Finger millet is a potential staple crop cultivated mostly in Eastern and Central Africa and India. It ranks fourth in importance among millets in the world after sorghum, pearl millet, foxtail millet and commonly referred as ragi, mandua, bird’s foot millet, caracan millet and African millet (Upadhyaya et al., 2007). Nutritionally, finger millet is an excellent sources of nutrients especially calcium, other minerals and dietary fiber. The mineral composition of finger millet grains is highly variable. The mineral content of food grains is affected by the presence of genetic factors and environmental conditions prevailing in particular growing region (Singh and Raghuvanshi, 2012; Singh et al., 2014). Finger millet contains a fair amount of protein (7.3%) (Malleshi and Klopfenstein, 1998), dietary fiber (15–20%) (Chethan and Malleshi, 2007), and a rich source of calcium (344 mg/ 100 g) (Gopalan et al., 1999; Bhatt et al., 2003) and iron (3.7–6.8 mg/ 100 g) (Barbeau and Hilu, 1993). Ravindran (1991) estimated the protein content of finger millet to be 9.8%, that of calcium, oxalate and phytic acid to be 0.24, 0.44, and 0.48%, respectively. In several studies on estimation of calcium content in different genotypes of finger millet, high calcium values have been reported. Calcium content varied from 162 to 487 mg/100 g with a mean value of 320.8 mg/100 g grain in 36 genotypes of finger millet (Vadivoo et al., 1998), 293–390 mg/100 g in six varieties of finger millet (Babu et al., 1987); 50–300 mg/100 g in another set of six varieties (Admassu et al., 2009). Furthermore, very high calcium content, 450 mg/100 g (Panwar et al., 2010) and 489 mg/100 g (Upadhyaya et al., 2011) has been reported in few finger millet genotypes. It has also been found that the average calcium content (329 mg/100 g grain) in white genotypes was considerably higher than the brown (296 mg/100 grain) genotypes (Seetharam, 2001). Besides high nutritional value, finger millet has high grain yield potential in the range of 4-5 tons/ha (Food and Agriculture Organization [FAO] of the United Nations, 2008). Thus, finger millet is up-coming as an important food crop due to its exceptionally high calcium (Ca²⁺) content. It is not only an excellent source of dietary calcium (Ca²⁺) but also an excellent model to explore the genetic control and molecular mechanisms contributing to high grain calcium (Ca²⁺) content.

The millet is also well known for its health benefits such as hypocholesterolemic, hypoglycaemic and anti-ulcerative properties (Chethan and Malleshi, 2007). Further, the crop is productive in wide range of environments and growing conditions from Karnataka in South India to foothills and middle hills of Himalayas in North India and Nepal, and throughout the middle-elevation areas of Eastern and Southern Africa (Kumar et al., 2016). Finger millet being a promising source of micronutrients and protein (Malleshi and Klopfenstein, 1998), can play a significant role in alleviation of micronutrient and protein malnutrition, which affects more than one half of the world’s population, especially women and preschool children in most countries of South-east Asia and Africa (Underwood, 2000).

Calcium (Ca²⁺) is normally acquired by root from the soil solution in free ionic form. The outer layers of root, the epidermal cells and their elongated projection (root hairs) get in touch with the soil solution where Ca²⁺ are present in milli-molar range. Through a variety of Ca²⁺ permeable channels, which includes cyclic nucleotide gated channels (CNGCs), glutamate receptor like (GLR) proteins, two-pore channels (TPCs) and mechano-sensitive Ca-permeable channels (MSCCs) calcium (Ca²⁺) enters inside the root epidermal cells, where it moves both apoplastically and symplastically to the cortex and then to the stele. Further, it may reach xylem either apoplastically through extracellular spaces or symplastically, by entering root cells and moving from cell to cell through plasmodesmata.

When the root is young, metaxylem and protoxylem are the main routes of Ca²⁺ transport while in later stage when the vessels are fully conductive, central xylem becomes the major sink. After being released from root, Ca²⁺ are transported in the apoplast and xylem vessels of the shoot where the cation exchange capacity (CEC) of xylem walls and intensity of transpiration is the major factor for Ca²⁺ transportation (McLaughlin and Wimmer, 1999). Ca²⁺ present in the xylem sap exchanges with bound Ca²⁺ from the xylem walls; the movement of an individual Ca²⁺ will be in a series of jumps between exchange sites (Atkinson et al., 1992). Trunk xylem tissues deliver Ca²⁺ inside the leaf tissue via vein extensions. The calcium (Ca²⁺) delivery and distribution in leaves is mostly dependent on the pathway of water flow to and through the leaves (Gilliham et al., 2011). After separation of mineral cations from water, Ca²⁺ moves slower in apoplast. This could be due to the fact that Ca²⁺ transport within the leaf is not only by mass flow but also via an extracellular pathway (Canny, 1993). Growing part of leaf tissue requires high Ca²⁺ concentration, hence providing a sink for Ca²⁺ movement.

Members of calcium signaling and transport genes are reported to be involved in calcium uptake, transport and accumulation at cellular levels in plants. These include various types of calcium sensors viz., calcium dependent protein kinases (CDPKs), Calcinurin- B like protein kinases (CIPKs), calmodulin dependent protein kinases (CaMKs), calcium/calmodulin dependent protein kinases (CCaMKs) and transporters genes viz., Ca²⁺ -ATPases, Calcium/cation exchangers and calcium channels (Singh et al., 2015). Calcium transporters are actively involved in the uptake and transport of calcium in the cells, while calcium sensors are involved in the regulation of these transporters (Table 1). Understanding the role of these genes and the availability of the calcium in rhizosphere might help in development of calcium biofortified plants. Mirza et al. (2014) analyzed the expression of calcium transporter and their regulator genes in different tissues at different growth stages in two finger millet genotypes differing in calcium content and reported higher expression of CAX1 gene in roots of high calcium genotype GPHCPB45 in addition to two more genes (TPC1 and ATPase) in most of the tissues at vegetative growth and developing spikes except flag leaf. Higher expression of CAX1 in roots suggests that calcium uptake is taking place at high rate probably under the regulation of calmodulin independent pathway as the expression of calmodulin is invariably low in all vegetative tissues (Mirza et al., 2014). The expression of CAX1 was observed to be low in root tissue as compared to those in other tissues as reported earlier (Carter et al., 2004; Cocozza et al., 2008; Conn and Gilliham, 2010). This suggests that although calcium uptake is taking place at high rate, it is not getting stored in the vacuole rather it is effluxed in the root apoplast and is trafficked to the cells by means of the water transpiration stream. The calcium content in the leaves was higher than those in the root and stem tissue with higher content in GPHCPB45 leaves as compared to GPHCPB1. This indicates that higher transpiration might attribute to higher calcium (Ca²⁺) accumulation in GPHCPB45 genotype. Interestingly, the expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf tissue indicating that 14-3-3 might interact with the CAX1 for calcium content regulation in leaves. Further in the developing spike (S1 – S4 stage), all the transporters exhibited an increased expression with higher expression in GPHCPB45 and hence correspond to the higher calcium (Ca²⁺) content (Mirza et al., 2014). Higher expression of TPC1 and CAX1 genes in GPHCPB45 indicates higher uptake and accumulation of calcium (Ca²⁺) in comparison to GPHCPB1 in the developing spikes. All the regulatory proteins also exhibited expression patterns similar to the transporter genes in developing spike with generally a higher expression in GPHCPB45 genotype. CaM exhibited a similar expression pattern as Ca²⁺ ATPase gene indicating that this CaM isoform might be specific to developing spike and activating the Ca²⁺ ATPase through binding to the CBD domain (Amtmann and Blatt, 2009). CAM1 was strongly expressed during developing spikes of high grain calcium genotype along with CAX1. Insilico analysis showed that EcCAM interacts with aquaporin indicating calcium is probably delivered to developing spikes via mass flow of water (Kumar et al., 2014). The results indicate that calmodulin independent and dependent pathways cause greater stimulation of transport machinery operative in vegetative and spike tissues leading to higher accumulation of calcium from source to sink. A CAX–CaM dual gene construct may be designed for targeted co-expression under grain endosperm cell-specific promoters to fortify cereals with bioavailable calcium. In flag leaf, the expression of all the transporter and regulatory genes was found to be down regulated, however, the calcium content in flag leaf was highest among all the tissues in both the genotypes. This indicates that the calcium accumulation in flag leaf is largely dependent on the transpirational pull and isoforms.

Briefly, the differential expression of genes shows differential spatial and temporal accumulation of calcium (Ca²⁺) in the two genotypes of finger millet. Studies on finger millet CAX1 and ATPase might offer further insights in understanding their auto-inhibition and strategic regulation. However, higher transcript abundance does not always corroborate with higher gene product abundance and activity. There are many post translational modifications and other bottle necks that may alter the fate of transcriptional activity. The results of transcriptome needs further validation using knock in and knock out approaches. Thus, finger millet plant might as well befall as a model system for better understanding of the underlying genetic control and molecular physiological mechanisms contributing to high grain calcium. Figure 2 depicts the role of potential transporters and regulatory genes involved in calcium (Ca²⁺) transport from source to sink in finger millet.

Seed consists of filial tissues (aleurone and endosperm cell) and maternal tissues (seed coat and nucellar cell) with no direct vascular connection between them (Wolswinkel, 1992). Generally, differentiated vascular systems are restricted to seed tissues and are primarily comprised of phloem with no or limited xylem (Patrick, 1997). Phloem immobility of Ca²⁺ forces it to take different route for delivery in seed. The possibilities include the non-vascular symplastic pathway from funiculas, viz., xylem transfer cell, pigment strands, nucellar cell, or apoplastic (cell wall) routes (Van Bel, 1990). The calcium concentration in phloem-fed tissues, such as fruits, seeds and tubers is in general low in many crops (Karley and White, 2009), but finger millet seeds have been found to be accumulating very high calcium varying from 100 to 450 mg/100 g (Panwar et al., 2010). Therefore, it becomes an important target crop toward elucidating the genetic and epigenetic basis of calcium uptake, transport and accumulation in plant. The information generated through this could be utilized for biofortification of staple crops through transgenic approaches which relies on improving the uptake, translocation and accumulation in edible tissues or through adequate availability in their roots.

Based on the existing knowledge about calcium (Ca²⁺) transport and accumulation, a hypothetical pathway of seed calcium accumulation is proposed in Figure 3. In this model, it is proposed that Ca²⁺ from xylem passes through the pedicel tissues (Xylem transfer cell and pigmented strand) and then enters the maternal tissue (seed coat) with the help of transporters. It gets pumped toward the apoplast from seed coat, where it is absorbed by aleurone and then in endodermal cell. So, prior to uptake by the embryo, Ca²⁺ are deposited in the seed-coat apoplastic space abutting the surface of the embryo (Patrick, 1990). The seed coat is major Ca²⁺ storage tissue followed by aleurone layer and the endosperm (Nath et al., 2012). The contrasting distribution pattern of Ca²⁺ in different tissues of seed during seed development indicates the role of physiological and molecular mechanisms of their accumulation. The presence of prominent, insoluble calcium oxalate crystals (especially in seed coat) and calcium phytates in the embryo of mature seed has been reported to be an important factor for high grain calcium in plants (Barnabas and Arnott, 1990; Ilarslan et al., 2001).

Spatial distribution of calcium transporters within the cell provides the path for storage and movement of calcium (Ca²⁺). There is good evidence that Ca uptake from the apoplast is mediated by activity of Ca channels, which would be the most energetically efficient mechanisms because they utilize the large inward directed electrochemical potential gradient for Ca (Franceschi and Nakata, 2005). The occurrence of abundant Ca²⁺ channels in the cell membrane toward the xylem (funiculus) and Ca²⁺-ATPases toward endosperm might be responsible to pump Ca²⁺ in the seed (Figure 2). Once Ca²⁺ enters inside the seed it might get stored inside cell organelles viz., vacuole, ER etc. Vacuole is reported as the major reservoir of Ca²⁺ and up to 80% or more of the cellular volume of plant cells is occupied by vacuole (Pittman, 2011). The abundance of Ca²⁺ transporters especially antiporters on tonoplast contribute toward high calcium (Ca²⁺) in seeds (Conn et al., 2011; Punshon et al., 2012). Higher expression of Ca²⁺ transporters and calcium binding proteins (CaBPs) in the tonoplast membrane has been reported for the uptake and storage of Ca²⁺ in vacuole of leaf cell (Blumwald and Poole, 1986; Wang et al., 1995).

The mechanism of Ca transport inside seed is still not known, as Ca is phloem immobile and seed is fed mainly by phloem. Our preliminary study based on rice Ca sensor and transporter data suggests that Ca transporters (especially Ca Exchanger) are the main Ca transporting proteins that pump Ca inside seed. Based on the structural and expression analysis of the calcium transporter genes and available data in literature, a hypothetical model for accumulation of calcium in the cereal grains was proposed (Goel et al., 2011, 2012). The nine calcium transporter genes identified through the analysis of rice MPSS and microarray data were tested and one calcium ATPase and one calcium exchanger was found to be highly expressed in high seed calcium finger millet genotype. In finger millet, out of the five genes encoding calcium transporters and sensors (Ca ATPase-01, CAX-01, TPC-01, CaMK-02 and one gene of 14.3.3) isolated from finger millet, CAX1 showed strong expression in the later stages of spike development indicating its role in accumulating high amounts of calcium in seeds (Mirza et al., 2014). Therefore, it can be speculated that the same mechanism of grain calcium accumulation exists in finger millet as is found in rice. However, the model is probabilistic and experimental evidence like gain or loss of function studies are required to validate this model. Figure 4 depicts a hypothetical model showing the possible mechanism of calcium accumulation in various stages of cereal grains development.

Transcriptome sequence data of finger millet showed high expression of Ca²⁺ transporters including CAX-1, CAX-3 and calcium sensor protein such as CIPK-24 and CaM in high seed calcium finger millet genotype (GPHCPB45). Transcript profiling (qPCR) study of such identified candidate genes indicated that Ca²⁺ transporters including CAX-1, CAX-3 and calcium sensor proteins such as CIPK-8 and CaM are highly expressed in high seed calcium finger millet genotype (GPHCPB45) than GPHCPB1. Based on expression analysis and insilico interaction studies a speculative model is shown in Figure 5 which shows tri-partite interaction of calcium transport and sensor genes leading to activation of transport machinery that pumps calcium from cytosol to vacuole in developing seeds in finger millet. It is suggested that SOS3/CBL4 and SOS2/CBL10 strongly interact with CIPK-24 in either vegetative or developing spikes stage. Such SOS/CBL complexes bind and activate CIPK-24 kinase activity by relieving self-inhibitory folding within the regulatory and kinase domain of the CIPK-24 protein (Halfter et al., 2000) and thus modulating the efficiency of CAX1 and CAX3 channel transporters. Ion transporters present at the tonoplast, including an H⁺ pump and Ca²⁺/H⁺ exchanger are the targets of CIPK-24 for their activation (Verslues et al., 2007). The calcium transport machinery regulated by calmodulin dependent and calmodulin independent pathways operative in seed tissues is leading to not only differential accumulation of calcium in seeds but also varied accumulation of calcium in different seed tissues viz. higher accumulation in aleurone layer, followed by seed coat and least in endosperm as reported by SEM-DEX analysis (Nath et al., 2012).

Fr From the last 20 years, molecular biology has revolutionized conventional breeding techniques in all areas. Biochemical and molecular techniques have shortened the duration of breeding programs from years to months or eliminated the need for them all together. The use of molecular markers in conventional breeding techniques have also improved the accuracy of crosses and allowed breeders to produce genotypes with combined traits that were very difficult before the advent of DNA technology. DNA markers can be generated in large numbers and can prove to be very useful for a variety of purposes relevant to crop improvement. Figure 6 gives a schematic representation of genomics and molecular breeding approaches for developing calcium biofortified finger millet.

Low level of polymorphism has been reported in most of the studies on diversity analysis using molecular markers in cultivated finger millet (Muza et al., 1995). For the first time Dida et al. (2007) developed genomic SSRs by isolating di- and trinucleotide SSRs from random genomic HindIII, PstI and SalI libraries of finger millet. They developed first genetic map of finger millet with 31 genomic SSRs as well as RFLP, AFLP and EST markers. The sudden increase in the volume of sequence data generated from EST projects in several plant species facilitated the identification of genic SSRs in large numbers. Since the genomes of minor grasses like finger millet is yet to be sequenced, markers in minor grasses can be fetched from the major cereals. Comparative genetic mapping of cereal crops has shown that both gene contents and/or gene orders are largely conserved over the evolutionary history of the grasses (Moore et al., 1995) to the extent that grass genomes represent a ‘single genetic system’ (Bennetzen and Freeling, 1997).

Assessment of genetic diversity in finger millet revealed important information that South Indian and the African genotypes are close together and genetically distinct from North Indian genotypes including Uttarakhand (Panwar et al., 2010). Some of these genotypes of this crop possess very high grain calcium (Ca²⁺) content (450 mg/100 g) which is 10–30 times higher than wheat and rice (Panwar et al., 2010). In a study of Uttarakhand finger millet genotypes, molecular marker analysis differentiated the genotypes into three distinct clusters according to calcium (Ca²⁺) content indicating that variation in calcium content is also genetically controlled (Panwar et al., 2010). Markers have been utilized extensively for marker assisted selection, based on their association with genes/QTLs controlling grain calcium trait (Table 2). In order to identify the markers associated with high grain calcium trait, 146 genic SSR markers were assessed for cross species transferability across a diverse panel of grass species. The average transferability of genic SSR markers from sorghum to other grasses was highest (73.2%) followed by rice (63.4%) with an overall average of 68.3% which establishes the importance of these major crops as a useful resource of genomic information for minor crops. The genic SSR primers (69.7%) failed to detect variations across the finger millet germplasm, indicating that the mineral transport and storage machinery remain conserved in plants and even SSR variations in them remain suppressed during the course of evolution (Yadav et al., 2014). Development and molecular characterization of genic molecular markers for grain protein and calcium content has also been done (Nirgude et al., 2014). Of the 86 SSRs used in linkage mapping study, only 6 primers were polymorphic among the two parents PRM 801 (low calcium genotype) and GE 86 (high calcium genotype). Further, 20 polymorphic primers used across the association mapping panel of 238 genotypes led to the identification of 5 SSR markers viz. ugep67, ugep24, ugep77, ugep12 and ugep 10, which were significantly associated with calcium trait. For identifying QTLs for calcium content marker trait associations have been explored through association mapping studies and two minor QTLs associated with grain calcium (Ca²⁺) content on linkage group 3 and 8 respectively have been identified (Yadav et al., 2014). Linkage group 8 has been found to harbor a minor QTL for the trait and high levels of conserved co-linearity between rice and finger millet genomes (Srinivasachary et al., 2007) reveals that finger millet chromosome 8 might also contain genes/regions responsible for effective mineral accumulation. Similarly, finger millet LG 3 shares co-linearity with rice chromosome 3 and Ca²⁺ QTLs have also been mapped on chromosome 3 of rice. Furthermore, these results indicate that broad genome-wide search will be required to identify all the genes that control this complex trait and variation in a population.

The putative QTLs for calcium accumulation and increased calcium uptake have been explored in several plant species by different workers (Guzmán-Maldonado et al., 2003; Lexer et al., 2003; Zhang et al., 2009). In rice, QTLs for calcium accumulation have been identified and mapped on chromosomes 1, 3, 4, 5, 9, 10, 11, and 12 with phenotypic variation ranging from 5 to 18% in rice (Garcia-Oliveira et al., 2009; Du et al., 2013). Owing to sufficient variation for calcium content in finger millet germplasm, putative QTLs could be located using comparative genomics approach due to high co-linearity between rice and finger millet genomes.

One of the greatest challenges of biology in the post genomic era understands the functional connections between genes, transcripts, proteins, metabolites and nutrients (Kumar et al., 2015a). This understanding will highly beneficial for the plant biofortification processes. Storage of nutrients in different plant organs such as leaves, roots or seeds is governed by a specific subset of genes which ultimately control whole plant concentrations for numerous elements. The field of genomics has significantly contributed to this information, as it has helped in identifying the genes and genomes of several food plants. As it is already known that some genes are responsible for the uptake and transport of more than one mineral, thus, affecting the mineral homeostasis in a coordinated way. Therefore, alternate tools for expediting the prioritization of candidate genes that control mineral accumulation in plants are desirable (Conn et al., 2012). Therefore, the field of high throughput sequencing/transcriptomics has been fundamental in accelerating gene discovery.

Since, the finger millet crop is valued for its high calcium content, studies have been focused to characterize calcium (Ca²⁺) sensing, transport and accumulation mechanisms across genotypes differing in their grain calcium (Ca²⁺) content with the use of high-throughput transcriptome sequencing (Kumar et al., 2014; Singh et al., 2014). To generate the nucleotide sequence information resource and gain deep insights about the developing grain transcriptome and identify the genes responsible or involved in the process of high grain calcium (Ca²⁺) accumulation, Kumar et al. (2015b) used Illumina HiSeq-2000 high-throughput RNA sequencing technology to identify differentially expressed targets for calcium (Ca²⁺) content variation in two contrasting finger millet genotypes differing in grain calcium (Ca²⁺) content (GPHCPB45 – a high calcium genotype and GPHCPB1 – a low calcium genotype). The differential expression analysis on the basis of FPKM value resulted in 24 genes highly expressed in GPHCPB45 genotype and 11 were highly expressed in GPHCPB1 genes (Kumar et al., 2015b). The results obtained from transcriptome analysis were validated on spikes of finger millet grown on different concentration of calcium (Ca²⁺) (0.1, 5.0, 10, and 20 mM) at four stages of spike development. Significant correlation between the expression of selected Ca²⁺ sensor genes and amount of exogenous Ca²⁺ supply was observed (Singh et al., 2014).

Through peptide mass finger printing, two Ca²⁺ binding protein viz., ‘Calcineurin-B’ and ‘Calreticulin’ were identified in finger millet seeds (Singh et al., 2016). Following the information about the involvement of calcineurin and calreticulin proteins in binding calcium (Ca²⁺) in seed, expressions of 5 each calcineurin and calreticulin genes of finger millet were studied during grain filling. EcCRT1, which shows high expression in the S1 stage of seed development, was partially cloned, sequenced and its full length cloning is under progress (Singh et al., 2014). The mechanism of how Ca²⁺ are moving/transporting inside seed is still not known, as Ca²⁺ in phloem is immobile and seed is mainly fed by phloem. The preliminary results based on rice Ca²⁺ sensor and transporter data suggest that Ca²⁺ transporters (especially Ca Exchanger) are the main Ca²⁺ transporting proteins that pump Ca²⁺ inside seed (Li, 2006). The activities of these Ca²⁺ transporters are governed by Ca²⁺ sensor proteins such as CaM. Southern hybridization results showed the presence of at least four copies of the previously isolated CaM, that is located on different regions of the finger millet genome. Immuno-detection using mono-specific polyclonal anti-EcCaM antibodies revealed that EcCaM is localized in the embryo, close to the aleurone layer and accumulates in higher amounts in the high grain calcium genotype. Abundance of CaM around aleurone layer in high grain Ca²⁺ genotype suggests that CaM might be responsible for high grain calcium accumulation (Kumar et al., 2014). However, all studies on Ca²⁺ sensor and transporter genes were based on cloning of gene by designing primers from conserved region of related species/genus that may miss important gene of major effects. A detailed transcriptome wide study of calcium (Ca²⁺) sensors and transporters was carried out to identify the differentially expressed genes during developing spikes of finger millet and can serve as potential candidate genes having role in seed calcium accumulation (Singh et al., 2014, 2016). Transgenics for Ca²⁺ Exchanger (CAX), an important Ca²⁺ transporter that pump Ca²⁺ inside vacuoles were developed, which resulted in significant Ca²⁺ increase but deleterious effects like mineral imbalance, stunted plant growth and other structural changes were also seen (Kumar et al., 2015a). GPHCPB45 was found to be more efficient calcium (Ca²⁺) accumulator at low external calcium (Ca²⁺) concentrations, whereas GPHCPB1 was found to be more responsive to increased external calcium (Ca²⁺) concentration (Singh et al., 2014). Comprehensive sequencing efforts and analysis of gene function in the developing spikes transcriptome of finger millet (E. coracana) represents the most extensive expressed sequence resource available for finger millet to date (Kumar et al., 2014). Analysis of transcriptome sequence data of finger millet has indicated that Ca²⁺ transporter genes including CAX-1, CAX-3 and calcium (Ca²⁺) sensor protein such as CIPK-24 and CaM are highly expressed in high seed Ca²⁺ finger millet genotype. In order to understand the role and interrelationship expression of these genes in high seed calcium (Ca²⁺) accumulation, comparative tissue wide transcript profiling study of potential candidate genes, viz. CAX-1, CAX-3, CIPK-24 and CaM at vegetative stage and developing spikes stage have shown mostly higher expression in high grain calcium (Ca²⁺) genotype. The up-regulation of CAX-1 in vegetative tissues and developing spikes and CAX-3 in developing spikes provide most plausible clue explaining the accumulation of high calcium (Ca²⁺) in finger millet. The genetic information generated from these studies can be deployed in calcium (Ca²⁺) biofortification program by using genetic engineering or marker assisted selection strategies. Breeding for high grain Ca²⁺ in finger millet can be accelerated by using the closely linked markers to identified genes (EcCIPKs). This is just beginning, and it needs further studies in cloning and validation of the above genes using a set of different contrasting genotypes.

Recently, an effort has been also made to study the genetic make of diverse world finger millet collection and population structure analysis of 113 finger millet accessions using genotyping by sequencing (GBS) (Kumar et al., 2016). This resulted in a genome wide set of 23000 Single Nucleotide Polymorphisms (SNPs) segregating across the entire collection and several thousand SNPs segregating within every accession. It will provide a better opportunity to dissect complex traits and identification of superior alleles/ genes contributing to the target traits.

Considering the examination of mineral/micronutrients and their complex networks, ionomics or ionome profiling has emerged as a striking area that facilitates the genome-scale understanding of dynamics of elemental accumulation in the living systems (Baxter, 2010). In the context to ionomics, several techniques driven by the electronic or nuclear property of the element are currently being used to investigate elemental composition in different organisms (Singh et al., 2013). These techniques include AAS, ion beam analysis (IBA), X-ray fluorescence spectroscopy (XRF), ICP mass spectroscopy (ICP-MS) and neutron activation analysis (NAA) (Pfeiffer and McClafferty, 2007; Djingove et al., 2013; Singh et al., 2013).

Metabolomics is another area to better explore the chemical components which are present in food grains, how they are synthesized and the genetic and environmental factors that are involved in determining food composition and stability. Metabolomics is much more than just a diagnostics tool (Schauer and Fernie, 2006) and can help us greatly to improve our understanding of the complexity of metabolic regulation and explain how minor perturbations can have a myriad of biochemical end-points. This creates the potential to provide us with the knowledge necessary to facilitate a more targeted approach to crop improvement, specifically in terms of biochemical composition and nutritional value (Robert and Jennifer, 2010).