Phytochemical profiling of the stem bark of Betula utilis from different geographical regions of India using UHPLC-ESI-MS/MS.

A simple, rapid, sensitive, and reliable ultra-high-performance liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry method was developed and validated for simultaneous determination of 10 bioactive compounds in stem bark of Betula utilis grown in high altitude of Himalaya, India. The objective of the study is to develop and validate ultra-high-performance liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry for investigation of geographical variations of triterpenoids, phenolics, and flavonoids contents in stem bark of B. utilis. The validated method was successfully applied to investigate geographical variations of triterpenoids, phenolics, and flavonoids in stem bark of B. utilis. The contents of betulinic acid and oleanolic acid were detected higher among selected analytes. The present variation study reveals great importance for the application and overall assessment of B. utilis.

The rediscovery of Uraria lacei Craib (Leguminosae) after 67 years from India.

Manipur is one of the biodiversity-rich states in the North-Eastern region of India, and it is also part of the Indo-Burma biodiversity hotspot with rich plant diversity and endemism. Recent field exploration in the area has resulted in the rediscovery of Uraria lacei Craib after 67 years from its last collection in 1952. The rediscovery of this beautiful species fills a gap in the current distribution knowledge and should pave the way for its immediate conservation and propagation.

Estimation of genetic diversity and population structure in Tinospora cordifolia using SSR markers.

Thirty polymorphic SSRs, derived from RNA sequencing of Tinospora cordifolia (willd.), were utilized for genetic diversity and population structure evaluation among 96 accessions collected from ten different geographical regions of India. A total of 7611 SSRs were identified from 268149 transcripts. Of all SSR loci, 69.07% of them were tri-nucleotide repeat motifs, followed by di-nucleotide repeat motifs (12.82%). A total of 230 alleles were generated by 30 SSRs with an average of 7.67 alleles per locus with comparatively higher polymorphic information content (average 0.68). The expected (He) and observed (Ho) heterozygosity means were 0.71 and 0.12, respectively. All the loci showed significant deviation from Hardy-Weinberg Equilibrium (HWE). The neighbor joining clustering based on jaccard's coefficient grouped all the 96 accessions into three major cluster which was also in congruence with model-based structure plot. The result of molecular variance (AMOVA) revealed higher genetic variance within populations than among populations. The result reflects an existence of high level of genetic diversity in the collected accessions of T. cordifolia. The accessions Tc131, Tc31, Tc129, Tc38, Tc16, Tc59, Tc60, Tc17, Tc106 and Tc130 was found to be potential and diverse in nature and the SSRs TCSSR-18, TCSSR-37, TCTSSR-59, TCTSSR-92, TCTSSR-123 and TCTSSR-126 as potential markers. These accessions and newly developed SSR markers provide valuable resource and could be strategically utilized for further genetic improvement of T. cordifolia.

Molecular analysis of genetic diversity and population genetic structure in Ephedra foliata: an endemic and threatened plant species of arid and semi-arid regions of India.

Studies on the genetic diversity and structure in endangered and threatened species are of utmost importance to design and promote effective conservation and management programs. Ephedra foliata, an endemic and threatened species growing naturally in arid and semi-arid regions of north western India, was investigated to estimate genetic variability and population structure using inter-simple sequence repeats (ISSR) and directed amplification of mini-satellite DNA (DAMD) markers. Twenty-five (ISSR 15; DAMD 10) markers produced 449 fragments, of which 382 were polymorphic in nature, revealing 84.59% polymorphism. ISSR markers revealed higher levels of polymorphism, polymorphic information content, marker index, diversity index and effective multiplex ratio than DAMD markers. Higher values of polymorphism, genetic diversity and Shannon information index at species level than at population level revealed that E. foliata possess high genetic diversity. AMOVA revealed much higher variance within populations than among the populations. The three clustering approaches viz., UPGMA, PCoA, and STRUCTURE, grouped the eleven investigated populations into two clusters revealing two genetic populations and the patterns of clustering of populations was in accordance with their geographic distribution, suggesting that these populations have evolved in response to their local environments. The high level of genetic differentiation (G ST = 0.31) and moderate gene flow (N m = 1.11) among populations could be due to geographic isolation, regional climatic conditions, over-exploitation and improper seed setting. To the best of our knowledge, this study is the first endeavour to estimate genetic diversity and population structure of E. foliata using molecular markers.

Major bioactive phenolics in Bergenia species from the Indian Himalayan region: Method development, validation and quantitative estimation using UHPLC-QqQLIT-MS/MS.

Bergenia species are important medicinal plants used in indigenous systems of medicine for their antilithiatic and diuretic properties. An ultra-high performance liquid chromatography coupled to hybrid linear ion trap triple quadrupole mass spectrometry (UHPLC-QqQLIT-MS/MS) method has been developed and validated for the estimation of quantitative variation of eight major bioactive phenolics in the rhizomes (150 samples) of four species of this herb, Bergenia (B. ciliata, B. ligulata, B. purpurascens and B. stracheyi). Chromatographic separation was obtained on a Waters ACQUITY UPLCTM BEH (ethylene bridged hybrid) C18 column with a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile under a gradient elution manner. A hybrid linear ion trap triple quadrupole mass spectrometer was operated in negative electrospray ionization mode with multiple reactions monitoring for detection and quantification of the eight compounds. The validated method demonstrated good linearity (r2 ≥ 0.9991), precision (RSD ≤ 1.87%) and accuracy (95.16-102.11%, RSD ≤ 1.83%) for all reference analytes. The quantitative results revealed that B. ligulata contains the highest amount of the major active marker-bergenin. The results also suggest that sensitive UHPLC-QqQLIT-MS/MS method, a sensitive, accurate and convenient one, could be helpful in identification of potential accession(s), rapid quality control and establishing authenticity of Bergenia species as raw material for pharmaceutical industries.

Nucleotide diversity and phylogenetic relationships among Gladiolus cultivars and related taxa of family Iridaceae.

The plastid genome regions of two intergenic spacers, psbA-trnH and trnL-trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA-trnH region was higher than trnL-trnF region of chloroplast. We employed Bayesian, maximum parsimony (MP) and neighbour-joining (NJ) approaches for phylogenetic analysis of Gladiolus and related taxa using combined datasets from chloroplast genome. The psbA-trnH and trnL-trnF intergenic spacers of Gladiolus and related taxa-like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out group species (Hymenocallis littoralis and Asphodeline lutea) were used in the present investigation. Results showed that subfamily Iridoideae have sister lineage with subfamily Ixioideae and Crocoideae. H. littoralis and A. lutea were separately attached at the base of tree as the diverging Iridaceae relative's lineage. Present study revealed that psbA-trnH region are useful in addressing questions of phylogenetic relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and have more phylogenetically informative sites than the trnL-trnF spacer, and therefore, are suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are extensively used as an ornamental crop and showed high potential in floriculture trade. Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover, one of the most popular methods for generating new cultivars is hybridization. Hence, information on phylogenetic relationships among cultivars could be useful for hybridization programmes for further improvement of the crop.

Molecular differentiation of Chenopodium album complex and some related species using ISSR profiles and ITS sequences.

The present study was undertaken to understand the genetic differentiation and relationships in various components of C. album complex, C. giganteum and some related species using inter simple sequence repeats (ISSR) profiles and internal transcribed spacer (ITS) sequences. The relationships based on UPGMA dendrograms have shown the heterogenous nature of C. album complex. The 2x taxa while showing close relation among themselves are sharply segregated from 4x and 6x taxa belonging to C. album and C. giganteum. Among the three cytotypes from North Indian plains the 4x shows greater similarity to 6x than to 2x which is corroborated by the karyotypic studies. Furthermore, the 6x C. album and C. giganteum accessions of American and European origin are clearly segregated from those of Indian origin which may show their separate origin. Other related species show relationships according to their taxonomic position. The present study based on ISSR profiles and ITS sequences has therefore been very useful in explaining the relationships between various components of C. album complex and related species. However, more work needs to be done using different CpDNA loci to define correct species boundary of the taxa under C. album complex from Himalayas and North Indian Plains.

Genetic variability and population structure in Sapindus emarginatus Vahl from India.

Sapindus emarginatus is an economically important tropical tree species sparsely distributed in different geographical provinces like Gangetic Plains, Western Ghats, and Deccan Plateau in India. In the present paper estimation of genetic variability within and among 41 accessions representing five populations was carried out using 3 single primer amplification reaction (SPAR) methods viz. RAPD, DAMD and ISSR. The cumulative data analysis was carried out for all three SPAR methods, and showed 82.32% polymorphism across all the accessions of S. emarginatus. Jaccard's similarity values among 41 accessions ranged from 0.15 to 0.49 with an average value of 0.37. The intra-population genetic diversity revealed highest values of Nei's genetic diversity (0.19,) Shannon information index (0.29) and polymorphic loci (55.18%), among the accessions of Gujarat (GJ) population, while the corresponding lowest values were (0.10), (0.15) and (26.40%) respectively among the accessions of Rajasthan (RJ) population. The maximum inter-population average genetic distance (0.20) was between Karnataka (KA) and RJ, while the corresponding least genetic distance (0.06) was between Allahabad (AL) and Varanasi (VS) populations. The analysis of molecular variance (AMOVA) revealed maximum percentage of variation among individuals of populations (72%) followed by 16% among regions and 12% among populations. Principal coordinate analysis (PCA) of cumulative data also supported the clustering pattern in the UPGMA dendrogram. These results suggest that genetic diversity is corroborating with the geographical diversity. Mantel's test was performed which revealed a highly significant correlation between cumulative vs RAPD, and showed the maximum (0.93) correlation coefficient, followed by cumulative vs ISSR (0.78) and cumulative vs DAMD (0.91) respectively, and this clearly indicates that the SPAR methods (RAPD, DAMD and ISSR) are sufficiently informative and are suitable to analyze the genetic variability within and among the populations of S. emarginatus.