Genetic and Genomic Drivers of Iron and Zinc in Rice Grains

Review Article
Published: 2025-12-22
https://doi.org/10.20935/AcadMolBioGen8075
Genetic and genomic drivers of iron and zinc levels in rice
grains
Yheni Dwiningsih1, *
Academic Editor: Brian D. Gregory
Abstract
Rice (Oryza sativa L.) is the primary staple food for more than half of the world’s population, containing essential human nutrients.
Essential micronutrients such as iron (Fe) and zinc (Zn) are very important in human diets, but bioavailability of Fe and Zn is lacking
in the rice grains, contributing to global malnutrition and a need for these micronutrients to be biofortified into rice grains. Most of the
popular rice varieties only contain 5.0–6.0 mg/kg of Fe and 14.0 mg/kg of Zn, which is not sufficient for rice consumers. This article
reviews the phenotypic variation in Fe and Zn concentrations of diverse rice genotypes, association between environmental conditions
and accumulation of Fe and Zn in the rice grains, correlation of Fe and Zn concentrations with other agronomic traits, and also genomic
analysis of Fe and Zn concentrations in rice. All of these factors are crucial to developing rice varieties with sufficient amounts of Fe and
Zn to improve rice grain quality and address mineral deficiency, which can be accomplished through biofortification, conventional rice
breeding, and genetic engineering methods.
Keywords: rice, micronutrients, phenotypic variance, genomics
Citation: Dwiningsih Y. Genetic and genomic drivers of iron and zinc levels in rice grains. Academia Molecular Biology and Genomics
2025;2. https://doi.org/10.20935/AcadMolBioGen8075
1. Introduction
Rice (Oryza sativa L.) is consumed by over half of the global
population and is the most important cereal grain, providing
carbohydrate and other important essential minerals for human
body [1, 2]. About 49 minerals are required by the human body,
which can be supplied by balanced diets [3]. Because of the high
rice consumption, 190 kg/capital/year, rice grains are expected
to become a key mineral source for human body [4–6]. Environmental conditions and genetic factors influence the mineral
concentrations in rice grains [7, 8]. Iron (Fe) and zinc (Zn) are
essential minerals that lack bioavailability in rice and are commonly fortified into the grains [9–11]. Fe is the crucial element in
hemoglobin within human red blood cells that plays an important
role in immune function, DNA synthesis, and energy metabolism.
Fe deficiency leads to anemia, with the symptoms of less energy,
fatigue, decreased cognitive function, and an impaired immune
system, that affects about 60% of the world population. Deficiency
of Fe during pregnancy contributes to maternal mortality and also
impairs child health. Zn is the primary element for enzymes associated with protein synthesis, cell division, and growth [12, 13].
Zn is also a co-factor for more than 300 enzymes. Zn deficiency
was correlated with immune system disfunction and slower mental and physical development in over 30% of the global population [14–22]. The recommended dietary intake of Fe for adults
is approximately 10–15 mg per day, whereas polished rice grains
contain only about 2 mg of Fe per kilogram of grain. Additionally,
the recommended daily Zn intake is around 12–15 mg of Zn per
kilogram of grain, while polished grains only have 12 mg of Zn
per kilogram of grain [16–18]. Hence, mineral deficiency generally
happens in rice-dependent populations, affecting children and
adolescents in developed, developing, and underdeveloped countries globally [19]. Up to 17% of the population in developed
countries, more than 30% in developing countries, and about 60%
of the total population in underdeveloped countries suffer from
Fe and Zn deficiency. It was also detected that more than 50%
of children under five years old and up to 60% women of reproductive age suffer from the micronutrient deficiency. The WHO
also reported that deficiencies in Fe and Zn are the sixth most
important factor causing human illness in low-income countries.
Furthermore, these mineral deficiencies may lead to dysfunction
in human organs and serious illness [23–27]. Therefore, rice
biofortification with Fe and Zn becomes the most practical and
economical solution to address these deficiencies, particularly in
vulnerable populations, including pregnant women, children, and
individuals in low-income countries [28–33].
Traditional rice breeding and genetic engineering approaches are
promising solutions that can enhance Fe and Zn concentrations
in rice grains without reducing crop productivity [33, 34]. Several
aromatic rice genotypes, such as Xua Bue, Jalmagna, and Zuchem
contain high concentrations of Fe and Zn in their grains [35]. Rice
varieties that contain Fe over 30 mg/kg include Nagina22, ASD16,
CH45, Dragon Eyeball 100, HKR126, and Annada [36]. Moreover,
rice genotypes that have high Zn concentrations include CR Dhan
311, Chattishgarh Zinc Rice-1, Norungan, and DRR Dhan 45 [15].
The Manipuri black rice variety contains high Fe concentration,
up to 37.6 mg/kg, and Zn concentration, reaching 17.98 mg/kg.
Several studies also reported that japonica rice shows higher
Fe concentrations than indica rice varieties [36, 37]. Moreover,
1 Department of Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR, USA.
∗ email: [email protected]
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brown rice grains have higher Fe and Zn concentrations compared to polished rice grains [38]. Zn-enriched indica inbred highyield varieties (HYVs) were released by the Bangladesh Rice Research Institute (BRRI), including BRRI dhan42, BRRI dhan43,
BRRI dhan62, BRRI dhan64, BRRI dhan72, BRRI dhan74, BRRI
dhan84, and BRRI dhan104, which contain 19–27.5 mg/kg of
Zn [39]. Based on grain measurement using X-ray fluorescence
and histochemical techniques, the aleurone layer of the rice grain
is the most abundant in terms of Fe and Zn concentrations [40–
42]. Cultivated rice showed lower concentrations of Fe and Zn
compared to wild rice accessions, indicating that wild rice species
are a better genetic source for conventional rice breeding to increase Fe and Zn concentrations [36, 43].
Understanding the genomic mechanisms of Fe and Zn concentrations in rice grains is essential to developing rice varieties
with enhanced Fe and Zn contents [1]. Concentrations of Fe and
Zn in rice grains are the quantitative traits because of the wide
range of concentrations among diverse rice genotypes [1, 44].
Thus, association mapping, quantitative trait loci (QTL) mapping,
and genome-wide association studies (GWASs) have generally
been used to identify loci or candidate gene locations on the rice
genome associated with Fe and Zn concentrations, using many
rice mapping populations. For example, genomic regions relevant
to Zn concentration were identified by a GWAS in the RDP1
rice population [45–47]. Recently, Fe and Zn concentrations in
rice grains has become a crucial selection trait in rice breeding
programs [48]. In conventional rice breeding, screening of various rice germplasms with high Fe and Zn concentrations is very
important in order to find the optimum donor parents [49, 50]. O.
officinalis, O. latifolia, and O. nivara showed high concentrations
of Fe and Zn [36, 51]. Many QTL for Fe and Zn contents have
been identified by using doubled-haploid rice populations under
controlled environmental conditions [52], as well as being found
in introgression rice line populations under field conditions [53].
Furthermore, detection of genomic regions regulating the Fe and
Zn concentrations is the first step in developing marker-assisted
selection for these traits.
Enhancing the concentrations of Fe and Zn in rice grains would
significantly increase rice quality in terms of addressing mineral
deficiencies in rice consumers, and can be accomplished by adopting plant breeding programs and genetic engineering approaches.
The HarvestPlus program is one of the successful programs for
the solution of mineral deficiencies which is trying to improve essential micronutrient concentrations in human diets [9]. Recently,
with the benefit of traditional breeding and genetic engineering
methods, different agronomically and genetically developed rice
varieties with improved Fe and Zn concentrations have emerged.
This article reviews the phenotypic variation in Fe and Zn concentrations of diverse rice genotypes, association between environmental conditions and accumulation of Fe and Zn in rice grains,
correlation of Fe and Zn concentrations with other agronomic
traits, and genomic analysis of Fe and Zn concentrations in rice.
All of these factors are crucial to developing rice varieties with
sufficient amounts of Fe and Zn to improve rice grain quality in
terms of addressing mineral deficiency.
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2. Phenotypic variation in Fe and Zn
concentrations of diverse rice genotypes
The concentrations of Fe and Zn in rice grains are influenced
by genetic factors, environmental growth conditions, and their
interactions [7]. Grain position on the rice panicle, soil properties, and harvest time also affect Fe and Zn accumulation in
grains. Interactions among the ions in the rhizosphere, where ion
competition happens for absorption, transport, and accumulation
in the plant tissue, also affects the Fe and Zn concentrations
in grains. A number of methodologies have been used to determine the concentrations of Fe and Zn in rice grains, including
an energy dispersive X-ray fluorescence spectrometer (ED-XRF),
inductively coupled plasma mass spectrometry (ICP-MS), and
Atomic Absorption Spectrophotometry (AAS) [7]. ED-XRF analysis is commonly used for rapid screening, and it is less precise for
Fe and Zn quantification, particularly when validating biofortified
varieties. AAS and ICP-MS are the recommended standard analytical methods for accurate Fe and Zn determination. Many studies
reported that Fe and Zn concentrations showed a wide variation
among rice genotypes (Table 1). Under different growth conditions, the concentrations of Fe and Zn displayed a continuous
distribution, which provides the possibility to select donor parents
with high Fe and Zn concentrations for rice nutrient breeding
programs [48]. Fe and Zn concentrations in diverse rice genotypes
classified as high broad-sense heritability, with H2 > 0.5, indicate
the possibility of the acceleration of Fe and Zn improvement in
rice grains [54, 55].
Wild rice species, such as Oryza rufipogon, Oryza longistaminata, Oryza nivara, and Oryza meridionalis, have become an
important gene pool for potential genetic resources for mapping
rice populations in order to develop rice varieties with enhanced
Fe and Zn concentrations. Many studies have already exploited
the gene pool of O. rufipogon [16]. The genes associated with Fe
and Zn from wild rice species can be transferred to popular rice
cultivars and potentially enhance Fe and Zn accumulation in the
grains of popular varieties. Moreover, Fe and Zn concentrations in
the different rice accessions of indica, japonica, and aus showed
wide variation, suggesting different genetic aspects regulating
these traits [21]. Indica has the lowest Zn concentration compared
to japonica and aus. Meanwhile, temperate japonica and aus
have been reported to accumulate high Zn concentrations, such as
in Bulgare (temperate japonica), Jamir (aus), Khao Tot Long 227
(aus), DZ 192 (aus), and Estrela (admix) [56]. Variations in the
phenotypic and genotypic characteristics of indica and japonica
rice accessions indicate a huge potential to improve micronutrient
concentrations in rice grains by crossing different accessions.
Molecular mechanisms of micronutrient accumulation in rice
grains are therefore very important to elucidate.
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Table 1 • Variation in Fe and Zn Concentrations in Rice Grains.
Rice accessions
Fe concentration
(mg/kg)
Zn concentration
(mg/kg)
Landraces
0.40–147 [57]
15.1–124 [57]
Landraces and diverse rice genotypes
0.561–4.48 [15]
9.7–26.96 [15]
Wild-type and transgenic rice accessions
10 [58]
23 [58]
Wild-type and transgenic rice of japonica (Nipponbare)
25–56 [59]
40–59 [59]
Wild-type and transgenic rice
2–13 [21]
16–28 [21]
Wild-type and transgenic rice of japonica cultivar Tsukinohikari
0.9 [60]
12 [60]
Diverse rice genotypes
0.98–26.78 [61]
53.53–83.66 [62]
Diverse rice genotypes
37.02–51.99 [62]
7.99 [63]
Diverse rice genotypes
6.5–15.6 [40]
13.5–58.4 [24]
Diverse rice genotypes
6.03 [63]
13.32–43.65 [61]
Diverse rice genotypes
6.3–24.4 [24]
13.7–22 [24]
Diverse rice genotypes
14.5–31.4 [64]
24.4–44.7 [64]
Diverse rice genotypes
6.2–71.6 [36]
26.2–67.3 [36]
Diverse rice genotypes
11.2–16.1 [65]
19.3–24 [65]
Diverse rice genotypes: USDA Rice Mini-Core Collection—211 O. sativa, 5 O.
glaberrima, 2 O. rufipogon, 1 O. nivara
17.35 [66]
32.36 [66]
Diverse rice genotypes
119.26 [7]
28.7 [1]
Diverse rice genotypes
8.1–16.7 [67]
24.29 [68]
Diverse rice genotypes
11.45 [68]
3.15–5.54 [69]
Diverse rice genotypes
19.44 [1]
14.5–35.3 [70]
Diverse rice genotypes
6.63–9.23 [69]
47.0 [20]
Diverse rice genotypes
6.9–22.3 [70]
19.7–23.3 [67]
Diverse rice genotypes
23.1 [20]
25.69 [7]
Aromatic and non-aromatic rice accessions
37.6 [70]
17.98 [71]
F2 population derived from cross (Anjana Dhan as female parent and Nipponbare
as male parent)
18 [72]
44 [72]
F2 population derived from cross between PAU201 (high-yielding) and Palman
579 (iron-rich) indica rice varieties
0–150 [73]
4.4–157.4 [73]
Two BC2F3 mapping populations derived from crosses of O. sativa cv Swarna with
two different accessions of O. nivara
1.6–22.2 [55]
7.1–64.7 [55]
Doubled-haploid (DH) population derived from cross between CJ06 (japonica)
and TN1 (indica)
5–18 [48]
13–42 [48]
Backcross inbred lines (BILs) derived from cross between Sasanishiki (japonica)
and Habataki (indica)
7.80–14.6 [74]
19.6–47.5 [74]
Introgression line (IL) population derived from Teqing (indica) as recipient parent
and wild rice Oryza rufipogon as donor parent
4.9–20 [53]
13.3–60.1 [53]
F6 RIL population derived from cross between Bala (indica) and Azucena
(japonica)
29.1–30.6 [75]
29.7–36.5 [75]
F6 recombinant inbred lines (RILs) derived from Madhukar and Swarna cross
0.2–224 [36]
0.4–104 [36]
RIL population derived from cross between two indica cultivars: Zhengshan 97
and Minghui
18.93 [76]
22.33 [76]
RIL population derived from cross between Lemont (japonica) and TeQing (indica)
7.3–22.0 [77]
18.0–33.0 [77]
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A strong positive correlation was reported between Fe and Zn
concentrations in rice grains, indicating that high Fe will occur
simultaneously with high Zn [66]. Many studies also reported that
Fe has a significant positive correlation with the Zn concentration in brown rice varieties, and the genetic factors that regulate
increased Fe accumulation co-segregate with the genetic factors
that enhance Zn content in grains [34, 35, 41]. Fe and Zn also
showed a positive correlation with Ca, Cd, K, Mg, Mn, Na, and
Pb [61, 78–82]. Meanwhile, Zn has a negative correlation with
Ni [83]. During the grain filling stage (R3), mineral elements
including Fe and Zn are transported by the phloem from the flag
leaf to the grains [84]. The accumulation of Fe and Zn in the grains
is influenced by molecular mechanisms of root uptake transport
and partitioning of the elements to grains and plant tissues [54].
3. Association between environmental
conditions and accumulation of Fe and
Zn in rice grains
Concentrations of Fe and Zn in grains are also influenced by
environmental growth conditions, such as water availability, pH,
salinity, commercial fertilizers, and manure application [85–88].
Metabolism of rice plants, including the rate of Fe and Zn transport, also directly affect the concentration of these microelements.
Most of the Fe and Zn in the grains is supplied by root uptake,
being translocated from vegetative tissues to grains during the
grain filling reproductive stage [89–91]. Therefore, Fe and Zn
concentrations are strongly influenced by nitrogen fertilizer application [92] and spraying of Fe and Zn fertilizers on rice leaves [65].
Rice is usually grown in rice fields under anaerobic conditions,
in which Fe2+ is easily absorbed by plants due to its low redox
potential [60]. The uptake of Zn from the rhizosphere is influenced
by the molecular size of organic acids [61]. The variation in Fe
and Zn concentrations in rice grains is also influenced by the
homeostasis of Fe and Zn, which is correlated to the uptake,
transport, and accumulation processes [62].
Geographical regions in different countries also significantly influence the Fe and Zn concentration. Hence, landraces and adapted
rice cultivars show different transportation mechanisms of Fe and
Zn, developed in order to adapt to their particular growth conditions [54]. Environmental stress conditions such as soil acidity,
salinity, and drought significantly decrease Fe and Zn concentrations [35]. Accumulation of Fe and Zn in grains starts from the
uptake of Fe and Zn from the roots, followed by nutrient loading
to the xylem, mineral transportations, partitioning, sequestration
in the vacuoles, and accumulation in the grains. Fe and Zn uptake
from the soil to the grains is also significantly affected by the
soil characteristics, including clay content, organic carbon composition, and cation exchange capacity [92, 93]. Application of
N and P fertilizers also showed a significant effect on Fe and Zn
accumulations in grains [94]. N and P elements in the soil may
affect root morphology, which directly influences the mineral concentrations in the grains. Maintaining soil fertility with manure,
compost, and biochar is also important as it allows the rice plants
to grow optimally. Therefore, field water management and soil
fertility also affect Fe and Zn concentrations in rice grains.
Milling, polishing, storing, and cooking processes of the rice grains
also affect the Fe and Zn concentrations. Due to the milling process,
the Fe concentrations of red rice varieties, including Jalmagna,
ACADEMIA MOLECULAR BIOLOGY AND GENOMICS
Xue Bue Nuo, and Tong Lang Mo Mi, is significantly reduced.
The Fe content of popular brown rice commercial variety IR64 is
also decreased because of its 15 min milling process [20]. In the
process of grain polishing, about 10% of Fe and Zn were removed
due to these elements being abundant in the aleurone layer [95].
Rinsing the rice grains with excess water also decreases the Fe
and Zn concentrations. Excessive heat in the cooking process also
potentially reduces the mineral concentrations. Storing the grains
for more than one year also significantly decreases Fe and Zn.
4. Correlation of Fe and Zn concentrations
with others agronomic traits
A strong positive correlation was observed among the Fe and Zn
concentrations with heading date and flowering time [66, 96].
Heading date is one of the important agronomic traits associated
with the adaptation ability of the rice plant to various environmental conditions during the vegetative growth stage. Rice plants
with a late heading date will have a longer time to absorb mineral
elements from the soil and accumulate them in the grains during
the reproductive grain filling stage. Thus, extending the heading
date is one of the effective strategies that can enhance Fe and
Zn concentrations in rice grains [54, 97, 98]. Meanwhile, Fe and
Zn accumulation showed no correlation with grain yield, panicle
length, grain weight, or plant height [73, 99, 100].
Fe and Zn accumulations displayed a significant positive correlation with grain shape [62]. Fe has direct correlation with
grain weight [54]. Moreover, Zn exhibited strong relationship
with grain length [36]. Grain width is significantly associated
with both Fe and Zn. Fe and Zn concentration in the aromatic
long-grain Basmati variety is higher compared to short-grain rice
varieties [101, 102]. Phytate (inositol-hexa-phosphate) concentration in grains also has a strong correlation with Fe and Zn concentrations [5]. Grain color is also associated with high Fe and Zn
concentration [103]. Pigmented rice varieties are excellent source
of flavonoids, anthocyanin, carotenoids, vitamins, polyphenols,
and antioxidants. Pigmented rice varieties are reported to accumulate high amounts of Fe [104]. Purple-bran grains have higher
Fe accumulation compared to white grains [54].
Concentrations of Fe and Zn in the rice grains are also linked to several rice quality characteristics, such as amino acid contents, protein
compositions, amylose content, gel consistency, and aroma. There
are 17 kinds of amino acid associated with these microelements,
including aspartic acid, threonine, serine, glutaminic acid, glycine,
alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine,
phenylalanine, lysine, histidine, arginine, and proline [61].
5. Genomics analysis of Fe and Zn
concentrations in rice grains
Many genes regulating the uptake, transport, and accumulation of
Fe and Zn in the rice grains have been identified, and the expressions of these genes are different in the vegetative and reproductive
stages. These genes were detected by integrating traditional rice
breeding, molecular markers, transgenic assisted breeding, bioinformatics analysis, and other genetic engineering approaches [97].
Because Fe and Zn concentrations are both quantitative traits,
the quantitative trait loci (QTL) method becomes an effective tool
for detecting the genes controlling these elements. Furthermore,
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knockout and over-expression of the candidate genes are very useful tools to validate the candidate genes within QTL regions [7].
QTL identification is significantly influenced by the donor parents,
population size, type of population, markers, and environmental
growth conditions, leading to more precise and accurate QTL.
Recently, more than 200 QTL associated with the Fe and Zn concentrations have been identified. Gene networks associated with
the Fe and Zn contents also crucial to be analyzed.
Genes associated with Fe and Zn concentrations have been identified by using landraces, diverse rice populations, popular rice varieties, backcross populations, doubled-haploid populations, introgression line populations, recombinant inbred line populations,
and other biparental rice populations. Advanced molecular markers like single-nucleotide polymorphisms (SNPs) have been generally employed to cover the whole rice genome [20]. Currently,
many genes controlling Fe and Zn have been detected by QTL
mapping, transgenic approaches, and genome-wide association
studies (GWASs) (Table 2). QTL controlling Fe and Zn accumulation have been identified on seven chromosomes, and some of
these QTL are co-localized with loci regulating the grain yield,
plant height, biomass, and sheath blight resistance [76]. These
candidate genes are OsYSL1 and OsMTP1, correlated with Fe
concentration, and OsARD, OsIRT1, OsNAS1, and OsNAS2, controlling Zn accumulation [36].
Table 2 • Genetic analysis of Fe and Zn concentrations in rice grains.
Fe and Zn concentrations in rice grains
Chromosome
Loci
References
Fe
1
LOC_Os01g0238700 (OsYSL1)
[55]
LOC_Os01g0503400 (OsNRAMP6)
[55]
LOC_Os01g13710 (OsYSL1)
[36]
qGFe1.1
[105]
LOC_Os02g0306401 (OsNAAT1)
[55]
qGFe2.1
[105]
OsMIT
[20]
LOC_Os03g0307300 (OsNAS1)
[55]
LOC_Os0307200 (OsNAS2)
[55]
LOC_Os04g39380
[7]
LOC_Os04g0542800 (OsYSL16)
[55]
LOC_Os04t0578600-02 (OsFRO2)
[55]
LOC_Os04g0444800 (OsFRO1)
[55]
LOC_Os04g39380
[7]
LOC_Os05g16290 (OsYSL4)
[36]
LOC_Os05g10940 (OsZIP7)
[36]
LOC_Os05g07210 (OsZIP6)
[36]
LOC_Os05g03780 (OsMTP1)
[36]
OsNRAMP3
[20]
LOC_Os04g39380
[7]
LOC_Os04g39380
[7]
LOC_Os07g12890 (OsZIP8)
[36]
LOC_Os07g43040
[36]
LOC_Os07g15460 (OsNRAMP1)
[36]
LOC_Os07g48980 (OsNAS3)
[36]
LOC_Os08g0290300 (OsYSL17)
[55]
LOC_Os08g0207500 (OsZIP4)
[55]
qGFe8.1
[105]
10
LOC_Os10g02340
[7]
11
LOC_Os11g0184900 (OsNAC5)
[55]
12
LOC_Os10g02340
[7]
LOC_Os12g39860 (APRT)
[36]
qGFe12.1
[105]
qGFe12.2
[105]
2
3
4
5
6
7
8
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Table 2 • Cont.
Fe and Zn concentrations in rice grains
Chromosome
Loci
References
Zn
1
LOC_Os01g0834400 (OsHAP3)
[55]
LOC_Os01g0816100 (OsNAC4)
[55]
LOC_Os03g19420 (OsNAS2)
[36]
LOC_Os03g19427 (OsNAS1)
[36]
LOC_Os03g46470 (OsIRT1)
[36]
LOC_Os03g06620 (OsARD2)
[36]
LOC_Os03g19427 (OsNAS1)
[36]
LOC_Os03g19420 (OsNAS2)
[36]
4
OsZIP3
[20]
5
LOC_Os05g41070
[7]
LOC_Os05g0111300
[55]
LOC_Os06g39960
[7]
LOC_Os06g0676000 (OsNRAMP3)
[55]
LOC_Os06g39960
[7]
LOC_Os07g12890
[36]
LOC_Os07g43040
[36]
LOC_Os07g15460 (OsNRAMP1)
[36]
LOC_Os07g48980 (OsNAS3)
[36]
qZN-7
[98]
8
LOC_Os06g39960
[7]
9
LOC_Os06g39960
[7]
10
qZN10.1
[98]
12
LOC_Os12g39860 (APRT)
[36]
Myb transcription factor
[55]
qGZn12.1
[105]
3
6
7
Identifying candidate genes regulating Fe and Zn concentrations
has been accelerated by GWASs. Many genomic regions controlling Fe and Zn have been identified by GWASs that use 192 rice
genotypes from landraces and several popular rice varieties, including Basmati [20]. GWASs have also detected genes relevant to
Fe and Zn by using a RIL population derived from crossing Swarna
and Madhukar with 168 lines [36]. A total of 529 diverse rice
genotypes and 6.4 million SNPs have been employed to identify
genes that control Fe and Zn contents [7]. Over 200 candidate
genes related to Fe and Zn concentrations were detected [98].
Genetic analysis of Fe and Zn concentrations in rice grains has
become the most important knowledge needed in order to develop
rice varieties with increased Fe and Zn contents, as well as the
most efficient and cost-effective approach for alleviating micronutrient deficiencies and human malnutrition. Many studies have
been using wild rice species as the donor parents to transfer genes
related to Fe and Zn concentrations from the wild rice species
to popular rice varieties. A total of six genes were detected on
chromosomes 6, 8, and 10 from the introgression line population
(96 lines) derived from a cross of wild rice Oryza rufipogon and an
ACADEMIA MOLECULAR BIOLOGY AND GENOMICS
elite Korean cultivar “Hwaseong”, grown in the two different locations [7]. The increased Fe and Zn contents came from the O. rufipogon alleles qFe10 and qZn10, located between the SSR markers RM1873 and RM25612. LOC_Os10g33960, as the candidate
gene, regulated the Fe and Zn concentrations and was identified
based on the gene expression data, whole-genome sequencing,
and spatiotemporal expression profile. According to the gene expression data, LOC_Os10g33960 and LOC_Os10g33930 showed
high levels of expression in the leaves, while LOC_Os10g33930
and LOC_Os10g33920 displayed high expression levels in roots,
and LOC_Os10g33960 had the highest gene expression level in
the rice seeds. Based on whole-genome sequencing, 17 genes out
of 26 genes showed at least one non-synonymous InDel or SNP in
the DNA coding sequences. According to the spatiotemporal expression profiles, LOC_Os10g33960 and LOC_Os10g33940 were
highly expressed in the rice endosperm, while LOC_Os10g33920
was expressed highly in the roots, and LOC_Os10g33960 showed
the highest expression in the leaves. Identification of these candidate genes from O. rufipogon could be crucial in developing rice
varieties with high Fe and Zn concentrations without impairing
other agronomic traits.
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About 31 QTL related to Fe and Zn concentrations were identified
from a cross between the wild rice O. rufipogon and indica cultivar
Teqing [55]. Four QTL associated with high Zn in rice grains also
detected by using backcrossed recombinant inbred lines derived
from crossing wild rice O. merionalis with O. sativa “Nipponbare” [98]. O. nivara, that contains Fe and Zn concentrations
three times higher than O. sativa Swarna, has also been used
to identify QTL responsible for Fe and Zn by developing two
BC2F3 populations derived from two different rice accessions of
O. nivara (IRGC81832 and IRGC81848) and Swarna, identifying
30 QTL [55]. Backcross inbred lines of O. longistaminata have
also been used to detect 33 QTL related to Fe and Zn concentrations in rice grains [19]. All of the QTL identified across different
environments have been determined to be stable and showed
important roles in regulating Fe and Zn accumulation [48].
QTL associated with Fe and Zn showed co-localization with the
QTL for Mn and Ca concentrations in grains. The co-localization
occurs because of the pleiotropic effect in a single gene, indicating
that the same mechanisms control the metabolisms of these minerals [51]. The absorption, transport, and storage of these minerals may be controlled by the same genomic regions and follow
similar pathways. In rice metabolism, the transport of Fe from
the rhizosphere to the grains is regulated by Fe chelators, such as
nicotianamine (NA), deoxymugineic acid (DMA), and citrate [52].
Similarly, NA and DMA play an important role in Zn uptake from
the rhizosphere [54]. Because of this similarity, the homeostasis
of Fe and Zn are connected [55]. This QTL co-localization will
accelerate the enhancing of Fe and Zn in the rice grains.
Nicotianamine synthase (NAS) family genes, including OsNAS1
and OsNAS2, are involved in increasing Fe and Zn concentrations in grains [66]. Over-expression of the NAS genes is directly
related to 2-to-3-fold increases in the level of these micronutrients [58, 59]. Meanwhile, low Zn concentration in grains correlated to the over-expression OsZIP8 [99]. Knock-down of OsMIT
on chromosome 3 resulted in the higher Fe concentration in rice
grains [20]. The CRISPR/Cas9 genome editing technique has also
been employed for improving Fe accumulation in grains [100–
103]. Furthermore, Fe and Zn uptake, transport, and accumulation in rice grains are regulated by key transporters such as HMA,
ZIP, YSL, and IRT [103]. These metal transporter genes regulate
Fe and Zn homeostasis, which is influenced by environmental
factors. Bioavailability of Fe and Zn significantly was increased
in grains by manipulation of transcription factors, ferric-chelate
reductase, and ferritin genes. Through over-expression of NAS
genes, ferritin genes, and metal transporter genes, Fe and Zn
bioavailability in grains reaches optimal concentrations.
Identification of new genes correlated to Fe and Zn and their
potential benefits for future rice breeding programs is important
to accelerating the optimization of micromineral content in rice
grains. Several important genes responsible for enhanced Fe and
Zn content in rice grains have been cloned, such as Oryza sativa
nicotianamine synthase (OsNAS), Oryza sativa yellow-like stripe
2 (OsYSL2), and Oryza sativa ferritin 2 (Osfer2). There are three
NAS genes that have been detected in rice: OsNAS1, OsNAS2,
and OsNAS3. Identification of these OsNAS genes was achieved
using a GWAS with a diverse indica rice population, using 5.2
million SNP markers. The OsNAS genes were stable in several
different growing environments. Over-expression of the OsNAS
gene for the biofortification of rice grains resulted in an up to
four-fold increase in Fe concentration and a two-fold increase
ACADEMIA MOLECULAR BIOLOGY AND GENOMICS
in Zn concentration [66]. Moreover, OsYSL2 is highly expressed
in phloem cells and involved in Fe translocation to grains [67].
The over-expression of Osfer2 resulted in an increased in Fe
accumulation up to 2.09-fold, and a Zn increase reaching 1.37fold [103]. Several genes associated with Fe and Zn have also been
identified using map-based cloning, such as Oryza sativa short
postembryonic root 1 (OsSPR1), nicotianamine aminotransferase
(NAAT1), SUS1, and GBSS1 [104]. In order to validate these candidate genes, further gene functionality studies would be useful for
rice breeding programs. Currently, a total of 300 biofortified rice
varieties have been released in more than 40 developing countries,
including Zn-biofortified rice with moderate concentrations of Zn,
suggesting the urgent need for further enhancement [105]. An
effective strategy for developing Fe-dense rice is using a transgenic
approach which over-expresses NAS genes and produces significant improvement in Fe levels.
A meta-analysis of 155 QTL associated with Fe and Zn from 26
studies has been conducted. The results showed that 11 metaquantitative trait loci (MQTL) were co-localized with the 20
known major genes involved in element uptake, transport, and
storage in rice grains [105]. These MQTL are distributed on
chromosomes 3, 4, 5, 6, 7, 11, and 12, and belong to ZIP family
genes (OsZIP5, OsZIP7, and OsZIP9), transcription factor of Fe
homeostasis (OsbHLH133), metal-regulated genes (OsNRAMP7),
metal transporter genes (OsFRDL1, OsMTP1, OsMTP7, and
OsFRO1), metallothioneins (OsMTId, OsMT1g, OsMT1a, and
OsMT1f ), GTPase for Fe homeostasis (OsRab6B2), and mugineic
acid family phytosiderophores (OsTOM1, OsTOM2, OsTOM3, OsNAS3, OsDMAS1, and OsSAMS1). These genes showed low expression in reproductive tissue but were highly expressed in roots,
leaf sheathes, and leaf blades. OsMT1g, OsMT1f, OsDMAS1, and
OsRab6B2 were highly expressed in rice endosperm. OsTOM3
was expressed in all of the stages in endosperm development,
including leaf sheathes, roots, and ovaries. Meanwhile, OsTOM1
was expressed in the root system and leaf sheaths. OsZIP5 was
expressed in vegetative and reproductive tissues, such as the stem,
leaf sheath, leaf blade, roots, ovary, pistil, palea, and lemma. Several genes were highly expressed in the stem, including OsMT1a,
OsZIP5, and OsFRDL1. Many genes were also high expressed in
the ovary, including OsTOM2, OsNAS3, OsDMAS1, and OsFRDL1.
Some of these genes were also strongly expressed in the anther
(OsMT1a, OsMT1g, OsFRO1, OsDMAS1, and OsRab6B2), pistil
(OsZIP5), and inflorescence (OsFRO1).
Co-expression analyses and in silico expression revealed that the
gene networking complex was involved in Fe and Zn homeostasis.
Identification of superior haplotypes is important for developing
rice with Fe and Zn biofortification as the most cost-effective
and the sustainable food program to address malnutrition. Along
with the increasing number of superior haplotypes, the Fe and Zn
concentrations of rice lines will increase. The superior haplotypes
with the highest Fe and Zn contents were OsMT1g (21.92 mg/kg),
OsNRAMP7 (21.17 mg/kg), OsDMAS1 (19.77 mg/kg), OsZIP7
(18.35 mg/kg), OsFRO1 (18.35 mg/kg), OsRab6B2 (18.13 mg/kg),
OsZIP9 (17.77 mg/kg), OsZIP5 (17.66 mg/kg), and OsMT1f (17.63
mg/kg). These superior haplotypes were distributed in different
rice sub-populations. The superior haplotypes for OsDMAS1 and
OsZIP9 were found in indica, whereas OsMT1g was identified in
temperate japonica, tropical japonica, and aromatic subgroups.
OsMT1f and OsZIP5 were identified in indica and aus, whereas
OsRab6B2, OsFRO1, and OsZIP7 were distributed in the aus
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subgroup. Meanwhile, OsNRAMP7 was detected in aus at a low
rate. Currently, a number of biofortified rice varieties with higher
Fe and Zn concentrations have been commercialized and made
available to communities in targeted countries in Asia based on
bio-efficacy studies showing that consuming biofortified rice was
effective in enhancing nutritional status.
6. Conclusions
Rice varieties with optimum Fe and Zn concentrations could be
developed by employing several approaches, such as conventional
rice breeding programs and genetic engineering methods that
provide promising solutions to combat deficiencies in Fe and
Zn. A wide variation in Fe and Zn concentrations in diverse rice
genotypes is potentially useful for their use as donor parents in the
rice breeding program in order to develop grains with higher Fe
and Zn accumulations. Understanding the molecular mechanisms
of Fe and Zn accumulation and also identifying genes associated
with Fe and Zn could be useful for genetic engineering approaches
by over-expressing or knocking-out relevant genes to accelerate
the development of higher Fe and Zn level in grains to address
micronutrient deficiencies in rice consumers. It is important to
integrate multi-omics studies, such as ionomics, transcriptomics,
and proteomics, to resolve contradictions in gene function across
growth stages of rice plants related to Fe and Zn accumulation
mechanisms. The important challenges in developing rice varieties with enhanced Fe and Zn concentrations are the stability of
these mineral concentrations under various environmental conditions, government regulations about transgenic rice varieties, and
public perception related to the genetically modified organisms
(GMOs). Therefore, interdisciplinary collaboration and advanced
technologies need to be developed. In the future, developing rice
varieties from white rice and pigmented rice with higher yield,
good grain quality, and high micronutrient concentrations will
become an important research area.
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article distributed under the terms and conditions of the Creative
Commons Attribution (CC BY) license (https://creativecommon
s.org/licenses/by/4.0/).
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