RAPD and ISSR Marker-Based Comparative Evaluation of Genetic Diversity Among Indian Germplasms of <Emphasis Type="Italic">Euryale ferox</Emphasis>: an Aquatic Food Plant

Euryale ferox Salisbury is an important aquatic food plant cultivated largely in eastern India. E. ferox is a monotypic genus, and breeding programmes have mostly relied on the variability present in the primary gene pool. Knowledge of the genetic structure of the population is limited, and there are very few reports available on the genetic diversity of E. ferox. In this study, comprehensive research on the genetic diversity of 16 germplasms of E. ferox was carried out using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 320 RAPD and 95 ISSR primers screened initially, 61 primers (40 RAPD and 21 ISSR) gave reproducible bands and were selected for further work. Amplification of the 40 RAPD primers gave 533 polymorphic bands with an average of 13.32 polymorphic bands per primer. The percentage of polymorphism ranged from 37.5 to 100, with an average of 88.3 %. The 21 ISSR primers produced 259 bands, of which 214 were polymorphic, with an average of 10.19 polymorphic bands per primer. The percentage of polymorphism using ISSR primers ranged from 50 to 100, with a mean of 82.6 %. Jaccard’s coefficient ranged from 0.45 to 0.69 (RAPD), 0.50 to 0.77 (ISSR) and 0.48 to 0.71 (RAPD and ISSR). Molecular characterization of different germplasms of E. ferox not only is essential for its conservation but also can be used in further breeding programmes.     

Studies on the genetic variation of the green unicellular alga Haematococcus pluvialis (Chlorophyceae) obtained from different geographical locations using ISSR and RAPD molecular marker

Haematococcus pluvialis (Flotow) is a unicellular green alga, which is considered to be the best astaxanthin-producing organism. Molecular markers are suitable tools for the purpose of finding out genetic variations in organisms; however there have been no studies conducted on ISSR or RAPD molecular markers for this organism. The DNA of 10 different strains of H. pluvialis (four strains from Iran, two strains from Finland, one strain from Switzerland and three strains from the USA) was extracted. A genetic similarity study was carried out using 14 ISSR and 12 RAPD primers. Moreover, the molecular weights of the bands produced ranged from 0.14 to 3.4 Kb. The PCA and dendrogram clustered the H. pluvialis strains into various groups according to their geographical origin. The lowest genetic similarity was between the Iran2 and USA2 strains (0.08) and the highest genetic similarity was between Finland1 and Finland2 (0.64). The maximum numbers of bands produced by the ISSR and RAPD primers were 35 and 6 bands, respectively. The results showed that ISSR and RAPD markers are useful for genetic diversity studies of Haematococcus as they showed geographical discrimination.     

Assessment of Genetic Stability in Traditional Ginger Cultivated in Manipur,India Based on Molecular and Chemical Markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyze the genetic stability of ten local cultivars collected from nine districts of Manipur, India with the released ginger variety Nadia. A total of 15 RAPD and 8 ISSR primers resulted in 107 and 53 distinct and reproducible bands, respectively. A lack of polymorphism revealed the genetic stability among the local cultivars. Unlike molecular markers, analysis of essential oil composition by gas chromatography–mass spectrometry (GC-MS) revealed variation among 11 clones. Among eight major constituents obtained by GC-MS technique, cinnamyl acetate was found only in IBSD/Z-41d cultivars, whereas, in IBSD/Z-41o no trace of trans-geraniol was observed. Moreover, concentration of 6-gingerol determined by high-performance liquid chromatographic (HPLC) method shows that IBSD/Z-41r contains the highest and IBSD/Z-41i contains the lowest gingerol percentage.     

An Improved Micropropagation of Arnebia hispidissima (Lehm.) DC. and Assessment of Genetic Fidelity of Micropropagated Plants Using DNA-Based Molecular Markers

An efficient and improved in vitro propagation method has been developed for Arnebia hispidissima, a medicinally and pharmaceutically important plant species of arid and semiarid regions. Nodal segments (3–4 cm) with two to three nodes obtained from field grown plants were used as explants for shoot proliferation. Murashige and Skoog’s (MS) medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) or naphthalene acetic acid was used for shoot multiplication. Out of different PGRs combinations, MS medium containing 0.5 mg l^?1 6-benzylaminopurine and 0.1 mg l^?1 IAA was optimal for shoot multiplication. On this medium, explants produced the highest number of shoots (47.50?±?0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under the greenhouse condition when the base (2–4 mm) of shoots was treated with 300 mg l^?1 of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhouse with 85–90 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro-regenerated plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened, 15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR) reproducible amplicons. The amplified products were monomorphic across all the micropropagated plants and were similar to the mother plant. To the best of our knowledge, it is the first report on the assessment of the genetic fidelity in micropropagated plants of A. hispidissima.     

Correlation between the genetic diversity and variation of total phenolic acids contents in Fructus Xanthii from different populations in China

Fructus Xanthii (Cang-Er-Zi) is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. For the effective quality control of its medicinal values, reflected by chemical variation patterns, in addition to the relationship with genetic diversity, analyses based on UV spectrophotometry, HPLC fingerprinting and inter-simple sequence repeat (ISSR) molecular markers were carried out, involving 16 Xanthium populations from different locations in China. The HPLC data showed considerable variation of chemical constituents among the 16 Xanthium populations, and they were classified to three chemotypes by hierarchical clustering analysis. Abundant genetic diversity was detected among the Xanthium populations, which were also clustered into three groups based on their ISSR data and varied according to different species. Combining the genetic divergence and chemical differences showed an important result that, in the two chemotypes, the higher contents of total phenolic acids (TPA) in Fructus Xanthii showed greater genetic diversity (I). We suggest that genetic diversity affects the contents of TPA. Since variable phenolic acid contents may affect therapeutic efficacy, it is important to point out that combining the use of genetic base with chemotype will help control the favourable chemotypes and breed new cultivars with more desirable chemical constituents.     

In Vitro Propagation and Assessment of the Genetic Fidelity of Musa acuminata (AAA) cv. Vaibalhla Derived from Immature Male Flowers

An efficient in vitro propagation method has been developed for the first time for Musa acuminata (AAA) cv. Vaibalhla, an economically important banana cultivar of Mizoram, India. Immature male flowers were used as explants. Murashige and Skoog’s (MS) medium supplemented with plant growth regulators (PGRs) were used for the regeneration process. Out of different PGR combinations, MS medium supplemented with 2 mg L^?1 6-benzylaminopurine (BAP) + 0.5 mg L^?1 α-naphthalene acetic acid (NAA) was optimal for production of white bud-like structures (WBLS). On this medium, explants produced the highest number of buds per explant (4.30). The highest percentage (77.77) and number (3.51) of shoot formation from each explants was observed in MS medium supplemented with 2 mg L^?1 kinetin + 0.5 mg L^?1 NAA. While MS medium supplemented with a combination of 2 mg L^?1 BAP + 0.5 mg L^?1 NAA showed the maximum shoot length (14.44 cm). Rooting efficiency of the shoots was highest in the MS basal medium without any PGRs. The plantlets were hardened successfully in the greenhouse with 96 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro regenerated plantlets of M. acuminata (AAA) cv. Vaibalhla. Eight RAPD and 8 ISSR primers were successfully used for the analysis from the 40 RAPD and 30 ISSR primers screened initially. The amplified products were monomorphic across all the regenerated plants and were similar to the mother plant. The present standardised protocol will find application in mass production, conservation and genetic transformation studies of this commercially important banana.     

Genetic diversity and identification of Chinese-grown pecan using ISSR and SSR markers

Pecan is an important horticultural nut crop originally from North America and now widely cultivated in China for its high ecological, ornamental and economic value. Currently, there are over one hundred cultivars grown in China, including introduced American cultivars and Chinese seedling breeding cultivars. Molecular markers were used to assess the genetic diversity of these cultivars and to identify the pedigrees of fine pecan plants with good characteristics and no cultivar-related data. A total of 77 samples grown in China were studied, including 14 introduced cultivars, 12 domestic seedling breeding cultivars, and 49 fine pecan plants with no cultivar data, together with Carya cathayensis and Juglans nigra. A total of 77 ISSR and 19 SSR primers were prescreened; 10 ISSR and eight SSR primers were selected, yielding a total of 94 amplified bands (100% polymorphic) in the range of 140-1,950 bp for the ISSR and 70 amplified bands (100% polymorphic) in the range of 50-350 bp for SSR markers. Genetic diversity analyses indicated Chinese-grown pecan cultivars and fine plants had significant diversity at the DNA level. The dengrograms constructed with ISSR, SSR or combined data were very similar, but showed very weak grouping association with morphological characters. However, the progeny were always grouped with the parents. The great diversity found among the Chinese cultivars and the interesting germplasm of the fine pecan plants analyzed in this study are very useful for increasing the diversity of the pecan gene pool. All 77 accessions in this study could be separated based on the ISSR and SSR fingerprints produced by one or more primers. The results of our study also showed that ISSR and SSR techniques were both suitable for genetic diversity analyses and the identification of pecan resources.     

Inter-Simple Sequence Repeat (ISSR) Marker Analysis in Parkia timoriana (DC.) Merr. Populations from Northeast India

Genetic variation in three populations of Parkia timoriana (DC.) Merr. grown in the Manipur state of northeast India was analysed using inter-simple sequence repeat (ISSR) markers. A total of 30 individual trees representing three populations were sampled and studied using 22 University of British Columbia (UBC set no. 9) primers in the present study. Of the total 22 primers, 19 primers produced distinct, reproducible and well-resolved fragments. Overall, a total number of 111 fragments were generated by the 19 primers and of which, 51 were polymorphic (45.94 %). The average number of loci and polymorphic loci generated per primer were 5.84 and 2.68, respectively. The genetic variation generated by ISSR markers within the three populations studied ranges from 33.33 to 18.92 %. The overall genetic differentiation (Gst) among populations was estimated to be 0.29, and the number of gene flow (Nm) was estimated to be 1.23 per generation between populations. Of the total genetic variance, 70.04 % was attributed to within-population diversity while 4.72 % differences to the among-populations. The genetic similarity across the individuals belonging to the three populations was represented by the dendrogram showing the grouping of the individuals into three major groups which is also supported by the principle component analysis. The present finding asserts the effectiveness of ISSR procedure for assessing genetic variations of P. timoriana populations and provides valuable genetic information that can be utilized for breeding and conservation strategies.     

Melledonal and melledonol,sesquiterpene esters from armillaria mellea

Isolation of two new sesquiterpene aryl esters melledonal (6) and melledonol (8) from culture broth extract of Armillaria mellea are reported. Their structures were deduced from spectral and chemical data.     

Genetic Diversity Caused by Environmental Stress in Natural Populations of Niupidujuan as Revealed by RAPD Technique

Multiplex environmental factors are generally expected to have significant effects on genetic diversity of plant populations.In this study,randomly amplified polymorphic DNA(RAPD) technique was used to reveal the genetic diversity in the same species of four populations collected from Niupidujuan(Rhododendron chrysanthum) at different altitudes,an endangered species,endemic to Northeast China.Initially,twenty informative and reproducible primers were chosen for final RAPD analysis.A total of 152 clear bands...     

Transfer of <Emphasis Type="Italic">Ephedra</Emphasis> Genomic DNA to Yeasts by Ion Implantation

The genomic DNA from Ephedra glauca was randomly transferred to Saccharomyces cerevisiae and Hansenula anomala by argon and nitrogen ion implantation. Through repeated subculturing and using reversed phase high-performance liquid chromatography analysis to quantify the concentrations of the secondary metabolites, l-ephedrine and d-pseudoephedrine, 12 recombinant strains of genetically stable yeast were obtained, each using glucose as a carbon source, NaNO₃ as a nitrogen source and producing l-ephedrine and/or d-pseudoephedrine. After culturing in liquid medium for 72 h, extracellular l-ephedrine and d-pseudoephedrine concentrations of 18.85 and 4.11 mg/L, respectively, were detected. Using l-ephedrine and d-pseudoephedrine as the target products, the transformation efficiencies of the genomic DNA from E. glauca transferred to S. cerevisiae and H. anomala were 1.15% (1/87) and 2.13% (8/376), respectively. The addition of the amino acid, L-Phe, to culture media substantially changed the amount of l-ephedrine and/or d-pseudoephedrine produced by the recombined yeasts. However, the change in metabolite production was not consistent among strains, rising in some, while dropping to nondetectable levels in others. After random amplification of polymorphic DNA (RAPD) analysis, four RAPD primers were obtained from the initial 100 RAPD primers, each amplifying different fragments with the recombined yeast Ar_Han0458 genome. Using one primer as polymerase chain reaction primer, the result showed that the recombined yeast Ar_Han0458 genome matched E. glauca genomic DNA at 150 bp, indicating a successful transfer of genetic information, facilitated by ion implantation.     

Evaluation of Genetic Homogeneity in Tissue Culture Regenerates of Jatropha curcas L. using Flow Cytometer and DNA-based Molecular Markers

The present investigation aimed to evaluate the reliability of in vitro propagation methods for elite genotypes of Jatropha curcas L., that maintain genetic integrity of tissue culture (TC) regenerates among two regeneration systems developed through direct shoot bud regeneration using nodal/apical shoot segments (protocol-A) and in vitro-derived leaves (protocol-B) as explants. Random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), simple sequence repeat (SSR) molecular markers, and flow cytometery (FCM) were employed to evaluate genetic homogeneity in TC-regenerates at different passages of subcultures. RAPD markers showed genetic homogeneity in fifth-generation TC-regenerates of both protocols. ISSR markers showed genetic stability of leaf regenerates (protocol-B) at 10th generation. FCM analysis of TC-regenerates at 10th generation in protocol-B and at 20th generation in both protocols, showed stability of ploidy level. SSR assessment of TC-regenerates at 20th generation in both protocols confirmed genetic homogeneity. The results confirmed the genetic stability of the TC-regenerates and demonstrated the reliability of the regeneration systems developed so far using explants of two different origins, for large-scale multiplication of elite genotypes of Jatropha.     

Sequence‐related amplified polymorphism,an effective molecular approach for studying genetic variation in Fasciola spp. of human and animal health significance

In the present study, a recently described molecular approach, namely sequence‐related amplified polymorphism (SRAP), which preferentially amplifies ORFs, was evaluated for the studies of genetic variation among Fasciola hepatica, Fasciola gigantica and the “intermediate” Fasciola from different host species and geographical locations in mainland China. Five SRAP primer combinations were used to amplify 120 Fasciola samples after ten SRAP primer combinations were evaluated. The number of fragments amplified from Fasciola samples using each primer combination ranged from 12 to 20, with an average of 15 polymorphic bands per primer pair. Fifty‐nine main polymorphic bands were observed, ranging in size from 100 to 2000 bp, and SRAP bands specific to F. hepatica or F. gigantica were observed. SRAP fragments common to F. hepatica and the “intermediate” Fasciola, or common to F. gigantica and the “intermediate” Fasciola were identified, excised and confirmed by PCR amplification of genomic DNA using primers designed based on sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair‐group method with arithmetic averages clustering algorithm categorized all of the examined representative Fasciola samples into three groups, representing the F. hepatica, the “intermediate” Fasciola, or the F. gigantica. These results demonstrated the usefulness of the SRAP technique for revealing genetic variability between F. hepatica, F. gigantica and the “intermediate” Fasciola, and also provided genomic evidence for the existence of the “intermediate” Fasciola between F. hepatica and F. gigantica. This technique provides an alternative and a useful tool for the genetic characterization and studies of genetic variability in parasites.     

Assessment of Genetic Stability and Instability of Tissue Culture-Propagated Plantlets of Aloe vera L. by RAPD and ISSR Markers

Efficient plantlet regeneration with and without intermediate callus phase was achieved for a selected genotype of Aloe vera L. which is sweet in test and used as a vegetable and source of food. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) marker assays were employed to evaluate genetic stability of plantlets and validate the most reliable method for true-to-type propagation of sweet aloe, among two regeneration systems developed so far. Despite phenotypic similarities in plantlets produced through both regeneration systems, the differences in genomic constituents of plantlets produced through intermediate callus phase using soft base of inflorescence have been effectively distinguished by RAPD and ISSR markers. No polymorphism was observed in regenerants produced following direct regeneration of axillary buds, whereas 80% and 73.3% of polymorphism were observed in RAPD and ISSR, respectively, in the regenerants produced indirectly from base of the inflorescence axis via an intermediate callus phase. Overall, 86.6% of variations were observed in the plantlets produced via an intermediate callus phase. The occurrence of genetic polymorphism is associated with choice of explants and method used for plantlet regeneration. This confirms that clonal propagation of sweet aloe using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. On the other hand, a high degree of variations were observed in plantlets obtained through indirect regeneration and thus cannot be used for the mass multiplication of the genotype; however, it can be used for crop improvement through induction of somaclonal variations and genetic manipulations.     

Four New Alkaloids from the Fermentation Broth of Armillaria mellea

Four compounds with similar structures and UV spectra were isolated from the fermentation broth of Armillaria mellea by means of preparative HPLC. Their structures were established as methyl (2S)‐1‐[2‐(furan‐2‐yl)‐2‐oxoethyl]‐5‐oxopyrrolidine‐2‐carboxylate ( 1 ), (2S)‐1‐[2‐(furan‐2‐yl)‐2‐oxoethyl]‐5‐oxopyrrolidine‐2‐carboxylic acid ( 2 ), 1‐[2‐(furan‐2‐yl)‐2‐oxoethyl]pyrrolidin‐2‐one ( 3 ) and 1‐[2‐(furan‐2‐yl)‐2‐oxoethyl]piperidin‐2‐one ( 4 ) on the basis of their 1D‐ and 2D‐NMR, and HR‐MS data. The absolute configurations of compounds 1 and 2 were determined by comparison of the experimental and calculated electronic circular dichroism (ECD) data. Additionally, four known compounds, 5 – 8 , were also isolated.     

Screening of Tea (Camellia sinensis) for Trait-Associated Molecular Markers

This study was done to identify random amplified polymorphic DNA (RAPD) markers that may associate with seven important traits in tea. Sixty RAPD primers were first screened using 18 cultivars under each of the 7 traits, followed by confirmatory screening of 20 promising primers with 32 tea cultivars. Six RAPD primers generated a total of nine specific bands that associated with six desired traits: black tea quality and tolerance to drought, high temperature, low temperature, Phomopsis theae, and high yield. These markers would allow early identification of plant material with the desired traits that can be advanced to the next stage of selection and enhance targeted choice of breeding stocks with the desirable traits. The nine RAPD markers identified in this study could improve precision and efficiency in tea breeding and selection and are an important contribution towards the establishment of marker-assisted selection in tea breeding programmes.     

Retrotransposon‐microsatellite amplified polymorphism,an electrophoretic approach for studying genetic variability among Schistosoma japonicum geographical isolates

In the present study, retrotransposon‐microsatellite amplified polymorphism (REMAP) was used to examine genetic variability among Schistosoma japonicum isolates from different endemic provinces in mainland China, using S. japonicum from Japan and the Philippines for comparison. Of the 50 primer combinations screened, eight produced highly reproducible REMAP fragments. Using these primers, 190 distinct DNA fragments were generated in total, of which 147 (77.37%) were polymorphic, indicating considerable genetic variation among the 43 S. japonicum isolates examined. The percentage of polymorphic bands (PPB) among S. japonicum isolates from mainland China, Japan, and the Philippines was 77.37%; PPB values of 18.42% and 53.68% were found among isolates from southwestern (SW) China and the lower Yangtze/Zhejiang province in eastern (E) China, respectively. Based on REMAP profiles, unweighted pair‐group method with arithmetic averages (UPGMA) dendrogram analysis revealed that all of the S. japonicum samples grouped into three distinct clusters: parasites from mainland China, Japan, and the Philippines were clustered in each individual clade. Within the mainland China cluster, SW China isolates (from Sichuan and Yunnan provinces) grouped together, whereas worms from E China (Zhejiang, Anhui, Jiangxi, Jiangsu, Hunan, and Hubei provinces) grouped together. These results demonstrated that the REMAP marker system provides a reliable electrophoretic technique for studying genetic diversity and population structures of S. japonicum isolates from mainland China, and could be applied to other pathogens of human and animal health significance.     

Extraction optimization,total phenolic,flavonoid contents,HPLC-DAD analysis and diverse pharmacological evaluations of Dysphania ambrosioides (L.) Mosyakin & Clemants

The present study aims to evaluate phytochemical and pharmacological potentials of Dysphania ambrosioides (L.) Mosyakin & Clemants previously known as Chenopodium ambrosioides L. Extraction was carried out using 14 solvents with wide range of polarity to find out the best solvent system for each bioactivity. Total phenolic and flavonoids contents were measured colorimetrically and polyphenolics were quantified via HPLC-DAD analysis. The samples were screened for inhibitory potentials against free radicals, leishmania, cancer cell lines, protein kinase, α-Amylase enzymes and microbial strains. Among all solvents, maximum percentage of extract was recovered from methanol-water fraction of leaves. HPLC analysis exhibited the presence of rutin, myricetin and quercetin. In DPPH assay, methanolic leaf extract exhibited IC₅₀ value of 130.7 ± 0.57 μg/mL. Considerable α-amylase inhibitory, cytotoxic, leishmanicidal and antimicrobial potentials were exhibited by plant samples. D. ambrosioides revealed significant antioxidant, cytotoxic, antimicrobial and anti-diabetic potentials and thus warrant further detailed studies to find novel drugs.     

Electrophoretic detection of genetic variability among Schistosoma japonicum isolates by sequence-related amplified polymorphism

In the present study, sequence‐related amplification polymorphism (SRAP) was utilized to study the genetic variability among Schistosoma japonicum isolates from different provinces in China, using Schistosoma mansoni from Puerto Rico for comparison. Five out of ten tested SRAP primer combinations displayed significant polymorphisms among S. japonicum isolates from China, namely ME2/EM1, ME4/EM1, ME4/EM6, ME5/EM4 and ME5/EM5. Analysis of the 61 S. japonicum samples from China with five SRAP primer combinations identified a total of 83 reproducible polymorphic fragments. The number of fragments using each primer combination ranged from 14 to 19, with an average of 16 polymorphic bands per primer pair, and the size of fragment ranged approximately from 100 to 1000 bp. Representative‐specific SRAP fragments were excised from the gels, and confirmed by PCR amplification of genomic DNA using primers designed and based on the sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair‐group method with arithmetic averages (UPGMA) dendrogram was constructed. UPGMA clustering algorithm categorized S. japonicum isolates from China into nine clades and two lineages (representing the mountainous and lake/marshland regions). These results indicate the usefulness of the SRAP technique for revealing genetic variability among S. japonicum isolates from China, and the SRAP technique should be applicable to other living organisms.     

Analysis of Genetic Diversity of Certain Species of Piper Using RAPD-Based Molecular Markers

The utility of RAPD markers in assessing genetic diversity and phenetic relationships of six different species of Piper from Northeast India was investigated. Polymerase chain reaction (PCR) with four arbitrary 10-mer oligonucleotide primers applied to the six species produced a total of 195 marker bands, of which, 159 were polymorphic. On average, six RAPD fragments were amplified per reaction. In the UPGMA phenetic dendrogram based on Jaccard’s coefficient, the different accessions of Piper showed a high level of genetic variation. This study may be useful in identifying diverse genetic stocks of Piper, which may then be conserved on a priority basis.