Next Generation Nutrition: Genomic and Molecular Breeding Innovations for Iron and Zinc Biofortification in Rice

doi:10.1016/j.rsci.2024.04.008

Abstract

Abstract:

Global efforts to address malnutrition and hidden hunger, particularly prevalent in low- and middle-income countries, have intensified, with a focus on enhancing the nutritional content of staple crops like rice. Despite serving as a staple for over half of the world’s population, rice falls short in meeting daily nutritional requirements, especially for iron (Fe) and zinc (Zn). Genetic resources, such as wild rice species and specific rice varieties, offer promising avenues for enhancing Fe and Zn content. Additionally, molecular breeding approaches have identified key genes and loci associated with Fe and Zn accumulation in rice grains. This review explores the genetic resources and molecular mechanisms underlying Fe and Zn accumulation in rice grains. The functional genomics involved in Fe uptake, transport, and distribution in rice plants have revealed key genes such as OsFRO1, OsIRT1, and OsNAS3. Similarly, genes associated with Zn uptake and translocation, including OsZIP11 and OsNRAMP1, have been identified. Transgenic approaches, leveraging transporter gene families and genome editing technologies, offer promising avenues for enhancing Fe and Zn content in rice grains. Moreover, strategies for reducing phytic acid (PA) content, a known inhibitor of mineral bioavailability, have been explored, including the identification of low-PA mutants and natural variants. The integration of genomic information, including whole-genome resequencing and pan-genome analyses, provides valuable insights into the genetic basis of micronutrient traits and facilitates targeted breeding efforts. Functional genomics studies have elucidated the molecular mechanisms underlying Fe uptake and translocation in rice. Furthermore, transgenic and genome editing techniques have shown promise in enhancing Fe and Zn content in rice grains through the manipulation of key transporter genes. Overall, the integration of multi-omics approaches holds significant promise for addressing global malnutrition and hidden hunger by enhancing the nutritional quality of rice, thereby contributing to improved food and nutritional security worldwide.

Key words: biofortification, grain quality, iron, phytic acid, rice, zinc

Kunhikrishnan Hemalatha Dhanyalakshmi, Reshma Mohan, Sasmita Behera, Uday Chand Jha, Debashis Moharana, Ahalya Behera, Sini Thomas, Preman Rejitha Soumya, Rameswar Prasad Sah, Radha Beena. Next Generation Nutrition: Genomic and Molecular Breeding Innovations for Iron and Zinc Biofortification in Rice[J]. Rice Science, 2024, 31(5): 526-544.

Figures/Tables 8

References 162

[1]	Adeyeye E I, Arogundade L A, Akintayo E T, Aisida O A, Alao P A. 2000. Calcium, zinc and phytate interrelationships in some foods of major consumption in Nigeria. Food Chem, 71(4): 435-441.
[2]	Agarwal S, Tripura Venkata V G N, Kotla A, Mangrauthia S K, Neelamraju S. 2014. Expression patterns of QTL based and other candidate genes in Madhukar × Swarna RILs with contrasting levels of iron and zinc in unpolished rice grains. Gene, 546(2): 430-436. DOI PMID
[3]	Al Hasan S M, Hassan M, Saha S, Islam M, Billah M, Islam S. 2016. Dietary phytate intake inhibits the bioavailability of iron and calcium in the diets of pregnant women in rural Bangladesh: A cross-sectional study. BMC Nutr, 2(1): 24.
[4]	Ali N, Paul S, Gayen D, Sarkar S N, Datta K, Datta S K. 2013a. Development of low phytate rice by RNAi mediated seed-specific silencing of inositol 1, 3, 4, 5, 6-pentakisphosphate 2-kinase gene (IPK1). PLoS One, 8(7): e68161.
[5]	Ali N, Paul S, Gayen D, Sarkar S N, Datta S K, Datta K. 2013b. RNAi mediated down regulation of myo-inositol-3-phosphate synthase to generate low phytate rice. Rice, 6(1): 12.
[6]	Amirabdollahian F, Ash R. 2010. An estimate of phytate intake and molar ratio of phytate to zinc in the diet of the people in the United Kingdom. Public Health Nutr, 13(9): 1380-1388. DOI PMID
[7]	Anilkumar C, Sah R P, Muhammed A T P, Sunitha N C, Behera S, Marndi B C, Sharma T R, Singh A K. 2022. Genomic selection in rice:Current status and future prospects. In: Elias A A, Goel S. Genomic Selection in Plants:A Guide for Breeders. Boca Raton, USA: CRC Press: 68-82.
[8]	Anuradha K, Agarwal S, Rao Y V, Rao K V, Viraktamath B C, Sarla N. 2012. Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished rice of Madhukar × Swarna RILs. Gene, 508(2): 233-240. DOI PMID
[9]	Anusha G, Rao D S, Jaldhani V, Beulah P, Neeraja C N, Gireesh C, Anantha M S, Suneetha K, Santhosha R, Prasad A S H, Sundaram R M, Madhav M S, Fiyaz A, Brajendra P, Tuti M D, Bhave M H V, Krishna K V R, Ali J, Subrahmanyam D, Senguttuvel P. 2021. Grain Fe and Zn content, heterosis, combining ability and its association with grain yield in irrigated and aerobic rice. Sci Rep, 11(1): 10579.
[10]	Aoyama T, Kobayashi T, Takahashi M, Nagasaka S, Usuda K, Kakei Y, Ishimaru Y, Nakanishi H, Mori S, Nishizawa N K. 2009. OsYSL18 is a rice iron(III)-deoxymugineic acid transporter specifically expressed in reproductive organs and phloem of lamina joints. Plant Mol Biol, 70(6): 681-692. DOI PMID
[11]	Aung M S, Kobayashi T, Masuda H, Nishizawa N K. 2018. Rice HRZ ubiquitin ligases are crucial for response to excess iron. Physiol Plant, 163(3): 282-296.
[12]	Aung M S, Masuda H, Nozoye T, Kobayashi T, Jeon J S, An G, Nishizawa N K. 2019. Nicotianamine synthesis by OsNAS3 is important for mitigating iron excess stress in rice. Front Plant Sci, 10: 660.
[13]	Aung M S, Masuda H. 2020. How does rice defend against excess iron? Physiological and molecular mechanisms. Front Plant Sci, 11: 1102.
[14]	Azharudheen M T P, Kumar A, Anilkumar C, Sah R P, Behera S, Marndi B C. 2022. Understanding natural genetic variation for grain phytic acid content and functional marker development for phytic acid-related genes in rice. BMC Plant Biol, 22(1): 446.
[15]	Bailey R L, West Jr K P, Black R E. 2015. The epidemiology of global micronutrient deficiencies. Ann Nutr Metab, 66(Suppl 2): 22-33.
[16]	Banerjee S, Sharma D, Verulkar S, Chandel G. 2010. Use of in silico and semiquantitative RT-PCR approaches to develop nutrient rich rice (Oryza sativa L.). Indian J Biotechnol, 9: 203-212.
[17]	Bashir K, Inoue H, Nagasaka S, Takahashi M, Nakanishi H, Mori S, Nishizawa N K. 2006. Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants. J Biol Chem, 281: 32395-32402. DOI PMID
[18]	Bashir K, Ishimaru Y, Shimo H, Kakei Y, Senoura T, Takahashi R, Sato Y, Sato Y, Uozumi N, Nakanishi H, Nishizawa N K. 2011. Rice phenolics efflux transporter 2 (PEZ2) plays an important role in solubilizing apoplasmic iron. Soil Sci Plant Nutr, 57(6): 803-812.
[19]	Bashir K, Takahashi R, Akhtar S, Ishimaru Y, Nakanishi H, Nishizawa N K. 2013. The knockdown of OsVIT2 and MIT affects iron localization in rice seed. Rice, 6(1): 31.
[20]	Bashir K, Nozoye T, Nagasaka S, Rasheed S, Miyauchi N, Seki M, Nakanishi H, Nishizawa N K. 2017. Paralogs and mutants show that one DMA synthase functions in iron homeostasis in rice. J Exp Bot, 68(7): 1785-1795. DOI PMID
[21]	Bhavya M S P, Manju RV, Viji M M, Roy S, Anith K N, Beena R. 2023. Impact of biofertilisers on iron homeostasis and grain quality in the rice variety Uma under elevated CO₂. Front Plant Sci, 14: 1144905.
[22]	Bollinedi H, Yadav A K, Vinod K K, Gopala Krishnan S, Bhowmick P K, Nagarajan M, Neeraja C N, Ellur R K, Singh A K. 2020. Genome-wide association study reveals novel marker-trait associations (MTAs) governing the localization of Fe and Zn in the rice grain. Front Genet, 11: 213. DOI PMID
[23]	Boonyaves K, Gruissem W, Bhullar N K. 2016. NOD promoter- controlled AtIRT1 expression functions synergistically with NAS and FERRITIN genes to increase iron in rice grains. Plant Mol Biol, 90(3): 207-215. DOI PMID
[24]	Bouis H E, Saltzman A. 2017. Improving nutrition through biofortification: A review of evidence from HarvestPlus, 2003 through 2016. Glob Food Sec, 12: 49-58.
[25]	Bregitzer P, Raboy V. 2006. Effects of four independent low- phytate mutations on barley agronomic performance. Crop Sci, 46(3): 1318-1322.
[26]	Budhlakoti N, Kushwaha A K, Rai A, Chaturvedi K K, Kumar A, Pradhan A K, Kumar U, Kumar R R, Juliana P, Mishra D C, Kumar S. 2022. Genomic selection: A tool for accelerating the efficiency of molecular breeding for development of climate- resilient crops. Front Genet, 13: 832153.
[27]	Calayugan M I C, Formantes A K, Amparado A, Descalsota-Empleo G I, Nha C T, Inabangan-Asilo M A, Swe Z M, Hernandez J E, Borromeo T H, Lalusin A G, Mendioro M S, Diaz M G Q, Viña C B D, Reinke R, Swamy B P M. 2020. Genetic analysis of agronomic traits and grain iron and zinc concentrations in a doubled haploid population of rice (Oryza sativa L.). Sci Rep, 10(1): 2283.
[28]	Champagne E T, Bett-Garber K L, Fitzgerald M A, Grimm C C, Lea J, Ohtsubo K, Jongdee S, Xie L H, Bassinello P Z, Resurreccion A, Ahmad R, Habibi F, Reinke R. 2010. Important sensory properties differentiating premium rice varieties. Rice, 3(4): 270-281.
[29]	Che J, Yamaji N, Ma J F. 2021. Role of a vacuolar iron transporter OsVIT2 in the distribution of iron to rice grains. New Phytol, 230(3): 1049-1062. DOI PMID
[30]	Cheng C H, Chung M C, Liu S M, Chen S K, Kao F Y, Lin S J, Hsiao S H, Hsing Y I C, Wu H P, Chen C S, Shaw J F, Wu J Z, Matsumoto T, Sasaki T, Chen H H, Chow T Y. 2005. A fine physical map of the Oryza sativa chromosome 5. Mol Genet Genomics, 274: 337-345.
[31]	Cheng L J, Wang F, Shou H X, Huang F L, Zheng L Q, He F, Li J H, Zhao F J, Ueno D, Ma J F, Wu P. 2007. Mutation in nicotianamine aminotransferase stimulated the Fe(II) acquisition system and led to iron accumulation in rice. Plant Physiol, 145(4): 1647-1657. DOI PMID
[32]	Descalsota G I L, Mallikarjuna Swamy B P, Zaw H, Inabangan- Asilo M A, Amparado A, Mauleon R, Chadha-Mohanty P, Arocena E C, Raghavan C, Leung H, Hernandez J E, Lalusin A B, Mendioro M S, Diaz M G Q, Reinke R. 2018. Genome-wide association mapping in a rice MAGIC plus population detects QTLs and genes useful for biofortification. Front Plant Sci, 9: 1347. DOI PMID
[33]	Development Initiatives. 2018. Global Nutrition Report:Shining a Light to Spur Action on Nutrition. Bristol, UK: Development Initiatives.
[34]	Dixit S, Singh U M, Abbai R, Ram T, Singh V K, Paul A, Virk P S, Kumar A. 2019. Identification of genomic region(s) responsible for high iron and zinc content in rice. Sci Rep, 9(1): 8136.
[35]	Egli I, Davidsson L, Zeder C, Walczyk T, Hurrell R. 2004. Dephytinization of a complementary food based on wheat and soy increases zinc, but not copper, apparent absorption in adults. J Nutr, 134(5): 1077-1080. DOI PMID
[36]	Garcia-Oliveira A L, Chander S, Ortiz R, Menkir A, Gedil M. 2018. Genetic basis and breeding perspectives of grain iron and zinc enrichment in cereals. Front Plant Sci, 9: 937. DOI PMID
[37]	Garutti M, Nevola G, Mazzeo R, Cucciniello L, Totaro F, Bertuzzi C A, Caccialanza R, Pedrazzoli P, Puglisi F. 2022. The impact of cereal grain composition on the health and disease outcomes. Front Nutr, 9: 888974.
[38]	Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F. 1999. Iron fortification of rice seed by the soybean ferritin gene. Nat Biotechnol, 17(3): 282-286. DOI PMID
[39]	Gregorio G B. 2002. Progress in breeding for trace minerals in staple crops. J Nutr, 132(3): 500S-502S. DOI PMID
[40]	Gross J, Stein R J, Fett-Neto A G, Fett J P. 2003. Iron homeostasis related genes in rice. Genet Mol Biol, 26(4): 477-497.
[41]	Hariprasanna K, Agte V, Elangovan M, Patil J V. 2014. Genetic variability for grain iron and zinc content in cultivars, breeding lines and selected germplasm accessions of Sorghum [Sorghum bicolor (L.) Moench]. Indian J Genet Plant Breed, 74(1): 42-49.
[42]	Hu S B, Du B B, Mu G M, Jiang Z C, Li H, Song Y X R, Zhang B L, Xia J X, Rouached H. Zheng L Q. 2024. The transcription factor OsbZIP48 governs rice responses to zinc deficiency. Plant Cell Environ, 47(5): 1526-1542.
[43]	Hurrell R F, Reddy M B, Juillerat M A, Cook J D. 2003. Degradation of phytic acid in cereal porridges improves iron absorption by human subjects. Am J Clin Nutr, 77(5): 1213-1219. DOI PMID
[44]	Inoue H, Higuchi K, Takahashi M, Nakanishi H, Mori S, Nishizawa N K. 2003. Three rice nicotianamine synthase genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long- distance transport of iron and differentially regulated by iron. Plant J, 36(3): 366-381.
[45]	Inoue H, Takahashi M, Kobayashi T, Suzuki M, Nakanishi H, Mori S, Nishizawa N K. 2008. Identification and localisation of the rice nicotianamine aminotransferase gene OsNAAT1 expression suggests the site of phytosiderophore synthesis in rice. Plant Mol Biol, 66(1): 193-203.
[46]	Inoue H, Kobayashi T, Nozoye T, Takahashi M, Kakei Y, Suzuki K, Nakazono M, Nakanishi H, Mori S, Nishizawa N K. 2009. Rice OsYSL15 is an iron-regulated iron(III)-deoxymugineic acid transporter expressed in the roots and is essential for iron uptake in early growth of the seedlings. J Biol Chem, 284(6): 3470-3479. DOI PMID
[47]	Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S, Matsuhashi S, Takahashi M, Nakanishi H, Mori S, Nishizawa N K. 2006. Rice plants take up iron as an Fe³⁺-phytosiderophore and as Fe²⁺. Plant J, 45(3): 335-346. DOI PMID
[48]	Ishimaru Y, Bashir K, Fujimoto M, An G, Itai R N, Tsutsumi N, Nakanishi H, Nishizawa N K. 2009. Rice-specific mitochondrial iron-regulated gene (MIR) plays an important role in iron homeostasis. Mol Plant, 2(5): 1059-1066. DOI PMID
[49]	Ishimaru Y, Masuda H, Bashir K, Inoue H, Tsukamoto T, Takahashi M, Nakanishi H, Aoki N, Hirose T, Ohsugi R, Nishizawa N K. 2010. Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese. Plant J, 62(3): 379-390.
[50]	Ishimaru Y, Kakei Y, Shimo H, Bashir K, Sato Y, Sato Y, Uozumi N, Nakanishi H, Nishizawa N K. 2011. A rice phenolic efflux transporter is essential for solubilizing precipitated apoplasmic iron in the plant stele. J Biol Chem, 286(28): 24649-24655. DOI PMID
[51]	Isidro J, Jannink J L, Akdemir D, Poland J, Heslot N, Sorrells M E. 2015. Training set optimization under population structure in genomic selection. Theor Appl Genet, 128(1): 145-158. DOI PMID
[52]	Islam M Z, Arifuzzaman M, Banik S, Hossain M A, Ferdous J, Khalequzzaman M, Pittendrigh B R, Tomita M, Ali M P. 2020. Mapping QTLs underpin nutrition components in aromatic rice germplasm. PLoS One, 15(6): e0234395.
[53]	Itai R N, Ogo Y, Kobayashi T, Nakanishi H, Nishizawa N K. 2013. Rice genes involved in phytosiderophore biosynthesis are synchronously regulated during the early stages of iron deficiency in roots. Rice, 6(1): 16.
[54]	Jatav H S, Singh S K, Singh Y, Kumar O. 2018. Biochar and sewage sludge application increases yield and micronutrient uptake in rice (Oryza sativa L.). Commun Soil Sci Plant Anal, 49(13): 1617-1628.
[55]	Jiang M, Liu Y, Liu Y H, Tan Y Y, Huang J Z, Shu Q Y. 2019. Mutation of inositol 1,3,4-trisphosphate 5/6-kinase6 impairs plant growth and phytic acid synthesis in rice. Plants, 8(5): 114.
[56]	Johnson A A T, Kyriacou B, Callahan D L, Carruthers L, Stangoulis J, Lombi E, Tester M. 2011. Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm. PLoS One, 6(9): e24476.
[57]	Juliano B O. 1992. Rice starch properties and grain quality. J Jpn Soc Starch Sci, 39(1): 11-21.
[58]	Kawakami Y, Bhullar N K. 2018. Molecular processes in iron and zinc homeostasis and their modulation for biofortification in rice. J Integr Plant Biol, 60(12): 1181-1198. DOI
[59]	Kies A K, De Jonge L H, Kemme P A, Jongbloed A W. 2006. Interaction between protein, phytate, and microbial phytase: In vitro studies. J Agric Food Chem, 54(5): 1753-1758.
[60]	Kim S I, Andaya C B, Goyal S S, Tai T H. 2008a. The rice OsLpa1 gene encodes a novel protein involved in phytic acid metabolism. Theor Appl Genet, 117(5): 769-779.
[61]	Kim S I, Andaya C B, Newman J W, Goyal S S, Tai T H. 2008b. Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK. Theor Appl Genet, 117(8): 1291-1301.
[62]	Kobayashi T, Suzuki M, Inoue H, Itai R N, Takahashi M, Nakanishi H, Mori S, Nishizawa N K. 2005. Expression of iron-acquisition- related genes in iron-deficient rice is co-ordinately induced by partially conserved iron-deficiency-responsive elements. J Exp Bot, 56: 1305-1316. DOI PMID
[63]	Kobayashi T, Ogo Y, Itai R N, Nakanishi H, Takahashi M, Mori S, Nishizawa N K. 2007. The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants. Proc Nat Acad Sci USA, 104: 19150-19155.
[64]	Kobayashi T, Ogo Y, Aung M S, Nozoye T, Itai R N, Nakanishi H, Yamakawa T, Nishizawa N K. 2010. The spatial expression and regulation of transcription factors IDEF1 and IDEF2. Ann Bot, 105(7): 1109-1117.
[65]	Kobayashi T, Nagasaka S, Senoura T, Itai R N, Nakanishi H, Nishizawa N K. 2013. Iron-binding haemerythrin RING ubiquitin ligases regulate plant iron responses and accumulation. Nat Commun, 4: 2792. DOI PMID
[66]	Kobayashi T, Nagano A J, Nishizawa N K. 2021. Iron deficiency- inducible peptide-coding genes OsIMA1 and OsIMA2 positively regulate a major pathway of iron uptake and translocation in rice. J Exp Bot, 72(6): 2196-2211. DOI PMID
[67]	Koike S, Inoue H, Mizuno D, Takahashi M, Nakanishi H, Mori S, Nishizawa N K. 2004. OsYSL2 is a rice metal-nicotianamine transporter that is regulated by iron and expressed in the phloem. Plant J, 39(3): 415-424. DOI PMID
[68]	Kumar A, Sahu C, Panda P A, Biswal M, Sah R P, Lal M K, Baig M J, Swain P, Behera L, Chattopadhyay K, Sharma S. 2020. Phytic acid content may affect starch digestibility and glycemic index value of rice (Oryza sativa L.). J Sci Food Agric, 100(4): 1598-1607.
[69]	Kumar A, Nayak S, Ngangkham U, Sah R P, Lal M K, Azharudheen T P, Behera S, Swain P, Behera L, Sharma S. 2021. A single nucleotide substitution in the SPDT transporter gene reduced phytic acid and increased mineral bioavailability from rice grain (Oryza sativa L.). J Food Biochem, 45(7): e13822.
[70]	Kumar A, Dash G K, Sahoo S K, Lal M K, Sahoo U, Sah R P, Ngangkham U, Kumar S, Baig M J, Sharma S, Lenka S K. 2023a. Phytic acid: A reservoir of phosphorus in seeds plays a dynamic role in plant and animal metabolism. Phytochem Rev, 22(5): 1281-1304.
[71]	Kumar A, Lal M K, Sahoo U, Sahoo S K, Sah R P, Tiwari R K, Kumar R, Sharma S. 2023b. Combinatorial effect of heat processing and phytic acid on mineral bioavailability in rice grain. Food Chem Adv, 2: 100232.
[72]	Kuwano M, Ohyama A, Tanaka Y, Mimura T, Takaiwa F, Yoshida K T. 2006. Molecular breeding for transgenic rice with low- phytic-acid phenotype through manipulating myo-inositol 3- phosphate synthase gene. Mol Breed, 18(3): 263-272.
[73]	Kuwano M, Mimura T, Takaiwa F, Yoshida K T. 2009. Generation of stable ‘low phytic acid’ transgenic rice through antisense repression of the 1d-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter. Plant Biotechnol J, 7(1): 96-105. DOI PMID
[74]	Larson S R, Rutger J N, Young K A, Raboy V. 2000. Isolation and genetic mapping of a non-lethal rice (Oryza sativa L.) low phytic acid 1 mutation. Crop Sci, 40(5): 1397-1405.
[75]	Lau W C P, Rafii M Y, Ismail M R, Puteh A, Latif M A, Ramli A. 2015. Review of functional markers for improving cooking, eating, and the nutritional qualities of rice. Front Plant Sci, 6: 832. DOI PMID
[76]	Lee S, An G. 2009. Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice. Plant Cell Environ, 32(4): 408-416.
[77]	Lee S, Jeon U S, Lee S J, Kim Y K, Persson D P, Husted S, Schjørring J K, Kakei Y, Masuda H, Nishizawa N K, An G. 2009. Iron fortification of rice seeds through activation of the nicotianamine synthase gene. Proc Natl Acad Sci USA, 106(51): 22014-22019. DOI PMID
[78]	Lee S, Ryoo N, Jeon J S, Guerinot M L, An G. 2012a. Activation of rice Yellow Stripe1-Like 16 (OsYSL16) enhances iron efficiency. Mol Cells, 33(2): 117-126.
[79]	Lee S, Kim Y S, Jeon U S, Kim Y K, Schjoerring J K, An G. 2012b. Activation of rice nicotianamine synthase 2 (OsNAS2) enhances iron availability for biofortification. Mol Cells, 33(3): 269-276.
[80]	Lestienne I, Icard-Vernière C, Mouquet C, Picq C, Trèche S. 2005. Effects of soaking whole cereal and legume seeds on iron, zinc and phytate contents. Food Chem, 89(3): 421-425.
[81]	Li C Y, Park D S, Won S R, Hong S K, Ham J K, Choi J K, Rhee H I. 2008. Food chemical properties of low-phytate rice cultivar, Sang-gol. J Cereal Sci, 47(2): 262-265.
[82]	Li C Y, Li Y, Xu P, Liang G. 2022. OsIRO3 negatively regulates Fe homeostasis by repressing the expression of OsIRO2. Plant J, 111(4): 966-978.
[83]	Li L, Ye L X, Kong Q H, Shou H X. 2019. A vacuolar membrane ferric-chelate reductase, OsFRO1, alleviates Fe toxicity in rice (Oryza sativa L.). Front Plant Sci, 10: 700.
[84]	Li Q, Chen L, Yang A. 2019. The molecular mechanisms underlying iron deficiency responses in rice. Int J Mol Sci, 21(1): 43.
[85]	Liang J F, Li Z G, Tsuji K, Nakano K, Robert Nout M J, Hamer R J. 2008. Milling characteristics and distribution of phytic acid and zinc in long-, medium- and short-grain rice. J Cereal Sci, 48(1): 83-91.
[86]	Liu F, Chang X J, Ye Y, Xie W B, Wu P, Lian X M. 2011. Comprehensive sequence and whole-life-cycle expression profile analysis of the phosphate transporter gene family in rice. Mol Plant, 4(6): 1105-1122. DOI PMID
[87]	Liu Q L, Xu X H, Ren X L, Fu H W, Wu D X, Shu Q Y. 2007. Generation and characterization of low phytic acid germplasm in rice (Oryza sativa L.). Theor Appl Genet, 114(5): 803-814.
[88]	Liu S L, Zou W L, Lu X, Bian J M, He H H, Chen J G, Ye G Y. 2021. Genome-wide association study using a multiparent advanced generation intercross (MAGIC) population identified QTLs and candidate genes to predict shoot and grain zinc contents in rice. Agriculture, 11(1): 70.
[89]	Ludwig Y, Slamet-Loedin I H. 2019. Genetic biofortification to enrich rice and wheat grain iron: From genes to product. Front Plant Sci, 10: 833. DOI PMID
[90]	Mahesh S, Pavithra G J, Parvathi M S, Rajashekara R, Shankar A G. 2015. Effect of processing on phytic acid content and nutrient availability in food grains. Int J Agric Sci, 5: 771-777.
[91]	Marounek M, Skřivan M, Rosero O, Rop O. 2010. Intestinal and total tract phytate digestibility and phytase activity in the digestive tract of hens fed a wheat-maize-soyabean diet. J Anim Feed Sci, 19(3): 430-439.
[92]	Masuda H, Suzuki M, Morikawa K C, Kobayashi T, Nakanishi H, Takahashi M, Saigusa M, Mori S, Nishizawa N K. 2008. Increase in iron and zinc concentrations in rice grains via the introduction of barley genes involved in phytosiderophore synthesis. Rice, 1(1): 100-108.
[93]	Masuda H, Usuda K, Kobayashi T, Ishimaru Y, Kakei Y, Takahashi M, Higuchi K, Nakanishi H, Mori S, Nishizawa N K. 2009. Over- expression of the barley nicotianamine synthase gene HvNAS1 increases iron and zinc concentrations in rice grains. Rice, 2(4): 155-166.
[94]	Mayer J E, Pfeiffer W H, Beyer P. 2008. Biofortified crops to alleviate micronutrient malnutrition. Curr Opin Plant Biol, 11(2): 166-170. DOI PMID
[95]	Meuwissen T H E, Hayes B J, Goddard M E. 2001. Prediction of total genetic value using genome-wide dense marker maps. Genetics, 157: 1819-1829. DOI PMID
[96]	Mitchikpe E C S, Dossa R A M, Ategbo E A D, van Raaij J M A, Hulshof P J M, Kok F J. 2008. The supply of bioavailable iron and zinc may be affected by phytate in Beninese children. J Food Compos Anal, 21(1): 17-25.
[97]	Mroz Z, Jongbloed A W, Kemme P A. 1994. Apparent digestibility and retention of nutrients bound to phytate complexes as influenced by microbial phytase and feeding regimen in pigs. J Anim Sci, 72(1): 126-132. PMID
[98]	Nishiyama R, Kato M, Nagata S, Yanagisawa S, Yoneyama T. 2012. Identification of Zn-nicotianamine and Fe-2ʹ-Deoxymugineic acid in the phloem sap from rice plants (Oryza sativa L.). Plant Cell Physiol, 53(2): 381-390. DOI PMID
[99]	Nozoye T, Itai R N, Nagasaka S, Takahashi M, Nakanishi H, Mori S, Nishizawa N K. 2004. Diurnal changes in the expression of genes that participate in phytosiderophore synthesis in rice. Soil Sci Plant Nutr, 50(7): 1125-1131.
[100]	Nozoye T, Nagasaka S, Kobayashi T, Takahashi M, Sato Y, Sato Y, Uozumi N, Nakanishi H, Nishizawa N K. 2011. Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants. J Biol Chem, 286(7): 5446-5454. DOI PMID
[101]	Nozoye T, Nagasaka S, Kobayashi T, Sato Y, Uozumi N, Nakanishi H, Nishizawa N K. 2015. The phytosiderophore efflux transporter TOM2 is involved in metal transport in rice. J Biol Chem, 290(46): 27688-27699. DOI PMID
[102]	Nozoye T, von Wirén N, Sato Y, Higashiyama T, Nakanishi H, Nishizawa N K. 2019. Characterization of the nicotianamine exporter ENA1 in rice. Front Plant Sci, 10: 502. DOI PMID
[103]	Ogo Y, Itai R N, Nakanishi H, Inoue H, Kobayashi T, Suzuki M, Takahashi M, Mori S, Nishizawa N K. 2006. Isolation and characterization of IRO2, a novel iron-regulated bHLH transcription factor in graminaceous plants. J Exp Bot, 57(11): 2867-2878. DOI PMID
[104]	Ogo Y, Itai R N, Nakanishi H, Kobayashi T, Takahashi M, Mori S, Nishizawa N K. 2007. The rice bHLH protein OsIRO2 is an essential regulator of the genes involved in Fe uptake under Fe-deficient conditions. Plant J, 51(3): 366-377. DOI PMID
[105]	Ogo Y, Kobayashi T, Nakanishi Itai R, Nakanishi H, Kakei Y, Takahashi M, Toki S, Mori S, Nishizawa N K. 2008. A novel NAC transcription factor, IDEF2, that recognizes the iron deficiency- responsive element 2 regulates the genes involved in iron homeostasis in plants. J Biol Chem, 283(19): 13407-13417.
[106]	Ogo Y, Itai R N, Kobayashi T, Aung M S, Nakanishi H, Nishizawa N K. 2011. OsIRO2 is responsible for iron utilization in rice and improves growth and yield in calcareous soil. Plant Mol Biol, 75(6): 593-605. DOI PMID
[107]	Ogo Y, Kakei Y, Itai R N, Kobayashi T, Nakanishi H, Takahashi H, Nakazono M, Nishizawa N K. 2014. Spatial transcriptomes of iron-deficient and cadmium-stressed rice. New Phytol, 201(3): 781-794. DOI PMID
[108]	Onogi A, Ideta O, Inoshita Y, Ebana K, Yoshioka T, Yamasaki M, Iwata H. 2015. Exploring the areas of applicability of whole- genome prediction methods for Asian rice (Oryza sativa L.). Theor Appl Genet, 128(1): 41-53. DOI PMID
[109]	Palanog A D, Nha C T, Descalsota-Empleo G I L, Calayugan M I, Swe Z M, Amparado A, Inabangan-Asilo M A, Hernandez J E, Sta Cruz P C, Borromeo T H, Lalusin A G, Mauleon R, McNally K L, Mallikarjuna Swamy B P. 2023. Molecular dissection of connected rice populations revealed important genomic regions for agronomic and biofortification traits. Front Plant Sci, 14: 1157507.
[110]	Paul S, Ali N, Gayen D, Datta S K, Datta K. 2012. Molecular breeding of Osfer2 gene to increase iron nutrition in rice grain. GM Crops Food, 3(4): 310-316.
[111]	Paul S, Ali N, Datta S K, Datta K. 2014. Development of an iron- enriched high-yieldings indica rice cultivar by introgression of a high-iron trait from transgenic iron-biofortified rice. Plant Foods Hum Nutr, 69(3): 203-208.
[112]	Peng H, Wang K, Chen Z, Cao Y H, Gao Q, Li Y, Li X X, Lu H W, Du H L, Lu M, Yang X, Liang C Z. 2020. MBKbase for rice: An integrated omics knowledgebase for molecular breeding in rice. Nucleic Acids Res, 48(D1): D1085-D1092.
[113]	Perera I, Seneweera S, Hirotsu N. 2018. Manipulating the phytic acid content of rice grain toward improving micronutrient bioavailability. Rice, 11(1): 4.
[114]	Pippal A, Bhusal N, Meena R K, Bishnoi M, Bhoyar P I, Jain R K. 2022. Identification of genomic locations associated with grain micronutrients (iron and zinc) in rice (Oryza sativa L.). Genet Resour Crop Evol, 69(1): 221-230.
[115]	Pontoppidan K, Pettersson D, Sandberg A S. 2007. Peniophora lycii phytase is stabile and degrades phytate and solubilises minerals in vitro during simulation of gastrointestinal digestion in the pig. J Sci Food Agric, 87(14): 2700-2708.
[116]	Pradhan S K, Pandit E, Pawar S, Naveenkumar R, Barik S R, Mohanty S P, Nayak D K, Ghritlahre S K, Rao D S, Reddy J N, Patnaik S S C. 2020. Linkage disequilibrium mapping for grain Fe and Zn enhancing QTLs useful for nutrient dense rice breeding. BMC Plant Biol, 20(1): 57.
[117]	Prom-u-Thai C, Rashid A, Ram H, Zou C Q, Guilherme L R G, Corguinha A P B, Guo S W, Kaur C, Naeem A, Yamuangmorn S, Ashraf M Y, Sohu V S, Zhang Y Q, Martins F A D, Jumrus S, Tutus Y, Yazici M A, Cakmak I. 2020. Simultaneous biofortification of rice with zinc, iodine, iron and selenium through foliar treatment of a micronutrient cocktail in five countries. Front Plant Sci, 11: 589835.
[118]	Raboy V. 2000. Low-phytic-acid grains. Food Nutr Bull, 21(4): 423-427.
[119]	Radha B, Sunitha N C, Sah R P, Azarudeen T P M, Krishna G K, Umesh D K, Thomas S, Anilkumar C, Upadhyay S, Kumar A, Manikanta C L N, Behera S, Marndi B C, Siddique K H M. 2023. Physiological and molecular implications of multiple abiotic stresses on yield and quality of rice. Front Plant Sci, 13: 996514.
[120]	Rakotondramanana M, Tanaka R, Pariasca-Tanaka J, Stangoulis J, Grenier C, Wissuwa M. 2022. Genomic prediction of zinc- biofortification potential in rice gene bank accessions. Theor Appl Genet, 135(7): 2265-2278.
[121]	Ramesh S A, Shin R, Eide D J, Schachtman D P. 2003. Differential metal selectivity and gene expression of two zinc transporters from rice. Plant Physiol, 133(1): 126-134. DOI PMID
[122]	Raza Q, Riaz A, Saher H, Bibi A, Raza M A, Ali S S, Sabar M. 2020. Grain Fe and Zn contents linked SSR markers based genetic diversity in rice. PLoS One, 15(9): e0239739.
[123]	Sanghamitra P, Sah R P, Bagchi T B, Sharma S G, Kumar A, Munda S, Sahu R K. 2018. Evaluation of variability and environmental stability of grain quality and agronomic parameters of pigmented rice (O. sativa L.). J Food Sci Technol, 55(3): 879-890.
[124]	Senguttuvel P, Padmavathi G, Jasmine C, Sanjeeva R D, Neeraja C N, Jaldhani V, Beulah P, Gobinath R, Aravind K J, Sai Prasad S V, Subba Rao L V, Hariprasad A S, Sruthi K, Shivani D, Sundaram R M, Govindaraj M. 2023. Rice biofortification: Breeding and genomic approaches for genetic enhancement of grain zinc and iron contents. Front Plant Sci, 14: 1138408.
[125]	Senoura T, Sakashita E, Kobayashi T, Takahashi M, Aung M S, Masuda H, Nakanishi H, Nishizawa N K. 2017. The iron-chelate transporter OsYSL9 plays a role in iron distribution in developing rice grains. Plant Mol Biol, 95(4/5): 375-387.
[126]	Singh V, Singh V, Singh S, Khanna R. 2020. Effect of zinc and silicon on growth and yield of aromatic rice (Oryza sativa) in north-western plains of India. J Rice Res Dev, 3(1): 82-86.
[127]	Stangoulis J C R, Huynh B L, Welch R M, Choi E Y, Graham R D. 2007. Quantitative trait loci for phytate in rice grain and their relationship with grain micronutrient content. Euphytica, 154(3): 289-294.
[128]	Stevens G A, Beal T, Mbuya M N N, Luo H Q, Neufeld L M, Group G M D R. 2022. Micronutrient deficiencies among preschool- aged children and women of reproductive age worldwide: A pooled analysis of individual-level data from population-representative surveys. Lancet Glob Health, 10(11): e1590-e1599.
[129]	Sun S B, Gu M, Cao Y, Huang X P, Zhang X, Ai P H, Zhao J N, Fan X R, Xu G H. 2012. A constitutive expressed phosphate transporter, OsPht1;1, modulates phosphate uptake and translocation in phosphate-replete rice. Plant Physiol, 159(4): 1571-1581. DOI PMID
[130]	Suzuki M, Tanaka K, Kuwano M, Yoshida K T. 2007. Expression pattern of inositol phosphate-related enzymes in rice (Oryza sativa L.): Implications for the phytic acid biosynthetic pathway. Gene, 405(1/2): 55-64.
[131]	Suzuki M, Morikawa K C, Nakanishi H, Takahashi M, Saigusa M, Mori S, Nishizawa N K. 2008. Transgenic rice lines that include barley genes have increased tolerance to low iron availability in a calcareous paddy soil. Soil Sci Plant Nutr, 54(1): 77-85.
[132]	Swamy B P M, Kaladhar K, Anuradha K, Batchu A K, Longvah T, Sarla N. 2018. QTL analysis for grain iron and zinc concentrations in two O. nivara derived backcross populations. Rice Sci, 25(4): 197-207. DOI
[133]	Swamy B P M, Marathi B, Ribeiro-Barros A I F, Calayugan M I C, Ricachenevsky F K. 2021. Iron biofortification in rice: An update on quantitative trait loci and candidate genes. Front Plant Sci, 12: 647341.
[134]	Tan L T, Zhu Y X, Fan,T, Peng C, Wang J R, Sun L, Chen, C Y. 2019. OsZIP7 functions in xylem loading in roots and inter- vascular transfer in nodes to deliver Zn/Cd to grain in rice. Biochem Biophys Res Commun, 512(1): 112-118.
[135]	Trijatmiko K R, Dueñas C, Tsakirpaloglou N, Torrizo L, Arines F M, Adeva C, Balindong J, Oliva N, Sapasap M V, Borrero J, Rey J, Francisco P, Nelson A, Nakanishi H, Lombi E, Tako E, Glahn R P, Stangoulis J, Chadha-Mohanty P, Johnson A A T, Tohme J, Barry G, Slamet-Loedin I H. 2016. Biofortified indica rice attains iron and zinc nutrition dietary targets in the field. Sci Rep, 6: 19792. DOI PMID
[136]	Tripathy S K. 2020. Genetic variation for micronutrients and study of genetic diversity in diverse germplasm of rice. J Crop Weed, 16(1): 101-109.
[137]	Visakh R L, Anand S, Arya S N, Sasmitha B, Jha U C, Sah R P, Beena R. 2024. Rice heat tolerance breeding: A comprehensive review and forward gaze. Rice Sci, 31(4): 375-400. DOI
[138]	Wang F, Itai R N, Nozoye T, Kobayashi T, Nishizawa N K, Nakanishi H. 2020. The bHLH protein OsIRO3 is critical for plant survival and iron (Fe) homeostasis in rice (Oryza sativa L.) under Fe- deficient conditions. Soil Sci Plant Nutr, 66(4): 579-592.
[139]	Wang J J, Meng X B, Hu X X, Sun T T, Li J Y, Wang K J, Yu H. 2019. xCas9 expands the scope of genome editing with reduced efficiency in rice. Plant Biotechnol J, 17(4): 709-711. DOI PMID
[140]	Wang Y D, Wang X, Wong Y S. 2013. Generation of selenium- enriched rice with enhanced grain yield, selenium content and bioavailability through fertilisation with selenite. Food Chem, 141(3): 2385-2393.
[141]	Wirth J, Poletti S, Aeschlimann B, Yakandawala N, Drosse B, Osorio S, Tohge T, Fernie A R, Günther D, Gruissem W, Sautter C. 2009. Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin. Plant Biotechnol J, 7(7): 631-644. DOI PMID
[142]	World Health Organization. 2002. World Health Report 2002:Reducing Risks Promoting Healthy Life. Geneva, Switzerland: World Health Organization.
[143]	Wu T Y, Gruissem W, Bhullar N K. 2019. Targeting intracellular transport combined with efficient uptake and storage significantly increases grain iron and zinc levels in rice. Plant Biotechnol J, 17(1): 9-20.
[144]	Xu Q, Zheng T Q, Hu X, Cheng L R, Xu J L, Shi Y M, Li Z K. 2015. Examining two sets of introgression lines in rice (Oryza sativa L.) reveals favorable alleles that improve grain Zn and Fe concentrations. PLoS One, 10(7): e0131846.
[145]	Xu Y J, Ying Y N, Ouyang S H, Duan X L, Sun H, Jiang S K, Sun S C, Bao J S. 2018. Factors affecting sensory quality of cooked japonica rice. Rice Sci, 25(6): 330-339.
[146]	Yamaji N, Xia J X, Mitani-Ueno N, Yokosho K, Ma J F. 2013. Preferential delivery of zinc to developing tissues in rice is mediated by P-type heavy metal ATPase OsHMA2. Plant physiol, 162(2): 927-939. DOI PMID
[147]	Yamaji N, Takemoto Y, Miyaji T, Mitani-Ueno N, Yoshida K T, Ma J F. 2017. Reducing phosphorus accumulation in rice grains with an impaired transporter in the node. Nature, 541: 92-95.
[148]	Yang A, Li Y S, Xu Y Y, Zhang W H. 2013. A receptor-like protein RMC is involved in regulation of iron acquisition in rice. J Exp Bot, 64(16): 5009-5020. DOI PMID
[149]	Yang M, Li Y T, Liu, Z H, Tian J J, Liang L M, Qiu Y, Wang G Y, Du Q Q, Cheng D, Cai H M, Shi L, Xu F S, Lian X M. 2020. A high activity zinc transporter OsZIP9 mediates zinc uptake in rice. Plant J, 103(5): 1695-1709.
[150]	Yang Z, Wu Y R, Li Y, Ling H Q, Chu C C. 2009. OsMT1a, a type 1 metallothionein, plays the pivotal role in zinc homeostasis and drought tolerance in rice. Plant Mol Biol, 70(1/2): 219-229.
[151]	Yokosho K, Yamaji N, Ueno D, Mitani N, Ma J F. 2009. OsFRDL1 is a citrate transporter required for efficient translocation of iron in rice. Plant Physiol, 149(1): 297-305. DOI PMID
[152]	Yokosho K, Yamaji N, Ma J F. 2016. OsFRDL1 expressed in nodes is required for distribution of iron to grains in rice. J Exp Bot, 67(18): 5485-5494. PMID
[153]	Yoshida K T, Wada T, Koyama H, Mizobuchi-Fukuoka R, Naito S. 1999. Temporal and spatial patterns of accumulation of the transcript of myo-inositol-1-phosphate synthase and phytin-containing particles during seed development in rice. Plant Physiol, 119(1): 65-72. PMID
[154]	Yuan F J, Zhao H J, Ren X L, Zhu S L, Fu X J, Shu Q Y. 2007. Generation and characterization of two novel low phytate mutants in soybean (Glycine max L. Merr). Theor Appl Genet, 115(7): 945-957.
[155]	Zhang C, Shinwari K I, Luo L, Zheng L Q. 2018. OsYSL13 is involved in iron distribution in rice. Int J Mol Sci, 19(11): 3537.
[156]	Zhang H M, Li Y, Yao X N, Liang G, Yu D Q. 2017. POSITIVE REGULATOR OF IRON HOMEOSTASIS1, OsPRI1, facilitates iron homeostasis. Plant Physiol, 175(1): 543-554. DOI PMID
[157]	Zhang H M, Li Y, Pu M N, Xu P, Liang G, Yu D Q. 2020. Oryza sativa POSITIVE REGULATOR OF IRON DEFICIENCY RESPONSE 2 (OsPRI2) and OsPRI3 are involved in the maintenance of Fe homeostasis. Plant Cell Environ, 43(1): 261-274.
[158]	Zhang M, Pinson S R M, Tarpley L, Huang X Y, Lahner B, Yakubova E, Baxter I, Guerinot M L, Salt D E. 2014. Mapping and validation of quantitative trait loci associated with concentrations of 16 elements in unmilled rice grain. Theor Appl Genet, 127(1): 137-165. PMID
[159]	Zhang Y, Xu Y H, Yi H Y, Gong J M. 2012. Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice. Plant J, 72(3): 400-410.
[160]	Zhao H J, Liu Q L, Fu H W, Xu X H, Wu D X, Shu Q Y. 2008. Effect of non-lethal low phytic acid mutations on grain yield and seed viability in rice. Field Crops Res, 108(3): 206-211.
[161]	Zhao Y N, Li C, Li H, Liu X S, Yang Z M. 2022. OsZIP11 is a trans-Golgi-residing transporter required for rice iron accumulation and development. Gene, 836: 146678.
[162]	Zheng L Q, Ying Y H, Wang L, Wang F, Whelan J, Shou H X. 2010. Identification of a novel iron regulated basic helix-loop- helix protein involved in Fe homeostasis in Oryza sativa. BMC Plant Biol, 10(1): 166.

QTL/Locus	Mapping population	Marker	Chromosome	Nutritional component	Reference
QTLFe9	Inbred	RM215 (SSR)	9	Fe	Islam et al, 2020
QTLZn4		RM551 (SSR)	4	Zn	Islam et al, 2020
qFe3.3	Inbred	RM7 (SSR)	3	Fe	Pradhan et al, 2020
qFe7.3		RM1132 (SSR)	7
qZn2.2		RM300 (SSR)	2	Zn
qZn8.3		RM80 (SSR)	8
qZn12.3			12	Zn
qFe1.1	BIL	RM562‒RM11943 (SSR)	1	Fe	Dixit et al, 2019
qFe1.2, qZn1.1		RM294A‒RM12276 (SSR)	1	Fe and Zn
qFe6.1, qZn6.1		RM8226‒RM400 (SSR)	6	Fe and Zn
qFe6.2, qZn6.2		RM400‒RM162 (SSR)	6	Fe and Zn
qFe2	F₅ RIL	RM555	2	Fe	Pippal et al, 2022
qFe12	F₅ RIL	RM12	12	Fe
qFe2	F₆ RIL	RM263	2	Fe
qFe12	F₆ RIL	RM327	12	Fe
qZn2.1	F₅ RIL	RM1092	2	Zn
qZn2.2	F₅ RIL	RM406	2	Zn
qZn2.1	F₆RIL	RM555	2	Zn
qZn2.2	F₆ RIL	RM263.2	2	Zn
qZn6	F₆ RIL	RM162	6	Zn
qZn10	F₆ RIL	RM474	10	Zn
qZn12.1	F₆ RIL	RM1080	12	Zn
qZn12.2	F₆ RIL	RM2734	12	Zn
qFe9.1	DH	SNP-9809545-9819278	9	Fe	Calayugan et al, 2020
qFe12.1	DH	SNP-12702072-12732307	12	Fe
qZn1.1	DH	SNP-id1008679-439764	1	Zn
qZn5.1	DH	SNP-4904312-4908650	5	Zn
qZn9.1	DH	SNP-9809545-9819278	9	Zn
qZn12.1	DH	SNP-c12p4887439-12172332	12	Zn

QTL/Locus	Mapping population	Marker	Chromosome	Nutritional component	Reference
QTLFe9	Inbred	RM215 (SSR)	9	Fe	Islam et al, 2020
QTLZn4		RM551 (SSR)	4	Zn	Islam et al, 2020
qFe3.3	Inbred	RM7 (SSR)	3	Fe	Pradhan et al, 2020
qFe7.3		RM1132 (SSR)	7
qZn2.2		RM300 (SSR)	2	Zn
qZn8.3		RM80 (SSR)	8
qZn12.3			12	Zn
qFe1.1	BIL	RM562‒RM11943 (SSR)	1	Fe	Dixit et al, 2019
qFe1.2, qZn1.1		RM294A‒RM12276 (SSR)	1	Fe and Zn
qFe6.1, qZn6.1		RM8226‒RM400 (SSR)	6	Fe and Zn
qFe6.2, qZn6.2		RM400‒RM162 (SSR)	6	Fe and Zn
qFe2	F₅ RIL	RM555	2	Fe	Pippal et al, 2022
qFe12	F₅ RIL	RM12	12	Fe
qFe2	F₆ RIL	RM263	2	Fe
qFe12	F₆ RIL	RM327	12	Fe
qZn2.1	F₅ RIL	RM1092	2	Zn
qZn2.2	F₅ RIL	RM406	2	Zn
qZn2.1	F₆RIL	RM555	2	Zn
qZn2.2	F₆ RIL	RM263.2	2	Zn
qZn6	F₆ RIL	RM162	6	Zn
qZn10	F₆ RIL	RM474	10	Zn
qZn12.1	F₆ RIL	RM1080	12	Zn
qZn12.2	F₆ RIL	RM2734	12	Zn
qFe9.1	DH	SNP-9809545-9819278	9	Fe	Calayugan et al, 2020
qFe12.1	DH	SNP-12702072-12732307	12	Fe
qZn1.1	DH	SNP-id1008679-439764	1	Zn
qZn5.1	DH	SNP-4904312-4908650	5	Zn
qZn9.1	DH	SNP-9809545-9819278	9	Zn
qZn12.1	DH	SNP-c12p4887439-12172332	12	Zn

Trait	Gene	Additional information	Reference
Fe	OsFER1, OsFER2	Indica variety genome from genomic BLAST	Gross et al, 2003
	OsYSL1, OsMTP1	F₆ recombinant inbred lines derived from the cross of Madhukar and Swarna	Anuradha et al, 2012
Zn	OsARD2, OsIRT1, OsNAS1, OsNAS2	F₆ recombinant inbred lines derived from the cross of Madhukar and Swarna	Anuradha et al, 2012
Fe and Zn	OsNAS gene family	Transformation of Nipponbare	Johnson et al, 2011
Fe and Zn	OsNAS3, OsNRAMP1, heavy metal Fe transport, APRT	F₆ recombinant inbred lines derived from the cross of Madhukar and Swarna	Anuradha et al, 2012

Trait	Gene	Additional information	Reference
Fe	OsFER1, OsFER2	Indica variety genome from genomic BLAST	Gross et al, 2003
	OsYSL1, OsMTP1	F₆ recombinant inbred lines derived from the cross of Madhukar and Swarna	Anuradha et al, 2012
Zn	OsARD2, OsIRT1, OsNAS1, OsNAS2	F₆ recombinant inbred lines derived from the cross of Madhukar and Swarna	Anuradha et al, 2012
Fe and Zn	OsNAS gene family	Transformation of Nipponbare	Johnson et al, 2011
Fe and Zn	OsNAS3, OsNRAMP1, heavy metal Fe transport, APRT	F₆ recombinant inbred lines derived from the cross of Madhukar and Swarna	Anuradha et al, 2012

Gene	Localization/Expression pattern/Function	Reference
Genes associated with Strategy I
OsIRT1, OsIRT2	Fe²⁺ transporters expressed in roots, leaves, and stems associated with Fe distribution and partitioning; Induced under low Fe conditions	Ishimaru et al, 2006; Lee and An, 2009
OsFRO1, OsFRO2	OsFRO1 is localized in the vacuolar membrane to reduce Fe³⁺ to Fe²⁺ and functions to maintain Fe homeostasis between vacuole and cytoplasm; OsFRO2 is a homolog of the Arabidopsis ferric chelate reductase (FRO) responsible for the reduction of Fe	Ishimaru et al, 2009; Li L et al, 2019
OsPEZ1	Localized in the stele of roots and associated with the transport of phenolics such as protocatechuic acid for the solubilization of apoplasmic Fe in the root xylem; Facilitate long-distance transport of Fe	Ishimaru et al, 2011
OsPEZ2	Expressed in the plasma membrane of root epidermis/exodermis; Solubilization of apoplasmic Fe; Secretion of protocatechuic acid and caffeic acid into the rhizosphere	Bashir et al, 2011
OsFRDL1	Plasma membrane-localized transporter for citrate; Expressed in most cells of enlarged vascular bundles, diffuse vascular bundles, and the interjacent parenchyma cell bridges of uppermost node I, as well as vascular tissues of the leaf blade, leaf sheath, peduncle, rachis, husk, and stamen; Essential for the distribution of Fe in the panicles through solubilizing Fe deposited in the apoplastic part of nodes in rice	Yokosho et al, 2016
Genes associated with Strategy II
OsNAS1, OsNAS2, OsNAS3	Biosynthesis of mugineic acid and long-distance transport of Fe; The expression pattern and tissue vary depending upon Fe sufficient and deficient conditions; The expression of OsNAS1 and OsNAS2 transcripts are markedly elevated in both roots and leaves in response to Fe deficiency, while the expression of OsNAS3 is induced in roots but suppressed in leaves	Inoue et al, 2003
OsYSL2	Transport of Fe²⁺-NA and Mn²⁺-NA; Predominantly expressed in phloem cells and associated with the phloem transport of Fe and Mn including its translocation to the grain	Koike et al, 2004
OsNAAT1	Mugineic acid biosynthesis in Fe-deficient roots; Expression induced in companion cells of Fe-sufficient shoots and Fe-deficient leaves indicates roles in Fe acquisition and long-distance transport via phloem	Inoue et al, 2008
OsYSL18	Fe³⁺-DMA transporter and is expressed in flowers and the phloem of lamina joints, indicating its role in the translocation of Fe in reproductive organs and phloem joints	Aoyama et al, 2009
YSL15	Fe³⁺-DMA transporter responsible for uptake of Fe from the rhizosphere and phloem transport; Expressed in root epidermis under Fe deficient condition, and in seeds	Inoue et al, 2009; Lee et al, 2009
OsNAS3	Biosynthesis of mugineic acid; 7-fold increase in bound Fe in seeds; Increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities; Expressed in roots, especially in vascular bundle, epidermis, exodermis, stem, and old leaf tissues under Fe excess condition; Mitigation of excess Fe	Lee et al, 2009; Aung and Masuda, 2020
OsTOM1	DMA efflux transporter in roots; Higher expression in roots, leaf sheaths, and leaves under Fe deficient conditions	Nozoye et al, 2011
OsYSL16	Constitutively expressed in the plasma membranes and is highly expressed in the vascular tissues of the root, leaf, and spikelet, and in leaf mesophyll cells and associated with the Fe-homeostasis within plants	Lee et al, 2012a
OsTOM2	Expressed in tissues involved in metal translocation; Strong expression in developing tissues during seed maturation and germination; Localized in the cell membrane; Transports DMA to cell exterior	Nozoye et al, 2015
OsDMAS1	Expression is restricted to cells participating in long distance transport and is highly up-regulated in the entire root under Fe-deficient conditions; In shoot tissue, its promoter drives expression in vascular bundles specifically under Fe-deficient conditions	Bashir et al, 2017
OsYSL9	Transport of Fe as both Fe²⁺-NA and Fe³⁺-DMA; Localized mainly in the plasma membrane; Induced in roots in response to Fe deficiency, and in the scutellum of the embryo and in endosperm cells surrounding the embryo during the grain filling stage	Senoura et al, 2017
OsYSL13	Highly expressed in leaves and leaf blades; Fe deficiency induces expression in both leaves and roots; Associated with Fe distribution within the plant and also to seeds	Zhang et al, 2018
OsENA1	ENA is primarily expressed in root epidermis and cortex, while in shoots, the expression was induced near the root-to-shoot junction and absent in leaves; Localized in the plasma membrane and vesicular structures; Cellular trafficking between plasma membrane and intracellular compartments, and to cell exterior; Probable role in Fe homeostasis	Nozoye et al, 2019
Transcriptional regulators
IDEF1	Strongly expressed in pollen, ovary, aleurone layer, and embryo; Positively regulates Fe utilization- related genes under Fe deficiency; IDEF1 dependent induction of late embryogenesis abundant proteins like Osem, indicates the function of seed maturation-related genes in Fe-deficient vegetative organs; Binds to Fe and other divalent cations to sense the Fe status	Kobayashi et al, 2007, 2010,
OsIRO2	Localized in the cytoplasm; Nuclear accumulation under Fe limited conditions; Positively regulates the expression of genes associated with the Strategy II Fe uptake; Positively regulated by IDEF1; Regulation of DMA uptake and translocation of Fe to grain during seed maturation	Ogo et al, 2007
IDEF2	NAC like transcription factor; Constitutively present in roots and leaves; Expressed in pollen, ovary, and the dorsal vascular region of the endosperm; Associated with Fe homeostasis	Ogo et al, 2008; Kobayashi et al, 2010
OsIRO3	Negative regulator of Fe uptake and translocation; Directly represses the expression of OsIRO2 by interacting with OsPRI1 and OsPRI2; Repressor function due to the presence of an EAR motif	Zheng et al, 2010; Li et al, 2022
OsbHLH33	Negative regulator of Fe uptake and translocation	Wang et al, 2013
OsPRI1/OsbHLH060	Binds to the promoters of OsIRO2 and OsIRO3 and positively regulate their expression; Ubiquitously expressed in roots and shoots; Required for Fe translocation from roots to shoots; A target of OsHRZ1, degraded by 26S proteosome pathway	Zhang et al, 2017
OsbHLH156	Regulator of Strategy II; Expressed in the roots, and induced by Fe deficiency. Required for the nuclear localisation of IRO2	Wang et al, 2020
OsPRI2/OsbHLH058, OsPRI3/OsbHLH059	Paralogs of OsPRI1; Bind to the promoters of OsIRO2 and OsIRO3; Induce the expression of OsYSL2 by associating with its promoter; Directly regulated by OsHRZ1 via 26S proteasomal degradation	Zhang et al, 2020
Subcellular sequestration
OsVIT1, OsVIT2	OsVIT1 expressed in flag leaf blade and sheath while OsVIT2 expressed in sheath; Fe translocation between source and sink organs. OsVIT2 is involved in the distribution of Fe to the grains through sequestering Fe into vacuoles in mestome sheaths, nodes, and aleurone layers	Zhang et al, 2012 Che et al, 2021
Other genes
OsNAS1, OsNAS2, OsNAAT1, OsDMAS1	Increased expression in both roots and shoots by Fe deficiency	Inoue et al, 2003, 2008; Bashir et al, 2006
OsZIP1, OsZIP3,	Induced by Zn deficiency; Expressed in the vascular bundles in shoots, vascular bundles, and epidermal cells in roots	Ramesh et al, 2003; Ishimaru et al, 2006
OsZIP4	Identified in rice plants, induced by Zn deficiency, expressed in the meristem of Zn-deficient roots and shoots, and also in vascular bundles of the roots and shoots	Ramesh et al, 2003; Ishimaru et al, 2006
RT1, OsIRT2	Involved in Zn uptake and transport	Ishimaru et al, 2006
MIR	Induced in roots and shoots in response to Fe-deficiency; Localized in mitochondria; Associated with Fe homeostasis	Ishimaru et al, 2009
OsZIP5	Involved in Zn uptake and transport	Yang et al, 2009
TOM1	Involved in DMA secretion, expressed in all root cells	Nozoye et al, 2011
HORZ1	Protein with hemerythrin domain; Antagonistic to HRZ functions	Kobayashi et al, 2013
OsHRZ1	Fe binding sensor; Negative regulation of Fe acquisition under Fe-sufficient conditions; 26S proteasome mediated degradation of OsPRI1, OsPRI2, and OsPRI3 as potential Fe sensors, playing a negative role in Fe homeostasis	Kobayashi et al, 2013; Aung et al, 2018
OsHRZ2	Fe binding sensor; Negative regulation of Fe acquisition under Fe-sufficient conditions	Kobayashi et al, 2013; Aung et al, 2018
OsHMA2	Delivery of Zn to growing tissues; Localized to the pericycle of the roots and at the phloem of enlarged and diffuse vascular bundles in the nodes	Yamaji et al, 2013
OsRMC	Receptor like protein; Involved in the regulation of Fe acquisition	Yang et al, 2013
OsNRAMP1	Associated with Fe uptake in ferrous form	Ogo et al, 2014
OsIBP1.1, IBP1.2	Bowman-Birk trypsin inhibitors; Interact with IDEF1 and prevent its degradation mediated by 26S proteasome-mediated pathway	Zhang et al, 2014; Li Q et al, 2019
OsZIP7	Involved in Zn transport to growing tissues; Expressed in parenchyma cells of vascular bundles in roots and nodes	Tan et al, 2019
OsZIP9	Uptake of Zn into roots; Expressed in the epidermal and exodermal cells of lateral roots; Localized to the plasma membrane	Yang et al, 2020
OsZIP11	Involved in Fe accumulation; Knocking out OsZIP11 by CRISPR/Cas9 approach lowered Fe accumulation in brown rice, besides reducing plant height and biomass, and causing chlorosis and over-accumulation of malondialdehyde in rice plantlets	Zhao et al, 2022
OsbZIP48	Involved in the regulation of the expression of Zn transporters OsZIP4 and OsZIP8	Hu et al, 2024