Random Amplified Polymorphic DNA Reaction Research Articles

ANALYSIS OF GENETIC DIVERSITY OF SOME OLIVEGENOTYPES USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS

This study aimed to use the Random Amplified Polymorphic DNA (RAPD) markers based on the PCR technique in the analysis of genetic variation among seven olive grown cultivar Olea europaea L. cultivars in Saladin province in Iraq Eighteen primers were used in RAPD reactions ,Results of RAPD experiments showed different loci of the bands. The total number of loci defined by the primer was 99 loci, of which 18 were general sites for all samples and 81 different locations. (OPP-01, OPM-15, OPK-13,1700bp) highest, and the lowest partial size in the primers [100 bpOPH-14, OPG-05] The variance between the samples studied was highest, as OPZ-08 (9.0), and less than in OPM-03 (2.1) The ability to discriminate for the primers was characterized by the OPZ-08 primer with a maximum discriminating higher of 9.0. The OPM-03 primer was characterized by the least discriminating of 2.1A total of 429 total bands bundles were produced, of which 18 were general main bands and 411 were polymorphic bands, The total number of distinctive bands resulting from in this study was 49. 21 absent band and( 28) Unique bands. The sample Santacatrina 5 received the highest number of missing bands, reaching (16 bands) Shamlaily is a sample 2 with the highest number of unique bands 7. The genetic distance values ​​ranged from (0.769-0.089), the lowest value of the genetic distance (0.089) and the difference between the four types (Frantoryo and 3 photo), and the highest genetic distance was between 5 (Santacaterina and 6) 0.769.While the analysis of the genetic relationship revealed that there are three main groups, including the first sample (5) Santacaterina, the second group including subgroups B1, B2 included the first B1 sample only one sample Krodsoo, while the second group B2 has included the remaining items Shamali, Sorani, Frantoyo Duhkan, Qaisi

Diallel analysis and genetic diversity of some yellow maize inbred lines (Zea mays L.) using RAPD and SSR markers

Information about combining ability and genetic diversity among maize inbred lines is fundamental in designing breeding strategies for improving grain yield. Twenty one F1 hybrids were generated by crossing seven yellow maize inbred lines in a half diallel mating scheme in 2013 season. The 21 F1 hybrids plus the two check hybrids (SC166 and SC173) were evaluated in a randomized complete block design with three replications during 2014 season, to estimate combining ability effects and identify type of gene action governing the inheritance of grain yield and other important agronomic traits. Results showed that both general (GCA) and specific (SCA) combining ability mean squares were highly significant for all the studied characters. The GCA/SCA ratio was more than unity for all the studied traits, except days to 50% silking, plant height and grain yield, indicating the preponderance of the additive gene effects in the inheritance of these traits. The inbred line P1 appeared to be the best general combiner for earliness, grain yield and its components. The crosses P1×P2, P3×P7, P4×P7, and P5×P6 had the best SCA effects for grain yield and most of its component traits. The cross P1×P2 significantly out-yielded the two check hybrids (SC166 and SC173). The genetic diversity among the seven yellow parental inbred lines was assessed using two types of molecular markers; Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSR). Seven random primers were used in the RAPD reactions, resulting in the amplification of 82 bands with 95.12% polymorphism. While, eight pairs of SSR primers were used and resulted in 49 fragments with 100% polymorphism. Genetic similarity among all possible pairs of inbred lines varied from 0.12 to 0.69, with an average of 0.34, for RAPD markers, and from 0.01 to 0.56, with an average of 0.24, for SSR markers. The similarity matrices for RAPD and SSR data were not significantly correlated (r=0.34, p >0.05). Genetic distances based on RAPD and SSR markers were insignificantly correlated with F1 hybrids grain yield, the correlation values being (r =0.02 and 0.429 P>0.05), respectively. Therefore, the parental genetic distance could not be used to predict the grain yield of the F1 hybrids in this study.

Extraction of total DNA and optimization of the RAPD reaction system in Dioscorea opposita Thunb.

Dioscorea opposita Thunb. has been used as health food and herbal medicinal ingredients in traditional Chinese medicine. In this study, the total DNA of D. opposita Thunb. was extracted using an improved cetyltrimethylammonium bromide (CTAB) method, and the extracted DNA was further used for random amplified polymorphic DNA (RAPD) reaction system by design of the L16 (4(4)) orthogonal diagram. The results showed that the improved CTAB method can be used to isolate high-quality and high-concentration DNA, and the optimized protocol can overcome the instability of RAPD reaction system. The knowledge stated here can be used to study the genetic diversity of D. opposita Thunb.

Optimization of RAPD-PCR Conditions for the Study of Genetic Diversity of Centella asiatica

Random amplified polymorphic DNA (RAPD) is one of the molecular marker tools available for detecting the polymorphism in plant species. It has been used extensively for genetic diversity studies. In the present investigation, the RAPD reaction and cycling conditions were optimized for generating RAPD fingerprints of twenty one ecotypes of Centella. asiatica (L.) urban collected from different locations of Nepal. To determine the optimum conditions for PCR amplification, different concentrations of master mix ( 2X mix , Promega USA), template DNA, primer and PCR cycling conditions were varied. Reproducible amplified products were observed using 0.5 ?M of primer, 12.5?l master mix (2X), and 100 ng of template DNA in 25?l reaction volume of PCR. Cycling conditions were optimized with varying annealing temperature and initial denaturation temperature. The best cycling condition comprised of an initial denaturation of 3 min at 94°C, followed by 30 cycles of denaturation for 45 sec at 94°C, annealing for 1 min at 37°C, and extension for 1 min at 72°C and final extension at 72°C for 7 min . Of the 21 random primers tested, only 8 primers produced the best and reproducible amplification products. The optimized RAPD-PCR conditions and selected primers were subsequently used for the study of genetic diversity in C. asiatica.DOI: http://dx.doi.org/10.3126/njst.v12i0.6482 Nepal Journal of Science and Technology 12 (2011) 69-74

Molecular-based marker for sex differentiation of jojoba in vivo and in vitro Iraqi cultivars using RAPD- PCR technique

The objective of this study was to distinguish between males and females of jojoba (Simmondisia chinensis) using Random Amplified Polymorphic DNA (RAPD) markers in order to detect any DNA variation. A callus was produced from both male and female jojoba leaves, using MS medium with BA 0.5 and 2 to 4, D 2.5 mg/l respectively. Genomic DNA of jojoba plant was extracted using CTAB method. The concentration of DNA ranging between 100 to 150 µg with DNA purity ranging from 1.01 to 1.19. About 0.7 g of male and female plant leaves, and calli were used. Eight different random primers were evaluated for their usefulness in detecting DNA variation between male and female of jojoba. This involved optimization of RAPD reaction condition including, DNA template, Taq polymerase, and primers. Five primers showed no amplification and represent a mean of distinguishing between male and female. One primer (C5) showed amplification and represent a mean of distinguishing between the two sexes from the fresh sample tissue; the callus results show no significant difference. The results demonstrated the feasibility of using RAPD-PCR in distinguishing between jojoba sexes. Key words: RAPD- PCR, Simmondsia chinensis, sex differentiation.

RAPD and ISSR Methods Used for Fingerprinting Selected, Closely Related Cultivars of Aronia melanocarpa

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic diversity between six genotypes of Aronia melanocarpa (Hugin, Nero, N, Viking, Galicjanka 1 and Galicjanka 2), collected at the Department of Pomology at the West Pomeranian University of Technology in Szczecin, Poland. Analysis of 24 RAPD and 25 ISSR primers showed very low genetic diversity between the compared cultivars. Analysis of 214 RAPD loci and 184 ISSR loci revealed that the following five genotypes: Nero, N, Viking, Galicjanka 1 and Galicjanka 2 are characterized by a very small range of diversity in comparison to the substantially different cultivar Hugin. For each genotype, specific products were generated, including 36 products generated in RAPD reactions for cultivar Hugin only, and 20 for the other five genotypes altogether. Specific products were amplified also with the use of the ISSR technique. Thirty-nine ISSR products were generated for the cultivar Hugin only, and 8 for the other five genotypes altogether. Results of the research indicate a very small range of diversity between the Aronia accessions. Analysis of mutual genotypic relationships between the compared accessions was carried out with the use of the Mantel test and found a significant correlation (r(AB)=0.531) between similarity matrices calculated on the basis of results obtained with the use of the RAPD and ISSR techniques.

Phylogenetic relatedness among Spirulina and related cyanobacterial genera

Molecular polymorphisms in a selected set of Spirulina and related genera using random primers based on repetitive sequences along with biochemical parameters, led to the unambiguous differentiation of the strains and understanding of their phylogenetic relationships. A combination of 10 sets of dual primers generated 100% distinct polymorphic bands ranging from 150 to 5,000 bp. Total number of fragments ranged from 68 to 159 whereas polymorphic bands ranged from 13 to 32 for different Random Amplified Polymorphic DNA (RAPD) reactions. Spirulina platensis strains, Sp-2 and Sp-3, possessed quite comparable chlorophyll and protein content besides having maximum similarity coefficient (0.88) between them on the basis of RAPD reactions, thus proved to be closely related. Sp-8 (Spirulina from Loktak Lake) having the highest protein content and protein: chlorophyll ratio, showed close similarity with the mutant of Spirulina platensis (Sp-7) on the basis of RAPD analysis. Duncan’s Multiple Range Test (DMRT) ranking for the biochemical parameters were quite closer for the strains of Spirulina and Arthrospira. This is also supported by the cluster analysis based on RAPD data, as the strains of Spirulina and Arthrospira are placed together in the same subcluster in the dendrogram. The comparative closeness among the strains of Lyngbya, Oscillatoria and Phormidium is reflected by the low content of protein and protein: chlorophyll ratio, which is also supported by the dendrogram based upon RAPD; thus, exhibiting the usefulness of multiplex RAPD along with biochemical parameters for the phylogenetic analysis of Spirulina and related genera.

Optimization of conditions in random amplified polymorphic DNA (RAPD) reaction for determination of plant pathogenic bacteria

Random Amplified Polymorphic DNA (RAPD) technique is a powerful tool for genetic studies. It can be also applied for determination of bacterial species. The goal of this work was to optimize conditions for RAPD reaction, and to obtain RAPD patterns specific for some plant-associated bacteria. RAPD could be a good choice for screening and preliminary bacterial determination. Application of RAPD can determine that some of bacterial species are not reason of infection, and make additional tests for them unnecessary. This can provide time, labor, and as well as money saving, which is important particularly if expensive tests are applied. Optimization of reaction conditions in this work made possible good repeatability of RAPD electrophoretic patterns for particular bacterial species.

RAPD Analysis for Determination of Components in Herbal Medicine

In this study, the RAPD (Random Amplified Polymorphic DNA) technique was employed for determination of the components in an Ayurvedic herbal prescription, Rasayana Churna. One-hundred-and-twenty decamer oligonucleotide primers were screened in the RAPD analysis to identify three Ayurvedic medicines, dried stem of Tinospora cordifolia, dried fruit of Emblica officinalis and dried fruit of Tribulus terestris, the Ayurvedic prescription. Primer OPC-6 simultaneously generated three distinct amplicons, each specific to one component. The marker with 600 bp is specific to Tinospora cordifolia; the marker 500 bp is specific to Emblica officinalis and the remaining marker >1000 bp was present in Tribulus terestris. Presence of three herbal medicines was determined when RAPD reaction with OPC-6 was performed. The technique was proved to contribute to the identification of components in Ayurvedic herbal preparation and thus helping to serve as a complementary tool for quality control.

Genetics Polymorphism Study of Ixodes Ricinus L. Populations in Lithuania using RAPD Markers

Ixodes ricinus ticks are common and widespread in Lithuania. They are the main vector in transmission of infectious diseases like tick-born encephalitis, Lyme borreliosis and human granulocytic ehrlichiosis. The aim of our study was to investigate genetic variability of I. ricinus ticks in Lithuania. I. ricinus ticks were collected from different locations of Lithuania. Data on genetic polymorphism of I. ricinus were used to determinate genetic distances within and among different Lithuanian populations in this species. At first, 103 collected ticks were analysed using specifics for I. ricinus new primers designed by A. Jenkins for taxonomic identification. PCR analysis data showed that all ticks were I. ricinus. Polymorphism of I. ricinus was analysed using the random amplified polymorphic DNA (RAPD) method. 47 DNA samples of I. ricinus ticks were used for RAPD reaction. Ten arbitrary sequence decamers were used as primers. Reproducible amplification patterns were obtained using five primers. RAPD analys...

Use of RAPD markers for identifying Nelore bulls with early reproductive maturation onset

Random amplified polymorphic DNA (RAPD) markers were used for identifying animals with early (precocious) or late (non-precocious) reproductive maturation onset. Animals ( n=34) were phenotyped according to spermatozoa appearance in ejaculates (group A, 20 animals) or to the expected progeny difference (EPD) values for scrotal circumference (group B, 14 animals). The RAPD markers were initially detected by amplifying two pooled samples of equimolar amounts of DNA from the eight precocious 12 and non-precocious animals of group A. Only 38 out of 320 random primers used for screening group A pooled samples detected polymorphisms. These polymorphic primers generated 443 distinguishable and reproducible bands, from which 174 were polymorphic and 269, monomorphic. These polymorphic primers were then used in RAPD reactions to amplify individual DNA samples from animals belonging to both groups, A and B. The dendrograms generated from RAPD patterns allowed phenotypic class differentiation in both cases. Therefore, RAPD markers can be used as a tool for identifying genotypes favoring early sexual maturation in Nelore breeding programs.

RAPD Analysis of peaches within Czech National collection

The Random Amplified Polymorphic DNA (RAPD) technique was used to study the genetic diversity and relationships within the collection of the Czech National Plant Genetic Resources (PGR) of peaches (Prunus persica L.). The aim of the work was to elaborate a dendrogram of genetic similarity and to divide collection into clusters. 46 primers were applied to 6 cultivars differing in the place of origin, the fruit shape, the fruit colour, and in some other morphological characteristics. 12 primers were chosen which gave polymorphic repeatable strong and middle strong bands. They were subsequently used for the RAPD reactions within the whole collection of peaches. The selected RAPD primers distinguished 28 peach cultivars and RAPD data were used to group the accessions analysed. Almonds and peach × almond hybrids were clearly separated in the frame of the whole collection. The grouping corresponded to the botanical system, to the available information about pedigree, and to the cultivars description.  

Evaluation of genetic diversity in 19 Glycine max (L.) Merr. accessions included in the Czech National Collection of soybean genotypes

The random amplified polymorphic DNA (RAPD) technique was used to evaluate both genetic diversity among 19 soybean accessions included in the Czech National Collection of Soybean Genotypes and their potential as a new source of genetic variations for soybean breeding programs. Only 22 of all the 40 random primers used in RAPD reactions showed polymorphism acceptable for an effective characterisation of these accessions. Altogether 122 highly reproducible RAPD fragments were generated, 55 of them were polymorphic (46%). However, because of the previously observed low degree of RAPD polymorphism in the case of Glycine max, fragments with low level of informativeness were evaluated, too. Presented results enable the selection of genetically distinct individuals. Such information may be useful to breeders willing to use genetically diverse introductions in soybean improvement process. 

DETECTION BY RAPD OF GENETIC ALTERATIONS IN VITRO: AMPLIFICATION AND CONSERVATION CONDITIONS OF DNA EXTRACTS

The technique of random amplified polymorphic DNA (RAPD) permits the study of genetic ecotoxicology without having to face the problem posed by the high number of chromosomes generally presented by the fish species used in traditional methods. Additionally, the use of in vitro systems allows the study of a large number of samples in environmental-risk assessment. Good standardization of the parameters involved in the RAPD reaction, such as primer concentration and the DNA template used and its integrity, is fundamental for obtaining reliable and repeatable results. This is especially important when the differences in DNA fingerprints between control cells and cells exposed to genotoxic agents are interpreted as toxic-dependent alterations. The use of more than one primer increases sensitivity in the detection of such differences, provided the amplification is carried out under optimum conditions. This article demonstrates that the conditions established for certain primers in previous studies can be acceptable for others, independent of the complexity of band patterns generated. Furthermore, the integrity of the DNA is shown to be stable for several months in the genomic extracts stored at 4°C, which to a large extent facilitates the application of this methodology.

RAPD PCR for Diagnosis of Phytophthora parasitica var. nicotianae Isolates which Cause Black Shank on Tobacco

Phytophthora parasitica var. nicotianae is the fungal pathotype of tobacco black shank (TBS, Disease severity ≥ 2.0). Random amplified polymorphic DNA (RAPD) analysis was used to differentiate isolates which cause TBS from those which do not. Greenhouse assays combined with zoospore inoculation were performed to assess the virulence of the fungal isolates, and the results were compared with the RAPD pattern analysis. The RAPD results exhibited total correlation with the virulence assay results. Amplification patterns generated by RAPD reactions were used to generate a phenogram depicting the genetically distinct nature of the cluster defined by the TBS isolates. This cluster was exclusive and distinct from P. parasitica var. nicotianae isolates which do not cause TBS. Thus, RAPD proved to be a sensitive and highly reliable method for quickly identifying fungal pathotypes which cause TBS.

Interlaboratory Study on Thermal Cycler Performance in Controlled PCR and Random Amplified Polymorphic DNA Analyses

Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cyclers to PCR performance as measured by interblock reproducibility and intrablock repeatability. Two standardized assays designed to minimize the introduction of non-thermal-cycler-dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cyclers of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems. All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cyclers. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cyclers, which decreased to 63% in noncalibrated instruments. Irrespective of the make and model of thermal cycler, temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance.

RAPD-PCR Amplification of DNA Extracted from Avian Blood Infected With Haemoproteus Fails to Produce False Positive Markers

Abstract We tested whether contamination of avian blood by the parasite Haemoproteus could cause the amplification of false positive genetic markers generated by random amplified polymorphic DNA (RAPD). DNA was extracted from blood taken from five Rock Doves (Columba livia) infected with Haemoproteus, and one uninfected control individual. A drug treatment was administered to clear the infection, and a second DNA sample extracted. The parasitized and unparasitized DNA samples were used in RAPD reactions with 20 different 10-nucleotide primers, which produced 178 well-resolved markers. In no case were any of the reaction products specific to the parasitized samples, and in almost all cases the products were found consistently across all replications of infected and non-infected samples. The high ratio of avian to parasite DNA and the greater genome complexity of the host probably prevents the amplification of markers unique to parasite species such as Haemoproteus.

RAPD-PCR AMPLIFICATION OF DNA EXTRACTED FROM AVIAN BLOOD INFECTED WITH HAEMOPROTEUS FAILS TO PRODUCE FALSE POSITIVE MARKERS

Abstract We tested whether contamination of avian blood by the parasite Haemoproteus could cause the amplification of false positive genetic markers generated by random amplified polymorphic DNA (RAPD). DNA was extracted from blood taken from five Rock Doves (Columba livia) infected with Haemoproteus, and one uninfected control individual. A drug treatment was administered to clear the infection, and a second DNA sample extracted. The parasitized and unparasitized DNA samples were used in RAPD reactions with 20 different 10-nucleotide primers, which produced 178 well-resolved markers. In no case were any of the reaction products specific to the parasitized samples, and in almost all cases the products were found consistently across all replications of infected and non-infected samples. The high ratio of avian to parasite DNA and the greater genome complexity of the host probably prevents the amplification of markers unique to parasite species such as Haemoproteus.

Structure of Genetic Diversity among Common Bean Landraces of Middle American Origin Based on Correspondence Analysis of RAPD

More than 60% of common bean production worldwide is derived from cultivars of Middle American origin. Understanding the diversity of these will facilitate their use in genetic improvement. The objective of this study was to analyze a collection of 269 landraces of common bean (Phaseolus vulgaris L.) by correspondence analysis of random amplified polymorphic DNA (RAPD) data to determine the genetic structure of the Middle American gene pool of cultivated bean. One hundred eighty landraces originating in Mexico, the remainder in Central America and secondary centers of diversity within the Americas, and two checks were studied. DNA was extracted, RAPD reactions carried out, and polymorphic bands were scored as present or absent on the basis of 39 primers. Groups were formed which in part corresponded to races defined previously by morphological and agroecological criteria. However, tropical small‐seeded Race M was composed of two groups: one largely Mexican that included most small‐seeded black beans of upright plant habit; and one Central American with landraces of various seed colors. Most non‐black small‐seeded germplasm of Race M phenotype from secondary centers grouped with the Central American landraces, except for cream‐seeded and purple‐seeded accessions from Brazil. Races D and J could be distinguished and within races D and J further divisions could be recognized which were related to geographic origin. The more commercial Race D landraces formed a genetic group that was predominant in the western part of the Mexican highland plateau. Another Race D group was concentrated at the eastern extreme of the neovolcanic axis and was differentiated morphologically as well. Guatemalan germplasm contained accessions of climbing bean that did not group with any of the previously defined races and should be considered a separate race. Thus, Middle American germplasm of common bean is more complex than previously thought, and contains diversity that remains to be explored for its practical value.

Random amplified polymorphic DNA (RAPD) typing of necrophageous insects (Diptera, Coleoptera) in criminal forensic studies: validation and use in practice

To permit quick identification of arthropods, random amplified polymorphic DNA typing (RAPD) was used to support classical morphological and medico-legal analysis of maggots on a human corpse. The method was employed to determine if maggots which were found on the inside of a body bag were identical (a) with maggots found on the outside of the bag, and (b) pupae found on the floor under the corpse. Pre-mixed RAPD reaction beads together with semiautomatic computer aided analysis of the PCR products are shown to discriminate between closely related necrophageous insect species (flies and beetles) found on corpses. From the 11 RAPD primers used, one alone was sufficient in resolving a practical forensic situation. This is the first report of a forensic application of RAPD DNA typing.