Assessment of genetic diversity, DNA barcoding and chemical analysis of neopicrorhiza scrophulariiflora (pennell) hong of Nepal

Abstract

Forty-three samples of Neopicrorhiza scrophulariiflora (Pennell) Hong collected from Eastern, Central and Western Nepal were studied for genetic diversity and DNA barcoding studies. Inter Simple Sequence Repeat (ISSR) marker system was employed for the assessment of genetic diversity within and among N. scrophulariiflora populations. Out of total 197 amplified bands generated using 10 ISSR primers, 180 bands (91.37%) were polymorphic while 17 (8.63%) were monomorphic. Five primers (UBC 807, 835, 848, 850 and 868) generated ten population-specific ISSR markers for three populations. Using NTSYS-pc version 2.21, Cluster analysis was performed and phenogram based on Jaccard’s similarity coefficient and UPGMA clustering method was found to be the best for deducing genetic relationship between N. scrophulariiflora accessions. The average value of Jaccard’s coefficient of similarity for all accessions was found to be 0.289. The mean genetic similarity between MCA and LNP was 20%, LNP and KCA was 15% and MCA and KCA was 20%. Accessions from Manaslu Conservation Area (MCA) and Kanchenjunga Conservation Area (KCA) clustered together while accessions from Langtang National Park (LNP) clustered separately in phenogram. The result was supported by 3D-plot and Principal Coordinate Analysis (PCoA). The first two axes of PCoA analysis showed 37.63% and 27.24% of total variation respectively with cumulative variance of 64.87%. Population genetic diversity parameters such as Nei’s gene diversity (H) and Shannon’s diversity index (I) were computed using POPGENE v. 1.32 and was found to be 0.2320 and 0.3636 respectively for MCA, 0.2284 and 0.3413 respectively for LNP and 0.2439 and 0.3836 respectively for KCA. At the species level, H and I values for N. scrophulariiflora was found to be 0.2070 and 0.3282 respectively. The mean genetic differentiation between populations (GST) over all loci was found to be 0.4676 indicating 46.76% variation among populations. The average gene flow (Nm) value was found to be 0.6501. AMOVA revealed the among population variation coefficient, ФPT, to be 0.501 indicating 50.1% variation among populations. DNA barcoding was performed using three chloroplast (cp) DNA loci, viz, matK, rbcL and trnH-psbA and one nuclear DNA loci the Internal transcribed spacer (ITS). Of the three cpDNA loci, trnH-psbA was found to have four variable sites within the sequence while rest of the loci lacked variable sites. Inter-specific and intra-specific divergences and Best Match/Best Close Match values were calculated using TaxonDNA software in order to identify potential barcode for N. scrophulariiflora. Analyses of matK, rbcL, trnH-psbA and ITS sequences showed high rate of species identification potential for matK (75%) followed by ITS (72%). Phylogenetic analyses were performed using model averaged phylogeny (jModelTest 2.1.4) and parsimony based method (MEGA 5.2). The best fitted nucleotide substitution model was computed based on corrected Akaike’s Information Criteria (AICc) using jModelTest and was found to be TVM+I for matK, TPM2uf+G for rbcL, TPM1uf for trnH-psbA and GTR+ for ITS. Maximum Parsimony (MP) trees were constructed for all loci using MEGA 5.2 with Consistency index (CI) value 0.933 for matK while MP trees for remaining loci lacked CI values. matK, trnH-psbA and ITS resolved all the accessions into a monophyletic clade but trnH-psbA sequence showed high intra-specific distance compared to matK and ITS thus matK and ITS are proposed as potential barcodes for N. scrophulariiflora. Antibacterial activity of methanolic rhizome extract of N. scrophulariiflora was assessed and was found that Staphylococcus aureus, Klebsiella pneumoneae, and Pseudomonas aeruginosa were sensitive. IC50 values were computed for extracts from three different locations using DPPH method and LNP sample was found to be more potent antioxidant (1.742) followed by KCA (2.398) and MCA (4.141). Quantification of Picroside I and II using HPLC method revealed that Picrosides I and II content were highest in MCA extract (678.705 µg/mL and 2015.947 µg/mL respectively) followed by KCA (426.501 µg/mL and 1029.281 µg/mL respectively) and LNP (203.623 µg/mL and 751.637 µg/mL respectively). Present study has produced valuable insights for the conservation and sustainable utilization of N. scrophulariiflora of Nepal Himalaya.

Keywords: Neopicrorhiza scrophulariiflora, Kutki, ISSR, DNA barcode, matK, rbcL, trnH- psbA, ITS.