Characterization of a major QTL for manganese accumulation in rice grain

Some diets lack sufficient manganese (Mn), an essential mineral. Increasing Mn in grain by biofortification could prevent Mn deficiency, but may increase levels of the toxic element cadmium (Cd). Here, we investigated Mn in rice (Oryza sativa) grains in recombinant inbred lines (RILs) from the cross of 93–11 (low grain Mn) with PA64s (high grain Mn). Quantitative trait locus (QTL) analysis to identify loci controlling grain Mn identified a major QTL, qGMN7.1, on the short arm of chromosome 7; qGMN7.1 explained 15.6% and 22.8% of the phenotypic variation in the RIL populations grown in two distinct environments. We validated the QTL with a chromosome segment substitution line (CSSL), CSSL-qGMN7.1, in the 93–11 background harboring qGMN7.1 from PA64s. Compared to 93–11, CSSL-qGMN7.1 grain had increased Mn and decreased Cd concentrations; CSSL-qGMN7.1 roots also showed enhanced Mn uptake. Fine mapping delimited qGMN7.1 to a 49.3-kb region containing OsNRAMP5, a gene responsible for Mn and Cd uptake. Sequence variations in the OsNRAMP5 promoter caused changes in its transcript level, and in grain Mn levels. Our study thus cloned a major QTL for grain Mn concentration in rice, and identified materials for breeding rice for high Mn and low Cd concentrations in the grain.


Results
The qGMN7.1 from PA64s significantly increased grain Mn concentration. The RIL population from the rice super hybrid LYPJ, and the hybrid parents were grown in two different environments, Hainan (110.0 E, 18.5 N) and Hangzhou (120.0 E, 30.1 N), China. Mature seeds were harvested for determining the grain Mn concentration. The concentration of Mn in the grains was significantly different between the parents in both Hainan and Hangzhou, with concentrations in PA64s approximately 2-fold higher than in 93-11 ( Fig. S1 and Table S1). The RIL population showed a wide range of phenotypic variation, in a continuous distribution (Fig. S1). Using the high-resolution SNP map, we detected 12 QTLs for grain Mn concentration distributed on all chromosomes except for chromosomes 10, 11, and 12 ( Fig. S2 and Table S2). Among those QTLs, 5 were identified in the RIL populations grown in both Hainan and Hangzhou, and 8 had additive effects coming from PA64s. One major QTL with the highest LOD value, qGMN7.1, was mapped between markers SNP7-53 and SNP7-64 on the short arm of chromosome 7 and explained 15.6% and 22.8% of the phenotypic variation in the RIL populations grown in Hangzhou and Hainan, respectively ( Fig. S2 and Table S2).
Physiological characteristics of CSSL-qGMN7.1. We performed a series of physiological experiments to determine the physiological mechanism underlying the increased grain Mn concentration conferred by qGMN7. 1. In a time-course experiment of grain Mn accumulation, no significant difference was found between 93-11 and CSSL-qGMN7.1 at the early grain-filling stage, although both lines showed decreasing accumulation with time ( Fig. 2a). At the 18th day after heading, the grain Mn concentration stabilized and then significantly increased from the 24th day after heading in CSSL-qGMN7.1 compared to 93-11. The difference remained significant through maturity (Fig. 2a). Overall, CSSL-qGMN7.1 and 93-11 had a similar pattern of Mn accumulation, and qGMN7.1 functioned during the late grain-filling stage.
We also measured Mn concentrations in flag leaves at different times after heading and found that Mn increased in both CSSL-qGMN7.1 and 93-11, with higher levels in CSSL-qGMN7.1, from the late filling stage to maturity (Fig. 2b). To determine whether Mn is transferred from other organs, such as the flag leaf, into the grain at the late filling stage, we removed flag leaves and measured the effect on Mn in the grains. Removal of flag leaves at the heading and filling stages did not affect Mn accumulation in the grains (Fig. 2c, d). These results suggested that Mn is not transferred from flag leaves to the grains at the grain-filling through maturity stages.
As the distribution of Mn in the aboveground parts has been reported to play an important role in grain Mn accumulation, we analyzed the Mn concentration in different organs at maturity. The highest Mn concentration was observed in flag leaf blades, with approximately 3,500 mg·kg −1 dry weight (DW), and the lowest in the grains with only about 50 mg·kg −1 DW in the rice plants (Fig. 3a). Compared to 93-11, CSSL-qGMN7.1 accumulated higher concentrations of Mn in the grains, lemma, panicle branches, and flag leaf blades. However, Mn concentrations in other leaves and stems were almost the same in both lines (Fig. 3a). An analysis of the proportion of Mn content in different organs to the whole plant showed that the proportion of grain Mn content/whole plant Mn content was similar between CSSL-qGMN7.1 and 93-11 (Fig. 3b).
These results indicate that the higher Mn concentration in the grains of CSSL-qGMN7.1 did not result from greater distribution from other organs, but from higher assimilation in the roots. To confirm this, we performed a short-term (30 min) uptake experiment using intact roots of 93-11 and CSSL-qGMN7.1. The Mn uptake at 4 °C was much lower than that at 25 °C in both lines, but CSSL-qGMN7.1 exhibited higher Mn uptake than 93-11 at both temperatures (Fig. 4a). The net Mn uptake, calculated by subtracting the Mn uptake at 4 °C from that at 25 °C, was significantly greater in CSSL-qGMN7.1 than in 93-11 (Fig. 4b). Although CSSL-qGMN7.1 and 93-11 had a similar affinity for Mn, the value of V max for CSSL-qGMN7.1 (118.6 mg·kg −1 root DW·h −1 ) was significantly higher than that for 93-11 (103.4 mg·kg −1 root DW·h −1 ) (Fig. 4). were identified (Fig. 5). Nine newly developed insertion/deletion (InDel) markers, well distributed within the interval, were used to further genotype the 12 recombinants ( Fig. 5b and Table S3). We tested the F 2:3 progeny of the recombinants in Hangzhou in 2016. Grain Mn concentrations showed no significant difference among the three genotypes of recombinants Line 1, Line 2, and Line 8 ( Table 1). For the other recombinant lines, qGMN7.1 segregated and significant differences in phenotypes were found in the three genotypes of qGMN7.1 (Table 1). Based on the phenotypes and genotypes of these recombinants, we delimited qGMN7.1 to a region of approximately 49.3-kb between markers L8857 and L8906 (Fig. 5c, d).   6,9,12,18,24,30, and 36 days after heading (a), grains were tagged and harvested until maturity after their corresponding flag leaves were sampled at the heading, grain-filling, and maturity stages (c). (b,d) Flag leaf Mn concentration of 93-11 (red) and CSSL-qGMN7.1 (blue). Flag leaves were harvested at 0, 3, 6, 9, 12, 18, 24, 30, and 36 days after heading (b), flag leaves were sampled at the heading, grain-filling, and maturity stages (d). Vertical bars represent the standard deviation (n = 4). *and **indicate a 5% and 1% significance level, respectively, according to the t test. We also measured the Cd concentration in the grains of the F 2:3 progeny of the recombinants. Three genotypes in the recombinants Line 1 to Line 4 and Line 6 showed no significant difference in grain Cd concentration, and recombinants Line 8 to Line 10 and Line 12 exhibited segregating phenotypes (Table 1). Combined with the genetic recombination sites of these recombinants (Fig. 5c), we concluded that qGMN7.1 had little influence on Cd accumulation in the grain. Furthermore, the Cd concentration in the grain of recombinant Line 6 was about 300 µg·kg −1 DW, much lower than that of recombinant Line 1 to Line 4 (500 to 600 µg·kg −1 DW), indicating that the allele from PA64s could greatly decrease grain Cd concentration.

OsNRAMP5 is the candidate gene for the grain Mn accumulation trait. The Rice Genome Annotation
Project (http://rice.plantbiology.msu.edu/) predicted five genes in the 49.3-kb target region of qGMN7.1 (Fig. 5d): LOC_Os07g15350 encoding a transposon, LOC_Os07g15360 and LOC_Os07g15390 encoding retrotransposons, LOC_Os07g15400 encoding an expressed protein, and LOC_Os07g15370 encoding a metal transporter. Because LOC_Os07g15370 has been reported previously as OsNRAMP5, encoding a major transporter for Mn and Cd 12 , it was considered the most likely candidate gene for the grain Mn accumulation trait in qGMN7.1.
Sequence alignment of OsNRAMP5 between the two parents, 93-11 and PA64s, revealed no polymorphisms in the coding sequence, but 12 variations in the promoter region (Figs 5e and 6a). These sequence variations might alter the transcript levels and be responsible for the different phenotypes. Therefore, we measured the expression levels of OsNRAMP5 in 93-11 and CSSL-qGMN7.1 at different developmental stages. At the seedling stage, CSSL-qGMN7.1 had significantly higher transcript levels of OsNRAMP5 than in 93-11, with the largest difference of 3.7-fold found in the roots. Higher transcript levels were also observed in CSSL-qGMN7.1 at the booting stage, particularly in the roots (Fig. 6b, c). We then compared the promoter activity of OsNRAMP5 between 93-11 and PA64s by transient expression in rice protoplasts. The green fluorescent signals of GFP driven by the PA64s promoter were stronger than those driven by the 93-11 promoter (Figs 6d and S4), and the GUS transcript levels driven by the PA64s promoter also showed a higher level compared to that driven by the 93-11 promoter (Fig. 6e). These results suggested that the OsNRAMP5 promoter from PA64s was stronger than the OsNRAMP5 promoter from 93-11. To validate that OsNRAMP5 is responsible for Mn accumulation in the grain, we overexpressed it in 93-11. A significantly larger abundance of OsNRAMP5 transcript was found in roots (2 fold) and shoots (2-3 fold) of the overexpression lines than in 93-11 (Fig. 6f). The overexpression lines accumulated more Mn in the grains than 93-11 when grown in pots (Fig. 6g). However, the Cd concentration in the grains was nearly equal in 93-11 and the overexpression lines (Fig. 6h). Therefore, we concluded that OsNRAMP5 is responsible for the grain Mn accumulation trait in qGMN7.1.
To gain further insight into the variations of the OsNRAMP5 promoters, we isolated and compared the 2-kb 5′-flanking regions of OsNRAMP5 from 30 different rice varieties. Based on the promoter sequences, three haplotypes were identified ( Fig. 7a and Table S4). Among the 30 rice varieties, 13 had the same haplotype as PA64s (designated as type I) and 14 coincided with that of 93-11 (designated as type II). The promoters from varieties TN1, NJ6, and No.565 were consistent with type II, with the exception of nucleotide variations at positions −1,866 (A → T) and −1,550 (G → T) from the start codon ATG (these were designated as type III) (Fig. 7a). Compared to type I, the rice varieties containing type II and III promoters exhibited lower expression of OsNRAMP5 and lower accumulation of Mn in the grains (Fig. 7b, c).

Discussion
To date, hundreds of QTLs related to grain mineral elements have been identified in rice, but few have been fine mapped or cloned. In this study, we analyzed the Mn concentration in the grains of the RILs derived from the rice super hybrid LYPJ and found 12 putative QTLs in two testing environments. Among them, qGMN7.1, detected on the short arm of chromosome 7 in both environments, accounted for the largest proportion of phenotypic variation (Fig. S2 and Table S2). In the same chromosomal region, a major QTL was also detected by Ishikawa Previous studies showed that Mn content was significantly correlated with the contents of other mineral elements in grains, such as Mg, Fe, Zn, or Cu, suggesting that the contents of these elements might be controlled by common genes 23,24 . However, the concentrations of Mg, Fe, Zn, and Cu in the grains were nearly equal between CSSL-qGMN7.1 and 93-11 ( Fig. 1d-g). Though the Cd concentration in the grains of CSSL-qGMN7.1 was much lower than that of 93-11, fine mapping of qGMN7.1 demonstrated that it had little influence on Cd accumulation in the grains (Table 1). These results implied that qGMN7.1 might be specialized for controlling Mn accumulation in the grains. Considering that agronomic traits could also affect the accumulation of elements in grains 27,28 , we investigated 9 traits of 93-11 and CSSL-qGMN7.1 and found no significant differences between them (Table S5). Therefore, CSSL-qGMN7.1 is an ideal material for rice breeding due to its improved Mn concentration and decreased Cd concentration in the grains without an accompanying loss of yield.
The role of OsNRAMP5 in controlling Mn uptake and transport has been reported in rice [12][13][14][15]29 . OsNRAMP5 was constitutively expressed in the roots and encodes a plasma membrane-localized protein that belongs to the natural resistance associated macrophage protein (NRAMP) family, whose members function as proton-coupled metal ion transporters that can transport Mn 2+ , Zn 2+ , Cu 2+ , Fe 2+ , Cd 2+ , Ni 2+ , Co 2+ , and Al 3+ 12,30 . OsNRAMP5 encodes a major transporter responsible for Mn uptake in rice; knockout of OsNRAMP5 resulted in a significant decline in grain Mn concentrations compared with the wild type 12 . Ishimaru et al. also suggested that OsNRAMP5 could play a role in Mn transport during flowering and seed development 15 .
Based on the RILs and the backcross population, we fine mapped qGMN7.1 to a 49.3-kb region containing OsNRAMP5 (Fig. 5d and Table 1). Although we did not find any alterations in the coding sequence of OsNRAMP5 between 93-11 and PA64s, we did find nucleotide differences in the promoter region (Figs 5e and  6a). These sequence variations lead to differences in the expression level of OsNRAMP5 and in Mn accumulation in the grains between CSSL-qGMN7.1 and 93-11 (Fig. 6b, c). Overexpression of OsNRAMP5 in the 93-11 variety increased the grain Mn concentration (Fig. 6f, g). Therefore, we inferred that the expression level of OsNRAMP5 contributed to Mn accumulation in the grains. Variations in promoter sequences commonly lead to phenotypic variation in rice [31][32][33][34][35] . The expression of OsNRAMP1 in roots was higher in high Cd-accumulating varieties (Habataki, Anjana Dhan, Jarjan) compared to low Cd-accumulating varieties (Sasanishiki, Nipponbare) due to a  Table 1. Progeny test of nine recombinants for confirmation of the fine-mapped region of qGMN7.1. Note: Type 1, type 2, and type 3 in each panel represent the segregated genotypes of the recombinants. '9' , 'P' , and 'H' represent the homozygote of 93-11 and PA64s, and the heterozygote of the parents, respectively. *and **indicate a 5% and 1% significance level compared to type I, respectively, according to the t test (n = 6).
400-bp deletion in the promoter region of OsNRAMP1 in the high Cd-accumulating varieties 31 . Consistent with previous reports, we also found that some low Mn-accumulating varieties had similar OsNRAMP5 promoter sequences as 93-11, which contains low concentrations of Mn in the grains, whereas high Mn-accumulating varieties, including Nipponbare, exhibited promoter sequences similar to PA64s, which is known for high grain Mn concentrations (Fig. 7). Four major transport processes are involved in the accumulation of mineral elements: (1) root uptake, (2) root-to-shoot translocation by xylem flow, (3) distribution in aboveground tissues, and (4) remobilization from leaves 9 . In our study, Mn content in the flag leaves increased from the heading stage to maturity in the time-course experiment (Fig. 2b). Removal of the flag leaves at the heading and grain-filling stages did not affect Mn accumulation in the grains (Fig. 2c, d). Therefore, we speculated that elevation of Mn concentrations in the grain did not occur due to remobilization from the leaves. In addition, CSSL-qGMN7.1 and 93-11 showed little difference in Mn distribution (Fig. 3). However, higher Mn uptake activity was found in CSSL-qGMN7.1 compared to 93-11 (Fig. 4). Considering that OsNRAMP5 was constitutively expressed in roots (Fig. 6b, c) and that its expression was higher in CSSL-qGMN7.1 compared with 93-11, we concluded that OsNRAMP5 is responsible for the increased Mn concentrations in the grain by enhancing Mn uptake in roots.
OsNRAMP5 has also been reported to function as a Cd/Fe transporter [12][13][14]36,37 . OsNRAMP5-knockdown rice lines accumulated more Cd in the shoots, but the total Cd content was lower than in the wild-type plants 36 . The OsNRAMP5 knockout line lost the ability to take up Mn and Cd concurrently 12 , and the osnramp5 mutant exhibited decreased Cd (as well as Mn) concentrations in the straw and grain 13 . These studies demonstrated that the entry of Cd into rice root cells occurred via this Mn transporter, OsNRAMP5. However, in our study, CSSL-qGMN7.1 accumulated lower amounts of Cd than 93-11, contrary to the higher amounts of Mn in CSSL-qGMN7.1 compared to 93-11 (Fig. 1b, c). The recombinants that showed segregation at the qGMN7.1 region exhibited no significant differences in grain Cd concentrations (Table 1), and the overexpression lines of OsNRAMP5 did not accumulate more Cd in the grains compared with 93-11 (Fig. 6h). A possible explanation is that when plants are grown on relatively high-Mn and low-Cd soils (Hangzhou, pH = 6.04 ± 0.02; 480.35 ± 51.02 mg/kg Mn; 0.64 ± 0.12 mg/kg Cd), Cd does not readily accumulate in the grains. Additionally, an antagonistic effect may exist between Mn and Cd uptake. That is, OsNRAMP5 is mainly responsible for the transport of Mn, but not Cd when Mn is abundant. Alternatively, another locus may exist for grain Cd concentration in the substituted segments of CSSL-qGMN7.1 (Table 1), which displayed lower Cd accumulation levels when compared with 93-11.  The chromosomal segment substitution line, CSSL-qGMN7.1, was selected from the advanced backcross population (BC 4 F 2 ) derived from a cross of the recurrent parent 93-11 and the donor parent PA64s (Table S3). In 2015, 93-11 and CSSL-qGMN7.1 were grown in the paddy field of Hangzhou and Hainan. Both lines were also grown in pots inside a net enclosure in Hangzhou. Each pot was filled with 4 kg of sterilized paddy soil and amended with 2 mg/kg −1 CdCl 2 . The soil was maintained in a flooded state before heading, then kept moist until maturity.

Materials and Methods
In the paddy field of Hangzhou (2016), flag leaf blades from 93-11 and CSSL-qGMN7.1 were sampled at 0, 3,6,9,12,18,24,30, and 36 days after heading, and the grains were reaped at 6,9,12,18,24,30, and 36 days after heading. At maturity, the aboveground parts of 93-11 and CSSL-qGMN7.1 were reaped and segregated as grain, lemma, panicle branches, flag leaf blade, flag leaf sheath, lower leaf blade, lower leaf sheath, node, and basal stem. In the pot experiment, the flag leaf blades were harvested at heading, filling, and maturity stages, while the grains were labeled and retained to maturity. All samples were dried at 65 °C for 3 d and then weighed before determination of Mn concentration.
Statistical analysis and QTL mapping. Statistical analysis was conducted by SAS (version 9.0).
Broad-sense heritability was estimated as described by Singh and Chaudhary 38 . QTL analysis was performed with the MultiQTL package (www.mutiqtl.com) using the maximum likelihood interval mapping approach for the RILs. For major effect QTLs, the LOD threshold was obtained based on a permutation test (1,000 permutations, P = 0.05) for each dataset. We followed the suggestions by McCouch for the QTL nomenclature 39 .
Fine mapping of qGMN7.1. To fine map qGMN7.1, CSSL-qGMN7.1 was crossed with 93-11. A total of 4,943 segregating F 2 individuals were grown in Hainan in 2015. Twelve recombinants were genotyped with previously developed insertion/deletion (InDel) markers supplied in Table S3. The progeny of these recombinants were grown and genotyped in Hangzhou (2016). Mature seeds were collected and prepared for mineral determination as described below.
Short-term Mn uptake experiment. To compare the transport activity for Mn between 93-11 and CSSL-qGMN7.1, we performed a short-term (30 min) uptake experiment according to a previous method 12 . The seedlings (28 d old) of these two lines were exposed to the nutrient solution without Mn for 1 week and then subjected to the uptake solution containing various concentrations of Mn (0.0, 0.1, 0.5, 5, 10, 50, or 100 mM) at 25 °C and 4 °C with four replicates per treatment. After 30 min, the roots were washed three times with 5 mM CaCl 2 and separated from the shoots. The roots were dried at 70 °C for 3 d and used for mineral determination as described below.
Determination of metals in plant tissues. The dried samples were digested with a mixture of HNO 3 (85%) and HClO 4 (15%) at a gradient temperature (60 °C for 1 h, 120 °C for 1 h, 150 °C for 1 h, and up to 190 °C). The concentration of the metals in the digest solution was determined by atomic absorption spectrometry (Z-2000; Hitachi) and an inductively coupled plasma-mass spectrometer (7700X, Agilent Technologies) after dilution.
Quantitative reverse transcription PCR (qRT-PCR) analysis. Seedlings of 93-11 and CSSL-qGMN7.1 were grown in 1/2 Kimura B solution 12 for 2 weeks, then the roots and shoots were harvested separately. OsNRAMP5 expression was investigated in different tissues from plants grown in the paddy field at booting stage, including root, basal stem, node, lower leaf sheath, lower leaf blade, flag leaf blade, flag leaf sheath, and panicle. Flag leaves of rice varieties were sampled at the heading stage. RNA was extracted by the Micro RNA Extraction kit (Axygen) and reverse transcribed into cDNA using a ReverTra Ace qPCR-RT kit (TOYOBA, Japan). Primers for qRT-PCR were described in a previous study 12 (Table S3). Quantitative PCR was conducted on an ABI PRISM 7900HT Sequence Detector (Applied Biosystems) according to the manufacturer's instructions. The relative expression of each transcript was obtained by comparison with the expression of rice actin1 (Table S3).
Relative promoter activity assays. The promoter fragments (2 kb) of OsNRAMP5 were amplified by PCR from the 93-11 and PA64s lines using the forward primer 5′-accatgattacgccaagcttGCGCATGTATCATTTGTTGT -3′ and the reverse primer 5′-aacgacggccagtgaattcCTCACTGCTCTCTCTCTCAA-3′, and were then cloned into the pCambia1391Z vector. The constructed pCAMBIA1391Z::93-11 p and pCAMBIA1391Z::PA64s p plasmids were co-transformed with eGFP into rice protoplasts and transiently expressed 40 . After 12 h of incubation at 25 °C, protoplasts were collected for RNA extraction. The GUS expression level was detected by qRT-PCR with eGFP expression as the endogenous control.
Plasmid construction and rice transformation. The cDNA of OsNRAMP5 was amplified by PCR with the forward primer 5′-AAGGTACCATGGAGATTGAGAGAGAGAGC-3′ and the reverse primer 5′-AATCTAGACTACCTTGGGAGCGGGATGTC-3′, which include the KpnI and XbaI restriction sites, respectively. The amplified fragment was cloned into the pCAMBIA1300S vector for overexpression. The constructed vector was sequenced and introduced into 93-11 by Agrobacterium tumefaciens-(EHA105) mediated transformation. Thirteen independent transgenic plants of pCAMBIA1300S::OsNRAMP5 in the 93-11 background were obtained. Seedlings of these transgenic plants (T 2 selected by hygromycin) and 93-11 were grown in 1/2 Kimura solution and transferred to pots at the four-leaf stage.