Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations

The random amplified polymorphic DNA (RAPD) assay and related techniques like the arbitrarily primed polymerase chain reaction (AP-PCR) have been shown to detect genotoxin-induced DNA damage and mutations. The changes occurring in RAPD profiles following genotoxic treatments include variation in band intensity as well as gain or loss of bands. However, the interpretation of the molecular events responsible for differences in the RAPD patterns is not an easy task since different DNA alterations can induce similar type of changes. In this study, we evaluated the effects of a number of DNA alterations on the RAPD profiles. Genomic DNA from different species was digested with restriction enzymes, ultrasonicated, treated with benzo[a]pyrene (B[a]P) diol epoxide (BPDE) and the resulting RAPD profiles were evaluated. In comparison to the enzymatic DNA digestions, sonication caused greater changes in the RAPD patterns and induced a dose-related disappearance of the high molecular weight amplicons. A DNA sample substantially modified with BPDE caused very similar changes but amplicons of low molecular weight were also affected. Appearance of new bands and increase in band intensity were also evident in the RAPD profiles generated by the BPDE-modified DNA. Random mutations occurring in mismatch repair-deficient strains did not cause any changes in the banding patterns whereas a single base change in 10-mer primers produced substantial differences. Finally, further research is required to better understand the potential and limitations of the RAPD assay for the detection of DNA damage and mutations.     

Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations

The random amplified polymorphic DNA (RAPD) assay and related techniques like the arbitrarily primed polymerase chain reaction (AP-PCR) have been shown to detect genotoxin-induced DNA damage and mutations. The changes occurring in RAPD profiles following genotoxic treatments include variation in band intensity as well as gain or loss of bands. However, the interpretation of the molecular events responsible for differences in the RAPD patterns is not an easy task since different DNA alterations can induce similar type of changes. In this study, we evaluated the effects of a number of DNA alterations on the RAPD profiles. Genomic DNA from different species was digested with restriction enzymes, ultrasonicated, treated with benzo[a]pyrene (B[a]P) diol epoxide (BPDE) and the resulting RAPD profiles were evaluated. In comparison to the enzymatic DNA digestions, sonication caused greater changes in the RAPD patterns and induced a dose-related disappearance of the high molecular weight amplicons. A DNA sample substantially modified with BPDE caused very similar changes but amplicons of low molecular weight were also affected. Appearance of new bands and increase in band intensity were also evident in the RAPD profiles generated by the BPDE-modified DNA. Random mutations occurring in mismatch repair-deficient strains did not cause any changes in the banding patterns whereas a single base change in 10-mer primers produced substantial differences. Finally, further research is required to better understand the potential and limitations of the RAPD assay for the detection of DNA damage and mutations.     

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.     

采用SSR和RAPD标记研究黄瓜属（葫芦科）的系统发育关系

野黄瓜Cucumis hystrix（2n=24）是在亚洲发现的第一个染色体基数为12的黄瓜属物种。这一发现对现行的以染色体基数和地理分布为基础的黄瓜属分类系统提出了质疑。采用SSR和RAPD两种分子标记对黄瓜属22份不同类型材料的亲缘关系进行了研究。结果表明,野黄瓜C.hystrix与黄瓜C.sativus var.sativus（2n=14）间的遗传距离（SSR:0.59,RAPD:0.57）小于其与甜瓜C.melo var.melo（2n=24）间的距离（SSR:0.87,RAPD:0.70）。SS     

Molecular Characterization of <Emphasis Type="Italic">Cyclamen</Emphasis> Species Collected from Different Parts of Turkey by RAPD and SRAP Markers

The genus Cyclamen (family Myrsinaceae) contains about 20 species, most of which occur in the Mediterranean region. Turkey has critically important Cyclamen genetic resources. Molecular characterization of plant materials collected from different regions of Turkey in which Cyclamen species grow naturally, namely Adana, Antalya, Ayd?n, Mu?la, ?zmir, Denizli, Kahramanmara?, Osmaniye, Eski?ehir, Trabzon, and Rize provinces, was performed using RAPD and SRAP markers. DNA was successfully amplified by 30 RAPD primers and 14 SRAP primer pairs. Among the 470 bands generated by the RAPD primers, 467 were polymorphic. The number of bands detected by a single primer set ranged from 11 to 22 (average of 15.6). The percentage polymorphism was 99.3 % based on the RAPD data. In the SRAP analysis, a total of 216 bands were generated, showing 100 % polymorphism. The number of bands detected by a single primer set ranged from 9 to 22 (average of 15.4). All data were scored and UPGMA dendrograms were constructed with similar results in both marker systems, i.e., different species from nine provinces of Turkey were separated from each other in the dendrograms with the same species being clustered together.     

Random amplification of polymorphic DNA with conserved sequences reveals genome-specific monomorphic amplicons: Implications in clad identification

The enzymatic amplification of genomic DNA with an arbitrary primer generates informative band profile useful for genome analysis. We used a set of synthetic oligodeoxyribonucleotide primers OAT15.2 (GACA)_3.75, OAT18. 2 (GACA)_4.5, OAT24.2 (GACA)₆, OAT36 (GACA)₉, comprising 4–9 consecutive units of GACA repeat, O33.15 (CACCTCTCCACCTGCC) and 033.6 (CCTCCAGCCCTCCTCCAGCCCT) for RAPD reactions of genomic DNA from different sources. The GACA based oligos of 15 and 18 base residues generated discernible genome specific amplicons whereas primers larger than 18 bases revealed smeary signals. The other oligos O33.15 and O33.6 also generated genome specific amplicons with more bands compared with those obtained from OAT15.2 or OAT18.2. The presence of OAT15.1 (GATA)_3.75 and OAT15.2 (GACA)_3.75 sequences in different genomes were ascertained by independent dot-blot hybridization prior to using them for RAPD reactions. The RAPD amplicons generated by evolutionarily conserved primer(s) or sequences shared by many species may be useful for clad identification in controversial systematics, comparative genome analysis, and for establishing the phylogenetic status of an organism.     

Deciphering genetic diversity analysis of saffron (Crocus sativus L.) using RAPD and ISSR markers

The existence of genetic diversity in Crocus sativus has globally remained a mystery till date. The study investigated PCR based DNA amplification profile of saffron using ISSR and RAPD based primers. A total of 38 amplicons were generated by ISSR primers in the range from 7 to 12 with an average of 9.50 bands per primer. 20 bands were found to be polymorphic and 18 were monomorphic with an average percentage of polymorphism as 52.48%. RAPD based amplification revealed a total 161 amplicons, 107 as polymorphic and 54 as monomorphic with an average percentage of polymorphism as 66.44%. Cumulative results of RAPD and ISSR demonstrated that Nei-Li’s similarity index ranged between 0.70 and 0.97. The results of AMOVA has revealed 9% of variance among populations and 91% of variance within populations, Φ PT was found as 0.089, which indicates existence of genetic differences though limited. In conclusion, the results indicate that saffron accessions are minimally genetically differentiated, which could be capitalized in future breeding programmes to ameliorate this precious crop.     

ISSR and RAPD Based Evaluation of Genetic Stability of Encapsulated Micro Shoots of Glycyrrhiza glabra Following 6?Months of Storage

In vitro grown axillary micro shoots of Glycyrrhiza glabra were encapsulated in alginate beads. Following 6?months of normal storage at 25?±?2°C the re growth of encapsulated G. glabra micro shoots, reached 98% within 30?days of incubation on MS medium supplemented with 0.1 mg/l IAA. Re growth was characterized by the development of both shoot and root from single encapsulated micro shoot. Healthy plants were established to glass house with 95% survival. The genetic fidelity of plants obtained after conversion of alginate beads was ascertained through 10 RAPD and 13 ISSR primers. Of the 10 RAPD primers tested, 6 of them produced 14 clear and reproducible amplicons with an average of 2.3 bands per primer out of which 28.57% were polymorphic generated by only two primers. Eight ISSR primers produced total 37 bands ranging between 300 and 3,500?bp length. Number of scorable bands for each primer varied from 3 to 8 with an average of 4.6 bands per primer. Cluster analysis from ISSR and RAPD showed that all the tested plants including the mother plant distributed in two major groups with similarity coefficient ranging from 0.91 to 0.96 for RAPD and 0.89 to 0.97 for ISSR.     

Sequence homology of polymorphic AFLP markers in garlic (Allium sativum L.).

Linkage mapping and genetic diversity studies with DNA markers in plant species assume that comigrating bands are identical, or at least that they have homologous sequences. To test this assumption in a plant with a large genome, sequence identities of 7 polymorphic amplified fragment length polymorphism (AFLP) markers of garlic, previously used to estimate similarity in genetic diversity studies, were characterized. Among 37 diverse garlic clones, 87 bands from these 7 polymorphisms were excised, amplicons were cloned, and 2 to 6 colonies were sequenced from each band, to yield a total of 191 DNA amplicons. Of these 87 bands, 83 bands (95.4%) contained AFLP amplicons that were identical or highly homologous to the typical marker of that band; only 4 bands contained amplicons with little homology to the same-sized amplicons of other garlic clones. Of these 83 bands, 64 (73.6%) contained only highly homologous amplicons (>90% sequence identity), whereas 19 (21.8%) contained both homologous and nonhomologous amplicons, with sequence identities less than 60%. Of the 37 nonhomologous amplicons identified, 25 (67.5%) differed in length from other amplicons in the band. Sequence conservation of AFLP amplicons followed patterns similar to phylogenetic relationships among garlic clones, making them useful for developing simple PCR-based markers in genetic mapping and diversity assessment.     

Molecular characterization of Yarrowia lipolytica and Candida zeylanoides isolated from poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.     

Variability and phylogenetic relationships of the Cucumis sativus L. species inferred from NBS profiling and RAPD analysis

Genetic variability of the Cucumis sativus species and its phylogenetic relationsips with other species of the genus were studied on the basis of RAPD marking and analysis of intra- and interspecific polymorphism of the nucleotide sequences of the NBS-LRR gene family in species of the genus Cucumis with the use of the NBS profiling method. According to RAPD analysis, cucumber cultivars from different geographic regions are highly similar, except for accessions k-3835 and k-3833 from Afghanistan. NBS profiling analysis revealed phylogenetically most distinct accessions expected to be characterized by specificity of resistance: k-3845 from Uzbekistan, k-3851 from Kyrgyzstan, line 701, k-3835 and k-3833 from Afghanistan, k-2757 and k-3079 from Netherlands, vr.k. 908 from Canada, k-2926 from Bulgaria, Russian cultivars Monastyrskii, Izyashchnyi, and Lel'. Three essentially different groups of species were distinguished, and the C. sativus species (subgenus Cucumis) was found to be distant from the species belonging to the subgenus Melo.     

七种蟋蟀基因组DNA的RAPD多态性研究（直翅目：蟋蟀总科）

应用10种随机引物,对西北地区常见的3属7种蟋蟀进行RAPD多态性检测,共筛选出2种引物S142,S8可以对7个种扩增出清晰稳定的多态性片段,多态性片段共计58条,相对分子质量在320bp-2400bp之间。应用UPGMA（非加权配对算术平均法）对多态性片段进行聚类分析,构建树状图,推测系统发生关系。每一种蟋蟀均先各自聚为一类,棺头蟋属与油葫芦属间的遗传距离最小,亲缘关系最近,斗蟋属4个种间的亲缘关系较为复杂,明显分为2个支系,与传统分类并不一致。     

Analysis of genetic diversity among garden- and field-pea genotypes of higher Indian Himalayas

Genetic diversity among 31 genotypes of field and garden pea including primitive cultivated forms and widely cultivated varieties of India was studied using 40 random decamer and 9 ISSR primers. A total of 274 amplicons were detected using both types of markers, which amplified 192 RAPD and 82 ISSR amplicons. Average number of bands amplified per primer was higher in case of ISSR (9.1) as compared to RAPDs (4.8). ISSR primers also exhibited higher average polymorphism (89.0%) and resolving power (4.50) than RAPDs (72.4%, 1.87, respectively). Genetic similarity estimates based on the pooled data of both types of markers using Jaccard??s coefficient ranged from 0.58 to 0.85 delineating considerable diversity among the pea genotypes studied. The 31 genotypes clustered in two major groups based on pooled data. Popular cultivars of garden and field pea of the region exhibited high similarities among themselves. However, primitive cultivated forms collected from the higher Indian Himalayas were diverse from the current varieties and hold potential in pea breeding programmes.     

Single primer amplification reaction (SPAR) reveals inter- and intra-specific natural genetic variation in five species of Cymbidium (Orchidaceae)

A total of 53 primers belonging to three SPAR methods, viz. RAPD, ISSR and DAMD, collectively produced 456 polymorphic amplicons with 96.6% polymorphism at inter-specific level in five species of Cymbidium, viz. C. aloifolium, C. mastersii, C. elegans, C. eburneum and C. tigrinum, whereas at intra-specific level, the observed polymorphism ranged from 51.2% to 77.1% among them. Three SPARs collectively revealed 25 unique species-specific amplicons; most of them were amplified with RAPD and DAMD primers besides few bands which were either missed (absent) or lost (heterozygosity). UPGMA clustering evidently distinguished the representatives of C. aloifolium and C. tigrinum, with distinct genetic distance, which may be due to their entirely different habitats as well as discrete morphological characteristics. Upon analysis of the data generated, all the three SPAR methods, either independently and/or in combination, revealed wide range of genetic variation between and within five species of Cymbidium. Comparison of matrix of individual SPAR method revealed that analysis of natural genetic variation using combination of SPAR methods, rather than an isolated approach, is highly effective. The critical analyses of the amplicon data are indicative of DAMD as the most powerful SPAR method by showing highest resolving power (Rp) followed by ISSR and RAPD. Alternatively, the total polymorphic information content was highest in case of RAPD followed by other two SPAR methods. Thus, the present investigation for the first time provides a valuable baseline data for genetic variation at inter- and intra-specific levels in horticultural Cymbidiums and also addresses conservation concerns.     

A new method for RAPD primers selection based on primer bias in nucleotide sequence data

Sequence analysis has proved that decamer nucleotides, used as primers of RAPD (random amplified polymorphic DNA), differ with each other greatly in number of annealing sites in the Arabidopsis thaliana genome. It is called the 'primer bias' by the authors. The biased primers produce a highly variable number of amplicons by polymerase chain reaction (PCR). The number of amplicons is proved to correlate with the number of annealing sites. Therefore, a statistical method is proposed for selecting efficient primers based on the primer bias in the genomic sequence. The method was tested by experiment in A. thaliana genome, and the results demonstrate that the method outperforms routine methods and can substantially increase the efficiency of RAPD methodologies. We also proved that the expressed sequence tags (ESTs) show a highly coincident bias pattern with that of the whole genomic sequence, and can therefore be used to assess efficiencies of primers for species whose genomic sequence data are currently unknown.     

Comparison of ISSR and RAPD markers to characterize three Chilean Nothofagus species

The present study is the first report of fingerprinting on three Chilean Nothofagus species using ISSR and RAPD markers; 61 Nothofagus nervosa, 32 Nothofagus obliqua and 32 Nothafagus dombeyi individual trees, sampled from collection and natural sites, were analyzed. Among 45 primers tested, the 6 ISSR and 6 RAPD primers selected for the analysis generated a total of 63 ISSR and 42 RAPD fragments. A high proportion of polymorphic bands, ranging from 97% and 80%, was found using both markers. A similar number of private and marker bands was generated by both markers in all the species examined and one discriminant ISSR fragment was obtained for N. dombeyi. Jaccard and Dice similarity indices were used to evaluate pairwise genetic divergence; cluster analysis of the similarity matrices was performed to estimate the intra- and inter-specific genetic diversity, and PCA analysis was employed to evaluate the resolving power of the markers to differentiate between the species. These analyses, carried out for both markers, allowed us to identify three main groups corresponding to the three Nothofagus species. The results of the present study can be seen as a starting point for future researches on the population and evolutionary genetics of these species.     

Applicability of RAPD markers on silver-stained polyacrylamide gels to ascertain genetic diversity in Peripatus acacioi (Peripatidae; Onychophora)

RAPD (random amplification of polymorphic DNA) molecular markers can be utilized for analyzing genetic variability in populations for which only a few or no molecular markers are available. They were used in a study of an endangered species, Peripatus acacioi, found in the Tripuí Ecological Station, in Ouro Preto, MG, Brazil. The ecological station was specifically created to protect this velvet worm species, the first of this group found in Brazil. For an initial evaluation of the genetic diversity of this species, DNA samples from the lobopods of four individuals, collected at random, were analyzed using RAPD. Each reaction was run with a different primer (Operon RAPD 10-mer Kits), totaling 13 primers (OPC2, OPC3, OPC4, OPC6, OPC8, OPC10, OPC11, OPL2, OPL7, OPL11, OPL13, OPL18, and OPL19). Due to the low amplification yield, RAPD fragments were separated in polyacrylamide gels and stained with silver nitrate. Numerous bands were observed. Fifty-five of the amplified bands proved to be reproducible, both in terms of presence and intensity. Among these, 27 were variable and 28 were constant. The average number of bands per gel was 4.2. Nine of the 13 primers tested allowed the identification of constant and variable bands among these four individuals. RAPD analysis of genetic variation using silver-stained polyacrylamide gel electrophoresis provided measures of band sharing among the individuals, and therefore could be used in population genetics studies of P. acacioi.     

Evidence of Sibling Species in the Brown Planthopper Complex (Nilaparvata lugens) Detected from Short and Long Primer Random Amplified Polymorphic DNA Fingerprints

The inheritance of 31 amplicons from short and long primer RAPD was tested for segregating ratios in two families of the brown planthopper, Nilaparvata lugens, and they were found to be inherited in a simple Mendelian fashion. These markers could now be used in population genetics studies of N. lugens. Ten populations of N. lugens were collected from five locations in Malaysia. Each location had two sympatric populations. Cluster and principal coordinate analyses based on genetic distance along with AMOVA revealed that the rice-infesting populations (with high esterase activity) at five localities clustered together as a group, and Leersia-infesting populations (with low esterase activity) at the same localities formed another distinct cluster. Two amplicons from primers OPD03 (0.65 kb) and peh#6 (1.0 kb) could be considered diagnostic bands, which were fixed in the Leersia-infesting populations. These results represent evidence of a sibling species in the N. lugens complex.     

Cryptic Species in the Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) Complex: Incongruence Between Random Amplified Polymorphic DNA-Polymerase Chain Reaction Identification and Analysis of Mitochondrial DNA COI Gene Sequences

Random amplified polymorphic DNA (RAPD) diagnostic bands are one tool used to differentiate cryptic mosquito species in the Anopheles albitarsis Complex. Monophyly of four species (A. albitarsis Lynch-Arribálzaga, A. albitarsis B, A. deaneorum Rosa-Freitas, and A. marajoara Galv?o & Damasceno) currently identified with the RAPD technique was assessed using sequences of the cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) gene. Maximum parsimony, maximum likelihood, and Bayesian analyses support monophyly for A. albitarsis s.s., A. albitarsis B, and A. deaneorum. Anopheles marajoara, as identified by RAPD banding patterns, was either polyphyletic or paraphyletic in all phylogenetic analyses. The phylogenetic pattern and within-species genetic distances observed in A. marajoara suggest the existence of a previously unidentified species (species E) in northern Brazil and Venezuela. Diagnostic RAPD bands were unable to distinguish between A. marajoara and species E, probably because of the low number of correlated bands used to identify species and weaknesses of the RAPD technique, in particular, violations of the untested assumption of homology of comigrating bands. A. marajoara (even without species E) is paraphyletic with respect to A. deaneorum; if A. deaneorum is a separate species from A. marajoara, then A. marajoara may consist of two or more species in Amazonian Brazil. Based on mtDNA COI sequences, there are at least four phylogenetic species within the Albitarsis Complex: A. albitarsis s.s., A. albitarsis B, A. marajoara, and species E; the species status of A. deaneorum is ambiguous.     

Evolutionary distances and identification of Candida species in clinical isolates by Randomly Amplified Polymorphic DNA (RAPD)

Fast and reliable identification of different species of the genus Candida is important to define adequate therapeutic decisions, because the different species have highly variable susceptibilities to antifungal drugs; azoles and amphothericin B. Accurate statistical records on case history and epidemiological studies also depend on effective identification. To address this problem we established a RAPD method that enabled direct identification of five very common species of Candida. Initially, reference band patterns were established for C. albicans, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. One of the primers, M2, showed remarkably conserved intra-specific patterns of approximately 10 bands each, ranging in size from 2.0 to 0.1 kb. These patterns were significantly different and species-specific. Few bands were conserved between different species of Candida, which was assumed to be consistent with their phylogenetic relatedness. In addition, band patterns were constant and reproducible and DNA isolated from single colonies yielded sufficient DNA for identification. The reference band patterns were then used, in blind experiments, to identify species of Candida in 50 randomly chosen samples, including clinical isolates and ATCC strains. RAPD results were 100% consistent with results obtained by conventional diagnostic methods and were achieved in one day instead of several days taken by conventional methods. Because ideal identification methods should be consistent with phylogeny and taxonomy we tested whether RAPD could be used to calculate genetic distances. Comparison of RAPD phylogenetic trees with 18S rRNA trees showed significant differences in tree topologies which indicated that RAPD data could not accurately measure the relative distances between different species. Also, computer simulations of RAPD random patterns were used to test whether the observed degree of RAPD band pattern similarities could occur at random. These simulations suggested that the level of inter-specific band pattern similarities observed in our data could be obtained at random, while intra-specific pattern similarities could not. RAPD would be helpful to discriminate between isolates but not to quantitate the differences. We suggest that the inaccurate estimate of genetic distances from RAPD is a general limitation of the technique and not a specific problem of our identification method. Because of the repetitive character of the target sequences, genetic distances calculated from RAPD could be affected by paralogy, namely, recombination and duplication events not parallel with speciation events. This revised version was published online in June 2006 with corrections to the Cover Date.