Transcriptome profiling of Elettaria cardamomum (L.) Maton (small cardamom)

Elettaria cardamomum (L.) Maton, known as ‘queen of spices, is a perennial herbaceous monocot of the family Zingiberaceae, native to southern India. Cardamom is an economically valuable spice crop and used widely in culinary and medicinal purposes. In the present study, using Ion Proton RNA sequencing technology, we performed transcriptome sequencing and de novo transcriptome assembly of a wild and five cultivar genotypes of cardamom. RNA-seq generated a total of 22,811,983 (92 base) and 24,889,197 (75 base) raw reads accounting for approximately 8.21GB and 7.65GB of sequence data for wild and cultivar genotypes of cardamom respectively. The raw data was submitted to SRA database of NCBI under the accession numbers SRX1141272 (wild) and SRX1141276 (cultivars). The raw reads were quality filtered and assembled using MIRA assembler resulted with 112,208 and 264,161contigs having N50 value 616 and 664 for wild and cultivar cardamom. The assembled unigenes were functionally annotated using several databases including PlantCyc for pathway annotation. This work represents the first report on cardamom transcriptome sequencing. In order to generate a comprehensive reference transcriptome we further assembled the raw reads of wild and cultivar genotypes which might enrich the plant transcriptome database and trigger advanced research in cardamom genomics. 

Citation
Nadiya, F., N. Anjali, Jinu Thomas, A. Gangaprasad, K. K. Sabu. 2017. Transcriptome profiling of Elettaria cardamomum (L.) Maton (small cardamom). Genomics Data Vol 11: 102–103. doi: 10.1016/j.gdata.2016.12.013

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Development of a New Pipeline for Identification and Characterization of MicroRNAs from Plants

Open source microRNA analysis pipelines for next generation sequencing data (NGS) often make necessary use and working knowledge of command line interface, massive data processing resources and expertise which is a daunting task for biologists. Further, the microRNA data generated from NGS platforms will not be in a form from which one could understand or make use of it. Hence a comprehensive pipeline has been developed by integrating several open source NGS tools along with a graphical user interface called sRNAbench. It is useful for expression profiling of small RNAs and prediction of microRNAs from NGS data. The pipeline features functionalities such as read processing, sequence identification, target prediction and enrichment analysis. It provides even prediction of novel microRNAs and its sequences. The pipeline will be very useful for plant genomics community and it does not require knowledge in computational biology in order to discover miRNAs and utilize the same in genomics studies.

Citation
Thomas, J. and K. K. Sabu. 2016. Development of a New Pipeline for Identification and Characterization of MicroRNAs from Plants. International Journal for Computational Biology 5(1): 21-27.

MiRNA Isolation from Plants Rich in Polysaccharides and Polyphenols

MicroRNAs (miRNAs) are a group of 18–22-nucleotide-long noncoding RNAs, which show wide array of roles in various biological and metabolic processes in both animals and plants. They are formed endogenously and have evolutionary conserved sequences. RNA quality is extremely important for sequencing miRNAs using next-generation sequencing platforms. However, isolation and quantification of miRNAs from plant samples are often technically difficult and recovery of miRNAs from total RNA might be problematic. Degradation of RNA weakens the true miRNAs present in the sample, hence it is crucial to use a protocol that effectively retains good integrity miRNAs. In this chapter, we outline few protocols that can be used to maximize the retrieval of good-quality miRNA and total RNA to be used in miRNA analysis for plants rich in polysaccharides and polyphenols.

Citation
Sabu, K.K., Nadiya. F. and N. Anjali. 2017. MiRNA Isolation from Plants Rich in Polysaccharides and Polyphenols. In: MicroRNA Profiling. Vol. 1509. Pp 25-36. Sweta Rani (Ed). Springer. ISBN:978-1-4939-6522-9. PMID: 27826915 DOI: 10.1007/978-1-4939-6524-3_4

Genome Size Estimation Using Flow Cytometry in Elettaria cardamomum Maton

The relative 2C genome size and total number of base pairs of small cardamom (Elettaria cardamomum Maton) was determined using flow cytometer. Seven accessions of cardamom were included in the present study. Zea mays L. CE-777 (2C=5.43 pg) was found to be the most suitable reference standard. Samples for analysis were prepared following the two step procedure described by Otto and involving Propidium iodide staining. The fluorescence intensity of 5000 particles was recorded. The mean amount of 2C nuclear DNA of the cardamom sample was calculated as 2.84 pg. Conversion between DNA content and genome size (1 pg DNA=980 Mbp) indicate that the diploid genome size of cardamom is 2783 Mbp. This is the first report of DNA content and genome size in cardamom. Low variation in genome size has been observed for various germplasm accessions including wild, released varieties and landraces.

Citation
Anjali, N., Nadiya, F., Shefeek, S. and K. K. Sabu. 2016. Genome Size Estimation Using Flow Cytometry in Elettaria cardamomum Maton. Indian Forester 142 (9), 878-881

Assessment of genomic diversity in cardamom by flow cytometry, cytological studies and ISSR analysis

Intraspecific variations in cardamom (Elettaria cardamomum Maton): Assessment of genomic diversity by flow cytometry, cytological studies and ISSR analysis

Article in SpringerPlus 5(1) · September 2016
DOI: 10.1186/s40064-016-3226-x

Background
The main goal of the work was to analyse intraspecific variation in Elettaria cardamomum Maton (cardamom) using genome size, cytological studies and molecular marker data. Nuclear DNA content and molecular marker details furnish data on genome size and genetic diversity respectively among the studied accessions and both complement each other for evolutionary and taxonomic studies.

Results
The relative 2C genome size and total number of base pairs of cardamom was determined through flow cytometric analysis using propidium iodide staining. The nuclear DNA content was estimated in various sections of the species representing individuals from wild and cultivar genotypes following Zea mays L. CE-777 (2C = 5.43 pg) as internal reference standard. Chromosome number from growing root tip was examined following standard protocols. Twenty-six ISSR primers that generated polymorphic bands were used for genetic diversity analysis of the thirty accessions of cardamom. Estimated nuclear 2C DNA content ranged from 2.57 to 3.22 pg demonstrating 1.25-fold variation. The mean amount of 2C nuclear DNA of the cardamom was calculated as 2.87 pg which is equivalent of 2806 Mbp as the diploid genome size. The chromosome number was found to be 2n = 48. Among the thirty accessions of cardamom studied using ISSR markers, C53 (feral from Bonacaud) showed a very prominent level of genetic diversity and was lowest for C96 (Avinash-I, a released variety from Indian Institute of Spices Research, Kozhikode).

Conclusion
These analyses revealed the existence of genetic variability within the studied cardamom accessions. The plant specimens also differed significantly in their genome size. However, the genetic variability parameters did not show any correlation with genome size.

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ISSR and SSR markers reveal sex-specific DNA sequences in three Calamus species from India

Calamus species yields the raw materials for the cane industry. However, the extraction of the cane from the forests is being carried out indiscriminately without considering the sustenance of the species. Plants of both sexes should co-exist for reproductive success. The sex of Calamus plants can be identified only after flowering and hence proper planning of managed forestry practices cannot be accomplished. A study was carried out in this background and male specific ISSR markers for C. tenuis and C. flagellumand SSR markers for C. thwaitesii were identified. The diagnostic potential of these markers can be exploited to sex the Calamus species at the seedling stage for proper breeding and agroforestry management.

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Title ISSR and SSR markers reveal sex-specific DNA sequences in three Calamus species from India Journal Agroforestry Systems, (), 1-5 DOI 10.1007/s10457-016-9952-9

This article is available as 'Online First':  http://link.springer.com/article/10.1007/s10457-016-9952-9

In Silico Analysis of Transcriptomes of medicinal plants

Lakshmi Priya, P. M. and K. K. Sabu. 

In Silico Analysis of Transcriptomes of Catharanthus roseus and Rauvolfia serpentina, two potent medicinal plants using a pipeline developed from publicly available tools.

American Journal of Bioinformatics Research 5(2): 21-25 (2015)

doi:10.5923/j.bioinformatics.20150502.01

Until recently, there was no data available on the genome sequences of medicinal plants. But now, public databases for the transcriptomes of important medicinal plants are available. But an analysis pipeline effectively combining publicly available tools is lacking which would otherwise enable in-depth analysis of the transcriptomes. In this context, we have developed an effective in silico analysis pipeline using tools such as FastQC, FastQ Groomer, TopHat, Cufflinks, Cuffmerge and Cuffdiff available in Galaxy platform and other tools such as DAVID, ExPASy, MetaCyc and PlantCyc. We have tested the pipeline for comparative analysis of the transcriptome of Catharanthus roseus (L.) G.Don and Rauvolfia serpentina (L.) Benth. ex Kurz, two well-known medicinal plants. This study identified genes that are similarly expressing in the roots of these plants leading to the formation of the same secondary metabolite, “Strictosidine” and also identified differentially expressing genes in the leaves of C. roseusand R. serpentina lead to the formation of different metabolites, “Vinblastine” and “Ajmaline”. The findings of the study indicated that the pipeline developed is effective and helped to analyze the transcriptomes and expression data.

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