全 文 ：Vol. 30 , No. 4
pp. 329～333 　Apr. , 2004
作 　物 　学 　报
ACTA AGRONOMICA SINICA
第 30 卷 第 4 期
2004 年 4 月 　329～333 页
Detection and Analysis of QTLs Associated with Resistance to Ferrous Iron Toxicity
in Rice ( Oryza sativa L. ) , Using Recombinant Inbred Lines
WAN Jian2Lin1 , ZHAI Hu2Qu2 , WAN Jian2Min1 , 3 , Hideshi Yasui3 , Atsushi Yoshimura3
(1 National Key Laboratory of Crop Genetics & Germplasm Enhancement , Jiangsu Plant Gene Engineering Research Center , Nanjing Agricultural University , Nan2
jing 210095 , Jiangsu , China ;2 Chinese Academy of Agricultural Sciences , Beijing 100081 , China ;3 Faculty of Agriculture , Kyushu University , Fukuoka 8122
8581 , Japan)
Abstract 　A mapping population of 81 F11 lines (recombinant inbred lines : RILs) , derived from a cross between a japoni2
ca variety Kinmaze and an indica variety DV85 by the single2seed descent methods , was used to detect quantitative trait lo2
ci (QTLs) for leaf bronzing index (LBI) and 　plant height ( PH) under Fe2 + stress condition. Two parents and 81 RILs
were phenotyped for the traits by growing them in Fe2 + toxicity nutrient solution. A total of three QTLs were detected on
chromosome 3 , with score LOD ranging from 3. 79 to 5. 89. Two QTLs controlling LBI was located at the region of X2792
C25 and X1442X362 , and their contributions to total variation were 17. 38 % and 22. 07 % , respectively. One QTL for
PH was located at the region of R1468A2R1468B , with contribution rate 23. 18 %. Comparing with the other mapping re2
sults , the QTL for LBI located at the region of X2792C25 on chromosome 3 was identical with the QTL for chlorophyll cont2
ent on a rice function map . Another QTL for LBI located at the region of X1442X362 linked with QTL for chlorophyll cont2
ent which located at the region of C1362C944 on a rice function map , the genetic distance between the two QTLs was
25. 6 cM. The results indicated that ferrous iron toxicity of rice was characterized by bronzing spots on the lower leaves ,
which spread over the whole leaves , causing the lower leaves to turn dark gray and producing chlorophyll catabolites or deri2
vatives which reduced cytotoxicity of some heavy metals , such as ferrous iron.
Key words 　Rice ( Oryza sativa L. ) ; Resistance to ferrous iron toxicity ; Recombinant inbred lines ( RILs) population ;
Quantitative trait loci (QTLs)
利用重组自交群体检测水稻抗亚铁毒胁迫的 QTLs
万建林1 　翟虎渠2 　万建民1 , 3 　安井秀3 　吉村醇3 Ξ
(1 南京农业大学作物遗传与种质创新国家重点实验室 ,江苏省植物基因工程研究中心 ,江苏南京 210095 ; 2 中国农业科学院 ,北京 100081 ; 3 日
本国立九州大学农学部 ,福冈 81228581 ,日本)
摘 　要 　潜育性水稻田广泛分布于中国、斯里兰卡、印度、印度尼西亚、塞拉里昂、利比亚、尼日利亚、哥伦比亚和菲律宾
等国 ,其中我国南方稻区就有 670 万公顷低产潜育性水稻田。该类水稻田还原性强 ,矿质营养失调 ,尤以 Fe2 + 过量积累 ,
对水稻生长发育产生不良的逆境胁迫作用。培育抗亚铁毒的水稻品种是简便、经济有效地提高稻谷产量的重要途径之
一。本文利用 KinmazeΠDV85 的 81 个重组自交家系 (RILs)的作图群体 ,采用水培鉴定方法 ,在亚铁毒条件下 ,检测叶片棕
色斑点指数 (LBI) 、株高 (PH)的数量性状位点 (QTLs) 。结果表明 ,控制叶片棕色斑点指数、株高的数量性状位点都位于第
3 染色体上 ,各 QTL 的 LOD 值为 3. 79～5. 89。检测到与亚铁毒胁迫直接有关的性状叶片棕色斑点指数 QTL 2 个 ,分别位
于第 3 染色体的 X2792C25 和 X1442X362 间 ,对应的贡献率分别为 17. 38 %和 22. 07 % , 其中位于第 3 染色体 X2792C25 间
的叶片棕色斑点指数 QTL 与水稻功能图谱第 3 染色体上的控制叶绿素含量的 QTL 位置一致 ; 另一个位于 X1442X362 间
的叶片棕色斑点指数 QTL 与水稻功能图谱第 3 染色体上的另一个控制叶绿素含量的 QTL 连锁 ,相距 25. 6 cM。表明在亚
铁毒胁迫条件下 ,水稻在其叶片表面出现棕色斑点 , 叶片衰老 ,产生一些叶绿素降解物或衍生物 ,以提高叶片细胞对亚
铁等重金属毒害的耐受力。
关键词 　水稻 ;抗亚铁毒 ;重组自交 (RILs)群体 ;数量性状位点 (QTL s)
中图分类号 : S511
Foundation items :The study was supported in part by a grant from National Science Fund for Distinguished Young Scholars Abroad(30228023) , foundation
for key teachers , Ministry of Education and“948”project , Ministry of Agriculture.
Biography :万建林 (1966 - ) ,男 ,江西南昌人 ,在职博士生 ,副研究员 ,研究方向 :水稻遗传育种。3 通讯作者 (Author for correspondence) :万建民。E2mail : wanjm @mail . njau. edu. cn 　Tel and Fax :025284396516
Received (收稿日期) :2002212202 ,Accepted (接受日期) :2003204210.

　　The gleyed paddy soils are widely distributed in Peo2
ple’s Republic of China , Srilanka , India , Indonesia , Si2
erra Leone , Libya , Nigeria , Columbia and Philippin2
es
[1 ,2 ]
. The gleyed paddy soils are estimated to cover 7. 5
to 8. 0 million hectares , in People’s Republic of China ,
where ferrous iron toxicity in the gleyed paddy soils is re2
ported to reduce rice yield by 10 %———20 % depending
on the intensity of toxicity and the resistance of the rice
cultivar[3 ] .
Rice genotypes differ widely in resistance to Fe2 +
toxicity , which makes it possible to develop rice cultivars
with enhanced resistance to Fe2 + through breeding pro2
gram[4 - 7 ] . The breeding effort could be facilitated by a
better understanding of genetic background on the tole2
rance mechanism in rice.
In this study , based on a genetic map from a recom2
binant inbred lines ( RILs) derived from KinmazeΠDV85
( F10 ) , QTLs associated with resistance to ferrous iron
toxicity in rice were identified by using molecular mark2
ers , and their contributions to the total variation were
evaluated , and the genetics and physiology of resistance to
ferrous iron were discussed.
1 　Materials and Methods
1. 1 　Plant materials
　　A set of recombinant inbred lines (RILs) was devel2
oped from F1 to F10 , by the method of single seed descent
(SSD) from a cross of KinmazeΠDV85 , where“Kinmaze”
was a variety belonging to japonica subspecies , and
“DV85”was a variety belonging to indica subspecies.
RILs (F10 ) were provided by Professor Atsushi Yoshimura
at Faculty of Agriculture , Kyushu University , Fukuoka ,
Japan. Two parents and eighty one plants of RILs were
planted at the experimental station of Jiangsu Academy of
Agricultural Sciences , People’s Republic of China , and
two parents and F11 seeds were harvested for evaluation of
resistance to ferrous iron toxicity. IR26 and IR64 suscep2
tible , IR74 and Mahsuri moderately tolerant and Suakoko
8 tolerant were used as control cultivars.
1. 2 　Solution culture experiment
The two parents , Kinmaze and DV85 , five control
cultivars , i . e. , IR26 , IR64 , IR74 ,Mahsuri and Suako2
ko 8 , and 81 RILs in F11 were evaluated for resistance to
their ferrous iron toxicity. Seeds were germinated after
sterilization , and transplanted into quartz sand. After one
week , six plants of each line and 12 plants each of pa2
rents and control cultivars were randomly selected and
transferred to plastic culture containers containing 5 L nu2
trient solution. Individual seedlings were planted in a per2
forated sheet placed on the surface of solution in each
container. Standard rice nutrient solution[8 ] was used as
the normal treatment . For Fe2 + stress treatment , 250 mg
Fe2 + ·L - 1 were added as FeSO4 to the standard nutrient
solution. The pH of the solution was daily adjusted to 4. 5
using 1 molΠL NaOH or 1 molΠL HCl , and the solution
was replaced every three days. The entire experiment was
conducted in a glasshouse at Nanjing Agricultural Univer2
sity from June 10 to July 7 , in 2002. The day temperature
was 35 ℃to 22 ℃and the night temperature was 22 ℃ to
14 ℃in the glasshouse during the experiment . On 7 July
(28 days after treatment) each plant was harvested to test
leaf bronzing index (LBI = bronzing leaf numberΠtotal leaf
number) and PH , which were viewed as a direct indica2
tor[9 ,10 ] and an indirect indicator of resistance to ferrous
iron toxicity in rice , respectively.
1. 3 　Genetic mapping and QTL detection
A linkage map used for QTL detection comprised 137
markers (1 386. 2 cM) with an average marker density of
10. 1 cM. One2way ANOVA (SASΠ6. 11 , GLM) interval
mapping analysis (MapmakerΠQTL) [11 ] was performed for
detecting molecular markers associated with the variations
in LBI and PH and most likely positions of the gene loci .
P < 0. 001 for Type Ⅰ error and log102likelihood ratio
(LOD) value of 3. 0 were used as criteria to detect the
significant association between marker loci and gene loci .
Correlation analysis between the measurements was per2
formed using correlation procedure of Microsoft Excel 97.
2 　Results
2. 1 　Phenotypic response of two parents and control
cultivars and correlation between traits
　　Ferrous iron toxicity of rice is characterized by
bronzing spots on the lower leaves , which spread over the
whole leaves , causing the lower leaves to turn dark gray.
The two parents and five control cultivars were evaluated
for resistance to ferrous iron toxicity in rice. The results
033 　　　作 　　物 　　学 　　报 30 卷 　

were summarized in Table 1. The LBI of Kinmaze (0. 20)
was approximately equal to that of control cultivar Suakoko
8 (0. 08) , and the LBI of DV85 (0. 82) was equal to that
of control cultivars IR26(0. 83) and IR64 (0. 85) . There2
fore , Kinmaze and DV85 were evaluated to be tolerant
and sensitive , respectively , to ferrous iron toxicity. Un2
der ferrous iron toxicity stress , the PH of Kinmaze and
DV85 were 54. 9 cm and 65. 9 cm , respectively.
Table 1 Mean LBI and PH of parents and CK
Cultivar Mean LBI Mean PH
Kinmaze 0. 20 54. 9
DV85 0. 82 65. 9
IR26 0. 83 23. 8
IR64 0. 85 27. 7
IR74 0. 40 21. 4
Mahsuri 0. 35 31. 2
Suakoko8 0. 08 43. 7
　　As the LBI was indicated as a phenotypic value for
resistance to ferrous iron toxicity[9 ,10 ] , the correlation co2
efficient between LBI and PH were - 0. 826. The LBI
and PH were significantly negatively correlated , which in2
dicated that LBI was associated with PH reduction due to
Fe2 + toxicity in this RILs population.
2. 2 　Phenotypic response of two parents and RILs
The distributions of the LBI and PH of the RILs were
shown in Fig. 1. For the LBI counted there is clear differ2
ence between Kinmaze and DV85 , but there wasn’t very
clear difference in PH between two parents.
2. 3 　The mapping of QTL for some traits associated
with resistance to ferrous iron toxicity
　　A total of three QTLs were detected on chromosome
3 (Fig. 2 , Table 2) , with LOD score ranging from 3179
to 5189. Two QTLs controlling LBI was located at the re2
gion of X2792C25 and X1442X362 , and their contribution
to total variation were 17138 % and 22107 % respective2
ly. Kinmaze alleles in these regions reduced the LBI on
the heterozygous condition by 0. 05 and 0. 06 , respective2
ly. One QTL controlling PH was located at the region of
R1468A2R1468B , with contribution of 23. 18 %.
Kinmaze alleles in these regions increased PH on the
heterozygous condition by 3. 64 cm.
Fig. 1 Normal distribution of leaf bronzing index and plant height in RILs population
Table 2 QTL mapping of LBI and PH in rice seedlings
Traits Marker interval Chromosome LOD score PVE ( %) Additive
LBI X2792C25 3 4. 24 17. 38 - 0. 05
X1442X362 3 3. 79 22. 07 - 0. 06
PH R1468A2R1468B 3 5. 89 23. 18 　3. 64
3 　Discussion
In this study , the LBI was indicated as a phenotypic
value for resistance to ferrous iron toxicity , the QTL locat2
ed at the region of X2792C25 for LBI coincided with the
QTLs for chlorophyll content which located at the region of
C5152C25 on a rice function map [12 ] . Another QTL locat2
ed at the region of X1442X362 for LBI on chromosome 3 linked with QTL for chlorophyll content which located atthe region of C1362C944 on chromosome 3 on a rice func2tion map [12 ] , the genetic distance between the two QTLswas 25. 6 cM. The results indicated that ferrous iron tox2icity of rice was characterized by bronzing spots on thelower leaves , which spread over the whole leaves , causingthe lower leaves to turn dark gray and producing chloro2phyll catabolites or derivatives which reduced cytotoxicity
133　4 期 WAN Jian2Lin et al . :Detection and Analysis of QTLs Associated with Resistance to Ferrous Iron Toxicity in111 　　　

of some heavy metals[13 ] , such as ferrous iron.
Yan et al . (1998) used a doubled haploid popula2
tion of 123 lines from IR64ΠAzucena to detect a condition2
al QTL for PH between markers CDO87 and RG910 on
chromosome 3 only after 30 days of transplanting[14 ] . In
this study a QTL was also detected for PH at the region of
R1468A2R1468B on chromosome 3 after 30 days of trans2
planting under ferrous iron toxicity condition. Whether the
QTL for PH at the region of R1468A2R1468B on chromo2
some 3 is identical with the conditional QTL at the region
of CDO872RG910 needs further study.
Fig. 2 QTLs identified for resistance to iron toxicity in KinmazeΠDV85 RIL population
　　A QTL was detected for PH at the region of R1468A2
R1468B on chromosome 3 under ferrous iron toxicity con2
dition . Wu et al . (1997) used a doubled haploid (DH)
population consisting of 123 lines derived from a japonica
cultivar , Azucena and an indica cultivar , IR64 and lo2
cated a QTL for PH at the region of RZ2482RZ394 on
chromosome 3 under potassium deficiency condition[15 ] .
This result verified that PH produced under Fe2 + toxicity
stress was viewed as an indirect indicator of resistance to
Fe2 + toxicity in rice , because a QTL for PH detected un2 der Fe2 + toxicity was also detected under potassium defi2ciency which appeared not related to resistance to Fe2 +toxicity.A large population may provide greater insight aboutgenetic basis of resistance to Fe2 + . We are developing asubstitution population derived from extremely tolerantlines with minimum Kinmaze genome for the furtherstudy. Our goal is to identify breeding materials for resist2ance to Fe2 + toxicity through marker2assisted selectionbased on the detected markers.
233 　　　作 　　物 　　学 　　报 30 卷 　

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333　4 期 WAN Jian2Lin et al . :Detection and Analysis of QTLs Associated with Resistance to Ferrous Iron Toxicity in111