Identification of quantitative trait loci for cold toleranceduring the germination and seedling stages in rice(Oryza sativa L.)

Zhoufei Wang Fuhua Wang Rong Zhou

Jianfei Wang Hongsheng Zhang

Received: 7 March 2011 / Accepted: 24 May 2011 / Published online: 12 June 2011

Springer Science+Business Media B.V. 2011

Abstract Low temperature is a serious abiotic

stress affecting rice production in subtropical and

temperate areas. In this study, cold tolerance of rice at

the germination and seedling stages were evaluated

using one recombinant inbred line (RIL) population

derived from a cross between Daguandao (japonica),

with highly cold-tolerant at the seedling stage, and

IR28 (indica), with more cold-tolerant at the germi-

nation stage, and the quantitative trait loci (QTL)

mapping was conducted using the multiple interval

mapping (MIM) approach. Continuous segregation in

low temperature germinability (LTG) and cold toler-

ance at the seedling stage (CTS) were observed

among the RIL populations. Most RILs were mod-

erately susceptible or tolerant at the germination

stage, but were susceptible at the seedling stage. No

significant relationship was found in cold tolerance

between the germination and seedling stages. A total

of seven QTLs were identified with limit of detection

(LOD)[3.0 on chromosomes 3, 8, 11 and 12, and theamount of variation (R2) explained by each QTL

ranged from 5.5 to 22.4%. The rice LTG might be

regulated by two minor QTLs, with the CTS

controlled by one major QTL [qCTS8.1 (LOD =

16.1, R2 = 22.4%)] and several minor loci. Among

these loci, one simultaneously controls LTG

(qLTG11.1) and CTS (qCTS11.1). Several cold-

tolerance-related QTLs identified in previous studies

were found to be near the QTLs detected here, and

three QTLs are novel alleles. The alleles from

Daguandao at six QTLs increased cold tolerance

and could be good sources of genes for cold

tolerance. In addition, only one digenic interaction

was detected for CTS, with a R2 value of 6.4%. Those

major or minor QTLs could be used to significantly

improve cold tolerance by marker-assisted selection

(MAS) in rice.

Keywords Rice Cold tolerance Seed germination Seedling establishment Quantitative trait loci

Introduction

Seed germination and seedling establishment are

critical phases in the life of a higher plant. By

definition, germination is the growth of an embryonic

plant contained within a seed; it begins with

Z. Wang F. Wang R. Zhou J. Wang (&) H. Zhang (&)The Laboratory of Seed Science and Technology, State

Key Laboratory of Crop Genetics and Germplasm

Enhancement, Nanjing Agricultural University,

Nanjing 210095, Peoples Republic of China

e-mail: wangjf@njau.edu.cn

H. Zhang

e-mail: hszhang@njau.edu.cn

F. Wang

Institute of Crop Science, Henan Academy of Agricultural

Sciences, Zhengzhou 450002, Peoples Republic of China

123

Euphytica (2011) 181:405413

DOI 10.1007/s10681-011-0469-z

imbibition and terminates with the appearance of the

radicle through the seed coat. Seeds then commence

seedling establishment, which ends when the seedling

has exhausted the seeds energy reserves and starts to

carry out photosynthesis (Ichie et al. 2001). The seed

germination and early seedling growth stages are

extremely important for successful stand establish-

ment and plant development, both of which directly

impact yield. However, seed germination and seed-

ling growth depend on both internal and external

conditions. The most important external factors

include temperature, water, oxygen availability and

sometimes light or darkness (Thompson et al. 1977;

Goode and Allen 2009). For example, seeds often

have a temperature range within which they will

germinate and establish seedlings; they will not do so

above or below this range (Rodino et al. 2007;

Alvarez et al. 2007).

Rice (Oryza sativa L.) originating from tropical or

subtropical area is one of the most important crops

worldwide. The optimum temperatures for rice ger-

mination and seedling growth are from 25 to 35C,and these processes are generally susceptible to

temperatures below 15C (Nakagahra et al. 1997).A number of observations indicate that low temper-

ature slows or prevents germination, and in turn leads

to poor seedling establishment in soybean (Bramlage

et al. 1979), corn (Stewart et al. 1990) and rape

(Nykiforuk and Johnson-Flanagan 1999). Similarly,

the occurrence of low temperatures will cause a series

of problems in germination and seedling growth in

rice; these include poor germination, seedling stun-

ting, seedling yellowing or withering, poor seedling

establishment and low seedling vigor (Fujino 2004;

Zhang et al. 2005; Andaya and Tai 2006; Lou et al.

2007). Recently, direct-seeding cultivation has

become important and popular in many Asian

countries due to its lower cost and its operational

simplicity (Fujino 2004; Jiang et al. 2006). Therefore,

improving cold tolerance of rice at the germination

and seedling stages is becoming an important objec-

tive in rice breeding programs.

Exposure of plants to low temperatures leads to a

number of biochemical perturbations, including

changes in membrane fluidity, the stability of RNA

and DNA secondary structures and the activity of

enzymes (Smallwood and Bowles 2002). Recently, a

large number of genes that function in the response to

low temperature have been revealed in the model

plants Arabidopsis and rice. However, the mechanism

of chilling injury in seeds is different from that in

hydrated tissues (Bedi and Basra 1993). Cold toler-

ance of plants is a complex quantitative trait, the

mechanism of which is difficult to illuminate by the

study of any single gene. Therefore, QTL mapping

has provided a powerful tool for investigating the

cold tolerance of plants. In rice, the various treatment

of cold tolerance with the different processing

temperature and duration were conducted, such as

low temperature germinability (LTG) was scored at

15C for 4 days (Miura et al. 2001) or 15 days (Jianget al. 2006), cold tolerance at the seedling stage

(CTS) was evaluated at 10C for 10 and 13 days(Zhang et al. 2005), or at 9C for 8, 14, 16 and18 days (Andaya and Mackill 2003). Several QTLs

for LTG and CTS have been reported. For example,

Miura et al. (2001) identified five putative QTLs

controlling LTG, on chromosomes 2, 4, 5 and 11,

using backcross inbred lines (BILs). Fujino et al.

(2004) detected three putative QTLs associated with

LTG, on chromosomes 3 and 4, using BILs. Zhang

et al. (2005) identified three main-effect QTLs

conferring CTS, on chromosomes 3, 7 and 11, using

RILs. Jiang et al. (2006) detected eleven putative

QTLs for LTG on seven chromosomes, using an F2population. Lou et al. (2007) found five main-effect

QTLs associated with CTS, on chromosomes 1, 2 and

8 using a DH population. Koseki et al. (2010)

detected three QTLs for CTS, on chromosomes 3, 10

and 11, using an F2 population.

Although a number of QTLs for cold tolerance at

the germination and seedling stages have been

identified in rice, the genetic relationship of cold

tolerance between the two stages is still unknown. It

is not easy to compare the QTLs due to the different

parental varieties and markers used in the different

populations. Moreover, previous studies showed that

cold tolerance is developmentally regulated and

growth stage-specific in tomato (Foolad and Lin

2001) and that the chilling sensitivity of rice varies

during the life cycle (Zhang et al. 2005). Therefore,

through one RIL population derived from a cross

between japonica Daguandao and indica IR28, with a

multiple interval mapping (MIM) approach, we

intend to reveal the genetic relationships between

cold tolerance at the germination and seedling stages

and identify the QTLs controlling LTG and CTS for

rice breeding by marker assisted selection (MAS).

406 Euphytica (2011) 181:405413

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Materials and methods

Plant materials and growth

Two rice cultivars, Daguandao (japonica) and IR28

(indica), and one RIL population (F10) consisting of

227 lines, derived from a Daguandao 9 IR28 cross

by single seed descent, were used in this study. In our

previous experiment, it was found that IR28 was

highly cold-tolerant at the germination stage, and

Daguandao was more cold-tolerant at the seedling

stage (data unpublished).

All lines and their parents were planted in the field

at the Experimental Station of Nanjing Agricultural

University in the summer of 2008. The seeds were

harvested when mature, and were dried at 50C for7 days to break seed dormancy (Jiang et al. 2006).

The original germination rate of all seeds at 30C for12 days was more than 98% (data not shown).

Evaluation of LTG

100 seeds per replication of each RIL or parent were

surface-sterilized with a 0.1% sodium hypochlorite

solution for 15 min and then rinsed three times with

sterile distilled water. The seeds were then placed in a

9-cm Petri dish with two sheets of filter paper, and

10 ml of distilled water were added (Wang et al.

2011). All Petri dishes were placed in an incubator at

14 1C for 12 days in the dark. A seed wasrecorded as germinated when its radicle had broken

through the seed coat, and the number of germinated

seeds was counted daily. Seed germination percent-

age after 12 days of cold treatment was calculated as

the low temperature germinability (LTG). At the end

of this test, the seeds were incubated at 30C for5 days. The germination percentage was greater than

80% in each RIL, which means that the seeds were

free of secondary dormancy (Fujino 2004). The

experiment was repeated three times.

Evaluation of CTS

For each line and parent in each replication, 50 seeds

were soaked in distilled water at 30C for 3 days toallow the seeds to germinate. Thirty pre-germinated

seeds with coleoptiles longer than 2 mm were selected

to be sown in a plastic box (40 cm 9 30 cm 9 18 cm)

filled with 2.5 kg of clay soil. They were then grown in

a growth chamber at 30C/22C day/night for 12 dayswith a 12 h photoperiod each day. At the three-leaf

stage, weak and dead plants were removed, and 20

healthy seedlings per line or parent were retained at

9C for an 8 days cold treatment. After the coldtreatment, the temperature was gradually adjusted back

to 30C to start the recovery process. CTS scoring wasdone after recovery, using a scale of 1 (tolerant, all

leaves normal, no apparent visual injury) to 9 (suscep-

tible, all leaves wilted, seedlings apparently dead) as

described by Andaya and Mackill (2003). The exper-

iment was repeated three times.

QTL mapping

DNA was extracted from rice seedlings by the SDS

method (Dellaporta et al. 1983). PCR was performed

using the procedure of Chen et al. (1997); the PCR

products were then separated on an 8% non-denatur-

ing polyacrylamide gel and visualized by the silver

staining method of Sanguinetti et al. (1994). The

computer program Mapmaker/EXP 3.0 was used to

construct a complete linkage map (Lander et al.

1987). Finally, a set of 167 SSR markers covering

most of the rice genetic map at an average interval of

11.1 cM was constructed. The data of germination

percentage and seedling cold tolerance were trans-

formed by arcsine transformation into a typical

quantitative trait distribution for QTLs detection

(Wang et al. 2011). The method of multiple interval

mapping (MIM) was used for QTL mapping (Chur-

chill and Doerge 1994), and a LOD score of 3.0 was

used as the threshold value to declare the presence of

a putative QTL. In addition, the proportion of

observed phenotypic variance explained by each

QTL or each pair of epistatic loci and the corre-

sponding additive effects were estimated. QTL

nomenclature followed the method of McCouch and

CGSNL (2008).

Data analysis

Experimental data were analyzed using Statistical

Analysis System (SAS) software, and traits of parents

were compared according to Fishers least significant

difference (LSD) test at 1% level of probability. The

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123

correlations of traits were computed using PROC

CORR by SAS software (Wang et al. 2010).

Results

Phenotypic variation

The cold tolerance of the two parents at the germi-

nation and seedling stages was different (Table 1;

Fig. 1). Significant differences in LTG and CTS were

observed between the two parental varieties at a level

of P \ 0.01. IR28 presented an LTG of 97.6%, whileDaguandao had an LTG of 76.8%. The CTS scores of

IR28 and Daguandao were 9 and 2, respectively. The

RILs displayed a continuous distribution in LTG

from 2.1 to 96.3% as well as CTS scores ranging

from 3 to 9 with statistically significant differences,

suggesting the involvement of multiple genes for both

traits (Table 1; Fig. 1).

According to the observed LTG and CTS values,

the RILs could be classified into nine types (Table 2).

Most of the RILs (124 lines) that were moderately

susceptible (MS) (4079%) to cold at the germination

stage were very susceptible (S) (79) at the seedling

stage (MS-S). Other RILs (65 lines) were susceptible

(S) (039%, 79) at both stages (S-S). Few RILs were

tolerant (T) (80100%, 13) to cold at the germina-

tion, seedling or both stages (T-MS, T-S, MS-T, S-T

and T-T, respectively). Therefore, more RILs were

moderately susceptible to cold stress at the germina-

tion stage, but most RILs were susceptible at the

seedling stage.

Table 1 Phenotypic values of low temperature germinability and cold tolerance at seedling stage among parents and RILspopulation

Traits Parentsa RILs

Daguandao IR28 Range Variance coefficient (%) Mean Fb

Low temperature germinability (%) 76.8 2.5 97.6 1.6** 2.196.3 45.1 54.7 11.49**

Cold tolerance at seedling stage 2.0 0.2 9.0 0.1** 3.09.0 13.4 8.1 95.03**

a Means SD (standard deviation); for low temperature germinability sample size n = 100, replications r = 3; for cold tolerance atseedling stage sample size n = 20, replications r = 3; ** significant at the level of 0.01 probability according to Fishers leastsignificant difference (LSD) testb F test of variance among RILs population; ** significant at the level of 0.01, F0.01 (226, 454) = 1.30

020406080

100120140

Cold tolerance at seedling stage

No.

of

lines

0

10

20

30

40

50

2 3 4 5 6 7 8 910 20 30 40 50 60 70 80 90 100

Low temperature germinability (%)

No.

of

lines

IR28Daguandao

Daguandao

IR28Fig. 1 Frequencydistribution of low

temperature germinability

and cold tolerance at

seedling stage

Table 2 Phenotype of cold tolerance at the germination and seedling stages among RILs

Phenotype No. of lines Phenotype No. of lines Phenotype No. of lines Total

T-T 0 MS-T 0 S-T 1 1

T-MS 4 MS-MS 8 S-MS 3 15

T-S 22 MS-S 124 S-S 65 211

Total 26 132 69 227

T Tolerant, MS moderately susceptible and S susceptible; T-T the former letter presented the phenotype of LTG (T: 80100%, MS:4079% and S: 039%), and the later letter presented the phenotype of CTS (T: 13, MS: 46 and S: 79), respectively

408 Euphytica (2011) 181:405413

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QTL mapping

A whole-genome scan was performed, and two

putative QTLs (qLTG11.1 and qLTG11.2) associated

with LTG were detected on chromosome 11 (Table 3;

Fig. 2). These two QTLs explained 5.9 and 8.5% of

phenotypic variation, respectively. The additive

effect of Daugandao allele qLTG11.1 and IR28 allele

qLTG11.2 increased the germination rate by 4.4 and

4.9%, respectively.

Five putative QTLs (qCTS3.1, qCTS8.1, qCTS11.1,

qCTS11.2 and qCTS12.1) for CTS were found on

chromosomes 3, 8, 11 and 12, respectively (Table 3;

Fig. 2). Phenotypic variance explained by each QTL

ranged from 5.5 to 22.4%. One major QTL, qCTS8.1

(LOD = 16.1), was identified on chromosome 8

flanked by RM6356 and RM22491, which explained

22.4% of the total phenotypic variation. The positive

alleles of Daugandao could decrease the scores of CTS.

Detection of epistatic interactions for cold

tolerance

One significant epistatic interaction for cold tolerance

was detected at the seedling stage but not at the

germination stage (Table 4). One locus located

between RM7179 and RM282 of chromosome 3

interacted with one locus located between RM6356

and RM22491 on chromosome 8 and affected CTS.

The interaction showed a small effect on CTS, with

R2 = 6.4%, and a positive effect on the trait was

contributed by the two-locus recombinants.

Table 3 Chromosome location, coefficient of determination (R2) and additive effect (AE) of the putative QTLs conferring lowtemperature germinability and cold tolerance at the seedling stage

Trait Locus Chra Marker interval LOD support interval/cM Peak LOD AEb R2c

LTG qLTG11.1 11 RM5704-RM3701 49.557.2 4.0 -4.4 5.9

qLTG11.2 11 RM229-RM254 104.3122.3 4.5 4.9 8.5

CTS qCTS3.1 3 RM7179-RM282 35.341.7 11.6 0.53 15.7

qCTS8.1 8 RM6356-RM22491 17.123.6 16.1 0.63 22.4

qCTS11.1 11 RM5704-RM3701 55.463.0 4.8 0.27 9.0

qCTS11.2 11 RM6091-RM26632 66.570.7 3.0 0.22 6.8

qCTS12.1 12 RM3739-RM6947 5.911.0 3.1 0.22 5.5

LTG Low temperature germinability, CTS cold tolerance at the seedling stagea Chromosome on which the QTL was locatedb Additive effect is the effect of substituting a IR28 allele for a Daguandao allele; its positive value indicates that IR28 has the

positive allelec Variation explained by each putative QTL

Fig. 2 Chromosomalpositions of QTLs for low

temperature germinability

and cold tolerance at the

seedling stage in rice. Map

distances (cM) are shown

on the left

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123

The phenotype and linked QTLs in selected RILs

Several selected RILs with relatively high LTG and/

or CTS were found in this study (Table 5). The RILs

of classes I and II were cold-tolerant at the germi-

nation stage but moderately susceptible or susceptible

at the seedling stage, respectively, and those of class

III were moderately cold-susceptible at the seedling

stage but susceptible at the germination stage. These

RILs had at least one of the seven or all of the

positive alleles of QTLs. For example, RIL-211 and

RIL-203, which have relatively high LTG and CTS,

had six QTLs as detected in our study.

Discussion

The effects of low temperature on seed germination

and seedling growth have been reported in several

plants, including oilseed rape (Nykiforuk and John-

son-Flanagan 1999), tomato (Foolad and Lin 2001),

rice (Andaya and Mackill 2003; Fujino et al. 2004)

and alfalfa (Dias et al. 2011). Low temperatures will

slow the rate of seed imbibition and prevent

germination, which results in poor stand establish-

ment and ultimately reduced yield (Bedi and Basra

1993; Nykiforuk and Johnson-Flanagan 1999).

Therefore, cold tolerance of the germination and

seedling stages is an essential characteristic of rice

varieties adapted to direct-seeding culture (Miura

et al. 2001; Jiang et al. 2006; Fujino 2004; Lou et al.

2007). Imbibition is the period of germination that is

most sensitive to low temperatures (Thompson et al.

1977). After germination, water uptake increases and

storage reserves are utilized to support seedling

growth until the reserves are exhausted; the seedling

then begins photosynthesis (Ichie et al. 2001), but this

is also affected by low temperature, which decreases

water absorption by roots and water transport in the

shoot (Smallwood and Bowles 2002). Therefore, cold

tolerance of germination and seedling development

play an important role in plant growth.

Typically, japonica rice exhibits better cold toler-

ance than indica rice, as japonica is cultivated in

temperate and/or high elevation areas (Zhang et al.

2005; Andaya and Tai 2007; Cheng et al. 2007;

Baruah et al. 2009). However, this was not observed

in cold tolerance at the germination stage. One study

Table 4 Digenic epistatic affecting cold tolerance in the Daguandao/IR28 RIL population

Trait Loci (i) Loci (j) Peak LOD AAa R2b

Chromosome Interval Chromosome Interval

CTS 3 RM7179-RM282 8 RM6356-RM22491 11.1 -0.5 6.4

CTS Cold tolerance at seedling stagea Additive 9 additive effect: its positive value indicates that two loci genotypes being the same as those in parent Daguandao (or

IR28) take the positive effects, while the two-loci recombinants take the negative effects. The case of negative values is just the

oppositeb Variation explained by each pair of epistatic loci

Table 5 Phenotype of low temperature germinability and cold tolerance at the seedling stage in selected RILs and their related QTLs

Type Selected RILs LTG (%) CTS Linked QTLs

I 211 91.1 5.7 qLTG11.1, qLTG11.2, qCTS8.1, qCTS11.1, qCTS11.2, qCTS12.1

203 88.3 4.7 qLTG11.1, qCTS3.1, qCTS8.1, qCTS11.1, qCTS11.2, qCTS12.1

II 173 96.3 9 qLTG11.2

47 96 9 qLTG11.1, qCTS3.1

III 102 21.1 3.7 qCTS3.1, qCTS8.1, qCTS11.1, qCTS11.2

68 10.6 5.8 qCTS3.1, qCTS8.1, qCTS11.2, qCTS12.1

Parents Daguandao 76.8 2 qLTG11.1, qCTS3.1, qCTS8.1, qCTS11.1, qCTS11.2, qCTS12.1

IR28 97.6 9 qLTG11.2

LTG Low temperature germinability, CTS cold tolerance at the seedling stage

410 Euphytica (2011) 181:405413

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reported that japonica had a higher level of LTG than

indica (Jiang et al. 2006), while Baruah et al. (2009)

found no significant difference in LTG between

indica and japonica, and Miura et al. (2001) found

that indica had a higher level of LTG. In this study,

the indica IR28 had a higher LTG than the japonica

Daguandao, similar to what was observed by Miura

et al. (2001). At the seedling stage, we found the

japonica Daguandao to be more cold-tolerant than

indica rice, supporting other previous studies (Zhang

et al. 2005; Andaya and Tai 2007; Cheng et al. 2007;

Baruah et al. 2009). Furthermore, among the RIL

populations, few lines were cold-tolerant at both

stages, and most of the RILs were moderately

susceptible at the germination stage but susceptible

at the seedling stage. This may be caused by a

distorted segregation detected in our segregation

populations. In our study, the number of japonica

alleles and indica alleles per locus among RILs was

conducted, respectively. The results showed that

indica IR28 alleles represented 52.9% of the alleles

among RILs; certain regions showed a significantly

distorted segregation ratio (data not shown). Corre-

lation analysis indicated that there was no significant

relationship between LTG and CTS (r = -0.00025,

P = 0.9972), suggesting that cold tolerance might be

growth stage-specific in rice, as has been observed in

previous studies in muskmelon (Edelstein et al. 1991)

and tomato (Foolad and Lin 2001). Therefore, it is

not appropriate to estimate the cold tolerance of the

seedling stage based on that of the germination stage.

With the successful application of QTL mapping

technology, QTL analyses of cold tolerance have

been reported in several plants, including maize

(Fracheboud et al. 2004), sorghum (Knoll et al.

2008), soybean (Ikeda et al. 2009) and rice (Koseki

et al. 2010). In rice, a number of QTLs for LTG and

CTS have also been identified (Miura et al. 2001;

Andaya and Mackill 2003; Fujino et al. 2004; Zhang

et al. 2005; Jiang et al. 2006; Lou et al. 2007; Koseki

et al. 2010). In this study, two and five QTLs for LTG

and CTS were identified, respectively. By comparing

these QTLs positions with those previously identi-

fied, it was found that the QTL qCTS3.1 coincided

with the QTL qCTB-3 region for cold tolerance at the

booting stage that was reported by Andaya and

Mackill (2003). The QTL qCTS8.1 was very close to

the region of QTL qCTB-8 (Kuroki et al. 2007), and

the QTLs qLTG11.1 and qCTS11.1 were near the

region of QTL qLTG-11-1 for low temperature

germinability (Jiang et al. 2006) and qSCT-11 for

seedling cold tolerance (Zhang et al. 2005), respec-

tively. However, the three QTLs qLTG11.2,

qCTS11.2 and qCTS12.1 were reported for the first

time in our study. Further study is needed to confirm

these QTLs by using near isogenic lines (NILs). With

the identification of increasing numbers of favorable

alleles at QTLs for cold tolerance by QTL analysis,

the MAS strategy could become a promising

approach for improving cold tolerance.

Cold tolerance of rice is a very complex trait, and

phenotype analysis showed that no relationships were

found between the cold tolerances of the germination

and seedling stages in this study. Upon further study

by QTL mapping, we found the QTL for cold

tolerance is time-specific at two stages. LTG was

regulated by two loci with minor effects, while that of

CTS was controlled by several minor-effect loci and

one major QTL, qCTS8.1. Although the IR28 has

high LTG but low CTS while Daguandao has low

LTG but high CTS, one interval on chromosome 11,

flanked by RM5704 and RM3701, contained common

QTL (qLTG11.1 and qCTS11.1) responsible for LTG

and CTS, which might play an important role in cold

tolerance at the rice early growth stage. This inter-

esting QTL need be further studied. In addition, six

positive alleles that increased cold tolerance came

from japonica Daguandao, while only one positive

QTL (qLTG11.2) allele was derived from indica

IR28. The japonica parent had more cold tolerance

QTLs than the indica parent at the seedling stage,

supporting the observation that the japonica Dag-

uandao was highly tolerant to cold stress at the

seedling stage. Moreover, epistasis, or interaction

between nonallelic genes, is an important factor

affecting the phenotypic expression of genes and

genetic variation in populations (Li et al. 1997). In

the present study, one significant epistatic interaction

was detected in CTS (Table 2), which partly indi-

cated that the regulation of CTS might be more

complex than that of LTG.

To better understand the genetic mechanism of

cold tolerance in rice, fine mapping QTLs of cold

tolerance has been reported. For example, Andaya and

Tai (2006) delimited the major QTL qCTS12 to a

region of about 55 kb, and the most likely candidates

for the gene(s) underlying qCTS12 are OsGSTZ1 and

OsGSTZ2. Andaya and Tai (2007) fine-mapped the

Euphytica (2011) 181:405413 411

123

qCTS4 to a 128-kb region containing eighteen puta-

tive genes, with the most likely candidate for qCTS4

being the peroxidase gene. Fujino et al. (2008) placed

qLTG3-1 in a 4.8-kb region of chromosome 3 by high-

resolution mapping, and revealed that qLTG3-1,

expressed in the embryo during seed germination,

encodes a protein of unknown function. Koseki et al.

(2010) fine-mapped qCtss11, which functions in cold

tolerance at the seedling stage, to a 60-kb candidate

region in which six genes were annotated, of which

Os11g0615600 and/or Os11g0615900 are the most

likely gene(s) to be involved in CTS. In this study, a

new major QTL, qCTS-8 on chromosome 8, was

detected at a high LOD score of 16.1, explaining

22.4% of the phenotypic variation in cold tolerance at

the seedling stage. To elucidate the biological func-

tions of qCTS8.1, fine mapping and development of

near isogenic lines (NILs) for this gene are now in

progress. The nearest markers linked to qCTS8.1 will

be useful for rice breeding by MAS.

Pyramiding of the QTLs involved in cold tolerance

is necessary for achieving a high level of cold

tolerance in rice (Suh et al. 2010). In this study, the

analysis of several selected RILs with high LTG and/

or CTS showed that these RILs had at least one, or

all, of the seven positive alleles of QTLs, indicating

that QTLs pyramiding by MAS is available in rice

breeding programs. In order to understand the roles of

the QTLs identified in our study, some cold-tolerant

RILs have been used to develop the NILs for fine-

mapping the QTLs and determining the precise effect

of each QTL.

Acknowledgments This work was supported by the NationalNatural Science Foundation of China (Grant No. 31000748),

the Natural Science Foundation of Jiangsu Province (Grant No.

BK2010452). We thank reviewers for the careful reading of the

manuscript and constructive comments.

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Identification of quantitative trait loci for cold tolerance during the germination and seedling stages in rice (Oryza sativa L.)AbstractIntroductionMaterials and methodsPlant materials and growthEvaluation of LTGEvaluation of CTSQTL mappingData analysis

ResultsPhenotypic variationQTL mappingDetection of epistatic interactions for cold toleranceThe phenotype and linked QTLs in selected RILs

DiscussionAcknowledgmentsReferences