Inter-simple Sequence Repeat Primers Research Articles

Genetic Diversity Assessment of Cupressus gigantea W. C. Cheng & L. K. Fu Using Inter-Simple Sequence Repeat Technique

Genetic Diversity Assessment of Cupressus gigantea W. C. Cheng & L. K. Fu Using Inter-Simple Sequence Repeat Technique

Morphological and molecular characterization of cubios (Tropaeolum tuberosum Ruiz & Pavón) collected in two municipalities in Boyaca - Colombia

In this study, we aimed to determine the extent of genetic variation among cubios (Tropaeolum tuberosum) in a diversity micro-center in the department of Boyacá, Colombia, with morphological and ISSR (Inter Simple Sequence Repeats) molecular markers. Twenty-five cubio morphotypes were collected across different farms within the municipalities of Belén and Ventaquemada in Boyacá and were sown for subsequent morphological and molecular characterizations. Tuber features were evaluated according to descriptors of the International Potato Center, namely predominant tuber surface color, secondary tuber surface color, secondary tuber surface color distribution, predominant tuber pulp color, secondary tuber pulp color, distribution of secondary tuber pulp color, tuber shape, and depth of tuber eyes. Molecular variation was assessed via eight ISSR primers. Morphological and molecular characterizations allowed us to identify 25 cubio morphotypes with significant genetic variation in the study area. This finding is likely attributed to mechanisms maintaining genetic variability, such as naturally occurring crosses between morphotypes modulated by environmental conditions and local agricultural practices, including seed exchange and a preference for given morphotypes.

Molecular delineation and genetic diversity of Fusarium species complex causing tea dieback in India and their sensitivity to fungicides

Fusarium dieback is a prevalent disease of tea (Camellia sinensis (L.) Kuntze) in India and China. The pathogen affects plucking shoots and causes complete crop loss under favourable conditions. Although, other fungal diseases that infect tea were widely explored for pathogen identification and characterization, little is known about Fusarium dieback in major tea growing regions of India. Therefore, in this study 24 isolates of Fusarium species causing dieback on tea in 24 tea gardens of Assam and North Bengal were isolated and characterized through micro-morphological and molecular analysis using multi-locus DNA sequences to determine whether multiple species of Fusarium are responsible for dieback disease. Molecular analysis revealed the association of four species of Fusarium, namely F. concentricum, F. solani, F. fujikuroi, and F. oxysporum. The most dominant species was F. concentricum (62.5%), whereas the occurrence frequencies of F. solani, F. fujikuroi, and F. oxysporum were 20.8%, 12.5%, and 0.04%, respectively. All Fusarium isolates were pathogenic to tea leaves when individually and co-inoculated, with F. concentricum HPE0F10 and NHT0F17 exhibiting 19.3 mm lesion length being the most virulent isolates. The growth rate of the pathogen was positively correlated with lesion lengths (P < 0.001). Co-infection of two or multiple Fusarium species enhanced the disease severity and quicker disease development compared to single inoculation. All the isolates showed diverse genetic backgrounds based on Inter Simple Sequence Repeat (ISSR) primers and principal coordinate analysis. In fungicide screening test, all isolates except HPE0F10, NDS0F18, and SNY0F22 were insensitive to M1 fungicide (copper oxychloride). Isolates GNP0F8 and LKW0F16 were insensitive to MBC + M3 (Carbendazim + Mancozeb), whereas HPE0F10, NLG0F11, IND0F13, and NLW0F19 were insensitive to DMI (respective each two to Hexaconazole and Valextra) fungicides. Notably, all the isolates were susceptible to propiconazole. Further, ISSR segregated isolates insensitive to MBC + M3 and M1 fungicides into two separate clusters, representing their distinctiveness. The fungicide screening results indicate that fungicide resistance can be managed by rotating different modes of action of fungicides against multiple Fusarium species causing tea dieback.

Assessment of genetic homogeneity of in-vitro propagated apple root stock MM 104 using ISSR and SCoT primers

Apple is an important fruit crop that is always in demand due to its commercial and nutraceutical value. Also, the requirement for quality planting material for this fruit crop for new plantations is increasing continuously. In-vitro propagation is an alternative approach, which may help to produce genetically identical high grade planting material. In this study, for the first time, an efficient and reproducible propagation protocol has been established for apple root stock MM 104 via axillary bud. Culturing axillary buds on Murashige and Skoog apple rootstock (MM 104) resulted in better in-vitro propagation. (MS) basal medium supplemented with 3.0% (w/v) sucrose and 0.8% (w/v) agar. The axillary buds were established in MS basal medium with BA (5.0 µM), NAA (1.0 µM) and further used to establish invitro propagation protocol. Plant Growth Regulators (PGRs), BA (1.0 µM) in combination with NAA (1.0 µM) was found most efficient for shoot multiplication (100%) and produced 9.8 shoots/explants with an average shoot length of (2.4 ± cm). All the shoots produced roots in 0.1 µM IBA with a 5-day dark period. Acclimatization of in-vitro raised plantlets was obtained with vermiculite: perlite: sand: soil (2:2:1:1) resulting in 76% survival under field conditions. The study showed that the use of axillary bud is efficient for multiple-shoot production of apple rootstock (MM 104). This is the first comprehensive report on in-vitro growth of apple root stock MM 104 with an assessment of genetic stability using DNA fingerprinting profiles based on Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT). The genetic stability of in-vitro-produced plants, as determined by SCoT and ISSR primers, demonstrated genetic closeness to the mother plant.

Genetic diversity and phylogeography of Japanese brome (Bromus japonicus Thunb. ex Murr.) populations in China

Japanese brome (Bromus japonicus Thunb. ex Murr.) is a pervasive annual weed with wide distribution in winter wheat fields across the North China Plain. In this study, we researched the genetic diversity and phylogeography of 24 B. japonicus populations in China based on total genomic DNA and chloroplast DNA. The results showed that 106 fragments were scored using 12 inter-simple sequence repeat (ISSR) primers, and 101 fragments (95.28%) were polymorphic. The findings revealed substantial genetic diversity and differentiation among populations from different locations (Ht = 0.2125, Hs = 0.0730, Gst = 0.6562 and Nm = 0.2619). Mean values of Nei’s genetic diversity (H) and Shannon index of diversity (I) were 0.0731 and 0.1068, respectively. In addition, 15 haplotypes were identified based on combined cpDNA regions among the 24 populations exhibiting abundant haplotype (gene) diversity and nucleotide diversity. The AMOVA based on ISSR and cpDNA both showed that genetic variation mainly exists among populations rather than within them. The STRUCTURE analysises of ISSR and cpDNA indicated that geographical location and genetic relationship had no significant correlation. The haplotype network also illustrated that the widespread haplotypes (H1, H2) might represent ancient polymorphism. The results obtained in this study demonstrate the presence of extensive genetic variability among and within the Chinese populations of B. japonicus, which is likely to contribute significantly to its adaptability and infestation as a weed species.

Genetic Assessment of Leucaena leucocephala (Lam.) de Wit Provenances by Using ISSR Markers

Inter Simple Sequence Repeats (ISSR) technique was deployed for the estimation of genetic diversity in18 Leucaena leucocephala provenances. Seeds of these provenances were collected from across the country and planted at a single location in South India. To estimate the genetic diversity/ polymorphism, DNA was extracted through Cetyl Trimethyl Ammonium Bromide (CTAB) method, visualized and analyzed the DNA bands on UV trans- illuminator gel doc (α-imager 3.2). Totally ten ISSR primers were used in this study. Among them, eight ISSR microsatellites showed a high degree of polymorphism in all the eighteen leucaena provenances used in our study. These primers successfully amplified a total of 645 bands, of which 577 bands (89 %) showed polymorphism. On average, 14.92 bands per accession were obtained and 68.28 % provenances were distinguished through these primers. The ISSR primer T(GT)9 was able to distinguish 100 % of the L. luecocephalla provenances. Cluster analysis based on the genotypic similarities assembled the 18 leucaena accessions in to three clusters. The dendrogram generated based on the 8 ISSR primers distinctively demarcated many of the L. leucocephalla accessions into different clusters indicating vast genetic diversity among the provenances. The high degree of genetic variability demonstrated by the ISSR molecular markers in L. leucocephalla can be used as a tool for assessing genetic diversity and in future leucaena genetic improvement programs.

Genotyping of Vanilla planifolia Jacks ex Andrews from the Totonacapan region using Inter-Simple Sequence Repeat markers

Vanilla is the only genus of plants in the Orchidaceae family used in the food industry, being the most widely used flavoring worldwide and the second most valuable economically. Cultivation is of great biocultural importance in the Totonacapan region, in the state of Veracruz, as this region is considered its center of origin and distribution. However, little is known regarding the number of genotypes present in this area, as well as the genetic diversity, which is vital for the improvement of this crop. Therefore, the objective of this study was to perform the genotyping of 60 Vanilla planifolia accessions from the Germplasm Bank of The National Institute of Forestry, Agriculture, and Livestock Research (Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias - INIFAP), Experimental Field Ixtacuaco, which were selected and collected in the Totonacapan region. For this purpose, the number of V. planifolia genotypes was determined based on ISSR (Inter-Simple Sequence Repeat) molecular markers. Three DNA extraction protocols from plants and fungi described in the literature were evaluated, and a fourth protocol was adapted with different procedures from the previous protocols, seeking to obtain a high concentration and good quality and integrity. Once quality DNA was obtained, fourteen ISSR primers were evaluated, of which three primers that showed the highest number of amplified bands were selected. Subsequently, the genotyping of the 60 accessions was carried out, resulting in nine genotypes with primer 808 and six genotypes with primers 812 and 856. These results allowed the genotyping of Vanilla planifolia accessions with ISSR markers, which will help evaluate the genetic diversity of this crop in the Totonacapan region in the future.

Investigation of genetic diversity among autochthonous grape cultivars grown in Türkiye using molecular primers

AbstractGrape is a type of fruit widely grown globally and in Türkiye thanks to its importance in human nutrition and diversity of usage areas. Türkiye has a long history of grape cultivation, and the genetic diversity of local grapes grown in some regions has still not been revealed. This study showed the genetic diversity between 60 local grape genotypes selected from north Central Anatolia and nine reference grape cultivars;10 inter simple sequence repeats (ISSR) and 7 inter-primer binding site (IPBS) retrotransposon primers were used. The mean values of genetic diversity indices in ISSR and IPBS retrotransposon primers were calculated as expected allelic frequency (p) 0.476/0.195, observed allelic frequency (q) 0.524/0.805, number of effective alleles (Ne) 1.326/1.378, Shannon’s information index (I) 0.308/0.367, expected heterozygosity (He) 0.199/0.232 and unbiased expected heterozygosity (uHe) 0.201/0.234, respectively. Unweighted pair group method with arithmetic mean (UPGMA) and model-based STRUCTURE analysis divided the studied germplasm into two groups. However, local genotypes were not grouped per the regions where they grew. As a result of the analysis of molecular variance (AMOVA) analysis, the inter-population variation was determined to be 11%, and the intra-population variation was determined to be 89%. The mean genetic differentiation value (Fst) between populations was 0.303. The results showed that ISSR and IPBS retrotransposon primers effectively explained the genetic diversity.

Comprehensive Tools of Alkaloid/Volatile Compounds-Metabolomics and DNA Profiles: Bioassay-Role-Guided Differentiation Process of Six Annona sp. Grown in Egypt as Anticancer Therapy.

Trees of the Annona species that grow in the tropics and subtropics contain compounds that are highly valuable for pharmacological research and medication development and have anticancer, antioxidant, and migratory properties. Metabolomics was used to functionally characterize natural products and to distinguish differences between varieties. Natural products are therefore bioactive-marked and highly respected in the field of drug innovation. Our study aimed to evaluate the interrelationships among six Annona species. By utilizing six Start Codon Targeted (SCoT) and six Inter Simple Sequence Repeat (ISSR) primers for DNA fingerprinting, we discovered polymorphism percentages of 45.16 and 35.29%, respectively. The comparison of the profiles of 78 distinct volatile oil compounds in six Annona species was accomplished through the utilization of GC-MS-based plant metabolomics. Additionally, the differentiation process of 74 characterized alkaloid compound metabolomics was conducted through a structural analysis using HPLC-ESI-MSn and UPLC-HESI-MS/MS, and antiproliferative activities were assessed on five in vitro cell lines. High-throughput, low-sensitivity LC/MS-based metabolomics has facilitated comprehensive examinations of alterations in secondary metabolites through the utilization of bioassay-guided differentiation processes. This has been accomplished by employing twenty-four extracts derived from six distinct Annona species, which were subjected to in vitro evaluation. The primary objective of this evaluation was to investigate the IC50 profile as well as the antioxidant and migration activities. It should be noted, however, that these investigations were exclusively conducted utilizing the most potent extracts. These extracts were thoroughly examined on both the HepG2 and Caco cell lines to elucidate their potential anticancer effects. In vitro tests on cell cultures showed a significant concentration cytotoxic effect on all cell lines (HepG2, HCT, Caco, Mcf-7, and T47D) treated with six essential oil samples at the exposure time (48 h). Therefore, they showed remarkable antioxidant activity with simultaneous cytotoxic effects. In total, 50% and 80% of the A. muricata extract, the extract with the highest migratory activity, demonstrated a dose-dependent inhibition of migration. It was strong on highly metastatic Caco cells 48 h after treatment and scraping the Caco cell sheet, with the best reduction in the migration of HepG2 cells caused by the 50% A. reticulata extract. Also, the samples showing a significant IC50 value showed a significant effect in stopping metastasis and invasion of various cancer cell lines, making them an interesting topic for further research.

Genetic diversity among Vanda celebica, Vanda dearei, and their hybrids based on ISSR markers

A cross between two different orchid species is a way to improve the horticultural performance of the hybrids. Inter-simple sequence repeat (ISSR) is a method of molecular markers that could be used to analyze genetic diversity among parents and their hybrid progenies. The purpose of this study was to examine the genetic diversity of the parent species of Vanda dearei and Vanda celebica, the hybrid progenies of (♀ Vanda dearei x ♂ Vanda celebica)-cross and the hybrid progenies of their reciprocal cross. DNA amplification using 5 ISSR primers, namely UBC 810, UBC 815, UBC 834, UBC 835, and UBC 880, produced 60 DNA bands with a size of 200-2800 bp, with the percentage of polymorphism ranging from 81.8%-100% and the percentage of monomorphism as much as 0%-18.2%. Analysis of genetic diversity showed that the genetic diversity among hybrid progenies of (♀ V. dearei x ♂ V. celebica) was 8%. In comparison, those among hybrid progenies of its reciprocal cross (♀ V. celebica x ♂ V. dearei) were 13%. In conclusion, deploying V. celebica as a female parent and V. dearei as a male parent will most likely produce higher diversity among its progenies, compared to the reciprocal cross.&#x0D; Keywords: DNA bands; diversity; marker, orchid; reciprocal cross

The population structure of the Fusarium brachygibbosum species complex in the grassland biome of South Africa

Fusarium brachygibbosum Padwick is a phytopathogen with a widespread distribution, infecting various host plants. In South Africa, there is a limited number of studies on the genetic diversity of fusaria, particularly in undisturbed soils. In the current study, the genetic diversity of F. brachygibbosum was investigated using inter-simple sequence repeat (ISSR) and mating type genes. The F. brachygibbosum isolates were collected from four different geographic regions within the grassland biome of South Africa. A total of ten ISSR primers produced 64 distinct and reproducible amplicons, with 100% polymorphism. The ISSR markers revealed high levels of intraspecific variation, which is depicted by various genetic diversity indexes. In the analysis of mating type genes, the MAT1-1-2 gene was found among the four populations, but the MAT1-2-1 gene was only present in the population from the Groenkloof Nature Reserve. The data from this study will contribute substantially to knowledge of grassland fusaria of South Africa and their population structure in the grassland biome.

Agro-Morphological Traits and Molecular Diversity of Proso Millet (Panicum miliaceum L.) Affected by Various Colchicine Treatments

Colchicine is a substance used to induce mutations in order to regulate important agronomic traits. The genotypes Pavlodarskoe 4, Quartet, and PI 289324, originating from Kazakhstan, the Russian Federation, and Hungary, respectively, were used as materials. The objective of this study was to investigate the effects of different colchicine concentrations (0.0, 0.04, 0.06, 0.08, and 0.1%) and treatment times (6, 12, and 24 h) on the agronomic traits of proso millet (Panicum miliaceum L.) and to assess the genetic diversity of the M2 generation using inter simple sequence repeat (ISSR) markers. The experiment was conducted in 2021 for the M1 generation and in 2022 for the M2 generation, from May to September. The percentage of field germination decreased with increasing colchicine concentrations and exposure durations. The mean field germination percentages were 48.57% in Pavlodarskoe 4, 43.28% in Quartet, and 53.14% in PI 289324 under colchicine treatment. Chlorophyll-defective M1-M2 plants were obtained using various colchicine concentrations and exposure periods. The highest number of mutational modifications was attained with the 0.08–0.1% concentrations of colchicine. Based on the research results, a total of 248 plants with chlorophyll-defective mutations were selected from 2214 plants. The growing seasons of M1 and M2 plants were shortened by higher colchicine concentrations (0.08–0.1%) combined with soaking times of 12 and 24 h. Thus, the longest growing season (84 days) was observed with a 6 h treatment time for PI 289324, while the shortest (78 days) was recorded for 12 and 24 h treatments. The possibility of obtaining morphological mutations using colchicine has been confirmed. The ISSR primers amplified a total of 1333 fragments; 1281 bands were found to be polymorphic, and 52 bands were monomorphic. The percentage of polymorphism varied from 80 to 100%, with an average of 96.11%. Most of the different allelic bands were detected when applying the 0.08% colchicine concentration. These positive variations are a great opportunity to use colchicine as a tool for improving agronomic traits in plant breeding.

Genetic variability of wild palms Euterpe precatoria, Euterpe oleracea and Mauritia flexuosa with molecular markers ISSR

Wild palms constitute a group of plants of particular economic importance in the tropics, being a valuable source for obtaining construction materials, fabrics, fuel, food, ornamental and medicinal plants. Despite their economic, social, and environmental importance, the genetic variability existing in wild palms, mainly of the genus Mauritia and Euterpe, is unknown, which limits their potential use and the identification of promising genotypes to be included in recombination programs. The genetic variability of three wild palm species (Euterpe oleracea, Euterpe precatoria, and Mauritia flexuosa) was evaluated using Inter-Simple Sequence Repeat (ISSR) markers. A total of 51 genotypes were analyzed with seven ISSR primers, which generated a total of 153 bands, with a percentage of polymorphic loci of 90.24%. The cluster analysis revealed the formation of six groups at a similarity level of 37%, grouping the palm genotypes by species and geographical origin. Molecular variance analysis revealed that within group variations contributed more to genetic diversity (89%) than between group variations (11%). The average value of Fst was 0.20, demonstrating moderate genetic differentiation. The high genetic variability found in this study indicates the valuable genetic potential present in palm germplasm, which could be used for future improvement programs of the species.

Agro-morphological, biochemical, and molecular markers of barley genotypes grown under salinity stress conditions

The aim of this study was to evaluate the impact of salt stress on morphological, yield, biochemical, and molecular attributes of different barley genotypes. Ten genotypes were cultivated at Fayoum Research Station, El-Fayoum Governorate, Egypt, during two seasons (2020–2021 and 2021–2022), and they were exposed to two different salt concentrations (tap water as a control and 8000 ppm). The results showed that genotypes and salt stress had a significant impact on all morphological and physiological parameters. The morphological parameters (plant height) and yield attributes (spike length, number of grains per spike, and grain yield per plant) of all barley genotypes were significantly decreased under salt stress as compared to control plants. Under salt stress, the total soluble sugars, proline, total phenol, total flavonoid, ascorbic acid, malondialdehyde, hydrogen peroxide, and sodium contents of the shoots of all barley genotypes significantly increased while the potassium content decreased. L1, which is considered a sensitive genotype was more affected by salinity stress than the tolerance genotypes L4, L6, L9, and Giza 138. SDS-PAGE of seed proteins demonstrated high levels of genetic variety with a polymorphism rate of 42.11%. All genotypes evaluated revealed significant variations in the seed protein biochemical markers, with new protein bands appearing and other protein bands disappearing in the protein patterns of genotypes cultivated under various conditions. Two molecular marker techniques (SCoT and ISSR primers) were used in this study. Ten Start Codon Targeted (SCoT) primers exhibited a total of 94 fragments with sizes ranging from 1800 base pairs to 100 base pairs; 29 of them were monomorphic, and 65 bands, with a polymorphism of 62.18%, were polymorphic. These bands contained 21 unique bands (9 positive specific markers and 12 negative specific markers). A total of 54 amplified bands with molecular sizes ranging from 2200 to 200 bp were produced using seven Inter Simple Sequence Repeat (ISSR) primers; 31 of them were monomorphic bands and 23 polymorphic bands had a 40.9% polymorphism. The techniques identified molecular genetic markers associated with salt tolerance in barley crop and successfully marked each genotype with distinct bands. The ten genotypes were sorted into two main groups by the unweighted pair group method of arithmetic averages (UPGMA) cluster analysis based on molecular markers and data at a genetic similarity coefficient level of 0.71.

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

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

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

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

Genetic characterization of superior durian (Durio zibethinus L.) accessions in Batang District, Central Java, Indonesia based on ISSR markers

Abstract. Amrullah MI, Rahayu ES, Puwantoyo E, Maharani RI, Solichin, Sriyadi, Hadiprasetya TY, Retnoningsih A. 2023. Genetic characterization of superior durian (Durio zibethinus L.) accessions in Batang District, Central Java, Indonesia based on ISSR markers. Biodiversitas 24: 4811-4819. Durian (Durio zibethinus L.) has the highest level of genetic diversity in the genus of Durio. The genetic diversity of D. zibethinus in Batang District, Central Java, Indonesia needs to be explored to obtain data on superior durian accessions that are adaptive in the area. This study aimed to analyze the genetic diversity of 40 superior durian accessions from 13 locations in Batang District using 10 Inter Simple Sequence Repeat (ISSR) primers. Genomic DNA was isolated from leaves using a modified CTAB method. PCR products were electrophoresed on 2% agarose and yielded 161 ISSR alleles with a percentage of 100% polymorphic alleles. The level of genetic diversity based on Nei's gene diversity and Shannon's Information index are 0.277 and 0.434, respectively. The durian accessions studied have a similarity coefficient of 0.22-0.86 and are divided into two main clusters. Siwatu 1 and Siwatu 2 accessions have the highest genetic similarity coefficient (0.86), while Butternuts and Watu Tembogo 1 accessions show the lowest genetic similarity coefficient (0.05). Specific alleles were found in 11 durian accessions. The molecular characterization results show the high genetic diversity of superior durian accessions in Batang District and add to the list of local superior durian accessions in Indonesia.

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

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

Phenological, morphological and genetic characterization of local grapevine (Vitis labrusca L.) genotypes grown in the Black Sea Region in Northern Turkey

Vitis labrusca has become widely naturalized in the Black Sea region of Northern Turkey. The aim of this work was to evaluate the morphological, phenological, and genetic characteristics of V. labrusca accessions grown in the Black Sea region during the last three growing seasons. Local genotypes were described according to the Organisation Internationale de la Vigne et du Vin (OIV) ampelographic descriptor, including shoot length (cm), mature leaf size, bunch length and width, berry length and width, berry weight, number of berries, Total Soluble Solids (TTS) and titratable acidity. Additionally, phenological data, including bud burst, flowering, veraison and harvest date, were recorded. The accessions were characterized at the molecular level, and genetic relationships were assessed by means of Inter Simple Sequence Repeat (ISSR) markers. Using 6 ISSR primers, seventy-seven bands were obtained, of which 69 were polymorphic with a mean polymorphic rate of 88.68%. These ISSR primers produced polymorphism information content (PIC) values ranging from 0.48 to 0.5. The genetic similarity ranged from 0.08 to 0.83 among the genotypes. According to the dendrogram based on the ISSR analysis, Accessions 8 and 5 were genetically related, with a coefficient of similarity of 0.83, while Accession 3 was the most distantly related genotype, with a coefficient of similarity of 0.08. These results demonstrated that ISSR markers can be used for genetic diversity analysis among V. labrusca genotypes. Our results also described characteristics of new V. labrusca genotypes that could be valuable for future Marker-Assisted Selection (MAS) and grapevine breeding.&#x0D; Key words: Diversity, foxy species, Inter-Simple Sequence Repeat markers (ISSRs), Breeding, polymorphism

Genetic Diversity of the Endemic Species Phanera sirindhorniae in the Mekong Basin of Thailand Based on ISSR Markers

Phanera sirindhorniae, an endemic species to the Mekong Basin’s upper northeastern region in Thailand, is notable for its therapeutic properties in alleviating pain and discomfort. The objective of this study was to assess the genetic diversity of P. sirindhorniae derived from four natural sources, specifically Sakon Nakhon, Nakhon Phanom, Bueng Kan and Mukdahan provinces, utilizing Inter-Simple Sequence Repeat (ISSR) markers. A total of 51 samples were analyzed, and 10 primers exhibited distinct, robust amplified fragments and polymorphism. The amplified fragment sizes ranged from 170 to 1,300 base pairs, identifying a total of 113 amplified fragments, of which 107 (94.69 %) displayed polymorphism. Polymorphic information content (PIC) values spanned between 0.112 and 0.429, while Nei’s gene diversity ranged from 0.2088 ± 0.2184 to 0.2994 ± 0.1912. Assessment of genetic diversity within and among populations revealed the total genetic diversity (Ht = 0.3443 ± 0.0225) and the genetic diversity within populations (Hs = 0.2617 ± 0.0161). The variability among populations (Gst = 0.2401) indicated a significant degree of gene flow (Nm = 1.580). The Analysis of Molecular Variance (AMOVA) results supported this, with 74 % of genetic differentiation occurring within populations and 26 % among populations. Using the Neighbor-Joining (NJ) generated from Nei’s genetic distance-based clustering analysis, Dendrogram construction successfully classified 2 distinct groups. The analysis revealed that P. sirindhorniae from Nakhon Phanom, and Bueng Kan constituted 1 group, whereas P. sirindhorniae from Mukdahan, and Sakon Nakhon formed another. These findings provide essential data for developing genetic conservation initiatives for this valuable genetic resource. HIGHLIGHTS This study presents the inaugural analysis of genetic diversity in wild Phanera sirindhorniae populations, a species designated as endangered, utilizing Inter-Simple Sequence Repeat (ISSR) markers. A group of 10 Inter Simple Sequence Repeat (ISSR) primers showed values for Polymorphism Information Content (PIC), with (AG)8YC (PIC= 0.445) standing out. These results highlight their potential for use in the investigation of genetic diversity. The examination of P. sirindhorniae resulted in the identification of 2 distinct genetic groups, which have the potential to inform and facilitate the development of genetic conservation strategies for this crucial genetic resource. GRAPHICAL ABSTRACT