Detected Genetic Markers for Three Varieties of Rice (Oryza sativa L.) under Nano- Particles

High gene flow and genetic diversity in three economically important Zanthoxylum Spp. of Upper Brahmaputra Valley Zone of NE India using molecular markers

BACKGROUND: Bitter gourd (Momordica charantia L.) is an important vegetable crop in tropical countries, including China and India. A wide range of genetic diversity exists in India with respect to fruit morphology such as colour, size and exocarp. A diversity assessment conducted using different DNA marker systems amplified fragment length polymorphism (AFLP), inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers) will be helpful in the establishment of a broad‐based description for improved germplasm curation and the identification of germplasm for genome mapping and breeding of bitter gourd.RESULTS: Genetic relationships between 38 bitter gourd accessions were determined with the aid of 29 RAPD, 15 ISSR and six AFLP markers. Greater polymorphism was detected by AFLPs when compared with RAPD and ISSR analyses using the same germplasm array (RAPD 36.5%, ISSR 74.5% and AFLP 78.5% polymorphism). The average marker index (MI) values derived from the three different marker systems differed dramatically, indicating that they vary in their discriminatory power (AFLP > ISSR > RAPD). The AFLP markers used were only weakly correlated with ISSR (r2 = 0.007) and RAPD (r2 = 0.04) marker analyses, whereas a comparatively high correlation (r2 = 0.77) was found between RAPD and ISSR marker systems.CONCLUSION: The studies using RAPD and ISSR markers were not able to uniquely discriminate all the bitter gourd accessions examined, whereas AFLP analysis was discriminatory and allowed for a more complete dissection of unique differences among accessions of bitter gourd within and between collection sites. Copyright © 2007 Society of Chemical Industry

Genetic variations of 15 Brahmi (Bacopa monnieri L.) accessions were evaluated using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. During RAPD analysis, amplification of genomic DNA of the 15 accessions by 22 primers generated 197 fragments, of which 187 were polymorphic with an average of 8.95 bands per primer. The amplified products varied in size from 2,200 to 250 bp. Twenty-five selected ISSR primers produced 284 bands across 15 accessions, of which 270 were polymorphic with an average of 10.80 bands per primer. The PIC value ranges from 0.363 to 0.908 for RAPD primers, while 0.419 to 0.836 in case of ISSR. The size of amplified bands ranged from 2,800 to 240 bp. Similarity index values ranged from 0.16 to 0.95 (RAPD), 0.18 to 0.98 (ISSR) and 0.179 to 0.945 for pooled ISSR and RAPD markers data. Mantel test revealed the similar distribution pattern of the polymorphism between RAPD and ISSR markers and the correlation co-efficient (r) was 0.71384. The results indicated that both of the marker systems RAPD and ISSR, individually or combined can be effectively used in determination of genetic relationship among B. Monnieri accessions collected from different parts of Central India. It could be concluded that the information of genetic similarities and diversity among Brahmi accessions is necessary for their conservation and breeding programs.

To control the genetic quality during the whole process of tissue culture of the traditional Chinese medicinal plant, Saussurea involucrate Kar. et Kir., DNA polymorphisms and genetic variations were investigated using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. The genetic stability/variation in tissue-cultured products, including three calli, three adventitious shoots, regenerated plantlets and 2 year-old regenerated plantlets cultivated in the planting base in Tianshan Mountain, were assessed compared with 1 year-old and 2 year-old seedlings cultivated in the same planting base using aseptic seedlings as reference. Apparent genetic variation was detected in the 11 type of plant materials. The percentages of polymorphic bands in the RAPD and ISSR analysis were, respectively, 35% and 33%. Cluster analysis indicated that the genetic similarity values calculated on the basis of RAPD and ISSR data among the 11 type of plant materials were respectively ranged from 0.823 to 0.995 with a mean of 0.878 and 0.825 to 0.974 with a mean of 0.885, which classified the samples into three groups. The similarity coefficient also revealed that differences among three calli were not remarkable by both RAPD and ISSR analysis, and only chemical components and growth properties needed consideration in the screening of callus used for the next redifferentiation studies. But there are remarkable differences among three adventitious shoots analyzed by ISSR markers. Therefore, RAPD and ISSR markers are efficient tools in genetic variation assessment and quality control in plant tissue culture process.

Genetic variation within and among population is the basis for survival of the population both in short and long term. Thus, studying the plant genetic diversity is essential for any conservation program. Indigenous medicinal plants like Justicia adhatoda L. which are facing high rate of depletion from the wild population need immediate attention. DNA-based dominant molecular marker techniques, random amplification of polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) were used to unravel the genetic variability and relationships across thirty-two wild accessions of J. adhatoda L., a valuable medicinal shrub widespread throughout the tropical regions of Southeast Asia. Amplification of genomic DNA using 38 primers (18 RAPD and 20 ISSR) yielded 434 products, of which 404 products were polymorphic revealing 93.11 % polymorphism. The average polymorphic information content value obtained with RAPD and ISSR markers was 0.25 and 0.24, respectively. Marker index (RAPD = 3.94; ISSR = 3.53) and resolving power (RAPD = 4.24; ISSR = 3.94) indicate that the RAPD markers were relatively more efficient than the ISSR assay revealing the genetic diversity of J. adhatoda. The Shannon diversity index obtained with RAPD and ISSR markers was 0.40 and 0.38, respectively. The similarity coefficient ranged from 0.26 to 0.89, 0.33 to 0.93 and 0.31 to 0.90 with RAPD, ISSR and combined UPGMA dendrogram, respectively. PCA derived on the basis of pooled data of both the markers illustrated that the first three principal coordinate components accounted 79.27 % of the genetic similarity variance. The mantel test between two Jaccard’s similarity matrices gave r = 0.901, showing the fit correlation between ISSR- and RAPD-based similarities. Based on the results, ex-situ methods may be the most suitable and efficient measure for long-term conservation.

Genetic diversity in a set of 31 Amaranthus accessions was analysed using RAPD, ISSR and SSR markers. Polymorphic Information Content (PIC) values of the polymorphic RAPD, SSR and ISSR markers ranged from 0.067 to 0.471, 0.178 to 0.452 and 0.11 to 0.362 with an average of 0.229, 0.371 and 0.441 per primer, respectively. Gene diversity for RAPD, SSR and ISSR markers respectively ranged from 0.032 to 0.487, 0.11 to 0.362 and 0.104 to 0.384.The similarity metrics values for the accessions ranged from 0.58 to 0.98 with RAPD, 0.70 to 1.00 with SSR and from 0.44 to 0.88 with ISSR markers. The dendrogram generated by the markers had five major clusters in case of RAPD markers, two distinct clusters in caseof SSR markers and sixclusters in caseof ISSR markers. Amongst all, ISSR markers provided greater confidence for the assessment of genetic diversity in comparison to SSR and RAPD markers. All the accessions studied were found genetically diverse which may be useful in breeding for improving the genotypes of Amaranthus.

Genetic diversity and relationships detected by ISSR and RAPD analysis among Aethionema species growing in Eastern Anatolia (Turkey)

Embelia ribes Burm f. (Primulaceae) is a medicinal and vulnerable woody liana distributed throughout India. Embelin, a well-recognized active phytoconstituents in berries, is commonly used in ayurvedic formulations. Due to over-exploitation, the status of the plant is vulnerable. Previous studies on this species mainly focused on its phytochemical analysis, which led to overexploitation and loss of the germplasm. In the present study, 20 RAPD and 18 ISSR markers were employed to assess genetic divergence in 40 genotypes of E. ribes collected from different parts of the Western Ghats of India. In RAPD analysis, all 40 accessions with 20 RAPD primers amplified 282 fragments, with 83.91% average polymorphism and with an average of 14.10 bands per primer. The size of amplicons varied from 200 to 2500bp. While, ISSR primers produced 203 fragments of which 161 were polymorphic with an average of 11.28 bands per primer with 73.25% average polymorphism. The size of amplicons ranges from 200 to 2500bp. RAPD and ISSR markers were also assessed by calculating polymorphic information content (PIC) to discriminate the genotypes; the average PIC value for RAPD, ISSR, and combined RAPD + ISSR markers obtained was more than 0.50 suggesting the informativeness of markers. UPGMA analysis based on Jaccard's similarity coefficient for RAPD, ISSR, and RAPD + ISSR data reveals that 40 accessions of E. ribes were depicted in four clusters. The clustering pattern of all individuals in PCoA analysis agreed with the UPGMA dendrograms, which further confirms the genetic relationships explained by cluster analysis. AMOVA analysis of RAPD, ISSR, and combined marker system revealed variation within the population, ranging from 41 to 44%, and among the population, it ranged from 56 to 59%. The present study provides an optimized method for evaluating the genetic diversity of Embelia ribes using RAPD and ISSR markers which are useful for further sustainable utilization and conservation of natural populations in the Western Ghats of India.

Poerba YS, Ahmad F (2010) Genetic variability among 18 cultivars of cooking bananas and plantains by RAPD and ISSR markers. Biodiversitas 11: 118-123. This study was done to assess the molecular diversity of 36 accessions (18 cultivars) of the plantain and cooking bananas (Musa acuminata x M. balbisiana, AAB, ABB subgroups) based on Random amplified polymorphic DNA (RAPD) and and Inter Simple Sequence Repeats (ISSR) markers and to determine genetic relationships in the bananas. RAPD and ISSR fingerprinting of these banana varieties was carried out by five primers of RAPDs and two primers of ISSRs. RAPD primers produced 63 amplified fragments varying from 250 to 2500 bp in size. 96.82% of the amplification bands were polymorphic. ISSR primers produced 26 amplified fragments varying from 350 bp to 2000 bp in size. The results showed that 92.86% of the amplification bandswere polymorphic. The range of genetic distance of 18 cultivars was from 0.06-0.67.Key words: RAPD analysis, Musa acuminata, Musa balbisiana, plantain, cooking bananas.

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the DNA polymorphism in elite blackgram genotypes. A total of 25 random and 16 ISSR primers were used. Amplification of genomic DNA of the 18 genotypes, using RAPD analysis, yielded 104 fragments that could be scored, of which 44 were polymorphic, with an average of 1.8 polymorphic fragments per primer. Number of amplified fragments with random primers ranged from two (OPA-13) to nine (OPK-4) and varied in size from 200 bp to 2,500 bp. Percentage polymorphism ranged from 16.6% (OPK-7) to a maximum of 66.6% (OPE-5, OPH-2, and OPK-8), with an average of 42.7%. The 16 ISSR primers used in the study produced 101 bands across 18 genotypes, of which 55 were polymorphic. The number of amplified bands varied from two (ISSR 858) to ten (ISSR 810), with a size range of 200-2,200 bp. The average numbers of bands per primer and polymorphic bands per primer were 6.3 and 3.4, respectively. Percentage polymorphism ranged from 25% (ISSR 885) to 100% (ISSR 858), with an average percentage polymorphism of 57.5% across all the genotypes. The 3'-anchored primers based on poly(GA) and poly(AG) motifs produced high average polymorphisms of 54.98% and 58.32%, respectively. ISSR markers were more efficient than the RAPD assay, as they detected 57.4% polymorphic DNA markers in Vigna mungo as compared to 42.7% for RAPD markers. The Mantel test between the two Jaccard's similarity matrices gave r=0.32, showing low correlation between RAPD- and ISSR-based similarities. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in ISSR and combined data of RAPD and ISSR.

Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers were used to compare genetic diversity of 17 accessions of Clitoria ternatea (Fabaceae) populations, collected from nine different states of India. The populations included three different flower colours viz., blue, white and white with blue tinge. Twenty-three RAPD primers and eighteen ISSR primers amplified a total of 137 and 105 reproducible DNA fragments, respectively with fragment sizes ranged from 150 to 3,000 bp. ISSR showed higher polymorphism (29.52 %) in comparison to RAPD (27.73 %). In RAPD analysis, maximum polymorphic information content value (0.66) was observed in primer OPC 10 but in ISSR study, it was (0.55) in primer UBC 889. Jaccard’s coefficient of similarity showed that pair-wise genetic similarity coefficients ranged between 81 and 97 % in RAPD analysis, whereas 80 and 98 % in ISSR analysis, which were in close range to each other. Similarly, clustering pattern showed same trend in both the markers analysis which also revealed that all the accessions were grouped according to their geographical locations rather than on the basis of different flower colours. Mental Z test indicated highly significant co-relation between geographical distance and genetic distance among the populations (r = 0.8321, P < 0.01). Genetic similarity between populations showed very high values (<80 %) indicating low genetic variation within and among the populations. The narrow genetic base within the populations was either due to the exotic origin or due to self pollinated behaviour of the species which restrict gene flow within and between populations.

Grapevine (Vitis vinifera L.) is extensively cultivated in Turkey and holds significant cultural and economic value. However, genetic characterization of grape genotypes from lesser-studied regions such as Inner Anatolia remains incomplete, limiting conservation and breeding efforts. This study addresses this critical knowledge gap by utilizing ISSR (inter simple sequence repeats) and RAPD (randomly amplified polymorphic DNA) molecular markers to comprehensively assess genetic diversity and relationships among grape genotypes from the Divriği district in Sivas province, Turkey. Eight ISSR and eight RAPD primers were used, revealing considerable genetic variation. The ISSR primers produced a total of 88 polymorphic bands, with primer UBC-810 producing the highest number of bands (15). Similarly, RAPD primers generated 68 polymorphic bands and the highest number of bands (13) were produced by OPA-18 marker. The UPGMA dendrogram analysis using ISSR data revealed two primary genetic clades, with similarity indices between 0.212 and 0.816. Similarly, RAPD analysis also produced two main clusters, exhibiting genetic similarity indices between 0.164 and 0.912. Principal component analysis (PCA) corroborated the dendrogram groupings from both ISSR and RAPD analyses, exhibiting consistent clustering and strengthening the dependability of both marker systems. Significant genetic differentiation was observed among the tested grape genotypes, highlighting the presence of genetically distinct individuals. Genotypes ‘G7’, ‘G1’, ‘G9’, and ‘G10’ demonstrated significant genetic divergence, suggesting them as crucial candidates for conservation efforts. This genetic diversity likely reflects historical cultivation practices, natural selection, mutation accumulation, and potential introgression from wild populations. The results indicate the significance of integrating molecular markers into grapevine breeding and conservation efforts. Both ISSR and RAPD markers proved successful in genetic characterization, yielding significant insights into grapevine genetic resources. These findings are essential for sustainable viticulture practices, the preservation of genetic diversity, and future grapevine improvement initiatives in Turkey.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-18463-3.

There is a lack of information on the molecular characterization of Ocimum species and hence, efforts have been made under the present study to characterize 17 Ocimum genotypes belonging to 5 different species (O. basilicum, O. americanum, O. sanctum, O. gratissimum and O. Polystachyon) through random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. PCR amplification using 20 RAPD primers generated a total of 506 loci, of which 490 (96.47 %) loci were found polymorphic. The PIC value for RAPD ranged from 0.907 (OPF 14) to 0.954 (OPC 11) with an average of 0.937. The ISSR primers generated a total of 238 loci, of them 234 (98.17 %) loci were polymorphic. The PIC value ranged from 0.892 (UBC 808) to 0.943 (ISSR A12) with an average of 0.923. The average Jaccard’s similarity coefficient based on RAPD and ISSR analysis was 0.58 and 0.52, respectively. Clustering pattern of dendrogram generated using the pooled RAPD and ISSR data showed all Ocimum genotypes in their respective species groups at a cutoff value of 0.49 and 0.42, respectively. Many unique species-specific alleles were amplified by RAPD and ISSR markers. In both marker systems, a maximum number of unique alleles were observed in O. sanctum. The results of the present investigation provided valid guidelines for collection, conservation and characterization of Ocimum genetic resources.

Agreat deal of research has been focused on oil crops of various plants, especially members of the mustard family (Brassicaceae) such as species of Brassica. Canola (rapeseed; Brassica napus L. genome AACC, 2n=38) arises from spontaneous hybridization between turnip (Brassica rapa) (AA, 2n= 20) and cabbage (Brassica oleracea) (CC, 2n=18). It is now the second largest oilseed crop over the world after soybean (Glycine max) providing 13% of the world supplies (Abbas et al., 2009). Canola is primarily used for food and feed, but has recently gained an increasing interest as a source for bio-products (e.g., biodiesel). Besides that, the Food and Drug Administration (FDA) approved canola oil with a Qualified Health Claim (QHC) due to its ability to reduce the risk of coronary heart disease (Miller-Cebert et al., 2009). Like any other crop species, to improve quality and quantity of Brassica spp., presence of sufficient genetic diver- sity is very important. In the breeding process, significant improvement of quality and production was achieved, as well as utilization of rapeseed oil in human nutrition. However, genetic variability in this important crop is restricted with regard to many characters of value for breeding process (Marjanovic-jeromela et al., 2009). The success in breeding programs of a crop species largely relies on the presence of sufficient genetic diversity in the germplasm and knowledge about the characteristics of the genotypes and their genetic relationship. Various methods have been elaborated for this purpose. Pedigree analysis is the most widely used method for estimating the degree of similarity between varieties or populations, but the necessary information on ancestry is not always accurate or available. Application of morphological traits is hindered by their limited number and by the modifying effect of environmental factors in some cases. The spread of DNA markers has allowed the genome to be analyzed directly, thus eliminating errors caused by environmental factors. By using these markers, the genome can be characterized with great accuracy. In addition to the estimation of degrees of relationship between different varieties, a further important use of these markers is to distinguish between genotypes. Numerous molecular markers have been used for variety identification in various plant species, which allow cultivar identification in early stages of plant development, being neutral to environmental effects (Mohammadi, 2002; Meszaros et al., 2007; Moghaddam et al., 2009). A variety of molecular markers including Restriction Fragment Length Polymorphism (RFLP) (Thormann et al., 1994), Inter-Simple Sequence Repeats (ISSR) (Carolyn et al., 2000; Rudolph et al., 2002), amplified fragment length polymorphism (AFLP) (Sandip et al., 1999; Seyis et al., 2003; Jiang et al., 2007) and random amplified polymorphic DNA (RAPD) (Ashik Rabbani et al., 1998; Lazaro and Aguinagalde, 1998; Divaret et al., 1999), have been used to study the extent of genetic variation among the diverse group of important crop species in the genus Brassica (Afiah et al., 2007; Marjanovic-jeromela et al., 2009). In this study, RAPD and ISSR markers based on the polymerase chain reaction (PCR) were applied. The value of RAPD analysis for efficient germplasm management in plants is already known (Young, 2000; Jaroslava et al., 2002). The technique is quick, easy and requires less time. This detects nucleotide sequence polymorphisms using a single primer of arbitrary nucleotide sequence (Welsh and McClelland, 1990; Williams et al., 1990). ISSR permits detection of polymorphisms in inter-microsatellite loci, using a primer designed from dinucleotide or trinucleotide simple sequence repeats. Only a few papers comparing re- sults obtained by different molecular genetic methods have been published in the case of Brassica napus L. The capability of individual methods to differentiate the analyzed canola genotypes is described here. The present study was therefore undertaken (1) to determine the efficiency of RAPD and ISSR markers for estimating the genetic diversity and (2) to estimate the genetic diversity of canola genotypes based on molecular characterization. For this purpose, 10 canola (Brassica napus L.) genotypes were analyzed and the results of genetic distances estimated by ISSR and RAPD markers were compared.

PCR based molecular markers such as RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeats) were employed for the evaluation of genetic diversity among twenty varieties of Brassica juncea. Mean polymorphism information content (PIC) value was greater for RAPD (0.4195) as compared to ISSR (0.2602). In RAPD analysis, 98.9% loci were polymorphic whereas in ISSR, 94.8 % were polymorphic. The number of loci in RAPD profile ranged from 7 to 10 with an average of 9.3 per primer whereas in ISSR, these were from 3 to 12 with an average of 6.8 loci per primer. RAPD based genetic similarity ranged from 0.224 to 0.842 whereas ISSR derived genetic similarity 0.467 to 0.880. The mental test between two Jaccard’s similarity matrices gave r = 0.89, showing good fit correlation in between ISSR‐ and RAPD‐based similarities. The results obtained from the consensus tree constructed from RAPD+ISSR marker more likely support the distribution of the twenty genotypes of B. juncea based on ISSR analysis. The twenty varieties were clustered into three main clusters 1, 2, and 3 respectively. In combined dendrogram study, each cluster has 13, 3, and 4 varieties.