Cold tolerance at the early growth stage in wild

and cultivated rice

Akhil Ranjan Baruah Noriko Ishigo-Oka
Mieko Adachi Yasuyo Oguma
Yoshiro Tokizono Kazumitsu Onishi
Yoshio Sano
Received: 12 May 2008 / Accepted: 16 June 2008 / Published online: 5 July 2008
Springer Science+Business Media B.V. 2008
Abstract The present study was conducted to
understand the pattern of variation and the genetic
bases for cold tolerance at the early growth stage in
Asian rice. The genetic variation was investigated at
the germination, plumule and seedling stages among
57 strains including cultivated rice (Oryza sativa ssp.
indica and ssp. japonica) and its wild progenitor
(Oryza rupogon). The signicant differentiation of
cold tolerance was observed among the taxonomi-
cally divided groups. At the germination stage, both
indica and japonica subspecies tended to be more
tolerant than O. rupogon, whereas at the plumule
and seedling stages, ssp. japonica tended to be more
tolerant than ssp. indica and O. rupogon. Further-
more, in cold tolerance at the plumule stage, the
clinal variation across the latitude of origins was
observed within O. rupogon and ssp. japonica,
suggesting that the current pattern of variation seems
to have been shaped by both their phylogenetic
histories and on-going adaptation to the local envi-
ronments. QTL analysis between O. sativa ssp.
japonica (tolerant) and O. rupogon (susceptible)
revealed ve putative QTLs for cold tolerance at the
plumule and seedling stages but not at the
germination stage. Substitution mapping was also
carried out to precisely locate the two major QTLs for
cold tolerance at the plumule stage, which could be
used for improvement of tolerance to cold stress in
ssp. indica.
Keywords Cold tolerance Early growth stage
Latitudinal cline Oryza rupogon O. sativa QTL
Introduction
Low temperature is a major limiting factor for
agricultural productivity in temperate areas and at
high altitudes in Asian cultivated rice (Oryza sativa)
(Mackill and Lei 1997; Vergara 1976). The cultivated
rice species occupying broad geographic ranges
consists of two subspecies, ssp. indica and ssp.
japonica, and exhibit considerable intraspecic var-
iation in tolerance to cold stress. The ssp. indica
cultivars are conned mostly to tropical lowland
areas, whereas ssp. japonica cultivars are grown in
tropical to temperate zones (Glaszmann 1987; Oka
1988; Vergara 1976). During the early growth stage,
the occurrence of low-temperature or cold stress
affects seed germination and seedling establishment.
Since ssp. indica shows a lower level of tolerance to
cold stress than ssp. japonica, considerable breeding
efforts have been made to improve the cold tolerance
of indica cultivars (Glaszmann et al. 1990; Mackill
A. R. Baruah (&) N. Ishigo-Oka M. Adachi
Y. Oguma Y. Tokizono K. Onishi Y. Sano
Laboratory of Plant Breeding, Department of Applied
Science, Research Faculty of Agriculture, Hokkaido
University, Sapporo 060-8589, Japan
e-mail: arbaruah@abs.agr.hokudai.ac.jp
1 3
Euphytica (2009) 165:459470
DOI 10.1007/s10681-008-9753-y
and Lei 1997). Although ssp. indica cultivars were
screened for cold tolerance, their isozyme patterns
indicated that most of the cold tolerant cultivars
presumed as ssp. indica were genetically similar to
ssp. japonica (Glaszmann 1987). It has been reported
that ssp. japonica tends to show a higher degree of
cold tolerance at germination (Oka 1954), plumule
(Morishima and Oka 1981; Oka 1988) and seedling
stages (Glaszmann et al. 1990; Mackill and Lei 1997)
than ssp. indica that suffers various types of injury
due to cold stress. Thus, cold tolerance at the early
growth stage is known to be one of the major
characteristics for distinguishing the two subspecies
of cultivated rice. Furthermore, the genetic variation
of cold tolerance has been reported to be associated
with their geographical distribution (Mackill and Lei
1997; Nagamine and Nakagahra 1990), giving a
question regarding to what extent the genetic differ-
entiation of cold tolerance has resulted from the
regional adaptation.
With regard to the genetic bases for cold tolerance
in cultivated rice, few major genes, such as Chs1
(Chuong and Omura 1982), Cts1 (Kwak et al. 1984)
and Cts 2 (Nagamine 1991) are reported to confer
cold tolerance at the seedling stage; however, recent
quantitative trait locus (QTL) analyses revealed that
numerous QTLs for cold tolerance are present on the
rice genome (Andaya and Mackill 2003; Fujino et al.
2004; Lou et al. 2007; Misawa et al. 2000; Miura
et al. 2001; Zhang et al. 2004), suggesting that
adaptation to cold climates might involve complex
features in physiological and genetic mechanisms.
The distribution of genetic variants in plants is
generally affected by both current patterns of micro-
evolutionary forces, such as selection and gene ow,
and by the phylogenetic history of populations
(Schaal et al. 2003). The wild progenitor of Asian
cultivated rice, O. rupogon is mostly conned to
tropical lowland regions like ssp. indica (Oka 1988;
Vaughan et al. 2003). Because of their limited
dissemination by human, the patterns of genetic
variation in wild rice species will provide useful
information on adaptive signicances for cold toler-
ance; however, our present knowledge is very limited
regarding cold tolerance in Asian wild rice.
The present study was conducted to understand the
pattern of variation and the genetic bases for cold
tolerance at the early growth stage in Asian cultivated
and wild rice. We rstly surveyed genetic variation of
cold tolerance at the germination, plumule and
seedling stages, using 34 cultivated and 23 wild rice
strains. Then, we investigated the relationship
between cold tolerance and latitude of their origin
to understand the signicances of cold tolerance for
the adaptation to local environments. Further, QTL
analysis was performed in a cross between ssp.
japonica and wild rice strains for comparing the
genetic bases for cold tolerance at the three growth
stages. Finally, we carried out substitution mapping
of the two detected major QTLs (qCT11P and
qCTP12) for cold tolerance at the plumule stage,
since this trait was revealed to be associated with the
local adaptation as well as the differentiation of two
subspecies.
Materials and methods
Plant materials
The genetic stocks used were 34 cultivated (Oryza
sativa) and 23 wild (Oryza rupogon) rice strains
which were chosen to represent the diverse origins
(Table 1), from the genetic stocks preserved at
National Institute of Genetics, Mishima and Plant
Breeding Laboratory, Hokkaido University, Japan. O.
sativa comprises of the two subspecies, ssp. japonica
and ssp. indica, which are distinguished from each
other by morphological and physiological character-
istics as well as molecular markers (Garris et al.
2005; Glazmann 1987; Oka 1988). Both tropical and
temperate japonica subpopulations were included in
ssp. japonica in this study. O. rupogon was used for
the Asian wild rice which contained annual, inter-
mediate and perennial types although an annual type
is sometimes called O. nivara (Vaughan et al. 2003).
All the strains were used after several times of self-
pollination for genetic purication.
Although ssp. indica distributes to tropical and
subtropical regions, ssp. japonica extends its distri-
bution to temperate regions, suggesting that ssp.
japonica might be more tolerant to cold stresses than
ssp. indica. It is known that the genetic improvements
and modernization of cultivation systems made rice
production possible in Hokkaido island of Japan (42
N45 N) which is one of the northernmost areas of
rice cultivation at present (Satake 1976). To compare
the degree of cold tolerance, ssp. japonica strains
460 Euphytica (2009) 165:459470
1 3
were roughly classied into I (Hokkaido island), II
(temperate) and III (tropical and subtropical), accord-
ing to the country of origins. O. rupogon distributes
predominantly to tropical and subtropical areas. The
latitudes of their collection sites were used to
examine the clinal variation of cold tolerance among
strains, as listed in Table 1.
Evaluation of cold tolerances
Plants differ in their tolerance to chilling (015C)
and freezing (\0C) temperatures. In this study, cold
tolerance refers to chilling tolerance. Plants from
temperate regions are chilling tolerant, although most
species are not very tolerant to freezing but are able
Table 1 List of strains used
Subspecies
a
Strain Origin
b
(a) Cultivated rice (O.sativa)
ssp. japonica Akage Japan (I)
Akaine do. (I)
Akamuro do. (I)
Bouzu do. (I)
Kitamurawase do. (I)
A58 (Kokushokuto-2) do. (I)
Norin 20 do. (I)
Iburiwase do. (I)
Koshihikari do. (II)
Norin 8 do. (II)
Nipponbare do. (II)
Acc 780
c
China (II)
Hanfeng do. (II)
Taichung 65 Taiwan (II)
Acc 840
c
do. (II)
Nabeshi do. (II)
Garumbalay Philippines (III)
Malagkit pirurutong do. (III)
C9064
c
Thailand (III)
Silewah Indonesia (III)
C9071
c
India (III)
ssp. indica Acc 775
c
China
Ying-Nien 36 do.
Pehkuh Taiwan
Hong-ka-chiu do.
Patpaku do.
Acc27590
d
Bangladesh
Kasalath India
PTB 10 do.
Paddy type 52 do.
Surjmukhi do.
C6320
c
Cambodia
Acc 609
c
Indonesia
IR36 Philippines
Strain
e
Type
f
Origin Latitude (N)
(b) Wild rice (O. rupogon )
W593 (P) Malaysia 3.1
Acc105416 (A) Sri Lanka 6.5
Acc105451 (A) Sri Lanka 6.5
W1807 (A) Sri Lanka 6.5
W1294 (P) Philippines 8.0
W1551 (A) Thailand 14.3
Table 1 continued
Strain
e
Type
f
Origin Latitude (N)
W2005 (P) India 15.4
W172 (P) Thailand 16.3
W2002 (A) India 16.9
W2007 (P) India 16.9
W1860 (A) Thailand 17.0
W130 (I) India 19.0
W630 (A) Myanmar 20.1
W1681 (P) India 20.1
W107 (A) India 20.1
W106 (A) India 20.3
W1952 (P) China 23.0
W1990 (A) India 23.2
W149 (P) India 24.3
W154 (I) Taiwan 25.0
W1945 (P) China 25.0
W1944 (P) China 27.0
W1943 (P) China 28.0
a
ssp. japonica includes temperate and tropical japonica
strains
b
I, II, III are three different regions for comparison of cold
tolerance within ssp. japonica (see text)
c
Accession number of National Institute of Genetics (NIG),
Japan
d
Accession number of International Rice Research Institute,
Philippines (IRRI)
e
Accession number of NIG, except for two strains
(Acc105416 and Acc105451), which are the accession
number of IRRI
f
A, P, and I indicate annual, perennial and intermediate type,
respectively
Euphytica (2009) 165:459470 461
1 3
to increase their freezing tolerance by being exposed
to chilling temperatures, a process known as cold
acclimation during which numerous physiological
and molecular changes occur (Thomashow 1999).
Most of the studies on plant responses to cold stress
are focused on the mechanism of cold acclimation
rather than chilling tolerance. Nevertheless, recent
evidence indicates that some of the molecular
changes that occur during cold acclimation are also
important for chilling tolerance (Gong et al. 2002).
The degree of cold tolerance depends upon temper-
ature and duration of exposure to cold stress. We
examined tolerance to chilling temperatures at three
different stages (see below). Statistical analyses for
phenotypic comparisons were conducted using
STATVIEW (SAS Institute, Cary NC).
Germination stage
Low-temperature germinability (LTG) was prelimi-
narily examined at 10C, 13C and 15C using two
ssp. japonica (A58 and Taichung 65), two ssp. indica
(Pehkuh and Patpaku) and three wild rice (W107,
W630 and W1944) strains. Since a broader range of
variation was observed among strains at 13C than at
10C or 15C, 13C was adopted to survey a number
of strains in this study. Ten hulled seeds of each strain
were incubated on moistened lter papers in petri
dishes at 13C in the dark, with two replications. The
number of germinated seeds was counted daily until
20 days after sowing. For comparison, the germina-
tion coefcient (GC) was calculated by percentage of
germinated seeds/the mean days to germination,
according to the methods of Lee and Taguchi
(1969). The degree of tolerance was evaluated using
the scale of 0 (GC = 0.0) to 4 (GC = 16.1). All the
used seeds germinated after two days at 30C,
conrming no seed dormancy.
Plumule stage
The cold tolerance at the plumule stage was inves-
tigated according to the methods of Oka (1958). In
each line, 12 seeds were germinated on moistened
lter papers in petri dishes at 30C with two
replications. The germinated seeds attaining a plu-
mule length of 1.01.5 cm were incubated at 01C
for two days (48 h) in the dark. Then, the germinated
seeds were transferred into a growth cabinet
(BIOTRON LPH200: Nippon Medical & Chemical
Instruments Co. LTD), where the light of about
300 lmol m
-2
s
-1
was supplied from uorescent
lamps, giving a 14-h daylength at 26C. After six
days, the degree of tolerance was evaluated quanti-
tatively based on the injury and growth of plumules
using the scores of 0 (susceptible) to 4 (tolerant) as
shown in Fig. 1a.
Seedling stage
The cold tolerance at the seedling stage was inves-
tigated according to the methods of Nagamine and
Nakagahra (1990) with slight modications. In each
line, seeds were germinated in petri dishes at 30C
and ve seedlings were transplanted into plastic pots
(6 cm in diameter) lled with soil with two replica-
tions. They were grown in a growth cabinet
(BIOTRON LPH200: Nippon Medical & Chemical
0
(a)
4 3 2 1
(b)
0 4 3 2 1
Fig. 1 Cold tolerance scores (04) based on the degree of
injury to cold temperature at the plumule (a) and seedling (b)
stages
462 Euphytica (2009) 165:459470
1 3
Instruments Co. LTD), where the light of about
300 lmol m
-2
s
-1
was supplied from uorescent
lamps, giving a 14-h daylength at 26C. The 12-day-
old seedlings were subjected to 5C for four days in a
growth chamber with a 14-h daylength. This condi-
tion of cold treatment is suggested to be most
effective to evaluate the genetic variation of cold
tolerance in cultivated rice (Nagamine and Nak-
agahra 1990). After the cold treatment, seedlings
were cut at 5 cm above soil and they were grown at
26C (14-h daylength) for ve days. Based on the
recovery of shoots, the degree of tolerance was
evaluated using the scores of 0 (susceptible) to 4
(tolerant) as shown in Fig. 1b.
QTL analysis and substitution mappings
QTL analysis was conducted in 79 recombinant
inbred lines (RILs) from a cross between A58 and
W107, according to Onishi et al. (2007). A58 is a
strain of ssp. japonica from Hokkaido, Japan and
W107 is a strain of O. rupogon from India
(Table 1). QTL analysis was conducted by simple
interval mapping and MQM mapping methods using
MapQTL version 4.0 (Van Ooijen et al. 2002). In the
rst step, putative QTLs were identied by interval
mapping. Then, one marker at each putative QTL was
selected as a cofactor and the selected markers were
used as genetic background controls in the approx-
imate multiple QTL model of MapQTL. To rene the
mapping, cofactor markers at each QTL were moved
one by one around the putative QTL position, nally
selecting the nearest markers to the QTL, i.e., those
maximizing the LOD score. The threshold for a
signicant QTL was estimated as 2.7 based on the
method of Lander and Botstein (1989). For substitu-
tion mapping, the regions containing each of major
QTLs (qCTP11 and qCTP12) were introgressed into
A58 by backcrosses. In the BC
3
F
2
and BC
4
F
2
generations, recombinants were selected from 262
and 212 segregating plants for qCTP11 or qCTP12,
respectively. To examine the phenotypic effect
precisely, the homozygote for the recombined seg-
ment was obtained after self-pollination.
Genotyping with molecular markers
Genomic DNAs were isolated from leaves collected
fromseedlings according to the methods of Murray and
Thomson (1980) or Monna et al. (2002) with slight
modications. For QTL analyses, a total of 263
markers were used for map construction in the RILs
from a cross between A58 and W107 (Onishi et al.
2007). For ne mapping of the putative QTLs,
additional seven markers (RM 229, RM 1341,
RM2191, RM 27533, RM 247, RM 3472, RM 7619)
were used based on public databases from Gramene
(http://www.gramene.org/markers/) and Rice Genome
Research Program (http://rgp.dna.affrc.go.jp/E/public
data/caps/index.html). In addition, SSR and indel
markers were also developed using SSR Identication
Tool (SSRIT) of Gramenedatabase(Temnykhet al. 2001,
http://www.gramene.org/db/searches/ssrtool) and Pri-
mer3 (Rozen and Skaletsky 2000, http://www.frodo.
wi.mit.edu/cgi-bin/primer3/primer3). They were F10
(5
0
-CATCGATCCGTATGGGTTCT-3
0
and 5
0
-AAAA
ATTACCCATGCGTTTAACT-3
0
), AC108 (5
0
-CCTC
CTCCAACCTCCTCTTC-3
0
and 5
0
-CTAGCCTACG
CGGTTGACAT-3
0
), AC137 (5
0
-GAGGACAGGCTG
TCGTCA-3
0
and 5
0
-ATGTGCCAGAGGAATGGT
TT-3
0
), J03 (5
0
-TGCATCGATACAATCTACGG-3
0
and 5
0
-TATGGCCGTTTCCTCTTCAC-3
0
), K21 (5
0
-
GTAGTGTGTGGTGCTGACC-3
0
and 5
0
-GGGACA
GGAATAGAACCGAAC-3
0
), P054 (5
0
-GCCTGAGT
GGAACGCTAGTT-3
0
and 5
0
-TCAGAATACGAAA
GCTGTAAGG-3
0
), 85C16A (5
0
-GGCACCACGGTC
TATCAAGT-3
0
and 5
0
-TGTGCCCCACCTCTAGT
ACC-3
0
), K22A (5
0
-CGGATGTCTCGTGGAGTAC
G-3
0
and 5
0
-TGCTTGATAACAAGGAAAAACC-3
0
),
K22C (5
0
-TTCTGAAACAGAGACGATAACCA-3
0
and 5
0
-GTCTACACACATGCCGATGC-3
0
) and B10
(5
0
-TTTCATTGTTTCTGGGCTAGG-3
0
, 5
0
-TCTGA
CCGCCTCCAATTATC-3
0
).
Results
Variation in cold tolerance at the early growth
stage in Asian rice
Cold tolerance was surveyed in 34 cultivated and 23
wild rice strains at the germination, plumule and
seedling stages (Table 2). The means of the cold
tolerance scores were compared among ssp. indica,
ssp. japonica and O. rupogon. The genetic variation
of low-temperature germinability (LTG) revealed that
cultivated rice showed a higher level of tolerance
than O. rupogon whereas no signicant difference
Euphytica (2009) 165:459470 463
1 3
was found between ssp. indica and ssp. japonica. In
contrast, cold tolerance scores at the plumule and
seedling stages were signicantly higher in ssp.
japonica than in ssp. indica and O. rupogon.
To examine whether cold tolerance plays a role for
local adaptation, strains of ssp. japonica were roughly
divided into I (Hokkaido island), II (temperate regions)
and III (tropical and subtropical regions) based on their
country of origins. The strains from Hokkaido island
tended to be cold tolerant at the plumule stage, while
the strains from Hokkaido island tended to be slightly
low in LTG unexpectedly, when compared with those
fromthe other regions (Table 3). At the seedling stage,
no difference was found in cold tolerance among
strains from the three regions. The results suggest that
the cold tolerance at the plumule stage might have
played a signicant role for the distribution to the
northern areas in ssp. japonica.
Variation for cold tolerance in O. rupogon
Since wild plants are less inuenced by dissemination
by human than cultivated plants, the genetic variation
in O. rupogon might reect contemporary processes
of adaptation under natural habitats, although O.
rupogon predominantly grows in tropical and sub-
tropical regions. The relationships between the
latitude (N) of the collection sites and the cold
tolerance scores were investigated in O. rupogon to
assess whether the clinal variation is involved in
genetic variation of cold tolerance (Fig. 2). At the
plumule stage, the cold tolerance scores were posi-
tively correlated with the latitudes (r = 0.59,
df = 22, signicant at 1%). No clinal pattern of
variation, on the other hand, was found at the
germination and seedling stages. These results were
consistent with the tendency observed in ssp. japon-
ica, where strains from the northernmost region
tended to show higher cold tolerance scores at the
plumule stage. This supports the assumption that the
cold tolerance at the plumule stage might have played
a role for their geographical distribution.
QTL analysis
At the early growth stage, the cold tolerance signif-
icantly differed between ssp. japonica and O.
rupogon (Table 2). A58, a strain of ssp. japonica
from Hokkaido, Japan, and W107, a strain of O.
rupogon from India, were chosen as their represen-
tatives for further examination. In A58, the degrees of
cold tolerance were 2.70, 4.00, and 3.30 at the
germination, plumule and seedling stages, respec-
tively, whereas the degrees were 0.35, 0.45, and 2.73
in W107. To know the genetic bases of cold tolerance
at the three stages, QTL analyses were conducted
using 79 RILs (F
6
generation) from the cross between
A58 and W107. Three putative QTLs for cold
tolerance at the plumule stage (qCTP1, qCTP11 and
qCTP12) were detected on chromosomes 1, 11 and
12, respectively (Table 4, Fig. 3). Among them,
Table 3 Differences in cold tolerance scores among the
strains of different regions in ssp. japonica
Stage Region
1
Mean Range
Germination I 2.6a 2.13.3
II 3.3b 2.63.9
III 3.3b 2.44.0
Plumule I 2.9a 2.34.0
II 2.6ab 0.84.0
III 1.4b 0.62.6
Seedling I 3.6a 2.74.0
II 3.8a 3.64.0
III 3.4a 1.34.0
1
Regions are divided into I (the northermost), II (temperate)
and III (tropical and subtropical)
Different letters indicate signicance at the 5% level (Scheffes
test). No. of strains tested is 8 in I, 8 in II and 5 in III,
respectively
Table 2 Differences in cold tolerance scores at the germina-
tion, plumule and seedling stages in cultivated and wild rice
strains
Stage Species or subspecies Mean Range
Germination ssp. japonica 3.0a 2.14.0
ssp. indica 2.5a 1.73.1
O. rupogon 0.8b 0.02.4
Plumule ssp. japonica 2.4a 0.64.0
ssp. indica 0.7b 0.03.6
O. rupogon 1.0b 0.02.5
Seedling ssp. japonica 3.6a 1.34.0
ssp. indica 2.3b 0.04.0
O. rupogon 2.8b 0.04.0
Different letters indicate signicance at the 5% level (Scheffes
test). No. of strains tested is 21 in ssp. japonica, 13 in ssp.
indica and 23 in O. rupogon, respectively
464 Euphytica (2009) 165:459470
1 3
qCTP11 and qCTP12 each explained more than 20%
of total phenotypic variation, showing LOD scores of
6.0 and 6.8 (Table 4). At the seedling stage, two
putative QTLs for cold tolerance (qCTS5 and
qCTS11) were detected on chromosome 5 and 11,
showing LOD scores of 2.7 and 3.1, respectively.
Both of qCTS11 and qCTP11 were found near C189
on chromosome 11. All the QTLs detected revealed
that the A58-derived QTL increased the degree of
cold tolerance. On the other hand, no signicant QTL
for cold tolerance at the germination stage was found,
although the parental strains signicantly differed in
LTG scores. Based on the results obtained, substitu-
tion mapping was carried out to precisely locate
qCTP11 and qCTP12 since the two QTLs greatly
contribute to cold tolerance.
Substitution mapping of qCTP11 and qCTP12
The region from RM209 to RM144 was introgressed
into A58 by backcrosses since qCTP11 was present
near C189 (Fig. 4a). In the backcross generations (BC
3
and BC
4
), recombinants between RM209 to RM144
were surveyed in 262 segregating plants, using six
markers (RM209, RM229, RM1341, C189, RM2191
and RM144). Four different genotypes (R11-1 to R11-
4) were investigated regarding cold tolerance at the
plumule stage, and were compared with the parental
genotypes (R11-A58 and R11-W107) and the hetero-
zygote (R11-H). A distinct difference between R11-
A58 and R11-W107 suggested that qCTP11 is present
on the introgressed fragments (Fig. 4a). Two recom-
bined genotypes (R11-1 and R11-2) were tolerant
while the other recombined genotypes (R11-3 and
R11-4) were susceptible, indicating that qCTP11
resides in the region between RM1314 and RM2191.
To locate qCTP11 precisely, the three homozygotes of
recombined genotypes (R11-5 to R11-7) were exam-
ined with additional seven markers. Out of the three,
only R11-6 was cold tolerant, showing that the position
of qCTP11 was delimited into 778 kb region between
F10 and J03 (Fig. 4a).
For mapping of qCTP12, the region from C62896
to K22A was introgressed into A58 by backcrosses.
In the BC
3
F
2
and BC
4
F
2
populations, recombinants
between C62896 and K22A were examined in 212
segregating plants, using ve markers (C62896,
RM27533, RM247, RM3427 and K22A). Regarding
cold tolerance at the plumule stage, two recombined
genotypes (R12-1 and R12-2) were compared with
Fig. 2 Relationship of cold
tolerance with the latitude
of origins at the germination
(a), plumule (b) and
seedling (c) stages in 23
wild strains. ** indicates
signicance at the 1% level.
ns indicates non-
signicance
Table 4 Putative QTLs for cold tolerances detected in RILs between A58 and W107
Stage QTL Nearest markers
a
LOD PVE
b
(%) Multi-QTL PVE (%)
Plumule
c
qCTP1 RM24 3.9 13.1
qCTP11 C189 6.0 22.2
qCTP12 RM247 6.8 27.0 53.5
Seedling qCTS5 RM289 2.7 15.0
qCTS11 C189 3.1 16.0 26.5
All the detected QTLs had the effect of increasing the tolerance score by the A58 allele
a
Nearest markers were used as cofacor markers for MQM mapping
b
Percentage of variance explained
c
The data based on Onishi et al. (2007)
Euphytica (2009) 165:459470 465
1 3
the parental genotypes (R12-A58 and R12-W107)
and the heterozygote (R12-H) (Fig. 4b). In contrast to
the case for qCTP11, cold tolerance scores consid-
erably overlapped in R12-A58 and R12-W107.
However, R12-1 and R12-2 clearly showed the
tolerant and susceptible phenotypes, respectively,
without overlapping, indicating that qCTP12 resides
in the region between RM27533 and K22A.
Discussion
The present study revealed that cultivated and wild
rice has a considerable level of genetic variation for
cold tolerance at the early growth stage. The pattern
of genetic variation differed at the germination,
plumule and seedling stages. QTL analysis using a
cross between A58 (ssp. japonica) and W107 (O.
rupogon) detected ve signicant QTLs for cold
tolerance, in which QTLs for cold tolerance at the
plumule and seedling stages were detected in differ-
ent locations while qCTP11 and qCTS11 were located
at a similar region on chromosome 11. Despite that
A58 is tolerant to low temperature at all the three
stages examined, these results suggested that the
genetic bases for cold tolerance also differed at the
three stages examined, at least in part, as reported by
Andaya and Mackill (2003).
RM499
C62003
S13654
C52190
RM24
E30745
S5756
DFR
C137
RM8278
RM472
sd1
RM1387
C112
Chr. 1
KS3
q
C
T
P
1
C62896
RM247
RZ869
S14025
RM260
E11861
RM17
Chr. 11 Chr. 12
S1284
RM286
RM5704
S21074
RM202
RM209
RM144
C189
q
C
T
P
1
1
q
C
T
P
1
2
RM153
RM413
RM289
C10987
RM249
E10886
RM430
RM161
RM334
Chr. 5
q
C
T
S
5
q
C
T
S
1
1
10 cM
c
d
d
d
b
a
b
b
b
a
Fig. 3 QTLs for cold tolerance at the early growth stages
located on chromosome 1, 5, 11 and 12 in rice. The framework
map is based on Onishi et al. (2007). The ovals represent the
centromeres. Black, dotted (black) and grey lines indicate the
QTL positions detected in the crosses between indica and
japonica subspecies for cold tolerance at germination (or low-
temperature germinability) (a. Miura et al. 2001), young
seedling (or plumule) (c. Zhang et al. 2004) and seedling stage
(b. Andaya and Mackill 2003, and Andaya and Tai 2006; d.
Lou et al. 2007), respectively. a indicates the 0.5-LOD support
interval. bd indicate the intervals in which LOD values were
larger than the threshold values. The relative positions of QTLs
were estimated based on the physical locations of markers (
http://gramene.org; http://rgp.dna.affrc.go.jp). All the QTLs
have the effect increasing the tolerance score by the ssp.
japonica locus, except two QTLs with underlined letters (a and
d) which have the decreasing effect
466 Euphytica (2009) 165:459470
1 3
The genetic differentiation observed among the
geographical regions as well as among the taxonomic
groups suggests that the pattern of variation might
reect not only the phylogenetic history but also the
on-going adaptation to local environments. Low-
temperature germinability (LTG) shows a distinct
1 2 3 4 0
No. of
lines
No. of Plants
(a) qCTP11
10
37 58
17 71
10
10 35 40 14 5 6
1 5 7
5
31 28
3
6 22
2 2 21
1 2 10
4 18 26
1 3 9
T
S
S
T
S
SEG
T
T
S
S
6
1
Mean Phenotype
Cold tolerance score
qCTP11
778 kb
R11-H
R11-1
R11-2
R11-3
R11-4
R11-5
R11-6
R11-7
R11-A58
R11-W107
9
5 22 48 28
13 44 45 1
9
2
2
9 20 18 16 18 6
1 14 9
5 18 1
T
S
S
SEG
T
R
M
2
0
9
R
M
2
7
5
3
3
R
M
2
4
7
R
M
3
4
2
7
K
2
2
A
K
2
2
C
B
1
0
R
M
7
6
1
9
1 2 3
No. of plants
Mean Phenotype
4 0
No. of
lines
(b) qCTP12
Cold tolerance score
R12-1
R12-2
R12-A58
R12-W107
500kb
R12-H
500 kb
C
6
2
8
9
9
R
M
2
2
9
R
M
1
3
4
1
R
M
2
1
9
1
R
M
1
4
4
C
1
8
9
R
M
2
1
9
1
8
5
C
1
6
A
F
1
0
A
C
1
3
7
K
2
1
C
1
8
9
P
0
5
4
J
0
3
A
C
1
0
8
R
M
1
3
4
1
3.61
0.81
1.07
3.58
3.45
1.00
1.08
0.83
3.59
0.75
2.90
1.30
1.60
3.30
0.83
8cM
qCTS12
qCTP12
W107
A58
Including a breakage point
Heterozygous region
Fig. 4 Substitution mapping of qCTP11 (a) and qCTP12 (b).
The map positions of markers are based on physical locations
along chromosome 11 (AP008217) and chromosome 12
(AP008218). T, S and SEG represent tolerant, susceptible
and segregating, respectively. The location of qCTS12 reported
by Andaya and Tai (2006) is shown by an arrow head (b)
Euphytica (2009) 165:459470 467
1 3
difference between cultivated and wild rice (Table 2),
suggesting that the differentiation of cold tolerance
might be associated with the domestication event
since the wild progenitor generally show a lower
level of tolerance than cultivated forms. Neverthe-
less, no signicant QTL for LTG was detected in this
study. It was reported that ssp. japonica has a higher
level of LTG than ssp. indica (Oka 1954), whereas
ssp. indica has a higher level of LTG (or a higher
level of germination speed under low-temperature)
after the effect of seed dormancy was removed,
reecting the complex physiological process of
dormancy and germination in seeds (Ikehashi 1973;
Miura et al. 2001). The present study showed that
LTG were not different between the two rice
subspecies (Table 2).
In contrast to the germination stage, ssp. japonica
is more tolerant at the plumule and seedling stages
than ssp. indica (Table 2), in agreement with the
ndings of the previous studies (Glaszmann et al.
1990; Mackill and Lei 1997; Morishima and Oka
1981; Oka 1958). Since ssp. japonica is cultivated in
temperate and/or high elevation areas, it has been
reported that a higher level of cold tolerance might
have contributed for adaptation to cold climates.
Recent molecular studies estimated that the diver-
gence of genomic sequences of two rice subspecies
indica and japonica have occurred 0.20.44 million
years ago, which is far before the domestication of
rice (Ma and Bennetzen 2004; Vitte et al. 2004; Zhu
and Ge 2005). This implies that the distinct difference
observed between two subspecies at present could be
related not only to on-going microevolutionary forces
but also to the genetic diversication pre-existed in
wild population before domestication.
A strong evidence of adaptive signicance for cold
tolerance is a clinal pattern of variation observed at
the plumule stage in ssp. japonica and wild rice
(Table 3; Fig. 2). Climatic factors such as tempera-
ture tend to show latitudinal gradients, therefore, the
latitudinal cline in specic phenotypes have long
been used to infer adaptation to climatic variation
(Mitchell-Olds et al. 2007; Skt et al. 2002; Zhen and
Unger 2008). Regarding cold tolerance at the
seedling stage, Nagamine and Nakagahra (1990)
reported that Asian cultivated rice exhibits a latitu-
dinal cline, although it remains unknown to what
extent the clinal variation is associated with the
differential distribution of the two subspecies in rice.
The present results show that there was signicant
difference in cold tolerance at the seedling stage
between ssp. indica and ssp. japonica, and within ssp.
japonica, strains in the northern areas are not
different from those in the southern areas for cold
tolerance (Table 3), suggesting that a latitudinal cline
in cultivated rice might largely reect the differential
distribution of the two subspecies. On the contrary,
strains of ssp. japonica from the northernmost areas
are more tolerant at the plumule stage. Furthermore,
in O. rupogon, the higher is the latitude of collection
sites, higher is the cold tolerance scores at the
plumule stage, suggesting that natural selection might
be associated with the pattern of geographical
variation in part. A similar clinal variation across
the latitude was also reported in photoperiod sensi-
tivity for oral transition in rice (Oka and Chang
1960). It is highly expected that a wide range of
variation found in O. rupogon might have contrib-
uted to the genetic differentiation observed in Asian
cultivated rice.
Recently, QTL mapping analysis revealed several
QTLs for cold tolerance at the early growth stage are
present in the rice genome (Andaya and Mackill
2003; Fujino et al. 2004; Lou et al. 2007; Misawa
et al. 2000; Miura et al. 2001; Zhang et al. 2004).
However, it was not easy to compare their locations
due to different strains and markers used. In addition,
the degree of cold tolerance is affected by the levels
of low-temperature as well as the duration of
exposure to cold stresses, suggesting involvement of
complex networks in physiology. In the present
study, two major QTLs, qCTP11 and qCTP12, for
cold tolerance at the plumule stage were found
between a ssp. japonica (A58) and a wild rice
(W107) strains, and they were successfully mapped
on chromosome 11 and 12, respectively. In the
crosses between ssp. indica and ssp. japonica, QTLs
of large effects for cold tolerance at the young
seedling (or plumule) stage (qSCT-11) and seedling
stage (qCTS12) were previously reported on chromo-
some 11 (Zhang et al. 2004) and chromosome 12
(Andaya and Mackill 2003; Andaya and Tai 2006),
respectively (Figs. 3, 4). The present results of
substitution mapping showed that qCTP11 and
qCTP12 are different from those previously reported.
In addition, Cai and Morishima (2002) investigated
the genetic differentiation of cold tolerance at the
plumule stage between ssp. indica and O. rupogon
468 Euphytica (2009) 165:459470
1 3
by means of QTL analysis. The tolerance score was
examined by the same method that was used in this
experiment. They found only a minor QTL for cold
tolerance on chromosome 8, which suggests that no
genetic divergence is present around qCTP11 and
qCTP12 between ssp. indica and O. rupogon.
Although it is not yet known whether qCTP11 and
qCTS11 are the same locus or not, it is expected that
both A58-derived qCTP11 and qCTP12 could be
used for improvement of tolerance to cold stress in
ssp. indica. Furthermore, detailed studies of the
candidate genes might provide the opportunity to
address the question how environmental factors has
shaped the genetic variation in Asian rice.
Acknowledgements This work was supported in part by
grants from the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
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