ISSR Technique Research Articles

Somaclonal Variation in Callus Cultures of Rose Periwinkle, <i>Catharanthus Roseus L</i>. Under Induced Salt and Osmotic Stresses

The experiment was carried out at the laboratories of the National Commission for Biotechnology in Syria during the period 2020-2022 to detect the somaclonal genetic variation occurring in callus cultures at different ages and to compare them with the <em>In vitro</em> growing plants<em> </em>and<em> </em>introduced species of Rose Periwinkle that grew up in the Syrian environment, in addition, exploring the effect of abiotic stresses in causing genetic variations in callus, by the Inter Simple Sequence Repeats technique (ISSR) using 21 primers. Seeds were germinated on an MS nutrient medium devoid of growth regulators; explants were transferred to a propagation medium fortified with Naphthalene Acetic Acid (NAA) (1 mg L<sup>-1</sup>) and 6-Benzyladenine (BA) (2 mg L<sup>-1</sup>). Callus was developed from Rose periwinkle <em>Catharanthus roseus L</em>. leaves using 5C01 medium fortified with NAA and Kinetin (Kin). After that, callus was exposed to gradually increasing levels of PEG-6000-induced osmotic stress and salt stress (NaCl) with a higher level every 30 days. The molecular study results proved the effectiveness of all the used primers (21 primers) in presenting polymorphism between the studied samples at a rate of 92.16%. The cluster analysis showed separating the analyzed samples into two main clusters. The first cluster contained the osmotic stressed callus, while the second cluster separated into two sub-clusters that had the rest of the studied samples. It was found that there were genetic variations between <em>In vitro</em> plants and the callus. In addition, the stressed callus (salt and osmotic) was compared to the unstressed callus, and this confirmed that callus cultures were an essential source of genetic variations, and the ISSR technique was an effective tool for detecting these variations.

Effects of Grafting on Genomic Stability in Salinity Stress Conditions in Cucumber (Cucumis sativus L.)

Cucumber is an important type of vegetable that is grown in high quantities. Abiotic stress factors can cause significant morphological, physiological and molecular changes in cucumber. Grafting on strong rootstocks can reduce this negative effect. This study was carried out to evaluate the genotoxic potential of salinity stress in cucumber and to determine the effect of grafting on genotoxicity. Non-grafted and grafted plants were treated with 100 mM NaCl and the ISSR profiles were compared after 15 days. Using 14 ISSR primers, 51.9% polymorphism was detected between all groups. While salinity stress decreased the GTS rate to 47.2%, it was determined that this rate could be increased (%72.4-79.5) with grafting. GTS and similarity indices gave similar results and it was concluded that the ISSR technique could be effective in determining genotoxicity. There were also differences in measurements between rootstocks. With this study, it was concluded that salinity stress may cause genomic template stability changes in cucumber, this parameter can be protected by grafting depending on the rootstock variety used, and the ISSR technique can be used to determine genotoxicity. This study provides a basis for investigating the molecular mechanisms underlying grafting in the cucumber response to salinity stress.

Mutagenic effect of acridine orange on the production of EPS in Bacillus sp., its applications as anticoagulant and genetic diversity by ISSR and SCoT technique

Mutagenic effect of acridine orange on the production of EPS in Bacillus sp., its applications as anticoagulant and genetic diversity by ISSR and SCoT technique

Mutagenic effect of acridine orange on the production of EPS in Bacillus sp., its applications as anticoagulant and genetic diversity by ISSR and SCoT technique

Mutagenic effect of acridine orange on the production of EPS in Bacillus sp., its applications as anticoagulant and genetic diversity by ISSR and SCoT technique

Evaluation of the Current State of Preservation of Vaccinio uliginosi-Pinetum Kleist 1929 in Eastern Poland

The study assessed the genetic variability and the possibility of Scots pine regeneration in marshy forest. The genetic parameters were determined using the ISSR technique. The relationships between herbaceous plants, pine regeneration density, and their genetic variability were determined. On average, per 1 m2, three regenerated pine seedlings with a mean height of 27.56 cm were inventoried. Based on genetic analysis, it was found that the proportion of polymorphic loci was 60.46%. The average number of alleles at the locus was 1.345, and the effective number of alleles at the locus was 1.345. The values of the expected heterozygosity and Shannon index were 0.200 and 0.301, respectively. No species competing with pine regeneration were found. A significantly negative correlation of the number of pine regenerations with the area covered with an herbaceous plant layer and tree canopy closure was found. There was a relation to the insufficient amount of light under the stand canopy. In conclusion, the condition of marshy forests was satisfactory and the genetic variability of pine seedlings was moderate. The vegetation was typical for this habitat, but the significant presence of dry habitat species could indicate the beginning of habitat drainage. It seemed that the amount of light under the stand canopy was insufficient. Nevertheless, more light probably reached the inside of the stand in the terminal stage, as a result of upper layer tree separation, which in turn may facilitate the effective regeneration of Scots pine in this habitat.

Population genetic diversity of Huperzia chinensis (Herter ex Nessel) Ching. in Northwest region of Vietnam revealed by ISSR and SCcoT markers

Genetic diversity of natural Huperzia chinensis population in Northwest region of Vietnam was analyzed based on the DNA fingerprint data induced by ISSR and SCoT methods separately and combined data from both methods. The genetic diversity parameters and genetic distances among individuals based on SCoT technique were higher than ISSR technique [expected heterozygousity (HeS) was 0.2812; Shannon index (IS) was 0.4153; Percentage of polymorphic bands (PPBS) was 75.44%; Genetic similarity coefficients (GSCsS ) were in range of 0.561 - 0.947 with the average value (AGSCS) of 0.758 revealed by SCoT technique compared to HeI was 0.2394; II was 0.3515; PPBI was 60.87%, GSCsI were 0.551 - 0.986 and AGSCI was 0.860 revealed by ISSR technique. Combined data showed that genetic diversity level of investigated population (He was 0.2583; I was 0.3804; PPB was 67.46%, higher than genetic diversity level of Huperzia serrata population in the same distribution area which was previously evaluated by the same markers. Combined data also indicated the genetic similarities among individuals (GSCs) were 0.579 - 0.960 with the average (AGSC) of 0.814. ISSR method reflected more loci in the investigated plants than SCoT method but SCoT method could differentiate the tested samples better and reflects genetic diversity at higher level than ISSR method.

Interaction between Growth Regulators Controls In Vitro Shoot Multiplication in Paulownia and Selection of NaCl-Tolerant Variants.

The interaction between cytokinin, auxin and GA controlled in vitro shoot multiplication in paulownia was influenced by a medium water potential (Ψ) modulation, where it was modulated using different textures or strengths of MS medium, media of different types (MS, WPM, SH and B5) or NaCl incorporation. The interaction between 2 mg/L BAP and 0.1 mg/L NAA expressed the highest shoot number on each media type, but it was better with media of lower water potential (MS and WPM), and MS medium was the best. Ψ of full-strength semisolid MS medium expressed the highest shoot multiplication. The opposite was detected when Ψ of MS medium was changed using half- or double-strength MS. Ψ of full-strength MS medium in semisolid form resulted in a valuable interaction between 2 mg/L BAP, 0.1 mg/L NAA and 0.1 mg/L GA, leading to efficient shoot formation, and it was associated with an increase in internode length and decrease in stem diameter, which facilitated obtaining synseeds with a high ability to convert. High genetic variation was recorded under long-term culture (14 subcultures). Polymorphism using the ISSR technique was higher than that of RAPD. A further increase in polymorphism was detected when NaCl was used, where five salt-tolerant lines were selected. Some salt-tolerant-selected lines showed one or more amplification products of a specific molecular weight that did not appear in the control. For example, with OPA-07 and OPG-02 RAPD primers, all the salt-tolerant-selected lines showed the appearance of amplification fragments (610 bp and 300 bp, respectively) that were not detected in control.

ASSESSMENT OF GENETIC VARIATION AMONG PISTACIA ATLANTICA DESF. REGARDING SEXUAL GENOTYPES (MALE, FEMALE AND HERMAPHRODITIC) USING ISSRS. MARKERS

All Pistacia species are dioecious, male and female flowers are born on separated trees. Our recent studies identified new hermaphroditic genotypes of P. atlantica with different structure of racemes and flowers at the south of Syria. Therefore, the current research aimed to assess genetic variation among 11 genotypes (3 female, 5 hermaphroditic, 3 male) across fifteen ISSRs primers in Sweida Research Center (2018-2019). All of the primers were able to detect the polymorphism, which revealed 214 bands, 205 of them were polymorphic (95.79%). The number of bands for each primer ranged from 6 to 33, with an average 14.27 bands for each Primer. Genetic similarity among all studied genotypes ranged from (0.27) between hermaphroditic genotype (PA52) with female genotype (FA3) as well as between MA3 male genotype and FA2 female genotype, while the highest genetic similarity was 0.77 between two hermaphroditic genotypes (PA37and PA52). Cluster analysis grouped all studied genotypes into three main clusters according to their sexual structure; the first cluster contained all of the hermaphroditic genotypes and the second cluster comprised of all male genotypes, while the third cluster included all female genotypes. The results demonstrated the importance and the efficiency of ISSR technique by revealing the genetic variation among P. atlantica genotypes and separating all of them into detached clusters according to their sexual structure. Farther more, some primers were able to detect common bands in each sexual structure which might help to understand the mechanism of sexual inheritance within the studied species.

Analysis of intraspecific polymorphism ofNitraria sibiricaPall. using the ISSR technique

The analysis of DNA polymorphism ofNitraria sibirica Pall. was carried out at 13 natural populations of the Republic of Altai and Altai Territory using the ISSR technique. Seven effective ISSR primers have been identified to analyze DNA polymorphism inN. sibirica. 99 DNA fragments were yielded at DNA amplification with these primers, 66 of them were polymorphic. The genetic distance Nei (D) between the studied populations ofN. sibiricaaveraged 0.32, at mean 0.09 - within populations. An identification ISSR marker has been revealed, which can be used to study the genetic variability of the genusNitraria L. (Nitrariaceae)species.

Study of the genetic diversity of tomato (Solanum spp.) with ISSR markers

ABSTRACT In this study, 55 tomato genotypes from different geographical regions were evaluated with ISSR markers (Inter Simple Sequence Repeats). The seven ISSR primers originated 63 amplified bands, of which 90.48% were polymorphic. The cluster analysis based on the Nei-Li similarity coefficient using the average genetic clustering method (UPGMA) revealed the conformation of five clusters at a level of similarity of 72%. The ISSR technique did not discriminate tomato genotypes according to the species or region of provenance. The STRUCTURE analysis and the dendrogram did not reveal a genetic structure in the population evaluated. The genotypes of the species of S. pimpinellifolium, S. l. cerasiforme, S. lycopersicum and S. peruvianum were found consistently grouped, showing a close genetic relationship among them. A high genetic variation among the individuals within each of the groups formed was suggested by the AMOVA. The ISSR markers were effective in assessing the genetic diversity and structure of populations of tomato genotypes. The high genetic variability found in this study indicates the valuable genetic potential present in tomato germplasm, especially of wild species, which could be used for future breeding programs of the species.

Infraspecific genetic variation and population structure of Salvia nemorosa L. (Lamiaceae) in Iran

Salvia nemorosa is widely distributed in different parts of Iran, while, there is no information about its population genetic structure and genetic diversity. The current information of its potential protection or conservation status in Iran is almost absent and unclear. Our investigation was the first molecular study of this medicinal species. We performed analysis of genetic variability and population structure of 11 populations of S. nemorosa in Iran using ISSR technique. We revealed intra and inter-population genetic diversity in the studied populations. Genetic parameters widely varied among the studied populations and confirmed their high genetic diversity. Moreover, AMOVA test showed significant molecular variation among and within the populations. The arrangement of populations and their individuals in NJ tree, PCA and MDS plots was in agreement with AMOVA results and individuals of five groups were overlapped. The Nm value showed low amount of gene flow among the populations. Based on STRUCTURE analysis and UPGMA tree of genetic distance, six genetic groups were identified among the studied populations, while two populations had significant differences and could be definite as ecotypes.

GENETIC DIVERGENCE PATTERNS OF TWO SIBLING SPECIES (MYOTIS MYOTIS, MYOTIS BLYTHII) AND MYOTIS CAPPACINII (MAMMALIA: CHIROPTERA) IN CENTRAL ANATOLIA REGION

Myotis myotis (Borkhausen, 1797) and Myotis blythii (Tomes, 1857) are genetically close species of bats with very similar morphology and karyology. In this study, specific descriptive markers of 3 species ( Myotis myotis, Myotis blythii, and M. cappacinii ) belonging to the genus Myotis were determined by using ISSR technique for the first time. In total, 91 bands including 86 polymorphic bands were obtained from the 7 best-optimized primers out of the analyzed 16 primers. UPMGA cluster analysis showed 4 separate clusters. According to these results, the M. myotis and M. blythii were divided into two close groups, whereas a single individual belonging to M. myotis colony formed the third group. M. cappacinii was in a more distinct and remote group with respect to sibling species. Genetic diversity for all populations was calculated as 94.5%. ISSR-PCR method was shown to be a reliable and useful technique for detecting genotypic similarities/differences of Myotis species.

Genotyping and molecular analysis of transgenic sequences in chrysanthemum (Chrysanthemum X morifolium Ramat)

Abstract Genotypes of the chrysanthemum cultivars Harman and Indianapolis were transformed using Agrobacterium tumefaciens strain LBA4404, which carries the binary plasmid pBIN19::Rd29A::ScTPS1TPS2::nos and pBIN19::35S::ScTPS1TPS2::nos, respectively. ISSR markers were used to discriminate two cultivars from each other, and independent transgenic plants generated in each cultivar. Firstly, the nptII gene and Tnos sequence in the transformed genotypes was confirmed by PCR. For genotyping, 10 ISSR profile markers produced 131 DNA bands. The percentage of polymorphism ranged from 20 to 82.4 %. Primer UBC 872 provided the highest percentage of polymorphic bands (PBP), polymorphic information content (PIC), marker index (MI) and resolving power (Rp). A positive correlation was found between the Rp value of each primer and the number of identified genotypes (r=0.822***). The UPGMA analysis generated two groups with a similarity coefficient of 0.67. The genotype grouping was confirmed by PCoA. The ISSR technique was efficient to discriminate transgenic cultivars from non-transgenic plants.

Genetic Diversity and Fine-Scale Genetic Structure in Syagrus oleracea (Mart.) Becc.: A Survivor Palm Species of the Semiarid

The species Syagrus oleracea is a native palm tree, present in highly impacted environments of the Brazilian semiarid. This species is exploited for use in the manufacture of folk crafts and for human and animal feeding. The objective of this study was to characterize the diversity and genetic structure of individuals from different populations, located in anthropic regions of the Brazilian semiarid region, in the state of Minas Gerais. This information is important for species management and conservation strategies. Thus, we used ISSR molecular markers in 157 individuals in seven populations of S. oleracea. Looking at the results, one can observe that amplifications of 109 locos occurred and that an average of 18, 17 alleles were found on each primer, values that demonstrated that the ISSR technique used was highly efficient. The genetic diversity indexes were observed, showing expected heterozygosity (He) and Shannon index (I) of 0.260 and 0.383, respectively. The number of alleles observed (na) among populations was 1.954 and the effective number of alleles (ne) was 1,606. Individuals of the populations are genetically structured in groups (demes) in a random and aggregate manner. We obtained the amplification of 109 ISSR loci, with a mean of 18.2 alleles per primer. The genetic diversity indexes revealed expected heterozygosity (He) and Shannon index (I) of 0.260 and 0.383, respectively. The number of alleles observed (na) was 1.954 and the effective number of alleles (ne) was 1.606. The value of the indirect gene flow found was 1.546. Evidence of high levels of kinship in populations was found, possibly due to low migration rates and geographical barrier. In addition, we observed signs of overexploitation in the areas, which consequently leads to a low rate of gene flow, occurring inbreeding among the sampled populations of Syagrus oleracea.

Genetic Diversity Based on Autogeny and ISSR Markers in Culex pipiens Mosquitoes from Northern Coast of Egypt

The heredity of autogeny in Culex pipiens from northern coast, Alexandria, Egypt, was investigated. Parental females laid more autogenous egg rafts than F1, F3, and F4 females. No significant difference was found in the autogeny rate when F3 female mosquitoes from an autogenous colony mated with males either from an autogenous or anautogenous colony. Similarly, when F3 males from an autogenous colony mated with females either from an autogenous or anautogenous colony, statistically comparable autogeny rates were recorded. However, when females from an anautogenous colony were allowed to mate with males from an autogenous colony, the autogeny rate was statistically higher than that when females from an autogenous colony mated with males from an anautogenous colony. Molecular analysis using ISSR was suitable for detecting polymorphic banding patterns between autogenous and anautogenous specimens of mosquitoes. The ISSR technique succeeded in showing 13 different molecular markers which can be relied upon in distinguishing between populations. Molecular distances among populations were low, ranging between 0.114 and 0.284. On the other hand, cluster and principal coordinate analyses showed that anautogenous specimens were contiguous in one group and separate from autogenous specimens. The ISSR technique is therefore able to detect genetic differentiation among the studied populations, and to distinguish between autogenous and anautogenous mosquitoes from the northern coast of Egypt.

DISTINGUISHING OF ZYGOTIC AND NUCELLAR SEEDLINGS IN CITRUS ROOTSTOCKS USING ISSR TECHNIQUE

The objective of this study is to evaluate the morpho-based method (common system) which used to distinguish between zygotic seedling and nucellar seedling of Cleopatra mandarin and Sour orange rootstocks using ISSR technique. Eight ISSR primers were used to check true-to-type of three mother-plants and nine seedlings of two citrus species, Cleopatra mandarin from Giza nursery and Sour orange from Benha nursery. Results showed that ISSR markers were a utility to identify nucellar or zygotic seedlings and check seedlings true to the type of mother plant. Three Cleopatra mandarin seedlings (33.3%) were identified as nucellar seedlings and six seedlings (66.7%) were identified as zygotic seedlings when using ISSR5 primer. While this percentages were different when using ISSR5 primer with Sour orange seedlings, which revealed one of them (11.1%) was identified as a nucellar seedling and eight seedlings (88.8%) were identified as zygotic seedlings. This suggested that the common method used until now for selection the nucellar seedlings in nursery is not perfect. Finally, that screening of seedlings for early identification of putative zygotic seedlings by ISSR analysis for random and various combinations is essential in citrus to maintain the genetic characteristics of the citrus rootstocks in orchards.

The physiological and genetic differences between flycatchers (Ficedula albicollis vs. Ficedula hypoleuca)

Abstract The paper studies interspecies physiological and genetic differences between the white-collared flycatcher and the pied flycatcher of the genus Ficedula. The fact that the flycatchers are capable of interspecies hybridization is one more reason for particular interest to these species. Using our own-developed method of taking venous blood from the eye’s sinus, we took blood samples from birds in the protected area of Homilshanski Forests, Kharkiv, Ukraine, to study their leukogram patterns. The bird feathers were also collected for genetic analysis -- to identify interspecies differences with application of the ISSR technique. It was revealed that the percentage of heterophiles in the nestlings of the pied flycatcher was lower than in the nestlings of the white-collared flycatcher. The micronucleus test did not reveal any significant difference in the interspecies groups. The spectra of amplification products obtained with the primer (AGC)6 G showed that the white-collared flycatchers had a more heterogeneous structure. The study of the leukogram, micronucleus test, and the ISSR analysis can be especially effective in the study of intra-species genetic differentiation.

Evaluation of chickpea (Cicer arietinum L.) irradiation-induced mutants for resistance to ascochyta blight in controlled environment

Induced mutation is one of the best alternatives for the improvement in chickpea resistance to ascochyta blight. This study was conducted to determine the lethal dose of 50% for the irradiation of chickpea seeds (LD50) and select chickpea mutant lines resistant to Ascochyta rabiei Pass. Irradiation of chickpea seeds with a range of gamma ray doses led to define the LD50 as 150 Gy. In addition, a genetic variability among plants derived by mutagenesis (M2 generation) was demonstrated by the ISSR technique. Among 36 studied mutant lines we selected three mutant lines resistant to A. rabiei in M2 and M3 generations.

Applicability of Different Molecular Markers Techniques for Genetic Distinguish Between Two Genera Cressa Linn. and Cuscuta Yunck. Family Convolvulaceae.

The DNA fingerprinting is used to determine the relationship between species in the same genus or between genera related to the same family. The aim of this study was to determine the relationships between two samples related to the same family Convolvulaceae, representing two genera, Cressa Linn. and Cuscuta Yunck. by RAPD, ISSR and SCoT molecular techniques (PCR based DNA fingerprint). The RAPD, ISSR and SCoT based DNA fingerprinting techniques were implemented to identify the fingerprint diversity between two genera, Cressa Linn. and Cuscuta Yunck-belonging to the family Convolvulaceae. Applying of RAPD technique revealed that using OP-A02, OP-A09, OP-A10, OP-C04 and OP-M01 primers recorded 60, 83.33, 100, 50 and 70.66% polymorphism, respectively. On the other hand, ISSR technique recorded 40, 50, 100, 66.67, 33.33 and 37.5% polymorphism with 44B, HB-08, HB-09, HB-10, HB-11 and HB-12 primers, respectively. However, amplification of SCoT technique, SCoT 1, SCoT 2, SCoT 3, SCoT 4, SCoT 6, SCoT 8, SCoT 10 and SCoT 12 primers recorded 33.33, 28.57, 14.28, 66.66, 25, 40, 42.85 and 50%, respectively . The total polymorphism recorded 73.33, 54.58 and 37.7% for RAPD, ISSR and SCoT techniques, respectively. The result of this study indicated that SCoT technique was more efficient and sustainable for distinguish between two genera under investigation.

In vitro, induction of salt tolerant potato (solanum tuberosum l.) Plants with gamma irradiation and characterization of genetic variations through sds-page and issr-pcr analysis

Salt tolerant mutants of potato (Solanum tuberosum L. ‘Sponta’) were obtained via gamma irradiation. bud explants of two strains of potato (Sponta and lady rosetta were treated with various dosages of gamma irradiation, and the clonal generations were developed. Selection of salt-tolerant mutants was accomplished by in vitro selection media containing 30, 60, 90 and 120 mM NaCl. Molecular-level differences between the control and mutant plants were elucidated using ISSR technique, and the polymorphism rate according to the selected primers was calculated as 89.66%. Genetic distances between the controls and mutants were also calculated, and related dendrograms were produced. On average the mutants were genetically 27.5% different from the control plants. The greatest difference encountered between the control and mutants was 47%, which was detected in mutant plants produced by 20 or 30 Gy gamma irradiation and regenerated in selection medium containing 100 mM NaCl.