Monday, December 10, 2018

Identification and characterization of drought responsive microRNAs and their target genes in cardamom

Anjali, N, Nadiya, F, Thomas, J, and K. K. Sabu. 2018. Identification and characterization of drought responsive microRNAs and their target genes in cardamom (Elettaria cardamomum Maton). Plant Growth Regulation. doi: 10.1007/s10725-018-0462-9 Online First: 2018-12-06

Plant miRNAs are found to be present throughout the genome and they regulate gene expression either by cleaving mRNA or inhibiting the translational process at the post transcriptional level. Drought is one of the major limiting factors that negatively affect productivity of plants. Cardamom cultivation is having good production potential but the plants are vulnerable to biotic and abiotic stress factors. To date, nothing is known about the regulatory roles of miRNAs in response to drought stress in cardamom. Ion Torrent sequencing of two small RNA libraries prepared from control (C) and treated (T) plants raised under well irrigated and drought stressed treatments respectively created 3,938,342 and 4,083,181 primary reads. A total of 150 conserved and 20 novel microRNAs were identified from both the control and treated libraries. Discovery of 17 differentially expressed miRNAs under drought stress suggests that these miRNAs might have involved in various biological processes to improve plant tolerance to water stress. Several target genes for drought stress regulating miRNAs were identified including miR156l and miR169c which cleave the target mRNA involved in response to water deprivation. miR530b and miR156a target mRNAs which respond to water deprivation and inhibit the translational process. The expression patterns of some of the miRNAs and their targets were validated by qRT-PCR. This study is the first report of drought responsive miRNAs and their targets in cardamom. The outcome of this research could provide insights into the miRNA based regulatory mechanisms in response to drought stress in monocot plants.

See at Journal website -

Sunday, October 14, 2018

Nadiya, F., N. Anjali, Jinu Thomas, A. Gangaprasad, K. K. Sabu. 2018. Deep sequencing identified potential miRNAs involved in defense response, stress and plant growth characteristics of wild genotypes of cardamom. Plant Biology. Published online: 11 August 2018


  • Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses.
  • In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer.
  • We identified 161 potential miRNAs representing 42 families, including monocot/tissue‐specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars.
  • Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT‐PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA–mRNA target pairing using RNA ligase‐mediated 5′ Rapid Amplification of cDNA Ends (5′RLM‐RACE) PCR.
Keywords: Cardamom, microRNA, next generation sequencing, RNA sequencing
Sakthipriya M., S. S. Vishnu, S. Sujith, P. Rajeesh Kumar, and K. K. Sabu. 2018. Analysis of genetic diversity of Centella asiatica using SSR markers. International Journal of Applied Sciences and Biotechnology Vol 6(2): 103-109.

Genetic diversity represents the heritable variation within and between populations of organisms. A better understanding of genetic diversity and its distribution is essential for its conservation and use. In this study the genetic diversity analysis was performed in 30 accessions of Centella asiatica (L.) Urb. using 10 SSR markers. C. asiatica is a tropical medicinal plant from Apiaceae family native to Southeast Asian countries. It is among the top herbs in the category of anti-aging and CNS drugs used worldwide. The whole plant and aerial parts are used widely in traditional and alternative medicines due to its ample pharmacological activities. The molecular screening through microsatellite markers showed low polymorphism (0.019) between the samples analyzed. Further studies, including the effect of environmental factors, genetic composition or possibility of inbreeding, are required to analyze the probable reason for the low variability exists in the species.

Int. J. Appl. Sci. Biotechnol. Vol 6(2): 103-109

Keywords: Centella asiatica; genetic diversity; SSR; microsatellites; medicinal plant

Transcriptome mining for EST-INDELs and development of EST-SSR markers in turmeric

Sabu K. K., M. D. Shehenaz and J. Amrutha. 2018. Transcriptome mining for EST-INDELs and development of EST-SSR markers in turmeric (Curcuma longa L.). International Journal of Agriculture, Environment and Biotechnology 11(3): 487-492.

Curcuma longa L. commonly known as turmeric is used as a culinary spice in India and many Asian countries. Turmeric has anti-fungal, anti-bacterial, anti-malarial and anti-cancer properties to mention a few. We have analyzed the transcriptome of C. longa retrieved from NCBI SRA database (SRR495223) for the development and validation of EST-SSR markers and identification of EST-InDels to be used for C. longa and related genera. A total of 337 primers were developed and 20 primers having the rating of 100% with the help of NetPrimer were selected and used for PCR validation in C. longa, Amomum subulatum Roxb. (large cardamom) and Elettaria cardamomum (L.) Maton (small cardamom). It was found that around 50% primers generated PCR products in both types of cardamom and 85% in C. longa. The developed primers worked with curcuma, large cardamom and small cardamom plants at varying levels. Besides, the transcriptome analysis detected high amount of deletion and 18 addition of bases which could be screened through the development of CAPS markers using the tool SNP2CAPS and it was found that 93 restriction enzyme can be used for screening these InDels.

  • Transcriptome sequence of Curcuma longa was used for the development of EST-SSR markers.
  • The EST-SSR primers were validated in Curcuma longa and its related taxa such as Amomum subulatum and Elettaria cardamomum
  • EST-InDels were also successfully identified in the transcriptome sequences of Curcuma longa.

Keywords: Curcuma longa, EST-SSR, EST-InDel, PCR, ClustalW, WebSat, NetPrimer, SNP2CAPS

Sunday, July 29, 2018

Stress analysis and cytotoxicity in response to the biotic elicitor, Piriformospora indica and its’ cell wall extract in Centella asiatica L. Urban.

Stress analysis and cytotoxicity in response to the biotic elicitor, Piriformospora indica and its' cell wall extract in Centella asiatica L. Urban


Oxidative stress analysis in Centella asiatica(L.) Urban on co-cultivation with Piriformospora indica and its extract is proposed.
The presence of Piriformospora indica had protective role in alleviating stress.
No variation in the cytotoxicity levels in control and treated groups, as evidenced by MTT assay.
The cell integrity appeared intact in P. indicacolonized C. asiatica.


Study reports prominent findings on the analysis of oxidative stress generated in Centella asiatica, a multifunctional medicinal plant on co-cultivation with P. indica and its extract, P. indica Cell Wall Extract. The presence of P. indica had protective role in alleviating stress, evidenced by lack of significant change in H2O2, increased total antioxidant capacity and phenolics in P. indica colonized and PiCWE treated plants. No variation was observed between IC50 values and the cell integrity in P. indica colonized C. asiaticaappeared intact by TEM analysis. The study preludes the possible role of P. indica in conferring drought tolerance to C. asiatica.


Centella asiatica, 
Oxidative stress, 
Piriformospora indica, 
Total antioxidant capacity

Genome-wide differential expression profiling in wild and cultivar genotypes of cardamom reveals regulation of key pathways in plant growth and development

Agri Gene

Volume 8, June 2018, Pages 18-27

Genome-wide differential expression profiling in wild and cultivar genotypes of cardamom reveals regulation of key pathways in plant growth and development


Transcriptome sequencing data of five cultivars and a wild genotype of cardamom was used to find the differential expression profiling of the cultivar and wild genotypes of cardamom.
Differential expression profiling of the cultivar and wild genotypes of cardamom exhibited 132 unigenes as differentially expressed (log2>4) with 105 up regulated and 27 down regulated genes in cultivars comparing with its wild genotype.
This work represents the first report on differential expression profiling and functional annotation of wild and cultivar genotypes which will enhance the genomic resources of the species.


Cardamom (Elettaria cardamomum Maton) is an economically valuable spice crop and the essential oil of cardamom possesses splendid medicinal properties. To get insight into the domestication related traits and varied gene regulation, differential expression profiling of wild and cultivar cardamom was performed by analyzing the transcriptome data available for cardamom. Functional annotation using seven different publicly available databases identified significant genes coding for enzymes participating in monoterpenoid biosynthesis. Differential expression profile of cultivar and wild genotypes of cardamom exhibited 132 unigenes as differentially expressed (log2 > 4) with 105 up regulated and 27 down regulated genes in cultivars comparing with its wild genotype. Expression analysis showed wild cardamom has increased drought stress tolerance, defense response, and various plant growth regulations. Most of the genes coding for enzymes participating in flavanoid biosynthesis were up regulated, while resistance related genes (cinnamoyl CoA reductase) and few genes involved in the monoterpenoid biosynthetic pathway were down regulated in cultivars when compared to its wild progenitor. Transcriptome data were validated and correlated using qPCR using 10 randomly selected differentially expressed genes. Our study is the first application of next generation RNA-seq to explore the genes involved in various metabolic pathways and quantification of transcript expression levels in cultivar and wild cardamom genotypes.
Differential expression, 
Up regulation, 
Down regulation, 
Essential oil biosynthesis

Piriformospora indica Cell wall Extract as the best elicitor for asiaticoside production in Centella asiatica (L.) Urban, evidenced by morphological, physiological and molecular analyses

 2018 Apr;125:106-115. doi: 10.1016/j.plaphy.2018.01.021. Epub 2018 Feb 7.

Piriformospora indica cell wall extract as the best elicitor for asiaticoside production in Centella asiatica (L.) Urban, evidenced by morphological, physiological and molecular analyses.


Vascular plants synthesise a multitude of organic molecules or phytochemicals, referred to as "secondary metabolites". These molecules are involved in a variety of roles in the life span of plants, ranging from structural ones to protection. Centella asiatica (L.) Urban has probably been used since prehistoric times and has been reported to have been used for various medicinal and cosmetic purposes. The plant contains several active constituents, of which the most important is asiaticoside, a triterpenoid. Asiaticoside content in C. asiatica can be enhanced by the use of biotic elicitors like Piriformospora indica. P. indica has been used as a model to study the mechanisms and evolution of mutualistic symbiosis. P. indica is similar to Arbuscular Mycorrhizal (AM) fungi in terms of plant growth promotional effects. The autoclaved fraction from P. indica (PiCWE) was found to be the most active fraction in promoting the plant biomass and asiaticoside content. To date, there are no reports on the potential role of PiCWE in enhancement of asiaticoside over the control and P. indica colonized plants, which was evidenced by the differential expression of key genes involved and final asiaticoside content along with the determination of phytohormones. Moreover, differential expression of selected miRNAs in PiCWE - C. asiatica root interactions over the control and P. indica treated C. asiatica leaf samples was also scrutinized. The important consequence of induction with PiCWE was the significant enhancement of asiaticoside in the PiCWE induced plants in comparison with the asiaticoside content in control and P. indica-C. asiatica interaction. In addition, the role of miRNAs in C. asiatica - PiCWE would enable more in-depth studies for deciphering the molecular and physiological mechanisms of the association and regulation of PiCWE - C. asiatica interactions.


Centella asiatica; HPLC; Piriformospora indica cell wall extract; β amyrin synthase

Sunday, January 14, 2018

Rapidly evolving sex-specific sequences in Calamus travancoricus Bedd. ex. Becc. and Calamus nagbettai R.R.Fernald & Dey

Gene sequences mediating sexual reproduction are more divergent within and between closely related species. Microsatellite or simple sequence repeat (SSR) markers are valuable molecular tools for analysis of genetic variability, phylogeny, and also for identifying sex at seedling stage in dioecious plant species. Calamus travancoricus Bedd. ex. Becc. and Calamus nagbettai R.R.Fernald & Dey are economically important rattan species. The dioecious nature of the Calamus spp. limits its breeding and cultivation. The sex of rattans can only be identified after attaining reproductive maturity which ranges from 5 to 15 years. A study was carried out in this background and 9 putative sex-specific PCR products were identified as sex markers for Ctravancoricus and Cnagbettai and sequenced by Sanger method. The sequence homology search revealed occurrence of identical sequences in many plant species across different families indicating the conserved nature of the sequences. However, these sequences were not present in opposite sex in the studied species, indicating divergent evolution favoring sex determination. Annotation of these sequences revealed that most of these are mediating sexual reproduction by and large. An adequate sex ratio is to be maintained for these dioecious palms in natural habitat for producing offsprings having equal gene complements for continual evolution and sustainable utilization. Developing scientific management strategies and improved utilization of canes could help to generate employment locally and thus contribute to the socioeconomics sustainably.

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