Random Amplified Polymorphic DNA Patterns Research Articles

Species Identification of Pufferfish Products Using RAPD Analysis.

A simple and rapid method was developed to identify the source species of pufferfish products. Randomly amplified polymorphic DNA (RAPD) analysis was applied to identify 8 species of pufferfish. Commercial kits were used for DNA extraction and amplification. Simultaneous identification was possible by polyacrylamide gel electrophoresis of PCR products. Two primers were chosen based on the result of pre-examination with 40 primers, and the PCR conditions were optimized. Characteristic RAPD patterns were obtained for each pufferfish species. The developed method was applied to identify the source species of 26 pufferfish products. The results suggest that the developed method would be useful for verification of the labeled species of pufferfish products.

Prevalence of high-level gentamicin-resistant Enterococcus faecalis in an Iranian hospital

This study was designed to determine the molecular characteristic and antimicrobial resistance of e nterococcal strains isolated from patients in an Iranian Hospital. Enterococcal strains were isolated from the burn patients. All strains were screened for genes encoding resistance to aminoglycoside [ aac(6 ' )-Ie-aph(2 '' )-Ia , aph (3') , ant (4' )], resistance to vancomycin ( vanA , vanB ), resistance to tetracycline ( tetK , tetL , tetM , tetO ), and resistance to erythromycin ( ermA , ermB , ermC ) by PCR and multiplex PCR–based methods. Genetic diversity was evaluated using Random Amplified Polymorphic DNA (RAPD)-PCR. All enterococcal isolates showed complete sensitivity to vancomycin with MIC ≤ 0.5µg/ml. Resistance to gentamicin, tetracycline, erythromycin, ciprofloxacin or quinopristin-dalfopristin was detected, whilst more than 96.2% of isolates were high-level gentamicin-resistant (HLGR) and multiresistant. The most prevalent aminoglycoside resistance gene was aac(6 ' )-Ie-aph(2 '' )-Ia, found in 96.2% (26/27) of the isolates. The most prevalent tetracycline resistance gene was tetM , found in 85.1% (23/27) of the isolates followed by tetL and tetO found in 7.4% (2/27) of the isolates. The ermA and ermB genes were detected in 33.3% (9/27) and 44.4% (12/27) of the isolates respectively. Random Amplified Polymorphic DNA (RAPD)-PCR analysis yielded 17 distinct profiles among 27 investigated isolates. One clusters of isolates shared the same RAPD patterns, while 16 isolates had unique RAPD patterns. we found that over the examined time period one RAPD genotype were common type and were disseminated among patients in the burn unit. Interestingly, most of these strains had an identical or very similar antibiotic and gene resistance pattern.

Analyses of somaclonal variation in endosperm-supported mature embryo culture of rye (Secale cereale L.)

ABSTRACTGenetic variations, and especially somaclonal variations, are undesirable in genetic transformation. In this study, random amplified polymorphic DNA (RAPD) and coupled restriction enzyme digestion-random amplification (CRED-RA) markers were used for detection of the variation in calli that were obtained from endosperm-supported mature embryo of rye on Murashige and Skoog (MS) medium containing different auxins (2,4-D, dicamba and picloram) at a range of different concentrations (2, 4, 6, 8, 10 and 12 mg/L). High level of auxins caused a reduction in the genomic template stability (GTS) value. While the highest GTS was observed in the calli maintained on MS medium with 2 mg/L dicamba (98.4%), the calli maintained on MS medium with 12 mg/L picloram were found to show the least GTS (81.7%) when RAPD patterns were analysed. Epigenetic changes were more frequent and variable than genetic changes when compared to RAPD and CRED-RA results. DNA hypermethylation was observed at higher concentrations of 2,4-D and picloram, whereas DNA hypomethylation was observed in dicamba. These results indicate that RAPD and CRED-RA techniques can be used for detection of somaclonal variation in in vitro cultures, which is a fundamental step in plant genetic transformation.

Genotoxicity evaluation of ionic liquid 1-octyl-3-methylimidazolium bromide in freshwater planarian Dugesia japonica using RAPD assay

The randomly amplified polymorphic DNA (RAPD) assay has been used to detect DNA alternation and mutation recently. However, the effectiveness of this method in detecting DNA damage in planarians, a model organism for assessing the toxicity of environmental pollutants is unknown. In the present study, RAPD assay was used to detect the DNA damage in planarians treated by the ionic liquid 1-octyl-3-methylimidazolium bromide ([C8mim]Br) for the first time. Among the 20 test RAPD primers, 13 primers with 60–70% GC content produced unique polymorphic band profiles. A total of 60 bands were observed in the untreated control planarians. In comparison with the control group, the [C8mim]Br-treated groups displayed differences in RAPD patterns in the band intensity, disappearance of normal bands and appearance of new bands. The variation of RAPD profiles showed both concentration- and time-effect relationships. Meanwhile, the genomic template stability (GTS) of treated planarians decreased and exhibited negative correlation to the exposure concentration and time of [C8mim]Br. Our results suggested that [C8mim]Br had genotoxic effects on planarians, and this DNA damage analysis would lay the foundation for further elucidating the toxicity mechanisms of ionic liquids on planarians. Furthermore, RAPD analysis was proved to be a highly sensitive method for the detection of DNA damage induced by environmental pollutants like toxic chemicals on planarians.

Hypervirulent Klebsiella pneumoniae induced ventilator-associated pneumonia in mechanically ventilated patients in China.

The purpose of this study was to investigate the clinical characteristics of hypervirulent K. pneumoniae (hvKP) induced ventilator-associated pneumonia (VAP) and the microbiological characteristics and epidemiology of the hvKP strains. A retrospective study of 49 mechanically ventilated patients with K. pneumoniae induced VAP was conducted at a university hospital in China from January 2014 to December 2014. Clinical characteristics and K. pneumoniae antimicrobial susceptibility and biofilm formation were analyzed. Genes of capsular serotypes K1, K2, K5, K20, K54 and K57 and virulence factors plasmid rmpA(p-rmpA), iroB, iucA, mrkD, entB, iutA, ybtS, kfu and allS were also evaluated. Multilocus sequence typing (MLST) and random amplified polymorphic DNA (RAPD) analyses were used to study the clonal relationship of the K. pneumoniae strains. Strains possessed p-rmpA and iroB and iucA were defined as hvKP. Of 49 patients, 14 patients (28.6%) were infected by hvKP. Antimicrobial resistant rate was significantly higher in cKP than that in hvKP. One ST29 K54 extended-spectrum-beta-lactamase (ESBL) producing hvKP strain was detected. The prevalence of K1 and K2 in hvKP was 42.9 % and 21.4%, respectively. The incidences of K1, K2, K20, p-rmpA, iroB, iucA, iutA, Kfu and alls were significantly higher in hvKP than those in cKP. ST23 was dominant among hvKP strains, and all the ST23 strains had identical RAPD pattern. hvKP has become a common pathogen of VAP in mechanically ventilated patients in China. Clinicians should increase awareness of hvKP induced VAP and enhance epidemiologic surveillance.

Characterization ofListeria monocytogenesfrom various sources by Random Amplified Polymorphic DNA (RAPD) and serotyping

Genetic diversity among Listeria monocytogenes isolates recovered from livestock food products (milk and meat products), vegetables and human clinical cases with bad obstetrics history were studied. A total of 259 samples were screened for isolation, identification and characterization of L. monocytogenes by serotyping PCR and RAPD profiling. In the present investigation, 11.1% prevalence of L. monocytogenes has been recorded. Of these, 5 (19.2%) isolates were from vegetables, 14 (17.5%) from milk, 2 (9.5%) from milk products, 3 (5.7%) from meat, 1 (8.3%) meat products and 4 (5.8%) from humans clinical cases with bad obstetrics history. Serotyping PCR study revealed presence of serotype 1/2b and 4b. RAPD profiling resulted into 5 clusters indicating five major patterns of L. monocytogenes. All clusters included the isolates from various sources. However, cluster 3 and 5 included isolates from human and milk origin, respectively. RAPD pattern 4 and 5 included the isolates belonging to 1/2b or 1/2b and 4b serovar, whereas, other pattern, included L. monocytogenes belonging to various serovars. Prevalence of L. monocytogenes in different food samples signifies its potential as food born pathogen and RAPD-PCR offers greater discrimination of strains.

Genotyping Of Lactococcus garvieae Isolated From Rainbow Trout By RAPD-PCR Using M13 And P5 Primers In Turkey

Event Abstract Back to Event Genotyping Of Lactococcus garvieae Isolated From Rainbow Trout By RAPD-PCR Using M13 And P5 Primers In Turkey Muhammed DUMAN1, Soner ALTUN1, Izzet B. SATICIOĞLU1* and Ayşe G. BÜYÜKEKİZ1 1 Uludag University, Department of Aquatic Animal Diseases, Türkiye Aquaculture production is considerably large and total aquaculture production was reached 260.000 tons in 2015, in Turkey. Rainbow trout (Oncorhynchus mykiss) production is about %50 of these total fish production. Especially, when water temperature is above 15 ° C, Lactococcosis infection occurs in rainbow trout farms (L. garvieae, synonym Enterococcus seriolicida). The aim of this study was to evaluate the efficiency of randomly amplified polymorphic DNA (RAPD) method and to compare the discriminatory powers of M13 and P5 primers for genotyping L. garvieae isolates. In this study, L. garvieae isolated on rainbow trout farms in different regions (Marmara, Aegean, Central Anatolia, Southeast and Eastern Anatolia) of Turkey in 2013-2015 years. Isolated bacteria were identified by classic microbiologic tests (Gram staining, oxidase, catalase, Oxidation/Fermentation), rapid identification kits (API 32 STREPT) and molecular analysis (PCR). RAPD PCR analysis was also applied for determining epidemiologic and genotypic relationship of 137 L. garvieae. 137 strains have been identified by PCR analyses. Strains were separated into groups by analyzing RAPD patterns. While 5 different genotypes have been identified by using M13 primer which determined 95 isolates (69.3%) were found similar profiles, 4 different genotypes have been identified using P5 primer which determined 96 isolates (70 %) were found similar profiles. Aegean and Central Anatolia regions’ strains found genetically similar to L. garvieae strains. While there were five different genetic groups of L. garvieae isolates in 2013, two different genetic groups were found isolates in 2014-2015 with M13 primer. As a result; similarity of L. garvieae strains which isolated from trout farms in Turkey have found at least 69% with M13 and P5 primers. When RAPD-PCR analysis results were compared with M13 and P5 primers, it was concluded that M13 primer had more binding sites on DNA and discriminatory power of the M13 primers was found to be higher than P5. Keywords: Lactococcus garvieae, genotyping, RAPD-PCR, M13 primer, P5 primer Conference: AquaEpi I - 2016, Oslo, Norway, 20 Sep - 22 Sep, 2016. Presentation Type: Poster Topic: Aquatic Animal Epidemiology Citation: DUMAN M, ALTUN S, SATICIOĞLU IB and BÜYÜKEKİZ AG (2016). Genotyping Of Lactococcus garvieae Isolated From Rainbow Trout By RAPD-PCR Using M13 And P5 Primers In Turkey. Front. Vet. Sci. Conference Abstract: AquaEpi I - 2016. doi: 10.3389/conf.FVETS.2016.02.00017 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 May 2016; Published Online: 14 Sep 2016. * Correspondence: MD, PhD. Izzet B SATICIOĞLU, Uludag University, Department of Aquatic Animal Diseases, Bursa, Türkiye, iburcinsat@uludag.edu.tr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Muhammed DUMAN Soner ALTUN Izzet B SATICIOĞLU Ayşe G BÜYÜKEKİZ Google Muhammed DUMAN Soner ALTUN Izzet B SATICIOĞLU Ayşe G BÜYÜKEKİZ Google Scholar Muhammed DUMAN Soner ALTUN Izzet B SATICIOĞLU Ayşe G BÜYÜKEKİZ PubMed Muhammed DUMAN Soner ALTUN Izzet B SATICIOĞLU Ayşe G BÜYÜKEKİZ Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Distribution of plasmid-mediated quinolone resistance in Gram-negative bacteria from a tertiary hospital in Nigeria.

Until recently, mechanisms of resistance to quinolones in Gram-negative bacteria were believed to be only chromosome encoded. However, emergence of plasmid-mediated quinolone resistance (PMQR) has been reported worldwide. This study investigated distribution of PMQR in Gram-negative bacteria from a tertiary hospital in eastern part of Nigeria. Seventy-one nonduplicate Gram-negative bacterial isolates of eight species were analyzed for antimicrobial susceptibility, genotypic detection of various PMQRs, typed by random amplified polymorphic DNA (RAPD) and analysis of plasmids present, including replicon typing. The minimum inhibitory concentrations showed MIC90values as high as 256 μg/ml for fluoroquinolones. Carriage of PMQR was found to be 35.2%. Twenty (28.2%) isolates carried various qnr genes, of which seven (9.9%) qnrA1; four (5.6%) qnrB1; eight (11.3%) qnrS1 while one (1.4%) encoded qnrD1. Eighteen (25.4%) isolates were positive for aac(6')-Ib-cr while carriage of multiple genes exists in some strains. Similarly, 13 isolates (18.7%) were found to carry PMQR efflux pump gene, qepA. Conjugation experiments revealed that the plasmids once transferred coded for fluoroquinolone resistance. The transconjugant strains carried a common plasmid estimated to be 65 kb. These plasmids were untypable for replicon/incompatibility. Typing revealed high diversity among all species tested with no identical RAPD pattern seen. This study further confirms high level resistance to many antimicrobials in different species of Gram-negative bacteria including fluoroquinolones and spread of PMQR genes in Southern Nigeria.

Application of the Random Amplified Polymorphic DNA (RAPD) Fingerprinting to Analyze Genetic Variation in Community Associated-Methicillin Resistant Staphylococcus Aureus (CA-MRSA) Isolates in Iran.

The aim of this study was to apply RAPD technique to analyze the genetic variability among the Iranian CA-MRSA isolates.The RAPD amplification was implemented on 25 strains isolated from the anterior nares of 410 healthy children using four randomly selected oligonucleotide primers from the stocks available in our laboratory, including the primers 1254, GE6, OLP6 and OLP13 from our stock. The amplified PCR products were detected on a 1.5% agarose gel and subjected to further analysis to establish the band profiles and genetic relationships using the Gel Compar® program.The Iranian CA-MRSA isolates produced distinct RAPD patterns which varied based on the primer used, however, the primer 1254 revealed highly polymorphic patterns consisting 5 discernable RAPD types (RT), “RT1” (12, 48%), “RT2” (8, 32%), “RT3” (3, 12%), and “RT4 and RT5”, (a single RAPD type each, 4%). Phylogenetic analysis based on RAPD profiles divided most of the CA-MRSA isolates into 2 distinct but related RAPD clusters, a small group and two single unrelated RAPD types.This study shows that the simple and cost-effective but rather difficult to optimize RAPD fingerprinting could be used to evaluate genetic and epidemiological relationships of CA-MRSA isolates on condition that the patterns are obtained from carefully optimized laboratory tests.

Disease Burden of Invasive Listeriosis and Molecular Characterization of Clinical Isolates in Taiwan, 2000-2013.

The information about disease burden and epidemiology of invasive listeriosis in Asia is scarce. From 2000 to 2013, a total of 338 patients with invasive listeriosis (bacteremia, meningitis, and peritonitis) were treated at four medical centers in Taiwan. The incidence (per 10,000 admissions) of invasive listeriosis increased significantly during the 14-year period among the four centers (0.15 in 2000 and >1.25 during 2010–2012) and at each of the four medical centers. Among these patients, 45.9% were elderly (>65 years old) and 3.3% were less than one year of age. More than one-third (36.7%) of the patients acquired invasive listeriosis in the spring (April to June). Among the 132 preserved Listeria monocytogenes isolates analyzed, the most frequently isolated PCR serogroup-sequence type (ST) was IIb-ST87 (23.5%), followed by IIa-ST378 (19.7%) and IIa-ST155 (12.1%). Isolation of PCR serogroups IIb and IVb increased significantly with year, with a predominance of IIb-ST87 isolates (23.5%) and IIb-ST 228 isolates emerging in 2013. A total of 12 different randomly amplified polymorphic DNA (RAPD) patterns (Patterns I to XII) were identified among the 112 L. monocytogenes isolates belonging to eight main PCR serogroup-STs. Identical RAPD patterns were found among the isolates exhibiting the same PCR serogroup-ST. In conclusion, our study revealed that during 2000–2013, listeriosis at four medical centers in Taiwan was caused by heterogeneous strains and that the upsurge in incidence beginning in 2005 was caused by at least two predominant clones.

Genetic characterization of antibiotic-resistant Staphylococcus aureus from milk in the North-West Province, South Africa.

Food borne diseases are a major public health concern worldwide. Staphylococcus aureus is one of the potential food borne pathogens which causes nosocomial and community acquired infections. In the present study, 74 representative strains of S. aureus isolated and characterized in previous study from different milk samples were subjected to random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR to generate fingerprints to determine the genetic relationships of the isolated strains. A total of 20 RAPD patterns were generated and the number of amplified fragments obtained ranged from 0 to 8 with molecular weight ranging from 250 to 2000 bp. A dendrogram based on fingerprinting pattern grouped isolates into twelve major clusters (I–XII). In the case of ERIC-PCR 9 banding patterns were obtained with amplicons ranging from 1 to 8 and band sizes ranging from 250 to 2000 bp. A total of four major clusters (I–IV) were observed in the dendrogram based on ERIC fingerprints. The discrete banding patterns obtained both from ERIC-PCR and RAPD-PCR showed remarkably the genetic diversity of S. aureus. The findings of this study indicate that raw, bulk and pasteurized milk in the North-West Province was contaminated with toxigenic and multi-drug resistant S. aureus strains. This emphasizes the need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains to reduce the burden of disease in humans.

English

Klebsiella pneumoniae is one of the major causes of nosocomial infection. Understanding the route and dynamics of dissemination in the outbreaks of infections relies on using accurate typing methods. This is achieved through molecular typing using random amplified polymorphic DNA (RAPD) analysis technique in correlation to the infection source and antibiotic resistance. In this study, a total of 300 clinical isolates were collected from different clinical sources among Mansoura University Hospitals, Dakahlia governorate, Egypt. Ninety six isolates were identified as K. pneumoniae. The antimicrobial susceptibility test showed high resistance to the majority of tested antimicrobials, especially to β-lactams. K. pneumoniae isolates were categorized into 24 different antimicrobial susceptibility patterns (A1-A24). Furthermore, RAPD analysis was applied as a molecular typing method using two individual primers, AP3 and OPA13. RAPD typing showed 51 distinct patterns (R1-R51) verified into 5 groups (A, B, C, D, and E). The most common patterns were D11, D18 and D19 included in groups B and C. On the other hand, more molecular variable isolates were distributed in groups D and E. Correlation between RAPD analysis and antibiotying established that specific RAPD pattern D11 was associated with multidrug resistant isolates. This confirms that cross acquisition can play an important role in the epidemiology of nosocomial colonization and infection with K. pneumoniae at Mansoura University Hospitals. In conclusion, this study emphasized the need for appropriate monitoring of K. pneumoniae infections, by using both traditional and molecular methods. RAPD proved to be effective technique in discriminating K. pneumoniae isolates. Key words: Klebsiella pneumoniae, random amplified polymorphic DNA (RAPD), antimicrobial susceptibility, typing, dendrogram.

Environmental surveillance and molecular epidemiology of waterborne pathogen Legionella pneumophila in health-care facilities of Northeastern Greece: a 4-year survey.

A 4-year proactive environmental surveillance of Legionella spp. in the water distribution and cooling systems of five health-care facilities was carried out as part of the strategy for the prevention of hospital-acquired Legionnaires' disease in Northeastern Greece. Legionella spp. were detected in 71 out of 458 collected samples. The majority of strains belonged to Legionella pneumophila serogroups 2-15 (75.0%), while all L. pneumophila serogroup 1 strains (23.6%) were isolated from a single hospital. The highest percentage of positive samples was found in distal sites (19.4%), while no Legionella strains were detected in cooling systems. Each hospital was colonized at least once with L. pneumophila, while remedial actions resulted in significant reduction of Legionella concentration. The molecular epidemiology of environmental L. pneumophila strains was also investigated using random amplified polymorphic DNA (RAPD) and multi-gene sequence-based analysis. Based on RAPD patterns, L. pneumophila serogroups 2-15 and serogroup 1 strains were classified into 24 and 9 operational taxonomic units (OTUs), respectively. Sequencing of housekeeping and diversifying pressure-related genes recommended by European Working Group for Legionella Infections (EWGLI) revealed not only a high intraspecies variability but also the circulation and persistence of one specific genotyping profile in the majority of hospitals. This study highlights the necessity for diachronic surveillance of Legionella in health-care facilities by adopting both cultural and molecular methods.

Genetic Diversity Study of Locally Isolated Bacillus thuringiensis Strains from Kuwait Using Random Amplified Polymorphic DNA Analysis

Introduction: Bacillus thuringiensis (Bt) is significant bacteria in the field of insect biological control due to their insecticidal properties and its importance in agriculture. Aim: The objective of this study was to analyze genetic variation of 15 native isolates of Bacillus thuringiensis and four reference strains (total 19) using the random amplified polymorphic DNA (RAPD) technique, this allowed the analysis the genetic diversity of this species in the microbial populations from different soil samples of Kuwait. Methodology: We isolated 109 Bacillus thuringiensis strains, out of which, 15 strains were subspecies thuringiensis, using culture and serological method. A rapid typing method of Bacillus thuringiensis local isolates from Kuwait soil was established using the RAPD technique. A single Original Research Article Qasem et al.; BMRJ, 7(4): 193-201, 2015; Article no.BMRJ.2015.111 194 decamer primer was used to study and characterize Bacillus Spp, Bacillus thuringiensis var. thuringiensis, to differentiate the isolated species. Based on RAPD pattern, data were subjected to cluster analysis using Alpha Ease software. Results: We found three groups each, with two strains that had a similar pattern of DNA and one group that had three subspecies that have a similar DNA pattern. The rest of the eight isolates each had a unique pattern of DNA. These isolates were classified according to the same chemical and physical characterization, but were different genetically. Conclusion: In conclusion, using molecular methods for comparison of genomic DNA between different bacterial species of the same genus is a good measurement of genetic relations between different species, which could lead to discovery of new species unique to the local environment.

Genetic Diversity and Relationship in Squash Using Morphological, Chemical and Molecular Analyses

Germplasm characterization is an important link between the conservation and utilization of plant genetic resources. A collection of thirteen summer, spaghetti and acorn squash ( Cucurbita pepo L .) and one winter squash ( Cucurbita moschata L .) germplasm accessions were screened using morphological (leaf area, fruit weight, fruit length, fruit firmness), chemical [Soluble Solids Content (S.S.C.), ascorbic acid] and molecular [Random Amplified Polymorphic DNA (RAPD)] analyses. A wide range of variability among genotypes was recorded for morphological and chemical characteristics. The percentages of variation were 217%, 532%, 175%, 166%, 148%, 520% for leaf area, fruit length, fruit weight, fruit firmness, S.S.C, ascorbic acid content, respectively. Both significant positive and negative correlations have been found between the morphological and chemical characteristics. The RAPD analysis produced 209 DNA fragments with 100% polymorphism in two or more squash genotypes while none of the fragments showed monomorphic behavior among squash genotypes. At a similarity level of 82% the genotypes were divided into two clusters. The first cluster consisted of eight genotypes (PI 506466- PI 292014- PI 518688- PI 615119- PI 136448- Butternut- Copi- Eskandrani). The second cluster contained only two genotypes (Yellow Crookneck and Shamamy). When the cluster analysis of RAPD patterns was associated with morphological and chemical evaluation of squash genotypes used in this study, there was a notable degree of agreement. The RAPD-PCR was found to be suitable for use with squash due to its ability to discriminate between genotypes and determine their genetic relationship. Results obtained in this investigation provide clear evidence that there is a considerable variation among summer squash genotypes. Present results support the development of breeding programs in squash since high genetic variability in its accessions and landraces has been found.

English

The taxonomy of Solanum melongena L., also known as brinjal eggplant, has remained difficult because previous studies to establish genetic relationships among taxa are mainly based on morphological features, which are insufficient to establish genetic affinities. In the present investigation, five highly polymorphic random amplified polymorphic DNA primers were used to describe the genetic similarity and diversity among its accessions in Nigeria. The results show a high level of polymorphism based on the banding patterns among the samples. This indicated a wide and diverse genetic base. Four distinct clusters were equally noticeable at a coefficient of 0.80 from the dendrogram generated. Generally, the grouping pattern clearly indicates that irrespective of variations in fruit traits among samples, they were still grouped together in different clusters with a high similarity coefficient value. This probably showed some genetic relatedness/closeness among the samples concerned. The study also revealed that there is no association between RAPD pattern and the geographic origin of accessions. These agreed with previously published data on the characterization of eggplant. The study disclosed that molecular methods coupled with morphological analyses could make proper classification of S. melongena and other Solanum species in Nigeria possible to achieve.   Key words: Eggplant, random amplified polymorphic DNA (RAPD), Data, polymorphism, taxonomy.

Using Random Amplified Polymorphic DNA (RAPD) analysis to investigation of genetic diversity, and relationships among a set of clinical Aspergillus fumigatus isolates

This study is an attempt to determine the genetic diversity and relationships among fourteen local isolate isolated from patients with Aspergillosis (Aspergillus fumigatus) by using the Random Amplified Polymorphic DNA (RAPD) technique. Twelve universal primers used in this study produced 94 bands across fourteen isolates. Of these bands, 67 bands or 71.2% were polymorphic. The size of the amplified bands ranged between 100-2000 bp. The genetic polymorphism value of each primer was determined and ranged between 33-100%. In terms of unique banding patterns, determine the finger print for six isolates the most characteristic banding pattern was for the (AFU1, AFU2, AFU3, AFU4, AFU8 and AFU14) with primer (OP F-16 , OP I-06, OP F-16, OP X-01, OP X-01and OP A-06). Genetic distances ranged from 0.12419 to 0.64404 among A. fumigatus isolates. Cluster analyses were performed to construct a dendrogram among studied A. fumigatus isolates. The cluster analysis places most of the A.fumigatus isolates isolated from patient come from yhe same area into a close relation (subcluster) showing a high level of genetic relatedness and were distinct from isolates from another area (the other subcluster). Interestingly, a number of isolates originating from the same sources did form well defined groups, indicating association between the RAPD patterns and the geographic origin of the isolates. The information generated from this study can be used in the future for controlling of Aspergillosis programs.

Characterization of the fungi associated with ascochyta blight of field pea in Alberta, Canada

Ascochyta blight, caused by a complex of Mycosphaerella pinodes, Phoma pinodella, Ascochyta pisi, and/or Phoma koolunga, is a devastating disease of field pea. In order to understand the composition of fungi associated with ascochyta blight in Alberta, Canada, a total of 157 single-pycnidiospore fungal isolates were obtained from diseased pea samples collected from central and northern Alberta in 2011. These isolates were characterized for species identity, aggressiveness, DNA sequence variation in the internal transcribed spacer (ITS) region, and random amplification of polymorphic DNA (RAPD) patterns. The ITS sequences obtained from 142 fungal isolates were all identical to the ITS sequences from M. pinodes and/or P. pinodella. Inoculation of the 157 isolates on a susceptible pea cultivar Midas indicated that most of the isolates were moderately to highly aggressive. Phylogenetic analysis based on the RAPD data revealed two main groups and six sub-groups, with one main group comprising 78% of the 157 isolates. Distinct RAPD patterns were associated with isolates from particular geographic locations, but were not generally associated with isolate aggressiveness.

Interspecific somatic hybrids Solanum villosum (+) S. tuberosum, resistant to Phytophthora infestans

The interspecific somatic hybrids 4x S. villosum (+) 2x S. tuberosum clone DG 81-68 (VT hybrids) were obtained and characterized molecularly and cytogenetically.The morphology of fusion-derived plants was intermediate in relation to the parental species. The expected ploidy level of the regenerants was 6x for the VT hybrids, but the real ploidy of the hybrids varied, with some of them being euploids, and others – aneuploids. The hybridity of the regenerants was verified by random amplified polymorphic DNA (RAPD) analysis. Despite the variation in ploidy, the RAPD patterns of the hybrids were mostly uniform, suggesting similarity of the genotypes of the VT clones. Genomic in situ hybridization (GISH) analysis discriminated between the chromosomes of both parental genomes in VT somatic hybrids and also confirmed their hybridity. The resistance of VT somatic hybrids to Phytophthora infestans was evaluated and all of the hybrids proved to be highly resistant. In search of the mechanisms involved in resistance of the Solanum species to P. infestans, the biochemical reactions occurring early after elicitor treatment were studied. The production of reactive oxygen species (ROS), as one of the earliest reactions induced by pathogens or their elicitors, was examined in the resistant wild species S. villosum, susceptible S. tuberosum clone DG 81-68 and in the VT hybrid, resistant to P. infestans. After treatment of the leaves with elicitor, the relative increase in ROS production was higher in leaves of the susceptible potato clone than in the resistant plants of S. villosum and the somatic hybrid.

Differentiation of Streptococcus thermophilus strains in commercial Direct Vat Set yoghurt starter

Based on inhibition of acid production, 100 individual colonies of Streptococcus thermophilus isolated from commercial Direct Vat Set yoghurt starter DVS-M were typed into 2 groups of sensitive and insensitive to phage φ001, which was isolated from the lysis culture of the turbidity test. The ratio between the sensitive and insensitive isolates in DVS-M was calculated at 3:2. Whole-cell protein profile (WCPP) and randomly amplified polymorphic DNA (RAPD) analysis showed that both WCPP and RAPD patterns between sensitive and insensitive isolates were distinct, whereas isolates with the same sensitivity or insensitivity shared common WCPP and RAPD patterns. It suggested that all the sensitive isolates belong to one S. thermophilus strain and all the insensitive isolates belong to another one. This study revealed that DVS-M yoghurt starter consists of 2 S. thermophilus strains with different phage sensitivity.