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. 2025 Aug 2;45(8):66. doi: 10.1007/s11032-025-01575-z

Navigating the genetic basis of cadmium accumulation: a comprehensive analysis of low-cadmium accumulation rice

Deyi Shao 2,#, Lixin Yin 2,#, Jian Zhao 2,#, Zhengliang Luo 1, Weiguo Li 2, Yongka Wang 2, Xiangjie Liu 2, Bingchuan Tian 2,✉, Xiaobo Long 2,✉, Hexing Yin 2,✉, Kun Zhou 1,✉
PMCID: PMC12317945  PMID: 40755490

Abstract

The presence of cadmium (Cd) in rice poses a significant health risk to consumers, highlighting the urgency of breeding rice varieties with low Cd accumulation. To identify genetic resources and potential genes for developing such rice varieties, a comprehensive genome-wide association study (GWAS) was conducted on 158 rice varieties, which tested between 2021 and 2023 in low cadmium accumulation testing framework, to identify candidate genes associated with cadmium content in brown rice. Based on their parental origin and genetic population structure analysis, we categorized these 158 varieties into four subgroups: Luohong, lcd1, intermediate and early indica series. Specifically, the four subgroups of low cadmium varieties were breeded based on OsNramp5 mutants Luohong 3A/4A, lcd1, Lian 1S and Shaoxiang 100, respectively. GWAS analysis identified sixteen loci significantly associated with cadmium content, twelve of which showed consistent associations across multiple environments, these loci were mapped to chromosomes 1, 2, 5, 7, 11, and 12, suggesting their potential for further fine mapping and functional validation. Through gene function annotation analysis, candidate genes related to cadmium content in these loci were identified, including Os05 g0382200, Os07 g0232800 (OsZIP8), Os07 g0232900 (OsHMA3), Os07 g0257200 (OsNramp5), Os07 g0258400 (OsNramp1), Os12 g0512100, Os12 g0512700, and Os12 g0514000. These genes are implicated in the absorption, transport, and accumulation of heavy metals, particularly cadmium. Haplotype analysis of key genes OsZIP8, OsHMA3, OsNramp5, and OsNramp1 identified specific low-cadmium dominant haplotypes. Notably, OsHMA3-Hap2 (GC), OsNramp5-Hap1 (DEL), and OsNramp1-Hap1 (DEL) were associated with Luohong-origin varieties, while OsHMA3-Hap1 (AC), OsNramp5-Hap2 (AA), and OsNramp1-Hap2 (GGG) were linked to lcd1-origin varieties. Overall, this study illustrated the genetic basis for breeding low-cadmium rice varieties and provided candidate loci to develop molecular markers to enhance food safety through reduced heavy metal content.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11032-025-01575-z.

Keywords: Low cadmium accumulation varieties, Quantitative trait locus, Cadmium content, Genome-wide association study, Haplotype, Candidate gene

Introduction

Rice (Oryza sativa L.) is a keystone crop in China, with the national production reaching nearly 207 million metric tons, which is about 28% of the global total (FAO 2023; NBSC 2023). This crop is vital for China’s food security and plays a significant role in the international rice market. Despite a decline in the cultivated area, China has maintained rice self-sufficiency by raising yield of over 50% since the 1980 s (Grassini et al 2013; NBSC 2023). The “cadmium rice” incidents in 2013 in Hunan, Guangdong, and Jiangxi provinces have raised public awareness of rice safety, as these incidents exposed a significant hazard to a staple food in China (Wang et al 2019a). The National Soil Pollution Survey Bulletin of 2014 reported that 16.1% of the country’s soil had high levels of heavy metals, with cadmium contamination being the most frequent at a rate of 7.0%, and this rate was observed to be increasing from north to south (Ministry of Environmental Protection 2014). This trend underlines the critical need to address soil contamination to preserve public health and agricultural productivity (Rai et al 2019; Song et al 2015).

Rice shows a substantially higher rate of cadmium influx through its roots in comparison to other cereals such as wheat (Triticum aestivum L.) and corn (Zea mays L.), with uptake rates being approximately 2.2 to 6.5 times higher (Sui et al 2018). This superior absorption and its subsequent distribution to grains not only marks a significant conduit into the food chain but also underlines the potential for Cd accumulation in human body through dietary, thus posing considerable health risks (Song et al 2015; Wang et al 2014). The bioaccumulation of Cd in rice thereby emphasizes the urgency for reducing Cd level to ensure food safety and protect public health (Xie et al 2015; Xue et al 2014; Aziz et al 2015). As such, the cultivation of low cadmium rice varieties is in harmony with sustainable agricultural goals by reducing the environmental impact of rice production. It not only defends consumers against the harmful effects of long-term exposure to this contaminant but also ensures the safety and quality of the food supply. The selection of rice plants with low cadmium accumulation enables the cultivation of this crop in contaminated soil, thereby optimizing land use and preserving arable land for future generations.

Rice varieties with cadmium levels below 0.2 mg/kg in brown rice, grown in soils with a total cadmium content not exceeding 1.5 mg/kg, are classified as low cadmium accumulation varieties according to the local standard DB43/T 2599–2023 of Hunan Province. Following the 2013 “cadmium rice incident”, the Hunan Academy of Agricultural Sciences and collaborators initiated extensive screening for low Cd accumulation rice varieties and developed an integrated control technology known as VIP + n in 2014 (Wang et al 2016; Tang et al 2020). This technology includes planting low Cd accumulation rice varieties (V), optimizing water management (I), adjusting soil pH (P), and applying foliar barrier agents (n). Despite being effective emergency measures to mitigate cadmium pollution in rice, these methods have limitations in terms of simplicity, sustainability, ease of operation, and environmental impact. To address cadmium contamination in medium to heavily polluted fields, breeding rice varieties with low cadmium accumulation is crucial. Various breeding methods, including molecular marker-assisted selection (MAS), mutation breeding, and gene editing, have been employed (Zhang et al 2019b; Wang et al 2019b; Shi et al 2019; Lin et al 2012; Chen et al 2018a). Notably, “Luohong 3 A” and “Luohong 4 A” had been identified as exceptional low-cadmium materials from a global pool of 275 rice genetic resources (Wang et al 2021). Furthermore, a 408 kb genomic deletion was discovered in these materials, which includes the OsNramp5 and OsNramp1 genes, potentially reducing cadmium uptake in rice grains (Lv et al 2020). Recent research resulted in the development of low-cadmium rice varieties “Lianliangyou 1” and “Shaoxiang 100” through heavy ion mutagenesis and M1TDS technology, which showed no significant yield or quality differences from their parent strains (Shao et al 2022; Mao et al 2023; Li et al. 2024). Meanwhile, researchers at the China National Rice Research Institute created a non-synonymous mutant “lcd1” by mutating the OsNramp5 gene, which significantly reduced cadmium uptake without impacting other agronomic traits (Cao et al 2019). Hunan Jinjian Seed Industry Technology Co., Ltd., in partnership with the China National Rice Research Institute, has developed a series of low Cd accumulation rice varieties, including “Zhonganzao 7”, “Anyou 2”, and “Hualiangyou Q6”, based on gene lcd1 (Wang 2023). The CRISPR/Cas9 gene-editing technique was also used to create an improved version of the hybrid rice variety “Longliangyouhuazhan” with very low cadmium content, reducing it by over 98% without affecting yield (Tang et al 2017). To expedite the approval and dissemination of low Cd accumulation rice varieties, the Ministry of Agriculture and Rural Affairs authorized the Hunan Academy of Agricultural Sciences in 2021 to establish a testing channel for special rice varieties with low cadmium accumulation. By 2023, the field test has expanded to 65 sites across 5 ecological groups, covering all major rice-growing regions in southern China. Among the 158 varieties tested, “Xizi 3” stands out as the first nationally approved low Cd accumulation rice variety. However, more efforts focusing on the genetic mechanisms of Cd resistance is still needed for further development and improvement of low Cd rice varieties.

The process by which rice absorbs and accumulates cadmium is intricate and governed by a network of genes. This process varies significantly across different rice varieties, highlighting the genetic diversity in cadmium management (Clemens et al 2013; Ding et al 2019). The journey of cadmium in rice can be broken down into three stages: absorption by root cells from the soil, translocation from roots to shoots, and finally, transport from shoots to grains through the phloem (Hu et al 2021). In recent years, leveraging a rich collection of rice varieties with varying cadmium phenotypes, researchers have identified and cloned several genes associated with cadmium accumulation. These genes are categorized into three functional groups: transporters, chelators, and regulatory genes. The NRAMP (Natural Resistance-Associated Macrophage Protein) family, a group of transmembrane transporters, plays a pivotal role in cadmium uptake and transport. Notably, OsNramp5, OsNramp1, and OsNramp2 are key players, with OsNramp5 being particularly significant in cadmium uptake by rice roots (Ishimaru et al 2012; Sasaki et al 2012; Takahashi et al 2011; Zhao et al 2018). Mutations in OsNramp5 can effectively block cadmium uptake, making it a prime target for breeding low-cadmium rice varieties (Takahashi et al 2014; Tang et al 2017; Wang et al 2019b). The P-type heavy metal ATPase (HMA) family, another set of transmembrane transporters, is crucial for cadmium transport. OsHMA3 and OsHMA2 are particularly important, with OsHMA3 being the first identified cadmium transporter in the tonoplast membrane of rice root cells (Miyadate et al 2011; Ueno et al 2010). It sequesters cadmium into root cell vacuoles, thereby reducing the translocation to above-ground tissues and resulting in lower cadmium levels in the grain, making it a promising candidate for low-cadmium breeding (Tezuka et al 2010; Satoh-Nagasawa et al 2012).

In addition, a bundle of transporter genes implicated in cadmium transport include OsLCT1 (Uraguchi et al 2011), OsZIP1 (Liu et al 2019), OsZIP3 (Ramesh et al 2003), OsZIP6 (Kavitha et al 2015), OsZIP7 (Tan et al 2019), OsIRT1 (Nakanishi et al 2006), OsIRT2 (Nakanishi et al 2006), OsMTP1/OZT1 (Lan et al 2013), OsABCC9 (Yang et al 2021), OsABCG36 (Fu et al 2019), OsCCX2 (Hao et al 2018), and OsCd1 (Yan et al 2019). Cadmium ions can also form chelates with proteins rich in cysteine, reducing their toxicity and cellular damage, including CAL1 (cysteine-rich defensin protein) (Luo et al 2018), OsMTI-1b (metallothionein) (Malekzadeh and Shahpiri 2017), OsCDT1 (cysteine-rich protein) (Kuramata et al 2009), OsPCS1, OsPCS2 (plant complexin) (Das et al 2017). Regulatory genes such as OsLCD (Shimo et al 2011), OsHB4 (Ding et al 2018), and OsHIR1 (Lim et al 2014) are involved in cadmium tolerance and accumulation. OsLCD, a dominant gene expressed in vascular bundle tissues, encodes a soluble protein that plays a role in response to cadmium stress and its transport and accumulation (Shimo et al 2011). Research into the molecular mechanisms behind cadmium uptake, transport, and accumulation in rice has provided a theoretical foundation for developing rice varieties with lower Cd content. Despite this, our understanding of the specific molecular mechanisms is still incomplete, which poses a challenge for the urgent need to breed rice with minimal cadmium accumulation. Therefore, identifying new Cd-related genes and loci, as well as systematically evaluating the molecular genetic characteristics of existing low Cd accumulation rice varieties, is crucial for both breeding and cultivating of new rice varieties.

In this study, we built the low cadmium accumulation testing framework by utilizing rice varieties tested between 2021 and 2023. Our investigations involved accurate identification of cadmium phenotypes through pool planting in regions along the Yangtze River, where cadmium levels in rice often exceed safety standards. The goal was to collect data on how these varieties perform under different cadmium background conditions. By resequencing the genomes of the tested rice varieties, the study employed Genome-Wide Association Studies (GWAS) to uncover loci associated with rice cadmium content and to identify potential candidate genes. This study aimed to reveal the genetic characteristics of various types of low Cd accumulation rice, thereby laying the groundwork for further research into the genetic mechanisms of cadmium accumulation and for the advancement of low Cd accumulation rice varieties.

Materials and methods

Experimental materials and cadmium phenotypic data

The experimental materials comprised 158 rice varieties from the 2021–2023 low-cadmium accumulation tests, provided by the Rice Research Institute of Hunan Academy of Agricultural Sciences. The collection included 23, 37, and 98 varieties tested in 2021, 2022, and 2023, respectively. Cadmium phenotypic data were collected through a combination of pool and field experiments, detailed in Tables S2 and S3. Pool-planting adhered to the local standard DB 43/T 2586–2023 of Hunan Province, with three replications at Hunan Biological Research Institute (HBRI), Hunan Rice Research Institute (HRRI), and Hunan Yahua Seed Research Institute (HYSRI) at cadmium levels of 1.32 mg/kg, 1.66 mg/kg, and 2.41 mg/kg, respectively. This approach yielded three distinct datasets of cadmium content in mature brown rice.

Field experiments were conducted according to the local standard DB43/T 2599–2023 of Hunan Province, across rice-growing regions with varying cadmium levels (0.3 ~ 2.5 mg/kg). Annually, five representative sites were selected to assess cadmium content in mature brown rice, ensuring coverage of low, medium, and high cadmium environments. Cadmium content was determined following the National Standard for Food Safety (GB 5009.268–2016). Three replications at Quality Inspection and Testing Center for Rice and Products of the China National Rice Research Institute, Inspection and Testing Center for Rice and Products of the Hunan Rice Research Institute, and Testing Center of Hunan Institute of Microbiology were carried out for cadmium determination. The average of their results defined the cadmium content for each variety at the test sites.

Test sites were categorized into low (0.3–0.5 mg/kg), medium (0.6–0.9 mg/kg), and high (≥ 1 mg/kg) cadmium content groups. Phenotypic data from these sites were averaged to obtain three distinct datasets representing cadmium content under low (Low-Cd, 0.43 mg/kg), medium (Medium-Cd, 0.85 mg/kg), and high (High-Cd, 1.48 mg/kg) environments. These six sets of cadmium phenotype data were utilized for GWAS analysis.

Whole genome resequencing

A total of 158 rice varieties were selected for whole genome resequencing. For each variety, about 100 seeds were germinated, and leaf tissue at the 3-leaves stage was harvested for DNA extraction using the CTAB method (Allen et al 2006). DNA from qualified samples was sequenced to a depth of 20 × on the DNBSEQ-T7 platform, providing comprehensive coverage of the rice genome. Post-sequencing, quality control was performed using the FASTP software to filter out low-quality reads (Chen et al 2018b). Reads with more than 50% of base mass values below 20 or more than 5 N-bases were discarded to ensure high-quality input for subsequent analyses.The sequencing data of the 158 rice varieties used in this study have been deposited to NCBI with BioProject accession numbers: PRJNA1165535.

The clean reads were aligned to the rice reference genome of Nipponbare release 7 (Kawahara et al 2013) (NCBI RefSeq assembly: GCF_001433935.1) using the Sentieon DNAseq (v202112.06) process (Kendig et al 2019), which identified mutations relative to the reference. The GATK (v4.1.7.0) software (McKenna et al 2010) was then employed for hard filtering of single nucleotide polymorphisms (SNPs), ensuring that only high-confidence variants were retained for further analysis. To select informative SNPs for population genetic analysis, VCFtools (v0.1.17) (Danecek et al 2011) were used to filter the initial SNP set based on minimum allele frequency (MAF) greater than 5% and a missing rate less than 30%. This stringent filtering process resulted in the identification of 3,532,155 high-quality SNP loci, which were deemed suitable for genetic analysis.

Analysis of population structure and kinship

To elucidate the genetic structure and diversity among the rice varieties, we employed ADMIXTURE (v1.3.0) software (Alexander et al 2009) to estimate the genetic components across a range of potential subpopulations (K values set from 1 to 10). The optimal number of subpopulations was determined by identifying the K value that corresponded to the minimum cross-validation (CV) error, providing the most accurate reflection of population structure. We utilized PLINK (v 1.9) software for principal component analysis (PCA) (Purcell et al 2007), which reduced the dimensionality of the genetic data and highlighted the primary sources of genetic variation among the varieties.

Further, to visualize the genetic relationships and construct a phylogenetic tree (NJ tree), we used the R package “ape” (Paradis and Schliep 2019). For the visualization of the phylogenetic tree, we utilized the online tool iTol (http://itol.embl.de/) (Letunic and Bork 2024), which offered an interactive and user-friendly platform to explore and present the complex genetic relationships among the rice varieties.

Genome-wide association analysis

We employed the mixed linear model (MLM) implemented in the GEMMA (v0.98.5) software to perform a genome-wide association study (GWAS) (Zhou and Stephens 2012). This model is robust for controlling population structure and relatedness in the genetic data of the rice varieties. To account for environmental variations that might affect cadmium uptake, we incorporated environmental covariates into the analysis, ensuring that the identification of genetic markers were as precise as possible. A stringent statistical threshold of P = 1.0 × 10–7 was applied to identify SNPs significantly associated with cadmium content. This threshold helps to minimize false positives and enhances the reliability of the association signals detected. To graphically represent the GWAS outcomes, we used the R package “CMplot” to generate a Manhattan plot and a QQ (quantile–quantile) plot (Yin et al 2021). These plots are essential tools for visualizing the distribution of association statistics across the genome, allowing for the quick identification of genomic regions that may harbor genes affecting cadmium content.

Candidate gene prediction and haploid analysis

We utilized PopLDdecay (v3.42) software (Zhang et al 2019a) to analyze the Linkage Disequilibrium (LD) decay among 3,532,155 SNPs. This analysis calculated the non-random associations of alleles across different loci. The LD decay was measured using the r2 statistic, and the decay plot was generated using a perl script provided by PopLDdecay. The LD decay was considered significant when the average r2 value dropped to 0.2 within a 100 kb window. Based on the LD decay analysis (Fig. S1), we defined QTL intervals as the regions 100 kb upstream and downstream of the peaks of the significantly associated SNPs. This approach allowed us to pinpoint the genomic regions likely contain genes influencing cadmium content.

Gene function annotation and candidate gene prediction were conducted within the identified QTL intervals, using the Nipponbare rice genome sequence as a reference (National Rice Data Center https://www.ricedata.cn/ gene/). The gene sequences of the 158 rice varieties were subjected to haplotype analysis. This analysis involved the use of the Wilcoxon rank sum test for significance assessment, ClustalX1.81 for sequence alignment (Thompson et al 2002), and DnaSP6 for nucleotide polymorphism and haplotype diversity analysis (Rozas et al 2017). The haplotype analysis aimed to identify specific genetic variations associated with low cadmium accumulation.

Statistical analysis

Experimental data were meticulously organized and initially analyzed using Microsoft Excel 2010. For a more in-depth statistical analysis, we utilized SPSS 19.0 to perform one-way ANOVA to assess the differences in cadmium content among various rice varieties and subgroups. Additionally, the Duncan multiple range test was employed to provide a post-hoc comparison of means, enabling us to identify which groups significantly differed from each other. Correlation analysis was also conducted to explore the relationships between cadmium content and other phenotypic or genetic variables. To graphically represent the distribution of phenotypic data, we used Origin9. These visualizations were crucial for understanding the variability in cadmium content across different rice varieties and environmental conditions.

Results

Varietal cadmium contents under different experimental conditions

In the 2021–2023 growing seasons, a comprehensive analysis was conducted to evaluate the cadmium content in rice varieties under varied soil Cd concentrations, through both pool-planting and large-field experimental conditions. The national standard GB2762 “Limits of Contaminants in Foods” was adhered to classify varieties with Cd content ≤ 0.2 mg/kg as low Cd (Y), and those exceeding this threshold as high Cd (N).

The findings revealed a broad spectrum of Cd content among the tested varieties (Fig. 1, Table 1). In the Hunan Biological Research Institute (HBRI) pool with a soil Cd concentration of 1.32 mg/kg, the Cd levels in the tested varieties ranged from 0.010 to 2.740 mg/kg, averaging at 0.304 mg/kg. This group included 107 low Cd (≤ 0.2 mg/kg) and 51 high Cd (>0.2 mg/kg) varieties. Similarly, in the Hunan Rice Research Institute (HRRI) pool with a high soil Cd level of 1.66 mg/kg, the Cd content of rice grains varied from 0.006 to 3.610 mg/kg, averaging 0.418 mg/kg, with an identical count of 107 low Cd and 51 high Cd varieties. In the Hunan Yahua Seed Research Institute (HYSRI) pool, characterized by a significantly higher soil Cd concentration of 2.41 mg/kg, the Cd content of rice grains spanned an even wider range, from 0.000 to 7.042 mg/kg, yet the average Cd content was 0.830 mg/kg, maintaining the same number of low and high Cd varieties at 107 and 51, respectively.

Fig. 1.

Fig. 1

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Cadmium content distribution in varieties. A, (B) and (C) are pool-planting with three replications at Hunan Yahua Seed Research Institute (HYSRI), Hunan Rice Research Institute (HRRI) and Hunan Biological Research Institute (HBRI) at cadmium levels of 2.41 mg/kg, 1.66 mg/kg and 1.32 mg/kg, respectively; (D), (E) and (F) are field experiments with three cadmium content under high (High Cd, 1.48 mg/kg), medium (Medium Cd, 0.85 mg/kg) and low (Low Cd, 0.43 mg/kg) environments

Table 1.

Analysis on the cadmium content in test varieties under various environments

Environment Soil cadmium content (mg/kg) Range
(mg/kg)		 Mean ± Standard Deviation Coefficient of Variation/% Number of low cadmium varieties Y(Cd ≤ 0.2 mg/kg) Number of high cadmium varieties N(Cd > 0.2 mg/kg)
HBRI 1.32 0.010 ~ 2.740 0.304 ± 0.456 b 150.00 107 51
HRRI 1.66 0.006 ~ 3.610 0.418 ± 0.669 b 160.05 107 51
HYSRI 2.41 0.000 ~ 7.042 0.830 ± 1.563 a 188.31 107 51
Low Cd 0.43 0.010 ~ 1.026 0.158 ± 0.217 c 137.34 112 46
Medium Cd 0.85 0.004 ~ 2.323 0.337 ± 0.552 b 163.80 111 47
High Cd 1.48 0.002 ~ 3.673 0.407 ± 0.732 b 179.85 109 49
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Different letters indicate significant differences by one-way ANOVA and Duncan’s test p ≤ 0.05

Shifting to large-field experiments, the Low-Cd trial with a soil Cd level of 0.43 mg/kg exhibited the most promising results, with Cd content of rice grains varying from 0.010 to 1.026 mg/kg and an average of 0.158 mg/kg, identifying 112 low Cd and 46 high Cd varieties. The Medium-Cd and High-Cd trials, with soil Cd concentrations of 0.85 mg/kg and 1.48 mg/kg, respectively, showed slightly higher average Cd contents of 0.337 mg/kg and 0.407 mg/kg, corresponding to 111 and 109 low Cd varieties, and 47 and 49 high Cd varieties.

Across the six experimental conditions, spanning soil Cd content from 0.43 to 2.41 mg/kg, a clear trend emerged: the Cd content in the tested varieties increased with the rising soil Cd levels. Notably, the low Cd accumulation varieties demonstrated minimal variation in Cd content, while the high Cd accumulation varieties showed substantial fluctuation. The overall ratio of low to high Cd accumulation varieties was approximately 2:1, indicating considerable genotypic diversity in Cd accumulation potential.

Genetic diversity and population structure of low Cd accumulation rice varieties

To delve into the genetic underpinnings of the low Cd accumulation rice varieties, we conducted a comprehensive molecular evaluation of the tested rice varieties in the 2021–2023 Cd accumulation trials. The DNA extracted from these varieties was sequenced, yielding 3,532,155 high-quality single nucleotide polymorphisms (SNPs) after stringent filtering criteria were applied. We proceeded with population structure analysis and principal component analysis (PCA) based on these SNP markers to uncover the genetic relationships among the rice varieties (Fig. 2). The PCA delineated the 158 varieties into four distinct subgroups, which we named according to their parental lineage: Luohong, intermediate, lcd1, and early indica series.

Fig. 2.

Fig. 2

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Principal component analysis and population structures of 158 rice varieties. A Principal component analysis; (B) Phylogenetic trees; (C) Population structure distribution

The “Luohong” series, developed from crosses involving “Luohong 4 A” or “Luohong 4B”, comprised 44 varieties, with 39 classified as low-Cd (88.6%). The intermediate type, a diverse group of 56 varieties, included 12 low-Cd varieties (21.4%). The lcd1 series, derived from crosses with the low-Cd mutant lcd1, stood out with all 39 varieties exhibiting low-Cd traits (100%). The early indica series, consisting of 19 varieties, had 17 low-Cd varieties (89.5%). The prevalence of low-Cd varieties across these subgroups followed the order: lcd1 series > early indica series > Luohong series > intermediate type.

The phylogenetic tree and population structure distribution were in concordance with the PCA findings, revealing a rich genetic diversity and distinct subpopulation structures among the tested varieties. These differences underscore the varied genetic backgrounds and molecular mechanisms associated with low-Cd accumulation, providing valuable insights for the genetic improvement of rice in the context of Cd contamination.

Genome-wide association analysis of cadmium content in rice grains

We conducted a genome-wide association study (GWAS) to identify genetic markers associated with cadmium content in rice. Utilizing a mixed linear model (MLM) within the GEMMA software, we analyzed 3,532,155 high-quality SNPs markers in relation to the cadmium levels measured in the brown rice of varieties grown under various soil cadmium conditions (Fig. 3). The threshold for significance was set at -log10(P) = 7. The GWAS revealed a total of 575 significantly associated SNPs across all 12 rice chromosomes, with chromosome 7 harboring the highest number of SNPs, totaling 145. The number of SNPs identified under different experimental conditions varied: 124 under the HBRI (1.32 mg/kg) pool experiment condition, 269 under the HRRI (1.66 mg/kg) pool condition, 38 under the HYSRI (2.41 mg/kg) pool condition, 58 under the Low-Cd (0.43 mg/kg) field experiment, 12 under the Medium-Cd (0.85 mg/kg) field experiment, and 74 under the High-Cd (1.48 mg/kg) field experiment.

Fig. 3.

Fig. 3

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Manhattan plots and QQ plots of for cadmium content in brown rice under pool and field conditions. A, (B) and (C) are Manhattan plot and QQ plot of cadmium content in brown rice planted in pools during 2021–2023; (D), (E) and (F) are Manhattan plot and QQ plot of cadmium content in brown rice under different field experiments during 2021–2023

Identification and annotation of candidate genes for cadmium accumulation

In our analysis, we utilized the principle of linkage disequilibrium (LD) to define quantitative trait loci (QTL) regions, we defined QTL regions with a decay distance of 200 kb by extending the SNP loci at the boundaries of identified QTLs by 100 kb on each side. This approach led to the identification of a total of 202 QTLs associated with cadmium content in rice, which were distributed across all 12 chromosomes, with chromosome 7 harboring the highest number of QTLs at 45 (Table S5). The environmental conditions under which these QTLs were identified varied, with 28 QTLs under the HBRI (1.32 mg/kg) pool condition, 83 under the HRRI (1.66 mg/kg) pool condition, 21 under the HYSRI (2.41 mg/kg) pool condition, 21 under the Low-Cd (0.43 mg/kg) field experiment, 9 under the Medium-Cd (0.85 mg/kg) field experiment, and 40 under the High-Cd (1.48 mg/kg) field experiment. Notably, 52 QTLs were consistently identified under two or more conditions.

By comparing to the reference genome sequence of Nipponbare, we predicted genes within the QTL intervals using the National Rice Data Center (www.ricedata.com), resulting in a total of 3,746 annotated genes (Table S6). Through functional annotation screening, we identified 16 QTLs that are potentially involved in the regulation of cadmium content in mature brown rice (Fig. 3, Table 2). These QTLs are associated with genes encoding proteins such as metallothioneins, nitrate ion transporters, zinc-iron transporters, cadmium-manganese transporters, and ABC transporters.

Table 2.

Candidate genes and their functional annotations for the QTLs releted to cadmium content of brown rice

QTL Environment Chromosome Position P-value Candidate gene Functional annotation Reference gene
qCd1.2 HRRI chr01 5504946 6.13E-08 Os01 g0200700 type 3 metallolthionein isoform; OsMTI-3a;OsMT-3a
qCd1 HYSRI chr01 42100524 5.27E-09 Os01 g0955700 CRT-like transporter 1 OsCLT1
qCd1 HYSRI chr01 42100524 5.27E-09 Os01 g0956700 Low cadmium LCD
qCd2.13 HRRI chr02 28287439 1.75E-09 Os02 g0689900 nitrate transporter gene; OsNPF7.9
qCd5 HBRI chr05 18423440 8.50E-08 Os05 g0382200 transporter, monovalent cation:proton antiporter-2 family, putative, expressed
qCd5 Low-Cd chr05 18500014 2.66E-08
qCd5.3 High-Cd chr05 18500014 2.09E-09
qCd7.3 HYSRI chr07 7358577 6.17E-10 Os07 g0232800 metal cation transporter; zinc transporter protein OsZIP8; OsZIP13
qCd7.3 Low-Cd chr07 7358577 1.25E-08
qCd7.3 HYSRI chr07 7358577 6.17E-10 Os07 g0232900 transporter for Cd; single recessive gene controls cadmium translocation OsHMA3; qCdT7
qCd7.3 Low-Cd chr07 7358577 1.25E-08
qCd7.2 HBRI chr07 8876106 1.21E-09 Os07 g0257200 natural resistance-associated macrophage protein; cadmium tolerance; cadmium uptake; cadmium transport; manganese translocation OsNramp5
qCd7.6 HYSRI chr07 8876106 2.09E-15
qCd7.6 Low-Cd chr07 8876106 1.08E-13
qCd7.1 Medium-Cd chr07 8876106 1.13E-12
qCd7.1 High-Cd chr07 8876106 5.91E-10
qCd7.2 HBRI chr07 8876106 1.21E-09 Os07 g0258400 natural resistance-associated macrophage protein OsNramp1
qCd7.6 HYSRI chr07 8876106 2.09E-15
qCd7.6 Low-Cd chr07 8876106 1.08E-13
qCd7.1 Medium-Cd chr07 8,876,106 1.13E-12
qCd7.1 High-Cd chr07 8876106 5.91E-10
qCd11.6 HRRI chr11 28884441 5.50E-09 Os11 g0704500 type 1 metallothionein; OsMT1a; OsMT1e,
qCd12.2 HBRI chr12 19887419 2.55E-09 Os12 g0512100 transporter family protein, putative, expressed
qCd12.6 HRRI chr12 19858172 5.06E-08
qCd12.2 HBRI chr12 19887419 2.55E-09 Os12 g0512700 ATP-binding cassette transporter OsABCG50; OsPDR23,
qCd12.6 HRRI chr12 19858172 5.06E-08
qCd12.2 HBRI chr12 19887419 2.55E-09 Os12 g0514000 transporter family protein, putative, expressed
qCd12.6 HRRI chr12 19858172 5.06E-08
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Specifically, QTL qCd5 (HBRI, Low-Cd) and qCd5.3 (High-Cd) on chromosomes 5, qCd7.3 (HYSRI, Low-Cd), qCd7.2 (HBRI), qCd7.6 (HYSRI, Low-Cd) and qCd7.1 (Medium-Cd, High-Cd) on chromosome 7, and qCd12.2 (HBRI) and qCd12.6 (HRRI) on chromosome 12 recurred in multiple environments. These four QTLs that showed significant associations in multiple environments were selected for further in-depth analysis. This analysis focused on 100 kb intervals upstream and downstream of each SNP locus, identifying a total of 8 associated genes: Os05 g0382200, Os07 g0232800, Os07 g0232900, Os07 g0257200, Os07 g0258400, Os12 g0512100, Os12 g0512700, and Os12 g0514000 (Table 1). Among these genes, Os07 g0232800 (OsZIP8), Os07 g0232900 (OsHMA3), Os07 g0257200 (OsNramp5), and Os07 g0258400 (OsNramp1) are known cadmium-related genes that participate in the regulation of cadmium ion uptake and transport. The other genes, Os05 g0382200 and Os12 g0512100 (transporter proteins), Os12 g0512700 (ABC transporter protein), and Os12 g0514000 (transporter protein family), are uncloned and are predicted to encode transporter proteins and ABC transporters, which may play a role in the regulation of cation and cadmium ion transport. It requires further functional analysis to confirm their involvement in cadmium accumulation.

Linkage disequilibrium and haplotype analysis of cadmium-related candidate genes

To further investigate the genetic basis of cadmium accumulation in rice, we conducted a detailed linkage disequilibrium (LD) and haplotype analysis for the candidate genes OsZIP8, OsHMA3, OsNramp5, and OsNramp1. The Manhattan maps were expanded to examine the regions harboring the SNP loci of these genes, and LD haplotype blocks were identified (Fig. 4). Our analysis revealed a close association between these candidate genes and the SNPs located at their respective loci. Notably, OsNramp5 was found to overlap with the significant SNP locus within a QTL, while OsNramp1 was situated just 3 kb away from the significant SNP locus, indicating their potential as important candidates for cadmium content in mature brown rice.

Fig. 4.

Fig. 4

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LD and haplotype analysis results of important candidate gene. A, (B), (C), (D)and (E) are the LD and haplotype analysis results of OsZIP8 and OsHMA3; (F), (G), (H), (I) and (J) are the LD and haplotype analysis results of OsNramp5 and OsNramp1

Haplotype analysis provided further insights (Fig. 4, Table 3). For OsZIP8, three haplotypes were identified: Hap1 (TTA), Hap2 (TTT), and Hap3 (CCT). There were no significant differences in cadmium content among these haplotypes in the pool environment, except under the HBRI pool condition. Hap1 was the predominant haplotype, present in 134 varieties (96.4%), with the low Cd accumulation varieties count for 73.1%, 33.3%, and 0% of the hyplotypes. For OsHMA3, three haplotypes were observed: Hap1 (AC), Hap2 (GC), and Hap3 (GG). The cadmium content of Hap1 (AC) was significantly lower compared to Hap3 (GG) across both pool and field environments. Hap2 (GC) also showed a significantly lower cadmium content than Hap3 (GG) in the field experiment. Hap1 and Hap2 were the primary haplotypes, found in 31 and 49 varieties (37.8% and 59.8%), with low Cd accumulation variety percentages of 83.9%, 75.5%, and 0% for the three haplotypes. Gene OsNramp5 exhibited four haplotypes: Hap1 (DEL), Hap2 (AA), Hap3 (GG), and Hap4 (AG). Significant differences in cadmium content were noted among these haplotypes. Hap1, Hap2, and Hap3 were the main haplotypes, present in 32, 54, and 41 varieties (24.1%, 40.6%, and 30.8%). The low Cd accumulation varieties count for 100%, 100%, 31.7%, and 16.7% of the hyplotypes. Gene OsNramp1 also showed four haplotypes: Hap1 (DEL), Hap2 (GGG), Hap3 (TCA), and Hap4 (TCG). Significant or highly significant differences in cadmium content were observed among these haplotypes. Hap1, Hap2 and Hap3 were the major haplotypes, found in 31, 60, and 35 varieties (24.2%, 46.9%, and 27.3%), with low Cd accumulation varieties count for 100%, 91.7%, 31.4%, and 0% of the hyplotypes.

Table 3.

Haplotype of candidate genes and number of rice varieties in each haplotype

Gene Haplotype Number of varieties in different subgroups Total/Number
Luohong series lcd1 series Early indica series Intermediate type Low Cd variety High Cd variety
OsZIP8-Os07 g0232800 Hap1 (TTA) 40 37 18 39 98 36
Hap2 (TTT) 1 0 0 2 1 2
Hap3 (CCT) 0 0 0 2 0 2
OsHMA3-Os07 g0232900 Hap1 (AC) 1 21 3 6 26 5
Hap2 (GC) 37 0 1 11 37 12
Hap3 (GG) 0 0 0 2 0 2
OsNramp5-Os07 g0257200 Hap1 (DEL) 30 0 2 0 32 0
Hap2 (AA) 2 37 15 0 54 0
Hap3 (GG) 5 0 2 34 13 28
Hap4 (AG) 1 0 0 5 1 5
OsNramp1-Os07 g0258400 Hap1 (DEL) 29 0 2 0 31 0
Hap2 (GGG) 3 37 15 5 55 5
Hap3 (TCA) 4 0 2 29 11 24
Hap4 (TCG) 0 0 0 2 0 2
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These findings underscore the importance of candidate genes in the context of cadmium accumulation in rice and provide a foundation for future functional validation studies aimed at developing rice varieties with reduced cadmium content, thereby enhancing food safety and reducing health risks associated with cadmium exposure through rice consumption.

Discussion

The “cadmium rice” incident has drawn widespread attention to the issue of heavy metal contamination in rice. The presence of cadmium in rice poses a significant risk to human health, thus breeding rice varieties with low Cd accumulation has become a priority. This study provides a comprehensive genetic analysis of low Cd accumulation rice varieties, utilizing genome-wide association studies (GWAS) to identify significant loci associated with Cd content. We evaluated the genetic diversity of low Cd accumulation rice varieties, identified molecular markers, and discovered candidate genes that could be used in breeding programs to reduce Cd levels in rice.

Since the “cadmium rice” incident, there has been a heightened social awareness of Cd in rice, emphasizing the need for solutions. Breeding low-Cd rice varieties is currently the most effective strategy. The available low-Cd accumulation germplasm resources, including Luohong 3 A/4 A, lcd1 mutants, Shaoxiang 100, and Lian 1S, are primarily based on the OsNramp5 gene and are crucial for direct use in breeding new varieties (Wang et al 2021; Lv et al 2020; Shao et al 2022; Mao et al 2023; Li et al 2024; Cao et al 2019; Chen et al 2019; Wang 2023). Our study categorized 158 tested rice varieties into four subgroups based on parental origin and population structure, including Luohong series, intermediate type, lcd1 series, and early indica series. The Luohong series, which includes the nationally-approved low Cd accumulation variety Xizi 3, showed a high proportion of low Cd accumulation varieties. The intermediate type, with a lower proportion of low Cd accumulation varieties, was primarily bred from crosses with Lian 1S or Shaoxiang 100. The lcd1 series, bred from the mutant lcd1, exhibited a 100% low-Cd rate, indicating stable low Cd accumulation characteristics. Early indica series, due to their genetic distance, were a separate subgroup with low Cd accumulation varieties mainly from the Luohong and lcd1 series.

GWAS analysis identified 16 significant QTLs associated with Cd content, with four loci detected in a single environment. Notably, qCd1.2 and qCd11.6 are consistent with known genes OsMTI-3a/OsMT-3a and OsMT1a/OsMT1e, which encode metallothioneins that enhance Cd tolerance (Mekawy et al 2018; Rono et al 2021). The QTL qCd1 (HYSRI) interval contains OsCLT1 (Yang et al 2016) and LCD (Shimo et al 2011), key genes in Cd detoxification and transport. QTL qCd2.13 (HRRI) aligns with OsNPF7.9, involved in nitrate distribution and Cd tolerance (Guan et al 2022). All of these loci were consistent with the cloned genes and were detected in a single environment, mainly enhancing cadmium tolerance in heavy metal stress. The other 12 loci, detectable in multiple environments, include seven associated with reported genes or loci. For instance, qCd7.3 (HYSRI, Low-Cd) contains OsZIP8 (Lee et al 2010) and OsHMA3 (Sasaki et al 2014), with the latter known to transport Cd2+ into vacuoles and reduce cadmium toxicity (Liu et al 2020). The qCd7.2 (HBRI), qCd7.6 (HYSRI, Low-Cd), and qCd7.1 (Medium-Cd, High-Cd) intervals include OsNramp5 (Ishimaru et al 2012; Sasaki et al 2012) and OsNramp1 (Takahashi et al 2011), significant for Cd uptake. These newly identified loci are robust across environments, indicating their potential in breeding for low-Cd rice varieties. To current knowledge, the OsNramp5 is the most important gene for Cd uptake in rice roots. In addition, we first reported several transporter-related genes Os12 g0512100, Os12 g0512700 and Os12 g0514000 in the qCd12.2 (HBRI) and qCd12.6 (HRRI) interval, as well as Os05 g0382200 in the qCd5 (HBRI, Low-Cd) and qCd5.3 (High-Cd) interval, which are significantly associated with cadmium content, but they have not been explored in Cd tolerance. The regulatory mechanism and function of these genes remain to be further investigated.

A large number of studies have reported that OsHMA3, OsNramp5 and OsNramp1 involve in the uptake and accumulation of cadmium (Ishimaru et al 2012; Sasaki et al 2012, 2014; Takahashi et al 2011; Tezuka et al 2010; Liu et al 2020; Lu et al 2019). OsNramp5 encodes a transmembrane transporter protein of cadmium and manganese ions. Knockout or mutation of OsNramp5 can significantly reduce the content of Cd in rice, and at the same time, may lead to the deficiency of the essential element Mn, further lead to premature senescence in plants. There are 7 nucleotide variants in the OsHMA3 promoter region with obvious indica-japonica differentiation. The fact that indica rice varieties carry low expression alleles of OsHMA3 and japonica rice varieties carry high expression alleles of OsHMA3 may be one of the reasons for the high accumulation of Cd in indica rice grains and low accumulation of Cd in japonica rice grains. The overexpression of OsHMA3 in indica rice varieties can effectively reduce the cadmium content of rice without significant effects on agronomic traits and the content of essential micronutrients (Lu et al 2019). Haplotype analysis of four cloned genes (OsZIP8, OsHMA3, OsNramp5, and OsNramp1) revealed low-Cd dominant haplotypes, such as OsHMA3-Hap2 (GC), OsNramp5-Hap1 (DEL) and OsNramp1-Hap1 (DEL) were low-cadmium accumulation dominant haplotypes of Luohong origin, and OsHMA3-Hap1 (AC), OsNramp5-Hap2 (AA) and OsNramp1-Hap2 (GGG) were low-cadmium accumulation dominant haplotypes of lcd1 origin, so the polymerization of low-cadmium dominant haplotypes of OsHMA3, OsNramp5, and OsNramp1 can reduce the uptake of cadmium without affecting the uptake of other essential elements, such as manganese, which can be directly used for the breeding of low cadmium varieties. In conclusion, this study contributes to the global effort to ensure food safety and security by providing a scientific foundation for the breeding of rice varieties with low Cd content. As we continue to confront the challenges of heavy metal contamination in agriculture, the genetic resources and molecular tools identified here will be invaluable for safeguarding public health and sustaining rice production in the face of environmental pressures.

Conclusions

In summary, this study systematically categorized the tested low-cadmium accumulation varieties from the perspective of molecular genetics. Based on the analysis of parental origin and genetic population structure, the tested varieties can be categorized into four subgroups: Luohong series, lcd1 series, intermediate type, and early indica series. The low cadmium accumulation varieties of the four subgroups were bred based on OsNramp5 mutants Luohong 3 A/4 A, lcd1, Lian 1S, and Shaoxiang 100, which is the main way of low-cadmium accumulation varieties breeding at present. 12 Cd-associated loci were identified in two or more environments through GWAS analysis, candidate genes for cadmium content including Os05 g0382200, Os07 g0232800 (OsZIP8), Os07 g0232900 (OsHMA3), Os07 g0257200 (OsNramp5), Os07 g0258400 (OsNramp1), Os12 g0512100, Os12 g0512700, and Os12 g0514000. These genes are implicated in the absorption, transport, and accumulation of heavy metals, particularly cadmium. The haplotype analysis revealed that OsHMA3-Hap2 (GC), OsNramp5-Hap1 (DEL) and OsNramp1-Hap1 (DEL) were low-cadmium accumulation dominant haplotypes of Luohong origin, and OsHMA3-Hap1 (AC), OsNramp5-Hap2 (AA) and OsNramp1-Hap2 (GGG) were low-cadmium accumulation dominant haplotypes of lcd1 origin, which can be directly used for low-cadmium accumulation varieties breeding and molecular marker development, contributing to low-cadmium accumulation rice breeding through polymerizing dominant haplotype alleles.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 11572 KB) (11.3MB, pdf)
Supplementary file2 (XLSX 389 KB) (389.3KB, xlsx)

Acknowledgements

The authors would like to express their sincere gratitude to Dr. Haomin Lyu for his invaluable contributions on manuscript revision.

Author contributions

Conceptualization, K.Z. and H.Y.; methodology, D.S., L.Y. and J.Z.; software, L.Y. and W.L.; investigation, D.S., Y.W., Z.L. and X.L. (Xiangjie Liu); resources, K.Z., H.Y., X.L. (Xiaobo Long) and B.T.; writing-original draft preparation, D.S., L.Y. and J.Z.; writing-review and editing, K.Z. and H.Y.; supervision, X.L. (Xiaobo Long) and B.T.; project administration, Z.L. All the authors read and approved the final manuscript. All authors have read and agreed to publish the manuscript.

Funding

This study was supported by the Hunan Agricultural Science and Technology Innovation Fund Project (Grant No. 2024 CX09、2023 CX15).

Data availability

Data is contained within the article and within Supplementary Material.

Declarations

Competing interests

The authors declare that they have no confict of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Deyi Shao, Lixin Yin and Jian Zhao contributed equally to this work.

Contributor Information

Bingchuan Tian, Email: tianbc@higentec.com.

Xiaobo Long, Email: xiaobo.long@higentec.com.

Hexing Yin, Email: hexing.yin@higentec.com.

Kun Zhou, Email: zhzhkp@163.com.

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Data Availability Statement

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