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Subject: Agricultural Biotechnology × Author: Chandra, Ajay Kumar × Start date: 2020 × Type: Thesis ×
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    ThesisItemOpen Access
    Genotyping-by-sequencing and functional genomics based approaches for identification and nano-minerals (Fe and Zn) mediated modulation of candidate genes involved in iron and zinc homeostasis in finger millet
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-08) Chandra, Ajay Kumar; Pandey, Dinesh
    Micronutrient deficiencies of iron and zinc are mainly responsible for significant amount of malnutrition among children and women across the world. One of the approaches to solve this problem is through biofortification of staple food crops provided that key candidate genes and markers associated with iron and zinc accumulation are identified. Being very good accumulator of minerals such as iron and zinc, finger millet is an ideal crop for identifying the molecular mechanism of iron and zinc homeostasis and molecular markers linked to high iron or zinc content in finger millet. With an aim to identify potential markers linked to grain iron and zinc content, initially phenotyping of 202finger millet genotype was done for fourteen morphological traits along with grain iron and zinc content at two different environments viz. Pantnagar (L1) and Almora (L2). Analysis of variance for both the environments resulted in highly significant differences among accessions for most of the traits, which showed the existence of sufficient variability. Also the wide range of variation in the agronomic performance of the accessions suggested that these traits could be considered as good candidates for marker-trait associations. The grain iron content varied from 26.72 to 78.59 mg.kg-1 seed with an average of 44.34 mg.kg-1 seed at L1 whereas, the GIC at L2 varied from 29.83 to 76.40 mg.kg-1 seed with an average of 46.14 mg.kg-1 seed. Similarly, the grain zinc content varied from 16.28 to 45.17 mg.kg-1 seed with an average of 32.18 mg.kg-1 seed at L1 whereas, the GZC at L2 varied from 12.40 to 47.18 mg.kg-1 seed with an average of 32.36 mg.kg-1 seed which indicates presence of sufficient variability for grain iron and zinc trait amongst the selected finger millet genotypes. Further, Genetic diversity and population structure analysis was conducted on a natural collection of 202 finger millet genotypes. Neighbour joining cluster analysis grouped the population into 3 clusters ‘A’, ‘B’ and ‘C’ based on their geographical location and grain iron and zinc content. The major cluster A consisted broadly of all the Indian genotypes whereas cluster B and C comprised of exotic genotypes. Thus, there was good congruence between the phylogenetic tree and the population structure. SNPs through GBS were used for association mapping to identify reliable marker(s) linked to grain iron and zinc content. A total of five SNP markers, three for grain iron [TP413281 associated with ferritin (Fer1), TP566120 with iron-regulated transporter-like protein (IRT2), and TP1431395 associated with yellow stripe-like 2 proteins (YSL2)] and two for grain zinc [TP1316808 associated with zinc transporter ZIP1 protein and TP784188 associated with zinc transporter ZTP29-like protein], showed homology to candidate genes of Oryza sativa and Setaria italica, which might play an important role in grain iron and zinc homeostasis process in finger millet. Further, a total of fifteen genes (EcDMAS1, EcFER1, EcIRT2, EcNAAT6, EcNAC2, EcNAS1NAS2, EcNRAMP2, EcNRAMP6, EcPDR9, EcTOM1, EcTOM2, EcYSL1, EcYSL2, EcZIP1, and EcZTP29) orthologous to CGs of Oryza sativa and Setaria italica putatively involved in iron and zinc homeostasis in finger millet were identified through Genome-Transcriptome transition approaches. Structural and functional annotation of identified genes further highlights its significance that the genes are putatively involved in iron and zinc homeostasis process in finger millet. Furthermore, it was studied whether nano-treatments have any influencing role in epigenetic behavior so that it could enhance the molecular machineries of Fe and Zn enrichment in the plants. Molecular modeling and docking potential of identified proteins with nano-minerals (Fe3O4 NPs and ZnO NPs) confirmed that these nano-minerals may significantly influence the binding potential and modulation of regulatory genes. In addition, comparative In vitro studies for nano-minerals augmentation using seed priming approach resulted that the lower concentrations of Fe3O4 NPs (100 ppm) and ZnO NPs (5 ppm) play a significant and promotory effect on fourteen quantitative traits. Besides, the mean iron content in nano seed primed harvested grains, significantly increased by 12.26% and, the zinc by 13.96% concerning controlled harvested grains. Thus to confirm the genetic and molecular basis of identified genes and their modulation in response to nano-minerals, the Transcriptome based expression profiling revealed that of these 15 genes, EcFER1, EcIRT2, EcYSL2, EcZIP1, and EcZTP29 are the major genes which might play an important and direct role while the rest genes may indirectly involved in differential iron and zinc homeostasis in finger millet. The result was further validated by quantitative real-time PCR analysis. Structural and functional validation of set of genes involved in strategy-I (EcIRT2, EcFER1, and EcZIP1), strategy-II (EcYSL2), combined i.e., strategy-I and strategy-II (EcIRT2, EcFER1, EcYSL2, and EcZIP1) and the expression of EcZTP29 (stress-responsive gene) in finger millet tissues suggested that being a stress-resilient crop finger millet utilizes a combination of strategies I and II of iron and zinc homeostasis pathway. Since, there is not yet available any information on genetic and molecular basis of Fe and Zn homeostasis in this crop, therefore the results of present investigations have unlocked new avenues for effective utilization of finger millet in future iron and zinc biofortification programs.