Random Amplification Of Polymorphic DNA Bands Research Articles

Genetic relationships and genome verification of Thai banana cultivars using Random Amplification of Polymorphic DNA (RAPD) markers

Abstract. Boonsrangsom T, Fuenghoi C, Premjet D, Suvittawat K, Ratanasut K, Sujipuli K. 2023. Genetic relationships and genome verification of Thai banana cultivars using Random Amplification of Polymorphic DNA (RAPD) markers. Biodiversitas 24: 3758-3765. Edible bananas and plantains, belonging to the family Musaceae, genus Musa, represent one of the most important fruit crops, with an annual production of more than 65 million tons worldwide. Bananas have several hybrid variations since they are descended from the two species Musa acuminata Colla (AA genome) and Musa balbisiana Colla (BB genome). Different morphological traits divide almost hybrid bananas into various genomic groupings. Banana genome categorization and identification, however, have always been challenging issues. This study aimed to assess the genetic relationships and verify the genomes of Thai banana cultivars using random amplification of polymorphic DNA (RAPD) markers. Using the 15 selected RAPD markers, 149 RAPD bands were found, with sizes ranging from 0.2 to 3.2 kb, and 88.6% were polymorphic. Polymorphic information content (PIC) values ranged from 0.18 to 0.42, averaging 0.30. Based on the Jaccard coefficient, the unweighted pair-group method arithmetic average (UPGMA) analysis showed that the banana samples had a similarity range of 0.27 to 1.00 with a mean of 0.56, demonstrating an abundance of viability across six banana genomes. The dendrogram generated from RAPD data revealed that all 18 Musa samples could be divided into two main groups (Group I and II). Three additional subgroups were created for each primary group (A, B, and C). The accurate identification and genetic data on the available genetic resources for bananas will be beneficial for breeding and conservation programs.

Investigation of Phages Infecting Escherichia coli Strains B and C, and Enterobacter cloacae in Sewage and Ebrié Lagoon, Côte d'Ivoire.

Background: Bacteriophages are a promising biotechnological against bacterial pathogens. Currently, phage research is garnering interest in sub-Saharan countries as bacterial resistance to antibiotics becomes widespread. They are sought in all environments as they offer the possibility of a sustainable alternative to antibiotics. Materials and Methods: Altogether 30 water samples from urban sewage and environmental water were screened for the presence of bacteriophages able to infect Escherichia coli and Enterobacter cloacae. Their genomic diversity was determined by random amplification of polymorphic DNA (RAPD)-PCR fingerprinting. Results: We isolated 35 phages including 9 polyvalent phages that infect simultaneously E. coli and E. cloacae. This study allowed first isolation of E. cloacae-specific phages in Côte d'Ivoire. All phages were distinct based on their RAPD band patterns. Conclusions: Sewage systems of Yopougon and the environmental water of Ebrié lagoon were a rich source of phages. The phage collection could be useful for phage application in Côte d'Ivoire.

GENETIC DIVERSITY IN ACCESSIONS OF INDIAN TURMERIC (CURCUMA LONGA L.) USING RAPD MARKERS

Objective: The present investigation was undertaken for identification and assessment of eight accessions of Curcuma longa collected from all ecological zones in India by random amplification of polymorphic DNA (RAPD) markers.Methods: DNA was isolated using modified cetyl trimethyl ammonium bromide (CTAB) method. Polymerase chain reaction (PCR) was performed according to the method based on Williams et al. and data analysis was done using Alpha Imager EC software.Results: Eleven out of twenty primers screened were informative and produced 150 amplification products among which 132 products (88%) were found to be polymorphic. The percentage polymorphism of all 08 accessions ranged from 44.44% to 100%. A total of 150 amplification products were scored with an average frequency of 13.63 bands per primer. Most of the RAPD markers studied showed a different level of genetic polymorphism. The data of 150 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA.Conclusion: Results shows that C. longa undergoes genetic variation due to a wide range of ecological conditions within distribution area of its population in India. This investigation as an understanding of the level and partitioning of genetic variation within the accessions and would provide an important input into determining efficient management strategies and will help to breeders for turmeric improvement program.

Draft Genome Comparison of Representatives of the Three Dominant Genotype Groups of Dairy Bacillus licheniformis Strains

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3' ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species.

Assessment of genetic diversity and relationships among Egyptian mango (Mangifera indica L.) cultivers grown in Suez Canal and Sinai region using RAPD markers.

DNA-based RAPD (Random Amplification of Polymorphic DNA) markers have been used extensively to study genetic diversity and relationships in a number of fruit crops. In this study, 10 (7 commercial mango cultivars and 3 accessions) mango genotypes traditionally grown in Suez Canal and Sinai region of Egypt, were selected to assess genetic diversity and relatedness. Total genomic DNA was extracted and subjected to RAPD analysis using 30 arbitrary 10-mer primers. Of these, eleven primers were selected which gave 92 clear and bright fragments. A total of 72 polymorphic RAPD bands were detected out of 92 bands, generating 78% polymorphisms. The mean PIC values scores for all loci were of 0.85. This reflects a high level of discriminatory power of a marker and most of these primers produced unique band pattern for each cultivar. A dendrogram based on Nei's Genetic distance co-efficient implied a moderate degree of genetic diversity among the cultivars used for experimentation, with some differences. The hybrid which had derived from cultivar as female parent was placed together. In the cluster, the cultivars and accessions formed separate groups according to bearing habit and type of embryo and the members in each group were very closely linked. Cluster analysis clearly showed two main groups, the first consisting of indigenous to the Delta of Egypt cultivars and the second consisting of indigenous to the Suez Canal and Sinai region. From the analysis of results, it appears the majority of mango cultivars originated from a local mango genepool and were domesticated later. The results indicated the potential of RAPD markers for the identification and management of mango germplasm for breeding purposes.

Omics-based biomarkers for the identification of six Korean cultivars of sweet potato (Ipomoea batatas L. Lam)

SummaryThis study was conducted to develop biomarkers to identify 26 cultivars of Korean sweet potato (Ipomoea batatas L. Lam) using omics-based methods. Random amplification of polymorphic DNA (RAPD) markers revealed that 75 random 10-mer primers generated 50 polymorphic RAPD markers from the 28 Korean sweet potato cultivars. Six sweet potato cultivars were selected for further analysis by proteomic and metabolomic approaches based on their flesh colour (two each having cream, orange, or purple flesh) and by a dendrogram generated using the polymorphic RAPD bands. Changes in the proteomes of these six sweet potato cultivars were investigated by two-dimensional gel electrophoresis (2-DGE) coupled with matrix-assisted, laser desorption/ionisation tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis. Overall, ten protein spots were expressed differentially between the six cultivars and identified by MALDI-TOF/TOF MS. Among these, an ATP-dependent zinc metalloprotease and the chaperone protein, ClpC1, accumulated specifically in the cultivars ‘Singeonmi’ and ‘Jami’, respectively. Furthermore, metabolomic analysis, using reversed-phase high performance liquid chromatography (HPLC) coupled with liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS), revealed that four polyphenolic compounds accumulated differentially in the six sweet potato cultivars. Members of the caffeoylquinic acid family were detected at the highest levels in ‘Juhwangmi’. These results suggest that the proteins and metabolites that accumulate differentially may be used as biomarkers to identify Korean sweet potato cultivars.

Genetic diversity in wild and hatchery populations of stinging catfish (Heteropneustes fossilis Bloch) revealed by RAPD analysis

Context: DNA fingerprinting using genetic markers such as Random Amplification of Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP), microsatellite (Simple sequence repeat), Amplified Fragment Length Polymorphism (AFLP) etc. can be successfully used to reveal genetic variation within and among different populations. Objective: The aim of the present study was to assess genetic diversity in two wild and one hatchery populations of stinging catfish Heteropneustes fossilis by RAPD fingerprinting. Materials and Methods: A total of 90 live fish (H. fossilis), 30 from each source, were collected from a beel in Patuakhali, a beel in Jessore and Rupali Hatchery, Mymensingh. Genomic DNA was extracted from fin tissues. The concentration of DNA was estimated using a spectrophotometer. Fifteen decamer primers of random sequence from three kits (six from kit A, seven from kit B and two from kit C) (Operon technologies, Inc., Alameda, CA, USA) were screened on sub-samples of one randomly chosen H. fossilis DNA sample from the each population to test their suitability for amplifying RAPDs. The amplified products from each sample were separated by electrophoresis on 1.4% agarose gel containing ethidium bromide. The sizes of the bands were calculated using the software DNAFRAG and the sizes in base pair (bp) were used for identification of the bands (RAPD markers). The similarity index values (SI) between the RAPD fingerprint of any two individuals on the same gel were calculated from RAPD band sharing. Results: A total of 28 RAPD bands were obtained using four decamer random primers, among which 21 bands were polymorphic. The percentage of polymorphic loci, intra-population similarity indices and Nei's gene diversity values were 85.71%, 78.75 and 0.304±0.183 for Jessore population, 83.71%, 82.62 and 0.280±0.159 for Patuakhali population, 82.14%, 85.25 and 0.271±0.165 for Rupali hatchery population, respectively. The overall gene flow (Nm) among the populations was 5.755. The highest inter-similarity (Sij) was found between Patuakhali - Rupali hatchery populations. Among the three populations, the highest genetic distance (0.069) was found between Jessore and Patuakhali population. Considering polymorphic loci, intrapopulation similarity index and gene diversity the genetic variation in the Jessore population was higher than the other two populations. The genetic variation of the hatchery population was found to be lower than the two wild populations. Conclusion: The result of the present study can be used as baseline information regarding the genetic variation and population structure before undertaking any breeding programme. Study indicated that the genetic variation in the hatchery populations were slightly lower than those of the wild populations. DOI: http://dx.doi.org/10.3329/jbs.v19i0.13005 J. bio-sci. 19 81-87, 2011

CHARACTERIZATION OF TYPICAL AND ATYPICAL MALASSEZIA SPP. FROM CATTLE AND DOG BY RANDOM AMPLIFIED POLYMORPHIC DNA ANALYSIS

ABSTRACT There are few numbers of biochemical tests for specie classification in the genus Malassezia and these can to fail in the identification of the atypical isolates. In this study, typical and atypical isolates were analysed by random amplification of polymorphic DNA (RAPD) to compare with biochemical-physiological characteristics of the Malassezia species from bovine and canine ears. RAPD band patterns using OPA4 primer clustered all isolates according its biochemicalphysiological characteristics in the species from cattle and dog. Malassezia nana and M. sympodialis isolates were sub-clustered in separated sub-branches and both were from a different branch of the other species. The DNA pattern of the two atypical lipid-dependent M. pachydermatis strains was similar with of other typical strains but it did not show the one specific band of 200bp. Future studies in the specific RAPD bands of genetic profiles can be important to corroborate the identification of typical and atypical isolates of the genus Malassezia.

Molecular Investigation of Streptococcus agalactiae Isolates from Environmental Samples and Fish Specimens During a Massive Fish Kill in Kuwait Bay

This study was undertaken to identify and characterize bacterial isolates obtained simultaneously from dead fish samples during a massive fish kill in Kuwait Bay and sewage-water samples running into Kuwait Bay using conventional and molecular techniques. Of the 71 bacterial isolates studied; 66 were recovered from 7 different fish species and 5 strains were isolated from sewage samples. The species-specific identity of the isolates was established by phenotypic characteristics and by PCR amplification of 16S rRNA by using Streptococcus agalactiae-specific primers. The genotyping of 12 isolates from fish samples and all 5 isolates from sewage samples was performed by random amplification of polymorphic DNA (RAPD) analysis. Culture methods identified 44 of 66 (67%) and 4 of 5 (80%) isolates obtained from fish and sewage samples, respectively, as S. agalactiae. The PCR amplification of 16S rRNA not only confirmed the results of conventional methods but also resulted in additional identification of 14 of 66 (21%) isolates obtained from fish samples and the remaining isolate recovered from sewage sample, as S. agalactiae. A total of 9 RAPD patterns were observed among the 17 isolates studied; these RAPD patterns were grouped into three clusters. Interestingly, four of the isolates recovered from sewage samples produced nearly identical RAPD band patterns (85-100% similarity) with some of the S. agalactiae strains isolated from Mullet kidney and brain indicting the possibility of sewage being the source of infection.

Molecular Identification of Tropical Tasar Silkworm (Antheraea mylitta) Ecoraces with RAPD and SCAR Markers

The tropical tasar silkworm, Antheraea mylitta, has several ecoraces, 10 of which are commercially exploited for the production of tasar silk. These ecoraces are identified by morphological markers that are greatly influenced by photoperiod, humidity, altitude, and host plants. The DNA markers, random amplification of polymorphic DNA (RAPD), and sequence-characterized amplified region (SCAR) are identified to complement the existing morphological markers. Seven RAPD bands are selected that identify 8 of the 10 ecoraces. These identified RAPD fragments are sequenced and primers are designed for SCAR markers. Of the seven sets of primers, a single primer pair produced polymorphic SCAR bands that diagnose 5 of the 10 ecoraces. All 10 ecoraces are identified by the use of RAPD and SCAR markers together.

Molecular identification of lizard by RAPD & FINS of mitochondrial 16s rRNA gene

In the present study, we identified the structure-less skeleton suspected to be of house lizard present in jaggery, consumption of which caused mass food poisoning using, RAPD (Random Amplification of Polymorphic DNA) with random primers and FINS (Forensically Informative Nucleotide Sequencing) with mitochondrial 16s rRNA gene. The NJ tree dendogram based on distance calculated from RAPD bands clearly identified the structure-less as Calotes versicolor (Garden Lizard). In FINS analysis of the mitochondrial 16s rRNA gene the NJ tree based on Kimura-2-parametere distance matrices clearly reveal that the unknown sample clustered with Agmidae family and closest to Calotes versicolor (Garden Lizard) with 100% bootstrap support, whereas all other species belong to Gekkonida family form a single distinct cluster including Hemidactylus fluviviridis (House Lizard). This is the first successful typing of mitochondrial 16s rRNA with FINS approach to identify the biological origin of a structure-less skeleton. Our analysis also sustained successful identification of unknown samples using RAPD method with optimized conditions in a laboratory setup with low resources.

Repetitive DNA alterations in human skin cancers

Repetitive sequences constitute landmarks for genome regulation, evolution, and chromatin architecture. Patterns of specific and non-specific repetitive sequences change in many types and stages of tumor cells, characterized by band loss, gain, and (de) increased staining of pre-existing bands. In this work, repetitive DNA was studied in search of genome instability of skin cancers: basal and squamous cell carcinomas (BCC and SCC), malignant melanoma (MM), melanocytic nevus (MN), and actinic keratosis (AK) lesions. DNAs were extracted from blood and tumor samples from 21 BCC, 7 SCC, 11 MM and 7 lesions. Banding patterns were obtained by random amplification of polymorphic DNA (RAPD), and specific D9S50 and D9S52 microsatellites (9p21). D9S50 patterns revealed microsatellite instability (MSI) and/or loss of heterozygosity (LOH) in 36% BCC, 25% SCC, and 57% MM tumors. D9S52 microsatellite showed 28.5%; 42.8%; and 71.4% altered tumors, respectively. No microsatellite alterations were found in MN and AK. On the other hand, genomic rearrangements detected by RAPD were present in 100% tumors. In BCC, the mean number of tumor DNA alterations showed predominant gain of bands. On the contrary, MM samples presented loss, or decreased intensity signal of RAPD bands. Genome alterations in skin cancers would result from chromosomal rearrangements, aneuploidy and/or polysomies. The low-cost and quick RAPD technique may reveal unknown genes or DNA sequences associated with tumor development and progression, and may be easily implemented in clinical diagnosis.

Isolation of DNA for RAPD analysis from dry leaf material of someHesperis L. specimens

An improved protocol for the isolation of DNA from dry material of someHesperis specimens is described. The isolated DNA is suitable for random amplification of polymorphic DNA (RAPD) analysis. Different DNA extraction protocols were examined to determine which might yield DNA from dry leaf tissue ofHesperis specimens. The methods examined include the protocols with hexadecyltrimethylammonium bromide (CTAB) described by Doyle and Doyle (1987); sodium dodecyl sulfate (SDS) by Dellaporta et al. (1983); and CTAB and SDS, the modified minipreparation, by Dellaporta et al (1983). None of these procedures yielded DNA of suitable purity for RAPD assay. We established an improved procedure involving CTAB and enzymatic digestion of proteins and RNA. The recovery of DNA with an average yield of 25 mg/g of leaf material was possible with this procedure. RAPD bands, which could be used to distinguish amongHesperis specimens, were generated.

Microsporum gypseum isolated from a feline case of dermatophytosis

The 1- to 2-month-old female cross-breed cat presented with alopecia, erythema and many crusts were present on the tail. Microscopic examination of crusts from the tail disclosed epithelial debris, exudate, mycelium, and arthrospores. Microsporum gypseum which is rarely isolated from cats as a causative agent of dermatophytsis was cultured from the crusts on a 1/10 Sabouraud glucose agar at 27 degrees C for 1 week. The isolate of M. gypseum from the cat was examined by random amplification of polymorphic DNA (RAPD), chitin synthase 1 gene (CHS1) sequence and mating experiments. The RAPD band patterns of the clinical isolate of M. gypseum was identical to those of tester strains of Arthroderma gypseum. Nucleotide sequence analysis of the CHS1 gene fragments from the isolate and a tester strain of A. gypseum showed 100% similarity. The mating experiments on the clinical isolate of M. gypseum completely agreed with the results from RAPD and CHS1 gene sequence. The isolate from the cat was confirmed to be A. gypseum (-) mating type, which was consistent with the result of mycological examination by molecular analyses.

Microsporum gypseum isolated from a feline case of dermatophytosis

Summary. The 1‐ to 2‐month‐old female cross‐breed cat presented with alopecia, erythema and many crusts were present on the tail. Microscopic examination of crusts from the tail disclosed epithelial debris, exudate, mycelium, and arthrospores. Microsporum gypseum which is rarely isolated from cats as a causative agent of dermatophytsis was cultured from the crusts on a 1/10 Sabouraud glucose agar at 27°C for 1 week. The isolate of M. gypseum from the cat was examined by random amplification of polymorphic DNA (RAPD), chitin synthase 1 gene (CHS1) sequence and mating experiments. The RAPD band patterns of the clinical isolate of M. gypseum was identical to those of tester strains of Arthroderma gypseum. Nucleotide sequence analysis of the CHS1 gene fragments from the isolate and a tester strain of A. gypseum showed 100% similarity. The mating experiments on the clinical isolate of M. gypseum completely agreed with the results from RAPD and CHS1 gene sequence. The isolate from the cat was confirmed to be A. gypseum (–) mating type, which was consistent with the result of mycological examination by molecular analyses.

Molecular heterogeneity in clinical isolates of Malassezia pachydermatis from dogs

Molecular investigation of 16 strains, conventionally identified to be Malassezia pachydermatis, isolated from dogs in Japan was carried out by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 ( CHS2) gene sequence analyses. The RAPD band patterns of 13 clinical isolates were identical to that of standard strain of M. pachydermatis (CBS-1879). The other three clinical isolates were different from the standard strain of M. pachydermatis in RAPD patterns, and two of the three isolates were identical. About 620 bp genomic DNA fragments of the CHS2 gene were amplified from the same 16 clinical isolates of M. pachydermatis by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of the 16 clinical isolates revealed that the 13 strains were genetically very close to the standard strain of M. pachydermatis and the other two isolates were genetically close to the standard strain of M. furfur rather than M. pachydermatis. The remaining one isolate was phylogenetically distinct from all the seven Malassezia species reported so far.

Identification of clinical isolates of Microsporum canis and M. gypseum by random amplification of polymorphic DNA (RAPD) and Southern hybridization analyses.

Clinical isolates of Microsporum canis and M. gypseum from humans, dogs and cats were examined by random amplification of polymorphic DNA (RAPD) and Southern hybridization analyses. The RAPD band patterns of six clinical isolates of M. canis were identical to those of standard strains of Arthroderma otae. Of nine clinical isolates of M. gypseum seven and two isolates showed RAPD patterns identical to those of standard strains of A. gypseum and A. incurvatum respectively. Southern blot analysis using a probe (C3) obtained from A. otae DNA revealed that six clinical isolates of M. canis showed specific bands identical to those detected in the standard strains of A. otae. Of nine clinical isolates of M. gypseum, seven and two isolates showed bands hybridized by the C3 probe identical to those detected in A. gypseum and A. incurvatum respectively. Furthermore, the results from mating experiments on these nine clinical isolates of M. gypseum showed complete agreement with the results from RAPD and Southern hybridization analyses. These findings clearly indicate that RAPD and Southern hybridization analyses are very useful in the identification of clinical isolates of M. canis and M. gypseum.

The use of species‐specific DNA probes for the identification of Mycosphaerella fijiensis and M. musicola, the causal agents of Sigatoka disease of banana

The polymerase chain reaction (PCR)‐based technique of random amplification of polymorphic DNA (RAPD) was used to differentiate DNA from species of the genus Mycosphaerella. DNA from two pathogens which cause Sigatoka leafspot diseases of banana, M. fijiensis and M. musicola, and two other Mycosphaerella species which are commonly found on banana, M. musae and M. minima, gave distinct RAPD banding patterns with all PCR primers tested. PCR, using primer RC07, amplified a 1250bp RAPD fragment from all isolates of M. fijiensis obtained from 11 geographical origins. This fragment was absent from the other species of Mycosphaerella. In Southern blots of genomic DNA, this band hybridized exclusively to DNA from M. fijiensis, and the pattern of hybridization suggested that it was binding to repeated DNA. A RAPD band amplified with primer PM06 obtained from M. musicola was also found to be species‐specific. Southern analysis suggested that the fragment hybridized to a single‐copy sequence in the M. musicola genome. Total genomic DNA from M. musicola was found to be a species‐specific hybridization probe. Dot‐blots confirmed the specificity of these probes, and could be used to identify isolates of Mycosphaerella which cause Sigatoka disease of banana in south‐east Asia.