Introduction
More than half of the world’s population, especially women and children in the developing countries, suffer from micronutrient malnutrition or ‘hidden hunger’ resulting from the consumption of meager bioavailable vitamins and minerals containing diets (UN SCN, 2004). Micronutrient malnutrition is the condition that develops when the body does not get the optimum amount of the vitamins, minerals and other micronutrients which are essential to maintain metabolic regulation and organ function. Among the major nutritional problems common in developing countries are: micronutrient (iron, zinc, vitamin A) and protein-energy malnutrition (Bouis et al., 2003). It is estimated that over 800 million people go to bed hungry everyday and approximately 3 billion people are suffering from micronutrient deficiency. Micronutrient malnutrition causes several diseases; the affected people are more prone to infection to other diseases resulting in further deterioration in quality of life.

Rice, the world’s most important food crop that feeds over half of the global population, is a model plant species for genomic research. Over the past few decades till 1990, most of the breeding research was concentrated on increasing the grain yield and to improve the resistance to environmental stresses, pests and pathogens (Borlaug, 2000), but little or no attention was given towards the enhancement of its nutritional quality. In1996, the International Rice Research Institute (IRRI) released the first high yielding rice variety ‘IR8’. In the subsequent decade, a small number of such high yielding varieties almost completely replaced the thousands of the traditional rice landraces previously cultivated by the farmers. This resulted in the immense ‘genetic erosion’ and loss of biodiversity.  Despite the loss, a lot of germplasm still exists and being maintained by the international such as International Rice Research Institute (IRRI, The Philippines) and national research institutions in various countries. Most of this germplasm is yet to be tested for the nutritional quality traits. Several groups have examined the feasibility of “Biofortification” approach for improving the micronutrient content of staple crops and found that: (i) substantial useful genetic variation exists in key staple crops (ii) breeding programs can readily manage nutritional quality traits, which have been reported to be highly heritable in some crops and desired traits are sufficiently stable across a wide range of growing environments and (iii) traits for high nutrient content can be combined with superior agronomic and high yield characteristics (Welch and Graham, 2004).

Thousands of SSR markers have been mapped and developed as molecular markers (McCouch et al., 2002; www.gramene.org). The microsatellite markers are supposed to be particularly suitable for evaluating genetic diversity and relationships among closely related plant accessions or individuals, such as different rice cultivars. Markers linked to the gene can be used to select plants possessing the desired trait, and markers throughout the genome can be used to select plants that are genetically similar to the recurrent parent (background selection). This approach is thought to be promising in rice because a number of rice cultivars are widely grown for their adaptation, stable performance, and desirable grain quality.

Alternatively, genetic transformation can be used to transfer useful genes in commercially important rice varieties without disrupting their otherwise desirable genetic make-up. The development of efficient protocols for in vitro culture and transformation of japonica as well as indica rice varietieswere reported (Jain 2003; Veluthambi et al., 2003).

1 Rice and Genetic diversity for mineral content
Oryza is an agronomically important genus containing species with highly diverse morphology. The genus Oryza includes cultivated rice species, O. sativa, which constitutes an important part of the diet of more than half of the world’s population. More than 90% of this cultivated rice is grown and consumed in Asia. India stands first in terms of area under rice cultivation and second in rice production after China. Worldwide, rice production has more than doubled in 35 year period from 257 million tons in 1996 to 600 million tons in 2000. Rice (Oryza sativa) has become a model cereal for genomic research because of its small genome size (~430 million bp) and diploid nature. International Rice Research Institute (IRRI, The Philippines) and national research institutions in various countries are maintaining > 200,000 germplasm accessions of rice. Rice is one of the most diverse crops being grown as far north as Manchuria (China) and far south as Uruguay and New South Wales in Australia. In the last twenty years, a rapid progress has been made towards the basic and applied research in rice biotechnology and molecular biology. In fact, progress towards development of molecular techniques has been more rapid with rice than any other cereal. Some of the milestones includes: (i) development of the first saturated restriction fragment length polymorphism (RFLP) map; (ii) the application of polymerase chain reaction (PCR) based markers such as simple sequence repeat (SSR) markers, amplified fragment length polymorphism (AFLP); (iii) the identification of genes/quantitative trait loci (QTLs) for many agronomically important traits and marker assisted breeding; (iv) development of efficient techniques for genetic transformation; (v) complete sequencing and annotation of indica and japonica rice; (vi) Development of new generation markers (SSR, SNP, InDel, etc) (Shen et al., 2004), (vii) synteny between genomes of rice and other cereals (Xu et al., 2005) and (viii) access to several genome databases that facilitate depositing, searching, querying and analyzing information about rice and other cereals (www.gramene.org; http://rgp.dna.affrc.go.jp/) that makes rice a good entry point for characterizing the genes of other cereals, and associating them with various agronomic traits.

Gregorio et al., (2000) reported wide range of Fe (6.3~24.4 µg/g) and Zn (13.5~58.4 µg/g) concentrations in brown rice within the eight sets of genotypes, which clearly indicates existence of genetic potential to increase the concentration of these micronutrients in rice grain. Genetic component analysis conducted for high-Fe trait in grain using four traditional high-Fe rice varieties (Azucena, Basmati 370, Xua Bue Nuo and Tong Lang Mo Mi), three advanced lines (IR61608, PP2462-11 and AT5-15), and three IRRI released varieties (IR36, IR64 and IR72) showed significant genetic effect on grain Fe concentration, suggesting that selection among F1 progenies is possible. Fageria (2001) identified several Zn-efficient rice genotypes, which had Zn concentration of 20~25 ppm. Results so far obtained from various micronutrient projects across the world indicate that the breeding parameters are not difficult and are highly likely to be low cost.

Liang et al. (2007) has been investigated the variation of phytic acid (PA), iron (Fe) and zinc (Zn) levels in 56 varieties of Chinese rice. Fe levels showed the biggest variation (9~45 mg/kg) and were not related with PA content or grain shape. Zn showed a moderate variability (13~39 mg/kg), which was narrower than for Fe, while broader than for PA (7.2~11.9 g/kg).

Banerjee et al. (2010) analyzed variability in grain protein and Fe/Zn levels in 46 rice lines including indica and japonica rice cultivars, germplasm assesions, advanced breeding lines and wild rice genotypes and reported large variation for grain protein and micronutrient levels among the tested rice genotypes, which ranged from 6.19 to 10.75% for grain protein content, 4.82 to 22.69 mg/kg (µg/g) for grain Fe and 13.95 to 41.73 mg/kg (µg/g) for grain Zn content. Ezeonu et al. (2002) analyzed Fe and Zn concentrations in water, soil and staple food samples including rice. Zinc and iron concentration in rice varied from 3.5.0~15.0 mg/kg (ppm/l) and 20.0~75.0 mg/kg (ppm/l) respectively.  

The molecular understanding of metal homeostasis in plants in general and rice in particular, with the knowledge of the physiology of metal uptake, translocation and movement across the cell membranes will provide a basis to design strategies for development of micronutrient rich staple foods (Chandel et al., 2010). This can be achieved by identification and critical functional characterization of genes involved in metal uptake and transport in rice. Several molecular players have been identified with speculated functions in transporting minerals into the plants such as those belonging to ZIP, NRAMP and YSL family of transporters (Maser et al., 2001, Gross et al., 2003, Kobayashi et al., 2005). Rice genes orthologous to NAS and NAAT genes of barley (Hordeum vulgare L) viz OsNAS1, OsNAS2 and OsNAS3 have also been isolated (Tomako et al., 2007) and characterized for functions in metal uptake and translocation. These genes, which are related to the phytosiderophore biosynthetic pathway, have been shown to be involved in iron acquisition during germination (Koike et al., 2004). Mineral-rich and mineral-poor rice genotypes identified in this study could be an ideal material for such molecular and physiological analyses.

2 Micronutrient malnutrition the hidden- hunger
Micronutrient malnutrition is the condition that develops when the body does not get the optimum amount of the vitamins, minerals and other micronutrients which are essential to maintain metabolic regulation and organ function. Among the major nutritional problems common in developing countries are: micronutrient (iron, zinc, vitamin A) and protein-energy malnutrition (Zimmermann and Hurrell, 2002; Bouis et al., 2003; Welch and Graham, 2004). It is estimated that over 800 million people go to bed hungry every day and approximately 3 billion people are suffering from micronutrient deficiency (Evans 1998). More than half of the world’s population, especially women and children in the developing countries, suffer from micronutrient malnutrition or ‘hidden hunger’ resulting from the consumption of meager bioavailable vitamins and minerals containing diets (UN SCN, 2004).

Plant biologists can provide a crucial input in this fight to reduce micronutrient malnutrition by producing staple foods whose edible portions are denser in bioavailable minerals (such as iron, zinc) and vitamins. “Biofortification” refers to the development of micronutrient-dense staple crops using the best traditional breeding practices and modern biotechnology (Gregorio, 2002; Pfeiffer and McClafferty, 2007; White and Broadley, 2005). This approach has multiple advantages. First, it capitalizes on regular daily intake of a consistent and large amount of staple food by all family members, because staple foods are predominantly consumed by the poor people. Second, after the one-time investment to develop seeds that fortify themselves, recurrent costs are low, and germplasm can be shared internationally. Third, biofortification provides a feasible mean of reaching undernourished populations in relatively remote rural areas, delivering naturally fortified foods to people with limited access to commercially marketed fortified foods that are more readily available in urban areas/developed nations (Nestel et al., 2006).

Iron deficiency is the most common micronutrient deficiency in the world Globally, anemia affects more than 1.6 billion people, or approximately 25% of the population. In developing countries, approximately 50% of anemia in the population is thought to be due to iron-deficiency but the proportion may vary among population groups and in different areas according to local conditions. According to WHO estimates, iron deficiency is most rampant in preschool children and pregnant women in developing countries. Iron deficiency results in anemia in human beings that reduce the immune competence, impairs the body homeostasis, affect the development brain. Billions of people are at risk for zinc deficiency. In fact, more than 400,000 children die each year due to zinc deficiency. Current estimates of the risk of zinc deficiency indicate that approximately one-third of the world’s population live in countries where the risk of zinc deficiency is high. Zinc is involved in more body functions than any other mineral. Zinc’s role include acting as necessary component of more than 200 enzyme systems, normal growth and development, the maintenance of body tissues, sexual function, and the immune system (Zimmermann and Hurrell 2002).

Some 127 million preschool children are vitamin A-deficient, which is about one-quarter of all preschool children in high-risk regions of the developing world (www: unicef.org/vitamina). Globally, approximately 4.4 million preschool-age children have visible eye damage due to vitamin A deficiency. Close to 20 million pregnant women in developing countries are also vitamin A deficient, of which about one-third are clinically night-blind. Nearly one-half of these cases occur in India (Flowers, 2000).

Animals, including humans, are incapable of synthesizing 10 of the 20 amino acids needed for protein synthesis, and these ‘‘essential’’ amino acids must therefore be obtained from the diet (WHO, 2000). Amino acid deficiency causes the disease “kwashiorkor” and its symptoms include apathy, diarrhoea, inactivity, failure to grow, and edema (Schwartz et al. 2003).

3 Biofortification
Biofortification, which refers to the breeding of staple plants/ foods products with high bioavailable micronutrient content has the potential to provide coverage for remote rural populations, where supplementation and fortification programs may not reach and it inherently targets the poor especially women, infants and children who consume high levels of staple foods and little else (Bouis et al., 2003). Possibility of evolving micronutrient dense crops using plant breeding and/or biotechnological (marker assisted selection, transformation, etc.) strategies exists within the genomes of staple food crops (Welch and Graham, 2004). A lot of variability does exist for micronutrient (Fe, Zn, Vitaimin A, etc) content and bioavailability in many crops including rice. Alternatively, several useful genes have been identified, which may be used to improve the nutritional quality of commercially important cereal crops via transformation (Bouis et al., 2003). Some of the landmark achievements in developing nutrient-dense crops include: (i) engineering of β-carotene biosynthesis pathway in rice (Paine et al., 2005), (ii) improvement of iron content in rice by transferring the soybean and Phaseolus vulgaris ferritin genes and (iii) positional cloning of GPC-B1, and a wild-wheat QTL associated with increased grain protein, Zn and Fe contents (Uauy et al., 2006). In addition, micronutrient element enrichment of seeds can increase crop yields when sowed to micronutrient-poor soils, assuring their adoption by farmers. A comprehensive program on “Biofortification” was started in last century mainly under the auspices of HarvestPlus (Pfiffer and McClafferty 2007).

4 Molecular and transgenic strategies for improving iron and zinc content and bioavailability in rice
Over the past few decades till 1990, most of the breeding research was concentrated on increasing the grain yield and to improve the resistance to environmental stresses, pests and pathogens (Borlaug, 2000), but little or no attention was given towards the enhancement of its nutritional quality. Recent developments in the area of genomics, transformation and molecular mapping have provided new tools for molecular dissection of complex polygenic traits, improving our understanding of factor regulating micronutrient efficiency, rapid discovery of genes/ QTLs involved, marker-assisted breeding and transfer of important genes with potential to improve mineral density.

Several Zn-efficient rice genotypes including Metica 1, Epagri 108, CNA 7550, and CAN 86, with Zn concentration of 20~25 ppm have also been identified (Fageria 2001). The Fe and Zn concentrations varied within the six sets of genotypes (n=939) were 7.5~24.4 μg/g for Fe, and 13.5~58.4 μg/g for Zn (Welch & Graham, 2000). Iron and zinc content in rice varied from 4.3~25.8 mg/kg and 8.6~43.0 mg/kg respectively (Meng et al., 2005). Wide variations for grain protein and micronutrient levels were recorded among the tested rice genotypes; grain protein content ranged from 6.19% to 10.75%, grain Fe from 4.82 mg/g to 22.69 mg/g and grain Zn content from 13.95 mg/g to 41.73 mg/g (Banerjee et al., 2010). The rice genotypes with high grain protein and micronutrients will provide the basis of bioavailability assay and will also serve as potential genetic material for molecular breeding of nutrient rich rice.

Brar et al. (2011) reported, large variation for iron and zinc contents in a collection of 220 rice genotypes; iron content varied between 5.1 (IR6387 2-4-2-2-1) - 441.50 µg/g (HKR95-157) and zinc content varied between 2.12 (KBR466) – 39.4 µg per g (Taraori Basmati). Notably, there was about eighty-fold difference in Fe content and nineteen- fold difference in Zn concentrations in the present set of 220 rice genotypes suggesting the existence of genetic potential to increase the concentrations of these micronutrients in rice grain. These results also indicate that there is significant genetic diversity for Fe and Zn in the available rice germplasm and it should be feasible to plan a breeding program to develop high-yielding, mineral-rich rice genotypes.

The large genotypic variation of mineral content (iron and zinc) in rice grains could be due to tightly controlled homoeostatic mechanisms that regulate metal absorption, translocation, and redistribution in plants allowing adequate, but non-toxic levels of these nutrients to accumulate in plant tissues (Welch and Graham, 2004). Iron and zinc contents in edible portions also depend on the efficiency of translocation of minerals from root tissues to edible plant organs and accumulation thereof. In addition, phloem sap loading, translocation and unloading rates within reproductive organs are important characteristics that imparts in the variability for iron and zinc contents in seeds.

4.1 Molecular approaches for developing iron and zinc dense crops
Molecular markers are valuable tools in both basic and applied research such as DNA fingerprinting, analyzing genetic diversity, marker-assisted breeding, phylogenetic analysis and map-based cloning of genes (Ni et al., 2002). The microsatellite markers are supposed to be particularly suitable for evaluating genetic diversity and relationships among closely related plant accessions or individuals, such as different rice cultivars. Genes/QTLs have been mapped for several agronomically important traits such as disease and insect resistance, yield, quality and abiotic stress tolerance traits (drought, sub-mergence tolerance, salinity tolerance, etc. (Khush & Brar, 2001). But, there have been only a few studies to map QTLs for mineral traits. A major QTL for high-Zn has been identified in rice on chromosome 5 flanking microsatellite markers, RM 167 and RM 87, using a selective phenotyping and genotyping approach via microsatellite marker analysis (unpublished data). Permanent mapping populations of F8 recombinant inbred lines were developed to map high-Fe and high-Zn traits and are being used to map genes/QTLs for high-micro- nutrient traits (Fageria, 2001). Bradbury et al. (2005) reported significant polymorphisms in the coding region of fragrant rice genotypes relative to nonfragrant genotypes for a gene with homology to the gene encoding betaine aldehyde dehydrogenase 2.

Garcia-Oliveira et al. (2009) reported substantial variation for Fe, Zn, Mn, Cu, Ca, Mg, P and K contents in 85 introgression lines (ILs) derived from a cross between an elite indica cultivar ‘Teqing’ and the wild rice (Oryza rufipogon L.) and all the mineral elements were significantly positive correlated or independent except for Fe with Cu. Zeng et al. (2009) showed that the iron content was significantly associated with the allele size of RM225; this marker was also linked with the nitrogen use efficiency. Cu content was significantly associated with the allele size of RM81A, RM60, and RM247, Mn content was correlated with the allele size of RM60 and RM225 and P content in brown rice showed a significant correlation with the allele size of RM81A, RM253, RM232, RM234, and RM244.
Nandakumar et al. (2004) reported that out of ten, nine STMS markers were found polymorphic across the hybrids and produced unique fingerprint for 11 rice hybrids. A set of four markers (RM206, RM216, RM258 and RM263) differentiated all the hybrids from each other, which can be used as referral markers for unambiguous identification and protection of these hybrids. Hashemi et al. (2009) reported that molecular markers RM1, RM263 and RM6344 markers for genetic purity test in hybrids except the first Iranian rice (IRH1). Pervaiz et al. (2010) reported that a total of 142 alleles were detected at 32 polymorphic SSR loci, while three loci were monomorphic in 75 Pakistani rice landraces. A dendrogram based divied the genotypes into four major clusters, differentiating tall, late maturing and slender aromatic types from the short, early and bold non-aromatic ones. Brar et al., (2014) reported, total of 258 alleles were detected at 48 SSR loci with an average number of 5.14 alleles per locus. These allelic frequency values quite comparable to those reported earlier keeping in view the lower number of rice genotypes used in the study. NTSYS-pc UPGMA tree cluster analysis showed the clustering of 14 rice genotypes into two major distinct groups.

4.2 Transgenic approaches for developing crops with increased iron and zinc bioavailability
Development of micronutrient (e.g. Fe and Zn) rich and efficient crops using novel molecular tools has gained attention now a day. Ye et al. (2000) reported the engineering of soybean ferritin gene and β-carotene (vitamin A) biosynthesis pathway in rice. Among the micronutrients, most of work has been done to tailor transgenic plants with high iron content and bioavailability (Jain et al., 2003; Guerinot 2007; Zhu et al., 2007). Takahashi (2003) reported the developments of transgenic rice plants expressing the barley nicotinamine aminotransferase (NAAT) gene, one of the genes of enzymes of MA biosynthetic pathway, which showed tolerance to low-Fe availability in calcareous soils. This phenomenon occurred because transgenic rice plants secreted higher amounts of MAs characteristic of strategy II than do non-transgenic rice plants. Vasconcelos et al. (2003) reported enhanced iron and zinc accumulation not only in brown grains but also in polished grains of transgenic rice expressing the soybean ferritin gene driven by the endosperm- specific glutelin promoter.

Ishimaru et al. (2009) reported that MIR transcripts were greatly increased in response to Fe deficiency in roots and shoot tissue. Growth in the MIR T-DNA knockout rice mutant (mir) was significantly impaired compared to wild-type (WT) plants when grown under Fe-deficient or -sufficient conditions. Furthermore, mir plants accumulated more than twice the amount of Fe in shoot and root tissue compared to WT plants, when grown under either Fe-sufficient or -deficient conditions and plays a significant role in Fe homeostasis.

5 Improving the protein content and amino acid quality
Improvement of nutritive value of crop plants, in particular the amino acid composition, has been a major long term goal of plants breeding programs because animals, including humans, are incapable of synthesizing 10 of the 20 amino acids needed for protein synthesis and these “essential” amino acids must therefore be obtained from the diet. The higher the quality of a specific protein, the more efficiently it is utilized and the less is needed to meet protein requirements. Thus changes in protein content or amino acid pattern of the resultant protein mix can have significant effects on the efficiency of protein utilization to meet nutritional requirements (Bouis et al., 2003).

To improve the nutritional value of rice, transgenic rice plants were developed with a lysine-feedback insensitive maize dhps gene under the control of CaMV 35S and the rice glutelin GluB-1 promoter for over-expression and seed-specific expression (Lee et al., 2001). The transgenic plants were fertile and expressed the dhps gene abundantly or specifically in rice seeds. Soybean glycine gene was successfully transferred into japonica rice variety Kitaake (Laiptan et al., 2005).

Chakarborty et al. (2010) produced transgenic potato expressing a seed albumin (AmA1) gene from Amaranthus hypochondriacus. These findings demonstrate the feasibility of using the AmA1 gene in genetic engineering to improve the nutritive value of other non-seed and grain crops. So, proteins that are already made in a particular plant can have new amino acids introduced into them or genes for new proteins that have the desired amino acid composition can be transferred in the target crops.

6 Improving the Vitamin A content
Besides increasing the crop productivity, plant genetic engineering has been/is being used to create crops that are tailored to provide better nutrition for humans and their domestic animals (Bouis et al., 2003). Micronutrient (iron, zinc, iodine, essential amino acids, etc) and vitamin (vitamin A) deficiencies are widely prevalent in Asia and Africa, adversely affecting the health of more than one-half of the population.

Vitamin A rich rice, commonly known as ‘Golden Rice’, contains the genes required to activate the biochemical pathway leading to -carotene. The intensity of the colour represents the concentration. It is estimated that in India 50,000 children become blind every year due to Vitamin A deficiency (Paarlberg, 2001). So, to provide adequate amounts of vitamin A to children in developing countries would save large numbers from night blindness or actual blindness. Golden rice is an excellent example of how genetic engineering of plant can be of direct benefit to the consumer, especially the poor in developing countries (Potrykus, 2001). Paine et al. (2005) reported the development of ‘Golden Rice 2’ by introducing maize psy in combination with the Erwinia uredovora crt1 gene. The research efforts are now being made by IRRI and various national institutions to transfer Golden rice 2 psy/crtI transgene combination from transgenic japonica rice into local rice cultivars via molecular breeding. Golden rice has been developed to deliver this nutrient to those populations who need it most (Potrykus, 2003).

7 Agrobacterium method for rice transformation
Recent advances in plant biotechnology have provided biologists with the tools to engineer desirable traits into rice plants with the capabilities far beyond than those provided by conventional plant breeding. One important application of genetic transformation is to transfer one or more useful genes into an elite cultivar without disturbing its original genetic background.

Since then, a steady progress has been made towards the development of efficient protocols for in vitro culture and transformation of japonica as well as indica rice varieties (Roy et al., 2000; Veluthambi et al., 2003; Bajaj and Mohanty, 2005; Nishimura et al., 2005; Toki et al., 2006). Rice can now be transformed efficiently using Agrobacterium method. An array of useful genes has been transferred in different rice varieties to improve their resistance/ tolerance against insect pests, fungal diseases, drought/salinity and to improve their nutritional quality (Jain et al., 2001). Rice is one of the nutritionally deficient crops especially with respect to protein content and essential amino acid composition and recently several efforts have been made to improve its nutritional quality (Bouis et al., 2003; Jain et al., 2004). Several factors affecting the transformation have been reviewed by Jain et al. (2003) and Yu et al. (2005). Ge et al., (2005) gave a new efficient tissue culture system suitable for highly recalcitrant indica varieties to improve their efficiency via Agrobacterium mediated transformation. The transformed lines of Basmati rice cultivar Pusa Basmati 1 were obtained through Agrobacterium transformation (Bhutani et al., 2006; Ignacimuthu et al., 2006).

In addition, micronutrient element enrichment of seeds can increase crop yields when sowed to micronutrient-poor soils, assuring their adoption by farmers (Welch and Graham, 2004). So, it is necessary to maintain the agronomic characters along with enriched micronutrient content of the crop. The yield/plant was reported to be positively correlated with 100 grains weight by Sharma and Shar¬ma (2007). Chakrabarty et al. (2010) reported that yield/plant showed significant positive genotypic correlation with plant height (0.21), panicles/plant (0.27), panicle length (0.53), effective grains/pani¬cle (0.57) and harvest index (0.86). Brar et al. (2015) also reported significant correlation for 13 of 96 pairs between allele size of molecular markers and mineral traits, 10 of 144 pairs between molecular markers and plant traits.

Conclusion
Notably, there was a large variation for mineral content in rice genotypes suggesting the existence of genetic potential to increase the concentrations of these micronutrients in rice grain. The present literature also indicate that there is significant genetic diversity for Fe and Zn in the available rice germplasm and it should be feasible to plan a breeding program to develop high-yielding, mineral- rich rice genotypes.

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