RAPD Research Articles

RAPD MARKERS ARE EFFECTIVE TOOL FOR THE DIFFERENTIATION OF COMMON AND TARTARY BUCKWHEAT GENOTYPES

Common and tartary buckwheat are important cultivated species of the genus Fagopyrum. Genetic polymorphism of thirty-five genotypes of common buckwheat (Fagopyrum esculentum Moench) and tartary buckwheat (Fagopyrum tataricum Gaertn.) was analyzed using 10 random amplified polymorphic DNA (RAPD) primers. A total of 119 DNA fragments were amplified using ten RAPD primers with an average of 11.9 fragments per primer. The number of amplified fragments ranged from 6 (OPB-08) to 16 (SIGMA-D-01). PIC values ranged from 0.782 (OPC-08) to 0.919 (SIGMA-D-01) with an average of 0.871 per primer. The marker index (10.364) and diversity detecting index (2.961) were high and presented the utility of used marker technique. To evaluate the genetic relationships among buckwheat genotypes a phylogenetic tree based on UPGMA algorithm was constructed. The genotypes of common and tartary buckwheat were separated independently into cluster I and II. Cluster I separated 14 genotypes of tartary buckwheat into two subclusters (Ia, Ib). Genotype 903016, which originated in Pakistan, was separated individually into the subcluster Ia. Cluster II included genotypes of common buckwheat and was further subdivided into subcluster IIa and IIb. Two genotypes of subcluster IIb (Bamby and Hruszowska) were genetically the closest. Bamby and Hruszowska reflected the maximum similarity value (0.605) according to Jaccard’s coefficient of similarity. Based on the presented data it is suggested that the RAPD technique is suitable for differentiation among genotypes of common and tartary buckwheat. RAPD primers have proven to be reliable and useful method for studying genetic variability of buckwheat genotypes.

Selection of starter lactic acid bacteria capable of forming biofilms on wooden vat prototypes for their future application in traditional Sicilian goat's milk cheese making

In this study, 327 presumptive lactic acid bacteria (LAB) were isolated from goats' milk acid curds produced at a Sicilian dairy farm with the aim to identify potential starter cultures for traditional cheeses. All isolates were first processed by randomly amplified polymorphic DNA (RAPD)-PCR analysis. This approach identified 63 distinct strains which were evaluated for their acidifying capacity. Only 15 strains specifically stood out for their acidification capacity and were identified through 16S rRNA gene sequencing as Lactococcus lactis (11 strains) Enterococcus faecalis (three strains), and Ligilactobacillus animalis (one strain). Notably, all 15 LAB isolates produced bacteriocin-like inhibitory substances and anti-biofilm compounds, against both planktonic and biofilm forms of Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli, and Staphylococcus aureus, albeit at varying levels. Among these 15 LAB, En. faecalis RGM25 and Lc. lactis RGM55, susceptible to five antibiotics tested, were put in contact with wooden vat prototypes, because all equipment used in traditional cheese production in Sicily are made of wood. Scanning electron microscopy and bacterial plate counts of the wooden vat prototypes showed the development of biofilms at levels of approximately 6.0 log CFU/cm2. Overall, this study contributes to establishing a custom-made LAB starter cultures with bio-preservatives properties for Sicilian cheese productions.

Virulence factors, antifungal susceptibility and molecular profile in Candida species isolated from the hands of health professionals before and after cleaning with 70% ethyl alcohol-based gel

Fungal infections in neonatal intensive care units (NICU) are mainly related to Candida species, with high mortality rates. They are predominantly of endogenous origin, however, cross-infection transmitted by healthcare professionals' hands has occurred. The aim of this study was to identify Candida species isolated from the hands of healthcare professionals in a NICU before and after hygiene with 70% ethanol-based gel and evaluate virulence factors DNase, phospholipase, proteinase, hemolysin, biofilm biomass production, and metabolic activity. In vitro antifungal susceptibility testing and similarity by random amplified polymorphic DNA (RAPD) were also performed. C. parapsilosis complex was the most frequent species (57.1%); all isolates presented at least one virulence factor; three isolates (Candida parapsilosis complex) were resistant to amphotericin B, two (Candida famata [currently Debaryomyces hansenii] and Candida guilliermondii [currently Meyerozyma guilliermondii]) was resistant to micafungin, and six (Candida parapsilosis complex, Candida guilliermondii [=Meyerozyma guilliermondii], Candida viswanathi, Candida catenulata [currently Diutina catenulata] and Candida lusitaniae [currently Clavispora lusitaniae]) were resistant to fluconazole. Molecular analysis by RAPD revealed two clusters of identical strains that were in the hands of distinct professionals. Candida spp. were isolated even after hygiene with 70% ethanol-based gel, highlighting the importance of stricter basic measures for hospital infection control to prevent nosocomial transmission.

Micropropagation and Genetic Fidelity of Fegra Fig (Ficus palmata Forssk.) and Grafting Compatibility of the Regenerated Plants with Ficus carica.

Ficus palmata is an important fig species that produces edible and nutritious fruit and possesses several therapeutic uses. This study reports an effective method for the micropropagation of F. palmata using nodal explants. In vitro shoots were cultured for 7 weeks onto MS medium fortified with different concentrations of cytokinins, light intensities, sucrose concentrations, and light/dark incubation treatments. Optimal axillary shoot proliferation (10.9 shoots per explant) was obtained on a medium containing 30 g/L sucrose and supplemented with 2 mg/L 6-benzylaminopurine (BAP) under 35 μmol/m2/s light intensity. Dark incubation limited the foliage growth but favored shoot elongation and rooting compared with light incubation. Elongated shoots, under dark conditions, were rooted (100%; 6.67 roots per explant) onto MS medium containing 1 mg/L indole-3-acetic acid (IAA) and 1.5 g/L activated charcoal. The micropropagated plantlets were acclimatized with a 95% survival rate. In this study, the genetic fidelity of micropropagated F. palmata clones along with their mother plant was tested using randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), and start codon targeted (SCoT) molecular markers. The genetic similarity between the micropropagated plantlets and the mother plant of F. palmata was nearly 95.9%, assuring high uniformity and true-to-type regenerated plants. Using micropropagated F. palmata plantlets as a rootstock proved appropriate for the grafting F. carica 'Brown Turkey'. These findings contribute to the commercial propagation and production of the fig crop.

Unveiling Genetic Variation in Garlic Genotypes in Response to Rust Disease Using RAPD Markers

Garlic (<em>Allium sativum</em>), cultivated worldwide for its medicinal and nutritional value, faces challenges due to diseases caused by various pathogens. In this study, eleven garlic genotypes from Iran and one from China were selected and sown under natural infection rendered by the rust fungus (<em>Puccinia alli</em>) over two consecutive years. Subsequently, disease distribution and severity, percentage of infection and susceptibility of different garlic genotypes to rust disease were investigated. The results showed that genotype Solan was the most susceptible, with disease severity of 30.81%. In comparison, genotypes Aliabad and Bahar were resistant against rust disease with the lowest infection percentages of 13% and 16.37%, respectively. Further, genetic diversity was assessed using random-amplified polymorphic DNA (RAPD) markers. Out of 10 primers used, 95 scorable bands were generated, of which 66 (69.48%) were found to be polymorphic. A dendrogram was constructed based on RAPD polymorphism using the UPGMA method, and the genotypes were separated into six distinct clusters based on Jaccard's coefficient of similarity. Additionally, it was observed that there is no genetic differentiation among the genotypes based on their geographical origin. This study highlights the significant diversity in resistance and susceptibility among garlic genotypes, which can be harnessed in plant breeding programs.

Zinc oxide and silver effects on the growth, pigment content and genetic stability of chrysanthemums propagated by the node culture method

ABSTRACT This article describes benefits of the application of zinc oxide submicron particles (ZnO SMPs), zinc oxide nanoparticles (ZnO NPs) and ZnO NPs combined with silver NPs (ZnO + Ag NPs) in chrysanthemum micropropagation. Single node explants of Chrysanthemum × morifolium (Ramat.) Hemsl. ‘UTP Burgundy Gold (UBG)’ and ‘UTP Pinky Gold (UPG)’ were inoculated on the Murashige and Skoog (MS) medium and treated with 100 mg · L−1, 200 mg · L−1, or 400 mg · L−1 ZnO SMPs, ZnO NPs (1.5% H2O), ZnO NPs (6% H2O), ZnO + 0.1% Ag NPs (1.5% H2O), ZnO + 0.1% Ag NPs (6% H2O), ZnO + 1% Ag NPs (1.5% H2O) and ZnO + 1% Ag NPs (6% H2O). Generally, the tested materials stimulated the growth and development of plantlets. In ‘UBG’, the most prominent treatments affecting increases in the number of leaves, micropropagation coefficient, shoot length and shoot FW/DW weight included 400 mg · L−1 ZnO SMPs and 100 mg · L−1 ZnO NPs (6% H2O). In ‘UPG’, the treatments with 200 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) and 200 mg · L−1 ZnO + 1% Ag NPs (6% H2O) were the most successful. The latter treatment stimulated an intensive development of root systems in the two studied cultivars. High values of leaf area, perimeter and width were reported in both cultivars for 400 mg · L−1 ZnO + 1% Ag NPs (6% H2O). As compared to the control, the treated plants were characterised by a similar or, most often, lower content of chlorophylls and carotenoids. The randomly amplified polymorphic DNA (RAPD) and start codon targeted polymorphism (SCoT) marker system analyses of the 400 mg · L−1 ZnO SMPs/ZnO NPs/ZnO + Ag NPs-treated chrysanthemums confirmed their genetic fidelity with the control plants. The obtained results can be implemented in the commercial large-scale production of chrysanthemums.

Randomly Amplified Polymorphic DNA (RAPD) Molecular Markers as a Valuable Tool to Confirm the Genetic Diversity in Soybean Germplasms (Glycine max)

Randomly Amplified Polymorphic DNA (RAPD) Molecular Markers as a Valuable Tool to Confirm the Genetic Diversity in Soybean Germplasms (Glycine max)

Molecular typing and virulence characteristics of Escherichia coli strains isolated from hospital and community acquired urinary tract infections.

The main causes of hospital- and community-acquired urinary tract infections (UTIs) are a group of Escherichia coli (E. coli) strains with multiple virulence factors known as uropathogenic E. coli. One hundred E. coli isolates from the urine specimens of hospital- and community-acquired UTI patients were characterized based on their virulence factors and genetic relatedness using PCR and RAPD‒PCR, respectively. Among all, the traT (71%), sitA (64%), ompT (54%), malX (49%), ibeA (44%), tsh (39%), hlyD (18%) and cnf1 (12%) genes had the highest to lowest frequencies, respectively. There was no significant difference between the frequency of tested virulence genes in E. coli isolates from inpatients and outpatients. The frequency of the hlyD gene was significantly greater in E. coli isolates from patients hospitalized in gynecology, dermatology and intensive care unit (ICU) wards than in those from other wards. Eight virulence gene patterns were common among the isolates of inpatients in different wards of the same hospital, of which five patterns belonged to the isolates of inpatients in the same ward. More E. coli isolates with similar virulence gene patterns and greater genetic similarity were found in female patients than in male patients. The analysis of the RAPD‒PCR dendrograms revealed more genetic similarities among the E. coli isolates from inpatients than among those from outpatients. Our findings indicate the presence of a wide variety of virulence factors in E. coli isolates and the possibility of spreading the same clones in different wards of the hospital.

RAPD PCR genotyping of <i>Enterococcus faecium</i> isolated from urinary tract infection and correlation between biofilm formation with antibiotic resistance and virulence genes

RAPD PCR genotyping of <i>Enterococcus faecium</i> isolated from urinary tract infection and correlation between biofilm formation with antibiotic resistance and virulence genes

The Genetic Homogeneity of Uganda’s East African Highland Bananas (Mutika/Lujugira) Does Not Match the Extensive Morphological Variation Identified in this Subgroup

The East African Highland banana (Mutika/Lujugira subgroup) is composed of triploid (AAA) cooking and beer banana varieties that are adapted to the high-altitude region of the Great Lakes region of East Africa. Banana production is affected by several biotic and abiotic factors. Breeding opportunities in bananas are limited due to female sterility and parthenocarpy. The genetic diversity of crops enables breeders to develop new germplasm. Molecular markers have been used widely to dissect crop plants’ genetic diversity. This study assessed the genetic variation in 27 varieties from the Mutika/Lujugira subgroup using random amplified polymorphic DNA (RAPD). No genetic variation was observed among the banana varieties, and the 18 ten-mer primers produced monomorphic banding profiles. The genetic homogeneity of this banana subgroup is not congruent with their extensive morphological variation. Domestication and the bottleneck effect are often cited as the cause of reduced diversity in crop plants. On the other hand, several mechanisms, including somatic mutations, transposable elements, polyploidy, genome plasticity, and epigenetic mechanisms, are known to increase plant phenotypic variability. Further in-depth research is needed to explain the puzzle between the genetic and morphological diversity in the Mutika/Lujugira subgroup.

Phenotypic and Molecular Diversity of Wild Populations of Acca sellowiana (Berg.) Burret in the Southern Area of Natural Distribution

Acca sellowiana is a subtropical tree in the myrtle family (Myrtaceae) native to southern Brazil and northeastern Uruguay. It is recognized for its value as a fruit-bearing, ornamental, and medicinal species. Based on distinctive characteristics of fruits, seeds, and leaves, as well as its geographical distribution pattern, two variants of the species are distinguished: the “Brazilian type” and the “Uruguayan type”. The objective of this study was to characterize, for the first time, the diversity of 202 individuals from four wild populations in Uruguay, representative of the species’ most southern natural distribution. Twenty-three morphological descriptors (leaf, flower, and fruit) and 204 RAPD (Random Amplified Polymorphic DNA) markers were used. The morphological data collected validated the main criteria that distinguish “Uruguayan type” populations from “Brazilian type” populations, such as lower seed weight and fruit size, thin and slightly rough skin, high pulp percentage, and hairy white abaxial leaf surfaces. Analyses of both morphological and molecular data indicated wide diversity and strong population structuring, which is relevant information for designing conservation plans, sustainable utilization, and genetic improvement of the plant genetic resources of this species.

Effects of Selenium on DNA Methylation and Genomic Instability Induced by Drought Stress in Wheat (Triticum aestivum L.)

The main purpose of the study was to clarify the effect of selenium (Se) on DNA damage and DNA methylation in wheat (Triticum aestivum L.) plants exposed to polyethylene glycol (PEG)-induced drought stress under in vitro tissue culture. Random amplified polymorphic DNA (RAPD) and coupled restriction enzyme digestion-random amplification (CRED-RA) were utilized to explain the DNA damage grade and variations in DNA methylation patterns, respectively. The outcomes indicate that drought stress gives rise to a rise in RAPD profile variations (as DNA damage) and a decrease in genomic template stability (GTS) rate and DNA methylation changes. According to the RAPD data, the greatest GTS value was computed at 56.9% (5% PEG 6000), and the lowest GTS value was 41.2% (15% PEG 6000), demonstrating the adverse effects of PEG 6000. However, DNA damage can be reduced by treatment with sodium selenate (2, 4, and 6 µM of Na2SeO4) together with PEG (5%, 10%, and 15% PEG 6000)-induced water deficits. Moreover, according to CRED-RA analysis, PEG-induced DNA methylation rates were changed after treating different doses of Se. These data demonstrate that Se dose-dependently modulates both DNA damage and methylation alterations induced by drought in wheat.

Genetic variability analysis of tomato varieties using rapd markers under heat stress

Heat stress threatens agricultural and food security by reducing yield and causing plant death. The use of intra-genetic diversity in heat stress responses is a potential avenue for harnessing tolerance to climate change circumstances. This study investigated the genetic diversity of twelve tomato genotypes in Bangladesh, focusing on their suitability for summer cultivation using seven Random Amplified Polymorphic DNA (RAPD) markers. The banding patterns obtained from the PCR results were used to identify genetic differences between individuals. The RAPD markers were found to show their effectiveness in discriminating the studied lines through the computation of average Polymorphism Information Content (PIC) values, particularly focusing on dominant markers with PIC values ranging from 0 to 0.5, emphasizing the informative nature of the employed primers. Cluster analysis grouped several genotypes with possible common ancestry. Overall, BARI Hybrid Tomato 4, BARI Hybrid Tomato 8, A12, D13, and RHS1 emerged as promising candidates for further genetic research and breeding programs, particularly in the context of heat-stress conditions during summer tomato cultivation in Bangladesh. Bangladesh J. Bot. 53(1): 91-100, 2024 (March)

Genetic diversity in see-see partridge (Ammoperdix griseogularis, Galliformes) populations from sub-Himalayan Mountain ranges of Pakistan

We used Random Amplified Polymorphic DNA (RAPD) markers to investigate the genetic structure of two populations of see-see partridge (Ammoperdix griseogularis, Galliformes) from the Suleiman range, in the Pakistani Himalayan region. The see-see partridge is a vulnerable species with a distribution in the Middle East and central Asia. The percentage of polymorphic bands (94.05%), Shannon Index (H = 0.455) and Nei’s average gene diversity (I = 0.298) of A. griseogularis at species level were rather high when compared with other avian species. 17% of polymorphic loci showed statistically significant differences in their allelic frequencies. The G (Nei’s coefficient of genetic variation) values indicated low levels of differentiation (G = 0.08). A genetic distance D of 0.05 indicated that both populations were to some degree in isolation but their differentiation was not significant. Overall, our genetic data can support action plans aiming to locally preserve differentiated genetic resources that, in the future, could potentially result in ecologically and behaviourally differentiated populations. In view of the rapid environmental changes that the Himalayan region has been experiencing in the last decade, this study could help in conservation plans.

Random Amplified Polymorphic DNA (RAPD) Markers Protocol of Bacterial Isolates from Two selected General Hospitals Wastewater (HWW)

In this research work, Random amplified polymorphic DNA (RAPD) markers Protocol was used to characterize bacterial isolates from two selected General Hospitals Waste Water (HWW). The hospital environment and its wastes accumulate diseases from both inward and outward patients. It is pertinent to investigate the wastewater and study its microbial community. Wastewater samples were collected from two major General Hospitals in Akoko area, namely, Ikare and Akungba-Akoko General Hospitals. Samples were microbiologically examined for the presence of bacterial colonies. Isolated bacterial species were preliminarily identified through conventional biochemical tests and were further characterized using 16s rRNA sequencing protocol. The bacterial gene was amplified using selected primers. DNA was isolated, purified, and amplified using 16s rRNA gene sequence protocol, and the results were analyzed and assessed with the standard bacteria sequence in the NCBI database. Molecular evolutionary analyses and Phylogenetic trees were mapped out from the results obtained. it was observed that the Bacterial counts in Cfu/ml range between 10.0 x 103Cfu/ml to 24.0 x 103Cfu/ml. The highest bacterial count was observed from the test sample from Ikare followed by Akungba Akoko General Hospitals with the Cfu/ml value of 24.0 x 103 and 18.0 x103 Cfu/ml respectively. Molecular data sequence, when compared with NCBI database using BLAST showed 99.60 –99.87% bactaria phylogenic identity and E-value equal to 0 for all closely related taxa. The following bacteria were characterized using Random amplified polymorphic DNA (RAPD) markers protocol namely: Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis. this study has revealed the presence of different bacterial species in hospital waste water which pose threat to public health. Hence, proper monitoring and sewage treatment should be encouraged in different hospital settings before the disposal of these wastewaters to the public water runways

Genetic diversity of maize resources revealed by different molecular markers

AbstractMaize (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.

Molecular Characterization of Extended Spectrum β-Lactamase-Producing Escherichia coli: Insights into the O25b-ST131 Clone in Mexican Urinary Tract Infections

Background: The urgent need for antimicrobial research to address the escalating global challenge of β-lactam antibiotic resistance, particularly in Escherichia coli-induced urinary tract infections (UTI), is underscored by the increasing resistance to ciprofloxacin in Latin America. This issue has led to a heightened dependence on alternative therapeutics, such as cephalosporins. The identification of extended-spectrum β-lactamase (ESBL)-producing E. coli, notably the O25b-ST131 clone, adds complexity to UTI management. The prevalence of ESBL-producing E. coli varies globally due to factors including regional antimicrobial usage practices. Objectives: The goal of this study was to identify and molecularly characterize ESBL-producing E. coli isolates to identify the pandemic O25b-ST131 clone related to UTIs in one healthcare institution in Mexico. Methods: Bacterial species identification and antibiotic susceptibility tests were performed using the VITEK 2. The ESBL genes were identified using polymerase chain reaction (PCR). The E. coli genotyping was carried out by the phylogenetic group analysis and the O25b-ST131 identification. Results: A total of 86 unique E. coli isolates were confirmed as ESBL, and 75% were obtained from UTIs. The most prevalent β-lactamase genes were blaCTX-M (66%), blaTEM (8.1%), blaCTX-M/SHV (5.8%), blaCTX-M/TEM (4.6%), and blaSHV (2.3%). The B2 phylogroup was most prominent (54.4%), with 46.5% identified as a globally pandemic O25b-ST131 clone. No evident relationship was observed using random amplified polymorphic DNA (RAPD) between nosocomial and community-acquired infections in ESBL-producing E. coli isolates. Conclusions: The obtained findings highlight the significance of monitoring molecular epidemiology in antibiotic resistance profiles of the O25b-ST131 E. coli clone.

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.

Identification and Biological Characterization of a New Cyst Nematode, Heterodera glycines sbsp.n. tabacum, Parasitizing Tobacco in China.

In an investigation of diseases from plant-parasitizing nematodes in Henan Province, a cyst nematode was found on tobacco roots and in rhizosphere soil. We identified this strain as a new cyst nematode subspecies, Heterodera glycines sbsp.n. tabacum. The cysts and second-stage juveniles (J2s) parasitizing Henan tobacco were larger than those of H. glycines. A single 345-bp fragment was amplified from H. glycines sbsp.n. tabacum, whereas the 345- and 181-bp fragments were amplified from the soybean cyst nematode. Thus, H. glycines sbsp.n. tabacum was distinct from H. glycines. There were base transversions at 504 sites and base transitions at 560, 858, 920, and 921 sites in the rDNA-ITS sequences of H. glycines sbsp.n. tabacum compared with H. glycines, and there were base transitions at 41, 275, 278, and 380 sites in the mtDNA-COI sequences. In the phylogenetic tree based on the rDNA-ITS and mtDNA-COI regions, H. glycines sbsp.n. tabacum was clustered on a single branch. Based on the randomly amplified polymorphic DNA (RAPD) technique, sequence characterized amplified region (SCAR)-PCR primers were designed. A single 1,113-bp fragment was amplified by specific primers (HtF1/HtR1) from H. glycines sbsp.n. tabacum, while no fragments were obtained from H. glycines. The H. glycines sbsp.n. tabacum can infect soybean plants but cannot complete its life cycle on soybean. Eleven tested tobacco cultivars were infected, with an average reproduction factor (Rf) of 9.74 and a maximum of 64.2 in 'K326'. The cumulative egg hatching rate of H. glycines sbsp.n. tabacum in the presence of tobacco root exudates was 42.6% at 32 days posthatching, which was significantly greater than that in the presence of soybean root exudates (30.3%) or sterile water (33.1%). In summary, the cyst nematode population parasitizing Henan tobacco was identified as a new subspecies, H. glycines sbsp.n. tabacum.

Random Amplified Polymorphic DNA (RAPD)-PCR analysis of genotypic and phenotypic characteristics of MethicillinResistant <i>Staphylococcus epidermidis</i> (MRSE) strains involved in biofilm formation

Purpose: The current study aims to investigate genotypic and phenotypic aspects of methicillinresistant Staphylococcus epidermidis (MRSE) strains involved in the biofilm formation and the attendance of the icaADB gene and genotypic characterization of this gene by random amplified polymorphic DNA (RAPD) PCR.Methods: 60 Staphylococcus epidermidis strains were isolated from clinical specimens, suspected of having the bacteria, from the laboratories of Isfahan. Biofilm formation was measured by the microtiter plate method. The attendance of biofilm formation genes was studied using PCR and all isolates producing biofilm (strong and moderate) were genetically classified by RAPD-PCR.Results: 37 isolates (61.7%) were MRSE and all positive biofilm strains. The prevalence of biofilmrelated genes in the isolates was SesC (100%), SesI (45.9%), icaA (29.7%), icaB (37.8%), icaC (81.08%), icaD (70.2%), arcA (81.08%), and opp3AB (70%). PCR analysis showed that among 30 isolates of strong and medium biofilm production, 70% (21/30) positive for the icaADB gene. The Dendrograms obtained from RAPD-PCR results showed that all nine main clusters were at an 80% similarity level, and there were four isolates of a single type.Conclusion: These findings confirmed the high genotypic diversity of biofilm-producing MRSE strains and the relative diffusion of specific clones among clinical samples.