Random Amplified Polymorphic DNA Assay Research Articles

High Genetic Diversity of 16 Indian lettuce (Lactuca indica L.) Accessions from Vietnam.

<b>Background and Objective:</b> Plant genetic resources provide the raw material for crop improvement and plant breeding program largely depends on it. Therefore, the evaluation of plant genetic resources plays a critical role in crop improvement and also in conserving valuable genetic resources for the future. In this study, the genetic diversity of 16 <i>Lactuca indica</i> L. accessions collected in Vietnam was investigated by using ISSR and RAPD markers. <b>Materials and Methods:</b> Genetic diversity of 16 <i>Lactuca sativa</i> L. genotypes collected in Vietnam were evaluated using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) molecular markers. <b>Results:</b> In this study, 42 RAPD and ISSR primers were initially used, of which 12 and 9 primers, respectively were finally selected as they produced scorable patterns. RAPD markers produced a total of 113 loci, out of which 52 loci (45.96%) were polymorphic. The average percentage of the polymorphic band for RAPD primer is 45.96% and the genetic similarity based on simple matching coefficient ranged from 69.0-94.7%. ISSR analysis detected a total of 60 loci, out of which 22 loci (36.32%) were polymorphic and the genetic similarity ranged from 56.7-95.0%. In general, ISSR markers amplified fewer loci and showed lower variation in the percentage of polymorphism compares to the RAPD assay. <b>Conclusion:</b> These results indicate that the 16 collected Indian lettuce genotypes are genetically diverse. Because of these genetic diversities, the collected genotypes could be used for preserving or crossing programs to improve this precious medicinal plant in Vietnam.

The Efficiency of Long Primers Compared to The Short Primer for RAPD Technique in Date Palm

Random Amplified Polymorphic DNA (RAPD) applies single arbitrary short primers (8-12nucleotides) to produce many amplified discrete DNA. Limited reports and studies were done onthe use of long primers (over 12 bases). This study was performed to investigate the potential valueof long primers (15-21 bases) for generating RAPD polymorphisms. We compared both short andlong primers in RAPD assays of two date palm cultivars grown in Malaysia: Ajwa and Barhi. Thenumber of produced polymorphic fragments ranged in order from 2 and 38 bands for short andlong primers in Ajwa. Meanwhile, more polymorphic fragments were generated by long primersin Barhi, which were 50 and only five bands for short primers. 18-mer GY107 and 20-mer CO4primers yielded 100% polymorphism in Ajwa and Barhi, respectively. Moreover, long primersproduced more DNA fragments and a wider range of DNA fragment sizes (from 140-1600 bp,with respect to 300-1000 bp obtained with 10-mer primers). Hence, a significant correlation wasobserved between primer length and the number of polymorphic fragments within the long primergroup, suggesting that increasing primer length above 15 bases may demonstrate enhancedproduction of more polymorphism.

Investigation of the genetic variation of anise (Pimpinella anisum L.) using RAPD and ISSR markers

Anise (Pimpinella anisum L.) is an annual spice and medicinal plant belonging to the family Apiaceae. Only a limited number of anise breeding studies are available. Therefore, genetic variation among anise landraces has not been extensively examined with molecular markers. In this study, the molecular diversity of 15 Turkish anise landraces collected from different regions of Turkey and four foreign landraces obtained from Cyprus, Syria and France were analyzed using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. The discriminatory power of these markers was assessed by calculating various marker parameters, namely percent polymorphism, polymorphism information content (PIC), resolving power (RP), and marker index (MI). The genomic DNA of 19 anise landraces was amplified with nine RAPD primers that generated 71 polymorphic bands and five ISSRs primers that produced 45 polymorphic bands. The percentage of polymorphic loci was 59.8% in RAPD profiling and 51.5% in ISSR profiling. The average PIC values obtained using RAPD and ISSR markers were 0.40 and 0.38, respectively. The mean RP (2.90 and 2.67) and MI values (1.23 and 1.05) of the RAPD assay were found to be somewhat higher than those of the ISSR assay, but ISSR was more favorable than the RAPD techniques in terms of presenting the phylogenetic relationships in anise landraces. A UPGMA cluster grouped the 19 anise landraces into two major clusters based on both markers. The RAPD and ISSR marker systems were found to be useful for determining genetic diversity in P. anisum L. and identifying variation among landraces.

Genetic diversity of South African sweetpotato germplasm using molecular markers

ABSTRACTSweetpotato (Ipomoea batatas L.(LAM)) is one of the most important food crops in developing countries, including South Africa. Most sweetpotato varieties in South Africa are cream or white fleshed and lack the essential beta-carotene of the orange-fleshed varieties. To develop new varieties with high beta-carotene, knowledge of the genetic variation of the crop is needed. The objectives of this study were to assess the genetic relationships among 30 orange- and cream-fleshed sweetpotato accessions by (i) examining the variation in leaf proteins and (ii) using random amplified polymorphic DNA (RAPD). The polymorphism levels of the proteins and RAPD assays were 55.6% and 98%, respectively. The dendrograms generated from all the analyses clustered the accessions according to their flesh color and country of origin. Analysis of molecular variance (AMOVA) found a significant difference between the South African and non-South African germplasm. The high genetic diversity in the South African sweetpotato germplasm is a positive indicator for a breeding program that has a number of targets, such as breeding for nutritional improvement, disease resistance and drought tolerance.

Evaluation of genotoxicity of the aqueous extracts from wild Artemisia herba-alba and Jasmina montana against Zucchini yellow mosaic virus (ZYMV) in Cucurbita pepo plants

Squash or cucurbit (Cucurbita pepo) is one of the famous and important vegetable plants in most world countries. The squash is infected by the critical Zucchini yellow mosaic virus (ZYMV) in Egypt. Though pesticides can protect the plants of pest infections, there are no effective compounds that can be applied as virucides. In this work, the effects of aqueous extracts from Jasmina montana and Artemisia herba-alba on ZYMV infection in the squash plants were examined. In addition, SDS-PAGE protein patterns and enzyme activities were evaluated and induction of resistance by plant elicitors against ZYMV was studied The results observed that using of the aqueous extracts from A. herba-alba or J. montana extracts prior to ZYMV inoculation recorded 100% inhibition of virus infection. Also, complete inhibition was obtained by treatment of A. herba-alba extract with the virus inoculum led to destroy the virus particles due to presence of the effective compounds (terpens, phenolics and the essential oils) in the plant extract. SDS-PAGE protein profiles and enzyme activities were studied in treated and untreated plants. Genomic DNA variation was studied using random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) loci. The alterations in RAPD and ISSR assays of representative squash plants can be applied to comprehend of induced systemic resistance. These inducers stimulated resistance in the squash plants. Therefore, elicitors should be taken into consideration in the breeding programs for ZYMV control.

Identification and characteristics of Testudinis Carapax et Plustrum based on fingerprint profiles of mitochondrial DNA constructed by species-specific PCR and random amplified polymorphic DNA

Traditional use of Testudinis Carapax et Plustrum (TCP) as a medicine and health food has been widely reported. We compared two DNA fingerprint profiles of mitochondrial (mtDNA) from TCP based on species-specific PCR and random amplified polymorphic DNA (RAPD) to identify their authority. A series of sequences from cytochrome b (Cyt b) of Chinemys reevesii and their counterfeits were downloaded from the Genbank, and Premier 5.0 software was used to design a set of primers. A species-specific PCR and RAPD were undertaken to obtain the different DNA fingerprints respectively. The mtDNA was successfully extracted from all samples using the modified salting-out method. A relative molecular mass of 16.6 × 103 bp was observed, and mtDNA was measured between 1.83 ± 0.02. Fragments of 78 bp were amplified from all the TCP samples tested (except adulterant animals) using species-specific PCR method. The RAPD showed different electrocardiogram between genuine and counterfeit tortoise shell goods along with stripe number and location. The salting-out method (as modified) was used to extract high-quality mtDNA from TCP. The species-specific PCR and RAPD assay proposed in this study could be used for quality control of TCP with more specificity, sensitivity, and applicability.

Genotoxicity evaluation of zinc oxide nanoparticles in Swiss mice after oral administration using chromosomal aberration, micronuclei, semen analysis, and RAPD profile.

The expanded uses of zinc oxide nanoparticles (ZnO NPs) have grown rapidly in the field of nanotechnology. Thus, rising production of nanoparticles (NPs) increases the possible risks to the environment and occupationally exposed humans. Hence, it is indispensable to appraise the safety toxicity including genotoxicity for these NPs. In the present study, we have evaluated the genotoxic effect of ZnO NPs after oral administration to Swiss mice at dose levels of 300 and 2000 mg/kg body weight. These doses were administered for 2 days at 24 h apart. Chromosomal aberration (CA) and micronucleus tests were conducted following Organization for Economic Co-operation and Development guidelines. DNA damage was evaluated at 0, 24, 48, and 72 h posttreatment using a randomly amplified polymorphic DNA (RAPD) assay; additionally, semen analyses were also performed at 34.5 days post oral exposure. The reactive oxygen species (ROS), 8-oxo-2'-deoxyguanosine and CAs were increased ( p < 0.05) at the highest dosage (2000 mg/kg) of ZnO NPs compared to controls. Aberrant sperm morphology with reduced sperm count and motility were also present ( p < 0.05) in the high-dose group. Based on the RAPD assay, the genomic template stability within the high-dose group (<90%) was less than the controls (100%). The results suggested that ZnO NPs are mildly genotoxic in a dose-related manner and this toxicity were induced by generation of ROS.

Characterization of Ornithobacterium rhinotracheale field isolates from Hungary

ABSTRACTOrnithobacterium rhinotracheale is a widely distributed rod-shaped Gram-negative bacterium that infects several avian species including chickens and turkeys. It is associated with respiratory signs, growth retardation, mortality, and reduced egg production, thus causing severe economic losses to the poultry industries. In this study, 37 field isolates of O. rhinotracheale, collected from various locations in Hungary between 1997 and 2015, were identified and characterized by the analysis of partial 16S rRNA gene sequences, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and random amplified polymorphic DNA (RAPD) PCR assays with the OPG11, OPH19, and M13 primers. Most of the field isolates were serotype A, one was serotype B, and four were serotype D. One isolate could not be typed with antisera against serotypes A–E. In a phylogenetic analysis of the 16S rRNA sequences, the isolates formed two clusters. Thirteen distinct patterns were identified with ERIC-PCR, and the RAPD assay with the M13 primer assigned the isolates to 10 different patterns. The other two RAPD assays were unsuitable for distinguishing and grouping the isolates. Neither ERIC type nor RAPD pattern correlated with the place or year of isolation. However, the strains isolated from chickens were more heterogeneous on ERIC-PCR than the isolates recovered from turkeys. In this study, ERIC-PCR was the most discriminatory method for investigating the genetic diversity of O. rhinotracheale isolates.

Genotoxicity Evaluation of an Urban River on Freshwater Planarian by RAPD Assay.

The aim of this study was to evaluate the genotoxic potential of an urban river - the Wei River in Xinxiang, China using randomly amplified polymorphic DNA (RAPD) assay in planarians. The results showed that the total number of polymorphic bands and varied bands in RAPD patterns of treated planarians decreased with the water sample site far away from the sewage outlet of a factory. In addition, the genome template stability of treated groups decreased and the degree of the decline was negatively related to the distance between the sample site and the sewage outlet, suggesting that the Wei River water had genotoxicity effects on planarians and strengthening the management of the Wei River was necessary. Furthermore, this work also indicated that RAPD assay in planarians was a very promising test for environmental monitoring studies.

Genotoxicity evaluation of ionic liquid 1-octyl-3-methylimidazolium bromide in freshwater planarian Dugesia japonica using RAPD assay

The randomly amplified polymorphic DNA (RAPD) assay has been used to detect DNA alternation and mutation recently. However, the effectiveness of this method in detecting DNA damage in planarians, a model organism for assessing the toxicity of environmental pollutants is unknown. In the present study, RAPD assay was used to detect the DNA damage in planarians treated by the ionic liquid 1-octyl-3-methylimidazolium bromide ([C8mim]Br) for the first time. Among the 20 test RAPD primers, 13 primers with 60–70% GC content produced unique polymorphic band profiles. A total of 60 bands were observed in the untreated control planarians. In comparison with the control group, the [C8mim]Br-treated groups displayed differences in RAPD patterns in the band intensity, disappearance of normal bands and appearance of new bands. The variation of RAPD profiles showed both concentration- and time-effect relationships. Meanwhile, the genomic template stability (GTS) of treated planarians decreased and exhibited negative correlation to the exposure concentration and time of [C8mim]Br. Our results suggested that [C8mim]Br had genotoxic effects on planarians, and this DNA damage analysis would lay the foundation for further elucidating the toxicity mechanisms of ionic liquids on planarians. Furthermore, RAPD analysis was proved to be a highly sensitive method for the detection of DNA damage induced by environmental pollutants like toxic chemicals on planarians.

Efficient management and maintenance of ultrasonic nebulizers to prevent microbial contamination

To seek the cause of Burkholderia cepacia complex (Bcc) infection outbreak and evaluate the efficacy of new methods for nebulizer maintenance. We investigated the annual number of Bcc isolates recovered from clinical samples in our hospital between 1999 and 2013. Swab samples were randomly collected for bacterial culture before patient use from 10 each of the two machine types in August 2001; these included 20 samples from each of the following: Drain tubes, operating water chambers, oscillators, and nebulizing chambers. In addition, 10 samples each of nebulizer solutions before and after use were cultured. For environmental investigation, 10 samples were collected from sinks in the nurse stations of the wards where patients positive for Bcc were hospitalized. Numbers of Bcc isolates were compared before and after introduction of new methods for nebulizer maintenance in October 2001. In addition, randomly amplified polymorphic DNA (RAPD) assay was applied to find the genetic divergence of the Bcc isolates obtained from clinical samples and nebulizers. From January 1999 to December 2013, a total of 487 Bcc isolates were obtained from clinical specimens from 181 patients. Notably, 322 (66.1%) Bcc isolates were obtained from clinical specimens from 1999 to 2001, including 244 (115 patients) from sputum and 34 (11 patients) from blood. During this period, 14 isolates were obtained from nebulizer components. Among these, six were derived from nebulizer drain tubes, five from operating water chambers, and one from the oscillator before patient use, and two from nebulizer solutions after patient use. When Bcc was isolated from the nebulizer solution after patient use, Bcc was simultaneously detected in other parts of the nebulizer. Bcc was not isolated from any nebulizer solution before use. RAPD assays revealed similar DNA profiles in isolates obtained from patients and nebulizers. Investigation revealed damaged diaphragms in many nebulizers. The new maintenance methods for nebulizers, including restriction of the usage period, thorough disinfection, and routine check for diaphragm breakage, remarkably reduced Bcc isolation (165 isolates from patients in 12 years and 0 isolate from nebulizers in periodical sampling). In particular, Bcc has been isolated from blood from only one patient since the new methods were introduced. Appropriate maintenance of ultrasonic nebulizers is crucial for preventing Bcc contamination of nebulizers and subsequent respiratory tract and blood infections.

How and why to monitor Pseudomonas aeruginosa infections in the long term at a cystic fibrosis centre

Pseudomonas aeruginosa is a major cystic fibrosis (CF) pathogen causing chronic respiratory infections and posing a risk for cross-infection between patients with CF. To propose an algorithm for long-term surveillance of P. aeruginosa and assess its suitability for monitoring the epidemiological situation at a CF centre with approximately 300 patients. Over a nine-year period, over 300 P. aeruginosa isolates from 131 infected patients were tested by multi-locus sequence typing (MLST) and/or random amplified polymorphic DNA (RAPD) assay. MLST analysis led to the identification of 97 different sequence types which were distributed among 17 RAPD-generated (pseudo)clusters. This indicates that the easy-to-perform RAPD assay is only suitable for intra-individual, not interindividual, strain analyses. No epidemic strains were observed. Longitudinal analysis revealed that 110 of the 131 patients were infected with the same strain over the observation period, whereas 21 patients had a strain replacement or a new infection. Chronic infection was found in 99 of the 131 patients, and the remaining 32 patients met the criteria for intermittent infection (as defined by the Leeds criteria). Eighteen of the 32 patients (56%) with intermittent infection were infected with the same strain for up to nine years. The strain type only changed in 16% of 131 patients with chronic or intermittent infection. As many as 56% of patients considered to have intermittent infection were actually chronically infected with the same strain for many years.

Effects of permissible maximum-contamination levels of VOC mixture in water on total DNA, antioxidant gene expression, and sequences of ribosomal DNA of Drosophila melanogaster.

In this study, we aimed to investigate the mutagenic and carcinogenic potential of a volatile organic compound (VOC) mixture with references to the response of D.melanogaster using selected antioxidant gene expressions, RAPD assay and base-pair change of ribosomal 18S, and the internal transcribed spacer, ITS2 rDNA gene sequences. For this purpose, Drosophila melanogaster Oregon R, reared under controlled conditions on artificial diets, were treated with the mixture of thirteen VOCs, which are commonly found in water in concentrations of 10, 20, 50, and 75 ppb for 1 and 5 days. In the random amplified polymorphic DNA (RAPD) assay, band changes were clearly detected, especially at the 50 and 75 ppb exposure levels, for both treatment periods, and the band profiles exhibited clear differences between the treated and untreated flies with changes in band intensity and the loss/appearance of bands. Quantitative real-time PCR (qRT-PCR) analysis of Mn-superoxide dismutase (Mn-SOD), catalase (CAT) and glutathione-synthetase (GS) expressions demonstrated that these markers responded significantly to VOC-induced oxidative stress. Whilst CAT gene expressions increased linearly with increasing concentrations of VOCs and treatment times, the 50- and 75-ppb treatments caused decreases in GS expressions compared to the control at 5 days. Treatment with VOCs at both exposure times, especially in high doses, caused gene mutation of the 18S and the ITS2 ribosomal DNA. According to this research, we thought that the permissible maximum-contamination level of VOCs can cause genotoxic effect especially when mixed.

The determination of physiological and DNA changes in seedlings of maize (Zea mays L.) seeds exposed to the waters of the Gediz River and copper heavy metal stress.

In this study, the effects of the heavy metal-polluted waters of the Gediz River, which flow into the Aegean Sea, and different concentrations of copper (Cu) solutions on maize (Zea mays L.) seedlings are investigated with physiological parameters and random amplified polymorphic DNA (RAPD) assay. Results displayed physiologically a significant difference in root and stem length between the control seedlings and the seedlings grown with the waters of the Gediz River. Also, the certain ascending concentrations of copper solution (80, 160, 320, 640, and 1280ppm) caused a significant decrease in root and stem length of seedlings compared to the control seedlings. As a result of the waters of the Gediz River and copper solution treatment, the changes occurred in RAPD profiles of seedlings observed as variations like increment and/or loss of bands compared with the control seedlings. And these changes were reflected as a decrease in genomic template stability (GTS, changes in RAPD profile) derived by genotoxicity. RAPD band profiles and GTS values showed consistent results with physiological parameter. In conclusion, the study revealed the environmental risk and negative effect of waters of the Gediz River on maize seedlings and the suitability of RAPD assay for the detection of environmental toxicology.

Determination of genotoxic effects of fipronil in Vicia faba using random amplified polymorphic DNA analysis.

This study was undertaken to appreciate genotoxic potential of fipronil herbicides in the roots of Vicia faba seedlings. Fipronil was clearly dose dependent on root growth inhibitors, total soluble protein, and random amplified polymorphic DNA (RAPD) assays. Increase in concentration of fipronil resulted in decreased amount of root length and increased level of protein. For the RAPD analyses, 15 RAPD primers were found to produce unique polymorphic band patterns and were subsequently used to produce a total of 110 bands of 45-1250 bp. Each primer generated 4-11 RAPD bands across 15 primers. The changes occurring in RAPD profiles following fipronil treatment included variation becoming evident as disappearance and/or appearance of DNA bands compared with the normal seedlings. These results indicated that genomic template stability was significantly affected at the above fipronil concentration. This study further confirmed that the RAPD assays are useful in determining potential genotoxicity of fipronil.

Evaluation of arsenic trioxide genotoxicity in wheat seedlings using oxidative system and RAPD assays.

Arsenic is a metalloid that is toxic to living organisms. It is known that high concentration of arsenic causes toxic damage to cells and tissues of plants. While the toxic effect of arsenic is known, limited efforts have been made to study its genotoxic effect on the crops. In the present study, effects of arsenic trioxide (As2O3) on seed germination, root length, reactive oxygen species (ROS), lipid peroxidation (malondialdehyde (MDA)), and activities of antioxidant enzymes, as well as DNA in wheat seedlings were investigated. Seedlings were exposed to different (10 to 40 mg/L) As2O3 concentrations for 7 days. Seed germination and root elongation decreased with increase of As2O3 concentration. The values of hydrogen peroxide (H2O2), superoxide anion (O2 (·-)), and MDA contents significantly increased by As2O3 concentrations. The highest values for H2O2, O2 (·-), and MDA were obtained in 40 mg/L treated wheat seedling. A significant increase of peroxidase (POX) and catalase (CAT) activity in seedlings were observed with increased concentration of As2O3, then decreased when reaching a value of 40 mg/L, whereas the activities of superoxide dismutase (SOD) were gradually enhanced with increasing As2O3 concentration. Alterations of DNA in wheat seedlings were detected using randomly amplified polymorphic DNA (RAPD) technique. The changes occurring in RAPD profiles of seedlings following As2O3 treatment included loss of normal bands and appearance of new bands in comparison to that of control seedlings. The results of our study showed that As2O3 induced DNA damage in a dose-dependent meaner, and the root cells of wheat studied showed a defense against As2O3-induced oxidative stress by enhancing their antioxidant activities.

Estimation of Genotoxic Effects from GSM Base Stations on Four Successive Generations (F1 F4) of Albino Mice, Mus Musculus Using RAPD Bands and Micronucleus Assay

The levels of radiofrequency radiations from the telecommunication base station and its potential genotoxic effects on DNA content and micronucleus induction in four generations of albino mice, Mus musculus were carried out. The level of radiofrequency radiations around the GSM base station ranged between 1.3 - 2.0⇥ 10 3 µW/m 2 respectively. The genotoxic effects of radiofrequency radiation on four mice generations were based on random amplified polymorphic DNA (RAPD) and gel electrophoresis, results showed gradual depreciation in DNA band intensity of exposed mice across generations when compared to control groups. The micronucleus assay using acridine orange stain showed increased in micronucleus count and cytotoxic effects on exposed mice being significantly (PCE/NCE P<0.05) higher when compared to the control . Statistical analysis also revealed that significant genotoxic effects were induced (p<0.05) in exposed mice compared with control over the four generations,the results indicated that the RAPD and micronucleus assay could be a suitable bio-molecular marker for the detection of potential long-term genotoxic effects of radiofrequency radiations on exposed mammalian organisms.

Assessment of arsenic-induced DNA damage in goldfish by a polymerase chain reaction-based technique using random amplified polymorphic DNA markers.

Arsenic is a groundwater contaminant of global concern. It is a potent human carcinogen, and its marked genotoxic effects have been reported in several human and animal studies. The present work investigates the applicability of the random amplified polymorphic DNA (RAPD) assay to study the DNA-damaging effects of arsenic at low-level exposure in goldfish Carassius auratus. Four experimental groups of fish, A, B, C and D, were exposed to 0, 10, 50, and 1,000 µg L(-1) of arsenic, respectively, in aquaria water for 15 consecutive days. Genomic DNA extraction was followed by RAPD-polymerase chain reaction amplification for each fish separately. One arbitrary decamer primer (PUZ-19) of 33 primers used appeared as the most informative and was capable of exhibiting marked alterations in RAPD profiles between arsenic-exposed and unexposed (control) samples. Different sets of 11 loci were amplified in various experimental groups with four clear polymorphic bands by the primer PUZ-19. The X and XIII amplification loci, which were prominent in the unexposed group, failed to appear in the arsenic-exposed groups. In contrast, the I and XI RAPD bands appeared as new amplification loci in all of the exposed groups. Such alterations in genomic DNA, however, did not exhibit a clear dose-dependent tendency. The RAPD assay, because of its efficacy to unmask alterations in genomic DNA induced by arsenic at low exposure level of 10 µg L(-1), appears to be a sensitive and potential tool for detecting arsenic genotoxicity.

Appraisal of RAPD and ISSR markers for genetic diversity analysis among cowpea (Vigna unguiculata L.) genotypes

The genetic variability and relationships among 11 cowpea genotypes representing two cultivars and nine elite genotypes were analyzed using 22 random amplified polymorphic DNA (RAPD) and nine inter-simple sequence repeat (ISSR) markers. ISSR markers were more efficient than RAPD assay with regards to polymorphism detection. But the average numbers of polymorphic loci per primer and resolution power were found to be higher for RAPD than for ISSR. Also, the total number of genotype specific marker loci, Nei’s genetic diversity, Shannon’s information index, total heterozygosity, and average heterozygosity were prominent in RAPD as compared to ISSR markers. The regression test between the two Nei’s genetic diversity indices showed low regression (0.3733) between ISSR and RAPD + ISSR-based similarities but maximum (0.9823) for RAPD and RAPD + ISSR-based similarities. The RAPD- and ISSR-generated cultivar- or genotype-specific unique DNA fingerprints able to identify the most diverse genotypes. A dendrogram constructed based on RAPD and ISSR combined data indicated a very clear pattern of clustering according to the groups (cultivars and elite genotypes). The results of principal coordinate analysis were comparable to the cluster analysis. Cluster analysis showed that most diverse genotypes (GP-125 — small size with good seed quality; GP-129, GP-90L — big size with poor seed quality) were separated from moderately diverse cultivars and genotypes. The genetic closeness among GP-129 and GP-90L, JCPL-42, and JCPL-107 could be explained by the high degree of commonness in these genotypes.

Role of antioxidant additives in the protection of DNA integrity of buck spermatozoa with RAPD assay

The role of antioxidants (reduced glutathione, ascorbic acid and α-tocopherol) on DNA integrity with the random amplified polymorphic DNA (RAPD) was studied. This assay relies on presence or absence of amplification product on ethidium bromide stained agarose gels. The size of amplification products ranged from 190 to 1,900 base pair. Antioxidants effectively protect sperm DNA from damage. Moreover, random amplified polymorphic DNA technique can be used for the studies of the molecular seminal characteristics of buck semen.