RAPD-PCR Analysis Research Articles

Cloning, sequencing, and characterizing of soil antibiotic active-producing Streptomyces species-specific DNA markers

Association of the antibiotic activity of the soil Streptomyces isolates to their genetic profiles analyzed through RAPD-PCR fingerprints prompted us here in this study to use the most common bands as specific markers to identify homologous proteins within these isolates by cloning, sequencing, and characterizing these markers. Six out of twelve DNA bands ranged between 600 and 1350 bp previously obtained by RAPD-PCR analysis were purified out of the RAPD gels, and then cloned into pGEM-T Easy vector system. Success of the cloning process was confirmed by digesting purified plasmids with EcoRI. The clones namely No. 54, 55, 20, 56, 57, and 58 were sequenced using the DNA BigDye Terminator Sequencing System utilizing the M13 primer. Results indicated that the size of the inserted sequences is 599, 566, 522, 870, 857, and 254 bp, in clones No. 54. 55, 20, 56, 57, and 58, respectively. Homologous proteins of the six cloned sequences generated by DNA blast software indicated that the highest score of protein homology was scored for clone No. 54 with 87 % homology to putative secreted pectate lyase [Streptomyces coelicolor A3(2)]. The other clones showed less homology with 77 % homology for the clones No. 55 and 56, 73 % homology for the clone No. 20, and 55 % homology for the clones No. 57 and 58. The association of homologous proteins to the reported RAPD pattern is confirmed here for the first time, and the resulting DNA cloned fragments deserve further molecular analysis.

Application of RAPD-PCR and Phylogenetic Analysis for Accurate Characterization of Salmonella spp. Isolated from Chicken and Their Feed and Drinking Water


 
 
 
 The aim of this study was ‎the‎ discrimination of Salmonella‎‎ isolated from chicken and their feed ‎and drinking water for the epidemiological control of salmonellosis. Totally, 289 samples, ‎including 217 chicken cloaca swabs, 46 water, and 26 feed samples were collected from five ‎different farms in Karbala governorate, Iraq. Conventional bacteriology tests, API 20E, Vitek 2, ‎and serology were used for bacterial identification. Random amplified polymorphic ‎DNA (RAPD)-polymerase chain reaction (PCR) was applied to analyze the genetic relationships ‎among Salmonella‎‎ isolates. The isolation rate of Salmonella‎‎ spp. was 21.1% (61/289). While the ‎water samples constituted the highest rate (30.4%), a rate of 21.7% was reported for the cloaca ‎swabs, with no isolate at all from chicken feed. Vitek 2 was able to identify some isolates to the ‎serotype level, such as S. Enteritidis, S. Paratyphi B, and S. Paratyphi C. However, the isolates ‎were diagnosed as S. enterica by API 20E, and as S. enterica subsp. arizonae through serology. ‎Analyzing the samples by the RAPD-PCR assay showed the presence of genetically different ‎Salmonella‎‎ spp. Dendrograms created by the GelJ software successfully delineated the genetic ‎relationships. Therefore, RAPD-PCR can be used as a surrogate tool for the fast, reliable, and ‎accurate detection of Salmonella‎‎ in epidemiological surveys when compared with other ‎biochemical-based identification methods.
 
 
 

Isolation and Identification of Lactic Acid Bacteria from Spontaneously Fermented Kunun-zaki using RAPD-PCR Analysis

Sixty lactic acid bacteria (LAB) were isolated from eighteen types of kunun-zaki produced naturally using millet, sorghum and maize each singly or in combinations with malted grains (millet, sorghum, maize, wheat and paddy rice) and characterized using conventional methods (biochemical and cultural) and molecular analyses. Seven morphotypes (Mt1-Mt7) were revealed among the 60 LAB isolates. Twenty LAB were selected based on the differences in their biochemical and physiological properties and these were subjected to DNA isolation and purification. The Agarose gel electrophoresis of the twenty (20) extracted DNA revealed very pure DNA electrophoretic pattern. The concentration of the DNA sample ranged between 0.7 mg/ml to 4.0 mg/ml and the purity index ranged from 1.5 to 1.9; indicating they were all very pure and can easily be used in Polymerase chain reaction (PCR) analysis. A highly polymorphic primer, OPB20 primer, was selected and used to analyze the 20 LAB DNA of which seven (7) isolates were identified to be Lactobacillus brevis, twelve (12) isolates were Lactobacillus plantarum and one isolate was identified as Lactobacillus acidophilus and these organisms could be potentially useful industrially.

Serotyping, RAPD PCR, Antibiotic Susceptibility Profiling of Listeria spp. Isolated from Raw Chicken Meat

The present study was aimed at finding the prevalence, serotyping, RAPD and Setyping of Listeria monocytogenes in raw chicken meat purchased from different retail outlets and local butcher shops across the Nagpur city. Cultural examination of 100 raw poultry meat samples, revealed the prevalence of Listeria spp. (35 %). L. grayi was predominant (15 %) among the isolates followed by L. monocytogenes (12 %), L. welshimeri (5 %), L. innocua (2 %) and L. seeligeri (1%) respectively. Among the majority of strains of L. monocytogenes, virulence marker genes hlyA and iap, all together or in combination were detected. Serotyping PCR study revealed that all L. monocytogenes isolates to be serotype 4b, which is the major serotype involved in the human listeriosis. RAPD PCR analysis of L. monocytogenes isolates revealed two types of banding patterns. All 35 strains of Listeria showed in-vitro susceptibility to antibiotics like vancomycin (93.88 %) ceftriaxone + Tazobactum (92.71%) and moderate sensitivity to enrofloxacin (38.78 %), ceftriaxone (34.90%) and ampicillin (34.69 %). Whereas, all the isolates were least sensitive to erythromycine (6.49%) and sulphasomidine (4.08 %). All the Listeriae isolates were refractory to penicillin. The present findings indicate the presence of multiple drug resistance among L. monocytogenes and other Listeria spp. isolated from raw poultry meat samples. Potentially pathogenic L. monocytogenes isolates recovered from raw poultry meat samples also signifies the zoonotic potential of listeriosis in the local area of Nagpur. High prevalence of L. monocytogenes and other Listeria spp. in raw poultry meat and their sensitivity to the antibiotics tested, clearly indicates that vancomycin and ceftriaxone +tazobactam should be used as a drug of choice for treatment of listeriosis. Keywords: Serotyping PCR, RAPD, Antibiotic Sensitivity test, ceftriaxone, sulphasomidine, tazobactam

Morphological and molecular comparison as a useful tool for identification of the three centric marine diatoms (Bacillariophyceae: Chaetoceros).

The objective of this study was to identify morphological and molecular comparison of three marine Chaetoceros species using microscopic observations, sequence analysis of 18S rDNA, random amplified polymorphic DNA (RAPD-PCR) barcoding and nuclear magnetic resonance (NMR) spectroscopy.Chaetoceros were obtained from three different algae laboratories: Center of Excellence for Marine Biotechnology (CEMB), Chanthaburi Coastal Fisheries Research and Development (CHAN) and Institute of Marine Science, Burapha University (BIM). Genomic DNA for the RAPD-PCR analysis was extracted using the phenol-chloroform method, followed by 18S rDNA amplification.The blast results of 18S rDNA sequence confirmed the significantly matched to C. gracilis for Chaetoceros BIM and CHAN and C. muelleri for Chaetoceros CEMB(e-value = 0.0, identity = 99%). The RAPD-PCR results revealed differences in the three Chaetoceros isolates with polymorphisms between 30.43% and 60.00%, and Chaetoceros CEMB showed high polymorphic bands. Scanning electron microscopy revealed that Chaetoceros CEMB were larger and had larger setae compared to the other isolates (P < 0.05). The results of the NMR characterization of metabolites were consistent with the results of the sequence and morphological analyses. The concentrations of several metabolites, including chlorophyll c1, chlorophyll a, Myo-inositol, fucoxanthin, astaxanthin, lutein and zeaxanthin, were lower in Chaetoceros CEMB than in Chaetoceros BIM and CHAN. However, high concentrations of fatty acids, such as oleic acid, linoleic acid, α-linolenic acid and arachidic acid, were observed in all isolates. Generally, the results of this study will aid future studies examining the diversity of Chaetoceros in various cultural environments.

Genetic Fidelity Studies using RAPD Marker on local fungal isolate of Jabalpur Region for over-expression of Glucoamylase

The present study was conducted to study genomic profile of wild and mutant isolates of local soil fungal isolate Aspergillus flavipes strain for over-production of glucoamylase using RAPD markers. The study involves application of mutational strategies using chemical and physical mutagens on the potent fungal isolate and their screening studies thereafter. The alteration in genetic makeup of the mutant fungal isolate (SSP#16M) with the wild type (SSP#16) was detected and confirmed by RAPD-PCR analysis of genomic DNA. Although various genotypic methods have been used for comparison of Aspergillus species but no comparable data is available for Aspergillus flavipes strain for increased enzyme production. In the study, the mutant fungal isolate designated NA3 (exposed to nitrous acid) showed 3.5 fold improved glucoamylase activity than wild isolate and showed consistency in the production of the enzyme after repeated subcultures. The data for genetic variability was analysed using software Dendro UPGMA wherein the similarity matrix was computed with Jaccard coefficient. This assessment study of mutant isolate of Aspergillus flavipes strain provides potential applicability in starch processing industries.

New Saccharomyces cerevisiae-Kluyveromyces marxianus fusant shows enhanced alcoholic fermentation performance.

Kluyveromyces marxianus has a promising enological potential because of strong extracellular pectinase activity and the copious production of specific higher alcohols and esters. However, K. marxianus is unable to complete alcoholic fermentation on its own. For this reason it is only used in co- or sequential fermentation in conjunction with Saccharomyces spp. Here we applied the protoplast fusion technique to two commercial Saccharomyces cerevisiae strains (Lalvin EC1118® and Lalvin QA23®) and K. marxianus isolate IWBT Y885, to obtain fusants that possess pre-selected, enologically relevant features of K. marxianus, but with improved fermentation performance. After an extensive selection process, performed using non-auxotrophic markers, we identified one fusant, BF2020, that shows enhanced fermentation performance compared to the parental strain K. marxianus Y885, but that still exhibits strong pectinase activity and may be suitable for industrial production. The genome of the selected hybrid was further investigated and characterized by RAPD-PCR analysis and whole genome sequencing.

Screening of Lactiplantibacillus plantarum Strains from Sourdoughs for Biosuppression of Pseudomonas syringae pv. syringae and Botrytis cinerea in Table Grapes.

Grapes (Vitis vinifera L.) are an essential crop for fresh consumption and wine production. Vineyards are attacked by several economically important bacterial and fungal diseases that require regular pesticide treatment. Among them, Pseudomonas syringae pv. syringae (Ps. syringae) and Botrytis cinerea (B. cinerea) infections cause huge economic losses. The fresh fruit market has shifted to functional natural foodstuffs with clear health benefits and a reduced use of chemicals along the production chain. Lactic acid bacteria (LAB) have a biopreservative effect and are applied to ensure food safety in response to consumers' demands. In the present study, the possibilities of using microorganisms with a potential antimicrobial effect against Ps. syringae and B. cinerea in the production of table grapes were investigated. LAB of the genus Lactiplantibacillus can be a natural antagonist of pathogenic bacteria and fungi by releasing lactic acid, acetic acid, ethanol, carbon dioxide and bacteriocins in the medium. The present study focuses on the characterization of nine Lactiplantibacillus plantarum (Lp. plantarum) strains isolated from spontaneously fermented sourdoughs. Species-specific PCR identified the isolated LAB for partial recA gene amplification with an amplicon size of 318 bp. RAPD-PCR analysis showed the intraspecific diversity of the individual strains. Thirteen plantaricin-like peptides (PlnA, PlnB, PlnC, PlnD, PlnEF, PlnG, PlnI, PlnJ, PlnK, PlnN, PlnNC8, PlnS, and PlnW) produced by isolated Lp. plantarum strains were detected by PCR with gene-specific primers. The key features for future industrial applications were their antimicrobial properties. The culture medium and cell-free supernatant (CFS) were used to establish in vitro antimicrobial activities of Lp. plantarum strains against Ps. syringae and B. cinerea, and inhibition of phytopathogen development was observed. The inhibitory effect of the CFS (cell-free supernatant) of all strains was assessed by infecting table grapes with these pathogens in in vivo experiments. Lp. plantarum Q4 showed the most effective suppression of the pathogens both in vitro and in vivo, which indicates its potential use as a biocontrol agent against berry rot and grey rot on grapes, caused by Ps. syringae and B. cinerea.

Establishing and characterising a new cell line from Calliphora vicina (diptera: calliphoridae) fly embryonic tissues

Insect cell lines represent a promising and expanding field as they have several research applications including biotechnology, virology, immunity, toxicology, cell signalling mechanisms and evolution. They constitute a powerful tool having a direct impact on human and veterinary medicine and agriculture. Although more than 1000 cell lines have currently been established from various insect species, Calliphora vicina-derived fly cell lines are lacking. This study was aimed at establishing a new C. vicina embryonic tissue-derived cell line. Adult flies were collected and embryonated eggs were mechanically homogenised and seeded in four types of culture media (L15, Grace's insect medium, Grace's/L15 and DMEM). Cell growth and morphological characteristics were recorded and cytogenetic and molecular patterns were determined. The CV-062020-PPB cell line was established and was shown to have optimal growth in Grace's/L15 medium. CV-062020-PPB cell monolayers that had been sub-cultured over 16 times consisted of firmly adhering cells having different morphologies; a fibroblast-like shape dominated and the karyotype had a 12-chromosome diploid number. RAPD-PCR analysis of the CV-062020-PPB cell line revealed a high similarity index and strong intraspecific relationship with C. vicina adult flies and a weaker relationship with the Lutzomyia longipalpis-derived cell line (Lulo). The CV-062020-PPB cell line constitutes the first cell line obtained from C. vicina embryonic tissues and represents an important basic and applied research tool.

Genotyping of Burkholderia pseudomallei Isolated From Patients in South-Western Coastal Region of India

Burkholderia pseudomallei is the causative agent of melioidosis. Various tools have been used to determine the genetic diversity in B. pseudomallei isolates. In this study, Random Amplified Polymorphic DNA (RAPD)-PCR and flagellin gene (fliC) based PCR-Restriction Fragment Length Polymorphism (RFLP) were used to genotype Indian clinical B. pseudomallei isolates. A total of 89 clinical isolates could be grouped in 6 groups (A through F) by RAPD-PCR analysis. Some of the isolates in various groups had identical banding pattern suggesting them to be epidemiologically related. The RAPD groups also correlated with MLST sequence types suggesting the utility of this easy to do typing method. The PCR- RFLP analysis suggested Type III to be the predominant type which is different from other RFLP types reported from Southeast Asia. In conclusion, the results of this study show that RAPD-PCR, a simple genotyping method, may be used for analyzing the B. pseudomallei isolates and also establish epidemiological relevant relatedness among them. The results of fliC PCR-RFLP further suggest the Indian isolates are different from other Southeast Asian isolates.

Genomic Characterization of (Diptera: Episyrphus balteatusSyrphidae) from Outer and Lower Himalayas, India

The present study deals with genomic characterization of a hover fly using Randomly Amplified Polymorphic Episyrphus balteatus DNA. RAPD-PCR analysis was carried out to compare genomic DNA of six geographically distinct populations of from Outer and E. balteatusLower Himalayas using decamer oligonucleotide primers. The RAPD-PCR method detected a number of discrete DNA bands with different intensities. Ten random primers produced 39 bands in the studied populations out of which 25 bands were polymorphic. These polymorphic bands were used to differentiate the six populations of . PIC (Polymorphism Information Content) and RP (Resolving Power) E. balteatus values was 0.3312 and 6.06 respectively. Genetic similarity between studied populations ranged from 0.564 to 0.872. Among the studied population the lowest genetic distance (0.128) was observed between population P1 and P3 whereas highest (0.436) was observed between P5 and P6. These changes in DNA showed the geographical accumulation of variations with the course of time that leads to differential adaptability towards the environmental pressures. Dendrogram Cluster analysis reveals various evolutionary was prepared using UPGMA. adaptations of populations. P1 and P3 are classified as most related populations whereas P6 is relatively distant from the other E. balteatus five populations.

Distinction of Paramecium strains by a combination method of RAPD analysis and multiplex PCR

Paramecium is employed as a valuable model organism in various research fields since a large number of strains with different characteristics of size, morphology, degree of aging, and type of conjugation can be obtained. It is necessary to determine a method for the classification and simple identification of strains to increase their utility as a research tool. This study attempted to establish a polymerase chain reaction (PCR)-based method to differentiate strains of the same species. Genomic DNA was purified from several strains of P. caudatum, P. tetraurelia, and P. bursaria used for comparison by the random amplified polymorphic DNA (RAPD)-PCR method. In P. tetraurelia and P. bursaria, it was sufficiently possible to distinguish specific strains depending on the pattern of random primers and amplification characteristics. For the classification of P. caudatum, based on the sequence data obtained by RAPD-PCR analysis, 5 specific primer sets were designed and a multiplex PCR method was developed. The comparative analysis of 2 standard strains, 12 recommended strains, and 12 other strains of P. caudatum provided by the National BioResource Project was conducted, and specific strains were identified. This multiplex PCR method would be an effective tool for the simple identification of environmental isolates or the management of Paramecium strains.

Potential of Seaweed Extract on Growth, Physiological, Cytological and Biochemical Parameters of Wheat (Triticum aestivum L.) Seedlings

Synthetic fertilizers are widely employed in agriculture relative to biofertilizers. Long-term use affects soil fertility, plant vigor, human and livestock health, and the soil microbial community. Seaweeds were employed as biofertilizers in sustainable agriculture to overcome this issue and boost the agricultural productivity. Therefore, the current study investigates the effect of wheat seeds priming with the aqueous extracts of Ulva linza or Corallina officinalis seaweeds at various concentrations (5, 10, 15, 20, and 30%). Wheat grains were primed with seaweed extracts and grown in petri dishes, and the germination rate, growth attributes, physiological parameters, cytological traits, and molecular analyses were evaluated. The physico-chemical characterization revealed that U. linza possessed higher levels of ash, soluble proteins, and sugars, as well as essential macronutrients and amino acids, than C. officinalis. Seedling growth parameters, physiological characteristics, and mitotic index all showed a substantial increase in low concentrations of both extracts when compared to the control. Higher concentrations, on the other hand, exhibited an inhibitory effect as well as certain chromosomal abnormalities. In the treated seedlings, new proteins were expressed, which could be owing to the activity of bioactive components in the extracts. As compared to untreated seedlings, PCR-RAPD analysis utilizing five markers revealed that both seaweed extracts triggered low and high molecular weight DNA fragments, as well as fragments specific to each extract. Seaweed extracts could be employed as an environmentally friendly and soil-safe alternative to typical chemical fertilizers.

The use of internal transcribe spacer (ITS) as a molecular diagnostic tool for understanding variation among population of Metarhizium spp.

Direct analysis of DNA polymorphisms now is a general approach to identify and compare fungi at intraspecific, species, genus, or higher level. In this research using molecular genetic techniques with RAPD-PCR technique with ITS1 and ITS2 as promers were used. The samples are Metrahizium from Indonesia (M4 and M5) and Philippines (M6) using RAPD-PCR amplification, DNA sequencing and Analysis of ITS DNA sequences and ITS DNA sequence homologous alignment by search similarities used Blast Biological Software and multi-aligned with CLUSTALW Biological Software. It confirmed three isolates are: M. anisopliae and by multiple sequence alignment revealed transmission translation and transversion and deletion among them. The phylogram showed biogeographic area and their virulence.

RAPD-PCR and phylogenetic analysis of E. coli isolated from human and cattle urinary tract infections

This study aimed at isolating uropathogenic Escherichia coli from urinary tract infections (UTIs) of human and cattle to examine the molecular diversity and phylogenetic relationship of the isolates. A total of 100 urine samples were collected from UTIs of human and cattle. The isolates identification was done using routine diagnostic methods and confirmed by Vitek2. Antimicrobial susceptibility was tested against 10 antimicrobials. Random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) was applied to identify the genetic diversity among E. coli isolates from human and animal origin by using five different octamer primers. The gelJ software for the phylogenetic analysis created Dendrograms. Out of 50 human urine samples, E. coli was isolated from 12 (24%) samples, and was positive in 5 out of 50 (10%) of cattle urine samples. Concerning the antimicrobial susceptibility test, both human and animal isolates revealed rather approximate results when tested mainly against Imipenem, Cefotaxime, and Ciprofloxacin. These antimicrobial data might indicate presence of a degree of similarity between the human and animal isolates. Using RAPD-PCR, three of the primers produced polymorphic bands; therefore, they were used for further analysis of the results. Either of P1, P3 or P4 primers showed presence of similarity between human and cow isolates. To conclude,RAPD-PCR and gelJ software might be of attractive use to identify and analyze the occurrence of genetic relationships, as this could assist in controlling the routes and sources of infection transmission between human and animal in order to prevent zoonotic infections.

Genetic Diversity of Orobanche cumana Populations in Serbia.

In this study, we report genetic characterization of Orobanche cumana, the causal agent of sunflower wilting in Serbia. The genetic diversity of this parasitic plant in Serbia was not studied before. Random amplified polymorphic DNA (RAPD) markers and partial rbcL gene sequences analysis were used to characterize the O. cumana populations at the molecular level. While phylogenetic analyses of RAPD-PCR amplicons were performed using unweighted pair-group Method analyses, rbcL gene sequences were analyzed using neigbor joining method and minimum spanning tree. Molecular analyses of RAPD-PCR analysis revealed high genetic diversity of O. cumana populations which indicated high adaptive potential of this parasitic weed in Serbia. Further analyses of rbcL gene using minimum spanning tree revealed clear differences among diverse sections of Orobanche genus. Although this molecular marker lacked the resolution to display intrapopulation diversity it could be a useful tool for understanding the evolution of this parasitic plant. Our results suggested that O. cumana has great genetic potential which can lead to differentiation of more virulent races which is important for determining crop breeding strategies for their control.

Development and Evaluation of Alternative Methods to Identify the Three Most Common Serotypes of Salmonella enterica Causing Clinical Infections in Kazakhstan.

In this study, we aimed to compare the performance of conventional PCR and real-time PCR assays as screening methods for identification of three frequent, clinically significant Salmonella serovars in Kazakhstan. We determined the diagnostic efficacy of three molecular methods for detection of S. enterica subsp. enterica and typing S. Typhimurium, S. Enteritidis, and S. Virchow. A total of 137 clinical samples and 883 food samples were obtained in Almaty in 2018–2019. All tests showed high analytical specificity for detecting S. enterica and its corresponding serovariants (100%). The sensitivity of real-time PCR for each of the tested targets was 1–10 microbial cells and in conventional PCR 10–100 microbial cells. The trials with conventional PCR and real-time PCR had a diagnostic efficacy (DE) of 100% and 99.71%, respectively. The DE of real-time PCR and conventional PCR for detecting S. Enteritidis and S. Typhimurium was 99.90%, while the DE of conventional PCR and real-time PCR for detecting S. Virchow was 99.31% and 99.80%, respectively. The RAPD-PCR analysis of the genomic DNA of Salmonella enterica showed the genetic kinship of S. Enteritidis isolates, and the genetic heterogeneity of S. Typhimurium and S. Virchow isolates. Thus, the developed methods can be considered as alternatives to classical serotyping using antisera.

Development of Molecular Markers Associated with Resistance to Gray Mold Disease in Onion (Allium cepa L.) through RAPD-PCR and Transcriptome Analysis

Onions (Allium cepa L.) are one of the most consumed vegetable crops worldwide and are damaged by several fungal diseases in the field or during storage. Gray mold disease caused by the necrotrophic pathogens Botrytis cinerea and Botrytis squamosa is a disease that reduces the productivity and storage life in onions. However, it is difficult to control gray mold disease in onions by using physical and chemical methods. Breeding resistant onions against gray mold disease can reduce the damage caused by pathogens, reduce the labor required for control, and reduce environmental pollution caused by fungicides. However, onions have a large genome size (16Gb), making them difficult to analyze, and have a biennial cycle, resulting in a very long breeding period. Therefore, in this study, markers were developed to shorten the onion breeding period. First, random amplified polymorphic DNA (RAPD) was performed to confirm the genetic relationship between the gray mold disease-resistant and -susceptible lines through a dendrogram. In addition, the sequence characterized amplified region (SCAR)-OPAN1 marker to select resistant lines was developed using a polymorphic RAPD fragment. Second, the RNA-seq of the gray mold-resistant and -susceptible onion lines were analyzed using NGS technology. Using the RNA-seq results and DEG and GO analyses were performed, and the variants, such as SNPs and indels, were analyzed to develop a selectable marker for the resistant line. This study developed the SNP-3 HRM marker for selecting gray mold disease-resistant lines by using the SNPs present in the aldo-keto reductase (AKR) gene with high expression levels in these lines. The SCAR-OPAN1 and SNP-3 HRM markers developed in this study could be used to select gray mold disease-resistant onions in breeding programs to reduce the damage caused by gray mold disease.

Quality and Safety Assessment of Edible Seaweeds Alaria esculenta and Saccharina latissima Cultivated in Scotland.

Within Europe over the last 10 years, there has been an increase in seaweeds cultivated for human consumption. For food safety reasons, it is important to assess the microbiological and nutritional quality of the biomass. The fresh and dried edible seaweeds Alaria esculenta and Saccharina latissima were assessed over two consecutive years for the presence of microorganisms. Seaweed samples supplied from Scotland were stored under isothermal conditions for specific time intervals depending on the sample’s condition (fresh, dried or rehydrated). During storage, microbiological analyses were performed for the enumeration of Total Viable Counts (TVC), Pseudomonas spp., Enterobacteriaceae and Bacillus spp., as well as yeasts and molds. Additionally, bacterial colonies from the Marine Agar growth medium were isolated and subjected to PCR-RAPD analysis for characterization of the bacterial diversity of seaweeds. Bacterial isolates with different fingerprint patterns were further subjected to sequencing (16S rDNA, V1–V4 region). The presence of human pathogenic bacteria was also investigated. Results showed that the initial population of TVC was differentiated depending on the year of seaweed harvest, being closer to the enumeration limit (1.0 log CFU/g) in fresh samples from 2020 and higher in samples from 2019 (6.7 and 3.9 log CFU/g in A. esculenta and S. latissima, respectively). DNA-based analysis revealed the presence of Psychrobacter, Cobetia and Pseudomonas species in A. esculenta, while Psychrobacter and Micrococcus species were present in S. latissima.

Callus induction and regeneration in sugarcane under drought stress.

Tissue culture methods are useful in assessing the tolerance of various stresses due to the ease of controlling stress under in vitro conditions. This study aimed to investigate the response of sugarcane genotyps to drought stress using calli as a model system. For inducing sugarcane callus, the medium of Murashige and Skoog (MS) was used with different mannitol concentrations (100, 200, and 300 mM) to measure their effects on callus frequency, the day of callus initiation, embryogenic potential, relative growth rate (RGR), water and proline contents, K+ and Na+ contents, as well as the formation of shoot and roots for three sugarcane genotypes (e.g., GT 54-9, G 84-47, and pH 8013). The RAPD-PCR analysis was carried out using five oligonucleotide primers to identify the genetic variation among sugarcane genotypes. The results indicated that the degree of callus proliferation varied from 70 − 86%. The highest value of callus proliferation, PGR, shoot formation was recorded for the genotype GT 54-9 compared to the other two genotypes (G 84-47 and pH 8013). Calli treated with 100 mM mannitol showed the highest RGR, proline and waer contents for the genotype GT 54-9, while, those treated with 300 mM recorded the lowest values of these parameters for the genotype pH 8013. The genotype G 84-47 collected highest Na+ content, while the genotype pH 8013 collected highest K+ content. The results of this study recommend preference for GT 54-9 genotype, which is considered the most promising genotype, showing more tolerance to drought stress based on all studied traits.