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Article

Genomics-Assisted Improvement in Blast Resistance and Low Cadmium Accumulation in an Elite Rice Variety

by
Zhi Xu
1,2,†,
Zhizhou He
3,†,
Yanglan He
1,
Hailong Chen
1,
Jihua Cheng
4,
Changrong Ye
1,
Zhouwei Li
1,
Le Li
1,
Hexing Yin
1,
Lijia Zheng
1,
Yuntian Wu
1,
Bingchuan Tian
1,* and
Junhua Peng
5,*
1
Huazhi Biotechnology Co., Ltd., Changsha 410125, China
2
Ministry of Agriculture Key Laboratory of Integrated Management of Pests on Crops in Southwest China, Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
3
Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 570100, China
4
Hubei Academy of Forestry, Wuhan 430075, China
5
Spring Valley Agriscience Co., Ltd., Jinan 250307, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(9), 2130; https://doi.org/10.3390/agronomy15092130
Submission received: 27 July 2025 / Revised: 27 August 2025 / Accepted: 4 September 2025 / Published: 5 September 2025
(This article belongs to the Section Crop Breeding and Genetics)
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Abstract

Xiangwanxian 13 (XWX13), an elite fragrant indica rice, is highly susceptible to rice blast and accumulates cadmium (Cd) in grain above the food safety limit in Cd-contaminated paddies, severely constraining its commercial use. Despite these shortcomings, the variety is widely grown for its high yield and superior grain quality. To overcome these limitations, we conducted marker-assisted backcrossing (MABC) complemented by genome-wide background selection. Four major genes, namely Pi1, Pi2, OsHMA3, and OsNramp5, were precisely introduced into XWX13. Two preferable BC3MF5 improved lines iXWX13-1 (stacking Pi1 + Pi2 + OsHMA3) and iXWX13-2 (stacking Pi1 + Pi2 + OsNramp5) were obtained with genomic background recovery rates of 94.44% and 94.63%, evaluated by using the RICE 1K SNP array, respectively. Seedling resistance spectrum assays demonstrated more than a 97% blast resistance rate against 39 Magnaporthe oryzae isolates, and both lines showed enhanced leaf and panicle neck blast resistance in natural nurseries. Multi-site field trials revealed grain Cd concentrations of 0.009–0.077 mg kg−1 in iXWX13-2, 90.98–98.87% lower than those in XWX13. Importantly, yield, major agronomic traits, and grain quality remained indistinguishable from the original variety. This study provides the first demonstration that MABC coupled with SNP array background selection can simultaneously enhance blast resistance and reduce grain Cd in XWX13 without yield or quality penalties, offering a robust strategy for pyramiding multiple desirable genes into elite cultivars.

1. Introduction

Rice (Oryza sativa L.) is one of the most important crops, serving as food supply for more than half of the world’s population [1]. To ensure food security, efforts have been made to achieve a sustainable increase in rice yield in the last decade [2]. However, high-yield cultivation of rice varieties has caused excessive use of pesticides due to a lack of resistance to pests and diseases, which has increased input costs and damaged the ecological environment [3,4,5]. In addition, industrial emissions, agro-chemical use, and anthropogenic activities have resulted in excessive heavy metals such as cadmium (Cd) in rice paddies, posing a serious threat to food safety [6,7,8]. To date, most reported improvement efforts have focused on a single trait, either pyramiding NLR genes to enhance blast resistance or introgressing OsHMA3 or OsNRAMP5 alleles to reduce grain Cd accumulation. However, systematic attempts to combine broad-spectrum blast resistance with low Cd accumulation in a single high-quality indica variety are lacking, limiting the realization of a ‘high-yield, high-quality, disease-resistant and safe’ ideotype in Cd-contaminated paddy areas.
Rice blast, caused by the pathogenic fungus Magnaporthe oryzae (M. oryzae), is one of the major diseases of rice, resulting in yield loss of 30% to 50% in large rice planting areas under favorable conditions [3]. Great efforts have been devoted to evaluating and characterizing the blast resistance of rice germplasm resources. To date, more than 100 blast resistance (R) genes have been identified and mapped on the rice genome, and 38 of them have been cloned and characterized [9,10]. Most of the genes are distributed in the gene clusters on chromosomes 6, 11, and 12, and very few of them have been introgressed into popular rice varieties [9]. The high variable nature of the pathogen M. oryzae leads to frequent emergence of new virulent races, resulting in a loss of resistance within 3–5 years of rice cultivation. Among the blast resistance genes, Pi1 is an allele at the Pik locus mapped at the end of chromosome 11, conferring dominant and durable resistance against geographically diverse M. oryzae isolates [11,12]. Likewise, gene Pi2 is an allele at the Piz locus located on chromosome 6 close to the centromere and also confers dominant and broad-spectrum resistance to various M. oryzae isolates [13,14,15,16]. Currently, several attempts had been made to deploy Pi1 and Pi2 in rice breeding [17,18,19,20], along with enhanced resistance to rice blast under high pressure in epidemic fields.
Among abiotic stresses, Cd contamination of soil poses a serious threat to sustainable agriculture and food safety. As the main source of Cd intake in human diets [21], rice generally poses a higher risk of Cd exposure compared to other crops [22,23]. To reduce the effects of Cd toxicity on human health, a maximum Cd concentration of 0.4 mg/kg in rice is allowed internationally, and an even stricter limit of 0.2 mg/kg has been established in China [24,25]. However, cases of excessive Cd accumulation in rice grain have occurred frequently in the past decade [26,27,28,29]. Some measures have been made to reduce the risk of excessive Cd accumulation in rice, such as soil Cd remediation, fertilizer control, irrigation management, and growing low-Cd-accumulating varieties [25]. Studies have shown that genetic improvement and development of low-Cd rice varieties are cost-effective in agricultural practice [22,30]. Currently, the physiological and genetic mechanisms of Cd accumulation in rice have been well studied, some quantitative trait loci (QTL) and genes associated with variation in grain Cd accumulation have been identified [31,32], and four Cd-QTL hotspots have been mapped on chromosomes 2, 3, 7, and 8 [33,34,35,36,37,38,39,40,41,42,43,44,45,46,47]. With a series of the associated QTLs and genes being found, genetic improvements in rice specifically targeting reducing grain Cd accumulation have become a top priority for rice breeders. Among the known cases, the natural resistance-associated macrophage protein gene OsNramp5, located on chromosome 7 and encoding a key transporter for Cd uptake in rice, is an ideal target gene to be manipulated to reduce Cd uptake [48,49,50,51]. Additionally, OsHMA3, encoding the P-type heavy metal ATPase, is another target gene that has been manipulated to sequester Cd into vacuoles of root cells, which subsequently reduced Cd transportation [52,53]. The use of OsNramp5 and OsHMA3 to generate low-Cd rice varieties was more effective when compared with genetic modification of genes such as OsNramp1, OsZIP5, OsZIP9, OsZIP7, and OsLCT1 [47].
Molecular breeding enables not only the improvement in specific traits in recipient materials, but also the development of novel varieties with superior characteristics through precise gene pyramiding. This approach offers greater accuracy and efficiency compared to conventional breeding methods. Among these, marker-assisted selection (MAS) has emerged as a highly efficient strategy due to its ability to enable rapid and precise identification of target genes [54]. Genomics-assisted backcrossing, in particular, is especially suited for tackling challenges such as the integration of multiple genes while recovering the recurrent parent genome in a limited number of generations. It allows breeders to overcome major constraints of traditional breeding, including low selection efficiency, environmental influence on phenotype, and the long timeframe required for gene stacking. The application of MAS has significantly accelerated rice breeding, despite limitations such as linkage drag and unpredictable genetic background effects [19]. Through MAS, foreground selection ensures the retention of target genes using informative markers [55], while background selection promotes the recovery of the recurrent parent’s genome [56]. The adoption of kompetitive allele-specific PCR (KASP) markers has further improved genotyping precision and efficiency in crop enhancement and variety development [57]. In rice, several SNP arrays, such as the 44K SNP chip, RICE6K, 50K SNP chip, Rice700K SNP array, Cornell 7K Array Infinium Rice (C7AIR), and Rice3K56 SNP array, have been developed and widely used for both foreground and background selection [58,59,60,61,62,63]. These genomic tools provide powerful means for trait selection and contribute to a deeper understanding and improvement of rice and other crops.
Xiangwanxian 13 (XWX13) is a late-maturing, fragrant, high-quality conventional indica variety developed jointly by the Hunan Provincial Rice Research Institute and Jinjian Rice Industry Co., Ltd. (Changde, China). It was approved in Hunan Province in 2001 [64]. Well accepted by farmers for its high yield and desirable grain quality, XWX13 has been cultivated commercially over a total area exceeding 600,000 hectares (https://www.ricedata.cn/variety/varis/605312.htm, accessed on 27 July 2025). However, the variety is highly susceptible to rice blast, and its grains were found to exceed the cadmium (Cd) safety standard of 0.2 mg/kg when grown in highly contaminated paddies, despite once being recommended as a low-Cd emergency variety [65]. These limitations pose production risks and restrict large-scale commercial use. Therefore, this study aimed to enhance blast resistance and reduce grain Cd accumulation in XWX13 through an efficient genomic breeding strategy. Using genomics-assisted selection, four major genes, Pi1, Pi2, OsHMA3, and OsNramp5, were precisely introgressed into XWX13 from three donor parents. The results demonstrate a successful case of genomics-assisted rapid and targeted improvement of XWX13 in both blast resistance and low Cd accumulation.

2. Materials and Methods

2.1. Plant Materials and M. oryzae Isolates

Rice plant materials used in this study include the blast resistance gene donor HZ02411 (possessing Pi1 + Pi2) and the low-Cd-accumulating gene donors HZ02415 (possessing OsHMA3) and HZ02416 (possessing OsNramp5); they were created and bred by Huazhi Biotechnology Co., Ltd. (Changsha, China). The original parent Xiangwanxian 13 (XWX13) was provided by Hunan Rice Research Institute (Changsha, China). The susceptible and resistant control varieties CO39 and Gumei4 were introduced from the National Germplasm Resource Bank (Hangzhou, China). Some of the 39 M. oryzae isolates were directly introduced from outside, and some were obtained through isolation of disease samples. All of the above materials are independently preserved by Huazhi Biotechnology Co., Ltd. (Changsha, China).

2.2. Population Development and Breeding Selection Procedure

The improved XWX13 lines (iXWX13-1 and iXWX13-2) were developed through marker-assisted backcrossing, as described in Figure 1. The late-maturing indica rice cultivar XWX13 was crossed with each of three different donors, and the foreground SNP markers were used to genotype XWX13, the corresponding donors, and the F1 plants. The F1 plants were then backcrossed with the recurrent parent XWX13. The BC1F1 plants were genotyped for foreground and background selections, and three plants with the target genes and the highest background recovery rate were selected to further backcross with XWX13. The same genotyping and selections were performed for the BC2F1 and BC3F1 populations. To pyramid genes for different traits, the BC3F1 plants with the target genes and the highest background recovery rate were used for multiple hybridization, and a double-cross F1 population was generated. Based on continuous self-pollination, foreground and background selections, and field phenotypic screening, two BC3MF5 improved lines iXWX13-1 and iXWX13-2 were successfully developed.

2.3. Marker-Assisted Selection and Background Analysis

To confirm the presence of the target genes, three gene donor lines were first screened with the corresponding molecular markers. Throughout the subsequent gene pyramid program, every backcross and self-pollination generation was detected with the same markers using the low-density KASP genotyping platform developed by Huazhi Biotechnology Co., Ltd. (Changsha, China). The Pi1, Pi2, OsHMA3, and OsNramp5 loci were all tracked with SNP-based KASP markers [66,67,68,69].
A total of 120 polymorphic SNP markers evenly distributed on 12 chromosomes were used for background selection. Leaf sampling, DNA extraction, and KASP genotyping were performed as described by He et al. [20]. The whole-genome SNP array RICE 1K mGPS was further used for detecting the genetic background between the recurrent parent XWX13 and the improved lines, iXWX13-1 and iXWX13-2. The SNP array RICE 1K mGPS was developed by Huazhi Biotechnology Co., Ltd. (Changsha, China). (https://www.higentec.com, accessed on 27 July 2025) and comprises 5456 loci in 1048 intervals uniformly distributed on the rice genome. According to similar methods previously reported [20], SNP array genotyping was performed and the background recovery rate was calculated.

2.4. Evaluation of Rice Blast Resistance

XWX13 and the improved lines were tested for resistance spectrum at the seedling stage in 2022 by Huazhi Biotechnology Co., Ltd. (Changsha, China). The 39 M. oryzae isolates were used for spraying inoculation in the greenhouse according to the method described by Liu et al. [70]. Disease reaction was assessed one week after inoculation following a standard 0–9 rating evaluation system [71,72]. The rice varieties CO39 and Gumei4 were used as the susceptible and resistant control, respectively. Three biological replicates for each line and control were used in the experiment. Resistance frequency was defined as the ratio of resistance-showing M. oryzae isolates to the total number of inoculated M. oryzae isolates on the rice genotype (line), and a higher frequency represents a broader resistance spectrum.
Leaf and panicle neck blast resistance of XWX13 and the improved lines were evaluated in 2022 in two natural blast nurseries, Dawei Mountain in Liuyang City and Taojiang in Yiyang City, Hunan, China. The experiment was set up with three biological replicates and randomly arranged in blocks. Blast resistance was assessed according to the evaluation system mentioned above [71,72].

2.5. Evaluation of Yield, Main Agronomic Traits, and Grain Quality

XWX13 and the improved lines were planted in multi-site trials in 2022. Five test sites were set up, namely Changsha and Yueyang in Hunan, Yichun and Shangrao in Jiangxi, and Hezhou in Guangxi. In the field layout, each variety or line was planted in a plot of 13.34 m2. Field cultivation and agronomic trait evaluation were conducted according to the agricultural industry standard ‘NY/T 1300-2007 Technical Specifications for Regional Trials of Rice Varieties’. The heading date was recorded for each plot. At maturity stage, ten individual plants in the middle of the central row in each plot were sampled for measurement of the main agronomic traits including plant height (PH, cm), panicle length (PL, cm), spikelets per panicle (SPP), filled-grain percentage (FGP, %), and 1000-grain weight (GW, g). The plot yield was measured for each variety or line at each site.
Bulked harvested seeds from each plot were used for grain quality analysis. The rice quality analysis was conducted according to the industry standard ‘NY/T 593-2021 Edible Rice Variety Quality, The Ministry of Agriculture, China’. The relevant indicators for assessment include brown rice percentage (BRP, %), milled rice percentage (MRP, %), head rice percentage (HRP, %), chalky rice percentage (CRP, %), chalkiness degree (CD, %), rice grain length (RGL, mm), grain length/width ratio (L/W), alkali spreading value (ASV), amylose content (AC, %), and gel consistency (GC, mm).

2.6. Determination of Grain Cd Concentration

The grain Cd concentration of XWX13 and the improved lines in multi-site trials was determined by using NX-100FA food heavy metal detector (NCS Testing Technology Co., Ltd., Suzhou, China). Bulk harvested seeds from each plot were randomly sampled three times, and the grain Cd concentration was measured following the instrument manual. The values of grain Cd concentration given are the mean of three biological replicates. Low Cd accumulation was judged against the rice cadmium limit (≤0.2 mg kg−1 brown rice) set by the China National Standard for Food Safety, GB 2762 [73].

2.7. Statistical Analysis

The experimental data were processed, calculated, and counted using WPS office, SPSS 19 for ANOVA, and a significance of difference test. The R language package RIdeogram [74] was used to generate high-resolution genetic background maps, enabling clear visualization of genome-wide data against chromosomal ideograms. The graph of grain Cd concentration was generated using WPS office. The letters a, b, c and the asterisk symbol indicate a significance of difference at the 5% probability level. All error lines in the graph of this paper are the standard deviations of three biological replicates of the data.

3. Results

3.1. Development of Improved Lines

All foreground and background SNP markers were employed to genotype XWX13, HZ02411 (donor of Pi1 + Pi2), HZ02415 (donor of OsHMA3), HZ02416 (donor of OsNramp5), and the F1 plants to confirm marker polymorphism.
F1 plants derived from crossing XWX13 with HZ02411 were genotyped using foreground SNP markers; eight F1 plants carrying the target genes were backcrossed to XWX13. In the BC1F1 population, 691 plants were genotyped with foreground markers, and 163 plants harboring both Pi1 and Pi2 genes were selected for background genotyping using 107 SNP markers. Background analysis indicated a recovery rate ranging from 70.7% to 88.6%. Three plants with the highest recovery rates were backcrossed to XWX13. In the BC2F1 population, 605 plants were genotyped with foreground markers, and 132 plants carrying Pi1 and Pi2 were selected for background genotyping using 30 SNP markers. The background recovery rate ranged from 86.2% to 94.8%, and three plants with the highest recovery were again backcrossed to XWX13. In the BC3F1 population, 91 plants were genotyped with foreground markers, and 24 plants with Pi1 and Pi2 were selected for background genotyping using nine SNP markers. The background recovery rate ranged from 96.5% to 97.3%.
Similarly, F1 plants from XWX13/HZ02415 were genotyped with foreground SNP markers, and eight target F1 plants were backcrossed to XWX13. In the BC1F1 population, 261 plants were genotyped with foreground markers, and 126 plants carrying OsHMA3 were selected for background analysis using 112 SNP markers. The background recovery rate ranged from 68.7% to 85.2%. Three plants with the highest recovery were selected for backcrossing to generate the BC2F1 population. In the BC2F1 population, 215 plants were genotyped with foreground markers, and 113 plants carrying OsHMA3 were selected for background analysis using 47 SNP markers. The recovery rate ranged from 81.4% to 96.6%. Three plants with the highest recovery were backcrossed to XWX13. In the BC3F1 population, 85 plants were genotyped with foreground markers, and 37 plants carrying OsHMA3 were selected for background analysis using 13 SNP markers. The recovery rate ranged from 94.1% to 98.5%.
For the XWX13/HZ02416 cross, F1 plants were genotyped with foreground SNP markers, and eight target plants were backcrossed to XWX13. In the BC1F1 population, 277 plants were genotyped with foreground markers, and 131 plants carrying OsNramp5 were selected for background analysis using 115 SNP markers. The recovery rate ranged from 69.2% to 86.3%. Three plants with the highest recovery were backcrossed to generate the BC2F1 population. In the BC2F1 population, 232 plants were genotyped with foreground markers, and 119 plants carrying OsNramp5 were selected for background analysis using 45 SNP markers. The recovery rate ranged from 82.6% to 96.9%. Three plants with the highest recovery were backcrossed to XWX13. In the BC3F1 population, 84 plants were genotyped with foreground markers, and 41 plants carrying OsNramp5 were selected for background analysis using 12 SNP markers. The recovery rate ranged from 94.6% to 98.7%.
To pyramid the genes Pi1 + Pi2 + OsHMA3/OsNramp5 in the XWX13 background, the best performing plants carrying OsHMA3 or OsNramp5 from the BC3F1 populations of XWX13/HZ02415 and XWX13/HZ02416 were crossed with the best plants carrying Pi1 and Pi2 from the BC3F1 population of XWX13/HZ02411 to produce multi-cross F1 (BC3MF1) seeds. BC3MF1 plants were subjected to foreground selection, and those carrying Pi1 + Pi2 + OsHMA3 or Pi1 + Pi2 + OsNramp5 were self-pollinated to generate BC3MF2 seeds. From foreground-screened BC3MF2 plants, individuals carrying the target genes and exhibiting agronomic traits comparable or superior to XWX13 were self-pollinated to produce BC3MF3 seeds. Subsequent foreground and field screening for target traits and overall agronomic performance was conducted across generations. Plants with homozygous genotypes for Pi1 + Pi2 + OsHMA3 or Pi1 + Pi2 + OsNramp5 and superior agronomic traits were repeatedly self-pollinated until BC3MF5 generation (Figure 1).
Four SNP-based KASP markers confirmed the parental genotypes (Table 1). The recurrent parent XWX13 carried none of the target alleles. HZ02411 donated the blast resistance genes Pi1 and Pi2, HZ02415 contributed the low-Cd allele OsHMA3, and HZ02416 supplied the low-Cd allele OsNramp5. Thus, four loci can be pyramided into XWX13 via crosses. The KASP genotype for target genes confirmed that the BC3MF5 improved line iXWX13-1 carried Pi1 + Pi2 + OsHMA3, and iXWX13-2 stacked Pi1 + Pi2 + OsNramp5 (Table 1).
The two improved lines, iXWX13-1 and iXWX13-2, and the original recipient parent XWX13 were further genotyped for genetic background analysis by using the SNP array RICE 1K mGPS. It was found that the genetic background recovery rates of iXWX13-1 and iXWX13-2 were 94.44% and 94.63%, indicating a high genomic similarity with XWX13 (Figure 2 and Figure 3).

3.2. Blast Resistance of the Improved Lines

The two improved lines, iXWX13-1 and iXWX13-2, the recurrent parent (XWX13), CO39 (susceptible control), and Gumei4 (resistant control) were scored at the seedling stage by spraying inoculations of 39 M. oryzae isolates in the greenhouse (Table 2), and the typical symptoms of two strains on five varieties or lines are shown in Figure 4. The two improved lines, iXWX13-1 and iXWX13-2, and the resistant control Gumei4 exhibited broad-spectrum resistance to rice blast, with a high resistance frequency of 97.44%, 100%, and 92.31%, respectively, whereas the recipient variety XWX13 and the susceptible control CO39 showed a low resistance frequency of 17.95% (Table 2). The results verify that the enhanced blast resistance occurs in the two improved lines (possessing Pi1 + Pi2).
The increased resistance of the two improved lines (iXWX13-1 and iXWX13-2) was also observed under two natural blast nurseries in comparison to the recurrent parent (XWX13). In detail, XWX13 showed level 7–8 leaf blast (S-HS) and a 15–18% panicle neck blast infection rate (MS) in the blast nursery of Taojiang County in 2022, and thus was susceptible to rice blast (Table 3, Figure 5). While the two improved lines (possessing Pi1 + Pi2) exhibited a higher-level resistance reaction, both iXWX13-1 and iXWX13-2 showed grade 2 leaf blast (R) and a 4–5% panicle neck blast infection rate (R). In the same year, iXWX13-1 and iXWX13-2 demonstrated resistance to leaf blast (0–1 grade) in the blast nursery of Dawei Mountain, whereas XWX13 showed grade 7 leaf blast (S) (Table 3). In short, the resistance to leaf and panicle neck blast under natural conditions was also effectively enhanced in the two improved lines (possessing Pi1 + Pi2).

3.3. Yield, Agronomic Traits, and Grain Quality of Improved Lines

To test whether the traits of the two improved lines, iXWX13-1 and iXWX13-2, were identical to those of the original recipient parent XWX13, the main agronomic traits of the lines were observed in a multi-site trial in 2022. Based on measurements from five test sites, the two improved lines iXWX13-1 and iXWX13-2 exhibited an average plant height (PH) of 112.7 cm and 112.3 cm and an average 1000-grain weight (GW) of 30.8 g and 30.6 g, respectively (Table 4). Minor differences were found for a few traits overall, including days to maturity (DTM), plant height (PH), and 1000-grain weight (GW). For example, DTM was slightly longer (123 d vs. 120.4 d) for the two lines compared to XWX13, and the GW of iXWX13-1 and iXWX13-2 was slightly higher (0.6–0.8 g) than that of XWX13 together with a decrease in plant height (2–3 cm) among iXWX13-1 and iXWX13-2. In general, no significant difference was observed between the two improved lines and XWX13 for most of the agronomic traits (Table 4), indicating that the main agronomic traits of the improved lines are highly similar to those of XWX13.
The grain yield of these tested lines in the multi-site trial was investigated. The average yields of XWX13, iXWX13-1, and iXWX13-2 from five test sites were 7.50 t/ha, 7.37 t/ha, and 7.51 t/ha, respectively (Table 4). Non-significant differences were found between XWX13 and the improved lines. In terms of each test site, the yield of iXWX13-2 was slightly higher than that of iXWX13-1, but the difference was not statistically significant, as indicated by the single-factor ANOVA test (Table 4).
To profile grain quality, rice grains from the five test sites were investigated for ten important rice quality traits, with a significant difference only found in grain shape (Table 5). For iXWX13-1 and iXWX13-2, grains were shorter (0.2–0.3 mm) compared to XWX13, thus resulting in relatively round grains. The other eight rice quality traits, BRP, MRP, HRP, CRP, CD, ASV, AC, and GC, showed statistically non-significant differences between XWX13 and the improved lines, revealing highly similar grain quality.

3.4. Grain Cd Accumulation in the Improved Lines

To evaluate the grain Cd content under Cd-polluted conditions, the grain Cd concentration of all the lines in the multi-site trial were examined, showing that Cd was hardly accumulated in the grains of iXWX13-2. Across five test sites, the grain Cd concentration in iXWX13-2 ranged from 0.009 to 0.077 mg/kg, significantly lower than that in XWX13 (0.618–1.052 mg/kg) and iXWX13-1 (0.569–0.864 mg/kg) (Figure 6). Compared with the original recipient parent XWX13, the grain Cd concentration in iXWX13-2 decreased by 90.98–98.87%. Regarding grain Cd accumulation in XWX13 and iXWX13-1, significant differences were only detected in two sites, Shangrao and Hezhou, where the grain Cd concentration in iXWX13-1 was relatively lower than that in XWX13 (Figure 6).
Of all tested samples, only the grain Cd concentration in iXWX13-2 (possessing OsNramp5) in each plot was obviously lower than the limit value of 0.2 mg/kg set by the China National Standard for Food Safety (GB 2762-2022), revealing that iXWX13-2 is a stable Cd-safe improved line and superior to iXWX13-1 (possessing OsHMA3). Therefore, iXWX13-2 (stacking Pi1 + Pi2 + OsNramp5) demonstrated enhanced blast resistance and low Cd accumulation without sacrificing the yield, main agronomic traits, and grain quality.

4. Discussion

4.1. Improvement Achieved by Genomic Marker-Assisted Selection

Phenotype-based selection in conventional breeding has played a great role in developing elite rice varieties in the past few decades, although with a long breeding cycle and low efficiency [75,76]. Alternatively, genomic marker-assisted breeding is an innovative approach that utilizes modern molecular tools and genomic information to improve the accuracy and efficiency of plant breeding [57], and it has been successfully applied to improve biotic or abiotic stress tolerance in rice varieties [20,47,77,78,79,80]. Also, high-throughput sequencing and genotyping platforms have accelerated the process of genomic marker-assisted breeding [57]. In this study, an efficient genomic breeding strategy for the integration of blast resistance and low Cd accumulation was adopted based on genomic marker-assisted foreground and background selection. Four major genes, namely Pi1, Pi2, OsHMA3, and OsNramp5, from three donors were precisely introduced into the genetic background of a good-quality fragrant rice variety XWX13 by using genomic marker-assisted selection. Eventually, two preferable improved lines iXWX13-1 (Pi1 + Pi2 + OsHMA3) and iXWX13-2 (Pi1 + Pi2 + OsNramp5) were obtained, with XWX13 background recovery rates of 94.44% and 94.63%. Compared to the traditional 6–8-year breeding cycle, our strategy shortens the cycle by up to 2–3 years, as reported in previous studies [19,20]. Therefore, this study has demonstrated a successful breeding practice using genomics-assisted rapid improvement of XWX13 in blast resistance and low Cd accumulation.

4.2. Pyramiding Genes to Improve Blast Resistance and Cd Accumulation

Blast resistance is easily lost after a few years of cultivation due to the variation and evolution of pathogen populations. For instance, Tang et al. [81] found that elite indica rice varieties from South China with Pi1 were not resistant to blast, while those with Pi2 exhibited an 83.3% resistance rate. Similarly, it was found that multi-gene pyramiding lines showed significantly higher resistance levels than monogenic lines [82]. So, there is an urgent need to pyramid multiple genes in breeding programs [83,84], and pyramiding of multiple resistance genes has proven to be an effective way to develop rice varieties with durable resistance to M. oryzae [20]. As found in our study, the two improved lines iXWX13-1 and iXWX13-2 carrying Pi1 + Pi2 exhibited more than 97.44% blast resistance under artificial inoculation at the seedling stage, indicating an obviously increased resistance spectrum compared with that of the original recipient variety XWX13. Furthermore, the two improved lines iXWX13-1 and iXWX13-2 showed grade 2 leaf blast (R) and a 4–5% panicle neck blast infection rate (R) in the blast nursery in Taojiang County, and thus possess enhanced resistance to leaf and panicle neck blast. The increased resistance to leaf blast was also observed in the blast nursery in Dawei Mountain. These results reveal that it is feasible to pyramid multiple broad-spectrum R genes into elite rice varieties, enhancing their resistance and durability against blast disease. However, under classical models, resistance half-life increases exponentially with gene number. Pyramiding R genes delays resistance breakdown, but we need to recognize that this durability is not absolute. Long-term success hinges on (i) choosing R genes with distinct AVR targets and high fitness costs for the pathogen, (ii) integrating quantitative resistance, and (iii) monitoring pathogen populations to permit timely deployment shifts.
Grain Cd accumulation poses serious safety concerns for the rice industry. To date, various approaches, including water management and administration of contaminated soil additives, have been used to alleviate Cd accumulation in rice grain [85,86]. Correspondingly, some low-Cd rice varieties have been identified and developed through mass selection, marker-assisted selection, and genetic manipulation [39,47]. Hu et al. [80] confirmed that the gene OsNramp5 was the most efficient in lowering Cd accumulation, and first generated low-Cd-accumulating restorer (R) lines by editing OsNramp5, OsLCD, and OsLCT1 in both japonica and indica rice. In the present study, two improved lines iXWX13-1 (Pi1 + Pi2 + OsHMA3) and iXWX13-2 (Pi1 + Pi2 + OsNramp5) were obtained through genomic marker-assisted selection. The evaluation of Cd accumulation in a multi-site trial showed that the grain Cd concentration in iXWX13-2 carrying OsNramp5 decreased by 90.98–98.87% compared with that in the original recipient variety XWX13, which was far below the threshold value of 0.2 mg/kg in China (GB 2762-2022), demonstrating a non-transgenic Cd-safe improved line; it also reconfirmed the stable efficiency of OsNramp5 in lowering Cd concentration [47,80]. Here, statistically significant differences in the grain Cd concentration between XWX13 and iXWX13-1 were observed in two test sites (Shangrao and Hezhou), but not in the other three sites (Changsha, Yueyang, and Yichun), and both of them exceeded the safe threshold value of 0.2 mg/kg, indicating that the improved line iXWX13-1 carrying OsHMA3 had no obvious effect on lowering Cd accumulation. The gene OsHMA3 is not as effective in lowering Cd accumulation as previously reported [47,87,88]. As a typical quantitative trait, the variations in grain Cd accumulation in rice are determined by both the genotypes and the environmental factors that determine Cd phyto-availability in soil [28,37,89], which makes it difficult to accurately evaluate the genetic effects of grain Cd variation [90]. It is usually accepted that there is natural variation in Cd accumulation among different rice varieties [91]. Previous studies have indicated that the indica rice varieties generally accumulate more Cd compared to the japonica rice varieties [92]. However, a consistent conclusion has not been obtained by comparing Cd accumulation in the same genetic background with different Cd-related genes. In our study, the two improved lines iXWX13-2 and iXWX13-1 have the similar genomic background of XWX13 (Figure 2 and Figure 3), and thus OsNramp5 is obviously more effective in lowering grain Cd accumulation than OsHMA3 in the same genetic background (Figure 6). Why might OsHMA3 be ineffective in this background? We speculate that the improved XWX13 harbors the weakly functional OsHMA3-R80H allele. Together with possible promoter-mediated downregulation and epistatic or epigenetic suppression inherent to the indica background, this makes OsHMA3 ineffective in lowering grain cadmium levels. It is suggested that further research be conducted. In addition, the performance of the two genes in other genetic backgrounds needs to be investigated in future studies.

4.3. Improvement in Traits Without Sacrificing Yield, Main Agronomic Traits, and Grain Quality

Plants face the constant challenge of allocating limited resources to immunity and growth to ensure survival and reproduction; therefore, one major aim of crop breeding is to balance biotic resistance and abiotic tolerance with yield [93]. The genomics-assisted breeding strategy can provide more powerful selection of quantitative traits and modify tradeoffs effectively. In this study, the characterization of the two improved lines iXWX13-1 and iXWX13-2 showed that their yield and main agronomic traits were almost identical to those of the recurrent parent XWX13, and the average values for each trait in a multi-site trial were not significantly different. In terms of rice quality, eight traits (brown rice percentage, milled rice percentage, head rice percentage, chalky rice percentage, chalkiness degree, alkali spreading value, amylose content, and gel consistency) showed no statistically significant difference between XWX13 and the improved lines. In view of the potential tradeoff between resistance and yield, multiple traits should be paid attention when screening the target plants selected based on the genomic marker-assisted foreground and background selections [94]. Successful cases selecting superior recombinants in breeding programs have been previously reported [79,95]. In general, when the donor line is a wild relative of the crop, linkage drag is a very common problem which can strongly limit the use of a particular gene in breeding [96]. Our study also provides an optimized strategy for overcoming linkage drag in crop breeding by using a commercial variety harboring favorable genes as an improved donor instead of the wild donor, which helps in further improving the background of the pyramided lines and shortening the breeding cycle.

5. Conclusions

This study successfully developed two improved rice lines, iXWX13-1 and iXWX13-2, using a genomic breeding strategy that integrated marker-assisted foreground and background selection along with phenotypic screening. These lines retain the high yield, agronomic performance, and grain quality of the elite variety XWX13 while introducing critical new traits. The iXWX13-1 serves as a blast-resistant version, and iXWX13-2 as a blast-resistant and low-grain-Cd version, providing safe and sustainable replacements for the original variety. To our knowledge, this is the first report demonstrating the successful application of genomics-assisted backcrossing to simultaneously improve blast resistance and reduce Cd accumulation in rice without sacrificing agricultural or quality traits. This strategy offers an efficient and precise framework for pyramiding multiple desirable genes into elite genetic backgrounds, significantly accelerating crop genetic improvement.

Author Contributions

Conceptualization, Z.X., Z.H. and J.P.; methodology, Y.H. and H.C.; validation, Z.X., Z.H. and Y.W.; formal analysis, Z.X., J.C. and C.Y.; investigation, Y.H., H.C., L.L. and H.Y.; resources, Z.L. and Z.H.; data curation, Z.X., Z.H. and Y.W.; writing—original draft preparation, Z.X. and Z.H.; writing—review and editing, Z.X., Z.H., J.C., C.Y. and J.P.; supervision, Z.H., Y.W. and B.T.; project administration, Z.H., Y.W. and B.T.; funding acquisition, Z.H., L.Z., Y.W. and B.T. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Biological Breeding-National Science and Technology Major Project (2023ZD04076), the Hunan Science and Technology Program (2017NK2022, 2023NK2001), the Sichuan Science and Technology Program (2024YFHZ0191), and the Suzhou Science and Technology Program (SNG2021003).

Data Availability Statement

The original contributions presented in this study are included in the article, and further inquiries can be directed to the corresponding authors.

Conflicts of Interest

Authors Zhi Xu, Yanglan He, Hailong Chen, Changrong Ye, Zhouwei Li, Le Li, Hexing Yin, Lijia Zheng, Yuntian Wu, Bingchuan Tian were employed by the company Huazhi Biotechnology Co., Ltd. Author Junhua Peng was employed by the company Spring Valley Agriscience Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. The scheme for the development of the improved lines iXWX13-1 and iXWX13-2 using molecular marker-assisted backcrossing (MABC). Hybridization began in Sanya, Hainan, in April 2017. XWX13 was used as the recurrent parent for three successive backcross generations, and each generation was detected with the foreground and background SNP markers. Following successive self-pollination of the BC3MF1 population, the BC3MF5 preferable improved lines with homozygous target genes were obtained in 2021, based on foreground selection and field screening of each generation.
Figure 1. The scheme for the development of the improved lines iXWX13-1 and iXWX13-2 using molecular marker-assisted backcrossing (MABC). Hybridization began in Sanya, Hainan, in April 2017. XWX13 was used as the recurrent parent for three successive backcross generations, and each generation was detected with the foreground and background SNP markers. Following successive self-pollination of the BC3MF1 population, the BC3MF5 preferable improved lines with homozygous target genes were obtained in 2021, based on foreground selection and field screening of each generation.
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Figure 2. Genetic background analysis for the improved line iXWX13-1 (Pi1 + Pi2 + OsHMA3) by using Rice 1K mGPS. Twelve chromosomes of rice are labeled from 1 to 12, and the triangle symbols indicate the positions of the target genes. The blue lines indicate the different and homozygous SNP loci between iXWX13-1 and the recurrent parent XWX13.
Figure 2. Genetic background analysis for the improved line iXWX13-1 (Pi1 + Pi2 + OsHMA3) by using Rice 1K mGPS. Twelve chromosomes of rice are labeled from 1 to 12, and the triangle symbols indicate the positions of the target genes. The blue lines indicate the different and homozygous SNP loci between iXWX13-1 and the recurrent parent XWX13.
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Figure 3. Genetic background analysis for the improved line iXWX13-2 (Pi1 + Pi2 + OsNramp5) by using Rice 1K mGPS. Twelve chromosomes of rice are labeled from 1 to 12, and the triangle symbols indicate the positions of the target genes. The green lines indicate the different and homozygous SNP loci between iXWX13-2 and the recurrent parent XWX13.
Figure 3. Genetic background analysis for the improved line iXWX13-2 (Pi1 + Pi2 + OsNramp5) by using Rice 1K mGPS. Twelve chromosomes of rice are labeled from 1 to 12, and the triangle symbols indicate the positions of the target genes. The green lines indicate the different and homozygous SNP loci between iXWX13-2 and the recurrent parent XWX13.
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Figure 4. The typical symptoms of seedling blast resistance of XWX13 and the improved lines. HR, highly resistant; S, susceptible; HS, highly susceptible. (A) The blast isolate 21DWS-2 originated from Dawei Mountain, Liuyang City, Hunan, China, in 2021. (B) The blast isolate 20CHTN1-2 originated from Chunhua Town, Changsha County, Hunan, China in 2020.
Figure 4. The typical symptoms of seedling blast resistance of XWX13 and the improved lines. HR, highly resistant; S, susceptible; HS, highly susceptible. (A) The blast isolate 21DWS-2 originated from Dawei Mountain, Liuyang City, Hunan, China, in 2021. (B) The blast isolate 20CHTN1-2 originated from Chunhua Town, Changsha County, Hunan, China in 2020.
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Figure 5. The performance for leaf blast of XWX13 and the improved lines in the field of Taojiang County, Yiyang City, Hunan, China, in 2022.
Figure 5. The performance for leaf blast of XWX13 and the improved lines in the field of Taojiang County, Yiyang City, Hunan, China, in 2022.
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Figure 6. Comparisons of grain Cd concentration in a multi-site trial. The values of grain Cd concentration given are the mean of three biological replications. ‘ppm’ is the abbreviation of ‘parts per million’ in English. The letters a, b, and c indicate the Student–Newman–Keuls significance at a 5% probability level in each test site based on the single-factor ANOVA test.
Figure 6. Comparisons of grain Cd concentration in a multi-site trial. The values of grain Cd concentration given are the mean of three biological replications. ‘ppm’ is the abbreviation of ‘parts per million’ in English. The letters a, b, and c indicate the Student–Newman–Keuls significance at a 5% probability level in each test site based on the single-factor ANOVA test.
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Table 1. KASP genotype for target genes.
Table 1. KASP genotype for target genes.
GeneMarkerChromosomeTypeKASP Genotype
HZ02411HZ02415HZ02416XWX13iXWX13-1iXWX13-2
Pi1K_11057311SNP+−−−++
Pi2K_0605026SNP+−−−++
OsHMA3K_0705207SNP−+−−+−
OsNramp5OS900248_K017SNP−−+−−+
The plus sign represents the favorable genotype, and the minus sign represents the unfavorable genotype.
Table 2. The lesion scores of rice lines at the seedling stage against 39 M. oryzae isolates.
Table 2. The lesion scores of rice lines at the seedling stage against 39 M. oryzae isolates.
Code of IsolatesPlace of OriginXWX13iXWX13-1iXWX13-2CO39Gumei4
110-2Hunan, China50050
236-1Hunan, China00050
2016CH-2Hunan, China80050
21TJ-9Hunan, China60030
21TJ-18Hunan, China80040
21TJ-23Hunan, China90030
21DWS-2Hunan, China90070
21DWS-4Hunan, China90050
21DWS-19Hunan, China90060
20JYR900-3Hunan, China60040
20JYR900-18Hunan, China70030
20JYR900-23Hunan, China70040
20CHR900-5Hunan, China60040
20CHTN1-2Hunan, China80070
20CHTN1-28Hunan, China80050
E2007046A2Hubei, China70286
E2007038A3Hubei, China70040
19-765-1-2Zhejiang, China40060
19-763-7-2Zhejiang, China70056
RB7Guangdong, China50070
CHL1743Guangdong, China90080
M2006123A3Fujian, China80080
M2006123A1Fujian, China90080
C8-3-1Sichuan, China50030
20DT-1Sichuan, China60060
20DT-6Sichuan, China70050
20DT-14Sichuan, China80060
12-1Sichuan, China40060
19-9-6-2Sichuan, China40020
CH-11391Yunnan, China33050
CH105aYunnan, China30050
2016ZY-8Unknown50050
95097AZC13Unknown50030
chnos60-2-3Unknown50030
ROR1South Korea20060
P06-6Philippines20050
Guy11France84050
ES6Spain30050
IC-17America30044
Number of resistant isolates73839736
Number of susceptible isolates3210323
Resistance frequency17.95%97.44%100.00%17.95%92.31%
The lesion scores of 0–3 are classified as resistant, and 4–9 are classified as susceptible against the isolates.
Table 3. Evaluation of leaf and panicle neck blast resistance of XWX13 and the improved lines in the blast epidemic fields in 2022.
Table 3. Evaluation of leaf and panicle neck blast resistance of XWX13 and the improved lines in the blast epidemic fields in 2022.
EntriesDawei Mountain, Liuyang CityTaojiang County, Yiyang City
Leaf Blast ScoreLeaf Blast ScorePanicle Neck Blast
Infection Rate
CO397750%
Fengliangyou47740%
XWX137818%
iXWX13-1024%
XWX137715%
iXWX13-2125%
Gumei4226%
Table 4. Yield and main agronomic traits of XWX13 and improved lines in multi-site trial in 2022.
Table 4. Yield and main agronomic traits of XWX13 and improved lines in multi-site trial in 2022.
SitesEntriesYDDTMPHPLSPPFGPGW
ChangshaXWX136.70116.0116.223.5128.281.129.6
iXWX13-16.53116.0115.923.8120.377.832.9
iXWX13-26.72116.0115.223.6125.379.631.8
YueyangXWX138.19115.0116.524.1120.384.133.2
iXWX13-18.15115.0115.324.3125.382.633.2
iXWX13-28.25115.0114.824.4124.882.433.1
YichunXWX137.99125.0133.023.8144.982.128.3
iXWX13-17.89130.0128.421.9140.774.227.6
iXWX13-28.05132.0128.822.8142.576.428.1
ShangraoXWX137.13128.095.521.2152.876.325.1
iXWX13-16.93136.091.522.4141.176.627.2
iXWX13-27.15134.091.221.8150.477.426.8
HezhouXWX137.49118.0115.223.9118.581.933.5
iXWX13-17.34118.0112.624.1127.479.333.0
iXWX13-27.37118.0111.623.6122.479.133.3
MeanXWX137.50120.4115.323.3132.981.130.0
iXWX13-17.37123.0112.723.3131.078.130.8
iXWX13-27.51123.0112.323.2133.179.030.6
F0.0760.1630.0710.0050.0441.5150.097
p-value0.9270.8510.9310.9950.9570.2590.909
Student–Newman–Keuls (p = 0.05)0.9360.8750.9350.9960.9620.2470.910
YD, yield (t/ha); DTM, days to maturity (d); PH, plant height (cm); PL, panicle length (cm); SPP, spikelets per panicle; FGP, filled-grain percentage (%); GW, 1000-grain weight (g). The single-factor ANOVA test was performed to examine the significance of variation in yield and agronomic traits using the software SPSS Statistics 19.
Table 5. The grain quality of XWX13 and the improved lines in the multi-site trial.
Table 5. The grain quality of XWX13 and the improved lines in the multi-site trial.
SitesEntriesBRPMRPHRPCRPCDRGLL/WASVACGC
ChangshaXWX1380.570.455.111.42.76.93.32.514.648.8
iXWX13-180.770.954.014.52.56.73.22.214.746.0
iXWX13-280.770.854.712.02.66.83.22.314.647.0
YueyangXWX1379.071.865.38.32.26.93.53.614.244.8
iXWX13-179.571.562.08.42.06.43.33.614.555.5
iXWX13-279.271.764.39.02.16.73.23.514.450.0
YichunXWX1379.070.663.07.22.36.83.42.214.051.5
iXWX13-179.370.963.94.41.36.63.22.016.055.0
iXWX13-279.470.863.65.01.56.63.32.115.554.0
ShangraoXWX1380.972.160.721.65.57.03.42.814.644.0
iXWX13-180.971.556.122.94.86.63.23.316.545.0
iXWX13-280.571.858.522.04.96.73.23.116.344.0
HezhouXWX1380.070.349.611.42.66.83.21.414.960.8
iXWX13-180.370.741.316.83.36.63.11.316.365.8
iXWX13-280.470.944.813.02.86.63.11.316.264.0
MeanXWX1379.8871.0458.7411.983.066.88 a3.36 a2.5014.4649.98
iXWX13-180.1471.1055.4613.402.786.58 b3.20 b2.4815.6053.46
iXWX13-280.0471.2057.1812.202.786.68 b3.20 b2.4615.4051.80
F0.1490.0880.2210.0700.07313.4625.5650.0033.1350.256
p-value0.8630.9160.8050.9320.9300.001 *0.019 *0.9970.0800.778
BRP, brown rice percentage (%); MRP, milled rice percentage (%); HRP, head rice percentage (%); CRP, halky rice percentage (%); CD, chalkiness degree (%); RGL, rice grain length (mm); L/W, grain length/width ratio; ASV, alkali spreading value; AC, amylose content (%); GC, gel consistency (mm). The letters a, b and the asterisk symbol indicate a significance of difference at the 5% probability level based on the single-factor ANOVA test.
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Xu, Z.; He, Z.; He, Y.; Chen, H.; Cheng, J.; Ye, C.; Li, Z.; Li, L.; Yin, H.; Zheng, L.; et al. Genomics-Assisted Improvement in Blast Resistance and Low Cadmium Accumulation in an Elite Rice Variety. Agronomy 2025, 15, 2130. https://doi.org/10.3390/agronomy15092130

AMA Style

Xu Z, He Z, He Y, Chen H, Cheng J, Ye C, Li Z, Li L, Yin H, Zheng L, et al. Genomics-Assisted Improvement in Blast Resistance and Low Cadmium Accumulation in an Elite Rice Variety. Agronomy. 2025; 15(9):2130. https://doi.org/10.3390/agronomy15092130

Chicago/Turabian Style

Xu, Zhi, Zhizhou He, Yanglan He, Hailong Chen, Jihua Cheng, Changrong Ye, Zhouwei Li, Le Li, Hexing Yin, Lijia Zheng, and et al. 2025. "Genomics-Assisted Improvement in Blast Resistance and Low Cadmium Accumulation in an Elite Rice Variety" Agronomy 15, no. 9: 2130. https://doi.org/10.3390/agronomy15092130

APA Style

Xu, Z., He, Z., He, Y., Chen, H., Cheng, J., Ye, C., Li, Z., Li, L., Yin, H., Zheng, L., Wu, Y., Tian, B., & Peng, J. (2025). Genomics-Assisted Improvement in Blast Resistance and Low Cadmium Accumulation in an Elite Rice Variety. Agronomy, 15(9), 2130. https://doi.org/10.3390/agronomy15092130

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