Characterization of genetic diversity in chickpea using SSR markers,Start Codon Targeted Polymorphism (SCoT) and Conserved DNA-Derived Polymorphism (CDDP)

Physiology and Molecular Biology of Plants - To evaluate the genetic diversity among 48 genotypes of chickpea comprising cultivars, landraces and internationally developed improved lines genetic...     

Genetic diversity of Hemarthria altissima and its related species by EST-SSR and SCoT markers

There is a need for an appropriate evaluation of Hemarthria germplasm resources using genetic analysis. Understanding their genetic background will promote effective development and utilization of its germplasm resources in plant breeding. We examined the genetic diversity and relationships among 46 Hemarthria germplasm resources from four continents. Expressed Sequence Tags-Simple Sequence Repeat (EST-SSR) and Start Codon Targeted (SCoT) markers were used to investigate species Hemarthria altissima (36), Hemarthria compressa (8) and Hemarthria uncinata (2). We selected 19 EST-SSR primers and generated 550 polymorphic (94.7%). Twenty one SCoT primers were selected and amplified to produce 597 bands with 89.4% of polymorphic bands. The Mantel test between EST-SSR and SCoT matrices revealed significant correlations (r = 0.854) and the data from both markers were combined for cluster analysis. The 46 materials were clustered into two main groups by UPGMA clustering with a similarity coefficient ranging from 0.573 to 0.940 and the dendrogram was basically concordant with geographical origins and species. Among the three Hemarthria species, H. altissima was genetically closest to H. compressa while it was not close to H. uncinata. When the utility of the two markers were compared, we found EST-SSR to be more efficient than SCoT in determining the genetic diversity study of Hemarthria species.     

Insight into the genetic variability analysis and relationships among some Aegilops and Triticum species,as genome progenitors of bread wheat,using SCoT markers

We assessed the molecular genetic diversity and relationships among some Aegilops and Triticum species using 15 start codon-targeted (SCoT) polymorphism markers. A total of 166 bands amplified, of which 164 (98.79%) were polymorphic. Analysis of molecular variance and inter-population differentiation (Gst) indicated high genetic variation within the studied populations. Our analyses revealed high genetic diversity in T. boeoticum, Ae. cylindrica, T. durum and Ae. umbellulata, low diversity in Ae. crassa, Ae. caudata and Ae. speltoides, and a close relationship among Ae. tauschii, T. aestivum, T. durum, T. urartu, and T. boeoticum. Cluster analysis indicated 180 individuals divided into 8 genome homogeneous clades and 11 sub-groups. T. aestivum and T. durum accessions were grouped together, and accessions with the C and U genomes were grouped into the same clade. Our results support the hypothesis that T. urartu and Ae. tauschii are two diploid ancestors of T. aestivum, and also that Ae. caudata and Ae. umbellulata are putative donors of C and U genomes for other Aegilops species that possess these genomes. Our results also revealed that the SCoT technique is informative and can be used to assess genetic relationships among wheat germplasm.     

Metabolomic profiling and antioxidant activity of some Acacia species

Metabolomic profiling of different parts (leaves, flowers and pods) of Acacia species (Acacia nilotica, Acacia seyal and Acacia laeta) was evaluated. The multivariate data analyses such as principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were used to differentiate the distribution of plant metabolites among different species or different organs of the same species. A.nilotica was characterized with a high content of saponins and A.seyal was characterized with high contents of proteins, phenolics, flavonoids and anthocyanins. A.laeta had a higher content of carbohydrates than A. nilotica and A. seyal. On the basis of these results, total antioxidant capacity, DPPH free radical scavenging activity and reducing power of the methanolic extracts of studied parts were evaluated. A.nilotica and A.seyal extracts showed less inhibitory concentration 50 (IC₅₀) compared to A.laeta extracts which means that these two species have the strongest radical scavenging activity whereas A. laeta extracts have the lowest radical scavenging activity. A positive correlation between saponins and flavonoids with total antioxidant capacity and DPPH radical scavenging activity was observed. Based on these results, the potentiality of these plants as antioxidants was discussed.     

GC-MS-employed phytochemical characterization,synergistic antioxidant,and cytotoxic potential of Triphala methanol extract at non-equivalent ratios of its constituents

Triphala is a famous triherbal drug, comprising three herb fruits, including Terminalia chebula (Haritaki), Terminalia bellirica (Bibhitaki), and Phyllanthus emblica (Amalaki). It is enriched with vitamin C, polyphenols, flavonoids, sterols, saponins, etc., and is well-documented for its potent antioxidant, anticancer, chemoprotective, antimicrobial, and anti-inflammatory effects. This research was conducted to evaluate the synergistic antioxidative and cytotoxic potential of mixtures of the individual constituents of Triphala at their nonequivalent ratios along with the chemical characterization of individual constituents of Triphala to identify and quantify individual compounds. The antioxidative potential was measured using total antioxidant capacity (TAC), DPPH free radical scavenging assay, and total phenolic content (TPC) tests. The cytotoxic potential was assessed on brain cancer cells (N4X4) using MTT assay, and phytochemical characterization was performed by GS-MS analysis. Nonequivalent ratios of Triphala constituents exhibited significantly higher synergistic antioxidant and cytotoxic potential than the equivalent ratios of them. Moreover, the nonequivalent ratio where the quantity of Amalaki was doubled than the other two constituents showed the highest synergistic antioxidant and cytotoxic effect. GC-MS analysis of individual constituents of Triphala identified and quantified the presence of a wide array of compounds, and fatty acid, fatty acid ester, triterpene, and aminoglycoside remained the predominant class of compounds. Thus, it can be inferred that the observed bioactivities can be attributed to the phytocompounds characterized and extracts at the nonequivalent ratio of Triphala constituents where Amalaki is doubled can be more effective in treating oxidative degenerative diseases and glioblastoma.     

Plant size,breeding system,and limits to reproductive success in two sister species of Ferocactus (Cactaceae)

Plant reproductive output can be limited by a variety of factors, bothintrinsic and extrinsic. I investigated the reproductive biologies of twospecies of unbranched short-columnar cacti, Ferocactuscylindraceus and F. wislizeni. I recordedfemalereproductive output (flowers produced, fruit set, seeds per fruit and seedmass), plant size and growth, and used hand-pollination experiments todeterminebreeding systems and pollen limitation. In both species, the ability to selfvaried among individuals, but self-pollination resulted in very few seeds,suggesting strong inbreeding depression. Neither species was pollen-limited.Numbers of flowers produced increases with plant size for both species, andseeds per fruit may also be related to plant size, although the relationship isunclear. Seed mass is not correlated with plant size. Flower production wassimilar in both species, but F. cylindraceus producedfewerseeds per fruit than F. wislizeni, and its seeds weighedless. Fruit set by F. cylindraceus was heavily impacted bya florivorous lepidopteran. Fruit set was very high (94 to 96%) inF. wislizeni, suggesting that architectural constraints(e.g., meristem limitation) are more limiting than resource levels or the levelof pollinator services. In F. cylindraceus, numbers ofseeds per fruit was positively correlated with seed mass, whereas inF. wislizeni, the relationship was negative (tradeoff).Thegrowth rates of F. wislizeni are affected by rainfall theprevious season, and growth rates increase as the plant ages.Ferocactus cylindraceus and F.wislizeni are thought to be sister species, meaning that observeddifferences between them are more likely to be the result of recentevolutionaryprocesses in their lineages rather than differing phylogenetic histories.     

Isolation and HPLC assisted quantification of two iridoid glycoside compounds and molecular DNA fingerprinting in critically endangered medicinal Picrorhiza kurroa Royle ex Benth: implications for conservation

Picrorhiza kurroa is a medicinally important, high altitude perennial herb, endemic to the Himalayas. It possesses strong hepato-protective bioactivity that is contributed by two iridoid picroside compounds viz Picroside-I (P-I) and Picroside-II (P-II). Commercially, many P. kurroa based hepato-stimulatory Ayurvedic drug brands that use different proportions of P-I and P-II are available in the market. To identify genetically heterozygous and high yielding genotypes for multiplication, sustained use and conservation, it is essential to assess genetic and phytochemical diversity and understand the population structure of P. kurroa. In the present study, isolation and HPLC based quantification of picrosides P-I and P-II and molecular DNA fingerprinting using RAPD, AFLP and ISSR markers have been undertaken in 124 and 91 genotypes, respectively. The analyzed samples were collected from 10 natural P. kurroa Himalayan populations spread across four states (Jammu & Kashmir, Sikkim, Uttarakhand and Himachal Pradesh) of India. Genotypes used in this study covered around 1000 km geographical area of the total Indian Himalayan habitat range of P. kurroa. Significant quantitative variation ranging from 0.01 per cent to 4.15% for P-I, and from 0.01% to 3.18% in P-II picroside was observed in the analyzed samples. Three molecular DNA markers, RAPD (22 primers), ISSR (15 primers) and AFLP (07 primer combinations) also revealed a high level of genetic variation. The percentage polymorphism and effective number of alleles for RAPD, ISSR and AFLP analysis varied from 83.5%, 80.6% and 72.1%; 1.5722, 1.5787 and 1.5665, respectively. Further, the rate of gene flow (Nm) between populations was moderate for RAPD (0.8434), and AFLP (0.9882) and comparatively higher for ISSR (1.6093). Fst values were observed to be 0.56, 0.33, and 0.51 for RAPD, ISSR and AFLP markers, respectively. These values suggest that most of the observed genetic variation resided within populations. Neighbour joining (NJ), principal coordinate analysis (PCoA) and Bayesian based STRUCTURE grouped all the analyzed accessions into largely region-wise clusters and showed some inter-mixing between the populations, indicating the existence of distinct gene pools with limited gene flow/exchange. The present study has revealed a high level of genetic diversity in the analyzed populations. The analysis has resulted in identification of genetically diverse and high picrosides containing P. kurroa genotypes from Sainj, Dayara, Tungnath, Furkia, Parsuthach, Arampatri, Manvarsar, Kedarnath, Thangu and Temza in the Indian Himalayan region. The inferences generated in this study can be used to devise future resource management and conservation strategies in P. kurroa.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00972-w.     

Phylogenetic relationships and DNA barcoding of nine endangered medicinal plant species endemic to Saint Katherine protectorate

A high degree of endemism has been recorded for several plant groups collectively in Saint Katherine Protectorate (SKP) in the Sinai Peninsula. Nine endangered endemic plant species in SKP were selected to test the variable abilities of three different DNA barcodes; Riboluse-1,5- Biphosphate Carboxylase/Oxygenase Large subunit (rbcL), Internal Transcribed Spacer (ITS), and the two regions of the plastid gene (ycf1) as well as Start Codon Targeted (SCoT) Polymorphism to find the phylogenetic relationships among them. The three barcodes were generally more capable of finding the genetic relationships among the plant species under study, new barcodes were introduced to the National Centre for Biotechnology Information (NCBI) for the first time through our work. The barcode sequences were efficient in finding the genetic relationships between the nine species. However, SCoT polymorphism could only cluster plant species belonging to the same genus together in one group, but it could not cluster plant species belonging to the same families except for some primers solely. RbcL was the most easily amplified and identified barcode in eight out of the nine species at the species level and the ninth barcode to the genus level. ITS identified all the species to the genus level. Finally, ycf1 identified six out of the eight species, but it could not identify two of the eight species to the genus level.     

Comparative assessment of ISSR, DAMD and SCoT markers for evaluation of genetic diversity and conservation of landrace chickpea (Cicer arietinum L.) genotypes collected from north-west of Iran

Morphological traits and three molecular markers techniques: start codon targeted (SCoT), inter-simple sequence repeat (ISSR) and directly amplified minisatellite DNA (DAMD) markers were compared for fingerprinting of 40 landraces chickpea genotypes collected from different geographical locations of north-west of Iran. Variance analysis of ten measured morphological traits showed significant differences existed between genotypes. Cluster analysis based on morphological traits, divided genotypes in three distinct clusters. Average polymorphism information content (PIC) for ISSR, DAMD and SCoT markers was 0.216, 0.232 and 0.232, respectively, and this revealed that SCoT markers were more informative, followed by ISSRs marker, than other markers for the assessment of diversity amongst genotypes. Cluster analysis for three different molecular types revealed that genotypes taken for the analysis can be divided in three and four distinct clusters. Accessions from same geographical regions mostly showed more genetic similarities than those from origins far isolated apart. These results suggest that efficiency of SCOT, DAMD and ISSR markers was relatively the same in fingerprinting of genotypes but SCOT and DAMD analysis are more effective in fingerprinting of chickpea genotypes. To our knowledge, this is the first detailed report of a comparison of performance among two targeted DNA region molecular markers (SCoT and DAMD) and the ISSR technique on a set of samples of chickpea. Overall, our results indicate that SCOT, ISSR and DAMD fingerprinting could be used to detect polymorphism for genotypes of chickpea.     

Molecular phylogeny of Trichomonadidae family inferred from ITS-1, 5.8S rRNA and ITS-2 sequences

The Trichomonads have been the subject of several molecular studies that reported some discrepancies both at the lower and higher taxonomic levels. The purpose of this study was to make an extensive phylogenetic analysis of the Trichomonadidae using ITS-1/5.8S/ITS-2 sequences, to better understand its phylogeny and the usefulness of this marker. ITS-1/5.8S/ITS-2 sequences of 36 strains from 14 species belonging to Trichomonadidae and Monocercomonadidae were analysed, in which 20 were newly determined. Maximum likelihood, maximum parsimony, neighbour joining, and Bayesian phylogenetic methods were employed in order to reconstruct and compare the evolutionary history of this group. Tetratrichomonas gallinarum and four strains of Tetratrichomonas sp. isolated from bull genital organs were found closely related, confirming the classification of the latter, probably as a new species. The monophyly of Tritrichomonadinae and Trichomonadinae subfamilies were corroborated, with the exclusion of Trichomitus batrachorum from the latter since it grouped consistently with Hypotrichomonas acosta. Tritrichomonas foetus, Tritrichomonas suis and potentially also Tritrichomonas mobilensis seemed to correspond to the same species. Monocercomonas sp. and Ditrichomonas honigbergii emerged as independent lineages, with their phylogenetic positions undetermined. Neither Trichomonadidae nor Monocercomonadidae were supported as monophyletic groups. The ITS-1/5.8S/ITS-2 seems to be a reliable locus for phylogenetic studies in the Trichomonadida, mainly at lower taxonomic levels, and at least up to the family level.     

In-vitro antibacterial,antioxidant potentials and cytotoxic activity of the leaves of Tridax procumbens

The present study explored the phytochemicals, antibacterial, antioxidant and cytotoxic effect of Tridax procumbens leaves. The leaves were dried and extracted with various organic solvents. The leaves contained the phytochemicals such as alkaloids, carbohydrates, polyphenols and tannins respectively. Antimicrobial potentials of the extracts were determined by performing the disc diffusion techniques. Results revealed that different organic solvents extracts namely methanol, ethanol and ethyl acetate extracts documented comparatively good activity against the studied microbial strains. The methanol extract of leaves of T. procumbens showed combatively better antioxidant potential. The tested plant leaf extract showed high activity against human lung cancer cells than breast cancer cell lines. 250 µg/ml plants extract showed 84 ± 2.8% toxicity against human lung cancer cells.     

Cytoplasmic diversity,phylogenetic relationships and molecular evolution of Tunisian Citrus species as inferred from mutational events and pseudogene of chloroplast trnL-trnF spacer

The genus Citrus L. is among the most important fruit trees in the world. In this report, cytoplasmic polymorphism of twenty seven Tunisian Citrus cultivars was explored using the chloroplast trnL-trnF intergenic spacer. Chloroplast sequences showed variation in length and nucleotide content. Haplotype and nucleotide diversity showed low variations. Molecular phylogenetic tree identifies Citrus maternal origins and demonstrates two major groups distinguishing between mandarin and pummelo groups. The trnL-trnF intergenic spacer showed one copy of pseudogene of the original trnF gene in 27 Citrus species at position 275 bp with a size varying from 49 to 63 bp. The anticodon domain was identified as the most conserved element, but one transversion (T−>C) was found in the D-domain. Meanwhile, one transversion (T−>A) and one transition (T−>G) were found in the T-domain. Neutrality tests (Tajima, Fu & Li and Fu) which revealed positive and non-significant values and Pi and θ_W assume a neutral model of evolution and advocated a constant population size. The study demonstrates the resolving power of trnL-trnF sequence data to prove both pummelo and mandarin gene pool’s contribution in the development of Tunisian secondary species and inferring their genetic and phylogenetic relationships.     

Neochilenin,a new glycoside of 3-O-methylquercetin,and other flavonols in the tepals ofNeochilenia,Neoporteria andParodia species (Cactaceae)

3-O-Methylated flavonols were isolated as crystals for the first time from the flowers ofNeochilenia, Neoporteria andParodia species belonging to the sub-family Cereoideae (Cactaceae), which are native to South America. The structures of three compounds were confirmed by chemical and spectral means. In the tepals of 7 species ofNeoporteria, 3-methyl ether of quercetin was found in the form of aglycone, whereas it was present as the 7-O-glucoside in the tepals ofParodia sanguiniflora and as the 4′-O-glucoside in the tepals of three species ofNeochilenia. Among those two glucosides of quercetin 3-methyl ether, the former has been found in a whole plant ofArtemisia transiliensis (Compositae), while the latter is new to the literature. Therefore, the term “neochilenin” may be assigned to this new pigment. Contribution from the Research Institute of Evolutionary Biology, No. 44.     

Antifungal, acetylcholinesterase inhibition, antioxidant and phytochemical properties of three Barleria species

This study was aimed at evaluating the antifungal, acetylcholinesterase inhibition and antioxidant activities of petroleum ether, dichloromethane, ethanol and methanol extracts from different parts of Barleria prionitis, Barleria greenii and Barleria albostellata. Their phytochemical properties and the possibility of plant-part substitution as a conservation strategy against destructive harvesting (use of aerial parts and roots) of these species for medicinal purposes were also investigated. Microtitre plate assays were used in determining their antifungal (against Candida albicans) and acetylcholinesterase inhibition activities. All the extracts demonstrated both fungistatic and fungicidal activities with the minimum inhibitory concentration ranging from 0.78 to 9.38 mg/ml and minimum fungicidal concentration ranging from 1.17 to 12.50 mg/ml. The higher inhibitory activity of B. greenii leaf extracts in most cases compared to similar extracts of the stems and roots suggest the potential of B. greenii leaves in plant-part substitution. At the lowest extract concentration (0.156 mg/ml), the leaf extract of B. greenii demonstrated a significantly higher acetylcholinesterase (AChE) inhibition than its stem and root extracts. In B. albostellata, the AChE inhibitory activity demonstrated by the stem was significantly greater than that recorded in its leaf extract. These findings suggest that the idea of plant part substitution may be species and/or biological activity dependent. In the DPPH radical scavenging assay, different parts of Barleria species showed free radical scavenging activity with EC₅₀ values ranging from 6.65 to 12.56 μg/ml. The ability of the extracts from different plant parts to reduce ferric ion/ferricyanide complex to the ferrous form and decrease carotenoid bleaching rate suggests the presence of antioxidant compounds capable of donating electrons and hydrogen atoms in their reaction mechanisms. Flavonoids, iridoids and tannins were detected in the different parts of these Barleria species. These phytochemicals might be responsible for the observed biological activities. The isolation of specific bioactive compounds through bioassay-guided fractionation and their characterization as well as studies evaluating their safety may be necessary in the exploration of these species for potential new therapeutic drugs or drug leads.     

Genetic diversity and phylogeny of the family Osteoglossidae by the nuclear 18S ribosomal RNA and implications for its conservation

The genetic structure of the family Osteoglossidae from different geographical regions was examined using the nuclear 18S ribosomal RNA (18S rDNA) sequence. The results showed that the partial length of 18S rDNA was 1277 bp, and they were relatively conserved in the species of the family Osteoglossidae. A total of 62 (4.83%) polymorphic sites were observed, and 28 haplotypes were defined, which were characterized by different haplotype diversity (0.00105–0.900) and nucleotide diversity (0.00321–3.000). Analysis of molecular variance (AMOVA) showed genetic divergence of these species (79.76%). Phylogenetic analysis revealed that 18S rDNA was a suitable genetic marker and was able to distinguish phylogenetic relationships among different Osteoglossidae species, and it also supported current taxonomy. In addition, low genetic diversity of several species provides genetic assessment information and should be taken into consideration to implement a conservation management planning for some species, such as Scleropages formosus green arowana.     

A comparative assessment of morphological and molecular characterization among three Ziziphus species

Genetic variability of 84 accessions of three Ziziphus species including Z. spina-christi, Z. nummularia and Z. mauritiana were analyzed using a combination of morphological traits and translation initiation codon (ATG) polymorphism. Both morphological and molecular data revealed a high level of inter and intra specific variations among the accessions. Accordingly, 90.49% of amplified fragments were polymorphic among the accessions with the mean values of 0.37 for polymorphic information content (PIC), 3.31 for resolving power (RP), and 1.95 for marker index (MI). The phylogenetic clustering clearly delineated the entire germplasm into three well supported distinct clusters according to the species sources. According to the Nei''s genetic identity, Z. spina-christi and Z. nummularia were the most similar species and had high differentiation with Z. mauritiana. Moreover, the highest values for Shannon’s information index (I = 0.505) and gene diversity (h = 0.347) were recorded in Z. spina-christi indicating there is higher genetic diversity compared with two other species. Four private alleles were identified in two species which could be beneficial for accessions authentication in argumentative situations. Moreover, results of the Mantel test showed there were moderate correlation between molecular and morphological matrices. In addition, estimation of bivariate correlations revealed there were significant positive and negative correlations between different variables, which offer a practical application of this information during phenotype based selection in ber improvement programs. The results of this investigation highlight the efficiency of translation initiation codon polymorphism for genetic characterization and accurate authentication of Ziziphus accessions as well as detecting and tagging morphologically important traits in this genus that would be helpful for implementation of effective conservation strategies and even broaden current genetic diversity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01000-7.     

Phytochemical,antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus L.) leaves

The phytochemical, antioxidant and mineral composition of hydroalcoholic extract of leaves of Cichorium intybus L., was determined. The leaves were found to possess comparatively higher values of total flavonoids, total phenolic acids. The phytochemical screening confirmed the presence of tannins, saponins, flavonoids, in the leaves of the plant. The leaf extract was found to show comparatively low value of IC₅₀ for 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition. The IC₅₀ value of chicory leaves extract was found to be 67.2 ± 2.6 μg/ml. The extracts were found to contain high amount of mineral elements especially Mg and Zn. Due to good phytochemical and antioxidant composition, C. intybus L., leaves would be an important candidate in pharmaceutical formulations and play an important role in improving the human health by participating in the antioxidant defense system against free radical generation.     

Allozyme diversity and morphometrics of Melocactus paucispinus (Cactaceae) and evidence for hybridization with M. concinnus in the Chapada Diamantina, North-eastern Brazil

BACKGROUND AND AIMS: Melocacatus paucispinus (Cactaceae) is endemic to the state of Bahia, Brazil, and due to its rarity and desirability to collectors it has been considered threatened with extinction. This species is usually sympatric and inter-fertile with M. concinnus, and morphological evidence for hybridization between them is present in some populations. Levels of genetic and morphological variation and sub-structuring in populations of these species were assessed and an attempt was made to verify the occurrence of natural hybridization between them. METHODS: Genetic variability was surveyed using allozymes (12 loci) and morphological variability using multivariate morphometric analyses (17 vegetative characters) in ten populations of M. paucispinus and three of M. concinnus occurring in the Chapada Diamantina, Bahia. KEY RESULTS: Genetic variability was low in both species (P = 0.0-33.3, A = 1.0-1.6, H(e) = 0.000-0.123 in M. paucispinus; P = 0.0-25.0, A = 1.0-1.4, H(e) = 0.000-0.104 in M. concinnus). Deficit of heterozygotes within the populations was detected in both species, with high values of F(IS) (0.732 and 0.901 in M. paucispinus and M. concinnus, respectively). Evidence of hybridization was detected by the relative allele frequency in the two diaphorase loci. High levels of genetic (F(ST) = 0.504 in M. paucispinus and 0.349 in M. concinnus) and morphological (A = 0.20 in M. paucispinus and 0.17 in M. concinnus) structuring among populations were found. CONCLUSIONS: The Melocactus spp. displayed levels of genetic variability lower than the values reported for other cactus species. The evidence indicates the occurrence of introgression in both species at two sites. The high F(ST) values cannot be explained by geographical substructuring, but are consistent with hybridization. Conversely, morphological differentiation in M. paucispinus, but not in M. concinnus, is probably due to isolation by distance.     

Comparative molecular and phytochemical study of the tree species Santalum austrocaledonicum (Santalaceae) distributed in the New-Caledonian archipelago

We have tried to elucidate the origin of phytochemical variation in trees by studying concomitantly the chemical and microsatellite variations in Santalum austrocaledonicum. Eight natural populations were sampled in the New-Caledonian archipelago, a total of 157 individuals being analyzed. The main components, as revealed by gas chromatography (GC), were alpha- and beta-santalol (as in other sandalwood species), although the level of (Z)-lanceol was particularly high. Most of the chemical variation was observed within populations (83.7%). With microsatellites, the variation between populations was more pronounced (32% of the total variation). Although the chemical variation between populations was small, we investigated the effects of genetic drift and migration by comparing the chemical- and molecular-differentiation patterns. The poor congruence between neighbor-joining trees, confirmed by the non-significant Mantel test between the molecular and chemical distance matrices (R=0.26, P=0.12), showed that genetic drift and migration are not the main evolutionary forces acting on chemical differentiation between populations. We could not find any effect of soil and rainfall conditions neither. Although the impact of drift and migration cannot be discounted in rationalizing between-population differentiation, the low variation among populations could result from a stabilizing selection caused by the same phytopathogen charge across the natural range.     

Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui

Ceropegias has acquired significant importance due to their medicinal properties, edible tubers, and its ornamental flowers. The aim of this study was to optimize direct shoot organogenesis (DSO), indirect shoot organogenesis (ISO) and plant regeneration of threatened medicinal plant Ceropegia santapaui, followed by analysis of genetic status and biochemical characterization of micropropagated plantlets. For optimization, cotyledonary nodes and cotyledons were used as source of explants in DSO and ISO respectively. The highest frequency of regeneration (88.0 %) for DSO with 8.1 ± 0.6 shoots per explant was obtained from cotyledonary nodes cultured on Murashige and Skoog’s (MS) medium containing 2.0 mg L^?1 2iP. The best response for callus induction and proliferation was achieved with 1.5 mg L^?1 PR (picloram) in which 97.5 % of cultures produced an average of 913 ± 10.9 mg (fresh weight) of callus. The highest frequency of shoot formation (92.5 %) with an average of 19.7 ± 0.3 shoots in ISO was obtained when calli were transferred to MS medium supplemented with 2.5 mg L^?1 BAP and 0.4 mg L^?1 IBA. Regenerated shoots were best rooted in half-strength MS medium with 2.0 mg L^?1 NAA. Plantlets successfully acclimatized were morphologically indistinguishable from the source plant. Micropropagated plantlets subjected to random amplified polymorphic DNA and inter simple sequence repeats (ISSR) marker based profiling reveled uniform banding pattern in DSO-derived plantlets which was similar to mother plant. ISSR fingerprints of ISO-derived plants showed low variation. Method of regeneration, plant part and solvent system significantly affected the levels of total phenolics, flavonoids and antioxidant capacity. Assay of antioxidant activity of different tissues revealed that significantly higher antioxidant activity was observed in ISO-derived tissues than DSO-derived and mother tissues. RP-HPLC analysis of micropropagated plantlets showed the presence of three major phenolic compounds which were similar to those detected in mother plant. Rapid multiplication rate, genetic stability and biochemical parameter ensures the efficacy of the protocol developed for the propagation of this threatened medicinal plant.