Molecular identification and genetic relationships among Alcea (Malvaceae) species by ISSR Markers: A high value medicinal plant

Alcea L. is one of the largest genera of Malvaceae family with nearly 70 species worldwide mainly distributed in SW Asia. According to the latest revision of the family, it is represented by 34 species in the Flora of Iran, among them, 15 species are endemic. It is tough to accurate germplasm/ plant recognition by using morphological characteristics because of its propagation, growing and using. We conducted a molecular data analysis on these plant species due to their importance. We examined 156 plants from 14 species in 16 regions that were selected randomly for this investigation. It has been 119 polymorphic bands (94.33%) were resulted from 128 bands of 10 primers in amplification of genomic DNA. ISSR primers have a great capacity to detect polymorphic loci among Alcea species, as evidenced by the high average PIC and MI values found. The genetic similarity of 14 species was calculated and ranged between 0.635 to 0.990. Inter-Simple sequence repeats (ISSR) markers research revealed that Alcea tarica Pakravan & Ghahreman and Alcea kopetdaghensis lljin had the least similarity, while Alcea semnanica Pakravan and Alcea mazandaranica Pakravan & Ghahreman had the most. The current study attempts to answer three questions: 1) can ISSR markers identify Alcea species? 2) what is the genetic structure of these taxa in Iran? and 3) what is the inter-relationship between these taxa? The current study discovered that ISSR markers can be used to identify species.

Genetic structure and diversity among individuals of Copaifera langsdorffii Desf. from Mato Grosso, Brazilian Amazon, using ISSR markers

The Amazon is the largest tropical forest in the world and is home to around 20% of all the biodiversity on the planet, among the species present in the Amazon is Copaifera langsdorffii, exploited mainly for the extraction of oil-resin and wood, often in ways incorrect, which can cause the loss of genetic variability. The aim of this study was to evaluate the genetic structure and diversity among individuals of C. langsdorffii located in Mato Grosso, Brazil, using ISSR markers. We sampled leaves from 27 adult individuals of C. langsdorffii, whose total genomic DNA was extracted. A total of 12 ISSR primers were used for the molecular characterization of the individuals. A grouping analysis was performed using the unweighted pair group method, Bayesian analysis and characterized by the genetic diversity. The genetic diversity among and within the groups was demonstrated by the AMOVA. As a result, 106 fragments were amplified and 98.11% were polymorphic. The polymorphic information content of each primer ranged from 0.45 to 0.81. The dendrogram showed the formation of 4 distinct groups. The greatest genetic variability is found within the groups and not between them. The percentage of polymorphism, genetic dissimilarity values and genetic diversity indexes indicate that there is high genetic variability among Copaifera langsdorffii individuals, suggesting that ISSR primers were efficient in detecting polymorphism in this species and that the individuals have potential for compose programs aimed at the preservation of the species and the ability to integrate germplasm banks.

Genetic identity of mint cultivars after in vitro conservation, assessed by ISSR primers

The species of the genus Mentha have been known since ancient times and have significant value in the pharmaceutical, cosmetic and food industries, as well as in medicine. For the widespread use of mint, including in a variety of breeding programs, and the preservation of genetic diversity, effective methods of maintaining cultivars and collection samples are required. Thanks to the development of biotechnological methods, in particular, the creation of slow-growing collections, are now actively used as an effective alternative to field collections. It is known that the cultivation of tissues and organs on artificial culture media can cause somaclonal variability. The purpose of this work is to study the effect of in vitro storage at 4-6°C without illumination after 3 and 4 in vitro conservation cycles on the genetic stability of three cultivars of mint Azhurnaya, Bergamotnaya and Zagrava using ISSR primers. 1 cycle: 1 year of in vitro conservation, microcutting and 2 subcultures of regrowth in a culture room. After conservation, the number of viable explants was 70.0-82.1%. Callus formation at the base of the shoots was not observed in any of the cultivars. After 3 and 4 cycles of in vitro conservation, genetic stability was assessed using 11 ISSR primers. It was found that all three mint genotypes showed full compliance (length and number of amplicons) with the profiles of control samples for all studied markers. It was also found that the markers used by ISSR are highly informative for mint cultivars.

Molecular marker for characterization of traditional and hybrid derivatives of Eleusine coracana (L.) using ISSR marker

Background Finger millet is the most important food grain in the world for its nutritional benefits. Finger millet is genetically and geographically diverse and widely spread in the African and Asian sub-continent. Therefore, the present study was undertaken to analyze the genetic diversity using ISSR genetic markers using 15 ISSR primers. Results About 23 genotypes of widely cultivated finger millet cultivars of economically important ones were characterized and the ISSR markers were critically analyzed for their performance with parameters such as polymorphic information content (PIC), effective multiplex ratio (EMR), marker index (MI), and resolving power (RP). In this study, 175 loci were scored across the 23 cultivars of finger millet, and out of these 173 loci (98%) were polymorphic, revealing the suitability of these loci for genetic diversity analysis with ISSR marker. The average number of polymorphic loci per primer was 11.50 with varying sizes from 100 bp to 2500 bp. ISSR primers that showed higher polymorphism were found to have higher EMR and MI values up to 15.30 and 13.44, respectively. Conclusion High degree of polymorphism supported with distinct differences of all the marker parameters revealed the suitability of ISSR markers for determining the genotypic differences based on ISSR markers among the 23 genotypes of finger millet. The possible application of the ISSR marker in the conservation and management of finger millet genetic resources is discussed.

Genetic diversity among asexual and sexual progenies of Phytophthora capsici detected with ISSR markers

The population structure of Phytophthora capsici among asexual and sexual progenies was analyzed using ISSR. Thirty asexual progenies of one parent and three sexual populations were assayed for genetic diversity using 5 ISSR primers and DNA from 120 offspring of P. capsici was amplified. In total, 71 reproducible ISSR fragments were obtained, of which 100% were polymorphic, revealing high polymorphism among the isolates. Among them, the percentages of polymorphism of sexual and asexual progeny isolates were 100.00 and 77.46%, respectively. Genetic similarity coefficients among all the isolates ranged from 0.54 to 0.73. The sexual offspring population showed much more variability than the asexual offspring population with 76.26% variability attributed to diversity within populations as compared with 23.74% among populations. This research reveals that the sexual progeny population of P. capsici contributes more genetic diversity than that of asexual progeny population.

Polymorphism in populations of Corydalis subjenisseensis s. l. (Papaveraceae) in the south of the Yenisei Siberia

Corydalis subjenisseensis (Antipova) is a tuberous ephemeroid characterized by a high morphological diversity. During the research work genetic polymorphism of 7 populations Corydalis subjenisseensis s. l., growing in the southof the Yenisei Siberia were analyzed using ISSR markers. The amplification of genomic DNA with 8 ISSR primers yielded100 DNA amplicons of which 78 were polymorphic. The number of amplified DNA fragments, depending on the primer,varied from 9 (ISSR-17) to 21 (HB14). The maximum level of genetic variation was observed for Western Sayan populations growing in aspen and fir forests. The genetic differentiation among populations (Gst) was 0.2415, indicating a highlevel of differentiation. The similarity dendrogram performed in the TFPGA program shows a division into 2 groups: thefirst group includes the Krasnoyarsk and Khakass populations of C. subjenisseensis, the second group includes the Tanzybei populations, which are characterized by a high level of polymorphism. A similar structure is observed when buildingclusters using the Bayesian approach. 69 genotypes are divided into a maximum of 7 genetic clusters. Among the populations of the Tanzybei, individuals of presumably hybridogenic origin are found, grouped around two centers of “attraction”.

Genetic Relationship in Sainfoin (Onobrychis viciifolia) Landraces Cultivated East Anatolia by using RAPD and ISSR Markers

Background: Sainfoin (Onobrychis viciifolia) is a forage crop that yields high in arid and calcareous soils and is cultivated in large areas. There aren’t many genetic diversity studies on the varieties of cultured sainfoin. This study was conducted to determine the genetic diversity and the degree of relationship between 23 cultivated landraces and one registered variety. Methods: To take samples from the populations, seeds were sown in the field in 2014. Samples were taken from the young leaves of the plants and preserved at -80oC in same year. RAPD and ISSR primers were used in the study. The bands obtained as a result of PCR were recorded and the data of both methods were also evaluated by combining them. Result: In the study, 5 RAPD and 4 ISSR primers were used and a total of 49 bands were obtained. Of 29 bands obtained using RAPD primers, 20 were found to be polymorphic and of 20 bands obtained using ISSR primers, 15 were found to be polymorphic. It was found that there was a very low correlation between the two methods. Using RAPD and ISSR markers and RAPD + ISSR combination, the similarity index among populations was found to be between 0.25-0.95, 0.5-1.00 and 0.45-0.91, respectively. The Nei’s genetic diversity index was found to be between 0.3365, 0.2656 and 0.3018 with RAPD, ISSR primers and RAPD + ISSR combination, respectively. Based on the dendrograms obtained using RAPD, ISSR primers and RAPD + ISSR combination, the populations under analysis were classified into 3, 3 and 5 groups, respectively. With this study, the closest populations were identified and a significantly high genetic diversity was detected.

Genetic diversity and relationships among Hypericum L. species by ISSR Markers: A high value medicinal plant from Northern of Iran

Hypericum L. species are generally known locally in Iran with the names “Hofariqun” which Ebn Sina (or Bo Ali Sina) called it. Plants of the genus Hypericum have traditionally been used as medicinal plants in various parts of the world. Hypericum perforatum L. is the source to one of the most manufactured and used herbal preparations in recent years, especially as a mild antidepressant. Therefore, due to the importance of these plant species, we performed a molecular data for this species. For this study, we used 175 randomly collected plants from 17 species in 9 provinces. Amplification of genomic DNA using 10 primers produced 141 bands, of which 127 were polymorphic (95.78%). The obtained high average PIC and MI values revealed high capacity of ISSR primers to detect polymorphic loci among Hypericum species. The genetic similarities of 17 collections were estimated from 0.617 to 0.911. According to Inter-Simple sequence repeats (ISSR) markers analysis, H. androsaemum and H. hirtellum had the lowest similarity and the species of H. perforaturm and H. triquetrifolium had the highest similarity. The aims of present study are: 1) can ISSR markers identify Hypericum species, 2) what is the genetic structure of these taxa in Iran, and 3) to investigate the species inter-relationship? The present study revealed that ISSR markers can identify the species.

DNA fingerprinting, fixation-index (Fst), and admixture mapping of selected Bambara groundnut (Vigna subterranea [L.] Verdc.) accessions using ISSR markers system

As a new crop in Malaysia, forty-four Bambara groundnut (Vigna subterranea L. verdc.) genotypes were sampled from eleven distinct populations of different origins to explore the genetic structure, genetic inconsistency, and fixation index. The Bambara groundnut, an African underutilized legume, has the capacity to boost food and nutrition security while simultaneously addressing environmental sustainability, food availability, and economic inequalities. A set of 32 ISSRs were screened out of 96 primers based on very sharp, clear, and reproducible bands which detected a total of 510 loci with an average of 97.64% polymorphism. The average calculated value of PIC = 0.243, RP = 5.30, H = 0.285, and MI = 0.675 representing the efficiency of primer set for genetic differentiation among the genotypes. The ISSR primers revealed the number of alleles (Na = 1.97), the effective number of alleles (Ne = 1.38), Nei's genetic diversity (h = 0.248), and a moderate level of gene flow (Nm = 2.26) across the genotypes studied. The estimated Shannon’s information index (I = 0.395) indicates a high level of genetic variation exists among the accessions. Based on Nei’s genetic dissimilarity a UPMGA phylogenetic tree was constructed and grouped the entire genotypes into 3 major clusters and 6 subclusters. PCA analysis revealed that first principal component extracted maximum variation (PC1 = 13.92%) than second principal component (PC2 = 12.59%). Bayesian model-based STRUCTURE analysis assembled the genotypes into 3 (best ΔK = 3) genetic groups. The fixation-index (Fst) analysis narrated a very great genetic diversity (Fst = 0.19 to 0.40) exists within the accessions of these 3 clusters. This investigation specifies the effectiveness of the ISSR primers system for the molecular portrayal of V. subterranea genotypes that could be used for genetic diversity valuation, detection, and tagging of potential genotypes with quick, precise, and authentic measures for this crop improvement through effective breeding schemes.

Morpho-molecular characterization of Bipolaris and Exserohilum spp. infecting various agricultural crops

The study was undertaken with major objective to differentiate fungal isolates belonging to various species of Bipolaris and Exserohilum infecting various agricultural crops. After routine isolation and purification of more than 35 attempts, 13 isolates were morphologically characterized into 8 species namely, Bipolaris cynodontis (IQBC-1 and DQBC-2), B. holmii (IQBH-1), B. micropus (IQBM-1), B. oryzae (DQBO-1, DQBO-2), B. sorghicola (DQBS-1, DQBS-2), B. tetramera (DQBT-1, DQBT-2), Exserohilum longirostrata (IQEL-1) and E. rostratum (DQER-1 and DQER-2). Molecular characterization of all the isolates using RAPD, ISSR and URP markers also revealed genetic variation. Out of 24 RAPD primers, only 9 (OPA-2, OPA-4, OPA-9, OPB-4, OPB-8, OPC-1, OPC-2, OPC-5 and OPE-5) could amplify the DNA sequence showing good polymorphism. Out of 20 ISSR primers, only 6 (ISSR 1, ISSR 13, ISSR 15, ISSR 17, ISSR 19 and ISSR 20) could show good polymorphism. Out of 13 URPs, only 5 (URP 4R, URP 6R, URP 9F, URP 13R and URP 38F) could amplify with a greater number of bands. In all the three makers, similarity matrix value using Jaccard’s coefficient ranged from 0.149 to 0.548 revealing genetic co-relation among isolates. Combined clustering of RAPD, ISSR and URP data showed relatively low genetic similarity coefficient ranging from 14.92 to 54.76 among isolates which revealed stronger co-relation among isolates. Cluster analysis based on UPGMA provided a clear resolution of relationship among all the 13 isolates belong to 6 Bipolaris and 2 Exserohilum species and confirmed the morphological characterization of the same. Further, molecular markers especially, RAPD, ISSR and URP have revolutionized the genetic analysis of plant pathogens including fungi and their application can greatly help in characterizing related genera and delineation of inter and intra-species complex of fungi.