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Barnyard millet comprises two different cultivated species, Japanese barnyard millet (Echinochloa esculenta L.) and Indian Barnyard millet (Echinochloa frumentacea L.) belongs to the plant family Poaceae.The other names of barnyard millet in Gujarati is moraiyo. Barnyard or sawa millet is the fastest growing crop of all millet, which can produce ripe grains within 45 days from the sowing time under optimal weather conditions. In addition to these agronomic advantages, the grains are valued for their high nutritional value and lower expense as compared to major cereals like rice, wheat, and maize. It is important minor millet in Japan, China, India and other South East Asian countries. Barnyard millet is a multi-purpose crop which is cultivated for food and fodder. Barnyard millet grains are a rich source of dietary fiber, iron, zinc, calcium, protein, carbohydrate, magnesium, fat, vitamins, some essential amino acids and, most notably, contains more micronutrients (iron and zinc) than other major cereals. Due to low glycemic index and high dietary fiber, it helps in preventing diabetes and cardio vascular disease with regular intake. Barnyard millet could be a good source of iron for vegetarians. Keeping these benefits in the mind present study was undertaken to find out candidate genes and metabolites responsible for Fe content in barnyard millet genotypes. In the current study, biochemical parameter (proximate and minerals) was collected out in 30 barnyard millet genotypes and result shown that total carbohydrate content was found in range between 53.51-73.98 %. The range for total protein (9.55- 11.99 %), crude fiber (11.14–16.64 %), moisture content (4.47-7.68 %), ash content (3.47 – 5.99 %), total fat content (3.38 - 6.95% ), energy (304.45–380.11 Kcal/mole), fatty acid profiling reveled Oleic acid content was observed higher (81.19 %) followed by Palmitic acid (69.33 %), Linolenic acid (50.96 %) and Linoleic acid (48.11 %), nitrogen (1.56-2.72 %), phosphorus (326.67-486 mg/100g), potassium (170-383 mg/100g), sodium (31.67-51 mg/100g), iron content (3.83-11.14 mg/100g), zinc (2.01- 3.88 mg/100g), manganese (1.33-2.85 mg/100g), calcium (35.83–209.18 mg/100g), copper (0.161-0.407 mg / 100g), magnesium (101.65 and 182.21 mg/100g) and boron (0.45-1.99 mg /100g) were observed in 30 barnyard millet genotypes. Two barnyard millet genotypes having high Fe (BAR-1433) and low Fe (BAR 1423) in their seeds were taken for transcriptome and metabolomic analysis during spike development stage. Total pool 30 HQ reads were considered for de novo assembly optimization using four standard assembler Trinity, Soapdenovo_trans, CLC and CAP3 assembler. The trimmed number of reads yielded a total of 138 million high-quality reads, which were assembled by trinity into 4,88,689 transcript. The trinity generated highest transcript with assembly size of 340 MB, N50 size of 1202 and N90 size of 611. CAP3 assembler was employed to reduce transcript redundancy and it generate 27,228 transcript with assembly size of 31.84 MB, N50 size of 1570 bp and N90 size of 723 bp. Moreover, more redundancy were removed by CD-HIT programme and assembler which generated 20,849 transcripts with size assembly of 24.42 MB, N50 size of 1604 bp and N90 size of 719 bp. Transcriptome analysis identified key genes regulating Fe accumulation like CIAO1, ISAM1 and UCRIA during different spike development stage. Further, Ferritin, ABC, Cytochrome, Metal, Zinc, Serine, Wall-associated and Cytochrome, Mitogen and Photosynthesis regulating differential gene also were identified which are also involved in iron content of Low Fe Vs High Fe barnyard millet genotype. Functional annotation was carried out for Gene Ontology (GO) enrichment and KEGG pathway enrichment from significant DEGs. Gene ontology (GO) and pathways analysis revealed that different metabolic pathway like glycolysis, purine and pyruvate metabolism based on expressed genes revealed the mechanism of minerals transportation. The resources generated in this study will facilitate discovery of new genes and further EST-SSR markers and thereby accelerate both genetic and functional genomic research in barnyard millet. Identified genes may be used for marker-assisted selection and breeding to develop minerals rich crops. The Comparison of transcript expression levels between transcriptome data and qRT-PCR depicted positive correlation. The validation through qRT-PCR was carried out using ACBL, ABA, RP8L, PDF1 and PHY which were among the up and down regulated transcripts in different stages of barnyard millet. Metabolome profiling of barnyard millet genotypes containing high Fe and low Fe was performed using GC-MS platform and results observed highest percentage of sugar and sugar alcohol (34%) followed by organic acid (26%), Amino acids, sterol, other compound (23%) and fatty acid (17%) in spike development stages of Low Fe Vs High Fe genotypes. Heat map revealed that during spike emergence the metabolic compounds like d-Ribose, D-Fructose, D-Glucose, Galactose, D-Turanose, Glucopyranose, D-Mannitol, Hexadecanoic acid, Docosanoic acid, alpha.- Glycerophosphoric acid and .beta.-Sitosterol were found high in high Fe genotype in comparison to Low Fe genotype. The hierarchical cluster analysis, revealed that high Fe five spike development stages shares close metabolite pool to each other and in low Fe four spike development stages also shares similarly metabolite pool to each other except spike emergence stage of Low Fe genotype. PCA showed that the expression patterns of the five developmental stages differed significantly.