Random Amplified Polymorphic DNA Profiles Research Articles

Effects of Selenium on DNA Methylation and Genomic Instability Induced by Drought Stress in Wheat (Triticum aestivum L.)

The main purpose of the study was to clarify the effect of selenium (Se) on DNA damage and DNA methylation in wheat (Triticum aestivum L.) plants exposed to polyethylene glycol (PEG)-induced drought stress under in vitro tissue culture. Random amplified polymorphic DNA (RAPD) and coupled restriction enzyme digestion-random amplification (CRED-RA) were utilized to explain the DNA damage grade and variations in DNA methylation patterns, respectively. The outcomes indicate that drought stress gives rise to a rise in RAPD profile variations (as DNA damage) and a decrease in genomic template stability (GTS) rate and DNA methylation changes. According to the RAPD data, the greatest GTS value was computed at 56.9% (5% PEG 6000), and the lowest GTS value was 41.2% (15% PEG 6000), demonstrating the adverse effects of PEG 6000. However, DNA damage can be reduced by treatment with sodium selenate (2, 4, and 6 µM of Na2SeO4) together with PEG (5%, 10%, and 15% PEG 6000)-induced water deficits. Moreover, according to CRED-RA analysis, PEG-induced DNA methylation rates were changed after treating different doses of Se. These data demonstrate that Se dose-dependently modulates both DNA damage and methylation alterations induced by drought in wheat.

Phytochemical Analysis and Genetic Characterization of Genus Blepharis in Rajasthan, India

Nature has been a source of therapeutic agents for thousands of years and a remarkable number of modern medications have been secluded from ordinary sources. Higher plants, as sources of therapeutic compounds, have sustained to play a leading role in the conservation of human health since pre-historic times. Blepharis is an Afro-asiatic genus including 129 species, belonging to the family Acanthaceae, widely distributed in dry and semi-dry habitats. The aim of this study was to identify primary metabolites quantitatively and flavonoid compounds qualitatively using thin-layer chromatography (TLC) from the leaves of two species of the Blepharis genus, and also to investigate the genetic analysis of these species using the random amplification of polymorphic DNA (RAPD). In quantitative analysis, primary metabolites such as protein, carbohydrates, lipid and phenol were found with significant values. In TLC characterization, Kaempferol and Rutin were identified as flavonoid compounds from both leaves samples. A total of 4 and 3 polymorphic bands were observed from OPA-2 primer in RAPD Profile of B. repens and B. repens var. rajasthanensis (a new varieties) leaves. The result concluded that these two different varieties of genus Blepharis can be used as an alternative to the conventional medicines due to having wide range of phytocemical activities. This study suggests that there is a need to know the possibility of using extracts and bio-active mixtures derived from Blepharis genus for the control of lasting ailments as claimed by traditional therapists.

Confirmation of mutation and genetic stability of the M4 generation of chili pepper’s (Capsicum frutescens L.) Ethyl Methane Sulfonate (EMS) mutant based on morphological, physiological and molecular characters

Abstract. Arumingtyas EL, Atiaturrochmah, Kusnadi J. 2023. Confirmation of mutation and genetic stability of the M4 generation of chili pepper’s (Capsicum frutescens L.) Ethyl Methane Sulfonate (EMS) mutant based on morphological, physiological and molecular characters. Biodiversitas 24: 531-538. The initial generation of Ethyl Methane Sulfonate (EMS) random mutations usually still shows high variation due to allele segregation. This research aimed to confirm the genetic differences between the M4 generation of chili pepper (Capsicum frutescens L.) mutant resulted from EMS mutations (G7/01) and the initial line (G7), and the stability of the mutant based on morphological, physiological, and molecular characters. The morphological characters and the capsaicinoid content of the mutants G7/01 with the initial line (G7) were compared. The capsaicinoid content and fruit spiciness were measured by spectrophotometer ? 280 nm. Molecular characterization was conducted using Random Amplified Polymorphic DNA (RAPD) genetic markers. Based on the morphological characters, the G7/01 mutants have some superior properties compared to the initial line G7. All the G7/01 mutant plants contain higher capsaicinoid compounds than the initial line plants. The dendrogram developed based on RAPD profile showed that all the mutant plants were positioned apart from the initial line plant, suggesting that there are some genomic changes in the mutants compared to the initial line plants. All the mutant plants, except T1, showed insignificant variation in morphological characteristic, capsaicin content, and RAPD profile, indicating genetic stability.

Genetic Polymorphism in Honey bee Apis cerana indica (F.) (Hymenoptera: Apidae) by Random amplified polymorphic DNA (RAPD) Markers

For pollination purpose and apiary by-products production Apis cerana indica is highly demanded honey bee in India. Honey bee is a polymorphic bee having three casts queen, workers and drones. In Apis cerana indica queen honey bee undergoes multiple mating with drones to gather genetic diversity in colony. Genomic DNA sample of adult drone and worker honey bees was used to study genetic polymorphism by Random amplified polymorphic DNA (RAPD) markers. Ten Primers of each containing different 10mer oligonucleotide sequence were used to generate RAPD profile. Present study observed the banding pattern for presence and absence of characters and find out the polymorphism information content (PIC) value generated by each primer. Low level of genetic polymorphism was found between workers and drones with 0.34984 PIC value.

Bacterial community analysis of infant foods obtained from Chinese markets by combining culture-dependent and high-throughput sequence methods

In this study, twenty-two baby foods including cereal-based products and powdered infant formula (PIF) obtained from local markets were comprehensively investigated for their bacterial contamination using culture-dependent and high-throughput sequence (HTS) methods. In addition, the genetic diversity and biofilm-forming capacity of the most abundant species were analyzed using random amplified polymorphic DNA (RAPD) and crystal violet staining assay, respectively. Results showed that 170 mesophilic isolates collected from 22 samples were clustered into 15 genera and 41 species. Bacillus (77.65%) was the most prevalent genus, followed by Paenibacillus (7.06%), Alkalibacillus (3.53%), and Lysinibacillus (2.35%). Bacillus licheniformis (49.41%) proved to be the most dominant species in infant foods, and a high genetic diversity with six different RAPD profiles was observed. A total of 87.5% of B. licheniformis isolates were identified as strong biofilm formers, and heterogeneous biofilm-forming ability was observed among the isolates sharing the same RAPD pattern. HTS analysis revealed an 18-fold higher biodiversity at the genus level, and a significantly different bacterial community of infant foods was dominated by Lactococcus, Streptococcus, and Bifidobacterium. Foodborne pathogens including Bacillus cereus, and potentially pathogenic microorganisms such as Acinetobacter baumannii, were identified in infant foods by HTS. The current results could expand the crucial information about bacterial contamination of baby foods.

Assessment of health risk, genotoxicity, and thiol compounds in Trigonella foenum-graecum (Fenugreek) under arsenic stress.

Arsenic (As) traces have been reported worldwide in vegetables and crops cultivated in As-polluted soils. Being carcinogenic, the presence of As in edibles is of great concern as it ultimately reaches humans and animals through the food chain. Besides, As toxicity adversely affects the growth, physiology, metabolism, and productivity of crops. In the present study, Trigonella foenum-graecum (Fenugreek) was exposed to the As stress (0, 50, 100, and 150μM sodium arsenate) for a week. Further, evaluation of As accumulation in roots and shoots, magnitude and visualization of oxyradicals, and thiol-based defence offered by Fenugreek was assessed. The root and leaf accumulated 258-453μgg-1 dry wt (DW) and 81.4-102.1μgg-1 DW of As, respectively. An arsenic-mediated decline in the growth index and increase in oxidative stress was noted. Arsenic stress modulated the content of thiol compounds; especially cysteine content increased from 0.36 to 0.43 µmole g-1 FW protein was noted. Random Amplified Polymorphic DNA (RAPD)-based analysis showed DNA damage in As-treated plants. Health risk assessment parameters showed that As concentration in the consumable plant shoot was below the critical hazard level (hazard quotient < 1). Moreover, T. foenum-graecum showed varied responses to As-induced oxidative stress with applied concentrations (150μM being more toxic than lower concentrations). In addition, the RAPD profile and level of thiol compounds were proved significant biomarkers to assess the As toxicity in plants. The conclusion of this study will help users of fenugreek to have a clue and create awareness regarding the consumption.

RAPD Profiles of Rhynchostylis gigantea (Lindl.) Ridl. Collected from Puspa Nirmala Orchids Banyumas, Central Java

Rhynchostylis gigantea (Lindl.) Ridl. is an orchid species spread over Southeast Asia countries. This species is very popular among ornamental plant collectors, especially due to its densely pack inflorescences. Hence, it is commercially found in many ornamental plant nursuries, such as Puspa Nirmala Orchids Banyumas, Central Java. Further development of the species should be supported by scientific data, particularly regarding the genetic variation. One of the molecular markers commonly used to study genetic variation is Random Amplified Polymorphic DNA (RAPD). This study aims to assess genetic variation of R. gigantea cultivars of Puspa Nirmala Orchids Banyumas collection  by RAPD profiles. Genomic DNAs were extracted from leaf samples of eight R. gigantea individuals, while RAPD markers were amplified using five random primers (OPA-15, OPK-16, OPP-15, OPP-08 and OPO-08). Descriptive analysis was employed on the data obtained. It was revealed that all of the primers resulted in a 100% monomorphism. This indicates an extremely low genetic variation among R. gigantea population of Puspa Nirmala Orchids collection, which is probably due to the same origin from a selected hybrid of the same crosses.

Genetic Diversity of Pathogenic Fungi Aspergillus flavus Isolates Using Random Amplified Polymorphic DNA-Polymerase Chain Reaction Technique

Aspergillus flavus is the second most public fungi that causing disease in human and causes diseases in several important agricultural crops. The aim of this study was to investigate the genetic variety among diverse isolates of Aspergillus flavus by the technique of polymerase chain reaction (PCR)-based random amplified polymorphic DNA (RAPD). Five isolates were obtained from Samarra hospital and outpatient clinic and were diagnosed morphologically and microscopically. Results of RAPD profile revealed a high genetic distance (0.244) between isolates number (1 and 5), while the isolates number (2 and 5) were joined with lower genetic distance (0.090). Unique and absent bands were displayed by certain isolates and can be taken as a positive marker for isolate identification. The dendrogram of banding patterns revealed a great relationship between isolates number (2 and 5) from one side and isolates number (1 and 3) from the other side, while the isolate number 4 was separated into particular subgroup which closer to the isolate number 1 and 3.

GENOTOXICOLOGICAL EVALUATION OF A BREWERY EFFLUENT IN BENIN CITY ON ALLIUM CEPA DNA USING THE RAPD TECHNIQUE

Industrial activities in breweries generate large quantities of wastewater, which if not properly treated, could pollute the receiving water bodies. In this study, the Random Amplified Polymorphic DNA (RAPD) technique was used to assess DNA integrity in Allium cepa L. roots exposed to varying concentrations [1, 5 and 10% (v/v)] of a ?treated? effluent from a brewery plant in Benin City. DNA polymorphism was discernable by changes in the RAPD profiles as variation in the intensity, disappearance of old and appearance of new bands compared to the control. A total of 89 bands were detected and 64 (71.9%) of these fragments were polymorphic. The loss and gain of bands increased with concentration of the effluent. Physicochemical analysis of the wastewater showed that BOD, nitrates, phosphates and conductivity were present at amounts higher than limits set by national (NESREA) and international (USEPA) for effluent discharge. Data obtained from the study imply that the current treatment given to the effluent in the brewery plant is insufficient to guarantee minimal risks on genotoxic effects in living cells associated with its discharge into the environment.

English

The application of bacteriophages biocontrol requires the formulation of genetically distinct bacteriophages in a phage cocktail. Random Amplified Polymorphic DNA (RAPD) - PCR is considered a cheap, reproducible, and readily applicable tool in detecting phage diversity compared to other molecular techniques such as whole-genome sequencing. We used in this study the RAPD-PCR technique to assess the genetic diversity of 28 bacteriophages infecting Pseudomonas aeruginosa and Staphylococcus aureus. According to their RAPD profiles, isolated phages were grouped into 2 main clusters which included phages from the same host. The typing by RAPD-PCR of newly isolated phages was useful to assess the genetic diversity bypassing previous whole-genome sequencing analysis. These genetically distinct phages lytic against P. aeruginosa and S. aureus could potentially be used in a phage cocktail for biocontrol against these clinically and industrially relevant bacteria. &nbsp; Key words: Phages, genetic diversity, RAPD PCR, P. aeruginosa, S. aureus.

Molecular characterization of Canna indica L. based on random amplified polymorphic DNA markers

Random amplified polymorphic DNA (RAPD) markers were employed for characterization, assessment of genetic variation and inferring relationships among six variants of Canna indica L. A total of 198 RAPD bands ranging from 200 bp to 3 kbp were generated by all the six variants. Among them, most of the bands were found to be polymorphic, four band were unique of which two bands (OPA022000 and OPA043000) were observed in the variant 2 (small red) and the other two (OPA013000 and OPA053000) were noticed in the variant 4 (orange), and the remaining bands were found to be monomorphic. The pair-wise genetic distance was determined among the six variants that ranged from 0.1446 to 0.6554. A dendogram was constructed based on the RAPD profiling to infer the relationship among the six variants of C. indica that resulted in two major clusters: the first one contained two variants, viz. variant 1 (local red) and variant 2 (small red), while the second cluster composed of the remaining four variants. The results as revealed from the RAPD analysis were found congruent with those of morphological and anatomical investigation of the species.&#x0D; Bangladesh J. Plant Taxon. 28(1): 75-81, 2021 (June)

Randomly amplified polymorphic DNA analysis of Staphylococcus chromogenes isolated from bovine and bubaline mastitis in Karnataka.

Background and Aim:In recent times, non-aureus staphylococci (NAS) have emerged as the major organisms isolated from mastitis cases in dairy animals, with a predominance of Staphylococcus epidermidis and Staphylococcus chromogenes. As compared to Staphylococcus aureus, much less is known about the molecular types or the spatiotemporal epidemiology of these NAS species. In the present study, randomly amplified polymorphic DNA (RAPD) was employed to detect genetic polymorphisms, intraspecies diversity, and epidemiology of S. chromogenes strains (n=37) isolated from bovine and bubaline mastitis cases in the state of Karnataka.Materials and Methods:Thirty-seven S. chromogenes isolates (14 from bovines and 23 from bubaline) isolated from subclinical mastitis cases, from organized and unorganized sectors, were subjected to RAPD typing. Further, methicillin resistance was determined by cefoxitin disk diffusion method.Results:The amplified DNA fragments ranged from 150 to 3000 base pairs and yielded several RAPD profiles. Further analysis using Digital Image Correlation Engine correlation coefficient and UPGMA method showed that the 37 isolates could be classified into 12 distinct RAPD types (A to L) at 62% similarity (D=0.889). Four of the most predominant RAPD types, B, A, C, and E, in that order, and together, represented 65% of the isolates. High diversity was observed among the isolates both within farms and between geographic locations. Most of the isolates exhibited methicillin resistance. This is the first such report from India.Conclusion:In the absence of defined multilocus sequence type protocols or sufficient sequences available in the public domain, RAPD can be employed to determine genetic diversity of S. chromogenes isolates.

Genetic diversity assessment in selected genetic resources of Gmelina arborea Roxb using RAPD markers

The genetic diversity and relationship among the fourteen accessions of Gmelina arborea were assessed using Random Amplified Polymorphic DNA (RAPD) markers. The RAPD profiles of all the accessions were compared and a total of 262 scorable bands were produced with fifteen primers, out of these 243 bands were polymorphic. The highest number of amplified products was observed in primers OPJ-19, OPM-02, OPN-02, OPN-05, OPN-16, OPN-18 and OPC-10 showed a lowest level of the polymorphic band. The similarity coefficient values were ranged from 0.58 to 0.74. The highest value similarity coefficient was found between the progenies of CPTs selected in Vazhachal and Kulathupuzha and the lowest values found between Vazhachal and Rosemala. Geographically closely related Gmelina accessions were also found to be related at the molecular level. A sizeable amount of inter-population diversity recorded, the Vazhachal and Rosemala accessions can be used as a parent in the Gmelina breeding program for improving productivity and wood quality.

Development of a RAPD protocol for typing strains of Phytophthora infestans in Mauritius

In Mauritius, substantial decrease in potato yields due to late blight are reported annually and it has become necessary to track the genetic identity of local Phytophthora infestans strains. The Random Amplified Polymorphic DNA (RAPD) technique is a low-cost and simple genetic characterisation tool that has been widely used for molecular fingerprinting but which requires extensive optimisation. The aim of this study was to carry out a series of experiments to optimise a RAPD protocol for routine P. infestans typing. Amplifications performed with DNA template concentration of 30-50 ng/μl, primer concentration of 2.0 − 3.0 μM and MgCl2 concentration of 3.0 mM gave the best RAPD profiles. These optimised conditions were used to carry out RAPD fingerprinting of 7 P. infestans isolates and codified data were used to construct a consensus dendogram, which grouped the 7 isolates into 3 distinct clusters, consistent with results obtained from other molecular characterisation techniques.

Microbial community diversity in the soil of Barrientos Island estimated by RAPD and Biolog Ecoplate methods

The diversity of soil microbial communities at Barrientos Island with  differents soil characteristics were evaluated using PCR-based method random amplified polymorphic DNA (RAPD) and community level physiological profiles (CLPP) of Biolog Ecoplate. The soils were selected from 17 different locations around Barrientos Island inhabited by different breeders. Shannon-Weaver index and multivariate analysis were performed to characterize variations of soil microbial communities. Both RAPD and CLPP methods exhibited that most soils with different type of rookery and characteristics could possibly affect the DNA sequence diversity and soil microbial diversity. The abandoned type of rookery had the highest Shannon-Weaver index as exhibited by soil sample 445 (3.4 for RAPD) and 450 (3.09 for CLPP). Higher coefficients of DNA sequence similarity were found in soil samples colonized by similar breeders, like soil 442 and 446 (both were active Chinstrap rookery) shared highest similarity in DNA sequences (73.53). The cluster analysis of RAPD profiles by UPGMA and principle component analysis (PCA) of Biolog Ecoplate exhibited similar influence of type of rookery and soil condition towards soil microbial community diversity. The results may suggest that the change in microbial community DNA composition is accompanied with the change in microbial functional properties.

Evaluation of a Potential Bacteriophage Cocktail for the Control of Shiga-Toxin Producing Escherichia coli in Food.

Shiga-toxin producing Escherichia coli (STEC) are important foodborne pathogens involved in gastrointestinal diseases. Furthermore, the recurrent use of antibiotics to treat different bacterial infections in animals has increased the spread of antibiotic-resistant bacteria, including E. coli, in foods of animal origin. The use of bacteriophages for the control of these microorganisms is therefore regarded as a valid alternative, especially considering the numerous advantages (high specificity, self-replicating, self-limiting, harmless to humans, animals, and plants). This study aimed to isolate bacteriophages active on STEC strains and to set up a suspension of viral particles that can be potentially used to control STEC food contamination. Thirty-one STEC of different serogroups (O26; O157; O111; O113; O145; O23, O76, O86, O91, O103, O104, O121, O128, and O139) were investigated for their antibiotic resistance profile and sensitivity to phage attack. Ten percent of strains exhibited a high multi-resistance profile, whereas ampicillin was the most effective antibiotic by inhibiting 65% of tested bacteria. On the other side, a total of 20 phages were isolated from feces, sewage, and bedding material of cattle. The viral particles proved not to carry genes codifying Shiga-toxins and intimin. No STEC was resistant to all phages, although some strains revealed weak sensitivity by forming turbid plaques. Three different bacteriophages (forming the “cocktail”) were selected considering their different RAPD (Random Amplification of Polymorphic DNA) profiles and the absence of virulence-encoding genes and antibiotic-resistance genes. The lytic ability against STEC strains was investigated at different multiplicity of infection (MOI = 0.1, 1, and 10). Significant differences (p < 0.05) among mean values of optical density were observed by comparing results of experiments at different MOI and controls. An effective reduction of bacterial population was obtained in 81% of cases, with top performance when the highest MOI was applied. The efficacy of the phage cocktail was tested on fresh cucumbers. Results showed a reduction in pathogenic E. coli by 1.97–2.01 log CFU/g at 25°C and by 1.16–2.01 log CFU/g at 4°C during 24 h, suggesting that the formulated cocktail could have the potential to be used in bio controlling STEC different serogroups.

Efficiency and reliability of random DNA markers (RDMs) for evaluation of genetic variability and relationship in Ocimum accessions

The genus Ocimum is characterized by a great variability in both morphological features and chemical composition because the cross-pollination appears leads to various species, subspecies, varieties and forms. In addition, the taxonomy of Ocimum is complicated by the existence of numerous botanical varieties, cultivar names, and chemotypes within the species that may not differ significantly in morphology. In the present study, genetic diversity and relationships within and among 24 Ocimum accessions from five different agro-climatic regions of West Bengal were developed using PCR-based Random Amplified polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers. The RAPD primers developed 191 robust loci, which revealed 69.42% polymorphism and 83 ISSR markers generated 67.46% polymorphism. Genetic diversity parameters [average and effective number of alleles, percent polymorphism, average heterozygosity, intralocus gene diversity, polymorphic information content (PIC)] for RAPD, ISSR, and RAPD+ISSR along with UPGMA clustering based on Jaccard's coefficient were estimated with a view to assess efficiency of the marker system in Ocimum. The UPGMA dendrogram generated using cumulative data revealed significant relationships among the genotypes. All 24 genotypes studied grouped into three clusters irrespective of their geographical affiliations with 100% bootstrap value except few genotypes, suggesting considerable diversity among the genotypes. Further the results demonstrate the efficiency and reliability of RAPD and ISSR markers in determining the genetic diversity and relationships among the Ocimum germplasm. RAPD and ISSR profiling have identified diverse Ocimum genotypes, which could be further utilized in various genetic improvement programmes including conventional as well as marker assisted breeding towards development of new and desirable Ocimum genotypes.

Growth, yield, genetic parameters and random amplified polymorphic DNA (RAPD) of five rice varieties treated with sodium azide and sown under different saline conditions

BackgroundRice is probably one of the most indispensable grain diets for the greater part of the world. It can be grown successfully under a wide range of climatic conditions; however, yields can be significantly diminished under moderate saline levels. Therefore, the employment of plant breeding techniques in enhancing plant survival and performance capacities under saline conditions becomes imperative. Mutation perhaps is one of the ultimate sources from which heritable variations could be selected from and hence are a useful tool to create raw materials for genetic improvement of rice. The effects of sodium azide (NaN3), as a chemical mutagen, on the growth, yield, genetic parameters, and RAPD profile of five rice varieties, FARO-44, FARO-52, FARO-57, NERICA L-34, and NERICA L-47, sown in soils maintained at a conductivity range of 4000 to 6000 μs/cm using sodium chloride solutions were investigated. The planting materials were treated with 0% NaN3 at pH 7 (control) and a concentration range of 0 to 0.032% NaN3 at pH 3 for 6 h.ResultsSodium azide treatment significantly reversed the negative effects associated with plant development under saline conditions. Consequently, NaN3 treatment significantly improved growth, yield, and genetic parameters of the rice varieties under high salinity compared with the controls. High heritability values were recorded for number of tillers per plant (78.06%), number of panicles per plant (78.74%), and 100-grain weight (98.15%) indicating the possibility of evolving higher yield variants through selection. Chlorophyll mutations observed were albino, viridis, and striata. The RAPD analyses with primers OPR 02 (5′–CAC AGC TGC C-3′), OPC 04 (5′-CCG CAT CTA C-3′), and OPC 05 (5′–GAT GAC CGC C-3′) revealed different DNA banding patterns between controls and NaN3-treated plants.ConclusionsThe study indicates that sodium azide is a potent mutagen, evident from information on genetic variability expressed by the DNA profiles of the rice varieties and their mutant counterparts as well as in the yield performances. The study thus indicated that the source of NaN3 induced variants among the treated progenies were genetic in nature and hence heritable. However, further screening on promising mutant plants from this research should be done through successive generations to identify stable traits and variant that can perform optimally under saline conditions.

DNA methylation and integrity in aged seeds and regenerated plants

AbstractSeed longevity is a complex process of key ecological and agronomic importance. DNA damage is a significant factor affecting seed ageing. Likewise, epigenetic changes can control gene expression and, therefore, seed response to ageing. The aim of the present work was to investigate the effect of ageing on nucleic acid stability and to identify reliable molecular markers that might help to monitor epigenetic changes within plant genetic resources during conservation. DNA profiles, evaluated by RAPD (random amplified polymorphic DNA), and methylation patterns, obtained by MSAP (methylation-sensitive amplification polymorphism), were compared in non-aged and agedMentha aquaticaseeds and plants produced by them. Germination decreased to 50% by storing seeds at 35°C and 12% wc for 28 days. RAPD profiles were 99% similar in these aged seeds compared to non-aged seeds. However, seedlings produced from the aged seeds showed a 13% dissimilarity compared to seedlings produced from the non-aged seeds. About 8% difference in the MSAP epigenetic profile was detected in seeds after storage and 16% difference was detected in the seedlings produced from them. This indicates that stress from high temperature and humidity during storage induced changes on the methylation state of seeds, and that changes were also detectable in the regenerated plants. Our results suggest that DNA integrity was compromised in seeds during ageing, and on seedlings produced by aged seeds. Genotype screening techniques such as RAPD and MSAP have the potential as markers of nucleic acid stability during seed ageing.

Microbial characterization of an artisanal production of Robiola di Roccaverano cheese

Robiola di Roccaverano, from the Piedmont region of Italy, is a Protected Designation of Origin soft cheese made with raw goat milk. The peculiarity of this cheese is that during the manufacturing process, a natural starter culture (NC) is added to raw milk. This study examined the viable microorganisms of technological interest, including lactic acid bacteria and fungal populations, in samples of raw milk, NC, and fresh and ripened cheese collected from one dairy using culture-dependent techniques. First, the isolated colonies were analyzed using random amplification of polymorphic DNA (RAPD) PCR, and strains with similar fingerprints were clustered together. Further, representative isolates of each group were subjected to 16S or 26S ribosomal DNA sequencing. Finally, species-specific PCR was conducted to distinguish the Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris. Among the studied lactic acid bacteria, 13 RAPD profiles were obtained, corresponding to 9 different bacterial species or subspecies. Concerning mold and yeast isolates, 5 species were found that coincided with 5 RAPD types. Observing the strains isolated in the study, Lc. lactis was the most prevalent species in raw milk and NC samples, and Leuconostoc mesenteroides was the predominant species identified in 5- and 15-d cheese isolates. Furthermore, whereas only these 2 species were detected in NC, Enterococcus and Lactobacillus genera were found in raw milk and cheese, respectively. Concerning the mold and yeast isolates, in NC Kluyveromyces spp. was mainly found, and in cheese samples the representative species were Geotrichum candidum and Yarrowia lipolytica. Finally, raw milk and cheese safety were evaluated, and the samples complied with the standard required by European Commission regulation number 2073/2005.