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Research

Blackwell Publishing, Ltd.

Chromosomal regions with quantitative trait loci controlling cadmium concentration in brown rice

(

Oryza sativa

)

Satoru Ishikawa

1

, Noriharu Ae

2

and Masahiro Yano

3

1

Department of Environmental Chemistry, Heavy Metal Group, Soil Biochemistry Unit, National Institute for Agro-Environmental Sciences, 3-1-3

Kannondai, Tsukuba, Ibaraki 305–8604, Japan;

2

Department of Biological and Environmental Sciences, Faculty of Agriculture, Kobe University, 1–1

Rokkodai, Nada, Kobe, Hyogo 657–8501, Japan;

3

Molecular Genetics Department, National Institute of Agrobiological Sciences, 2-1-2 Kannondai,

Tsukuba, Ibaraki 305–8602, Japan

Summary

• A novel mapping population consisting of 39 chromosome segment substitutionlines (CSSLs) was used to locate the putative quantitative trait loci (QTLs) forcadmium (Cd) concentration in brown rice (

Oryza sativa

). The mapping populationcarried a single chromosome segment of ‘Kasalath’ (

indica

) in each line overlappingwith neighbouring segments in a ‘Koshihikari’ (

japonica

) genetic background.• The parents and CSSLs were grown in pots filled with Cd-polluted soil until grainfilling. The brown rice of three of the 39 CSSLs had significantly lower Cd con-centrations than that of Koshihikari, and the brown rice of a further three hadsignificantly higher concentrations.• On the basis of graphical genotypes of CSSLs, putative QTLs controlling the Cdconcentration in brown rice were detected on chromosomes 3, 6 and 8.• Each of the CSSLs was nearly isogenic to Koshihikari, which is the most popularrice cultivar in Japan: they carried > 90% of the Koshihikari genetic background.Therefore, the development of a new Koshihikari with less Cd concentration inbrown rice would be feasible in the near future.

Key words:

brown rice (

Oryza sativa

), cadmium (Cd), chromosome segmentsubstitution lines (CSSLs), quantitative trait loci (QTLs).

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©

New Phytologist

(2005)

doi

: 10.1111/j.1469-8137.2005.01516.x

Author for correspondence:

Satoru Ishikawa Tel: +81 29 838 8148 Fax: +81 29 838 8199 Email: isatoru@niaes.affrc.go.jp

Received:

22 May 2005

Accepted:

16 June 2005

Introduction

Cadmium (Cd) is dispersed in natural and agricultural environ-ments principally through human activities. Japanese arablelands, especially paddy fields, are extensively contaminatedwith relatively low levels of Cd because of irrigation with riverwater originating from mines or because of emissions fromsmelters. Recently, international criteria for Cd concentrationsin foods have been proposed by the Codex AlimentariusCommission of FAO/WHO on account of the growing concernabout the safety of foods and human health. The threshold valuefor Cd in rice has been proposed to be either 0.2 or 0.4 mg kg

−

1

polished rice (Codex Alimentarius Commission, 2004). According

to a survey of Cd contamination in rice by the Ministry ofAgriculture, Forestry and Fisheries of Japan, 3.3% of ricecultivated in Japan exceeded 0.2 mg kg

−

1

and 0.3% exceeded0.4 mg kg

−

1

(http://www.maff.go.jp/cd/PDF/C11.pdf). Thedietary intake of Cd for the Japanese people is approx. 4 µg kg

−

1

body weight wk

−

1

, and nearly half (

c

. 2 µg) is derived fromrice (http://www.maff.go.jp/cd/html/A11.htm). Althoughour diet contains less than the current provisional tolerableweekly intake of Cd (7 µg kg

−

1

body weight wk

−

1

), rice is thelargest source of dietary intake of Cd for the Japanese people,so it is urgent to reduce the Cd concentration of rice.

Several alleviating techniques have been applied in Cd-polluted paddy fields to reduce the Cd levels of rice grains in

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Japan. Soil dressing, which is accomplished by removing theCd-polluted soil layer and replacing it with clean soil, is themost reliable technique. However, remediation of a large areaof low Cd-contaminated soils by soil dressing is not practica-ble because of its enormous cost ($300 000–500 000 ha

−

1

)and the shortage of clean replacement soil. The combinationof applying alkaline amendment and keeping soil flooded toreduce the bioavailability of Cd in the soil is widely practisedin Cd-polluted paddy fields in Japan. However, the techniqueis not always enough to reduce rice Cd contamination becausethe results depend on soil properties and weather conditions(e.g. rainfall).

Phytoremediation (the use of plants to restore contami-nated environments) has drawn much recent attention as apromising technique because it is environmentally friendlyand cost-effective. Although many studies have used hyperac-cumulator plants such as

Thlaspi caerulescens

(Baker

et al

., 1994;Brown

et al

., 1994), it is questionable whether such a hyper-accumulator plant is suitable for phytoremediation inCd-polluted paddy fields in Japan. The primary reason is thatappropriate agricultural practices such as planting, fertiliza-tion, water management and mechanical harvesting are notwell established for hyperaccumulator plants. In addition,technologies are needed for handling harvested plants, whichcontain large amounts of Cd.

The exploitation of rice cultivars that can absorb and trans-locate less Cd to the grain is a promising technology for reduc-ing Cd contamination in rice. Grain Cd concentration variesgreatly among rice cultivars (Morishita

et al

., 1987; Arao &Ae, 2003; Liu

et al

., 2005), suggesting the possibility of breed-ing cultivars with lower concentrations. Clarke

et al

. (1997)reported that the low grain Cd trait of durum wheat (

Triticumturgidum

L. var

durum

) is highly heritable and is controlled bya single dominant gene. Recently, they suggested that theCd uptake locus resides on chromosome 5B (Clarke & Knox,2003). They developed a marker linked in repulsion with alow-Cd allele for efficient wheat breeding (Clarke & Knox,2003). Recent progress in DNA markers and their linkagemaps has provided powerful tools for mapping of quantitativetrait loci (QTLs) (Yano, 2001). Many QTL analyses havebeen performed to identify the gene loci controlling agro-nomic and physiological traits in various crop plants. Tofacilitate genetic mapping and map-based cloning of QTLs,novel mapping populations as backcross inbred lines (BILs)(Lin

et al

., 1998; Ma

et al

., 2002; Nagata

et al

., 2002) or chro-mosome segment substitution lines (CSSLs) (Kubo

et al

., 2002;Ebitani

et al

., 2005) have been developed, especially in rice.Using these lines, researchers have identified QTLs relatedto agronomic traits such as heading date (Lin

et al

., 1998;Ebitani

et al

., 2005), ripening (Nagata

et al

., 2002) and grainsize (Kubo

et al

., 2002), or to physiological traits such astolerance of excess aluminium (Al) (Ma

et al

., 2002) andphosphorus (P) deficiency (Wissuwa

et al

., 1998). Althoughthe genetic analysis of rice is much more developed than that

of other crops, there is no useful genetic information oncontrolling the Cd concentration in brown rice.

The objective of this study was to locate QTLs controllingthe Cd concentration of brown rice by using a novel mappingpopulation consisting of CSSLs carrying overlapping chro-mosome segments of

indica

rice cultivar ‘Kasalath’ in a geneticbackground of the

japonica

rice cultivar ‘Koshihikari’.

Materials and Methods

Chromosome segment substitution lines

A unique mapping population consisting of 39 CSSLs in rice(

Oryza sativa

L.) was recently developed (Ebitani

et al

., 2005).Briefly, 49 BC

1

F

3

plants developed by self-pollinating BC

1

F

1

plants (Koshihikari/Kasalath/Koshihikari) by the single-seeddescent method were selected as the starting materials fordevelopment of CSSLs. Each BC

1

F

3

was crossed with Koshihikari,and then the resulting secondary F

1

(SF

1

) was crossed withKoshihikari to produce secondary BC

1

F

1

(SBC

1

F

1

) plants.Plants meeting the following requirements were selected fromthe SBC

1

F

1

, SBC

2

F

1

and SBC

3

F

1

lines by a maker-assistedtechnique: carrying homozygous chromosome segments forthe target region, covering the entire genome with overlappingsubstituted segments, and carrying a single intact or largechromosome segment to obtain a small set consisting oflines covering the entire donor genome. Finally, 39 lines wereselected from the self-pollinated progeny of selected popu-lations and used for QTL analysis.

Cd experiment

Soil was collected from the upper layer (0–15 cm) of a paddyfield that is contaminated with Cd from irrigation withriver water originating from mines. The Cd concentration ofbrown rice produced in this field had been over 1 mg kg

−

1

d.wt in 1998, but since then it has been kept at

c

. 0.4 mg kg

−

1

owing to the combination of alkaline amendment andcontinuous flooding during much of the growing season. Thesoil is classified as a Fluvisol. It has a relatively low pH (5.1),and the Cd concentration was 1.8 mg Cd kg

−

1

d. wt, asdetermined by 0.1

M

HCl extraction.A pot experiment was carried out in a glasshouse during the

rice-growing season (from early May to mid-October). Theparents and 39 CSSLs were sown in nursery boxes and grownfor 1 month. A set of two seedlings was then transplanted intoa 1/5000-a Wagner pot containing 2.5 kg of the soil. Theexperiment used a randomized block design with four repli-cates. Fertilizer was applied at a rate of 0.25 g each of N, P

2

O

5

and K

2

O as basal dressing in the form of ammonium sulfate,single superphosphate and potassium sulfate. Half of thoserates were also applied as top dressing at the panicle formationstage. The parents and 39 CSSLs were grown under oxidativeupland conditions with soil moisture at 60% of field capacity

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in order to reveal the intrinsic capacity for Cd uptake in eachof the CSSLs. Several CSSLs showing significantly lower orhigher grain Cd concentration than Koshihikari were then re-grown under continuously flooded conditions to confirm thepresence of several QTLs detected in upland cultivation.

After grain ripening, the plants were harvested by cuttingthe stems, and the brown rice was separated from the ricestraw and grain chaff. The brown rice was air-dried to 15%water content. Then 0.5 g of brown rice was digested in anacid mixture (HNO

3

: H

2

O

2

, 7 : 1 v/v) in a microwave oven(Ethos TC, Milestone, Bergamo, Italy). The Cd concentra-tion was determined in a Zeeman graphite furnace atomicabsorption spectrometer (SpectrAA 220Z, Varian, Mulgrave,Victoria, Australia). Certified standard material (rice flour)was used to ensure precision of analytical procedures (NationalInstitute of Standards and Technology, Gaithersburg, MD,USA).

Dunnett’s pairwise multiple comparison

t

-test was used todetect differences between the means of Koshihikari and eachCSSL for the Cd concentration of brown rice. A probabilitylevel of 0.1 was used as the threshold for the detection of aputative QTL.

Results

The graphical genotypes of all the chromosomes of the 39CSSLs are cited from the report of Ebitani

et al

. (2005) andshown in Fig. 1. These genotypes were determined by using129 restriction fragment length polymorphism (RFLP) markersdistributed evenly across the 12 rice chromosomes and areavailable at the Rice Genome Resource Center (http://www.rgrc.dna.affrc.go.jp/jp/ineKKCSSL39.html). The substituted

segments in the 39 CSSLs carry a homozygous genotype forthe target region, except for several small regions in SL-201(defined by C995 on chromosome 1), SL-216 (R1826 onchromosome 2) and SL-239 (S10637A on chromosome 12).The 39 CSSLs cover each chromosome with overlappingsubstituted segments, except for small regions at the distalends of the short arm of chromosome 8 and of the long armof chromosome 12. The net result is that single, intact, largechromosome segments from Kasalath are substituted in thegenetic background of Koshihikari. Therefore, this series of39 CSSLs almost completely fulfils our three requirements(see the Materials and Methods section). The averagesubstitution in each CSSL ranges from 2.3 to 9.4% of the entiregenome (Ebitani

et al

., 2005).The Cd concentrations in brown rice showed relatively

high values (> 1 mg kg

−

1

brown rice), owing to the cultivationunder aerobic soil conditions (Fig. 2). The Cd concentrationwas 1.55 mg kg

−

1

in Koshihikari and 2.03 mg kg

−

1

in Kasalath,and ranged from 0.85 to 4.39 mg kg

−

1

among the 39 CSSLs.Two CSSLs that carried a Kasalath chromosome segmenton chromosome 3 (SL-207 and SL-208) had significantlylower Cd concentrations than Koshihikari. These lines hadapproximately half the Cd concentration of Koshihikari. Ofthe three CSSLs in which chromosome 8 was substituted,SL-224 had significantly lower Cd concentration than Koshi-hikari. The average Cd concentration of SL-223 was similarto that of SL-224, although the probability level somewhatexceeded the threshold of 0.1 (

P =

0.141 for SL-223). On theother hand, three out of four CSSLs carrying a Kasalathsegment on chromosome 6 (SL-215, SL-217 and SL-218)had significantly higher Cd concentrations in brown rice thanin Koshihikari.

Fig. 1 Graphical genotypes of the chromosome segment substitution lines (CSSLs). The data are cited from the report of Ebitani et al. (2005). The grey regions indicate the chromosomal regions substituted with Kasalath genotype in a Koshihikari background.

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Under anaerobic flooded conditions, the brown rice of theparents and selected CSSLs showed much lower Cd concen-tration than under aerobic cultivation (Fig. 3). The averageCd concentration in brown rice was lower in SL-207, SL-223and SL-224 than in Koshihikari, but higher in SL-208. Onthe other hand, three CSSLs carrying a Kasalath segment onchromosome 6 (SL-215, SL-217 and SL-218) had higheraverage Cd concentrations than did Koshihikari. Thus, similargenetic variation was observed between the aerobic andanaerobic soils, with the exception of SL-208.

Using the graphical genotypes of CSSLs showing signifi-cant differences in Cd concentrations in brown rice relativeto Koshihikari under aerobic conditions, we mapped thechromosomal regions containing putative QTLs for Cd

concentration in brown rice by comparing the sizes of the sub-stituted segments (Fig. 4). Three putative QTLs were detectedon chromosomes 3, 6 and 8. A region defined by markersS1513 and R663 on chromosome 3 was shared in two lines,SL-207 and SL-208, which showed significantly lower Cdconcentrations in brown rice than Koshihikari. Of the threeCSSLs carrying Kasalath segments on chromosome 8, SL-224had significantly lower Cd concentrations in brown rice thanKoshihikari, but SL-225 did not. We could not exclude thepossibility that SL-223 possess a QTL for the low Cd traitbecause the probability level was close to the threshold of 0.1.Therefore, a putative QTL was mapped in the region definedby markers C390 and C1121 on chromosome 8, which wasshared by SL-223 and SL-224. On the other hand, a putativeQTL controlling a high Cd concentration of brown rice waslocated in the region defined by markers R2171 and R2549on chromosome 6, which is shared in three lines, SL-215,SL-217 and SL-218, which had significantly higher Cdconcentrations in brown rice than Koshihikari.

Discussion

A series of CSSLs is a powerful tool for QTL analysis in ricefor several reasons. As a population of CSSLs is much smallerthan a primary mapping population such as F2 and recom-binant inbred lines (RILs), it makes phenotype assaysmuch easier. Chromosomal regions with putative QTLs can besimply identified from graphical genotypes without statisticalanalysis for QTL detection. Because CSSLs normally carry 1substituted region, they can be used as near isogenic lines(NILs) themselves or as starting materials to develop NILs.The validity of using CSSLs for QTL analysis has been

Fig. 2 Cadmium (Cd) concentration in brown rice (Oryza sativa) of chromosome segment substitution lines (CSSLs) and parents grown under upland conditions. Bars are means of 4 replicates. The differences in Cd concentration in brown rice between each CSSL and the recurrent parent Koshihikari were evaluated by Dunnett’s pairwise multiple comparison t-test (P < 0.1). The asterisk (*) indicates that the probability level for SL-223 was close to the threshold of 0.1 (P = 0.141).

Fig. 3 Cadmium (Cd) concentration in brown rice (Oryza sativa) of chromosome segment substitution lines (CSSLs) and parents grown under continuously flooded conditions. Means and SE of 4 replicates are plotted.

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verified by a few reports in comparison with BILs (Ebitaniet al., 2005) and RILs (Kubo et al., 2002).

Under aerobic upland conditions, the Cd concentration inbrown rice of Koshihikari exceeded the threshold limit of1 mg kg−1 for brown rice as the Japanese national standard ofCd, and that of Kasalath was even higher (Fig. 2). Generally,the grain Cd concentration is higher in indica and indica ×japonica cultivars than in japonica cultivars under the samesoil conditions (Morishita et al., 1987; Arao & Ae, 2003; Liuet al., 2005). Nevertheless, several CSSLs substituted with achromosome segment of the indica cultivar Kasalath showedsignificantly lower Cd concentrations in brown rice than inthe japonica cultivar Koshihikari (Fig. 2). This suggests that wecan develop new cultivars with lower grain Cd than in Koshihikari.

By contrast, three out of four CSSLs carrying a Kasalathsegment on chromosome 6 had significantly higher Cd con-centrations in brown rice (Fig. 2). These results suggest thatKasalath might carry QTLs associated with both low and highCd concentration in brown rice. Using the same mappingpopulation of CSSLs, Ebitani et al. (2005) observed great var-iation in days to heading (DTH), ranging from 89.4 to 125 dunder natural conditions, even though the DTH value wasnearly the same in Koshihikari (101 d) and Kasalath (104 d).Thus, we found transgressive phenotypic variation relative tothe parents in several CSSLs.

It is well known that Cd uptake by rice varies greatlydepending on soil redox potential. Cd bioavailability in soilincreases under oxidative upland conditions owing to theformation of soluble cadmium sulfate (CdSO4) and decreasesunder reductive flooded conditions because of the formationof less-soluble cadmium sulfide (CdS). As a result, the grainCd concentration of rice was much lower under floodedconditions than under upland conditions (Figs 2 and 3). Thedifferent soil redox states may affect the phenotypic variationin grain Cd concentration of CSSLs. However, we observedsimilar variations between both soil conditions, indicatingthat the QTLs detected under aerobic conditions would beuseful in ordinary anaerobic paddy fields in Japan.

On the basis of the graphical genotypes of CSSLs, putativeQTLs for grain (brown rice) Cd concentration were detectedon chromosomes 3, 6 and 8 (Fig. 4). Putative QTLs related tolow grain Cd resided on Kasalath segments between markersS1513 and R663 on chromosome 3. The QTL detected onchromosome 8 (defined by C390 and C1121) could haveminor effects on the low grain Cd trait because it was closeto the threshold for detection of a putative QTL. A putativeQTL related to high Cd resided on a Kasalath segmentbetween markers R2171 and R2549 on chromosome 6. If theQTLs for grain Cd concentration are linked to those for someimportant agronomic traits, such as grain yield and quality,the development of a practical cultivar with low grain Cd con-centration might be beset with some difficulties. However, wefound no linkage between grain Cd concentration and grainyield, grain weight, grain shape or panicle number (datanot shown). The grain Cd concentrations of the 39 CSSLswere negatively correlated with DTH, and the chromosomalregions related to grain Cd concentration contained the QTLsfor DTH, that is, Hd8 on chromosome 3, Hd1 on chromo-some 6 and Hd5 on chromosome 8 (Yano et al., 2001; Linet al., 2003). This might imply a pleiotropic effect of theQTLs for grain Cd concentration. Whether or not QTLs forgrain Cd concentration are linked to those for DTH shouldbe verified by fine mapping of those QTLs after the develop-ment of NILs.

Koshihikari is the most popular rice cultivar amongJapanese people. Each CSSL is nearly isogenic to Koshihikari,carrying more than 90% of the Koshihikari genetic back-ground. Therefore, the development of a new Koshihikari

Fig. 4 Substitution mapping of regions containing quantitative trait loci (QTLs) associated with the cadmium (Cd) concentration in brown rice (Oryza sativa). The names of restriction fragment length polymorphism (RFLP) markers and their positions are indicated.

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with low grain Cd concentration should be feasible in the nearfuture. The combination of a new cultivar and agriculturalpractices such as water management and alkaline amendmentwould be promising techniques for reducing the Cd concen-tration of rice. To our knowledge, this is the first report ofthe mapping of putative QTLs associated with grain Cdconcentration.

Acknowledgements

This work was supported by Grants-in-Aid for Promotion ofScience and Technology from the Ministry of Education,Culture, Sports, Science and Technology, Japan. We are gratefulto Mr Daishi Misawa for his expert technical assistance.

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