Ritu Paliwal

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.

In the present study, novel genomic-SSR (g-SSR) markers generated in our laboratory were used to characterize Tinospora cordifolia and related species. The g-SSR marker was also compared with EST-SSR and SCoT markers used earlier in our laboratory to assess the genetic diversity of T. cordifolia. A total of 26 accessions of T. cordifolia and 1 accession each of Tinospora rumphii and Tinospora sinensis were characterized using 65 novel g-SSR markers. A total of 125 alleles were detected with 49 polymorphic g-SSR markers. The number of alleles per locus varied from 1–4 with a mean value of 2.55 alleles per locus. Mean PIC, gene diversity, and heterozygosity were estimated to be 0.33, 0.41, and 0.65, respectively. The two species, namely T. rumphii and T. sinensis, showed cross-species transferability of g-SSRs developed in T. cordifolia. The success rate of cross-species transferability in T. rumphii was 95.3% and 93.8% in T. sinensis, proving the usefulness of this marker in genetic diversity studies of related species. The Tinospora accessions were also used for molecular characterization using SCoT and EST-SSR markers and compared for genetic diversity and cross-species transferability. The PIC, gene diversity, heterozygosity, and principal coordinate analysis showed that g-SSR is the better maker for a genetic diversity study of T. cordifolia. Additionally, high cross-species transferability of g-SSRs was found (95.3% and 93.8%) compared to EST-SSRs (68.8% and 67.7%) in T. rumphii and T. sinensis, respectively.

Kalmegh (Andrographis paniculata (Burm. F.) Nees) is one of the most important medicinal plants and has been widely explored as traditional medicine. To exploit its natural genetic diversity and initiations of molecular breeding to develop novel cultivars or varieties, developments of genomic resources are essential. Four microsatellite-enriched genomic libraries—(CT)₁₄, (GT)₁₂, (AG)₁₅ and (AAC)₈—were constructed using the genomic DNA of A. paniculata. Initially, 183 recombinant colonies were screened for the presence of CT, GT, AG, and AAC microsatellite repeats, out of which 47 clones found positive for the desired simple sequence repeats (SSRs). It was found that few colonies had more than one desirable SSR. Thus, a sum of 67 SSRs were designed and synthesized for their validation among 42 A. paniculata accessions. Out of the 67 SSRs used for genotyping, only 41 were found to be polymorphic. The developed set of g-SSR markers showed substantial genetic variability among the selected A. paniculata accessions, with an average polymorphic information content (PIC) value of 0.32. Neighbor-joining tree analysis, population structure analysis, analysis of molecular variance (AMOVA), and principal coordinate analysis (PCoA) illustrated the considerable genetic diversity among them. The novel g-SSR markers developed in the present study could be important genomic resources for future applications in A. paniculata.