Random Amplified Polymorphic DNA Markers Research Articles

Gene Expression and Molecular Differences in Two Wheat Genotypes under Salt and Drought Stresses

T HIS STUDY was conducted to point out some highlights on the difference between two contrasting tolerant wheat varieties regarding to genetic expression and molecular characterization under salt and drought stress. Two different tolerant wheat genotypes namely, Gemmiza9 (sensitive) and Sakha93 (tolerant) were used to investigate the molecular and genetic differences under salt and drought stress. Stress treatments were used NaCl and poly ethylene glycol (PEG) of different levels. Sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS PAGE) analysis was revealed three disappeared bands with 15, 25, 90 kilodalton (KD) in salt treated plants of Gemmiza9 and appeared these bands as constant in controlled and salt treated plants of Sakha93. Contradict vision for protein profile was under drought stress that constant bands were disappeared and new synthetic bands were exhibited in both Gemmiza9 and Sakha93. Moreover, Random amplification of polymorphic DNA (RAPD) markers were produced several number of unique bands in Sakha93. Primer 2 generated unique bands in Gemmiza9. Simple sequence repeats (SSR) markers were 100% polymorphism. WMC_179 marker out of six markers was present unique alleles only in Sakha93. This marker might be practical and advantageous to the genotype that recognized to tolerance QTL markers alleles to utilize in marker assisted selection in further study. Comparison between RAPD and SSR markers was revealed that QND was less (0.06) in RAPD than in SSR (0.56). EMI was higher (0.31) in RAPD than in SSR (0.23). Nonetheless, the findings might be valuable and helpful to identify the genetic and molecular characterizations of different tolerant genotypes.

Evaluation of Genetic Diversity Among Exotic Sorghum (Sorghum bicolor L. Moench) Genotypes Through Molecular Based Analysis (RAPD-PCR)

There is a remarkable diversity among sorghum species; hence it requires a powerful marker system for genome characterisation. RAPD (randomly amplified polymorphic DNA) is a very favourable technique used to distinguish the sorghum genotypes due to its clarity and speed. We observed genetic diversity among 30 sorghum (Sorghum bicolor L. Moench) genotypes using RAPD markers. Sixteen RAPD markers produced a total of 148 bands with a mean of 9.25 fragments per loci. Out of the 148, 132 bands showed polymorphism (89.19%), and 16 bands showed monomorphism (10.81%). PIC (polymorphism information content) values were varying from 0.2035 to 0.3438 with a mean of 0.2792. Genetic distance was ranged from 0.013 (for genotype 4 and 8) to 0.807 (for genotype 10 and 27). Cluster analysis exhibited that dendrogram consists of four major groups. Results represent that genotype 1, 3, 5, 15, 6, 11, 4, 8, 26, 29, 20, 24, 14, 25, 19, 23, 2 and 13 were closely related to each other, and genotype 10 was the most diverse genotype among all the studied genotypes by making an independent cluster. The first two factors of principal component analysis (PCA) PC1 (15.06) and PC2 (10.98) had the highest contribution in variability as 10.18 and 7.42%, respectively. Thus, sorghum genotypes can be isolated from each other at the molecular level by using molecular markers.

Assessment of genetic diversity of different tomato genotypes using RAPD markers

The present investigation was carried out for assessment of genetic diversity among the 28 tomato genotypes though three random amplified polymorphic DNA (RAPD) markers. A total of 15 distinct DNA fragments ranging form 100-1000 bp were amplified by using three selected primers of which 5.00 polymorphic bands per primer and over all polymorphic loci was 100 percent. The extent of genetic diversity among these genotypes was computed through parameters of genetic diversity and Shanon’s information indices. The highest genetic distance was observed among the accession Cl-3d-0-99 (V93) vs. F1 (G X V12), F1 (G X V17), F1 (G X V29) and Durch fuegel (G) vs. F1 (G X V17) and F1 (G X V93) vs. F1 (G X V12), F1 (G X V17), while the lowest genetic distance was observed among the accessions Fut. Wed Abrid (V94) vs. Sunlight pole (V67) and F1 (V67 X VG) vs. F1 (V17 X VG), F1 (V93 X VG) and F1 (V93 X V17) vs. F1 (V17 X VG), F1 (V67 X VG), F1 (V93 X VG). The Unweighted Pair Group Method of Arithmetic Mean (UPGMA) dendrogram based on Nei’s genetic distance divided the genotypes into two main clusters: A & B. Cluster ‘A’ consists of 19 accessions and cluster ‘B’ consists of 9 accessions. The information generated from this study could be useful in gene mapping and marker assisted breeding for future tomato breeding programs.
 Progressive Agriculture 29 (4): 276-283, 2018

Evaluation of subculture ages on organogenic response from root callus and SPAR based genetic fidelity assessment in the regenerants of Hibiscus sabdariffa L.

The efficacy of an organogenesis based regeneration protocol and the occurrence of somaclonal variation has been studied up-to eighth subculture of Hibiscus sabdariffa L. var. HS4288. Initially, the induction of organogenic calli from 83.33% root explants was standardized in 2.26 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 μM kinetin (KIN) supplemented Driver-Kuniyuki Walnut (DKW) basal medium. The calli were maintained up to the eighth subculture (each with 6-weeks duration) in the same basal medium along with 1.13 μM 2, 4-D and 4.65 μM KIN. The highest regenerative efficiency of the root-derived calli was found in 1.08 μM α-Naphthaleneacetic acid (NAA) and 8.88 μM 6-Bezyladenine (BA) containing DKW medium. This particular medium produced 14.30 ± 0.47 numbers of visible shoots (>10 mm in length) at the first subculture from 425 ± 35 mg of callus through the process of organogenesis. The morpho-histological study confirmed the organogenic mode of regeneration. With the gradual increase of culture passage, the frequency of shoots was steadily declined. The genetic polymorphism of the regenerated plants from eight successive sub-cultures was assessed using three different single primer amplification reaction (SPAR) methods; viz. Random amplified polymorphic DNA (RAPD), Direct amplification of minisatellite DNA (DAMD) and Inter simple sequence repeat (ISSR) markers. The SPAR analysis revealed that up to fifth subculture all the three types of primers produced the monomorphic banding pattern among the regenerated plants with that of their mother plant. From the sixth subculture onwards there were some alterations in banding profiles both in RAPD and ISSR markers but DAMD marker did not show any polymorphism up to eighth subculture. In the present work, the highest polymorphism percentage was recorded among the plants of eighth subculture by using both RAPD (41.61%) and ISSR (31.65%) markers. The SPAR method ensured that the present protocol may be used for true to type plant production up-to five consecutive subcultures.

Genetic Diversity among some Egyptian Barley (Hordeum vulgare L.) Cultivars Based on Protein and RAPD Markers

Genetic variability of seven Egyptian barley cultivars was analyzed utilizing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and random amplified polymorphic DNA (RAPD) techniques. The results of SDS-PAGE produced 12 bands ranging from 135 to 11 KDa. Two polymorphic bands at molecular weight (MW) 13 and 12 KDs were detected with 16.6% polymorphism among barely cultivars. In the other hand, five RAPD primers were employed to assess the genetic diversity and relationships between the seven Egyptian barley cultivars. RAPD analysis exhibited 93 amplicons (86% polymorphism) with an average number of 18.6 amplicons per primer. Genetic similarity of RAPD and protein input ranged from 0.31 to 0.83. The dendrogram of combined data had clustered all the cultivars into two main clusters; the first one containing all barley cultivars except Giza 123 cultivar which formed a separate cluster indicating that the genetic background of this cultivar was distinct from all cultivars. The results obtained from RAPD and protein analysis exhibit different level of polymorphism. RAPD profile is more suitable technique than SDS-PAGE in assessing genetic variation among the seven barely cultivars. Moreover, the present results suggest that, the increasing number of primers and using more different markers could be more accurate to discriminate the genetic variance between barley cultivars. Subsequently, this could be useful to differentiate between barley cultivars in the breeding program.

Genetic analysis of selected Sargassum fusiforme (Harvey) Setchell (Sargassaceae, Phaeophyta) strains with RAPD and ISSR markers

Since the 1980s, Sargassum fusiforme has been cultivated in Zhejiang, South China, and nowadays it becomes one of the important commercial seaweeds in China. With traditions of eating habits in the East Asian countries, this brown alga is used as food, because it contained functional oligo/polysaccharides and chemical components, and was regarded playing roles in antioxidant activities and regulating immunology. Through over 15 years' selection, breeding and cultivation, we obtained three strains with good traits and testified their characters during the production, which included the cultivars with high yield and other two good characters, either all the selected strains were applied in the Sargassum production. To avoid confusion during the selection and nursery, it was preferred to establish one fingerprint for distinguishing the Sargassum cultivars from different strains. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) methods were adopted to analyze the genetic diversities of the selected S. fusiforme strains. With that, one fingerprint with RAPD markers was constructed, and one sequence characterized amplified region (SCAR) marker to S. fusiforme was obtained. It is indicated that the applied fingerprint could be valid in S. fusiforme genetic and germplasm justification, and will be positive to molecular marker assistance in its selection and cultivation.

Diversity Analysis of 15 Hibiscus Accession Based on RAPD Marker

Genus Hibiscus consist of 300 species in tropical and subtropical regions. Indonesia has many species of Hibiscus genus such as Hibiscus rosa-sinensis, kenaf, rosella, waru, sharon, and others. These species have similar flower morphology despite their different benefits. Flower morphology can be use as morphological marker characters to identify the genetic relationship in one genus of Hibiscus. However, morphological markers are less accurate because they are strongly influenced by the environment, requiring quite amount of time, and showing limited and inconsistent diversity. These limitations make researcher chooses molecular markers that are considered more accurate because the material used is plant DNA. One of the most used molecular marker is Random Amplified Polymorphic DNA (RAPD). This study used DNA extraction from the leaf of 15 Hibiscus genus. The next steps were DNA quantification, temperature and primer optimization, DNA amplification with PCR, electrophoresis with agarose gel, and data analysis. The result showed 10 primers operon used for Hibiscus to produce many polymorphic bands. Analysis result showed a high diversity in control population (Hibiscus cannabinus (K1), Hibiscus sabdariffa (K2) Hibiscus mutabilis (K3), Hibiscus syriacus (K4), Hibiscus schizopetalus (K5)) while in Hibiscus rosa-sinensis, the diversity was low especially on the genotypes of flowers with the same color. Molecular marker is a right way to identify the diversity in a population. Control group which consist of Hibiscus cannabinus, Hibiscus sabdariffa, Hibiscus mutabilis, Hibiscus syriacus and Hibiscus schizopetalus has high heterozygosity means control group has high diversity. From the PCA result, grouping in control plants is based on their age (perennial and annual), while in Hibiscus rosa-sinensis the grouping is based on the flowers color. Hibiscus rosa-sinensis is cross pollinated plants caused the position stigma higher than anthers, its prooved by this experiment that varians within population Hibiscus rosa-sinensis higher than among population.

Investigating the virulence and genetic diversity of Colletotrichum lindemuthianum populations distributed in the North Western Himalayan hill states

Bean anthracnose caused by Colletotrichum lindemuthianum is one of the devastating diseases in common beans possessing wide genetic and pathogenic variability. Sixty isolates of C. lindemuthianum collected from three North-Western Himalayan States were characterized on differential bean cultivars and analyzed by random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR). Based on pathogenicity tests and CIAT-defined binary race-classification system, 60 isolates belonged to 27 races. Out of 27 races, 19 races were identified from Himachal Pradesh, three from Jammu and Kashmir (J&K) and 13 from Uttrakhand. Predominant races present in North Western Himalayas were 17 and 503, although 25.9% (7 out of 27) of the races were detected repeatedly. In addition, this is the first study on characterization of C. lindemuthianum races from J&K and Uttrakhand. The virulence analysis on differential cultivars from Andean and Mesoamerican gene pool suggested four interaction types in accordance with the genetic origin of the cultivars that were attacked by a particular race. The interaction type I with race 0 isolates was avirulent on all the differential cultivars, while the races placed in interaction type IV attacked the genotypes from both the gene pools, thereby complicating the designing of resistance breeding strategies in this part of the world. The genetic diversity analysis using RAPD and ISSR markers revealed higher polymorphism with RAPD markers compared to ISSRs. The population STRUCTURE analysis revealed that most of the isolates from three States shared unique haplotypes based on Bayesian clustering. The RAPD and ISSR markers based analysis of C. lindemuthianum isolates grouped in different races revealed a very low level of similarity coefficient and thus their correlation with pathogenic races could not be established.

Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant.

Rauwolfia tetraphylla L. is an important medicinal plant species which is well known for its pharmaceutically important alkaloids. In the present study, we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol of indirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codon targeted (SCoT), inter simple sequence repeats (ISSR), and randomly amplified polymorphic DNA (RAPD) marker based analysis. Initially friable callus was induced in maximum amounts (378.7, 323.8, and 412.8 in mg) from leaf, root, and stem explants on Murashige and Skoog (MS) media supplemented with 5.0 mg/L, 3.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/L of naphthalene acetic acid (NAA), respectively. Shoot regeneration with the maximum number of shoot buds (25 and 20) was obtained from leaf and stem calluses on MS media supplemented with TDZ (0.25 mg/L) + BAP (2 mg/L). The regenerated shoots were rooted successfully with maximum rooting percentage of 98.0 on full strength MS media amended with IAA (1.0 mg/L) and IBA (1.0 mg/L). The regenerated plantlets were hardened using 2:1 ratio of sterile garden soil and sand, followed by acclimatization in field conditions with 86% of survival. SCoT, ISSR, and RAPD primers based polymerase chain reaction (PCR) analysis was carried out to check possible genetic variations in micro propagated plants in comparison with mother plant. Among the ten SCoT (S), ISSR (R), and RAPD (OPA) primers used, S2, R10, and OPA3 has given good amplification with scorable DNA bands. The results revealed that the regenerated plants did not have any polymorphism with mother plant. Hence, the in vitro regenerated R. tetraphylla plantlets were confirmed as true-to-type.

Analysis of Genetic Diversity Among ‘Misket’ Apple Clones Using AFLP, SSR and RAPD Markers

Knowledge of genetic relationship among genotypes is an important concern for utilization of germplasm resources in breeding. The need for knowledge in local apple cultivars is increasing and molecular marker analysis has contributed to understanding of the apple genetic diversity and phylogenetic relationship between genotypes. Three different DNA-based techniques, random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) were used for detecting genetic variation among the 30 local ‘Misket’ apple genotypes. A total of 423 bands amplified and 205 polymorphic band were obtained with three marker techniques. Thirty RAPD primers amplified 207 bands, of which 91 were polymorphic (40.1%), 10 SSR primers produced 33 bands and 26 of them were polymorphic (78.78%) and 5 AFLP combinations amplified 183 bands of which 88 were polymorphic (48.08%). The average numbers of polymorphic marker per primer combination were 17.6, 3.03 and 2.6 for AFLP, RAPD and SSR respectively. Our results indicate that SSR was less informative in characterizing closely related ‘Misket’ apple genotypes, compared with RAPD and AFLP markers. AFLP seemed to best suited for evaluating the genetic relationship among the apple genotypes. Closely related genotypes may require more than one marker technique. The findings will be useful to identify and evaluate the local growing apple genetic resources.

Genetic polymorphism among seven entomopathogenic nematode species (Steinernematidae) revealed by RAPD and SRAP analyses

The strength of sequence-related amplified polymorphism (SRAP) in comparison to random amplified polymorphic DNA (RAPD) in determining the genetic polymorphism among seven species of entomopathogenic nematodes (Steinernematidae) was tested. Nine RAPD and 12 SRAP primer pair’s combinations were used. The number of polymorphic bands and the polymorphism percentages was high in SRAP analysis (97 out of 107 bands were polymorphic (90.6%)) compared to RAPD (65 out of 89 bands were polymorphic (73%)). The highest number of RAPD bands was recorded for OPE-A-07 primer (14 band) while OPE-D-20 was scored the lowest band number (7 bands). The SRAP primers Me3-em2 registered the highest number of bands (13 bands) while Me3-em3and Me2-em2 showed the lowest band number (6 bands). The genotype-specific SRAP and RAPD markers for the different ENP species were recorded. The highest number of SRAP specific markers (7 markers) was scored for Steinernema glaseri, then 4 markers for S. abbasi 2 followed by S. riobrave (3 markers), 1 marker for S. abbasi 1, S. riobrave recorded the highest number of specific RAPD marker (4 specific markers) followed by S. carpocapsae DD 136 and S. glaseri (two specific markers), then only 1 specific marker for S. abbasi 1. Based on the data obtained from RAPD and SRAP analysis, the dendrogram was created to clarify the genetic distances among different species of the studied entomopathogenic nematodes. The present study indicated that SRAP was more powerful than RAPD analysis for detecting the genetic polymorphism among closely related species of nematodes. The genotype-specific markers detected from both RAPD and SRAP can be used in future biological control programs.

Enhanced bacoside content in polyamine treated in-vitro raised Bacopa monnieri (L.) Wettst

The study was designed to evaluate the role of various plant polyamines (PAs) on in-vitro multiplication and regeneration in the Ayurvedic memory enhancer Bacopa monnieri (L.) Wettst. Natural populations of B. monnieri were collected, sterilized and micropropagated in Murashige and Skoog (MS) media supplemented with different concentration and combination of plant growth regulators (PGRs) and PAs. MS media supplemented with 6-benzylaminopurine (BAP) (2.0 mg/l) + spermidine (spd) (1 mM) and in another set, kinetin (kin) (2.0 mg/l) + spd (1 mM) were found to be the best multiplication media whereas MS+ indole-3-butyric acid (IBA) (1.0 mg/l) + spermine (spm) (1.0 mM) produced highest average number of roots per shoot. After 4–6 weeks of incubation, nodal explants cultured in MS media supplemented with various PGRs and PAs produced green compact or white friable nodular base calli (NBC). The NBC showed organogenic potential when subjected to MS media supplemented with cytokinins and PAs. Clonal fidelity of the regenerated plants was assessed using inter-simple sequence repeat (ISSR) and random-amplified polymorphic DNA (RAPD) markers. Out of the 60 clear and reproducible bands recorded, 54 were found to be monomorphic displaying high degree of monomorphism (90%) and lower polymorphic level (10%) between the mother plant and in-vitro regenerants. High-performance liquid chromatography (HPLC) revealed total bacoside content in B. monnieri grown in-vitro (PA-treated) and the mother plant (MP) grown in nature to be 27.89 and 25.46 mg/g respectively. The phytochemical analysis of the PA-treated micropropagated plants also revealed significantly higher levels of phenolics and flavonoids with comprehensively higher antioxidant potentials in PA-treated in-vitro grown plants (PATP) and the subsequently acclimatized plants (PATP-A) over the PA-untreated in-vitro grown plants (with/no PGR) (PAUP) and the mother plant (MP).

Estimation of genetic variation in green tiger prawn, Penaeus semisulcatus by using random amplified polymorphic DNA, inter simple sequence repeat and simple sequence repeat markers

In this study, three molecular markers including random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) were used to evaluate genetic variation of green tiger prawn Penaeus semisulcatus collected from two geographically isolated environments; located in the Manifa, Arabian Gulf, Saudi Arabia and Ataka, Suez Gulf, Egypt. Genetic parameters included the percentage of polymorphism (P%), effective alleles (Ne), Nei genetic diversity (H) and Shannon index (I), which were calculated based on molecular data. All three marker systems distinguished genetic variation of P. semisulcatus in various levels. The highest polymorphism (91.30%) was obtained with SSR, followed by ISSR (82.26%) and RAPD markers (62.04%), respectively. Our results indicate that SSR appeared to be the best suited molecular assay for assessing the genetic variation between genotypes of P. semisulcatus. The present study indicated that Manifa and Ataka genotypes were closely related. Moreover, the analysis of variability could require more than one DNA‐based molecular marker techniques.

Molecular markers based genetic fidelity assessment of micropropagated Dendrobium chrysotoxum Lindl

Plant tissue culture is one of the most important methods used for effective conservation of many rare and endangered orchids. Genetic variation may appear in regenerants due to numerous factors associated with in vitro culture conditions. Assessment of genetic stability of in vitro regenerants is highly essential if true to type plants are the desired end product. In the present investigation, genetic homogeneity testing of micropropagated Dendrobium chrysotoxum was performed using 12 random amplified polymorphic DNA (RAPD) and 11 inter-simple sequence repeat (ISSR) primers. Molecular marker analysis unveiled high genetic monomorphism (96.30%) and low genetic polymorphism (3.6%) among the in vitro clones and mother plant. Nei's genetic distance ranged from 0.00 to 0.037 disclosing high genetic similarity between the clones. Dendrograms generated from RAPD and ISSR marker analysis also revealed high genetic proximity with mother plant and micropropagated clones. Principal coordinate analysis (PCoA) further confirmed the grouping of plants in accordance with the cluster analysis determined by unweighted pair group method using arithmetic averages (UPGMA) analysis. The study reported the successful clonal fidelity assessment of micropropagated D. chrysotoxum using RAPD and ISSR markers.

English

Tinospora cordifolia is a highly important medicinal plant in India, with a rich source of different secondary metabolites. In the present study, 12 samples of T. cordifolia (collected from different areas of Rayalseema region,Andhra Pradesh) were studied for genetic diversity using 15 random amplified polymorphic DNA (RAPD) and eight inter-simple sequence repeats (ISSR) markers. Out of them, 10 RAPD and 5 ISSR primers showed 122 polymorphic reproducible bands across the selected 12 samples of T. cordifolia. The average polymorphic index content (PIC) of RAPD and ISSR values was 0.27 to 0.41 and 0.17 to 0.41, respectively. The PIC with RAPD markers was the highest for primers OPV 17 (0.41) and OPW 17(0.41) followed by the primer OPB 20 (0.40). The PIC with ISSR markers was the highest for the primer 824 (0.41) followed by primer 867 (0.36). Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis of genetic similarity indices grouped all the samples into two major clusters. Jacquard’s coefficient of similarity varied from 0.51 to 0.77, indicating high levels of genetic variation across genotypes under study. The result of this study can be used for characterization of potential T. cordifolia genetic resources and their utilization as breeding materials, micropropagation and secondary metabolites screening and harvesting. T. cordifolia, being a medicinal plant, is very useful for discovery of new lead molecules from this plant for drug discovery and development studies. Key words: Tinospora cordifolia, genetic diversity, random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), unweighted pair group method with arithmetic mean (UPGMA).

Genetic diversity of Rhizopus microsporus from traditional inoculum of tempeh in Indonesia based on ITS sequences and RAPD marker

Abstract. Barus T, Halim R, Hartanti AT, Saputra PK. 2019. Genetic diversity of Rhizopus microsporus from traditional inoculum of tempeh in Indonesia based on ITS sequences and RAPD marker. Biodiversitas 20: 847-852. The main microorganism for tempeh fermentation is Rhizopus microsporus. These days, many tempeh producers use commercial inoculum, such as ‘Raprima’ as resource of R. microsporus. As a result, the genetic diversity of R. microsporus that had been reported in Indonesia has diminished. Information about genetic diversity is needed as a basis to select R. microsporus as tempeh inoculum. This research aims to investigate the genetic diversity of R. microsporus from waru leaves based on Internal Transcribed Spacer (ITS) Sequence and Random Amplified Polymorphic DNA (RAPD) markers. A total of 25 R. microsporus were isolated from traditional inoculum waru leaves (Inoculum 1) and traditional inoculum other than waru leaves (Inoculum 2). Amplification of ITS sequence was done using universal primer pairs of ITS-4 and ITS-5. Amplification of RAPD markers was done using primers OPC-08, OPC-19, OPQ-6, R-108, OPA-09 and OPJ-20. ITS sequence was not sufficient to compare the similarities among R. microsporus. On the other hand, RAPD markers successfully compared the similarities among 25 R. microsporus. A total of 25 R. microsporus were divided into 9 clusters. R. microsporus from Inoculum 1 grouped into Cluster 1, Cluster 3 and Cluster 4-8. Inoculum 2 grouped into Cluster 2 and Cluster 9. R. microsporus from tempeh grouped into Cluster 4 and was different from Inoculum 1 and Inoculum 2, except for TB3.

Genetic diversity and phylogenetic relationship of Nilgirianthus ciliatus populations using ISSR and RAPD markers: Implications for conservation of an endemic and vulnerable medicinal plant

Abstract Nilgirianthus ciliatus is of interest for its extensive applications in Ayurvedic medicine. The study focuses on the assessment of genetic diversity and phylogenetic relationships in N. ciliatus populations to aid conservation of this species at risk of extinction. Thirty five inter simple sequence repeats (ISSR) and thirty random amplified polymorphic DNA (RAPD) markers were employed in PCR yielding 298 and 99 bands, respectively. Using ISSR markers, bands of 200–3500 bp were obtained and accounted for 77.0% of polymorphism observed. The polymorphism information content (PIC) and resolving power (Rp) values were 0.59 and 20.8, respectively. RAPD analysis resulted in 99 total bands of which 89 were polymorphic (89.9%). The PIC value for RAPD was 0.61 and Rp value was 18.8. The two different cluster analyses (UPGMA and Neighbor Joining) and principal component analysis showed the genetic variation among the populations. Clustering of N. ciliatus populations from different geographical regions in distinct groups can be a good sign for clear separation that is supported by high bootstrap values. Overall, both ISSR and RAPD analyses were valuable for identifying genetic variations and phylogenetic relationships in N. ciliatus populations at the DNA level and these differences provides the basic knowledge for better conservation management policies and genetic identification of the different geographical populations of this plant.

Patterns of Genetic Variation in a Rare and Endangered Plant: Symplocos racemosa

Although comprehensive studies have been carried out on the genetic structure of rare and endangered plants, the populations of Symplocos racemosa from Odisha have never been studied. We collected 60 individuals from 6 natural populations of S. racemosa distributed in Odisha. We adopted random amplified polymorphic DNA (RAPDs) to investigate the genetic diversity within and among populations, and the correlation between the genetic and geographic distances. Twenty RAPD primers produced 191 polymorphic bands (83.04%) in about 60 individuals. The AMOVA analysis showed about 41.7% of the total genetic variation existed within populations and established possible geographic structure. In the study, it was noticed that S. racemosa has higher genetic diversity at species level and lower diversity at population level as revealed by RAPD markers. Out of all the populations, Daitari mines population is having highest genetic diversity. So this population is suitable for conservation and future propagation of this plant.

Molecular diversity in coriander (Coriandrum sativum) using RAPD and ISSR markers

Coriander (Coriandrum sativum L.) is an annual aromatic herb that belongs to Apiaceae family. Present investigation was carried out to explore the molecular characterization in 24 coriander genotypes. For molecular characterization two DNA based molecular marker techniques, viz. random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), were used. A total of 27 polymorphic primers (18 random and 9 ISSR) were used. Amplification of genomic DNA of 24 genotypes, using RAPD analysis, yielded 83 fragments, in which 57 (70.46%) were polymorphic. The 9 ISSR primers produced 37 bands across 24 genotypes, of which 17 (49.81%) were polymorphic. Based on the similarity matrix data dendrogram were prepared using UPGMA method. Genotypes were also classified into groups and several subgroups. Principal coordinate analysis (PCA) confirmed the separation of coriander genotypes into groups comparable to those from UPGMA analysis. The high rate of polymorphic lines generated by RAPD and ISSR markers indicated that the method is efficient to analyze molecular diversity in coriander genotypes and that the molecular divergence can be used to establish consistent heterotic groups between coriander genotypes.

Population genetic structure and diversity analysis in Hedychium coronarium populations using morphological, phytochemical and molecular markers

Hedychium coronarium (Zingiberaceae) is a perennial rhizomatous herb widely used for its aromatic, cosmetic and medicinal value. In the present research, the morphological, phytochemical and molecular diversity was assessed for 50 H. coronarium accessions collected from different states of Eastern India. The accessions were morphologically diverse with leaf number, leaf length, leaf width, rhizome diameter and rhizome dry weight characters showing the highest variation. A total of 44 constituents were identified in the essential oils with β-pinene (11.87–35.16%), eucalyptol (13.12–39.82%), linalool (1.58–41.33%) and coronarin-E (1.14–33.87%) as the predominant constituents. Using 10 Inter-simple sequence repeats (ISSR) and 5 Random amplified polymorphic DNA (RAPD) primers, a total of 71 and 34 bands were amplified, respectively. The average polymorphism, polymorphic information content, marker index were found to be 86.30%, 0.57, 3.26 and 87.58%, 0.55, 3.01 for ISSR and RAPD markers, respectively. Cluster analysis based on phytochemical and molecular characterization exhibited a clear relationship between diversity pattern and geographical origin. The percentage of variance explained using principal coordinate analysis (PCoA) was 70.31% and 71.91% for ISSR and RAPD markers, respectively. Analysis of molecular variance (AMOVA) analysis revealed high genetic variability occurred among the population than within populations. Bayesian analytical model and cluster analysis showed similar grouping patterns. The findings of the present research revealed a high degree of molecular and phytochemical variation among H. coronarium populations in relation to geographic locations. The results of the present research can be exploited for conservation and breeding programmes of H. coronarium for its genetic improvement.