RAPD Analysis Research Articles

An empirical study on the underutilized medicinal genus Kaempferia from India revealed cytological and genetic variability

The genus Kaempferia L., consisting of aromatic and ornamental species found in east and south-east Asia, have profound ethno-medicinal usages as antimicrobial and anticancer agents, attributable to presence of novel cyclohexane derivatives, flavonoids and di-terpenes. Represented by only 7 species in India, a comprehensive genetic study of Kaempferia from the country is lacking. We present here a study of 4 species of this genus from India, namely, K. rotunda, K. galanga, K. elegans and K. angustifolia. Cytological analyses revealed distinct inter-specific ploidy and consequent somatic chromosome number variations (2n = 22 to 2n = 54). High inter- and intra-chromosomal variability was observed with chromosome lengths ranging from 0.87 to 6.63 µm, constriction types varying from median to sub-median range and different secondary constrictions, along with contrasting values of asymmetry indices. RAPD and ISSR analyses of 8 accessions produced 1,398 polymorphic bands, their sizes ranging from 100 to 2000 bp; percentage polymorphism varying from 84 to 100%. UPGMA dendrogram generated using the polymorphic fragments showed Jaccard's similarity coefficient value varying between 0.07 and 0.93, producing 3 separate clusters for the studied Kaempferia species. ITS, matK and rbcL loci sequence analyses also confirmed high level of inter-specific variability of Kaempferia. Genetic variation revealed in this study could indicate chemotypic differences in Kaempferia and will direct our future research for a wider cytogenetic and phytochemical investigation including all the Indian species that would open up avenues for judicious exploitation of these medicinally important plants.

Molecular Genetic Diversity of Prunus armeniaca L. (Rosaceae) Genotypes by RAPD, ISSR-PCR, and Chloroplast DNA (cpDNA) trnL-F Sequences

ABSTRACT In this study, phylogenetic analysis of some Turkish Prunus armeniaca L. genotypes was conducted based on RAPD-PCR, ISSR-PCR, and chloroplast DNA (trnL-F) sequence analyses. Prunus armeniaca genotypes were obtained from Malatya province, and brought to the laboratory for genomic DNA isolation. Eleven RAPD and fifteen ISSR primers were used to determine molecular characterization of Prunus armeniaca genotypes. For the amplification of the trnL-F region, trne and trnf primers were used in Polymerase Chain Reaction (PCR). A total of 46 and 95 bands were obtained with RAPD and ISSR analysis, respectively. The trnL-F sequences varied from 398 to 403 nucleotides. Average nucleotide composition of trnL-F sequences was 31.0% T, 14.4% C, 37.4% A and 17.2% G. The genetic distance between the five apricot genotypes is 0.00. A maximum likelihood tree was generated to determine genetic relationships among Prunus armeniaca genotypes. Analysis of Prunus armeniaca genotypes based on RAPD, ISSR, and trnL-F sequences revealed that the rate of polymorphism obtained using RAPD and ISSR were higher than those obtained through the trnL-F sequences.

Genetic Characterization Based on Rapd-Pcr in Cultured Strains of Clarias gariepinus (Siluriformes: Clariidae)

The genetic polymorphisms of catfish populations collected from four fish farms in four Southwestern states of Nigeria were determined by Randomly Amplified Polymorphic DNA Polymerase Chain Reaction technique. This was to characterize the strains genetically and provide information on the studied catfish. Following standard procedures, polymerase chain reaction was performed by mixing 15 μl reaction mixture containing FIREPOL® Taq DNA polymerase, MgCl2, dNTPs, distilled water, dyes and glycerol, with 1.0 μl DNA sample of 16-week old C. gariepinus strains using OPA03, OPA04, OPC02, OPB08, OPC11, OPG16, OPG19 and OPA19 primers. A sum of 1708 loci having 3072 bands was amplified in all the samples. The RAPD analysis revealed significant genetic variability (P<0.05) among the four sampled populations. The unweighted pair group method with average (UPGMA) dendrogram based on Nei’s unbiased genetic distance matrix separated the Clarias gariepinus populations into two clades. The first clade was made up of populations from Ibadan and Abeokuta while the second clade consisted of populations from Ado-Ekiti and Ile-Ife. Thus, it is imperative to determine the genetic variation and population structure of the stocks of C. gariepinus in advance before commencing on breeding programmes.

Agromorphologic Characterization and Molecular Markers of some Teosinte (Zea mexicana) Genotypes الصفات المورفولوجیة والمحصولیة والمعلمات الجزیئیة فى بعض التراکیب الوراثیة من الذرة الریانة

Two field experiments was conducted during the summer seasons of 2017 and 2018 to evaluate and identify five teosinte genotypes. A total of morphological characterization depends on stem anthocyanin coloration of sheath of the first leaf (ACS), shape of tip of the first leaf (ShT), wavy surface of blade (WSB), blade attitude of leaf just above upper ear (BAL), anthocyanin coloration of sheath in the middle of plant (ACSh), brace roots(ACBR), fresh anthers and flag leaf angle also, sparse types of spikelet. Values of quantitative traits i.e. fresh and dry yield (kg/plot) and its total, leaf ear width and length, plant height, No. of leaves/plante, No. of tillers and 100 grains weight were varied from trait to other mainly due to genetic background. Molecular marker RAPD analysis polymorphisms among genotypes were detected by five random primers. The high level of polymorphism was occurred with primer OP-C4 which showed 57.1% polymorphism, while the low level of polymorphism was 16.7 % in primer OP-A10. Cluster analysis showed differences between the genotypes which separated into two main clusters, the first cluster was further divided into two sub-clusters (IA and IB), sub-cluster IA Included Balady, Sakha and Dammietta, sub-cluster IB included Gemmeiza3 while, Gemmeiza4 was in a separate cluster. The results of this research of great importance to select the right material which can be used in plant breeding programs as they help for introducing a new variety. Farmer field trails are suggested before the submission of these new varieties to registration testing and its release.

Establishment of meristem culture for virus-free and genetically stable production of the endangered plant Hosta capitata

Hosta species endemic to Korea are currently suffering from infection with Hosta virus X (HVX), and it has been difficult to produce virus-free plants when using infected plants as a source of material for in vitro and vegetative propagation in continuous production processes. Genetic variation in in vitro-regenerated plants has also been detected in some Hosta cultivars. Therefore, we aimed to develop an in vitro propagation method for the production of virus-free and genetically stable plants. First, we collected Hosta capitata showing symptoms of viral infection and confirmed the presence of an HVX virus gene using reverse transcription (RT)–PCR, followed by optimization of the sterilization of in vivo bulbs. Meristems derived from sterilized bulbs were cultured in media containing different concentrations of 1-naphthaleneacetic acid (NAA) with either benzyl aminopurine (BA) or thidiazuron (TDZ). The combination of 0.1 mg/l NAA and 3.0 mg/l BA generated the highest number of shoots per explant, and these shoots had a higher fresh weight. The addition of nano-silver (NAg) particles to the regeneration medium distinctly stimulated plant growth. According to the results of RAPD analysis, the meristem-derived plants were genetically stable compared with donor plants grown in a greenhouse, and absence of the HVX gene was also observed, indicating freedom from HVX infection. Therefore, they would be highly valuable for use in the landscape industry. We expect that the method proposed in this study will also prove helpful for generating virus-free, genetically stable plants in the commercial production of other Hosta species.

Assessment of genetic variation and chemical composition of Origanumspp

The study was carried out on 23 entries of Origanumcollected from different areas of south Italy. The 23 entries were characterizedvia determining the chemical composition of their essential oils and genetic variability. The gas-chromatography of the essential oils of oregano accessions allowed the detection of 44 components with the predominance of carvacrol, thymol, linalyl acetate, γ-terpinene, o-cimene, s-caryophylleneand cis-ocimene. A high variability in the main components concentration was revealed except in the case of the accessions 13, 14 and 15 where the linalyl acetate ranging between 51.27 and 60.93%, outlining a new oregano chemotype.Using hierarchical cluster analysis, four main groups of samples were observed. Genetic variability using the RAPD analysis was not able to reveal clear polymorphism PCR patterns useful to distinguish the entries.So that, we decided to conduct further molecular analyses to determine the genetic variation among the entries under investigation, using AFLPs a powerful tool to perform phylogenetic analysis. This technique shows a high capability in detecting genetic variation. Combination between fluorescent system and polyacrylamide gels allows obtaining large number of bands (225 to 557). Finally, thecurrent study shows thatthe Dendrogram of genetic similarity of Origanum is widely variable amongst genotypes of this plant.

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Bioremediation of malachite green dye by two bacterial strains isolated from textile effluents.

Globally, water pollution from the textile industries is an alarming issue. Malachite Green dye of the triphenylmethane group is an extensively used dye in the fabric industries that is emitted through textile wastewater. This study aimed to isolate and characterize potential Malachite Green (MG) dye degrading bacteria from textile effluents. Different growth and culture parameters such as temperature, pH and dye concentration were optimized to perform the dye-degradation assay using different concentrations of MG dye in the mineral salt medium. A photo-electric-colorimeter was used to measure the decolorizing activity of bacteria at different time intervals after aerobic incubation. Two potential bacterial strains of Enterobacter spp. CV-S1 (accession no: MH450229) and Enterobacter spp. CM-S1 (accession no: MH447289) were isolated from textile effluents exhibiting potential MG dye decoloring efficiency. Further, the RAPD analysis and 16S rRNA sequencing confirmed the genetic differences of the isolated strains. Enterobacter sp CV-S1 and Enterobacter sp CM-S1 can completely decolor MG dye up to 15 mg/L under shaking condition without any requirement of sole carbon source. Thus, these two bacteria have the potency to be utilized in the textile wastewater treatment plant.

Phylogenetic Relationships between Ten genotype of Hordeum vulgare L. Using Molecular Markers

Phylogenetic relationships between ten Hordeum vulgare L. , seven cultivars (Giza 123, Giza 126, Giza 127, Giza 128, Giza 129, Giza 130 and Giza 2000) and three landraces from Sinai (El-Kheroba, El-Sheikh Zuwaid, and Wadi Sedr) were carried out using two molecular genetic markers (RAPD-PCR and ISSR-PCR). The genetic distance between Ten genotype was also estimated from banding patterns Twenty two random primers were used in RAPD revealed 316 bands while 159 bands were detected of ISSR analysis using 10 primers. RAPD analysis among ten genotypes showed 41.77% polymorphism, while ISSR analysis showed 62.02% polymorphism. It was found that, ISSR was a more viable marker than RAPD in the detection of the genetic variability among Hordeum vulgare (barley) varieties. The genetic distance tree was detected using UPGMA based on both molecular markers (RAPD and ISSR) and analysis of combined data.In addition, the Band Shearing index (BSI) factor was calculated shows a marked difference between the ten genotypes, seven cultivars and three landraces of studied Hordeum vulgare where BSI average reach 1.42% with RAPD markers while reaching 0.93 % in ISSR. The obtained data indicated that both RAPD and ISSR markers are efficient in identification and differentiation between selected taxa, but the efficiency of ISSR was the best one. Also, the present results, enhancing the available knowledge of Hordeum vulgare genetic resources in Egypt, which may contribute to their conservation and utilization in breeding programs.

Impact of Environmental Diversity in Egypt on Catharanthus roseus Cultivars Genome and Assessment that by Different DNA Markers

The genetic diversity relationship was studied among ten cultivars of Catharanthus roseus depending on geographic locations in Egypt (five governorates: Marsa Matruh, Kafr ash-Shaykh, Port Said, Cairo, and Bur-Safajah) and two colors of flowers (white and pink) by using RAPD and ISSR assay. In the RAPD assay, four primers amplified gave low polymorphism (30%) and (18%) in ISSR markers. The highest value of similarity was 1, while the lowest value of similarity indexed was 0.539. In ISSR analysis, 17.6% polymorphism was observed, the highest value of similarity was 1 and the lowest value of similarity index recorded was 0.106. The combined RAPD and ISSR analysis data revealed the highest similarity was observed (0.984) but, the least similarity was (0.448). The primer OP-L13 only was given two specific markers in RAPD assay. The present study revealed RAPD would be a useful assay compared to the ISSR phylogenetic assay for studies to identify the genetic diversity of the genome. The Principle component analysis (PCA) of RAPD and ISSR assays by using 4 primers (both of them) was studied among ten genotypes cultivars of Catharanthus roseus plant. The results showed that the RAPD assay was given three clusters with 72.66 and 25.35% of the variance, the ISSR assay given two clusters with 75.55 and 24.26% of the variance. On the other hand the combined RAPD and ISSR assays were given the three clusters with 67.01 and 20.74% of the variance.

Droplet-vitrification of Aranda Broga Blue orchid: Role of ascorbic acid on the antioxidant system and genetic fidelity assessments via RAPD and SCoT markers

A droplet-vitrification cryopreservation protocol has been successfully developed for Aranda Broga Blue orchid hybrid using protocorm-like bodies (PLBs). However, maximum growth regeneration percentage was recorded at 5% only based on previous report. Thus, to improve growth recovery of cryopreserved PLBs, cryopreservation stages were supplemented with ascorbic acid, tested at 50, 100 and 150 mg/L. However, results demonstrated that exogenous ascorbic acid was not favorable in regeneration of cryopreserved explants (maximum value of 1.67 % with 50 mg/L ascorbic acid supplementation). Total soluble protein and various antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were evaluated after each cryopreservation stages in conjunction with the application of exogenous ascorbic acid. Addition of antioxidant must be carefully evaluated and its application may not guarantee successful growth recovery. RAPD and SCoT molecular analysis confirmed the genetic stability of regenerated cryopreserved PLBs as no polymorphism was detected compared to control PLBs culture.

Genetic and chemotypic diversity of two lineages of Aspergillus flavus isolated from maize seeds of different agroclimatic niches of India

Maize is susceptible to colonization by Aspergillus flavus, which is the major aflatoxins producing fungus. Aflatoxins are fungal secondary metabolites that have been reported to adversely affect normal cell metabolism and seed germination. In this study, 106 maize seed samples were sampled across 13 states throughout India, and were screened for incidences of fungal infection. Among the seed samples collected across India nearly all maize seed samples were found to be infected with Aspergillus. All morphologically confirmed isolates belonging to Aspergillus section Flavi were subjected to various aflatoxin, biochemical, physiological and genotype assays, as well as phylogenetic analyses. Among 60 isolates tested, eight were non-aflatoxigenic in nature as determined by icELISA, and seven of those eight isolates showed absence of one or more aflatoxin cluster genes in PCR assay. The morphological, physiological and biochemical characterizations failed to show variation based on their toxigenicity. The phenetic and molecular genetic diversity could be observed based on RAPD and ISSR analyses and little isolate diversity could be determined based on geography or aflatoxigenicity. An inferred ITS sequence phylogeny segregated isolates into only two lineage groups: a large group I (including Ia and Ib) and small group II contained A. flavus isolates closely related to A. parasiticus. The current study highlights inherent diverse A. flavus infections, exhibiting a range of aflatoxin concentrations, that are present in maize seeds and they are important because of the potential threat to agricultural production and consumption.

KERAGAMAN GENETIK MUTAN M-2 CABAI MERAH KERITING (Capsicum annuum L.) BERDASARKAN PENANDA RAPD

Chili is an important horticultural plant in Indonesia. This research aims to carry out RAPD analysis on Mutant M2 of chili pepper (Capsicum annuum L.). Six M2 genotypes of chili irradiated by gamma ray and control plants were amplified by 16 random primers. The amplification results of M2 chili with 16 primers produced 118 loci, with fragment sizes ranging from 150-2000 bp. The number of polymorphic loci was 96 loci and the percentage of polymorphic loci was 83.23%. The DNA fragment polymorphism produced in this research was relatively high and it showed that the gamma ray mutagen applied produced high chili genetic diversity. The value of genetic similarity between control plants and mutant plants ranged from 0.7474 to 0.4874. UPGMA dendogram classified seven genotypes tested into three groups, the first group consisted of mutants R2U6 and R2U17, the second group was mutants R1U14 and R1U17, and the third group was mutants R2U8, mutants R2U2 and control plants. The finding of this research can be used as a basic selection of genetic material for chili’s breeding in the future.

Assessment of genetic diversity in fenugreek (Trigonella foenum-graecum) genotypes using morphological and molecular markers

Fenugreek (Trigonella foenum-graecum) is one of the most important seed spices possessing aromatic and medicinal properties. Commonly conventional methods based over phenotypic variability are being used by breeders for identification of superior germplasm which are laborious and time consuming. A study was carried out at Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University Agriculture and Technology, Udaipur, during 2014–16 to assess the genetic diversity among 20 diverse fenugreek genotypes using morphological and molecular markers for the identification of superior entries. Mean squares due to genotypes were highly significant and wide mean range performance was observed for plant height, pods per plant, test weight, biological yield per plot and harvest index. RAPD analysis was carried out with 25 primers and 21 primers produced 76 bands, in which 37 were polymorphic bands. Average polymorphism was 48.60% and Jaccard’s Similarity Coefficient lies between 0.71 to 0.99. From the results based over morphological and molecular studies it could be concluded that 20 fenugreek entries were classified in 2 clusters through morphological characterization while the same entries were more precisely classified in 5 clusters through RAPD analysis. Superior fenugreek entries were identified for seed yield and its components. Based on the results promising and diverse fenugreek entries will be useful for further breeding programmes.

Biochemical and Molecular Characterization of Bamboo Variants

Classical taxonomic studies of the bamboos are based on floral morphology and growth habit, which can cause problems in identification due to erratic flowering. Identification and genetic relationships in 18 species of bamboo were investigated using SDS-PAGE and RAPD analysis. Protein profiles of 18 bamboo species revealed clear polymorphism which can be used to identify species. Dendrogram based on SDS-PAGE data separated 18 species into two major clusters with Bambusa nutans & B. polymorpha, Gigantochloa andamanica & Dendrocalamus longispathus, G. andamanica & D. gigangteus showed maximum similarity of 71.4% in each case while genotype B. tulda and B. nutans (10.4%) showed lowest similarity. On the other hand, RAPD analysis proved to be a better tool with 14.67 as an average number of bands per polymorphic primers. Dendrogram based on RAPD separated 18 species into 2 major clusters with D. membranaceous and B.multiplex showed maximum similarity of 83% while genotype D. asper and B. balcooa showed least similarity (10.6%).

Morphological characteristics as a key attribute for a successful determination of selected Cotoneaster species

Article Details: Received: 2019-12-17 | Accepted: 2019-01-24 | Available online: 2020-03-31 https://doi.org/10.15414/afz.2020.23.01.15-23 In this paper, morphological features, such as the number of pyrenes in pome and the number of pomes in infructescence, were used for determination of closely related tetraploid Cotoneaster species. Samples were collected from various localities in the Western Carpathians. The collection of samples, designed for counting of pyrenes in pome, included 2353 pomes of >130 individuals. Number of pyrenes in pome ranged from 1 to 5. Statistical analysis revealed a significant difference in pyrenes per pome mean values between C. integerrimus (3.01), C. melanocarpus agg. (2.46; including C. matrensis) and C. tomentosus (3.93). The collection of samples, designed for counting of pomes in infructescence, included 1019 infructescences of 141 individuals. Number of pomes in infructescence ranged from 1 to 5. Statistical analysis also revealed a significant difference in pomes per infructescence mean values between C. integerrimus (1.14) and C. melanocarpus agg. (1.54; including C. matrensis), and between C. integerrimus and C. tomentosus (1.50). Keywords: pyrenes, pomes, infructescence, Cotoneaster, Western Carpathians

Diversity analysis in soybean (Glycine max [L.] Merrill) mutant lines grown in saline soil using agronomic traits and RAPD markers

Soybean is one of the strategic food crops in Indonesia, but its production is far below the demand due to inadequate area for soybean cultivation. Shifting soybean cultivation to marginal land such as saline soil has been suggested as a realistic solution to increase soybean production. Development of soybean variety tolerant to salinity is the key step to support the cultivation of soybean in saline soil. The objective of this research was to evaluate the genetic diversity in mutant soybean lines generated using gamma rays based on agronomic traits and RAPD markers. A total of 200 irradiated seeds of cultivar Detam-3 were planted in saline soil with electrical conductivity of 1.2–4.3 dS/m. Agronomic traits were evaluated on plants until harvesting time. Genetic analysis using two RAPD markers (OPAA-02 and OPAA-14) was done on 11 plants of each radiation treatment. The results showed that 54 plants survived in saline soil. High level of variation based on agronomic traits was observed in these plants. RAPD analysis revealed 60% and 83.3% polymorphism among 11 plants for OPAA-02 and OPAA-14 markers, respectively.

Biochemical studies on genotypic characterization of Pseudomonas fluorescens isolates by PCR-RAPD analysis

Plant growth promoting rhizobacterial strains belonging to fluorescent Pseudomonads were isolated from the rhizosphere of citrus in Vidarbha region of Maharashtra, India. On the basis of morphological and biochemical studies, they were categorized as Pseudomonas fluorescens, P. putida and P. aurigonosa. Genotyping of these P. fluorescens strains was made by PCR–RAPD analysis since differentiation by biochemical methods was limited. Among the 16 primers screened, six primers (OPA-16, OPB-18, OPC-15, OPC-19, OPG-05 and OPG-10) were polymorphic showed better amplification of DNA while other were recorded monomorphic. Similarity co-efficient between each pair of accessions was used to construct a dendrogram using the unweighted paired group method with arithemetic average (UPGMA).

Effect of different plant extracts and nanoparticles on Thrips tabaci (Lind.) (Thysanoptera: Thripidae) under field conditions and their allelopathic potential on the onion, Allium cepa L. using bioassays and RAPD analysis

The present study was conducted to investigate the toxicity of Aerosil 200® (fumed silica nanoparticles) and leaf extracts of four plants, Cinnamomum camphora, Matricaria chamomilla, Mentha arvensis, and Trigonella foenum-graecum against Thrips tabaci (Lind.) (Thysanoptera: Thripidae) in onion fields, as well their allelopathic effects on onions; moreover, the chlorophyll, phenol, and protein contents were determined in onions. This study was performed in completely randomized plots. After a growth period of 1 month, bioassay investigations and molecular polymorphism in T. tabaci by RAPD-PCR were performed, and total chlorophyll, phenol, and protein concentrations were investigated in onion plants posttreatment as well. The initial reduction% of the T. tabaci population in onion fields after application of a high concentration of nanoparticles (Aerosil 200® (4 ml/l)) and 8000 ppm concentrations of the four plant extracts were 83.66, 81.08, 86.92, 74.49, and 91.38%, respectively, whereas their persistence effects were 73.18, 67.78, 71.46, 66.94, and 78.29%, respectively. Furthermore, the total chlorophyll contents in onions treated with the nanoparticles and four plant extracts were 1.35, 1.17, 1.09, 1.07, and 1.18 mg/g, respectively; additionally, the concentrations of phenols were 4.65, 3.15, 3.15, 2.85, and 3.70 mg/g in onions treated with C. camphora, M. chamomilla, M. arvensis, T. foenum-graecum, and Aerosil 200®, respectively. The C. camphora extract was the most potent, as it increased the protein content in the onion plants, while the Aerosil 200® decreased the protein content in onions. In addition, DNA-RAPD showed that the polymorphism percentages were 73, 71, and 67% when treated with high concentrations of C. camphora and M. arvensis extracts and Aerosol 200®, respectively. T. foenum-graecum and M. chamomilla extracts induced the least polymorphism (17 and 16%, respectively). Overall, this study indicated that these plant extracts as well as the nanoparticles in Aerosil 200® could be used to reduce onion infestations of T. tabaci in the field environment.

Nitrogen ion beam implantation for enhanced lipid accumulation of Scenedesmus obliquus in municipal wastewater

Breeding of high-lipid content microalgae in municipal wastewater is critical for large-scale microalgal biodiesel production. In this study, a strain of biodiesel promising microalga Scenedesmus obliquus with good stain resistance was obtained by N+ ion implantation, which was named as S1-A3. In the selenite enrichment (SE) medium, the lipid content of S1-A3 was up to 46.92%, which was increased by 24.1% than the original strain. Meanwhile, after 7 days of cultivation, the removal rates of TP,TN, NH4+-N and COD in wastewater attained 95.72%, 80.30%, 87.25% and 85.43% by S1-A3, respectively. S1-A3 had a good genetic stability in both municipal wastewater and SE medium, and its lipid production capacity was improved after four successive cultivation in wastewater. RAPD analysis showed that the mutant and the original strain were interspecific variations. Therefore, N+ ion implantation was proved to be an approach that can significantly improve the lipid content and adaptability of Scenedesmus obliquus.