Genetic analysis of arsenic accumulation in maize using QTL mapping

Arsenic (As) is a toxic heavy metal that can accumulate in crops and poses a threat to human health. The genetic mechanism of As accumulation is unclear. Herein, we used quantitative trait locus (QTL) mapping to unravel the genetic basis of As accumulation in a maize recombinant inbred line population derived from the Chinese crossbred variety Yuyu22. The kernels had the lowest As content among the different maize tissues, followed by the axes, stems, bracts and leaves. Fourteen QTLs were identified at each location. Some of these QTLs were identified in different environments and were also detected by joint analysis. Compared with the B73 RefGen v2 reference genome, the distributions and effects of some QTLs were closely linked to those of QTLs detected in a previous study; the QTLs were likely in strong linkage disequilibrium. Our findings could be used to help maintain maize production to satisfy the demand for edible corn and to decrease the As content in As-contaminated soil through the selection and breeding of As pollution-safe cultivars.

population from a Bala × Azucena population 28 . In maize, many studies have focused on the physiological and biochemical responses to As accumulation. The majority of these studies demonstrated a trend of decreasing As content from the roots to the aerial parts, including the leaves, stems and seeds 1 . Maize takes up the arsenic naturally present in the soil or arsenic that is added through groundwater irrigation or by soil additives contaminated with arsenic. Several studies have described a significant relationship between the As concentration in the irrigation water or soil and the total As content accumulated by maize plants 29 . Gulz et al. observed that the correlation between the total accumulated As in maize plants and the water-soluble As fraction in the soil was higher than the total As content in the soil 30 . Several factors, including pH, redox potential, organic matter content, interaction/ competition with other elements and chemical forms of the pollutant, can affect As solubility in soils 31 .
Maize is the most cultivated cereal in the world and is used as an important animal feed or a staple food crop for humans in many developing countries in Africa, Asia and Latin America 1 . Hence, maize grown on As-contaminated land could accumulate As and pose a risk to human health. Thus, methods to reduce As accumulation in maize are urgently needed. However, there have been few reports of mapped QTLs associated with As accumulation and distribution in maize. In the present study, a RIL population derived from parents with contrasting As tolerances was studied at two locations where the soil As levels substantially differed. The accumulation and distribution of As in different maize tissues were examined. Additionally, we generated data that may aid QTL mapping for important traits in breeding populations for the genetic improvement and production of maize pollution-safe cultivars.

Results
Performance of the measured traits at two locations. The soil As concentration at Xixian was 20.70 ± 0.37 mg/kg (pH = 6.5) because of irrigation with As-rich surface water. While the soil As concentration at Changge was 12.24 ± 0.21 mg/kg (pH = 6.5), which was used as a control. In terms of As concentration of the two parents and the hybrid, the five measured tissues showed higher levels at Xixian compared with Changge (Table 1, Fig. 1). The As content in the five maize tissues examined varied widely in the RIL population, which showed two-way transgressive segregation. In addition, the average As concentration in the RIL population was higher at Xixian than at Changge. The results indicated that soil As concentration is an important factor affecting the As content in maize tissues.
Variance analysis of As accumulation in different maize tissues. The trend of As concentration in different maize tissues at the two locations was as follows: kernels < axes < stems < bracts < leaves. Analysis of variance indicated the As concentration in the five measured tissues in the RIL population was significantly affected by environment, block, genotype and genotype-by-environment factors ( Table 2). The As concentration in the kernels largely differed by genotype and genotype-by-environment factors, explaining 32.03% and 39.86% of the total variation, respectively. However, the environment and block factors only contributed 8.43% and 1.16% of the total variation, respectively. The same trend was observed for the As concentration in the axes, stems, bracts and leaves. These results indicated that the differences in As concentrations in different maize tissues mostly depended on the genotype and the interaction between the genotype and the environment. The Pearson correlation coefficient was used to calculate the correlation of As content among the different tissues. The results showed that there were no significant relationships among the As concentration in the five measured tissues at the two locations (Table 3).

QTL analysis of As content in different maize tissues. QTLs identified in the two locations.
Twenty-eight QTLs related to the As concentration in the different maize tissues were detected in plants at the two locations (Table 4, Fig. 2). The QTLs were found on all chromosomes, except chromosomes 3 and 6. Six QTLs    QTLs identified by best linear unbiased predictions. Eleven QTLs were detected in a joint analysis of the two locations (see the best linear unbiased predictions (BLUP) section of Table 4). Six QTLs including one for the axes, three for the stems, one for the bracts and one for the leaves were also detected in a single environment analysis in common marker intervals. Additionally, some QTLs detected in the joint analysis were not detected in the single environment analysis (e.g., BAsK8, BAsA4a, BAsA10, BAsB7b and BAsL4). These may have been only minor QTLs and were not stable across different environments.

Discussion
In the present study, we found that As levels in maize tissues followed the trend: leaves > bracts > stems > axis > kernels. Regarding environmental As content, Gulz et al. observed that the As content in maize roots was positively correlated with the total As content in the soil 30 . In a study conducted in Thailand, Prabpai et al. reported that there was a direct linear relationship between As accumulation in maize tissues and the total soil As content 29 . Additionally, in the present study, we observed that the maize tissue As concentrations were significantly affected by the genotype, environment and genotype-by-environment interactions. However, there were small environment-related differences in the As concentrations in the various maize tissues, and the genotype and genotype-by-environment interactions contributed more to the total variation. These results suggest that As concentration in different maize tissues could be reduced through genetic improvement.
To the best of our knowledge, only one article has reported QTLs related to As accumulation in different maize tissues. Ding et al. detected 11 QTLs for As accumulation in four different maize tissues 24 . In our study, 14 QTLs were identified at each location and 11 QTLs were identified in a joint analysis. Some of these QTLs were identified in different environments and were also detected by joint analysis. Compared with the B73 RefGen v2 reference genome, the distributions and effects of certain QTLs were closely linked to those of QTLs detected in previous studies. Most QTLs clustered at 27. found two QTLs related to drought tolerance using a RIL population derived from the parents, Zong3 and 87-1 33 . XAsL4a was found in common regions with a QTL for Hg accumulation, which had been identified using the same population in a previous study 34 . There may be strong linkage disequilibrium between QTLs and a genome-wide association study (GWAS) will be conducted to test the linkage disequilibrium in the future. Our results and those reported in previous studies  identified certain chromosomal regions that should be analyzed further. These regions may represent targets for marker-assisted selection of maize cultivars with low As concentrations. In maize, kernels are the main edible parts for humans and animals. In this study, the kernels contained the lowest As concentration, while the main biomass products including the leaves, bracts, stems and axes had relatively high As concentrations. Additionally, the As concentration in the kernels was considerably lower than the limit of 200 μ g/kg specified in the National Standard of China (GB2762-2005). Maize is capable of adapting to its environment and is widely planted globally. Therefore, it is important to ensure maize production continue to satisfy the global demand for edible corn and to decrease the As content in As-contaminated soil by selecting and breeding As pollution-safe cultivars.

Materials and Methods
Experimental locations. The    Determination of the As concentration in maize. All plant materials were harvested when they reached physiological maturity and five consecutive plants from each plot were selected for further analysis. Mature plants were dissected into five parts: kernels, axes, stems, bracts and leaves. The collected plant materials were dried and ground into fine powder using a mortar and pestle. Powdered samples (0.5 g) were added to polypropylene tubes and digested with 5 mL HNO 3 /HClO 4 (80/20 v/v) using a heating block (AIM500 Digestion System, A.I. Scientific, Australia). The concentrations of As in the different plant materials were then determined using atomic fluorescence spectrometry (AFS-3000, Beijing Haiguang Analytical Instrument Co., Beijing, China).

Statistical analysis.
The expected genotypic variance (G), block variance (B), environmental variance (E) and G × E interaction were estimated by two-way ANOVA using the IBM SPSS Statistics package. The broad-sense heritabilities were calculated for genotype variance of each effect by the total sum of genotype variance and environmental variance. In the present study, 194 F8 generation RILs, which were derived from a cross between inbred line Zong3 and 87-1, were used as the materials. The two parents have contrasting levels of As accumulation. The soils of the test locations also have contrasting As contents. The materials and test locations were selected such that the G, E, and G × E terms were fitted as a fixed effect.
Linkage map construction and QTL analysis. The genetic linkage map, consisting of 263 simple sequence repeat markers, was constructed using Mapmaker 3.0 and covered 2,361 cM, with an average interval of 9 cM between markers 35 . The QTLs were identified with Model 6 of the Zmapqtl module of QTL Cartographer 2.5 using the composite interval mapping method 36 . The logarithm of odds thresholds for all measured traits were calculated by 1,000 random permutations at a significance level of α = 0.05, scanning intervals of 2 cM between markers and putative QTLs, with a 10 cM window 37 . The number of marker cofactors for background controls was determined by stepwise regression with five controlling markers. The phenotypic data for each measured material were based on the average values of three replicates. BLUP of arsenic concentration for each plant material at the two locations was calculated by a random effects model using the MIXED procedure in SAS. The QTLs were annotated as follows: for example, for XAsK1a, the "X" indicates the location at which the QTL was detected (Xixian, Changge and BLUP were abbreviated as X, C and B, respectively), "AsK" represents the arsenic concentration of the kernels (axes, stems, bracts and leaves were abbreviated as A, S, B and L, respectively), the number "1" is the serial number of the chromosome and "a" represents the serial number of the identified QTL.