De Novo Transcriptome Profiling for the Generation and Validation of Microsatellite Markers, Transcription Factors, and Database Development for Andrographis paniculata

Abstract

Andrographis paniculata belongs to the family Acanthaceae and is known for its medicinal properties owing to the presence of unique constituents belonging to the lactones, diterpenoids, diterpene glycosides, flavonoids, and flavonoid glycosides groups of chemicals. Andrographolide, a major therapeutic constituent of A. paniculata, is extracted primarily from the leaves of this plant and exhibits antimicrobial and anti-inflammatory activities. Using 454 GS-FLX pyrosequencing, we have generated a whole transcriptome profile of entire leaves of A. paniculata. A total of 22,402 high-quality transcripts were generated, with an average transcript length and N50 of 884 bp and 1007 bp, respectively. Functional annotation revealed that 19,264 (86%) of the total transcripts showed significant similarity with the NCBI-Nr database and were successfully annotated. Out of the 19,264 BLAST hits, 17,623 transcripts were assigned GO terms and distributed into three major functional categories: molecular function (44.62%), biological processes (29.19%), and cellular component (26.18%) based on BLAST2GO. Transcription factor analysis showed 6669 transcripts, belonging to 57 different transcription factor families. Fifteen TF genes that belong to the NAC, MYB, and bHLH TF categories were validated by RT PCR amplification. In silico analysis of gene families involved in the synthesis of biochemical compounds having medicinal values, such as cytochrome p450, protein kinases, heat shock proteins, and transporters, was completed and a total of 102 different transcripts encoding enzymes involved in the biosynthesis of terpenoids were predicted. Out of these, 33 transcripts belonged to terpenoid backbone biosynthesis. This study also identified 4254 EST-SSRs from 3661 transcripts, representing 16.34% of the total transcripts. Fifty-three novel EST-SSR markers generated from our EST dataset were used to assess the genetic diversity among eighteen A. paniculata accessions. The genetic diversity analysis revealed two distinct sub-clusters and all accessions based on the genetic similarity index were distinct from each other. A database based on EST transcripts, EST-SSR markers, and transcription factors has been developed using data generated from the present study combined with available transcriptomic resources from a public database using Meta transcriptome analysis to make genomic resources available in one place to the researchers working on this medicinal plant.

Patterns of Genetic Variation of Nepeta nuda L. from the Central Balkans: Understanding Drivers of Chemical Diversity

“…To elucidate the fundamentals of species’ natural chemical diversity, we assessed the genetic structure of wild populations, adopting a set of EST-SSR molecular markers mined from a leaf transcriptome of N. nuda and a pair of previously published SSR markers. Microsatellites, being highly polymorphic codominant markers with a neutral evolutionary history, provide a basic knowledge of the species’ genome variation through the estimation of the allelic fixation index, as well as the structure of populations, offering outstanding benefits because of their low cost and user-friendly nature [ 68 ]. EST-SSRs markers, being transcriptome-derived, are considered less variable as compared to genomic microsatellites, but meticulous selection of loci to be used can result in a greater discriminating power in comparison to genomic SSRs [ 69 ].…”

Comprehensive Analysis of Differentially Expressed lncRNAs in Papaver somniferum L.

“…Despite the availability of a wide variety of molecular markers in plants, lncRNA-derived SSR markers have high potential due to their high level of cross-transferability and polymorphism. These markers can make important contributions to genetic diversity studies as well as breeding programmes (Singh et al 2023). The MISA tool was used to determine where the SSR markers were derived from opium poppy DELs with default parameters (Beier et al 2017).…”

Isolation, Characterization, and Expression Analysis of NAC Transcription Factor from Andrographis paniculata (Burm. f.) Nees and Their Role in Andrographolide Production

“…Sequences for NAC domain-contained transcripts were retrieved from the transcript and SSR database of A. paniculata [57]. Ten transcripts were selected to identify the NAC gene's core fragment and were confirmed using an expasy translator for amino acid frame identification (https://web.expasy.org/translate/; accessed on 5 May 2023).…”

“…A total of ten putative transcript sequences were initially selected to identify the core NAC fragments from the Andrographis transcripts and SSR database [57], using expasy translation and the protein basic local alignment search tool (BLASTP) for homology. Out of these, eight transcripts demonstrated homology with NAC TF of different plant species, revealed from the National Centre for Biotechnology Information (NCBI) database.…”