De novo transcriptome profiling and development of novel secondary metabolites based genic SSRs in medicinal plant Phyllanthus emblica L. (Aonla).

Phyllanthus emblica (Aonla, Indian Gooseberry) is known to have various medicinal properties, but studies to understand its genetic structure are limited. Among the various secondary metabolites, ascorbic acid, flavonoids, terpenoids, phenols and tannins possess great potential for its pharmacological applications. Keeping this consideration, we assembled the transcriptome using the Illumina RNASeq500 platform, generating 39,933,248 high-quality paired-end reads assembled into 1,26,606 transcripts. A total of 87,771 unigenes were recovered after isoforms and unambiguous sequences deletion. Functional annotation of 43,377 coding sequences against the NCBI non-redundant (Nr) database search using BlastX yielded 38,692 sequences containing blast hits and found 4685 coding sequences to be unique. The transcript showed maximum similarity to Hevea brasilensis (16%), followed by to Jatropha curcas (12%). Considering key genes involved in the biosynthesis of flavonoids and various classes of terpenoid compounds, thirty EST-SSR primer sequences were designed based on transcriptomic data. Of which, 12 were found to be highly polymorphic with an average of 86.38%. The average value for marker index (MI), effective multiplicity ratio (EMR), resolution power (Rp) and polymorphic information content (PIC) was 7.20, 8.34, 8.64 and 0.80, respectively. Thus, from this study, we developed newly EST-SSRs linked to important genes involved in the secondary metabolites biosynthesis that will be serving as an invaluable genetic resource for crop improvement including the selection of elite genotypes in P. emblica and its closely related Phyllanthaceae species.

Development and characterization of microsatellite markers for Phyllanthus emblica Linn., important nontimber forest product species.

Phyllanthus emblica and P. indofischeri, commonly known as the Indian gooseberry, are important nontimber forest product (NTFP) species widely distributed across the Indian subcontinent. The fruits of these species are rich in vitamin C and are used in the preparation of a number of herbal medicines for treating a wide range of disorders. Due to the increased demand, they have been harvested extensively and form a major source of income for the forest-dwelling communities living in southern India. There are limited studies to understand the impact of harvesting on the genetic structure of these species. In this study, 15 polymorphic microsatellite markers have been developed for P. emblica and were characterized by screening 20 individuals each of P. emblica and P. indofischeri. The number of alleles per locus ranged 2-9 for P. emblica and 2-11 for P. indofischeri. The observed and expected heterozygosity of P. emblica ranged 0-1 and 0.401-0.825, respectively. Similarly, the observed and expected heterozygosity of P. indofischeri ranged 0.5-1 and 0.366-0.842, respectively. Cross-amplification of the designed primers was assessed with seven related Phyllanthus species. The microsatellite markers developed can be used for studying the population genetic structure, gene flow and genetic diversity of P. emblica and P. indofischeri.

Ectopic expression and functional characterization of type III polyketide synthase mutants from Emblica officinalis Gaertn.

KEY MESSAGE: Functional characterization and ectopic expression studies of chalcone synthase mutants implicate the role of phenylalanine in tailoring the substrate specificity of type III polyketide synthase. Chalcone synthase (CHS) is a plant-specific type III polyketide synthase that catalyzes the synthesis of flavonoids. Native CHS enzyme does not possess any functional activity on N-methylanthraniloyl-CoA, which is the substrate for acridione/quinolone alkaloid biosynthesis. Here, we report the functional transformation of chalcone synthase protein from Emblica officinalis (EoCHS) to quinolone and acridone synthase (ACS) with single amino acid substitutions. A cDNA of 1173 bp encoding chalcone synthase was isolated from E. officinalis and mutants (F215S and F265V) were generated by site-directed mutagenesis. Molecular modeling studies of EoCHS did not show any active binding with N-methylanthraniloyl-CoA, but the mutants of EoCHS showed strong affinity to the same. As revealed by the modeling studies, functional analysis of CHS mutants showed that they could utilize p-coumaroyl-CoA as well as N-methylanthraniloyl-CoA as substrates and yield active products such as naringenin, 4-hydroxy 1-methyl 2(H) quinolone and 1,3-dihydroxy-n-methyl acridone. Exchange of a single amino acid in EoCHS (F215S and F265V) resulted in functionally active mutants that preferred N-methylanthraniloyl-CoA over p-coumaroyl-CoA. This can be attributed to the increase in the relative volume of active sites in mutants by mutation. Moreover, metabolomic and MS analyses of tobacco leaves transiently expressing mutant genes showed high levels of naringenin, acridones and quinolone derivatives compared to wild-type CHS. This is the first report demonstrating the functional activity of EoCHS mutants with N-methylanthraniloyl-CoA and these results indicate the role of phenylalanine in altering the substrate specificity and in the evolution of type III PKSs.

Studies on bio-chemical profiling of Indian gooseberry (Emblica officinalis) for genetic diversity.

Biochemical profiling of physiologically mature fruits of 51 diverse Indian gooseberry (Emblica officinalis Gaertn) germplasm accessions was collected from Vindhyan hill region of Madhya Pradesh, with a view to select nutraceutically rich genotypes based on important biochemical traits. The mean ascorbic acid and total phenol (tannin) content amongst different accessions was recorded as 496.47 mg 100 g⁻¹ and 4.88% with highest value found in CISH A-12 (654.50 mg 100 g⁻¹) and CISH A-30 (7.18%), respectively. Apart from the above, wide range of variability in the composition of other important biochemical attributes viz., total soluble solids (8.60-17.70°Brix), acidity (1.61-2.94%), total sugar (4.15-9.17), reducing sugar (2.19-4.45%) and TSS/acid ratio (3.89-8.33) was also recorded. Highest significant and positive correlation was observed between total sugar and TSS (0.895) followed by reducing sugar and TSS (0.882). Significant positive correlation between ascorbic acid and tannins (0.551) was an indication to be associated with binding capacity of ascorbic acid over a longer period of storage.

Isolation and characterization of microsatellites in a medicinal plant, Phyllanthus emblica (Euphorbiaceae).

PREMISE OF THE STUDY: Microsatellite markers were isolated and characterized in a medicinal plant, Phyllanthus emblica, to study population genetics for designing an effective in situ and ex situ conservation of genetic resources of the species. • METHODS AND RESULTS: Six microsatellite markers were developed using an enrichment and magnetic separation protocol. They were characterized in two natural populations of P. emblica. Out of the six microsatellites, five showed polymorphism, with the number of alleles ranging from four to seven. Observed and expected heterozygosities ranged from 0.360 to 0.760 and 0.499 to 0.806, respectively. • CONCLUSIONS: The five polymorphic microsatellite markers will be useful for studying the genetic structure, reproductive biology, and for identification of clones and provenances of this important medicinal plant.

Isolation of high quality RNA from Phyllanthus emblica and its evaluation by downstream applications.

Next generation sequencing is a high-throughput technique widely used for transcriptome profiling. Isolation of high quality RNA is a prerequisite for such large scale transcriptome analysis. Phyllanthus emblica is an important medicinal plant having high amount of metabolites like vitamin C, flavonoids, polyphenolic compounds, tannins, which are responsible for its wondered medicinal properties. High concentration of secondary metabolites like polysaccharides and polyphenols proved to be an obstacle in isolating RNA of good quality. Any compromise with quality of RNA affects the downstream applications and requires extra cleaning steps that further reduce RNA quantity. We have developed a protocol for isolation of high quality RNA from P. embilca. RNA was successfully assessed for downstream applications like reverse transcription polymerase chain reaction, rapid amplification of cDNA ends, mRNA library preparation, and sequencing using HiSeq(™) 2000 sequencing technology. The protocol is simple and can be completed in 4-5 h.

Development and application of RAPD-SCAR marker for identification of Phyllanthus emblica LINN.

Correct genotype identification of medicinal plant material remains important for botanical drug industry. Limitations of chemical and morphological approaches for authentication have generated need for newer methods in quality control of botanicals. The present study was carried out to develop DNA based marker for identification of Phyllanthus emblica LINN. A putative marker (1.1 kb) specific for P. emblica was identified by Random Amplified Polymorphic DNA (RAPD) technique. Sequence Characterized Amplified Region (SCAR) marker was developed from the RAPD amplicon. The SCAR marker was found useful for identification of P. emblica in its commercial samples and Triphalachurna, a multi-component Ayurvedic formulation.