Intraspecific genetic variability, differentiation and evolutionary relationships revealed through microsatellite loci in seven economically important species of Calamus L.

Kurian, B., A. S. Hemanthakumar, Jacob J, Wickneswari R, Choong C Y, Sarmah P, Shefeek S, Vishnu V N, Sajithkumar S V, and K K Sabu. 2019. Intraspecific genetic variability, differentiation and evolutionary relationships revealed through microsatellite loci in seven economically important species of Calamus L. Journal of Forestry Research DOI: 10.1007/s11676-019-00984-z

Abstract
Population density, species richness and critical population parameters are crucial in determining the levels of gene diversity in dioecious species of the genus Calamus. The extent of intraspecific and intrageneric genetic variability in Calamus from the southern Western Ghats of India was studied using 26 microsatellite markers by sampling 227 individuals belonging to seven economically important species. The heterozygosity of microsatellite loci ranged from zero to 0.78. Average gene diversity within species was 0.13; in all species it was 0.18 and amongst species was 0.06. The Shannon Information Index was the lowest for Calamus metzianus (0.11), whereas it ranged from 0.16 to 0.26 for other species. The expected heterozygosity varied from 0.08 to 0.18. Calamus hookerianus and Calamu travancoricus showed the highest genetic differentiation (44%) revealed through Fst values, whereas the lowest (22%) was observed between Calamus gamblei and Calamu thwaitesii. Population structuring and phylogenetic analysis differentiated the seven species. Due to overexploitation and loss of rare alleles, small populations could lead to fertilization between closely related individuals, resulting in inbreeding and increasing the risk of extinction. This could be important for species such as C. metzianus where allelic polymorphism was 23%, whereas for all other species it was 38% to 46%. Genetic diversity “micro-hotspots” were identified from the protected area network of the southern and central Western Ghats with highest observed heterozygosity. Four micro-hotspots from the Agasthyamalai Biosphere Reserve and the Pushpagiri Wildlife Sanctuary may be possible for long-term conservation programs. The findings of this study lay a strong foundation for strengthening protected area networks, especially areas with intermediate levels of disturbance.

Keywords
Genetic diversity Differentiation Population structure Conservation Microsatellites Calamus spp. 

Expression profiling of selected microRNAs coupled with molecular and biochemical analyses of asiaticosides in Centella asiatica (L.) Urb in vitro

Jisha, S., Hemanthkumar A. S., Gouri P. R. and K. K. Sabu. 2019. Expression profiling of selected microRNAs coupled with molecular and biochemical analyses of asiaticosides in Centella asiatica (L.) Urb in vitro. International Journal of Pharmacy and Pharmaceutical Sciences Vol. 11, no. 5, Mar. 2019, https://innovareacademics.in/journals/index.php/ijpps/article/view/32097

ABSTRACT
Objective: Centella asiatica (L.) Urb from Umbelliferae is a potential source of secondary metabolites having immense medicinal value. Asiaticoside is the major therapeutic compound. In the present study, Identification of a possible relationship between concentration/transcript level expression of asiaticoside and concentrations of growth hormones at different growth stages was observed. The current study includes molecular and biochemical evaluation of stress generated in C. asiatica at different time intervals in vitro.

Methods: The enhancement in auxin, cytokinin and final asiaticoside content were determined using immunoassay kits for auxin, cytokinin and HPLC analysis respectively. Transcript level expression at different growth phases was carried out using real-time RT-PCR. For isolation of stress-related miRNAs, reverse transcription of total RNA using miScript II RT Kit PCR System was carried out as per instructions. The differential expression of five selected miRNAs was done by Real-Time RT-PCR. The analysis of stress in vitro was done by quantification of Hydrogen Peroxide (H2O2), total phenolics and total antioxidants by H2O2 assay kit, total antioxidant assay kit and Folin Ciocalteau reagent respectively. The final asiaticoside content was determined by HPLC.

Results: Differential expression of key genes involved in asiaticoside pathway showed significantly higher transcript expression, which is in correlation with the final asiaticoside content. The enhanced expression of miRNAs and the analysis of H2O2, total antioxidant capacity and total phenolics are suggestive of generation of oxidative stress under controlled conditions.

Conclusion: The present study shows a direct correlation between oxidative stress and transcript/phytochemical estimation of asiaticoside content under in vitro conditions.

KEYWORDS:  Asiaticoside, Centella asiatica, In vitro, microRNA, Real-time RT-PCR

The protective role of Piriformospora indica colonization in Centella asiatica (L.) in vitro under phosphate stress

Jisha, S., K. N. Anith and K. K. Sabu. 2019. The protective role of Piriformospora indica colonization in Centella asiatica (L.) in vitro under phosphate stress. Biocatalysis and Agricultural Biotechnology Vol. 19. Article 101088. https://doi.org/10.1016/j.bcab.2019.101088

Abstract
The core of present study was to determine potential role of Piriformospora indica, mutualistic Basidiomycete endosymbiont on co-cultivation with Centella asiatica (L.) Urb., a neutraceutical plant. The analysis of major secondary metabolite asiaticoside and differential expression of key genes involved in pathway under varying phosphate (P) concentrations maintained in vitro were carried out. Maximum asiaticoside content was documented in plants maintained in MS medium containing lowest level (62.5 μM) tried (p < 0.001). At phosphate concentration of 125 μ M (10% of normal P), significant stimulation of plant growth and asiaticoside production were observed in colonized plants. P levels at 125 μM however favored maximum fresh weight, root and shoot length, root and leaf number of plants. The transcript accumulation of β Amyrin Synthase, the key gene in asiaticoside pathway was recorded at the P level of 62.5 μM, which is significantly higher (p < 0.001) than those at other P concentrations tried which was evidenced by overproduction of auxin, Indole-3-Acetic Acid (IAA). A significantly higher level of IAA was recorded in P. indica colonized plants maintained in low (125 μM) P medium. The presence of P. indica under low phosphate concentrations has protective role in mitigating effects of stress, as evidenced by non-significant hydrogen peroxide production, acid/alkaline phosphatase activity, total phenolics and increased super oxide dismutase (SOD) activity in colonized plants. The study clarifies positive correlation of the asiaticoside production in P. indica colonized plants under low P conditions with significant up regulation of asiaticoside pathway gene transcripts with less stress.

Multifunctional aspects of Piriformospora indica in plant endosymbiosis

Jisha, S. and K.K. Sabu. 2019. Multifunctional aspects of Piriformospora indica in plant endosymbiosis. Mycology https://doi.org/10.1080/21501203.2019.1600063 Published online: 31 Mar 2019

ABSTRACT
Piriformospora indica (Hymenomycetes, Basidiomycota) is an endophytic fungus that colonises plant roots, and was originally isolated from Rajasthan desert. It is comparable to Arbuscular Mycorrhizal (AM) fungi in terms of plant growth promotional effects. P. indica has been used as an ideal example to analyse the mechanisms of mutualistic symbiosis. Major benefit of P. indica over AM fungi is that it is axenically cultivable in different synthetic and complex media. A preliminary attempt was made to scrutinise the role of P. indica co-cultivation on seedling vigour of common vegetables like Cucumis sativus L., Abelmoschus esculentus (L.) Moench, Solanum melongena L. and Capsicum annuum L. The positive effect of P. indica co-culture on seedling performance was compared to the effects of growth hormones like indole acetic acid and benzyl amino purine when supplemented to the MS medium at a concentration of 0.1 mg ml−1. An exogenous supply of auxin resulted in enhanced production of roots and cytokinin supplement favoured shoot production, whereas P. indica co-culture favoured simultaneous production of shoot and root over the control. P. indica colonisation inside the roots of C. sativus L. was also successfully established. These preliminary results indicate the prospective role of P. indica in vegetable farming through its favourable effect on plant growth.

KEYWORDS: Piriformospora indica, endosymbiosis, Cucumis sativus, phytohormones