Next generation sequencing-based cloning of Zea mays mutants and MIRNA-guided regulation of heat stress response in peanut (Arachis hypogaea)



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Strategies for crop improvement rely heavily on the genetic variation in plants that can arise through spontaneous mutations or induced mutagenesis. With the advent of next generation sequencing (NGS), it has become easier to identify and map mutations within a short period of time and with relative low cost. NGS has enabled identification of genetic markers associated with a particular trait by using the mapping by sequencing approach in forward genetics screens. The conventional methods of plant breeding can be complicated, require long breeding cycles and have narrow gene pool which can lead to reduced vigor and fertility (Suprasanna and Jain, 2017). However, induced mutagenesis, on the other hand, can play a greater role in crop improvement by creating huge genetic variations and new alleles within a short period of time for agronomically important traits (Oladosu et al., 2016). The plant breeders use mutant techniques to discover new genetics sources for herbicide resistance, increasing the crop yield, climate resilience and biotic/abiotic stress tolerance (Chaudhary et al., 2019). In this study, I explore various NGS techniques in conjunction with forward genetics approach to identify the causal mutated genes for a recessive spontaneous mutant albino (alb) of maize (Zea mays), ethyl methane sulfonate (EMS)-induced dominant Wilty mutants (Wilty 2 (Wi2) and Wilty 3 (Wi3)) of maize and exploring the role of small RNA during various stages of pod development under heat stress in peanut (Arachis hypogea). The majority of plant genes are of unknown function. Further, studying only dicot genetic models is insufficient: ~30% of proteins in maize are not found in Arabidopsis where 40% of genes are unclassified, & vice versa. The genetic approach has intrinsic value over other experimental (viz. correlative) studies; pleiotropic phenotypes and dominant gain-of-function alleles like Wilty maize mutants can reveal genes involved in processes otherwise hidden and open new vistas of understanding, insight, and experimentation. Functional identification and molecular cloning of pleiotropic mutants affecting vascular development, cell wall synthesis and drought-stress adaptation responses will provide fundamental insights into the processes of cell expansion, morphogenesis, and homeostasis. Elucidation of Wilty gene functions is not only important for basic plant science to meet the "genotype to phenotype challenge," but also can contribute to a knowledge foundation for better utilization of plant biomass for human needs. Dominant alleles in particular are the 'gift that keeps on giving'. Dominant mutations from EMS-mutagenized maize are ~200 times rarer than recessive mutations. By virtue of revealing functionally redundant pathways and gene families, dominant mutants have been particularly important for agriculture and elucidation of genetically redundant plant signaling pathways. The knowledge gained from cloning the Wilty genes and elucidating their structures (and thus function) will contribute to understanding plant function and identification of markers for potential accelerated molecular breeding of crops not limited to corn. In addition to the forward genetics screens which have benefitted largely from the NGS techniques, enormous amount of small RNA (sRNA) sequencing data has helped researchers investigate the role of microRNA (miRNAs) in various biological processes in plants and revolutionize the field of small RNA on a genome-wide scale. NGS is a powerful tool for analysis of sRNA as it allows for discovery of novel sRNA with better signal to noise ratios. miRNAs have been demonstrated to be important gene regulators involved in plant growth and development and response to biotic/abiotic stress response. Thus, miRNA-based technology is among one of the best approaches which can contribute to the crop improvement strategies to produce superior crop cultivars. miRNAs negatively regulate their mRNA target and strategies to manipulate miRNA-based control on gene expression include creation of artificial target mimics (Gupta, 2015) and overexpression of miRNA resistant targets. These are excellent strategies for generating transgenics for stress tolerance when miRNA of interest operates as negative regulators of stress in transgenic plants (Franco-Zorrila et al., 2007). Artificial miRNAs (amiRNAs) suppress the gene expression of the target protein coding mRNA of interest. The amiRNA technology has been successfully used in various plant species to generate transgenic plants with improved tolerance to various biotic and abiotic stresses. The amiRNA targeting the cucumber mosaic virus suppressor 2b efficiently inhibited the gene expression of 2b conferring resistance to this virus in the transgenic tobacco (Qu et al., 2007). My study is amongst the first to explore the heat induced miRNAs during various stages of peanut pod development and knowledge gained from this study will contribute to designing miRNA-based strategies to improve crop productivity not limited to peanut.

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Wilty, Maize, miRNA, Heat, Peanut, NGS