Differential biosynthesis and accumulation of picrosides in an endangered medicinal herb Picrorhiza kurroa

Picrorhiza kurroa Royle ex Benth (Family: Scrophulariaceae) is a medicinal herb, mainly found in the North-Western Himalayas. Extensive harvesting for pharmaceutical purposes, lack of organized cultivation and unorganized methods of uprooting the plants because of unawareness has brought an endangered status to this important herb in nature. The medicinal property of this plant is attributed to monoterpenoid picrosides. The influence of developmental status of different growth stages on picrosides content is poorly understood in Picrorhiza kurroa. Picroside-I (P-I) content increased from 0.05 % to 0.76 % in different growth stages of shoots. Significant increase in the contents of P-I (0.15–0.50 %) and Picroside-II (P-II) (0.1–0.45 %) was observed in rhizomes of different developmental stages. Highest amounts of P-I (8.7 %) and P-II (5.3 %) was detected in uppermost part of mature dried rhizomes compared to bottom part with 2.9 % and 2.2 % of P-I and P-II, respectively. P. kurroa grown at high altitude (Sairopa, 4,500 amsl) showed 1.75-folds increase in P-I in leaves whereas exponential increase in the P-I content was detected (0.05–1.7 %) in the leaves of different developmental stages (L1-L5) of P. kurroa grown at lower altitude (Jagatsukh, 1,900 m). Variable amounts of P-I and P-II in different growth and developmental stages of P. kurroa imply importance of selection of plant material (rhizomes and roots). The study undertaken explored the status of metabolites accumulation and biosynthesis in the field grown plants of P. kurroa where not only environmental parameters but different morphogenetic stages of its developmental cycles, different age groups and different parts of plantlets were extensively analysed and estimated for medicinally important picrosides.     

Tracking dynamics of enzyme activities and their gene expression in <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> with respect to picroside accumulation

Picrosides, the terpenoids synthesized by Picrorhiza kurroa, have ample usage in medicine. Identification of the regulatory enzymes involved in picroside biosynthesis needs to be explored for improving the level of these secondary metabolites. Current efforts are based on the analysis of secondary metabolism in picroside biosynthesis but its interpretation is limited by the lack of information on the involvement of primary metabolic pathways. The present study investigated the connection of primary metabolic enzymes with the picrosides levels in P. kurroa. The results showed changes in the catalytic activities as well as in the gene expression profiles of hexokinase, pyruvate kinase, isocitrate dehydrogenase, malate dehydrogenase, and NADP⁺-malic enzyme in congruence with picroside-I content under different conditions of P. kurroa growth, which indicates the role of these enzymes in the accumulation of picrosides. The significant correlation coefficients (p?<?0.05) observed between gene expression and enzyme activity underline the role of integrative studies for a better understanding of connecting links between metabolic pathways leading to picroside biosynthesis. This is apparently the first report on the involvement of glycolytic and TCA cycle enzymes in the accumulation of picrosides in P. kurroa.     

Comparison of RAPD,ISSR, and AFLP Molecular Markers to Reveal and Classify Orchardgrass (Dactylis glomerata L.) Germplasm Variations

Three different DNA-based techniques, Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) markers, were used for fingerprinting Dactylis glomerata genotypes and for detecting genetic variation between the three different subspecies. In this study, RAPD assays produced 97 bands, of which 40 were polymorphic (41.2%). The ISSR primers amplified 91 bands, and 54 showed polymorphism (59.3%). Finally, the AFLP showed 100 bands, of which 92 were polymorphic (92%). The fragments were scored as present (1) or absent (0), and those readings were entered in a computer file as a binary matrix (one for each marker). Three cluster analyses were performed to express–in the form of dendrograms–the relationships among the genotypes and the genetic variability detected. All DNA-based techniques used were able to amplify all of the genotypes. There were highly significant correlation coefficients between cophenetic matrices based on the genetic distance for the RAPD, ISSR, AFLP, and combined RAPD-ISSR-AFLP data (0.68, 0.78, 0.70, and 0.70, respectively). Two hypotheses were formulated to explain these results; both of them are in agreement with the results obtained using these three types of molecular markers. We conclude that when we study genotypes close related, the analysis of variability could require more than one DNA-based technique; in fact, the genetic variation present in different sources could interfere or combine with the more or less polymorphic ability, as our results showed for RAPD, ISSR and AFLP markers. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationship among genotypes of Dactylis glomerata.     

ISSR and RAPD based evaluation of genetic fidelity and active ingredient analysis of regenerated plants of Picrorhiza kurroa

Genetic stability and phytochemical analysis of in vitro established plants of Picrorhiza kurroa Royle ex Benth, have been carried out. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers were used to assess the genetic fidelity of tissue culture products including three adventitious shoots from three calli and 6 months old tissue culture raised plants growing in green house condition with mother plant. Apparent genetic variation was detected in the five types of plant materials. The percentage of polymorphic bands in the RAPD and ISSR analysis were 16.25 and 14.54 %, respectively. The genetic similarity was calculated on the basis of RAPD and ISSR data among the five types of plant materials and were ranged from 0.5 to 1.0 (mean 0.75) and 0.47 to 1.0 (mean 0.73), respectively. The similarity coefficient by both RAPD and ISSR analysis revealed that differences between tissue culture raised plants and mother plant was not remarkable, but notable differences were observed among three adventitious shoots regenerated from three calli. The phytochemical analysis of tissue culture raised products showed higher secondary metabolite (picrotin and picrotoxinin) content as compare to mother plant. The information gained on genetic stability/variability will be valuable for the large scale propagation and secondary metabolite production of P. kurroa.     

Assessment of genetic diversity through RAPD,ISSR and AFLP markers in Podophyllum hexandrum: a medicinal herb from the Northwestern Himalayan region

Total synthesis of podophyllotoxin is an expensive process and availability of the compound from the natural resources is an important issue for pharmaceutical companies that manufacture anticancer drugs. In order to facilitate reasoned scientific decisions on its management and conservation for selective breeding programme, genetic analysis of 28 populations was done with 19 random primers, 11 ISSR primers and 13 AFLP primer pairs. A total of 92.37 %, 83.82 % and 84.40 % genetic polymorphism among the populations of Podophyllum were detected using RAPD, ISSR and AFLP makers, respectively. Similarly the mean coefficient of gene differentiation (Gst) were 0.69, 0.63 and 0.51, indicating that 33.77 %, 29.44 % and 26 % of the genetic diversity resided within the population. Analysis of molecular variance (AMOVA) indicated that 53 %, 62 % and 64 % of the genetic diversity among the studied populations was attributed to geographical location while 47 %, 38 % and 36 % was attributed to differences in their habitats using RAPD, ISSR and AFLP markers. An overall value of mean estimated number of gene flow (Nm) were 0.110, 0.147 and 0.24 from RAPD, ISSR and AFLP markers indicating that there was limited gene flow among the sampled populations.     

Nuclear DNA assay of the wild endangered medicinal and aromatic Indian Himalayan Valeriana jatamansi germplasm with multiple DNA markers: implications for genetic enhancement,domestication and ex situ conservation

Population genetic analysis in the important endangered medicinal and aromatic plant species, Valeriana jatamansi, provided, first time, insights into the identification of novel sources of genetic variation as an aid for improvement and domestication, and for optimizing conservation strategies. The 75 genotypes of V. jatamansi were collected from 36 locations across northeast to northwest Indian Himalayas of ~1,000 km, harbouring variable climatic and ecological conditions and rugged rocky terrain. The known protocols for DNA extraction failed to yield quality DNA in good quantity. A new protocol was standardized for this purpose. All the three (RAPD, ISSR, AFLP) DNA markers were successful in detecting polymorphism in V. jatamansi genotypes, and the ISSR marker, vis-à-vis RAPD and AFLP markers, generated the highest level of polymorphism. The RAPD, ISSR and AFLP fingerprints with 23 and 15 primers and 8 primer combinations, respectively, revealed 85.8, 89.0 and 67.7 % polymorphism among 141, 91 and 37 genetic loci amplified from the 75 genotypes, respectively. The AMOVA analysis of AFLP (55.0, 8.3, 36.7 %), RAPD (57.4, 11.9, 30.6 %) and ISSR (76.0, 4.8, 19.1 %) data indicated that more variation existed in differences in genotypes within populations than between populations within a region and between regions, respectively. The present comprehensive input will assist in effective management and (or) devising conservation strategies of this important medicinal plant species. This study reports the start of a molecular biology programme targeting nuclear genome of V. jatamansi, the genetics of which is very little known.     

Analysis of genetic variability and population structure of the endemic medicinal Limonium sinense using molecular markers

Limonium sinense is an endemic medicinal herb used to treat fever, hemorrhage and other disorders. In the present study, population genetic diversity was elucidated using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) primers. Percentage of polymorphic bands, Nei's gene diversity and Shannon's information index revealed a high level of genetic diversity at species level. The analysis of molecular variance revealed that 69.88% (RAPD), 71.19% (ISSR) and 70.97% (AFLP) of variability were partitioned among individuals within populations, which indicated the coherent trend by Gst (0.3849/0.3577/0.3670). Gene flow number (Nm) was 0.581/0.618/0.612, which indicated that there was a limited gene exchange between populations. The UPGMA clustering results showed that the genetic distance had no significant correlation with geographic distance. These results indicate that these markers were reliable tools for the differentiation and determination of the genetic diversity among the populations of L. sinense and the conservation of existing natural population is necessary.     

Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers

The genetic stability of in vitro propagated potato microtubers was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Microtubers were developed through in vitro from potato microplants using standardized protocols. The microtubers were conserved for 1 year under three different culture media and consequently microplants were regenerated for the DNA analyses. During the study, a total of 38 (10 RAPD, 11 ISSR, 12 SSR and 5 AFLP) primers produced a total of 407 (58 RAPD, 56 ISSR, 96 SSR and 197 AFLP) clear, distinct and reproducible amplicons. Cluster analysis revealed 100 % genetic similarity among the mother plant and its derivatives within the clusters by SSR, ISSR and RAPD analyses, whereas AFLP analysis revealed from 85 to 100 % genetic similarity. Dendrogram analysis based on the Jaccard’s coefficient classified the genotypes into five clusters (I–V), each cluster consisting of mother plant and its derivatives. Principal component analysis (PCA) also plotted mother plant and its genotypes of each cluster together. Based on our results, it is concluded that AFLP is the best method followed by SSR, ISSR and RAPD to detect genetic stability of in vitro conserved potato microtubers. The in vitro conservation medium (T2) is a safe method for conservation of potato microtubers to produce true-to-type plans.     

Conservation genetics of Heritiera littoralis (Sterculiaceae), a threatened mangrove in China,based on AFLP and ISSR markers

AFLP and ISSR markers were used to determine the genetic variations in eight mangrove and non-mangrove populations of Heritiera littoralis (Sterculiaceae), a threatened species in China. Our results showed a moderate to high level of genetic variation in this species (P = 63.69%, H_T = 0.20 for AFLP; P = 76.07%, H_T = 0.22 for ISSR), and a relatively high level of genetic differentiation among populations (G_ST = 0.24 for AFLP and 0.27 for ISSR). Life history traits, long-distance dispersal of floating seeds, and local environments may play important roles in shaping the genetic diversity and genetic structure of this species. Investigation of the plant’s reproductive capacity showed that the natural seed production of H. littoralis was low, as was the germination rate and the transformation rate from juvenile to adult. H. littoralis is seriously threatened and is in urgent need of conservation in China.     

Genetic variability and population structure of Bergenia ciliata (Saxifragaceae) in the Western Himalaya inferred from DAMD and ISSR markers

Genetic variability and population structure of Bergenia ciliata (Haw.) Sternb., commonly known as “Pashanbheda” (Stone-breaker), collected from the Western Himalayan region of India were estimated using two DNA fingerprinting methods viz., directed amplification of minisatellite DNA (DAMD) and inter simple sequence repeats (ISSR). The cumulative data analysis of DAMD and ISSR markers for 74 accessions from eight populations showed 86.1% polymorphism. Analysis of molecular variance (AMOVA) showed highest percentage of variation within individuals of populations (73.6%) and 21.7% among populations. STRUCTURE and PCoA analyses on the hierarchical partitioning of genetic diversity showed strong admixture of individuals among the eight assumed geographical populations of B. ciliata. The data suggests that high genetic flow is one of the major factors responsible for low genetic differentiation. Preservation of genetic diversity of B. ciliata is important, both to promote adaptability of the populations to changing environment as well as to preserve a large gene pool for future prospection. The present study using DAMD and ISSR markers, therefore, provide the means of rapid characterization of accessions within the populations, and thus enable the selection of appropriate accessions for further utilization in conservation and prospection programmes.     

ISSR and RAPD Markers Assessed Genetic Variation of Aerides maculosum — an Epiphytic Orchid from Goa, India

Aerides maculosum Lindl is one of the most important orchids valued for its beautiful inflorescence/flowers. The present study aimed to understand the level of genetic variation among the populations of A. maculosum using RAPD and ISSR markers. Among the 35 primers tested, 13 RAPD and 6 ISSR primers were selected for the analysis. Total of 101 RAPD and 40 ISSR fragments were generated. High level of polymorphism was recorded in RAPD (90.45%) compared to ISSR (72.85 %). Nei’s average genetic identity values for different populations of A. maculosum- ranged from 0.465 to 0.762 (RAPD), while for ISSR it ranged from 0.475 to 0.975. The present study provides important insights about genetic variation in A. masculosum and may facilitates the conservation and management of this species.     

Genetic diversity and structure in fragmented populations of the endangered species Ranunculus cabrerensis (Ranunculaceae): implications for conservation

Ranunculus cabrerensis is an endemic and endangered species of the Northwestern Iberian Peninsula. The molecular markers AFLP and ISSR were used to investigate the genetic diversity and population structure of four populations across its known distribution. Fifteen selective primer combinations of AFLP and seventeen ISSR primer combinations produced a total of 2830 and 103 unambiguously repeatable fragments respectively, of which 97.57 and 81.38% were polymorphic for both markers. The genetic diversity of R. cabrerensis at species level was high (H _E = 0.294 by ISSR and H _E = 0.191 by AFLP) and differentiation between sampled locations was also relatively high (G _ST = 0.316 and 0.158 by ISSR and AFLP analysis respectively) compared to other studies of endangered and rare species using the same techniques. The analysis of molecular variance (AMOVA) indicated that the main genetic variation was within sampled locations (73% by AFLP; 52% by ISSR), even though the variation among locations was also significant. Principal Coordinates, NeighborNet and Bayesian analyses revealed a weak but significant relationship between the genetic structures of different populations in R. cabrerensis, with gene flow acting as a homogenizing force that prevents stronger differentiation of populations. Finally, suggestions for conservation strategies to preserve the genetic resources of this species are outlined.     

Modulation of Picroside-I Biosynthesis in Grown Elicited Shoots of <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> In Vitro

Elicitors are considered as biostimulants for growth improvement and enhancement of secondary metabolite content. To date, only seaweed extract (SWE) powder has been studied for its effect on picroside-I (P-I) production in in vitro grown Picrorhiza kurroa plants. However, little is known at the molecular level about P-I production in P. kurroa plants upon SWE treatment. Here, we investigated the relative effects of supplying different elicitors including methyl jasmonate (MeJa), sodium nitroprusside (SNP), and abscisic acid (ABA) with SWE on plant growth and P-I production in addition to their effects at the molecular level reflecting the metabolic status of P-I biosynthesis. Our results indicated that only SWE, ABA, and SNP stimulated P-I production by 2.60-, 2.01-, and 1.35-fold, respectively, whereas MeJa decreased P-I content. Interestingly, SWE modulated all four integrating secondary metabolic pathways, covering almost all critical steps in the methylerythritol phosphate (MEP), mevalonate (MVA), iridoid, and phenylpropanoid pathways to stimulate P-I biosynthesis. SNP targeted the MVA/MEP pathways in conjunction with the iridoid pathway, whereas ABA modulated the phenylpropanoid pathway to increase the P-I content in P. kurroa. This is apparently the first report on treatment of different elicitors in in vitro grown P. kurroa plants for eliciting P-I content and exploring the role of different elicitors at the molecular level.