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Fine mapping of a minor-effect QTL, DTH12, controlling
heading date in rice by up-regulation of florigen genes
under long-day conditions
Zhengzheng Zhong •Weixun Wu •Hongjun Wang •Liping Chen •
Linglong Liu •Chunming Wang •Zhigang Zhao •Guangwen Lu •
He Gao •Xiangjin Wei •Chuanyuan Yu •Mingjiang Chen •Yingyue Shen •
Xin Zhang •Zhijun Cheng •Jiulin Wang •Ling Jiang •Jianmin Wan
Received: 5 July 2013 / Accepted: 23 January 2014/ Published online: 11 March 2014
ÓSpringer Science+Business Media Dordrecht 2014
Abstract Heading date is a key determinant of
regional and seasonal adaptation in rice (Oryza sativa
L.). A minor-effect quantitative trait locus (QTL), QTL
for Days to heading 12a (qDTH-12a), with unknown
genetic action was previously coarsely detected in a
recombinant inbred line population. The study
reported here was designed to better define the
qDTH-12a locus (designated as DTH12) in advanced
segregating populations. DTH12 was initially verified
in chromosome segment substitution line CSSL84.
A CSSL84/Asominori//Asominori BC
4
F
2
population
was then developed, and a near-isogenic line (NIL),
NIL(DTH12), was subsequently selected from this
population using marker-assisted tracking that headed
8 days later than Asominori under long-day (LD)
conditions but which was not significantly different in
heading date in short-day environments. Using 358
Asominori/NIL(DTH12)F
2:3
families grown under LD
conditions, we were able to initially map DTH12 to a
26-cM interval between markers InDel12-1 and
RM6296. F
3
individuals heterozygous for the DTH12
regions were then chosen, and 2,388 F
4:5
families were
used for fine mapping. DTH12 was finally dissected as
a single gene and delimited to a 153-kb genomic region
with 32 open reading frames. Compared with Asomi-
nori, NIL(DTH12) showed reduced transcription of the
florigen genes Heading date 3a and RICE FLOWER-
ING LOCUS T 1, suggesting that DTH12 functions as
an up-regulator of florigen genes during floral induc-
tion under LD conditions. DTH12 was also found to
have an important role in rice adaptation and breeding
for precise control of seed maturity. These findings
provide a firm basis for cloning this minor-effect QTL
involved in rice flowering.
Keywords Oryza sativa DTH12 Heading
date Minor-effect QTL Near-isogenic line
Introduction
As sessile organisms, the ability of plants to adapt to
natural environments is essential for ecological prosper-
ity. Flowering time (i.e., heading date in rice) represents
Zhengzheng Zhong, Weixun Wu and Hongjun Wang have
contributed equally to this work.
Electronic supplementary material The online version of
this article (doi:10.1007/s11032-014-0035-1) contains supple-
mentary material, which is available to authorized users.
Z. Zhong X. Zhang Z. Cheng J. Wang J. Wan (&)
National Key Facility for Crop Gene Resources and
Genetic Improvement, Institute of Crop Sciences, Chinese
Academy of Agricultural Sciences, Beijing 100081, China
e-mail: wanjm@caas.net.cn
Z. Zhong W. Wu H. Wang L. Chen
L. Liu C. Wang Z. Zhao G. Lu H. Gao
X. Wei C. Yu M. Chen Y. Shen L. Jiang J. Wan
National Key Laboratory for Crop Genetics and
Germplasm Enhancement, Jiangsu Plant Gene
Engineering Research Center, Nanjing Agricultural
University, Nanjing 210095, China
123
Mol Breeding (2014) 34:311–322
DOI 10.1007/s11032-014-0035-1
an important ecological trait for adaptation to specific
growing seasons and regions. Photoperiodic sensitivity
determines the transformation from vegetative to repro-
ductive stages and is one of the most important regulators
of flowering in rice (Hayama and Coupland 2004;
Putterill et al. 2004). Allelic variation in genes controlling
these traits in rice is vital for adaptation to specific
environments (Izawa 2007;Xueetal.2008;Wuetal.
2013). For example, at higher latitudes, alleles control-
ling early flowering and photoperiod insensitivity are
essential to enable harvesting before the onset of cold
weather prevents successful seed setting and completion
of the life cycle (Izawa 2007).
Quantitative trait locus (QTL) analysis is used as a
powerful tool to decipher genetic control of heading
date in rice. Many QTLs underlying natural variation in
heading date have been identified (Hd1-Hd3a, Hd3b-
Hd17), and some of these have been cloned by map-
based cloning strategies (Yano et al. 2001; Matsubara
et al. 2008a; Komiya et al. 2009; Tsuji et al. 2011;
Brambilla and Fornara 2013). Among these, Heading
date 3a (Hd3a) and RICE FLOWERING LOCUS T 1
(RFT1, a close paralog of Hd3a), were recently
identified as a short-day (SD) and long-day (LD)
florigen in rice, respectively (Kojima et al. 2002;
Tamaki et al. 2007; Komiya et al. 2009; Tsuji et al.
2011,2013). Under SD conditions, the clock-compo-
nent OsGIGANTEA (OsGI) activates the zinc-finger
transcription factor Heading date 1 (Hd1), which in
turn promotes heading by inducing the florigen Hd3a
only under SD conditions—under LD conditions
activation of flowering is changed to suppression
(Yano et al. 2000; Hayama et al. 2003). In addition, rice
has a unique Hd1-independent flowering pathway
mediated by Early heading date 1 (Ehd1), which
positively regulates the expression of Hd3a and RFT1
to promote flowering (Doi et al. 2004). Under LD
conditions, expression of the florigen Hd3a/RFT1 is
regulated by an ‘‘LD-suppression pathway’’ and an
‘‘LD-promotion pathway’’ (Tsuji et al. 2011). In
addition to the OsGI-Hd1-Hd3a suppression pathway
under LD conditions, other suppression pathways
(Ghd7,DTH8/Ghd8,OsMADS56,OsCOL4,PHYB,
SE5,Ghd7.1/OsPRR37,Ef7/OsELF3/Hd17, and EL1/
Hd16) or promotion pathways (RID1/OsID1/Ehd2,
Ehd3,Ehd4,OsMADS50,OsMADS51, and SDG724)
integrate at Ehd1 (Lee et al. 2004; Kim et al. 2007;
Matsubara et al. 2008b,2011,2012; Park et al. 2008;
Wu et al. 2008; Xue et al. 2008; Andre
´s et al. 2009; Ryu
et al. 2009; Lee et al. 2010; Wei et al. 2010; Ishikawa
et al. 2011; Yan et al. 2011,2013; Saito et al. 2012; Sun
et al. 2012; Zhao et al. 2012; Gao et al. 2013; Koo et al.
2013; Kwon et al. 2014). DTH2 (for Days to heading on
chromosome 2) also promotes heading under LD
conditions by inducing the florigen genes and acts
independently of Hd1 and Ehd1 (Wu et al. 2013).
Although these studies have contributed significantly
to our understanding of the genetic control of flowering
in rice under LD conditions, there may be additional,
unknown genes/minor-effect QTLs and genetic vari-
ations that contribute to the wide adaptability of
cultivated rice throughout the world.
Yu (2005) identified a minor-effect QTL, QTL for
Days to heading 12a (qDTH-12a), in a recombinant
inbred line (RIL) population derived from a cross
between Asominori (japonica) and IR24 (indica). The
qDTH-12a locus was located on the short arm of
chromosome 12 (LOD score 3.96) and explained
9.29 % of the phenotypic variance in the RIL popu-
lation. Due to the small size of the RIL population (only
71 lines) used for this QTL analysis and possible
masking effects from several other major-effect QTLs
(Yu 2005), the specific genetic effects of qDTH12 were
not fully elucidated.
The objective of this study was to define qDTH-12a
more precisely by marker-assisted backcrossing. A set
of chromosome segment substitution lines (CSSLs)
and an advanced secondary population was developed
to dissect qDTH12. We resolved qDTH-12a as a single
gene and designated it as DTH12. Moreover, we found
that DTH12 induced heading by up-regulating Hd3a
and RFT1. Our study provides a new opportunity for
the isolation of a minor-effect QTL controlling rice
flowering.
Materials and methods
Isolation of the near-isogenic line
Asominori (a temperate japonica variety from
Japan), as recurrent parent, was backcrossed with
IR24 (an indica variety from the International Rice
Research Institute) using marker-assisted selection
(MAS) and following the scheme presented in
Electronic Supplementary Material (ESM) Fig. 1.
312 Mol Breeding (2014) 34:311–322
123
First, 71 F
7
RILs were derived from a cross between
Asominori and IR24 by single-seed descent (Tsu-
nematsu et al. 1996). Nineteen RILs were then
selected and backcrossed to Asominori until the
BC
3
F
1
generation to produce a series of CSSLs with
the Asominori background; 66 CSSLs were subse-
quently selected from the BC
3
F
2
(Kubo et al. 1999).
CSSL84, a CSSL with several IR24 introgressed
segments in the Asominori genetic background
(Kubo et al. 1999), also contains the DTH12 locus.
To further reduce the size of the introgressed
segment, CSSL84 was backcrossed four times to
Asominori, and the resulting BC
4
F
1
plants were self-
pollinated to construct a CSSL84/Asominori BC
4
F
2
population with 288 plants. From this population, a
near-isogenic line (NIL) [NIL(DTH12)] harboring a
homozygous 26-cM segment of IR24 at the DTH12
locus in the Asominori background was selected by
screening molecular markers on all chromosomes. In
NIL(DTH12), the IR24 fragment was flanked by the
markers InDel12-1 and RM6296 on chromosome 12.
Growth conditions
Asominori and NIL(DTH12) were grown under four
different growth regimes: Beijing natural LD (NLD)
conditions (northern China, 116.4°E, 39.9°N, day
length [15 h), Hainan natural SD (NSD) conditions
(southern China, 110.0°E, 18.5°N, day length \12 h),
controlled LD (CLD) conditions (14 h light, 30 °C/
10 h darkness, 25 °C), and controlled SD (CSD)
conditions (10 h light, 30 °C/14 h darkness, 25 °C).
Light in the artificially controlled growth cabinets was
provided by fluorescent lamps at an intensity of
300 lmol m
-2
s
-1
; humidity was set at 70 %.
Phenotypic evaluation
Days to heading of individual plants were recorded as
the number of days from seeding to heading. In
segregating populations, a late-heading plant was
defined as a plant which headed later than
NIL(DTH12), and a late-heading family was one in
which all 20 of the randomly selected plants headed at
the same time as, or later than, NIL(DTH12). Seed
maturity percentages were measured as described
previously (Wu et al. 2013).
DNA marker analysis
Total DNA was extracted from leaves using the
method described previously (Murray and Thompson
1980). Polymorphisms between CSSL84 and Asomi-
nori were screened using simple sequence repeat
(SSR) primers (McCouch et al. 2002; International
Rice Genome Sequencing Project 2005) and insertion/
deletion (InDel) markers which covered the genomic
region harboring the IR24 segments. Polymorphic
markers and the phenotypic data were used for QTL
analysis. Based on the distributions of the SSR and
InDel markers and initial mapping of DTH12, more
InDel markers were designed for the fine mapping of
DTH12. Primer pairs were designed with Primer
Premier ver. 5.0 (PREMIER Biosoft International,
Palo Alto, CA) software. The sequence information is
listed in ESM Table 1.
The PCR assay of the SSR and InDel markers was
performed in a 20-lL reaction volume containing
2lL of template DNA (20 ng lL
-1
), 1 lL of each
primer (10 lML
-1
), 1 lL dNTP (10 mM L
-1
), 19
buffer (Mg
2?
plus), and 0.2 lL Taq DNA polymerase
(5 U lL
-1
). Amplifications were carried out in a
070–951 Thermocycler (Biometra GmbH, Go
¨ttingen,
Germany) under the following conditions: one cycle at
98 °C for 3 min, followed by 35 cycles of 30 s at
94 °C, 30 s at 55 °C, and 1 min at 72 °C, with a final
extension of 7 min at 72 °C. Amplified DNA products
were separated by gel electrophoresis on 8 % poly-
acrylamide gels in 0.59TBE buffer and visualized by
silver staining (Bassam et al. 1991).
QTL analysis
The linkage map of the Asominori/IR24 RIL popula-
tion, including 375 restriction fragment length poly-
morphis markers (Tsunematsu et al. 1996), was used
for QTL analysis. An SSR marker linkage map of the
CSSL84/Asominori BC
4
F
2
population was con-
structed using MAPMAKER/EXP ver. 3.0 (Lander
et al. 1987). Additive QTLs for heading date were
detected by inclusive composite interval mapping
(ICIM) (Li et al. 2007). In the first step of ICIM,
Pvalues of 0.01 for entering variables (PIN) and 0.02
for removing variables (POUT) were used to select
significant markers. In the second step, a threshold
limit of detection (LOD) of 3.0 was used to declare a
Mol Breeding (2014) 34:311–322 313
123
QTL significant. The gene action of an identified QTL
was evaluated by the ttest to identify significant
differences between phenotypic values of the recur-
rent parent Asominori and those of lines harboring the
target QTL alleles derived from the donor parent IR24.
Fine mapping
NIL(DTH12) and Asominori were crossed, and the
resulting F
1
was self-pollinated to produce a popula-
tion of F
2:3
families. Late-heading F
2:3
families were
selected for the preliminary mapping of DTH12.F
4:5
families were further developed from the F
3
individ-
uals to expand the mapping populations and fine-map
the DTH12 locus.
Quantitative reverse transcriptase-PCR analysis
For the diurnal expression analysis, penultimate leaves
were collected from 68- and 36-day-old plants grown
under CLD and CSD conditions, respectively. Three
plants each of Asominori and NIL(DTH12)were
harvested at 4-h intervals during a 24-h period. Total
RNA was extracted from leaves usingthe RNeasy Plant
Mini kit (Qiagen, Hilden, Germany), and then 20 lLof
cDNA was synthesized using 1 lgRNAinaQuanti-
Tect
Ò
Reverse Transcription kit (Qiagen). Quantitative
restriction transcriptase (RT)-PCR (qRT-PCR) assays
of the 20-lL reaction volumes were carried out using
0.5 lL cDNA, 0.2 lM of each gene-specific primer, and
SYBR
Ò
Premix Ex Taq
TM
Kit (TaKaRa, Otsu, Japan) in
an ABI PRISM 7900HT (Applied Biosystems, Foster
City, CA). The rice Ubiquitin (UBQ)gene
(LOC_Os03g13170) was used as the internal control.
Primers for the qRT-PCR assay of UBQ,Hd3a,RFT1,
Hd1,Ehd1,RID1/OsID1/Ehd2,Ehd3,OsMADS50,
OsMADS51,Ghd7,DTH8/Ghd8,PHYB,andSE5 were
obtained from previous studies (Wu et al. 2013); those
for Ehd4 were obtained from Gao et al. (2013). The
primers for OsGI were 50-CCTTGAAGCCATCATC
TGTTG-30and 50- GGCCTGGATCGACGAAATA-30;
those for OsMADS56 were 50-GCCCCCAGCCTACA
GAAA-30and 50-TTTTCCGTCTGGACTCATCAAG-30;
those for OsCOL4 were 50-GAACACTCCATGGCC
CACAG-30and 50-CCCTCTCCTTCCCCTTGCT-30.
The qRT-PCR conditions consisted of one cycle of
95°C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C
for 30 s; at the disassociation stage, the conditions
were 95 °C for 15 s, 60 °C for 1 min, and 95 °C for
15 s. Data were analyzed according to the relative
quantification method (Livak and Schmittgen 2001).
Statistical analysis
Data for comparison included at least ten samples.
ttests were carried out using Microsoft Excel 2007 and
significance levels of P=0.01 or P=0.05.
Results
Detection of QTLs for heading date
and development of a NIL
The CSSL line CSSL84 which carried a chromosome
segment of IR24 containing the DTH12 locus in the
Asominori genetic background was selected to vali-
date DTH12 (Fig. 1a). A linkage map for the short arm
of chromosome 12 was constructed from a BC
4
F
2
population comprising 80 individuals derived from a
cross between CSSL84 and Asominori. DTH12 was
mapped in a 26-cM region flanked by markers
InDel12-1 and RM6296, co-segregating with RM20
(Fig. 1b); it had a LOD value of 7.79 and explained
31.71 % of the phenotypic variance (ESM Table 2).
The allele from IR24 contributed to an increased
number of days to heading.
The NIL line NIL(DTH12) was selected from the
CSSL84/Asominori//Asominori BC
4
F
2
population
using SSR and InDel markers. This line contained
only a 26-cM homozygous IR24 segment in the
Asominori background and harbored the DTH12 locus
Fig. 1 Coarse mapping of DTH12.aSketched map of the
CSSL84 genotype. White and black bars Asominori background
and IR24 insertion fragments, respectively. bGenetic map of
DTH12 on chromosome 12 constructed from 80 plants in a
CSSL84/Asominori BC
4
F
2
population
314 Mol Breeding (2014) 34:311–322
123
between markers InDel12-1 and RM6296. Genotype
analysis indicated that NIL(DTH12) was 96.875 %
isogenic with Asominori.
Phenotypes of Asominori and NIL(DTH12) in four
environmental regimes
Field examination showed that the heading date for
NIL(DTH12) (134.5 ±0.5 days) was 8.3 days later
than that for Asominori (126.2 ±0.4 days) under
NLD conditions. In contrast, there was no significant
difference in heading date between NIL(DTH12)
(68.7 ±0.9 days) and Asominori (66.9 ±1.1 days)
under NSD conditions (Fig. 2a, b). Similar patterns for
the two genotypes were observed in the controlled
conditions (Fig. 2b). Under CLD conditions, days to
heading for NIL(DTH12) was 96.0 ±1.7, an increase
of 9.1 days compared with Asominori (86.9 ±0.9).
Under CSD conditions, there was no significant
difference in days to heading between NIL(DTH12)
(55.8 ±0.9 days) and Asominori (54.5 ±0.7 days).
Under NLD conditions, 81.5 % of Asominori grains
had reached maturity (judged by yellow seed coat
pigmentation) at harvest, compared with only 24.8 %
of NIL(DTH12) grains (Fig. 2c, d). This difference
was possibly due to the lower temperature during seed
ripening of NIL(DTH12) than of Asominori, as
NIL(DTH12) headed 8.3 days later than Asominori
when cold weather is approaching. In contrast, the seed
maturity percentages of Asominori and NIL(DTH12)
were both 100.0 % under NSD conditions (Fig. 2d).
These results indicate that in the Asominori genetic
background the Asominori allele of DTH12 is better
adapted to NLD (especially low temperature) environ-
ments in terms of reproductive fitness.
Comparative leaf emergence rates for Asominori
and NIL(DTH12) were investigated to determine
whether a prolongation of the plastochron and a
decreased growth rate might lead to the late heading
of NIL(DTH12). An almost consistent leaf emergence
rate was observed between Asominori and
NIL(DTH12) under both CLD and CSD conditions
(ESM Fig. 2). Hence, growth rates were not affected in
the NIL(DTH12) plants, suggesting that the major role
of DTH12 in rice is to control floral transition under LD
conditions.
Fine mapping of the QTL DTH12
For fine mapping, NIL(DTH12) was initially crossed
with Asominori. Days to heading of the F
1
(128.5 ±0.7 days) was intermediate between that of
Asominori (126.2 ±0.4 days) and that of
NIL(DTH12) (134.5 ±0.5 days), but was slightly
skewed toward earliness. Using a single-locus additive
and dominance quantitative genetics model, we
Fig. 2 Comparison of the parental lines used for fine mapping
of DTH12.aPhenotypes of Asominori (Aso) and its near-
isogenic line (NIL) with DTH12 under natural long-day (NLD)
conditions in Beijing. Photo was taken when Aso was heading.
bDays to heading of Aso and NIL plants under four different
growth regimes. NSD Natural short-day, CLD control long-day,
CSD control short-day. Probabilities of difference (P) values
were obtained in two-tailed ttests. cBrown rice grains from Aso
and NIL plants harvested 60 days after heading of Aso under
NLD conditions. dSeed maturity percentages of Aso and NIL
plants grown under NLD and NSD conditions, respectively.
Numbers in columns Number of plants. Data are given as
mean ±standard error
Mol Breeding (2014) 34:311–322 315
123
determined the additive (a) and dominance (d) effects
to be -4.2 and -1.9 days, respectively, and the degree
of dominance (d/a) to be 0.45. The negative additive
effect indicated that the Asominori allele of DTH12
reduced the number of days to heading. The dominance
effect had the same direction as the additive effect,
suggesting that the Asominori allele of DTH12 was
partially dominant to the IR24 allele. Among 248 F
2
plants, the observed phenotypes (2 homozygous early
heading plants: 117 heterozygous plants: 129 homo-
zygous late heading plants; Fig. 3) did not fit a 1:2:1
segregation ratio (v
2
=130.86 [v
20.05 (2)
=5.99,
P=3.83 910
-29
). The extreme segregation distor-
tion was likely due to linkage between DTH12 and a
gametophytic reproductive factor (Harushima et al.
2001).
Two hundred late-heading families were selected
from 358 Asominori/NIL(DTH12)F
2:3
families under
NLD conditions. DTH12 was initially mapped to a
26-cM interval between markers InDel12-1 and
RM6296 (Fig. 4a). To enlarge the mapping popula-
tions, we developed 2,388 F
4:5
families from the F
3
individuals harboring heterozygous DTH12 regions,
and ultimately selected 1,227 late-heading lines to
fine-map the DTH12 locus. With the markers InDel12-
1 and RM6296, we chose 58 and 186 recombinants,
Fig. 3 Distribution of days to heading in a NIL(DTH12)/
Asominori F
2
population under natural long-day conditions.
Black bars Homozygous early heading plants, white bars
homozygous late heading plants, shaded (gray)region hetero-
zygous plants
Fig. 4 High-resolution mapping of DTH12.aThe DTH12
locus was initially mapped between markers InDel12-1 and
RM6296 on chromosome 12S. Gray bar The qS12 location
(Zhang et al. 2011). bFine mapping of the DTH12 locus using
1,227 recessive families. Number of recombinant individuals is
given below the marker name.cThe DTH12 locus was delimited
to a 153-kb region between markers W020 and W018 on
Nipponbare bacterial artificial chromosome contigs OS-
JNBb00119L20 and OJ1085_G07. d32 open reading frames
were predicted between the W020 and W018 markers;
arrowheads gene directions
316 Mol Breeding (2014) 34:311–322
123
respectively (Fig. 4b). Eleven markers (ESM Table 1)
were developed to identify further recombinants, and
five and six recombination events were identified with
InDel markers W005 and W014, respectively
(Fig. 4b). Finally, DTH12 was narrowed to a 153-kb
genomic region in Nipponbare (japonica reference
sequence) spanning contigs OSJNBb00119L20 and
OJ1085_G07 (Rice Genome Annotation Project,
http://rice.plantbiology.msu.edu/; Fig. 4c) and
flanked by InDel markers W020 and W018 (Fig. 4b).
The six molecular markers surrounding DTH12 in the
key recombinants and phenotypes of their progeny are
shown in ESM Table 3. Based on the Rice Genome
Annotation Project, 32 putative open reading frames
(ORFs) were predicted in this region (ESM Table 4).
The corresponding region of the indica genome rep-
resented by the sequenced cultivar 93-11 spans
174 kb. Due to possible different genome structure
among genotypes, sequencing of this genomic region
in Asominori and IR24 will be needed to predict the
gene models in the region.
Relation between DTH12 and other photoperiodic
genes
The photoperiodic response of DTH12 suggested that
it could participate in a photoperiodic pathway playing
a central role in control of flowering time in rice
(Komiya et al. 2009). To understand the molecular
basis of DTH12 in photoperiodic control of flowering,
we performed qRT-PCR assays to examine mRNA
abundance of several previously reported photope-
riod-related genes in Asominori and NIL(DTH12)
plants under both CLD and CSD conditions. Interest-
ingly, two florigen genes, Hd3a and RFT1, showed
higher expression levels in Asominori than in
NIL(DTH12) under CLD conditions (Fig. 5a, b).
However, no significant differences were observed
under CSD conditions (Fig. 5f, g). This result is
consistent with the absence of significant phenotypic
differences in number of days to heading between
Asominori and NIL(DTH12) under CSD conditions.
Hd1 and Ehd1 are known to be independent
regulators of Hd3a and RFT1 (Komiya et al. 2009).
We examined their mRNA levels in Asominori and
NIL(DTH12) to investigate whether both genes were
regulated by DTH12. However, the expression levels
of Hd1 and Ehd1 were not significantly different
between Asominori and NIL(DTH12) under both CLD
and CSD conditions (Fig. 5c, d, h, i). Moreover, six
positive regulators (OsGI,RID1/OsID1/Ehd2,Ehd3,
Ehd4,OsMADS50, and OsMADS51) and six negative
regulators (Ghd7,DTH8/Ghd8,OsMADS56,Os-
COL4,PHYB, and SE5) were examined to test whether
DTH12 affected the expression of other regulators of
the Hd1 or Ehd1 pathways. Similarly, no significant
differences in the expression of these regulators were
detected between Asominori and NIL(DTH12) under
both CLD (ESM Fig. 3) and CSD (ESM Fig. 4)
conditions. These results suggest that DTH12 up-
regulates the florigen genes, but has no effect on the
Hd1 and Ehd1 pathways.
DTH2 was recently identified as a floral activator
independent of Hd1 and Ehd1 (Wu et al. 2013).
Therefore, we then checked whether the DTH12
regulation of florigen genes was mediated through
DTH2. However, the expression of DTH2 was not
different in Asominori and NIL(DTH12) (Fig. 5e, j),
suggesting that DTH2 is not downstream of DTH12 in
the gene regulatory network of flowering. Thus, our
observations suggest that DTH12 could be a floral
activator promoting heading by up-regulating Hd3a
and RFT1 under LD conditions, while the upstream
genes of DTH12 remain as yet unknown.
Discussion
DTH12 is a QTL controlling heading date
on the duplicated segment of chromosome 12
There is a conserved segmental duplication between
the distal ends of the short arms of chromosomes 11
and 12 of approximately 2 Mb (Jiang et al. 2007;
Jacquemin et al. 2009). It is difficult to perform fine
mapping of genes on the distal ends of these chromo-
some arms due to their high similarity. Recent
progress in the complete genome sequencing of rice
provides opportunities to explore SSR and InDel
markers exhibiting polymorphisms between highly
similar sequences. In the study reported here, we
developed polymorphic SSR and InDel markers and
successfully mapped a QTL named DTH12 control-
ling heading date on the short arm of chromosome 12.
The QTL was delimited to a 153-kb genomic region
between InDel markers W020 and W018 (Fig. 4b, c).
Mol Breeding (2014) 34:311–322 317
123
Fig. 5 Diurnal expression
analysis of Hd3a,RFT1,
Hd1,Ehd1, and DTH2 in
Asominori (Aso) and its NIL
with DTH12 (NIL) under
controlled long-day (CLD)
(a–e) and controlled short-
day (CSD) conditions (f–
j) using qRT-PCR. Open
and filled bars Light and
dark periods, respectively.
UBQ Ubiquitin. The
mean ±standard deviations
were obtained from three
technical and two biological
replications
318 Mol Breeding (2014) 34:311–322
123
The target DNA segment is located in the 2-Mb
duplicated segment of chromosome 12.
Segregation distortion is a common feature in
crosses between indica and japonica cultivars due to
gametophytic reproductive barriers (Harushima et al.
2001). In the NIL(DTH12)/Asominori F
2
population,
segregation at the DTH12 locus did not fit the expected
segregation for a single Mendelian locus. Recently,
Zhang et al. (2011) reported that a major QTL (qS12)
causing male semi-sterility was a segregation distor-
tion locus and was located in an approximately 400-kb
DNA segment between markers MS062 and MS102 in
the distal part chromosome 12S. Because W020 is
located only 64 kb from MS102 (Fig. 4a; Zhang et al.
2011), qS12 should be tightly linked to DTH12 and
could, therefore, be the cause of the severe segregation
distortion of days to heading in our F
2
population.
There is an abundance of published studies on
heading date in rice (see review Komiya et al. 2009;
Tsuji et al. 2011; Brambilla and Fornara 2013). The
chromosome locations and genetic effects of numer-
ous heading date QTLs have been deposited in
Gramene (http://www.gramene.org). Using the same
Asominori/IR24 RIL population, Yu (2005) identified
12 heading date QTLs; in our study, we focused on
only one of these, DTH12. However, another gene
(SPL11) regulating flowering was isolated on chro-
mosome 12 which is a premature-termination muta-
tion that results in delayed flowering in rice under LD
conditions but has no effect under SD ones, similar to
DTH12 allele from IR24. Because DTH12 resides in
the physical position between 1,086,934 bp and
1,239,880 bp of the short arm of chromosome 12,
whereas SPL11 resides between 23,616,936 bp and
23,621,605 bp of the long arm, they should obviously
be different genes. The mutation of SPL11 showed
spotted leaves, and it negatively regulated pro-
grammed cell death and disease resistance (Vega-
Sa
´nchez et al. 2008); we observed no such phenotypes
in plants carrying DTH12 alleles (Fig. 2a). In addition,
a heading date QTL (Hd13) was detected using a
BC
2
F
2
population derived from a cross between indica
cultivar Nona Bokra and japonica cultivar Koshihikari
under NLD conditions. Hd13 was identified in an
approximate 1.9-Mb interval between markers R3375
and R1869 on chromosome 12 (Yano et al. 2001; Uga
et al. 2007). Although W018 is located approximately
4.4 Mb from R3375 in japonica cultivar Nipponbare
(Uga et al. 2007), we could not rule out the possibility
that DTH12 might be the same as Hd13 due to the
different populations used in mapping the two QTLs.
Further analyses, such as map-based cloning and
sequence comparison, will be required to determine
the relationship between these two loci.
DTH12 promotes heading by up-regulating Hd3a
and RFT1 under LD conditions
The northern geographic limit of the wild rice
progenitor Oryza rufipogon is around 28°N, whereas
cultivated rice is now grown between latitudes 55°N
and 36°S (Khush 1997; Izawa 2007). Izawa (2007)
proposed that two independent floral pathways (Hd1-
dependent and Ehd1-dependent) controlled flowering
in rice. Recently, Wu et al. (2013) indicated that DTH2
functions independently of Hd1 and Ehd1 to induce
flowering under LD conditions. In our study, we
showed that DTH12 specifically promotes heading
under LD conditions by inducing Hd3a and RFT1, but
that it has no effect on Hd1,Ehd1,orDTH2 (Fig. 5a–
e). To clarify whether DTH12 is downstream or
independent of Hd1,Ehd1, and/or DTH2, we need to
isolate DTH12 and to check its expression in NILs
carrying non-functional Hd1,Ehd1 or DTH2 alleles.
Our results do, however, provide some promising
candidate genes for future work.
DTH12 is a minor-effect QTL and useful in rice
breeding
Heading dateis one of the mostimportant traits enabling
rice to adapt to seasonal differences and specific growth
conditions (Izawa 2007). Successful sexual reproduc-
tion in flowering plants depends on the appropriate
timing of flowering, which is an important means of
plant adaptation to environmental changes. Asominori
is a temperate japonica cultivar adapted to the temperate
region of Japan,and IR24 is an indica cultivar adapted to
the tropical region of Philippines. Cropping regions of
Asominori are more northerly than IR24, suggesting
that some traits involving regional adaptability, such as
heading date, differ between them. In this study, we
found that DTH12 is a minor-effect QTL with a
relatively small effect on heading date under NLD
conditions (affecting heading date by about 8 days;
Fig. 2a, b). However, it is notable that the change in
heading date by several days may lead to a large
difference in grain yield and reproductive fitness as
Mol Breeding (2014) 34:311–322 319
123
indicated by higher percentages of mature seeds
(Fig. 2c, d). These results suggest that the Asominori
allele of DTH12 is better adapted to LD conditions.
Many researchers have found that flowering time is
a complex trait determined by numerous minor-effect
QTLs (Buckler et al. 2009; Huang et al. 2011). Since
the effect of DTH12 among F
2
individuals was small
and easily affected by environment, we used back-
cross-derived F
2:3
and F
4:5
families as the final
mapping populations in order to identify genotypes
and finally to locate DTH12 to a region of 153 kb. A
similar strategy was used to map a minor-effect QTL
DTH2 controlling heading date (Wu et al. 2013).
Despite its minor effect, DTH12 could be useful in
breeding by changing heading date and seed maturity
in the same way as DTH2. Using MAS, if we wanted to
cultivate in northern China an elite germplasm origi-
nating in southern China that carries the IR24 allele of
DTH12, we could introduce the Asominori allele of
DTH12 into this variety to replace the IR24 allele of
DTH12 in order to achieve earlier heading and better
fitness under the growing conditions in northern China.
In conclusion, we have identified a minor-effect
QTL with more precision and fine-mapped it to a
153-kb genomic region. Further study of this new
locus for flowering time in rice will contribute to our
basic understanding of adaptation and may be useful
for marker-assisted breeding.
Acknowledgments We thank Dr. Atsushi Yoshimura
(Kyushu University, Japan) for providing the CSSL
population. This work was supported by grants from the 973
Program of China (Grants 2010CB125904-4).
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