Current Genomics (v.17, #6)

Reverse Genetics and High Throughput Sequencing Methodologies for Plant Functional Genomics by Anis Ben-Amar, Samia Daldoul, Götz M. Reustle, Gabriele Krczal, Ahmed Mliki (460-475).
In the post-genomic era, increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of genes gives rise to a complex organism. With the advent of the next generation sequencing associated with effective computational approaches, wide variety of plant species have been fully sequenced giving a wealth of data sequence information on structure and organization of plant genomes. Since thousands of gene sequences are already known, recently developed functional genomics approaches provide powerful tools to analyze plant gene functions through various gene manipulation technologies. Integration of different omics platforms along with gene annotation and computational analysis may elucidate a complete view in a system biology level. Extensive investigations on reverse genetics methodologies were deployed for assigning biological function to a specific gene or gene product. We provide here an updated overview of these high throughout strategies highlighting recent advances in the knowledge of functional genomics in plants.

RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops by Qigao Guo, Qing Liu, Neil A. Smith, Guolu Liang, Ming-Bo Wang (476-489).
Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology. Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway. These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3' UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies. Some of these RNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture. For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products. In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops.

Chromatin-Based Epigenetic Regulation of Plant Abiotic Stress Response by Garima Pandey, Namisha Sharma, Pranav Pankaj Sahu, Manoj Prasad (490-498).
Plants are continuously exposed to various abiotic and biotic factors limiting their growth and reproduction. In response, they need various sophisticated ways to adapt to adverse environmental conditions without compromising their proper development, reproductive success and eventually survival. This requires an intricate network to regulate gene expression at transcriptional and post-transcriptional levels, including epigenetic switches. Changes in chromatin modifications such as DNA and histone methylation have been observed in plants upon exposure to several abiotic stresses. In the present review, we highlight the changes of DNA methylation in diverse plants in response to several abiotic stresses such as salinity, drought, cold and heat. We also discuss the progresses made in understanding how these DNA methylation changes might contribute to the abiotic stress tolerance.

Mutagenesis can be random or targeted and occur by nature or artificially by humans. However, the bulk of mutagenesis employed in plants are random and caused by physical agents such as x-ray and gamma-ray or chemicals such as ethyl-methane sulfonate (EMS). Researchers are interested in first identifying these mutations and/or polymorphisms in the genome followed by investigating their effects in the plant function as well as their application in crop improvement. The high-throughput technique called TILLING (Targeting Induced Local Lesion IN Genomes) has been already established and become popular for identifying candidate mutant individuals harboring mutations in the gene of interest. TILLING is a non-transgenic and reverse genetics method of identifying a single nucleotide changes. The procedure of TILLING comprises traditional mutagenesis using optimum type and concentration of mutagen, development of a non-chimeric population, DNA extraction and pooling, mutation detection as well as validation of results. In general, TILLING has proved to be robust in identifying useful mutant lines in diverse economically important crops of the world. The main goal of the current mini-review is to show the significance role played by mutagenesis and TILLING in the discovery of DNA lesions which are to be used in the improvement of crops for the trait of interest.

In recent years, the genomic sequence of numerous plant species including the main crop species has been determined. Computational tools have been developed to deal with the issue of which plant has been sequenced and where is the sequence hosted. In this mini-review, the databases for genome projects, the databases created to host species/clade projects and the databases developed to perform plant comparative genomics are revised. Because of their importance in modern research, an in-depth analysis of the plant comparative genomics databases has been performed. This comparative analysis is focused in the common and specific computational tools developed to achieve the particular objectives of each database. Besides, emerging high-performance bioinformatics tools specific for plant research are commented. What kind of computational approaches should be implemented in next years to efficiently analyze plant genomes is discussed.

Toxicogenomics to Evaluate Endocrine Disrupting Effects of Environmental Chemicals Using the Zebrafish Model by Karina Caballero-Gallardo, Jesus Olivero-Verbel, Jennifer L. Freeman (515-527).
The extent of our knowledge on the number of chemical compounds related to anthropogenic activities that can cause damage to the environment and to organisms is increasing. Endocrine disrupting chemicals (EDCs) are one group of potentially hazardous substances that include natural and synthetic chemicals and have the ability to mimic endogenous hormones, interfering with their biosynthesis, metabolism, and normal functions. Adverse effects associated with EDC exposure have been documented in aquatic biota and there is widespread interest in the characterization and understanding of their modes of action. Fish are considered one of the primary risk organisms for EDCs. Zebrafish (Danio rerio) are increasingly used as an animal model to study the effects of endocrine disruptors, due to their advantages compared to other model organisms. One approach to assess the toxicity of a compound is to identify those patterns of gene expression found in a tissue or organ exposed to particular classes of chemicals, through new technologies in genomics (toxicogenomics), such as microarrays or whole-genome sequencing. Application of these technologies permit the quantitative analysis of thousands of gene expression changes simultaneously in a single experiment and offer the opportunity to use transcript profiling as a tool to predict toxic outcomes of exposure to particular compounds. The application of toxicogenomic tools for identification of chemicals with endocrine disrupting capacity using the zebrafish model system is reviewed.

Meta-analysis Reveals No Association of DNMT3B -149 C>T Gene Polymorphism With Overall Cancer Risk by Raju Kumar Mandal, Shafiul Haque, Mohd Wahid, Arshad Jawed, Naseem Akhter, Md. Ekhlaque Ahmed Khan, Aditya Kumar Panda, Mohammed Yahya Areeshi, Sajad Ahmad Dar (528-537).
Background: DNA methyltransferase-3B (DNMT3B) plays a key role in establishment and maintenance of genomic methylation patterns. Polymorphism in promoter region -149 C>T (C46359T) of DNMT3B gene may alter DNMT3B activity which leads to increased susceptibility to cancer. Inconsistent results regarding this have been reported in a number of studies.
Objective: To carry out a meta-analysis of the studies reported to assess the precise relationship between the DNMT3B -149 C>T polymorphism and the overall cancer risk.
Method: PubMed (MEDLINE) web database was searched for the studies concerning DNMT3B -149 C>T polymorphism and its association with cancer risk. The pooled odds ratios (ORs) along with 95% confidence intervals (95% CIs) were calculated for all the genetic models, from the selected case-control studies, by meta-analysis.
Results: Overall eighteen studies containing 5583 cancer cases and 7618 controls were analyzed. No significant risk was observed overall for T allele carrier (T vs. C: p=0.303; OR=1.032, 95% CI=0.972- 1.097), homozygous (TT vs. CC: p=0.336; OR=1.063, 95% CI=0.939-1.204), heterozygous (CT vs. CC: p=0.802; OR=1.022, 95% CI=0.860-1.216), dominant (TT vs. CC+CT: p=0.298; OR=1.101, 95% CI=0.919-1.319) and recessive (TT+CT vs. CC: p=0.656; OR=1.021, 95% CI=0.931-1.121) genetic models. Subgroup analysis of Asian and Caucasian populations also did not demonstrate any cancer risk in all the genetic models studied.
Conclusion: Our meta-analysis proposes that the DNMT3B -149 C>T polymorphism may not be an independent predisposing factor for the risk of cancer. However, larger sample size and expression studies are required to confirm the observation.