Article

Crop Genetics

October 2010 Vol.55 No.29: 32833287

doi:10.1007/s11434-010-4031-5

SPECIAL TOPICS:

Fine mapping of qHUS6.1, a quantitative trait locus for silicon

content in rice (Oryza sativa L.)
GONG JunYi, WU JiRong, WANG Kai, FAN YeYang & ZHUANG JieYun*
Chinese National Center for Rice Improvement/State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006,
China
Received January 20, 2010; accepted April 30, 2010

Silicon is essential for optimal growth of rice (Oryza sativa L.). This study was conducted to fine map qHUS6.1, a quantitative
trait locus (QTL) for rice hull silicon content previously located in the interval RM510RM19417 on the short arm of chromosome 6, and to analyze the effect of this QTL on the silicon content in different organs of rice. Selfed progenies of a residual heterozygous line of rice were detected using 13 microsatellite markers in the vicinity of qHUS6.1. Three plants with overlapping
heterozygous segments were selected. Three sets of near isogenic lines (NILs) were developed from the selfed progenies of the 3
plants. They were grown in a paddy field and the silicon contents of the hull, flag leaf, and stem were measured at maturity. Based
on analyses of the phenotypic distribution and variance among different genotypic groups in the same NIL set, a significant genotypic effect was shown in the NIL set that was heterogenous in the interval RM19410RM5815, whereas a significant effect was
not found in the remaining 2 NIL sets that were heterogenous in either of the intervals RM4923RM19410 or RM19417RM204.
On comparison among the physical positions of the 3 heterogenous segments, qHUS6.1 was delimited to a 64.2-kb region flanked
by RM19410 and RM19417 that contains nine annotated genes according to the genome sequence of Nipponbare. This QTL
showed strong effects on all of the three traits tested, and the enhancing alleles were always derived from the paternal line Milyang 46. The present study will facilitate the cloning of qHUS6.1 and the exploration of new genetic resources for QTL fine mapping.
rice (Oryza sativa L.), silicon content, quantitative trait locus (QTL), fine mapping, residual heterozygous line (RHL), near
isogenic line (NIL)
Citation:

Gong J Y, Wu J R, Wang K, et al. Fine mapping of qHUS6.1, a quantitative trait locus for silicon content in rice (Oryza sativa L.). Chinese Sci Bull,
2010, 55: 32833287, doi:10.1007/s11434-010-4031-5

Dissection of quantitative trait loci (QTLs) in rice has been

conducted extensively since the 1990s. More recently, considerable progress has been achieved in QTL fine-mapping
and map-based cloning of various traits such as heading
date [1], yield traits [2], abiotic stress responses [3] and major domestication traits [4]. Differing from mutative traits
that are prevalently used in the cloning of major genes,
natural variation involving a large number of QTLs is usually the target of QTL fine-mapping and cloning. However,
fine-mapping of QTLs with smaller effects remains a challenging task [3].
*Corresponding author (email: jz1803@hzcnc.com)

Science China Press and Springer-Verlag Berlin Heidelberg 2010

Rice is a typical silicon-accumulating species. Silicon

accumulation in rice is helpful to alleviate the adverse impact of various biotic and abiotic stresses [5]. It also enhances rice yield by increasing the fertility of spikelets
[6,7]. Significant genotypic differences on the uptake, distribution and accumulation of silicon have been reported in
rice [810], which provides a basis for the study of genetic
mechanisms of silicon content in rice and the breeding of
rice cultivars with high silicon content. To date, QTL analysis on silicon content has been reported in three rice populations. Using 244 recombinant inbred lines (RILs) derived
from the indica/indica cross Zhenshan 97B/Milyang 46,
QTLs for silicon content in different rice organs under
csb.scichina.com

www.springerlink.com

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GONG JunYi, et al.

Chinese Sci Bull

paddy field conditions were detected on chromosomes 1, 5,

6, 11 and 12 [11]. Using 81 RILs derived from the indica/japonica cross Kinmaze/DV85, QTLs for silicon uptake of rice seedling in nutrition solution were detected on
chromosomes 1, 3, 7, 8 and 9 [12]. Using 79 RILs derived
from the indica/japonica cross Bala/Azucena, QTLs for
silicon content in the leaves under paddy field condition
were detected on chromosomes 5 and 10 [13].
In our previous studies, qHUS6, a QTL for silicon content in the hull located on the short arm of rice chromosome
6 [11] and validated in different environments and genetic
backgrounds [14], was dissected into 3 QTLs using segregating populations in an isogenic genetic background [15].
These QTLs are qHUS6.1 located in the 147.0 kb interval
RM510RM19417, qHUS6.2a in the 1.9 Mb interval
RM19706RM19795, and qHUS6.2b in the 2.0 Mb interval
RM314RM19665. According to the physical positions of
these intervals in the rice genome, it was found that
qHUS6.1 and qHUS6.2a were located in regions where no
QTLs or genes for rice silicon content have been reported in
other studies [15]. In the present study, 3 sets of near isogenic lines (NILs) with overlapping heterogenous segments
covering qHUS6.1 were constructed. This QTL was delimited to a 64.2-kb region, showing significant effects on silicon content in the hull (HUS), flag leaf (FLS) and stem
(STS).

1 Materials and methods

October (2010) Vol.55 No.29

plants named TF6-2, TF6-15 and TF6-17 were selected.

They carried heterozygous segments overlapping in the
qHUS6.1 region and paternal homozygotes at qHUS6.2a
and qHUS6.2b. Using 13 SSR markers covering qHUS6.1,
comprising the flanking markers RM510 and RM19417, 5
markers within the interval and 6 markers outside it, heterozygous segments carried by TF6-2, TF6-15 and TF6-17
were determined to be RM4923RM19410, RM19410
RM5815 and RM19417RM204, respectively (Figure 1).
Three F2 populations were derived from the selfed seeds
and genotyped with the 13 SSR markers. A total of 100
non-recombinants were selected, including 10 maternal
homozygotes and 10 paternal homozygotes from TF6-2, and
10 maternal homozygotes, 10 paternal homozygotes and 20
heterozygotes from TF6-15 and TF6-17, respectively. Three
sets of NILs were derived from the selfed seeds and named
TF6-2, TF6-15 and TF6-17 following the respective source
plant.
1.2

DNA extraction and SSR markers survey

DNA was extracted from a 3-cm fresh leaf from a single

plant at 7 d after transplantation following the method described by Zheng et al. [17]. The SSR markers were selected from Gramene (www.gramene.org). The amplification was performed according to Chen et al. [18]. PCR
products were detected on a 6% non-denaturing polyacrylamide gel using silver staining.

1.1 Rice materials

1.3

In previous studies, a residual heterozygous line (RHL) of

rice was selected from Zhenshan 97B/Milyang 46 F7 population based on genotyping with 208 polymorphic simple
sequence repeat (SSR) markers, and the resultant F2:3 population was used for the validation and dissection of QTLs
for yield traits and silicon content on the short arm of rice
chromosome 6 [15,16]. The RHL is homozygous except for
a 7.3-Mb heterozygous segment in the target region on the
short arm of chromosome 6 and 4 other segments of 3.8,
1.1, 2.0 and 0.6 Mb on chromosomes 1, 2, 4 and 5, respectively [16]. The RHL was selfed for 3 generations, and 3

The 3 NIL sets were grown in Fuyang, Zhejiang Province,

in the summer of 2007. Twelve plants per line were planted
in 2 replications at a spacing of 20 cm 23 cm. At maturity,
main stems of the middle 10 plants of each line in each replication were harvested, separated into panicle, flag leaf and
stem (culm and leaf sheath), and dried. A sample of 100 full
grains was husked. The hull, flag leaf and stem were ground
and dried to a constant weight at 60C. Silicon content was
determined by the colorimetric molybdenum blue method and
measured as the percentage amount of SiO2 [19]. Every sample
was measured twice and averaged. The SAS GLM procedure

Figure 1

Phenotyping and data analysis

Genotypes of RHLs TF6-2, TF6-15 and TF6-17 in the interval RM4923RM225 on the short arm of rice chromosome 6.

GONG JunYi, et al.

Chinese Sci Bull

was used to test the phenotypic variations among different genotypic groups in the same NIL set [14,20].

2 Results and analysis

2.1 Phenotypic distribution and variation in the 3 NIL
sets
In each of the 3 NIL sets, average phenotypic values of the
rice line showed considerable variation for all of the three
traits analyzed (Figure 2), but the frequency distribution of
different genotypic groups varied greatly among the three
NIL sets. A single-gene segregation pattern was observed in
TF6-15. A discrete distribution was observed for FLS, in
which the maternal, paternal and heterozygous line ranges
were 6.85%8.35%, 9.31%10.37% and 8.38% 10.19%,
respectively. Frequency distributions of HUS and STS in
TF6-15 were less distinguishable among different genotypes, but the maternal, paternal and heterozygous lines
tended to have lower, higher and intermediate values, respectively. In TF6-2 and TF6-17, different genotypic groups
had similar distributions in which the maternal and paternal
lines showed scattered, low to high values. These results
indicate that different genotypic groups of TF6-15 might
carry different alleles for HUS, FLS and STS, while those in
TF6-2 and TF6-17 carried the same allele, respectively.
2.2 Fine mapping of qHUS6.1 and analysis of its effect
on different organs
In each of the 3 NIL sets, different genotypic groups differ

October (2010) Vol.55 No.29

3285

only in a portion of the target region containing qHUS6.1.

Significant phenotypic variation among different genotypic
groups in the same NIL set would be an indication of the
presence of a QTL in the given portion. Correspondingly,
insignificant phenotypic differences among the genotypic
groups would be an indication of the absence of a QTL in
the given portion. On comparison among the heterogenous
regions presented in different NIL sets and the overlapping
of the heterogenous segments, the location of qHUS6.1
could be narrowed down. In the meantime, the effect of
qHUS6.1 on HUS, FLS and STS could be determined.
Results of the two-way ANOVA on the phenotypic differences among different genotypic groups in each NIL set
were presented in Table 1. It was shown that TF6-15 exhibited significant variation whereas the variations in TF6-2
and TF6-17 were not significant. This was in accordance
with the results indicated by the frequency distribution, providing evidence for the existence of allelic variation at
qHUS6.1 in TF6-15 and the absence of allelic variation at
qHUS6.1 in the remaining 2 NIL sets. In other words,
qHUS6.1 could be located in a region that is heterogenous
in TF6-15 but homogenous in TF6-2 and TF6-17.
The heterogenous region in TF6-15 was the heterozygous
interval RM19410RM5815 in its source RHL TF6-15
(Figure 1), and the two flanking cross-over intervals
RM3414RM19410 and RM5815RM19417 may also be
heterogenous. Thus qHUS6.1 was located within the region
RM3414RM19410RM5815RM19417. The heterogenous interval in TF6-2 showing no significant allelic effects
was RM4923RM19410, which covered the interval
RM3414RM19410, indicating that qHUS6.1 was not located

Figure 2 Phenotype distributions of silicon content in the NILs TF6-2 (a), TF6-15 (b) and TF6-17 (c). HUS, silicon content in the hull; FLS, silicon content in the flag leaf; STS, silicon content in the stem.

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Table 1

GONG JunYi, et al.

Chinese Sci Bull

October (2010) Vol.55 No.29

Allelic effects on silicon content of the 3 intervals on the short arm of rice chromosome 6 in 3 isogenic genetic backgrounds

NILs

Heterogenous
intervals

TF6-2

RM4923-RM19410

Traits

Genotype (MeanSD)a)
ZZ

MM

ANOVA
ZM

Ab)

Dc)

D/[A]d)

R2e)

HUS
8.131.15
7.781.07
n.a.
1.99
0.1749
FLS
8.031.21
7.991.15
n.a.
0.00
0.9960
1.19
0.2893
STS
7.940.99
7.870.75
n.a.
18.46 <0.0001 0.67 0.13
0.19
26.2
TF6-15 RM19410-RM5815
HUS
10.410.77
11.750.90
10.950.81
72.50 <0.0001 1.05 0.62
0.59
35.6
FLS
7.690.99
9.781.31
9.351.04
8.85
0.0007 0.45 0.04
0.08
15.5
STS
7.030.88
7.920.72
7.510.69
0.99
0.3829
TF6-17
RM19417-RM204
HUS
11.311.04
11.610.85
11.650.87
0.50
0.6086
FLS
8.940.88
9.170.85
9.080.96
0.71
0.4991
STS
7.830.57
7.940.62
7.960.68
a) ZZ, Zhenshan 97B homozygote; MM, Milyang 46 homozygote; ZM, heterozygote; n.a., not available. b) Additive effect. The genetic effect when a
maternal allele is replaced by a paternal allele. c) Dominance effect. d) Degree of dominance. e) The proportion of phenotypic variance explained by the
given QTL.

in RM3414RM19410. Thus the region for qHUS6.1 could

be narrowed down to RM19410RM5815RM19417. The
heterogenous interval of TF6-17 that also showed no significant allelic effects was RM19417RM204. Because this
region was not included in the qHUS6.1 region, it is reasonable that no significant variation was detected in this
NIL set. In conclusion, qHUS6.1 was delimited to a 64.2
region flanked by RM19410 and RM19417, corresponding
to the physical positions of 29140822978314 and 3453590
3517784 bp on chromosome 6 in the Nipponbare and 9311
genome, respectively (www.gramene.org).
In TF6-15 showing allelic variation at qHUS6.1, significant genotypic effects were detected on all of the 3 traits
analyzed. In all cases the paternal genotype had the highest
value, the maternal genotype had the lowest value and the
heterozygous genotype was intermediate. On HUS, FLS and
STS, the additive effects were 0.67%, 1.05% and 0.45%, the
dominance effects were 0.13%, 0.62% and 0.04%, and the
proportion of phenotypic variance explained was 26.2%,
35.6% and 15.5%, respectively (Table 1). These results indicate that qHUS6.1 have strong additive effects on all of
the three traits with the enhancing alleles derived from the
paternal line Milyang 46.

3 Discussion
Silicon is one of the most abundant elements in soils and
plays an important role in the growth and development of
rice. However, few genetic analyses of rice silicon have
been undertaken until recently when QTL mapping for
natural variation between different genotypes [1113] and
gene cloning using low silicon rice mutants [2124] were
published. In the present study, a QTL controlling silicon
content in different organs of rice, qHUS6.1, was delimited
to a 64.2-kb region flanked by RM19410 and RM19417.
According to the available Nipponbare sequence annotation
in Gramene (www.gramene.org), there are 9 annotated
genes in the 64.2-kb region. In addition to forming a foundation for qHUS6.1 cloning, this study has demonstrated
that a new approach of stepwise QTL fine-mapping pro-

posed previously [25,26] is feasible. Firstly, a RHL that

carries a relatively large heterozygous segment covering the
target region was identified. One or more segregating populations were derived and used to dissect linked QTLs of
which the effect of a single QTL may be low. Secondly,
sub-RHLs carrying smaller and overlapping heterozygous
segments were selected. Using the resultant segregating
populations or NILs with a sample size that is similar to
those used in a primary QTL analysis, a QTL is delimited to
a region containing a few candidate genes.
Nine annotated genes were located in the qHUS6.1 region.
LOC_Os06g06330 and LOC_Os06g06370 were functionally
unknown. LOC_Os06g06300 and LOC_Os06g06320 encode phosphatidylethanolamine-binding proteins and correspond to the genes RFT1 and Hd3a for heading date [1],
respectively. LOC_Os06g06310 encodes a putative porinlike protein belonging to voltage-gated proteins involved in
anion-selective membrane transportation in plant chloroplasts and mitochondria [27,28]. LOC_Os06g06340 encodes a nonspecific lipid transfer protein involved in the
transportation of phospholipids, glycolipids, fatty acids and
steroids between membranes [29]. LOC_Os06g06350 encodes a putative AMP-binding enzyme that is a super-family
containing peptide antibiotic synthetases, polyketide synthetases, 4-coumarate-CoA ligases, acetyl-CoA synthetases
and luciferases [30]. LOC_Os06g06360 encodes WRKY
transcription factors involved in the plant defense response,
senescence and development [31]. LOC_Os06g06380 encodes a putative NBS-LRR protein correlated to disease
resistance in plants [32]. Work is underway to identify the
most likely candidate gene for qHUS6.1.
Genotypic differences in silicon uptake and accumulation
have been reported for silicon content in the root and shoot
of rice seedlings [10] and on silicon content in the root, leaf
and stem at harvesting [8]. When the data were analyzed
between different organs, no significant correlations were
found. Of the three genes cloned, Lsi1 [21] and Lsi2 [22]
show uptake and transport activity in rice roots, but they do
not affect silicon distribution in different organs. On the
other hand, Lsi6 [23,24] is responsible for intervascular
transfer of silicon in rice, but it does not affect silicon up-

GONG JunYi, et al.

Chinese Sci Bull

take by the root. These results suggest that genes for rice
silicon content may or may not exert similar impact on different rice organs. In the present study, qHUS6.1 was shown
to simultaneously control HUS, FLS and STS, and the paternal allele always had the enhancing effect. This suggests
that qHUS6.1 might play a similar role in silicon accumulation in various organs of rice. At the same time, variation in
the genetic action mode and proportions of phenotypic
variance explained were observed among the three traits,
indicating that other factors affecting qHUS6.1 might be
involved. Presumably, a proportion of genes for rice silicon
content in the genetic background could have different effects on HUS, FLS and STS, and some of these may interact
with qHUS6.1. Consequently, the effects measured at
qHUS6.1 varied among HUS, FLS and STS. Another factor
that might also contribute partly to the variation is differences in phenotyping error among different rice organs.
More work is needed to clarify the cause.
This work was supported by the National High Technology Research and
Development Program of China (2009AA101101), and the National Natural Science Foundation of China (30571062).

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