes might influence the morphological responses against Pb stress.