Study of arsenic genotoxicity in a freshwater fish (Channa punctatus) using RAPD as molecular marker

Arsenic, the proven genotoxic carcinogen in humans, is a groundwater contaminant of global concern. The present work investigates the applicability of randomly amplified polymorphic DNA (RAPD) as molecular marker to demonstrate arsenic genotoxicity in the freshwater fish, Channa punctatus. Fish specimens, segregated into three groups, were exposed to 10, 50 and 500 µgL− 1 of arsenic respectively for 20 consecutive days. DNA extracted from same specimens of each group before and after exposure was amplified by polymerase chain reaction (PCR) using single arbitrary primers. Marked changes in RAPD profiles of fish DNA were observed upon exposure to arsenic compare to RAPD profiles of their pre-exposure state, resulting from loss or gain of certain bands. Altogether a total of 41 loci were amplified with 37–41 bands in each group. Band 1 of pre-exposure state was lost in all post-exposure samples while bands 4, 6 and 7 appeared as new bands after exposure to arsenic. The changes in the RAPD banding patterns upon exposure to arsenic reflect alterations in fish DNA. The RAPD bands, therefore, appeared as potential markers, capable of revealing the genotoxicity induced by arsenic in this piscine model.

Optimum vegetative growth conditions and genetic diversity in different strains of Pleurotus salmoneastramineus L.J.N. Vassilzeva

This experiment was undertaken to depict the favourable condition for mycelial growth, molecular identification and phylogenetic relationship of the selected strains of Pleurotus salmoneostramineus. Suitable temperature and pH were obtained at 25ºC and 6, respectively. Mushroom complete, glucose peptone and yeast malt extract culture media were favorable, while Hennerberg and Hoppkins were unfavorable. Dextrin was the best and xylose was the less effective carbon sources. Inorganic nitrogen sources were less effective for the mycelial growth of P. salmoneostramineus. The sequences of internal transcribed spacer (ITS) region of selected strains revealed that the total length ranged from 614 to 663 bp. The size of the ITS1 and ITS2 regions varied among the strains. Sequence analysis showed that 5.8S of rDNA sequences were identical. Phylogenetic tree of the ITS region sequences indicated that strains of P. salmoneostramineus belong to same cluster. The reciprocal homologies of the ITS region sequences ranged from 98 to 100%. The strains of P. salmoneostramineus were also analyzed by random amplification of polymorphic DNA (RAPD) with 20 arbitrary primers. RAPD results suggested that tested strains of P. salmoneostramineus were genetically similar with some variations, thus it could be concluded that RAPD and ITS techniques were well competent for detecting the genetic diversity of all tested strains of P. salmoneostramineus.

Effectiveness of six molecular typing methods as epidemiological tools for the study of Salmonella isolates in two Colombian regions

Aim: The aim of this study was the genotypic characterization of the strains of Salmonella spp. isolated from broiler chickens and humans with gastroenteritis from two regions of Colombia, by BOXA1R-polymerase chain reaction (PCR) and random amplification of polymorphic DNA (RAPD)-PCR methods. Materials and Methods: Forty-nine strains of Salmonella were assessed, 15 from poultry farms in Santander region, and 34 from Tolima region isolated from poultry farms (n=24) and the stool samples of people with gastroenteritis (n=10). BOXA1R primers were selected for repetitive element-based PCR (REP-PCR) and five arbitrary primers, namely, GTG 5, OPB 15, OPP 16, OPS 11, and P 1254 were used for RAPD-PCR to generate DNA fingerprints from the isolates. Fingerprint data from each typing method were under composite analysis and the diversity of the data was analyzed by grouping (clustering). The dendrogram was generated by the unweighted group method with analysis of the arithmetic mean based on the Dice similarity coefficient. In addition, Simpson's index was evaluated to discriminate the power of the methods. Results: OPP 16 primer and composite analysis proved to be superior compared to other REP-PCR typing methods. The best discriminatory index was observed when GTG 5 (0.92) and OPP 16 (0.85) primers were used alone or combined with RAPD-PCR and BOX-PCR (0.99). Conclusion: This study indicated that OPP 16 and GTG 5 primers provide suitable molecular typing results for the discrimination of the genetic relationship among Salmonella spp. isolates and may be useful for epidemiological studies.

Genotypic diversity of Streptococcus mutans isolated from preschoolers with and without early dental care

Introduction Streptococcus mutans (S. mutans) exhibits extensive genotypic diversity, but the role of this variation is poorly understood. Objective To evaluate the genotypic diversity of S. mutans in preschoolers with and without early dental care and to associate it with dental caries experience. Material and method Twenty children, aged five year old, with history of dental care since they were one year old (G1) and 20 children without history of dental care (G2) were included. Their oral health status was assessed by the dmft index. S. mutans samples were isolated from the saliva and analysed by polymerase chain reaction. A total of 339 S. mutans isolates (173 from G1 and 166 from G2) were genotyped by random amplified polymorphic DNA with arbitrary primers OPA-02 and OPA-13. Result The results revealed 75 distinct genotypes of S. mutans in G1 and 73 in G2. Furthermore, G1 and G2 subjects harboured one to eight and one to seven distinct genotypes of S. mutans, respectively. A statistically significant association (P=0.021) and a moderate correlation (r= 0.503) were observed between dental caries experience and genotypic diversity of S. mutans in G1. Conclusion From the limitations of the study design, we just can hypothesize that actions of early dental care carried out by an educative-preventive program can contribute to a distinct oral microbiota.

Arsenate-Resistant Genes in Egyptian Rice Cultivars as Soil Pollution Sensors

The main objective of the present study was to investigate arsenate [As (V)] resistance genes in rice cultivars grown in arsenic contaminated Egyptian soil in order to genetically induce resistance against arsenic in the local rice varieties as well as defining contaminated rice grains and/or soil. Three local rice cultivars; Sakha 102-104 were cultivated on modified Murashige and Skoog Basal Medium (MS medium) containing elevated concentrations of arsenate (0.1, 1 and 10 mg/l). The three varieties showed different resistant attitudes against arsenate with Sakha 104 being the most resistant. Extracted messenger RNA (mRNA) from treated and untreated Sakha 104 plantlets was scanned using differential display to demonstrate the arsenate resistant genes using three different arbitrary primers. About 100 different RNAs with (1500 bp - 50 bp) were obtained from which seven were up-regulated genes, subjected to DNA cloning using TOPO TA system and the selected clones were sequenced. The sequence analysis described four genes out of the seven namely disease resistance protein RPM1, Epstein-Barr virus EBNA-1-like, CwfJ family protein and outer membrane lipoprotein OmlA while the other three genes were hypothetical proteins. It is concluded the four induced genes in the resistant rice cultivar considered as a direct response to arsenic soil pollution. Genes detected in the present study can be used as geno-sensors for rice grains and soil contamination with As (V). Moreover, local rice cultivars may be genetically modified with such genes to induce high resistance and to overcome arsenic soil pollution.

Arsenate-Resistant Genes in Egyptian Rice Cultivars as Soil Pollution Sensors

The main objective of the present study was to investigate arsenate [As (V)] resistance genes in rice cultivars grown in arsenic contaminated Egyptian soil in order to genetically induce resistance against arsenic in the local rice varieties as well as defining contaminated rice grains and/or soil. Three local rice cultivars; Sakha 102-104 were cultivated on modified Murashige and Skoog Basal Medium (MS medium) containing elevated concentrations of arsenate (0.1, 1 and 10 mg/l). The three varieties showed different resistant attitudes against arsenate with Sakha 104 being the most resistant. Extracted messenger RNA (mRNA) from treated and untreated Sakha 104 plantlets was scanned using differential display to demonstrate the arsenate resistant genes using three different arbitrary primers. About 100 different RNAs with (1500 bp - 50 bp) were obtained from which seven were up-regulated genes, subjected to DNA cloning using TOPO TA system and the selected clones were sequenced. The sequence analysis described four genes out of the seven namely disease resistance protein RPM1, Epstein-Barr virus EBNA-1-like, CwfJ family protein and outer membrane lipoprotein OmlA while the other three genes were hypothetical proteins. It is concluded the four induced genes in the resistant rice cultivar considered as a direct response to arsenic soil pollution. Genes detected in the present study can be used as geno-sensors for rice grains and soil contamination with As (V). Moreover, local rice cultivars may be genetically modified with such genes to induce high resistance and to overcome arsenic soil pollution.

Morphometric and Molecular Analysis of the Three Arbutus Species of Greece

Arbutus andrachne, Arbutus unedo and Arbutus × andrachnoides found in the Greek macchia are promising species for reforestations, ornamental use, as well as for medicinal use and the food industry. Μorphological traits and molecular markers (RAPD) were used to identify and distinguish these Arbutus species to facilitate their exploitation. Since there are no descriptors established for Arbutus spp., 23 qualitative morphological characteristics of crown, foliage, bark, flowering, fruiting, and four quantitative morphological characteristics of leaf and fruit were selected and used to define differences and similarities between sampled individuals of A. andrachne, A. unedo and individuals with intermediate characteristics sampled as A. × andrachnoides. Twenty eight individuals representative of three Arbutus taxa were sampled in two typical macchia forest areas of the prefecture of Attica, Greece. Cluster analysis based on morphological characteristics separated the individuals in three distinct groups, and this was confirmed by molecular analysis. Τhe intermediate form was indicated as A. × andrachnoides, a natural hybrid of A. andrachne and A. unedo. Fifteen 10-mer oligonucleotide arbitrary primers used to amplify genomic DNA generated 166 reproducible polymorphic fragments, which revealed that A. × andrachnoides has higher degree of genetic similarity with A. andrachne than A. unedo. The applied morphometric characteristics are suggested as a basis to develop a complete list of discriminating descriptors for Arbutus genus.

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).

IDENTIFICATION OF CDNA FRAGMENT TIGHTLY LINKED TO NV AND TM-2 LOCI IN TOMATO

Tm-2 is a resistance gene in tomato to Tomato Mosaic Virus (ToMV), located in heterochromatic region of chromosome nine. Since map based cloning difficult to perform for identify the gene on that region, we apply differential display approach by using two near-isogenic tomato lines (NILs), one without Tm-2 and the other with Tm-2 to identify cDNAs of the transcripts from the region surrounding the Tm-2 locus. Among the 150 combinations of three anchor primers and fifty arbitrary primers, 10 combinations generated cDNA polymorphic bands. Out of them, only one combination of CA6, exhibited polymorphic band under southern blot analysis, subsequently a genetic experiment showed that the CA6 locus tightly linked to the Tm-2 locus. The CA6 fragment also hybridized to genomic DNA fragments from a tomato line carrying Tm-2a, a line of L. peruvianum from which Tm-2a originated, and a tomato line carrying another Tm-2-like gene. A northern hybridization blotting result suggested that the gene corresponding to CA6 fragment was constitutively transcribed.

Genetic diversity analysis of rice (Oryza sativa L.) landraces through RAPD markers

The molecular marker is a useful tool for assessing genetic variations and resolving cultivar identities. Information on genetic diversity and relationships among rice landraces from Bangladesh is currently very limited. Thirty-five rice genotypes including 33 landraces and 01 HYV of Bangladesh and 1 Indian landrace of particular interest to breeding programs were evaluated by means of random amplified polymorphic DNA (RAPD) technique. For molecular characterization, RAPD markers viz., OPC 03, OPC 04 and OPA 01 gave reproducible and distinct polymorphic amplified products. A total of 20 RAPD bands were scored of which 15 polymorphic amplification products were obtained by using these arbitrary primers. The size of amplified fragments were ranged from 550 to 1775 bp. Based on analysis performed on a similarity matrix using UPGMA, 35 genotypes were grouped into 2 main clusters. Landrace Sylhet balam and Mota aman was totally different from other genotypes. The information will facilitate selection of genotypes to serve as parents for effective rice breeding programs in Bangladesh. DOI: http://dx.doi.org/10.3329/ijarit.v4i1.21099 Int. J. Agril. Res. Innov. & Tech. 4 (1): 77-87, June, 2014