Genome-wide identification of conserved and novel microRNAs in one bud and two tender leaves of tea plant (Camellia sinensis) by small RNA sequencing, microarray-based hybridization and genome survey scaffold sequences.

BACKGROUND: MicroRNAs (miRNAs) are important for plant growth and responses to environmental stresses via post-transcriptional regulation of gene expression. Tea, which is primarily produced from one bud and two tender leaves of the tea plant (Camellia sinensis), is one of the most popular non-alcoholic beverages worldwide owing to its abundance of secondary metabolites. A large number of miRNAs have been identified in various plants, including non-model species. However, due to the lack of reference genome sequences and/or information of tea plant genome survey scaffold sequences, discovery of miRNAs has been limited in C. sinensis. RESULTS: Using small RNA sequencing, combined with our recently obtained genome survey data, we have identified and analyzed 175 conserved and 83 novel miRNAs mainly in one bud and two tender leaves of the tea plant. Among these, 93 conserved and 18 novel miRNAs were validated using miRNA microarray hybridization. In addition, the expression pattern of 11 conserved and 8 novel miRNAs were validated by stem-loop-qRT-PCR. A total of 716 potential target genes of identified miRNAs were predicted. Further, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the target genes were primarily involved in stress response and enzymes related to phenylpropanoid biosynthesis. The predicted targets of 4 conserved miRNAs were further validated by 5'RLM-RACE. A negative correlation between expression profiles of 3 out of 4 conserved miRNAs (csn-miR160a-5p, csn-miR164a, csn-miR828 and csn-miR858a) and their targets (ARF17, NAC100, WER and MYB12 transcription factor) were observed. CONCLUSION: In summary, the present study is one of few such studies on miRNA detection and identification in the tea plant. The predicted target genes of majority of miRNAs encoded enzymes, transcription factors, and functional proteins. The miRNA-target transcription factor gene interactions may provide important clues about the regulatory mechanism of these miRNAs in the tea plant. The data reported in this study will make a huge contribution to knowledge on the potential miRNA regulators of the secondary metabolism pathway and other important biological processes in C. sinensis.

Challenges in diagnosing and treating snakebites in a rural population of Tamil Nadu, India: The views of clinicians.

Snakebites cause death, disability and economic devastation to their victims, people who live almost exclusively in rural areas. Annually an estimated two million venomous bites cause as many as 100,000 deaths worldwide as well as hundreds of thousands of deformities and amputations. Recent studies suggest that India has the highest incidence of snakebite and associated deaths worldwide. In this study, we interviewed 25 hospital-based clinicians who regularly treat snakebites in Tamil Nadu, India, in order to gauge their opinions and views on the diagnostic tools and treatment methods available at that time, the difficulties encountered in treating snakebites and improvements to snakebite management protocols they deem necessary. Clinicians identified the improvement of community education, training of medical personnel, development of standard treatment protocols and improved medication as priorities for the immediate future.

Mechanism of Fenpropathrin Resistance in Red Spider Mite, Oligonychus coffeae (Acarina: Tetranychidae), Infesting Tea [Camellia sinensis L. (O. Kuntze)].

Red spider mite (RSM), Oligonychus coffeae (Nietner) (Acarina: Tetranychidae), has gained special attention in view of their widespread occurrence as a pest on tea [Camellia sinensis L. (O. Kuntze)]. The development of acaricide (fenpropathrin) resistance has been screened in field populations (FPs) of RSMs from different tea-growing regions of south India and compared with a laboratory-susceptible population (SP) based on toxicity bioassay, detoxifying enzyme activities, analysis of acetylcholine esterase gene (AChE, 2064 bp), and their expression pattern using semiquantitative RT-PCR. The increased resistance ratio (RR, 1.39 to 2.13) in LC50 of fenpropathrin observed in field populations of RSM provides a baseline for screening the development of resistance to fenpropathrin. This resistance developed due to hyperexpression of detoxifying enzymes, i.e., esterase (RR of 1.43 to 2.53) and glutathione S-transferase (RR of 1.11 to 1.86), and overexpression of AChE gene at 1.4 to 2.7-fold. These results necessitate molecular studies and warrant the continuous monitoring of acaricide susceptibility and resistance pattern in order to analyze the usefulness of AChE gene as target for developing alternate pest control strategies and management of pesticide resistance in tea ecosystem.

Differential expression of microRNAs in dormant bud of tea [Camellia sinensis (L.) O. Kuntze].

KEY MESSAGE: Expression analysis of miRNAs and understanding their target genes function in dormant tea bud might be used to identify molecular network panel and novel approaches for modulating dormancy in tea. Tea [Camellia sinensis (L) O. Kuntze, Theaceae] is an important commercial beverage crop manufactured from the apical bud and two leaves immediately below the bud. The yield and quality of tea depend on the vegetative growth of shoots and bud dormancy. The dormancy of bud is being regulated by many factors, such as mechanical, environmental and molecular mechanisms. MicroRNAs (miRNAs) are a newly identified class of small non-protein coding regulatory RNAs in both plants and animals which regulates gene expression at post-transcriptional level either by cleavage or translational inhibition of targeted mRNA transcripts. With these importances, the expression pattern of tea miRNAs was analyzed in active and dormant bud using stem-loop pulse RT-qPCR method. The results demonstrated the following expression pattern for highly up-regulated miRNAs, cs-miR 414[csmiR 408[cs-miR782[cs-miR169, and down-regulated miRNAs, cs-miR828[cs-miR1864[cs-miR852[csmiR1425 in dormant bud of tea. Furthermore, the role of target transcripts regulated by these miRNAs in relation to bud dormancy was discussed in detail. Therefore, the present study on the miRNA expression in tea will provide basis and considerably broaden the scope of understanding the function of miRNAs within the bud tissues and can serve as an initial point for RNA interference-based controlling strategies of bud dormancy in tea.

Snakebite and its socio-economic impact on the rural population of Tamil Nadu, India.

BACKGROUND: Snakebite represents a significant health issue worldwide, affecting several million people each year with as many as 95,000 deaths. India is considered to be the country most affected, but much remains unknown about snakebite incidence in this country, its socio-economic impact and how snakebite management could be improved. METHODS/PRINCIPAL FINDINGS: We conducted a study within rural villages in Tamil Nadu, India, which combines a household survey (28,494 people) of snakebite incidence with a more detailed survey of victims in order to understand the health and socio-economic effects of the bite, the treatments obtained and their views about future improvements. Our survey suggests that snakebite incidence is higher than previously reported. 3.9% of those surveyed had suffered from snakebite and the number of deaths corresponds to 0.45% of the population. The socio-economic impact of this is very considerable in terms of the treatment costs and the long-term effects on the health and ability of survivors to work. To reduce this, the victims recommended improvements to the accessibility and affordability of antivenom treatment. CONCLUSIONS: Snakebite has a considerable and disproportionate impact on rural populations, particularly in South Asia. This study provides an incentive for researchers and the public to work together to reduce the incidence and improve the outcomes for snake bite victims and their families.

Identification of differentially expressed genes in dormant (banjhi) bud of tea (Camellia sinensis (L.) O. Kuntze) using subtractive hybridization approach.

Growth regulation associated with dormancy is an essential element in plant's life cycle that leads to changes in expression of large number of genes. Forward and reverse suppression subtractive hybridization (SSH) libraries were developed to identify and characterize the genes associated with bud (banjhi) dormancy in tea (Camellia sinensis (L.) O. Kuntze). Efficiency of subtraction was confirmed by comparing the abundance of ß-actin gene. A total of 17 and 45 unique sequences were obtained from forward and reverse SSH library respectively. Many of the differentially regulated genes have unknown (41.1% and 26.7%) or hypothetical functions (11.7% and 2.2%) in forward and reverse SSH library respectively, while others have a role in cell growth and metabolism. Further, semi-quantitative RT-PCR was carried out for selected genes to validate the quality of ESTs from SSH library. Gene Ontology analysis identified a greater association of these ESTs in cellular metabolic pathways and their relevance to bud dormancy. Based on the EST data, the putative role of identified genes from tea is discussed in relation to dormancy, which includes various metabolic and signalling pathways. We demonstrated that SSH is an efficient tool for enriching up- and down-regulated genes related to bud dormancy in tea. This study represents an attempt to investigate banjhi dormancy in tea under field conditions, and the findings indicate that there is a potential to develop new approaches to modulate dormancy in this species.