Random Amplified Polymorphic DNA Fingerprinting Research Articles

Phenotypic and genotypic assessment of fluoroquinolones and aminoglycosides resistances in Pseudomonas aeruginosa collected from Minia hospitals, Egypt during COVID-19 pandemic

BackgroundOne of the most prevalent bacteria that cause nosocomial infections is Pseudomonas aeruginosa. Fluoroquinolones (FQ) and aminoglycosides are vital antipseudomonal drugs, but resistance is increasingly prevalent. The study sought to investigate the diverse mechanisms underlying FQ and aminoglycoside resistance in various P. aeruginosa strains particularly during the COVID-19 crisis.MethodsFrom various clinical and environmental samples, 110 P. aeruginosa isolates were identified and their susceptibility to several antibiotic classes was evaluated. Molecular techniques were used to track target gene mutations, the presence of genes encoding for quinolone resistance, modifying enzymes for aminoglycosides and resistance methyltransferase (RMT). Efflux pump role was assessed phenotypically and genotypically. Random amplified polymorphic DNA (RAPD) analysis was used to measure clonal diversity.ResultsQnrS was the most frequently encountered quinolone resistance gene (37.5%) followed by qnrA (31.2%) and qnrD (25%). Among aminoglycoside resistant isolates, 94.1% harbored modifying enzymes genes, while RMT genes were found in 55.9% of isolates. The aac(6')-Ib and rmtB were the most prevalent genes (79.4% and 32.3%, respectively). Most FQ resistant isolates overexpressed mexA (87.5%). RAPD fingerprinting showed 63.2% polymorphism.ConclusionsAminoglycosides and FQ resistance observed in this study was attributed to several mechanisms with the potential for cross-contamination existence so, strict infection control practices are crucial.

Random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) PCR of Vibrio cholerae from a foodborne outbreak in Limbang, Sarawak

Toxigenic and non-toxigenic V. cholerae strains can be monitored for changes in clones or serogroups, linkages between clinical and environmental isolates, genesis and clonal selection of epidemic strains, and population structure. Also, determining genetic relatedness among V. cholerae strains is critical for determining population genetic structure and evolutionary trends. In collaboration with the Sarawak Government Hospital, the present work was carried out on a total of 16 V. cholerae isolates in order to determine the genetic relatedness or heterogeneity of V. cholerae isolates from a foodborne outbreak among guests who attended a wedding ceremony in Limbang, Sarawak, Malaysia. The random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) were conducted to compare and determine strains and trace disease-causing microorganism. The RAPD fingerprinting was conducted using a total of 10-mer oligos 100 nmole random primers (OPAE1 - OPAE20). The primers OPAE7, OPAE10, OPAE14, and OPAE17 were selected because they were the most stable and discriminatory for V. cholerae. The PCR fingerprinting of ERIC-PCR was carried out using primer set of ERIC, ERIC1R (5’- ATGTAAGCTCCTGGGGATTCAC-3’), and ERIC2F (5’- AAGTAAGTGACTGGGGTGAGCG-3’). As a result, the 1 confirmed V. cholerae O1 samples were successfully fingerprinted. Based on the profiling results, the genetic fingerprint of some of the isolates from the clinical and environmental samples had 100% similarity, as indicated by the dendrogram. This indicated that the strains shared the same genetic profile. The smaller the genetic distance, the more homogeneous the strains are. The clinical and environmental strains shared some genetic characteristics. As indicated by the dendrogram, some strains were found to be closely linked to one another, while others were heterogeneous. Therefore, RAPD-PCR and ERIC-PCR produced the highest discrimination index. By combining these typing methods, evaluation of isolates' genetic diversity may be improved. The findings of the present work demonstrated the need for continued surveillance of V. cholera in Sarawak, Malaysia.

Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices.

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates ("IM" and "TA") were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen-derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.

In-vivo characterization of multidrug-resistant Salmonella enterica Serovar Enteritidis (SE) recovered from fertile eggs and baby chicks

The present study was conducted to isolate and characterize Salmonella spp. from hatching eggs and baby chicks. Additionally, the pathogenicity of the isolated Salmonella strains was assessed in one-day-old specific pathogen-free (SPF) chicks in-vivo. Samples from sick baby chicks from 14 broiler chicken farms (including 1 duck farm) and 150 egg batches from three breeder chicken farms were collected from 4 different governorates. Phenotypically identified Salmonella isolates were confirmed using species-specific multiplex-PCR targeting the inv -A gene for Salmonella genus, E -1 gene for Salmonella Enteritidis (SE) serovar, and Flic-C gene for Salmonella Typhimurium (ST) serovar. Confirmed SE isolates were further subjected to Random Amplified Polymorphic DNA (RAPD) fingerprinting. Phenotypic, multiplex-PCR, and RAPD fingerprinting confirmed six isolates (42.9%) from broiler chicken farms and two isolates from hatching egg batches (1.33%) as SE, of which eight were multidrug-resistant (MDR) strains with 0.214-0.786 MDR indices. In-vivo pathogenicity of selected multidrug-resistant (MDR) SE isolates was evaluated in one-day-old SPF chicks. Despite minor phenotypic diversity, most SE strains were highly invasive with variable mortality (50-100%). Interestingly, the lowest MDR indices were associated with high virulence in SE strains (mortality ≥85%) and vice versa. The study results showed the presence of SE in poultry in Egypt. The uncontrolled usage of antibiotics in poultry could be the reason for the increased prevalence of MDR Salmonella spp., which may limit Salmonella control measures and threaten public health.

RAPD Fingerprinting and Genetic Diversity of Salmonella Spp. Isolated from Broiler and Layer Flocks in Karbala, Iraq.

Salmonellosis in poultry is one of the most significant bacterial infections causing mortality, reduced production, and serious economic losses. This study aimed to study the molecular diversity among Salmonella isolates and investigate the epidemiological spread of these bacteria in broiler and layer chicken flocks in five different farms in Karbala, Iraq, using random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR). In total, 217 cloac a swabs were collected from the farms, out of which 129 and 88 swabs were taken from broiler and layer chickens. The samples were screened by PCR for S. enterica subsp. enterica using primers specific for the invA gene. Afterward, RAPD-PCR with uniplex or multiplex octamer primers was applied to genotype the isolates. The incidence rate of Salmonella infections in broilers and layers was estimated to be 27.9% and 12.5%. The uniplex primers P2 and P3, along with the multiplex primers yielded discriminatory patterns. Moreover, the RAPD typing showed a diverse range of banding patterns of Salmonella spp. Dendrograms created through GelJ software revealed various Salmonella genotypes in broilers and layers. The RAPD-PCR could be used as an accurate and fast tool to identify genetic relatedness among Salmonella spp. The obtained results would assist researchers in epidemiological studies and controlling salmonellosis in poultry fields.

Pathogenic and molecular variation of Fusarium species causing head blight on barley landraces

AbstractFusarium head blight (FHB) is consistently one of the most important barley diseases worldwide. This study aimed to evaluate the pathogenicity of 16 isolates of four Fusarium species under controlled conditions and their genetic variability using 22 random amplified polymorphic DNA (RAPD) markers. Pathogenic variation was characterized based on disease development rates and disease index on two Syrian barley landraces with varying resistance to FHB, Arabi Aswad (AS) and Arabi Abiad (AB). Significant differences in intra- and inter-Fusarium species pathogenicity and in susceptibility between the above-mentioned cultivars were highlighted. Overall, the two barley landraces showed moderately susceptible to moderately resistance levels to fungal infection and FHB spread within the head. Quantitative traits showed significant correlation with previous data generated in vitro and under field conditions, suggesting that growth chamber indices can predict fungal pathogenicity and quantitative disease resistance generated under various experimental conditions. Based on PCR amplification with seven different primers, the isolates showed genetic variation. Dendrogram generated by cluster analysis based on RAPD markers data showed two main groups, suggesting that a possible clonal origin could exist in the four Fusarium species. RAPD fingerprints are not useful to distinguish the 16 Fusarium isolates with different levels of pathogenicity.

Genetic diversity, virulence and distribution of antimicrobial resistance among Listeria monocytogenes isolated from milk, beef, and bovine farm environment.

Listeria monocytogenes is an opportunistic intracellular foodborne pathogen and is ubiquitous in nature. The occurrence of L. monocytogenes in animal production units coupled with their presence in milk, faeces, feed, water, sewage, and soil is a contributory factor for foodborne listeriosis in humans and animals. The study was aimed to characterize genotype and serogroup of L. monocytogenes recovered from different types of samples and also to study antimicrobial patterns by phenotypic and genotypic methods. Multiplex polymerase chain reaction (PCR) was used for the confirmation of L. monocytogenes, the identification of its serogroup and lineage, and the detection of virulence markers. Enterobacterial repetitive intergenic consensus (ERIC), and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize those isolates, and antimicrobial patterns were studied phenotypically by Kirby-Bauer method and genotypically by PCR. Out of the screened 474 samples (274 milk and 50 each of soil, feed, sewage, and beef), ten L. monocytogenes isolates (milk=8, soil=1, and beef=1) were confirmed by PCR targeting the hlyA gene and found to belong to the 1/2a, 3a serogroup and fall under type II lineage. Virulence potential assessment revealed that all the ten isolates harbored the iap gene while the presence of plcA and plcB genes were noticed in seven and eight isolates respectively. Six isolates from milk were found to group in the same cluster by ERIC and RAPD fingerprinting, suggesting both methods to be efficient molecular typing tools for L. monocytogenes. Genotypic characterization of antimicrobial resistance (AMR) genes revealed that seven isolates were positive for tetM, five for mefA, four for msrA, and one for lnuA genes while none of the isolates showed tetK, ermA, ermB, and lnuB genes. The presence of L. monocytogenes in bovine farm environments coupled with virulence markers, and multidrug resistance from the study area suggest a possible transmission from the environment to humans and animals which needs to be monitored regularly to ensure food safety and the well-being of animals and humans.

Development of a RAPD protocol for typing strains of Phytophthora infestans in Mauritius

In Mauritius, substantial decrease in potato yields due to late blight are reported annually and it has become necessary to track the genetic identity of local Phytophthora infestans strains. The Random Amplified Polymorphic DNA (RAPD) technique is a low-cost and simple genetic characterisation tool that has been widely used for molecular fingerprinting but which requires extensive optimisation. The aim of this study was to carry out a series of experiments to optimise a RAPD protocol for routine P. infestans typing. Amplifications performed with DNA template concentration of 30-50 ng/μl, primer concentration of 2.0 − 3.0 μM and MgCl2 concentration of 3.0 mM gave the best RAPD profiles. These optimised conditions were used to carry out RAPD fingerprinting of 7 P. infestans isolates and codified data were used to construct a consensus dendogram, which grouped the 7 isolates into 3 distinct clusters, consistent with results obtained from other molecular characterisation techniques.

DNA FINGERINTS OF TILAPIA SPECIES IN SHATT AL-AREB RIVER USING RAPD MARKERS

In the last decade, tilapia fish species distributed in the Iraqi inland waters. Three species; Nile tilapia Oreochromis niloticus (Linnaeus, 1758), Blue tilapia Oreochromis aureus (Steindachner, 1864) and Redbelly tilapia Coptedon zillii (Gervais, 1848) inhibiting Shatt Al-Arab River. They belong to family Cichlidae. They are very similar to differentiate among them using biometry. So, genetic markers used for species discrimination. Randomly amplified polymorphic DNA (RAPD) protocol used to examine genetic variation and to generate DNA fingerprints of tilapia fish species in Shatt Al-Arab River. Sixty-two specimens of tilapia fish collected from Shatt Al-Arab River at the governorate of Basrah. Seven universal decamer primers selected (OPA08, OPA10, OPA13, OPA17, OPA19, OPB08 and OPC02) to create RAPD DNA fingerprint. RAPD-PCR amplification carried out and electrophoresed with 100 bp ladder. DNA bands scored and molecular weight was calculated using PhotocaptMW software. Analog histogram drew using MS-Excel. The three RAPD DNA profiles apparently were different. DNA bands scored in the three species were 67 bands. The size of DNA bands was ranged from 64 bp to 2344 bp. RAPD fingerprints were efficient to distinguish the three species of tilapia fish. DNA markers of the three species of tilapia fish can use to achieve conservation programs of fish species in the future.

Interspecific hybridization between cultivated morels Morchella importuna and Morchella sextelata by PEG-induced double inactivated protoplast fusion.

The commercial production of Morchella mushrooms calls for urgent breeding of excellent varieties or strains with appropriate tools, such as protoplast fusion. However, the protoplast fusion in morels has not been studied. In this paper, interspecific hybridization between cultivated morels of M. importuna and M. sextelata by PEG-induced protoplast fusion was conducted. Apart from functional complementation of double inactivated protoplasts, the fusants were characterized by cultural and cultivated characters and molecular markers of random amplified polymorphic DNA (RAPD). The results suggested that the hybrids and their parents showed significant difference in their inoculum recovery time, mycelial growth rate, yield of cultivation and total amino acid content of ascocarps. Moreover, positive barrage reactions were observed between parental strains as well as between each parent and a hybrid line. A dendrogram created on the basis of RAPD fingerprints exhibited three major clusters, in which morel hybrids showed intra-cluster variations, M. sextelata #6 formed an out group, while M. importuna #4 was phylogenetically closer to morel hybrids. All the results demonstrated that real fusants were obtained in our study. Protoplast fusion may provide an ideal alternative for new strain selection, and thus will promote the healthy development of morel industry.

The analysis of virulence factors and antibiotic resistance between Helicobacter pylori strains isolated from gastric antrum and body

BackgroundIndividuals can be infected with multiple strains of Helicobacter pylori. However, the differences among co-infecting strains have not been well analyzed yet. This study aimed to investigate whether the virulence factors and antibiotic resistance patterns of H. pylori differ between strains isolated from different locations of the stomach in the same patient.MethodsH. pylori isolates were obtained from the antrum and body of the stomach. Genetic differences were examined by random amplified polymorphic DNA (RAPD) fingerprinting. Antibiotic resistance was assessed using the agar dilution method. Virulence factors were identified by polymerase chain reaction and DNA sequencing.ResultsAmong 80 patients, co-infection by two H. pylori strains was detected in 10 patients. Among the 10 pairs of H. pylori strains, differences in antibiotic resistance patterns were detected in 7 pairs (clarithromycin, 1 patient; quinolone, 3 patients; metronidazole, 4 patients) and differences in virulence factors were detected in 5 pairs. The cagA virulence gene was detected in all 10 patients, and 2 patients had H. pylori strains with different EPIYA motifs. Differences in vacA genotypes were detected in 4 patients.ConclusionsCo-infection by two H. pylori strains was confirmed by RAPD fingerprinting. Frequently, two H. pylori strains obtained from a single host differed in their virulence factors and antibiotic resistance patterns. Co-infection by multiple H. pylori strains could undermine the success of eradication therapy and should be considered when interpreting the results of antimicrobial susceptibility tests.

RAPD Fingerprinting and Genetic Relationships of Some Wheat Genotypes

The genetic variability and relationships among 5 Egyptian wheat genotypes representing Sakha8, Sakha69, Sakha93, Sids1 and Gemmiza7 were analyzed using 8 random amplified polymorphic DNA (RAPD). A total of 77 loci (73 % polymorphic) in all 5 wheat genotypes was amplified and discriminated all the wheat genotypes. PIC, RP, MI, DP values were evaluated and revealed degree of genetic divergence among the cultivars used. A cluster based on UPGMA (Un-weighted Pair-Group Method with Arithmetic Mean) analysis was used to determine genetic similarities. The five wheat genotypes were divided into two main clusters. Cluster 1 was divided into two groups. In subgroup 1 were included genotype 1 and genotype 2. They seemed very close which might depict sharing of the genetic background among the genotypes. In consequence, the close genetic relationships are entirely alarming and may hinder further plant improvement. Genotype 5 was in subgroup 2. The second cluster was included genotype 3 and genotype 4. The same genotypes were also assessed in field conditions for structural analyses, which were carried out based on six yield components. The dendrogram created was comparatively analyzed with the RAPD dendrogram. This study additionally indicates that RAPD markers are useful for distinguishing and characterizing wheat cultivars. The genetic relatedness among these genotypes could provide useful information for conservation and selection of cross parents in breeding.

Comparison of Genetic Variation of Anthurium (Anthurium andraeanum) Cultivars Using SCoT, CDDP and RAPD Markers

To evaluate the genetic diversity among 10 cultivars of anthurium were performed using three molecular markers such as Start Codon Targeted (SCoT) and Conserved DNA-derived Polymorphism (CDDP), and Random Amplification of Polymorphic DNA (RAPD). Polymorphism index content (PIC) was calculated 0.39, 0.42 and 0.37 for RAPD, SCoT and CDDP, respectively. This result showed all the three molecular markers had almost an identical potential in estimating genetic diversity. Cluster analysis using SCoT, CDDP and RAPD divided the cultivars to three distinct clusters. The similarity matrix obtained through SCoT and CDDP was positively significantly correlated (r = 0.76, p < 0.01). This is the first report in which the efficiency of two targeted DNA region molecular markers (SCoT and CDDP) together with RAPD technique have been compared with each other in a set of anthurium cultivras. Results suggested that SCOT, CDDP and RAPD fingerprinting techniques are of sufficient ability to detect polymorphism in anthurium cultivars.
 Plant Tissue Cult. & Biotech. 28(2): 171-182, 2018 (December)

Characterization of Xanthomonas citri subsp. CITRI isolated from grapefruit in Iran

Xanthomonas citri subsp. citri (Xcc) strains, obtained from cankers of grapefruit trees, were characterized by phenotypic, genotypic and pathogenicity features. In addition, some strains from Mexican lime were isolated to compare with Xcc strains from grapefruit. Specific polymerase chain reaction (PCR) analyses with three primer pairs and phylogenetic analysis based on sequencing of two housekeeping genes identified all Iranian isolates as Xcc-A*. The host range of the pathogen was assessed on four Citrus spp. Based on pathogenicity test, Iranian strains were divided into two pathogenic groups. The first group, corresponding to strains from Mexican lime, was only pathogenic to Mexican lime, whereas the second group, corresponding to strains from Mexican lime and grapefruit, was pathogenic to Mexican lime and grapefruit. However, reference strain was pathogenic to Mexican lime, grapefruit, tangerine and sweet lime. An insight into the genetic population features was attempted by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis. The combined RAPD and ERIC-PCR fingerprints divided the strains into four clusters/fifteen haplotypes. Strains isolated from grapefruit and some strains isolated from Mexican lime were grouped in cluster П. Based on our results, we determined that the sources of contamination of grapefruit trees were probably neighboring infected Mexican lime trees. To our knowledge, this is the first report of occurrence of citrus canker of grapefruit in the Fars province of Iran.

Genetic diversity within and among two-spotted spider mite resistant and susceptible common bean genotypes

Two-spotted spider mite (Tetranychus urticae C. L. Koch, 1836), is one of the most destructive herbivores of common bean. Very little is known about the diversity among resistant sources in this crop. The present study was conducted to characterize 22 resistant and susceptible common bean genotypes by 8 Simple Sequence Repeats (SSRs) and 8 Random Amplified Polymorphic DNA (RAPD) markers. These SSR and RAPD primers produced 100 % and 81.8 % polymorphic bands. Based on RAPD fingerprints and SSR profiles, pairwise genetic similarity ranged from 0.0 to 0.857 and from 0.125 to 1, respectively. The resistant and susceptible common bean accessions were grouped together in the dendrograms generated from RAPD and SSR clustering analyses. The results indicate that RAPD and SSR analysis could be successfully used for the estimation of genetic diversity among genotypes. SSR markers could group genotypes according to their resistibility and susceptibility to the spotted spider mite but RAPD could not. Therefore, the SSR markers can facilitate the development of resistant common bean cultivars through breeding programs against T. urticae.

Isolation of Probiotic Piliated Lactobacillus rhamnosus Strains from Human Fecal Microbiota Using SpaA Antiserum-Based Colony Immunoblotting.

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect 2.5 × 103 CFU/ml of pLR colonies spiked in 106 CFU/ml of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

Genetic divergence and allelic-specificity in relation to expression of voltinism in silkworm using ISSR and RAPD fingerprinting

The silkworm B. mori is a multicellular organism revealing genetic resources which makes an ideal model for lepidoptera for the present investigation. With the objective of targeting distinctive markers for utilization in future breeding programmes, Bivoltine and Polyvoltine silkworm strains were used by inter-simple sequence repeats (ISSR) and random amplified polymorphic-DNA (RAPD) fingerprinting to detect their genetic versatility and volatility. Six ISSR primers generated 99 markers, of which 76.76% were found to be polymorphic with an average number of observed alleles (Na) (1.86 ± 0.40), an effective number of alleles (Ne) (1.43 ± 0.30) as well as six RAPD primers that produced a total of 95 bands, developing 61.05% polymorphism with Na (1.93 ± 0.51) and Ne (1.18 ± 0.30). The dendrogram produced by UPGMA analysis, based on Dice’s coefficient, clustered four races into two major groups which accurately segregated them according to their inheritance of voltinism. In this research, the ISSR markers were more accurate than the RAPD markers and ISSR also displayed better polymorphism. The outcome showed that the bivoltine strains exhibited higher allelic expressions with ISSR primers when compared to the polyvoltine strains. Despite exhibiting their unique race by certain DNA markers, most of the primers represented voltinism-specificity. Hence molecular marker amplification is a beneficial approach to reveal genetic divergence among closely related strains, and molecular characterization of phylogenetic relationships in addressing evolutionary evidences of individuals.

Analysis of molecular variance and population structure in southern Indian finger millet genotypes using three different molecular markers

The genetic relationship among 42 genotypes of finger millet collected from different geographical regions of southern India was investigated using random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), and simple sequence repeats (SSR) markers. Ten RAPD primers produced 111 polymorphic bands. Five ISSR primers produced a total of 61 bands. Of these, 23 bands were polymorphic. The RAPD and ISSR fingerprints revealed 71.3 and 37.4% polymorphic banding patterns, respectively. Thirty-six SSR primers yielded 83 scorable alleles in which 62 were found to be polymorphic. Out of 36 SSR primers used, 14 primers (46.6%) produced polymorphic bands. The SSR primer UGEP7 produced a maximum number of six alleles. Mean polymorphic information content (PIC) of RAPD, ISSR and SSR were 0.44, 0.28, and 0.14, respectively. Molecular variances among the population were 2, 11, and 1% for RAPD, ISSR, and SSR markers, respectively. SSR produced 99% molecular variance within individuals. RAPD and ISSR markers produced a low level of molecular variance within individuals. The STRUCTURE (model-based program) analysis revealed that the 42 finger millet genotypes could be divided into a maximum of four subpopulations. Based on the Bayesian statistics, each RAPD and SSR marker produced three subpopulations (K=3), while ISSR marker showed four subpopulations (K=4). This study revealed that RAPD and SSR markers could narrow down the analysis of population structure and it may form the basis for finger millet breeding and improvement programs in the future.

GENETIC VARIABILITY OF THREE POPULATIONS OF FLYING FISH, Hirundichthy oxycephalus FROM MAKASSAR STRAIT

Flying fish, Hirundichthy oxycephalus is one of economically important marine species to Indonesia, particularly in Makassar Strait and Flores Sea. However, there is a limited published data on genetic variation in molecular marker level of this species. Random Amplified Polymorphic DNA (RAPD) was employed in this study to determine the genetic variability of three populations of flying fish collected from Takalar, Pare-Pare, and Majene in Makassar Strait. Genomic DNA was isolated from preserved muscle tissue using phenol-chloroform technique. Two selected arbitrary primers (CA-01 and P-40) were performed to generate RAPD finger printing of flying fish populations. The two primers generated a total of 81 fragments (loci) and 50 polymorphic fragments with size ranging from 125 to 1,250 bp. There were no significant differences in number of fragment and number of polymorphic fragment among populations. The high polymorphism (63.5±7.4%) was obtained from Takalar population followed by Pare-Pare (58.3±19.6%) and Majene population (57.7±0.8%). Similarity index of individuals was 0.60±0.17 for Takalar, 0.63±0.17 for Majene and 0.75±0.21 for Pare-Pare population. Seven fragments were identified as species-specific markers of H. oxycephalus. The UPGMA cluster analysis showed that the Takalar population was genetically closer to Pare-Pare population (D= 0.0812) than to Majene population (D= 0.1873).

Non-O157:H7 Shiga Toxin Producing Diarrhoeagenic Escherichia coli (STEC) in Southern India: A Tinderbox for Starting Epidemic.

Outbreaks due to non-O157:H7 Shiga toxin producing Escherichia coli (STEC) resulting in Haemolytic Uraemic Syndrome (HUS) have garnered much attention because of associated mortality transcending across continents and also because diarrhoea due to E.coli itself is rare in developed countries. The actual incidence of non-O157:H7 STEC in sporadic acute diarrhoea is not fully elucidated, both in developing as well as in developed countries. Due to larger extent of faecal-oral transmission in developing countries it is prudent to look for non-O157: H7 STEC in such epidemiological settings because of very high potential to spread across larger geographical regions and cause life threatening illness. To determine the extent of acute diarrhoea caused by Shiga toxin producing E. coli and measure their genotypic diversity. The study was designed as a cross-sectional study and conducted between 2009-2011 in department of Microbiology at JN Medical College Belgaum (Karnataka) and Regional Medical Research Center, Belgaum (RMRC-ICMR). Stool samples from 300 sporadic cases of acute diarrhoea were processed by microscopy, culture, for the identification of diarrhoeagenic pathogens viz. Vibrio cholera, Shigella spp., Salmonella spp. and protozoan parasites. PCR was performed for the detection of eae and stx genes in E. coli isolates. Their relatedness was determined by Random Amplification of Polymorphic DNA (RAPD). PCR detected stx along with eae in 23.2% culture isolates of E.coli isolated from diarrhoea samples. Only three isolates were identified as STEC by serology as O59, O60 and O69 serotypes. Eleven clones were detected by RAPD fingerprinting in the 46 STEC isolates. Non-O157:H7 STEC are prevalent in this region and laboratories shall look beyond O157:H7 serotype of E.coli. These isolates have potential of causing outbreaks transcending borders. Hence they shall be reported and efforts be made to identify their sources and prevent spread.