RAPD Research Articles

Revealing genetic diversity of wild Phalaenopsis orchids in Thailand through Random Amplified Polymorphic DNA markers

Abstract. Kate-ngam S, Jantasuriyarat C, Lakote P, Promchot T, Chueakaew C. 2023. Revealing genetic diversity of wild Phalaenopsis orchids in Thailand through Random Amplified Polymorphic DNA markers. Biodiversitas 24: 5409-5417. Wild Phalaenopsis orchids are widely distributed in Thailand, especially along the banks of the Mekong River. The tremendous decrease in the population of these orchids is a matter of concern for maintaining and preventing genetic loss in the natural ecosystem. This study aimed to determine the genetic diversity of 50 wild Phalaenopsis accessions collected from the northeastern of Thailand using random amplified polymorphic DNA (RAPD) markers. The 204 polymorphic bands generated from 27 RAPD primers were employed for fingerprinting these orchid accessions. Dice’s similarity coefficients ranged from 0.43 to 0.98, with an average of 0.65. In general, a dendrogram constructed based on the unweighted pair group method with arithmetic average (UPGMA) grouped these wild Phalaenopsis accessions into two clusters, namely P. pulcherrima and P. ubonensis clusters. The UPGMA clustering pattern and principal component analysis (PCA) corresponded well with their morphological classification and ploidy level. Our results suggest that this wild Phalaenopsis orchid exhibits a moderate level of relatedness. This might be a consequence of habitat destruction and human over-exploitation which limit gene flow and cause genetic drift among populations. The present finding supports the urgent need for a systematic conservation effort of this orchid species in Thailand.

Genetic variability of the pear lace bug Stephanitis pyri F. (Heteroptera: Tingidae) in Krasnodar Territory, Russia

Aim. Study of the genetic variability of the pear lace bug Stephanitis pyri F. depending on geographical location and trophic specialization.Material and Methods. The object of the study was insect samples (n=60) from the natural population of the pear lace bug Stephanitis pyri F. (Tingidae: Heteroptera) in the Krasnodar Territory: in Krasnodar, Kropotkin and Novorossiysk. The collection of S. pyri bugs was carried out on model trees of domestic apple (Malus domestica Borkh) and sour cherry (Prunus cerasus L.). Molecular genetic analysis was carried out in several stages: sample preparation, DNA extraction, and diagnostics based on RAPD (random amplified polymorphic DNA)‐PCR (DNA amplification).Results. With the help of molecular genetic markers, the genetic variability of the pear lace bug in the Krasnodar Territory was studied. The indicators of genetic differentiation between geographic (Gst=0.209) and trophic (Gst=0.049–0.302) intrapopulation groups of S. pyri were calculated. The levels of variability were determined within geographic (79.1 %) and trophic groups (70.0–95.1 %), as well as between them – 20.9 % and 4.9–30 %, respectively. The influence of the food factor on the genetic diversity and differentiation of insect populations is shown through the example of the Kropotkin trophic group of bugs that prefer the sour cherry P. cerasus as the main food plant.Conclusion. A small effect of isolation by distance was noted, which was characterized by a weak dependence of geographical and genetic distances between the population groups of the pear lace bug. The influence of the nutritional factor on the genetic variability of phytophages populations has been established, as is confirmed by other researchers.

Characterization of Trichoderma strains for novel species-specific markers by multiplex PCR and antagonistic property against Alternaria ricini in castor (Ricinus communis L.)

The study aimed at characterizing six Trichoderma strains isolated from the rhizospheric soil of castor growing region of Telangana, India. Microscopic examination and sequencing of ribosomal DNA (rDNA) using internal transcribed spacer (ITS) and D1-D2 region-specific primers revealed them as members of Trichoderma sp. Whereas the phylogenetic analysis revealed four of those strains as Trichoderma asperellum, two strains were confirmed as Trichoderma harzianum with a high sequence similarity of >99%. Further, multiplex PCR results with Trichoderma-specific primers; TEF1 (translation elongation factor 1) and rpb2 (the second largest subunit of RNA polymerase II) gene-specific primers confirmed the findings resulting from the phylogenetic analysis. To develop a set of sequence characterized amplified region (SCAR) markers, these strains were screened with 185 random amplified polymorphic DNA (RAPD) primers. A pairwise comparison of correlation of coefficient values determined from the RAPD-based genetic similarity analysis revealed that TV3 and TS4 strains were closely related to each other (0.98) confirming them as T. harzianum while N13, 7316, TV5, and TV2B16 strains formed a different cluster with T. asperellum strains. A set of novel SCAR markers were developed to distinguish these two species, T. asperellum and T. harzianum including the reported Trichoderma strains as Ex-type strains. T. harzianum strains had extremely low sporulation ability to be tested for biocontrol properties. Of the four T. asperellum strains, 7316 followed by N13 demonstrated high antagonistic ability against the fungal pathogen, Alternaria ricini indicating these strains as potential biocontrol agents to limit Alternaria infection in castor.

Evaluation of Biological Control of Sorghum Strains Using Bacillus Thuringiensis and Pseudomonas Aeruginosa Under Drought Stress

Background: Sorghum is an economically significant staple food crop for more than half a billion people in developing nations, especially in arid and semi-arid locations where drought stress is a significant limiting factor. Despite usually being regarded as tolerant, sorghum suffers severely from drought stress, which lowers its productivity and nutritional quality throughout its principal cultivation areas. Objective: Improvements in DNA fingerprinting by ISSRs, SSRs, and RAPD markers have also been employed in sorghum genetic modification (GMOs) to enhance the economic characteristics of this crop. Materials and methods: To provide a natural defence against pests, the most tolerant plants among the seven varieties of sorghum bicolour were selected and planted in the second season of 2020–2021 under treatment with two microorganisms, B. thuringiensis and P. aeruginosa. This study considered seven varieties of sorghum bicolour planted under 50% water deficiency in 2019–2020. Genetic variability analysis of sorghum genotypes was performed using seven Inter-Simple Sequence Repeat (ISSR) primers, six Simple Sequence Repeat (SSR) primers, and five Random Amplified Polymorphic DNA (RAPD) primers. Seven Sorghum bicolour accessions were collected from various regions of Egypt and their phylogenetic relationships were evaluated. Additionally, DNA fingerprinting and analyses of the genetic diversity and evolutionary linkages in the sorghum germplasm employed the (ISSR) molecular marker technique. Results and conclusion: The Fisher Least Significant difference test (LSD) at P < 0.05, based on RAPD, ISSR, and SSR markers demonstrated a significant connection. The findings demonstrated that 51 bands with a size range of 100–1500 bp and polymorphism percentage of 72.5% were created using five RAPD primers. Seven ISSR primers generated 45 bands With a 57.8(%) polymorphism percentage, ranging in size from 100 to 3000 bp. six SSR primers generated 28 bands with (67.86%) polymorphism percentage of 67.86 %, ranging in size from 100 to 1500 bp. Morphological characteristics and ISSR, SSR, and RAPD analyses were used to group the UPGMA Dendrogram into groups. Jaccard's coefficient was used to analyse the genetic similarity matrix. The maximum similarity was observed for ISSR between Hybrid Sh1 and Hybrid Sh306 (0.984%), SSR between Hybrid Sh306 and Sudan grass (0.964%), and RAPD between Giza 15 and Indian Millet (0.706%). The classification of sorghum germplasm, breeding initiatives, and conservation efforts rely heavily on the determination of the genetic diversity among sorghum species. Identification of genetic variants, morphological features, and genetic analysis of ISSR, SSR, and RAPD are useful techniques. These findings demonstrate a large ratio of variation in sorghum. This work could serve as a guide for future research on sorghum and aid in the understanding of species and breeding initiatives.

Detected Genetic Markers for Three Varieties of Rice (Oryza sativa L.) under Nano- Particles

Despite the enormous advancements in breeding, fresh and accessible methods of plant development are preferred. Many research have been done to determine the impact of zinc oxide nanoparticles (ZnO NPs) and magnesium nanoparticles (MgONPs) on plant growth. ZnO NPs and MgONPs have been used extensively in agriculture. Few research have looked into the potential impact of ZnO NPs on grain Zn content or cereal yield formation to date. The goal of the study was to demonstrate how varied concentrations of nanoparticles affected root germination in three different rice types (Giza 177, Sakha super 300, and Sakha 108) and the fungus Fusariummoniliforme, which causes foot rot in rice. Here, we performed a pot experiment to assess the effects that ZnO nanoparticles and MgONPs had on the shoot length, root length, and germination of rice. The experiment involved three dosages of Zn and Mg nanoparticle solution at varying concentrations of 10, 20, and 100 ppm. The outcomes showed that, when compared to the control treatment, ZnO NPs and MgONPs increased the length of the shoot, the length of the roots, and the rate of germination. Using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers, the genetic makeup of plants treated with 10, 20, and 100 ppm AgNPs was also examined. In vitro rooted shoots were acclimated in a greenhouse before being tested for biochemical stability and phenotypic stability. In vitro, callogenesis and caulogenesis were both reduced by AgNPs at the greatest dose (100 ppm). In order to maximise genome coverage, three rice cultivars from distinct origins were chosen, and they were tested using three RAPD markers and seven selected ISSR markers. AgNPs-treated plants using ISSR markers produced 87 amplified bands in total, 45 polymorphic allelic variants, and 36 monomorphic allelic variations. The Pearson correlation coefficient was used in the Mantel test to determine whether there was a significant association between the Jaccard's dissimilarity matrices based on ISSR and RAPD markers (r = 0.69; P 0.05). Morphological characteristics, ISSR, and RAPD analysis were used to partition the UPGMA Dendrogram into two groupings. Jaccard's coefficient was used to analyse the genetic similarity matrix. Sakha Super 300 and Giza 177 had the most genetic similarity (95%) while Sakha 108 and Giza 177 had the lowest genetic similarity (90%) according to ISSR and RAPD study. The classification of rice germplasm, breeding initiatives, and conservation efforts all heavily rely on the determination of genetic diversity within the rice species. To find genetic variations, morphological characteristics, ISSR, and RAPD analysis are useful techniques. The findings demonstrated the large ratio of variation in rice. This work successfully established the possibility of using MgONPs and ZnO NPs as high-performing fertilisers to improve rice output and quality.This study could serve as a guide for future research on rice and could support efforts to understand the species and improve breeding stock.

Involvement of Dynamic Adjustment of ABA, Proline and Sugar Levels in Rhizomes in Effective Acclimation of Solidago gigantea to Contrasting Weather and Soil Conditions in the Country of Invasion.

Giant goldenrod (Solidago gigantea Aiton) is one of the most invasive plant species occurring in Europe. Since little is known about the molecular mechanisms contributing to its invasiveness, we examined the natural dynamics of the content of rhizome compounds, which can be crucial for plant resistance and adaptation to environmental stress. We focused on rhizomes because they are the main vector of giant goldenrod dispersion in invaded lands. Water-soluble sugars, proline, and abscisic acid (ABA) were quantified in rhizomes, as well as ABA in the rhizosphere from three different but geographically close natural locations in Poland (50°04'11.3″ N, 19°50'40.2″ E) under extreme light, thermal, and soil conditions, in early spring, late summer, and late autumn. The genetic diversity of plants between locations was checked using the random amplified polymorphic DNA (RAPD) markers. Sugar and proline content was assayed spectrophotometrically, and abscisic acid (ABA) with the ELISA immunomethod. It can be assumed that the accumulation of sugars in giant goldenrod rhizomes facilitated the process of plant adaptation to adverse environmental conditions (high temperature and/or water scarcity) caused by extreme weather in summer and autumn. The same was true for high levels of proline and ABA in summer. On the other hand, the lowering of proline and ABA in autumn did not confirm the previous assumptions about their synthesis in rhizomes during the acquisition of frost resistance by giant goldenrod. However, in the location with intensive sunlight and most extreme soil conditions, a constant amount of ABA in rhizomes was noticed as well as its exudation into the rhizosphere. This research indicates that soluble sugars, proline, and ABA alterations in rhizomes can participate in the mechanism of acclimation of S. gigantea to specific soil and meteorological conditions in the country of invasion irrespective of plant genetic variation.

Comparative Study of Genomic DNA Extraction Methods for Common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is one of the significant grains used in the human diet, accounting for half of all grain legumes consumed globally. To enhance production, conventional breeding and molecular approaches have been used so far. An efficient and rapid genomic DNA extraction method is required for these molecular approaches. The aim of this study was to compare and optimize an efficient and rapid DNA extraction protocol for common bean. Modified cetyl trimethylammonium bromide (CTAB) and potassium chloride (KCL) extraction methods were used. The mean DNA yield per nanoliter was 209 µg from modified CTAB and 150.3 µg from the KCL method. The concentration of gDNA was significantly (P< 0.05) higher for the KCL method, which was 5.01 µg/µl and 2.09 µg/µl for the CTAB method. The obtained DNA was also pure, with an absorbance ratio at 260 nm to an absorbance of 280 nm (A260/280) of 1.75-2.23 for the KCL method and 1.86-2.09 for the modified CTAB method. Gel electrophoresis separation was used to evaluate the quality of the total DNA extracted by the present protocols. The results showed that intense bands close to the gel wells were obtained from both extraction methods. DNA isolated with the two methods was successfully used for PCR-based downstream analysis, which includes random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. In this study, it took approximately 150 minutes for KCL and 240 minutes for the CTAB for whole process. In contrast to the CTAB method, the KCL method uses inexpensive and less hazardous reagents and requires only ordinary laboratory equipment. Therefore, it is more convenient and economical than the traditional technique.

Variation of Fruit Color in Cakra Hijau, G1/M8 and HV-149 Chilli Pepper Cultivar: Physiology and Molecular Approach

The fruit color of chili pepper is an important characteristic in identification and classification and is often used as the basis for determining consumer preferences. Information on the relationship between chili fruit color and its molecular profile is very important in supporting selection activities in plant breeding. This study aims to identify genetic diversity associated with the fruit color of three genotypes of chili (Capsicum frutescens L.): Cakra Hijau, HV-149 and G1/M8, using Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR). Morphological confirmation was carried out according to Capsicum descriptors. Nineteen RAPD markers and six SSR markers were used for genetic variability assessment. Genetic variation was analyzed using the unweighted pair group method with the arithmetic mean and the Jaccard similarity index. The three chili genotypes had different fruit colors at each maturation stage. The immature Cakra Hijau fruit is dark green and turns dark red as it ripens. The immature fruits of the G1/M8 line are light green and turn red when ripe. Finally, immature HV-149 fruits are dark green and yellow when ripe. The SSR markers used in this study were unable to show polymorphism. On the other hand, the RAPD marker successfully detected genetic variation in the three chili genotypes and resulted in a total of 49 alleles. The average value of polymorphic information content of the RAPD primers used ranged from 0 to 0.296, with the highest index indicated by OPA-1. The dendrogram shows the separation of the three genotypes into two main clusters, with the first cluster consisting of the HV-149 variety and the second cluster consisting of Cakra Hijau and G1/M8 lines. This study revealed that there are genetic variations based on the morphological characteristics of fruit color at each ripening stage and RAPD band profile. The RAPD marker was more effective than the SSR marker for identifying the genetic diversity of fruit color in the three chilies studied.

Using Vitrification–Dehydration Method as a New Protocol for In-vitro Cryopreservation of Date Palm Shoot Tip Cultivars

This study was conducted to develop an in vitro method for the short-term conservation of the planting materials of date palm (Phoenix dactylifera). The conservation of the genetic resources of date palm species and germplasm represented by the cryopreservation of biological material, such as shoot tips of date palm dropped in LN (Liquid Nitrogen) at low temperatures, is a safe method. The main objective of this study was the collection and in vitro germplasm preservation of the date palm cultivars, and this study evaluated the in vitro preservation and genetic stability of date palm shoot tip explants using dimethyl sulfoxide (DMSO) in the medium for long-term storage. Shoot tip explants of about 2-3 cm in length were excised from in vitro cultures and were transferred to preservation media. The results showed that the highest rates of survival (80%) and recovery (75%) were observed with 1.2 M sucrose. To determine the effect of vitrification on freezing tolerances, cultures were exposed to a solution that dissolved the glaze for 60-80 minutes. The maximum survival rate obtained with exposed cultures was 85%. RAPD (randomly amplified polymorphic DNA) was used to explain the differences in the genetic characteristics of cryogenic tissue cultures and non-cryogenic tissues of date palm. Both were similar to the germinated date palm in the open field. Finally, the cryopreserved plants were able to adapt to free-living conditions after acclimatization, All aspects will contribute to the improvement of the currently available techniques for the in vitro germplasm conservation of date palms.

Role of Molecular Marker RAPD in Determining the Genetic Divergence between Hybrids and Inbreed Lines of Maize Using Full Diallel Cross

Field experiments were conducted during the spring and autumn seasons of 2021in farmer's fields on the right side of the Euphrates River – Ramadidistrict / the center of Anbar Governorate. In an entire reciprocal program, four inbreds of maize (Zm1, A119, Syn-33 and A105) were used and introduced to produce 12 single hybrids. The seeds of the parents and the resulting hybrids were planted according to randomized complete block design (RCBD)at four replications to estimate the heterosis. The reciprocal hybrid Syn-33 x A105 and the reverse hybrid Syn-33 x A105 were significantly superior. They gave the highest means of grain yield (215.78, 203.10 g), grain weight (99.74 and 98.56 g), number of grains in the ear (17.4 and 16.7 grains row-1 ) and heterosis (116.01 and 103.32%) in grain yield respectively. Randomly Amplified Polymorphic DNA (RAPD) technique and 15 primers were used. The highest genetic divergence was 0.69 between Zm1 and Syn-33 inbreds, while the highest genetic divergence between inbreds and hybrids was 0.75 between Zm1 inbred and A105 x A119 hybrid. Keywords: Primer, Heterosis, Genetic, Polymorphic, RCR.

Early detection of a possible multidrug-resistant Acinetobacter baumannii outbreak in the local hospital setting by using random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR), oxacillinase gene profiles, and antibiograms.

Detecting the source of a potential outbreak of multidrug resistant (MDR) Acinetobacter baumannii is necessary to be investigated. This study aimed to detect the possibility of A. baumannii outbreak in a hospital setting using a combination of random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR), antibiograms, and the presence of oxacillinase genes. The antibiogram of 31 clinical isolates and six environmental isolates of A. baumannii were determined by Vitek® 2 Compact. Oxacillinase genes (OXA-23, -24, -51, and -58) were detected by PCR, and RAPD-PCR was conducted using DAF-4 and ERIC-2 primers. The Similarity Index and dendrogram were generated using GelJ v2.3 software. The antibiograms showed that all MDR A. baumannii isolates has very limited susceptibility to cephalosporins, but mostly susceptible to tigecycline. All isolates were positive for bla OXA-51-like gene, thirty-two of 37 total isolates (86.5%) were positive for bla OXA-23-like gene, and none were positive for bla OXA-24-like and bla OXA-58-like genes. RAPD-PCR showed that the DAF-4 primer on average had more band visualization and lower Similarity Index's variation compared to the ERIC-2. The discriminatory power of DAF-4 was 0.906. There was a significant correlation between the DAF-4 dendrogram pattern with the antibiogram (r=0.494, p<0.001) and the presence of bla OXA-23-like gene (r=0.634, p<0.001) from all ICU A isolates. Six out of fourteen ICU A isolates belonged to the same cluster with >95% Similarity Index, while one clinical isolate having an identical dendrogram and antibiogram pattern with an environmental isolate within this cluster. There is a high probability of MDR A. baumannii outbreak within ICU A detected by multiple analysis of RAPD-PCR, antibiogram and the bla OXA-23-like gene profiles. This combinatorial approach is conceivable to mitigate possible outbreak situations of A. baumannii in the local hospital without sophisticated microbiology laboratory.

Genetic diversity and molecular analysis using RAPD markers of banana cultivars in the five regions of East Java, Indonesia

Abstract. Slameto. 2023. Genetic diversity and molecular analysis using RAPD markers of banana cultivars in the five regions of East Java, Indonesia. Biodiversitas 24: 5035-5043. The study explored banana plants in East Java, Indonesia, specifically in the districts of Jember, Banyuwangi, Bondowoso, Situbondo, and Lumajang, using Random Amplified Polymorphic Deoxyribonucleic Acid (RAPD) markers. Thirty accessions of Musa ssp. were collected from these five districts, revealing a high level of genetic diversity. Primers OPA-04 and OPC-05 were suitable for assessing genetic variation in banana plants. It was found that the OPD-07 primer produced the fewest DNA bands (8 bands and 7 polymorphic bands), while the OPA-04 primer produced the maximum number of DNA bands (14 bands, with 9 polymorphic bands). The OPB-08 primer had the lowest polymorphism percentage (56%), and the OPC-05 primer had the greatest percentage (92%). With genetic similarity scores ranging from 0.04 (Bigih Tanjung Glugur and Lilin Banyuwangi) to 0.94 (Agung Banyuwangi and Let). The phylogenetic tree of 30 banana plants formed two primary clusters: cluster I and II, included 26 and 4 banana varieties respectively. In addition, molecular variance (AMOVA) resulted a significant proportion (93%) of genetic diversity within the population. Further studies using more precise genetic markers are still needed to determine the exact identity of the banana plant genome.

Association between quantitative morphological traits and RAPD molecular markers in pomegranate (Punica granatum L.).

Pomegranate (Punica granatum L.) is very important in terms of horticulture and food around the world. The present research aimed to identify the random amplified polymorphic DNA (RAPD) markers associated with morphological traits in pomegranate genotypes. Significant differences were observed among the studied genotypes based on the recorded traits. The 18 RAPD primers produced a total of 154 polymorphic fragments among genotypes. Using multiple regression analysis between each of the morphological traits and 154 RAPD polymorphic bands, RAPD markers associated with each of the morphological traits were identified. In total, 11 markers showed significant correlations with fruit weight, 9 markers with 100-aril weight, 11 markers with anthocyanin, and 8 markers with total soluble solids. Some markers were associated with more than one morphological trait, showing that the association of a marker with more than one trait can be caused by the pleiotropic effects of quantitative trait loci related to each other in different traits. For instance, the BA6-1 marker showed positive correlations with fruit weight, fruit crown width, and leaf length. Also, OPG13-3 and BA6-10 markers showed positive correlations with total soluble solids and anthocyanin content. The informative markers identified related to morphological characteristics in pomegranate can be a suitable guide to identify the genotypes with valuable fruit traits. Also, these markers can be used in selecting suitable parents for population generation for mapping purposes.

The potential of gamma irradiation on antioxidant capacity and genomic alterations in Calendula officinalis

There are lines of evidence that ionizing radiations such as gamma rays can cause different biological effects on plants. Marigold (Calendula officinalis L.) is a member of the family Asteraceae. It possesses profound amounts of active ingredients. The aim of this study was to evaluate the changes imposed upon different dose levels of gamma radiation on some features of Calendula officinalis such as antioxidant activity, total phenolic compounds and flavonoid contents, antibacterial activity and genomic alterations. Calendula officinalis seeds were exposed to different doses of Gamma radiation (0, 10, 15, 20 and 25 GY). Total phenolics, flavonoids, antioxidant activity (measured by DPPH assay) using methanolic extracts of plants and antibacterial activity measured by the disc diffusion assay showed significant differences to the control samples. The samples treated with 10 GY gamma rays showed the highest total phenol and flavonoid contents. Antioxidant activity significantly differed between Gamma rays dose levels and it was the highest at 25 GY. Four bacterial strains including E. coli, Bacillus subtilis and Pseudomonas aeroginosa were used for the antibacterial assay. Extracts from plants treated with 25 GY gamma rays showed the highest antibacterial activity against the 4 bacterial strains. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the genetic variation. The polymorphism information content (PIC) for RAPD primers ranged from 3% to 13% and ranged from 6 to 13% for ISSR primers. Results indicated that ISSR markers were more efficient than RAPD markers, as they detected 25.57% polymorphic DNA bands compared to 21.31% polymorphism for RAPD markers.

DNA barcodes, ISSR, RAPD and SCAR markers as potential quality control tools for molecular authentication of black and white mulberry

Morus species, especially black mulberry (Morus nigra L.), are well known for their nutritive and economic values. Black mulberry is often a subject for deliberate and undeliberate substitution with the morphologically similar white mulberry (Morus alba L.). In the current study, the genetic diversity of Morus nigra L. and Morus alba L. was assessed using different genetic profiling and fingerprinting techniques namely namely DNA barcoding, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). DNA barcoding was implemented using 2 plastid loci (matK, and rbcL-1) and 2 nuclear spacers (ITS and ITS2) aiming at accurate authentication of the studied species. DNA barcoding, targeting the selected loci succeeded to identify and authenticate the studies Morus species but failed to differentiate them, owing to their close genetic resemblance. Out of 52 screened RAPD primers, 27 were able to produce reproducible and clear profiles with 115 amplified fragments of which 37 were polymorphic accounting for 32.17% polymorphism. The number of amplified bands varied from 2 to 7 ranging in size from 100 to 700 bp. Moreover, 55 ISSR primers were evaluated, 108 fragments were produced, using 20 primers, of which 28 were polymorphic accounting for 25.92% polymorphism with size range of 200–800 bp. Sequence Characterized Amplified Region (SCAR) marker was developed based on reproducible banding pattern obtained by RAPD primer (OPG-03), for the purpose of accelerating the initial screening of Morus batches and enabling the preventative rejection of adulterated samples. The polymorphic band obtained was sequenced and specific primers were designed to develop a SCAR marker of approximately 558 bp exclusively for M. nigra, allowing detection of down to 1 ng, of M. nigra. The SCAR marker showed 100% success in differentiating authentic M. nigra and M. alba samples. In addition, the application of the developed SCAR marker was extended to distinguish M. nigra from its four common adulterants namely Ficus carica L., Fragaria vesca L., Ficus racemosa L. and Vitis vinifera L.. The proposed protocol was successfully implemented for the evaluation of 47 commercial M. nigra samples collected from different Egyptian locations.

Comparative Assessment of SSR and RAPD markers for genetic diversity in some Mango cultivars.

Genetic improvement mainly depends on the level of genetic variability present in the population, and the degree of genetic diversity in a population largely determines the rate of genetic advancement. For analyzing genetic diversity and determining cultivar identities, a molecular marker is a useful tool. Using 30 SSR (simple sequence repeat) and 30 RAPD (randomly amplified polymorphic DNA) markers, this study evaluated the genetic divergence of 17 mango cultivars. The effectiveness of the two marker systems was evaluated using their genetic diversity characteristics. Additionally, the effects of SM (simple matching) and Dice similarity coefficients and their effects on mango clustering were evaluated. The findings showed that SSR markers generated 192 alleles, all of which were polymorphic (100%). With RAPD markers, 434 bands were obtained, 361 of which were polymorphic (83%). The average polymorphic information content (PIC) for RAPD and SSR was 0.378 and 0.735, respectively. Using SSR markers resulted in much higher values for other genetic diversity parameters compared to RAPD markers. Furthermore, grouping the genotypes according to the two similarity coefficients without detailed consideration of these coefficients could not influence the study results. The RAPD markers OPA_01, OPM_12 followed by OPO_12 and SSR markers MIAC_4, MIAC_5 followed by mMiCIR_21 were the most informative in terms of describing genetic variability among the cultivars under study; they can be used in further investigations such as genetic mapping or marker-assisted selection. Overall, 'Zebda' cultivar was the most diverse of the studied cultivars.

Exogenous melatonin enhances the tolerance of tiger nut (Cyperus esculentus L.) via DNA damage repair pathway under heavy metal stress (Cd2+) at the sprout stage

Heavy metal (HM) stress is a non-negligible abiotic stress that seriously restricts crop yield and quality, while the sprout stage is the most sensitive to stress and directly impacts the growth and development of the later stage. Melatonin (N-acetyl-5-methoxytryptamine), as an exogenous additive, enhances stress resistance due to its ability to oxidize and reduce. However, few reports on exogenous melatonin to tiger nuts under HM stress have explored whether exogenous melatonin enhances plants' resistance to heavy metals. Here, “Jisha 2″ was used as material, with a stress concentration of 5 mg/L and 100 μmol/L of CdCl2 to explore whether exogenous melatonin enhances plant resistance and molecular mechanism. The result revealed that stress limits growth, while melatonin alleviated the sprout damage under stress from the phenotypes. Moreover, stress-enhanced reactive oxygen species (ROS) accumulation and membrane lipid peroxidation, while melatonin-increased ROS reduce damage via the analysis of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) and malondialdehyde (MDA) content, hydrogen peroxide (H2O2), superoxide anion (O2-), and Electrolyte leakage (El). Further results indicated that HM leads to DNA damage while exogenous melatonin will repair the damage by analyzing random amplified polymorphic DNA (RAPD), DNA cross-linking, 8-hydroxy-20-deoxyguanine level, and relative density of apurinic sites. Furthermore, gene expression in the DNA-repaired pathway exhibited similar results. These results applied that exogenous melatonin released the hurt caused by HM stress, with DNA repair and ROS balance serving as candidate pathways. This study elucidated the mechanism of melatonin's influence and provided theoretical insights into its application in tiger nuts.

Влияние экологических факторов на генетический полиморфизм в природных популяциях Fragaria vesca

Genetic polymorphism of natural cenopopulations of wild strawberry (Fragaria vesca) from the Republic of Mari El was measured by randomly amplified polymorphic DNA (RAPD). The method of molecular genome marking based on RAPD-PCR makes it possible to determine the genetic status of populations and establish interpopulation relationships. The cenopopulations that we selected for the analysis were confined to different stages of the forest phytocenosis succession: the climatic phytocenosis and the area of the forest felling. These F. vesca cenopopulations differed in the ratio of sexual and vegetative reproduction during the maintenance of the cenopopulation size. In a felling area in conditions of greater illumination the intensity of sexual and vegetative reproduction increases. However, specimens of seed origin were not found in the felling area, since in conditions of high competition with ruderal and meadow plants, slowly developing strawberry seedlings died. The germination of seeds and plants of seed origin was noted under conditions of climatic phytocenosis. For the cenopopulation confined to the climax forest, Shannon’s diversity index and Nei’s genetic distance were 0.5179 and 0.3613 respectively. In a felling area vegetative reproduction processes predominate, so Shannon’s diversity index and Nei’s genetic distance were lower, 0.4573 and 0.3132 respectively. In a felling area revealed the loss of certain genetic clusters present in the climax forest. This indicates the loss of a strawberry genetic diversity in the transition to vegetative reproduction due to anthropogenic impact. Our data confirm the idea that climax communities with their more stable conditions can act as regenerative niches of the species.

Evaluation of the Genetic Stability of Amaranthus viridis L. Species in Selected Regions of Western Ghats using Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA is cheap and fast molecular technique to identify the genetic resemblances or variations in DNA in various plants. The proposed research work wasintendedto compare the genetic diversification of Amaranthus viridis species in selected regions of Western Ghats in Tamilnadu and Kerala by RAPD analysis.The biometric morphological data among the five A. viridis accessions were compared. Their genomic DNA was isolated and finger prints were obtained using three RAPD markers. The percentage polymorphism, polymorphism information content, effective multiplex ration and resolving power were calculated. UPGMA dendrogram was constructed and their genetic relatedness was compared using Jaccard coefficient.Upon finger printing, 66 bands were counted for the three RAPD primers used,among which 46 bands were polymorphic band numbers from 8 to 21. Percentage of polymorphic bands ranged between 44.44% and 80.77%. EMRvalues for 66 polymorphic loci ranged between 3.56 and 17.01. The UPGMA dendrogram comparing the genomic profiles of the 5 A. viridis accessions using Jaccard coefficient revealed their genetic distances where the maximum Jaccard’s coefficient value observed was 0.96 between AVKATN and AVNEK. Among the 5 selections the peak similarity index (0.9565) was witnessed between AVKATN and AVNEK. The cuurent study revealed that AVKATN and AVNEK had the highest their genetic distances, whereas AVKATN and AVNEK were genetically similar among the 5 A. viridis accessions. Several polymorphic bands which ranged up to 80.77% have proved their genetic variations.

Molecular characterization and differential effects of levofloxacin and ciprofloxacin on the potential for developing quinolone resistance among clinical Pseudomonas aeruginosa isolates.

Fluoroquinolones are some of the most used antimicrobial agents for the treatment of Pseudomonas aeruginosa. This study aimed at exploring the differential activity of ciprofloxacin and levofloxacin on the selection of resistance among P. aeruginosa isolates at our medical center. 233 P. aeruginosa clinical isolates were included in this study. Antimicrobial susceptibility testing (AST) was done using disk diffusion and broth microdilution assays. Random Amplification of Polymorphic DNA (RAPD) was done to determine the genetic relatedness between the isolates. Induction of resistance against ciprofloxacin and levofloxacin was done on 19 isolates. Fitness cost assay was done on the 38 induced mutants and their parental isolates. Finally, whole genome sequencing was done on 16 induced mutants and their 8 parental isolates. AST results showed that aztreonam had the highest non-susceptibility. RAPD results identified 18 clusters. The 19 P. aeruginosa isolates that were induced against ciprofloxacin and levofloxacin yielded MICs ranging between 16 and 256 μg/mL. Levofloxacin required fewer passages in 10 isolates and the same number of passages in 9 isolates as compared to ciprofloxacin to reach their breakpoints. Fitness cost results showed that 12 and 10 induced mutants against ciprofloxacin and levofloxacin, respectively, had higher fitness cost when compared to their parental isolates. Whole genome sequencing results showed that resistance to ciprofloxacin and levofloxacin in sequenced mutants were mainly associated with alterations in gyrA, gyrB and parC genes. Understanding resistance patterns and risk factors associated with infections is crucial to decrease the emerging threat of antimicrobial resistance.