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Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)
Journal of Integrative Agriculture
March 2012
2012, 11(3): 359-367
RESEARCH ARTICLE
Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.) CHENG Li-rui1*, WANG Jun-min2*, Veronica Uzokwe1, MENG Li-jun1, WANG Yun1, SUN Yong1, ZHU Linghua1, XU Jian-long1 and LI Zhi-kang1, 3 National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 2 Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, P.R.China 3 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines 1
Abstract A set of 240 introgression lines derived from the advanced backcross population of a cross between a japonica cultivar, Xiushui 09, and an indica breeding line, IR2061, was developed to dissect QTLs affecting cold tolerance (CT) at seedling stage and heat tolerance (HT) at anthesis. Survival rate of seedlings (SRS) and spikelet fertility (SF), the index traits of CT and HT, showed significant differences between the two parents under stresses. A total of four QTLs (qSRS1, qSRS7, qSRS11a and qSRS11b) for CT were identified on chromosomes 1, 7, 11, and the Xiushui 09 alleles increased SRS at all loci except qSRS7. Four QTLs for SF were identified on chromosomes 4, 5, 6, and 11. These QTLs could be classified into two major types based on their behaviors under normal and stress conditions. The first was QTL expressed only under normal condition; and the second QTL was apparently stress induced and only expressed under stress. Among them, two QTLs (qSF4 and qSF6) which reduced the trait difference between heat stress and normal conditions must have contributed to HT because of their obvious contribution to trait stability, and the IR2061 allele at the qSF6 and the Xiushui 09 allele at the qSF4 improved HT, respectively. No similar QTL was found between CT at seedling stage and HT at anthesis. Therefore, it is possible to breed a new variety with CT and HT by pyramiding the favorable CT- and HT-improved alleles at above loci from Xiushui 09 and IR2061, respectively, through marker-assisted selection (MAS). Key words: cold tolerance, heat tolerance, advanced backcross population, QTL mapping, rice
INTRODUCTION Rice is by far the world’s most important food crop for the poor, it supplies 27% of the calories in the lowand lower-middle-income countries, more than any other crop (Dawe et al. 2010; FAO 2010). With global climate changing, most rice growing regions are experiencing more extreme environmental fluctuations (Solomon et al. 2007). Rice is susceptible to a variety
Received 10 February, 2011
of abiotic stresses including cold and heat stresses. The chilling stress below 15°C often happen in early season rice growing regions in the lower Yangtze River and single season rice-growing regions in the Northeast, Northwest and Yungui altiplano of China. Cold damage below 15°C at seedling stage usually results in poor seedling establishment or increase of seedling mortality (Nakagahra et al. 1997; Jacobs and Pearson 1999; Ji et al. 2008), thus greatly reducing the rice yield. On the other hand, high temperature (>35°C) occurrence at flower-
Accepted 23 March, 2011
Correspondence XU Jian-long, Tel: +86-10-82105854, Fax: +86-10-82108559, E-mail: [email protected] * These authors contributed equally to this work. © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
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ing stage leads to spikelet sterility (Satake and Yoshida 1978; Matsui and Omasa 2002; Nakagawa et al. 2003; Jagadish et al. 2007; Rang et al. 2011). In 2003, large areas across Hubei, Anhui, Zhejiang, Hunan and Guangdong provinces in China, suffered high temperature stress above 38°C for a week, causing spikelet fertility lower than 50%, thus resulting in great yield deduction (Yang and Liu 2009). Consequently, in these areas, improvement of tolerance to stresses of low temperature at the seedling stage and high temperature at anthesis is important in rice breeding programs. However, identifying QTLs associated with cold tolerance (CT) and heat tolerance (HT) and elucidating their genetic relationship are the prerequisite for developing rice varieties with CT and HT. CT and HT are complex traits that are controlled by quantitative trait loci (QTL). Many QTLs related to CT at different stages such as germination, seedling, vegetative, reproductive and grain maturity have been identified by different researchers using recombinant inbred lines (RILs) (Andava and Tai 2006; Jiang et al. 2008; Suh et al. 2010), doubled haploid (DH) (Chen et al. 2006; Lou et al. 2007), F2:3 lines (Han et al. 2006), backcross (Li et al. 1997), and introgression lines (Liu et al. 2003; Xu et al. 2008; Zeng et al. 2009; Zhou et al. 2010). With regard to different stages, the genetic mechanism of CT during the early growth stage is highly essential because the evaluation of CT at seedling stage can be easily manipulated, which is also vital for the seedling establishment, seedling mortality and subsequent vigorous vegetative growth (Zhang et al. 2005; Lou et al. 2007; Jiang et al. 2008). However, the most sensitive growth stage for HT in rice is at anthesis. Recently, QTL mapping studies involving HT at flowering or grain-filling stages with seed set rate or grain quality traits as an evaluation index, revealed that HT are controlled by polygenes which were distributed on 12 chromosomes (Cao et al. 2002; Zhu et al. 2005; Zhao et al. 2006; Chen et al. 2008; Zhang et al. 2009; Jagadish et al. 2010). Both cold and heat stresses belong to the two extreme temperature stresses. Athough some CT or HT QTLs have been identified, our understanding of the genetic relationships between CT and HT is unknown. In the present study, we developed a set of BC2F6 introgression lines population derived from a cross between
japonica cultivar Xiushui 09, as a recurrent parent and indica cultivar IR2061, as a donor parent to dissect QTLs affecting CT at the seedling stage and HT at anthesis. The objective was to identify QTLs affecting the two stresses and analyze the genetic relationships between the two tolerances, provide some useful information for rice breeding of stress tolerance by MAS.
RESULTS Linkage map Among 550 SSR markers surveyed, 201 showed polymorphism between the two parents, and 142 polymorphic markers evenly distributed on 12 rice chromosomes were selected to construct the linkage map (Fig.). The linkage map spanned 1 591.1 cM with an average distance of 11.20 cM between adjacent markers. Most genome of the ILs was recovered to Xiushui 09 with an average of 91.4% of Xiushui 09 genome and a range of 73.2-98.8%. The average numbers of homozygous introgressed segments per line was 6.1.
Phenotypic variation Significant differences of SRS under cold stress treatment were observed between the two parents (Table 1), indicating that the japonica cultivar Xiushui 09 was more tolerant to cold stress than indica cultivar IR2061. The ILs expressed transgressive segregation and continuous variation which were suitable for QTL mapping. With regard to HT, significant difference was not observed for the SF between the parents under normal temperature. However, under high temperature, SF reduced to 26.3 and 47.9% for Xiushui 09 and IR2061, respectively, bringing about a significant difference between the two parents. This implied that IR2061 was more tolerant to heat stress at the anthesis than Xiushui 09 (Table 1). The ILs showed transgressive segregation for SF under high temperature.
QTLs detection of CT at the seedling stage Four QTLs (qSRS1, qSRS7, qSRS11a and qSRS11b) associated with SRS under cold treatment at seedling
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Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)
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Fig. The molecular linkage map and QTLs affecting survival rate of seedlings (SRS) under cold stress at the seedling stage and spikelet fertility (SF) under heat stress at anthesis identified in the advanced backcross population of Xiushui 09/ IR2061. , QTLs affecting SRS under cold stress at the seedling stage. , , , QTLs affecting SF in normal, heat stress and SF difference of stress to normal conditions, respectively.
Table 1 Phenotypic performance for the traits associated with cold tolerance and heat tolerance of the Xiushui 09/IR2061 BC2 F 6 introgression lines and their parents Traits 1)
Treatment2)
SRS (%) SF (%)
Cold stress Control Heat stress Difference
Xiushui 09 (P1 ) 42.8±6.4 82.1±0.8 26.3±6.5 -55.8±6.2
Parents IR2061 (P2 )
P 1 -P 2
22.1±3.0 80.6±0.3 47.9±8.0 -32.7±2.4
20.7 ** 1.5 -21.6 ** -23.1 **
Introgression lines Mean±SD Range 36.45±26.4 87.6±7.1 30.5±17.0 -69.6±20.2
0-100 70.6-97.2 1.0-77.6 12.7-93.6
1)
SRS, survival rate of seedlings at seedling stage; SF, spikelet fertility at anthesis. Difference, (Stress-Control) of the ILs between the stress and control conditions. ** , significance level at P<0.01. 2)
stage were detected on chromosomes 1, 7, and 11, accounting for a total of 33.99% of the phenotypic variance presented in the population. The IR2061 alleles at all loci except qSF7 decreased SRS (Table 2 and Fig.).
QTLs for HT-related traits at anthesis Four QTLs were identified and mapped for SF on chromosomes 4, 5, 6, and 11. Two of these QTLs were detected under normal and stress conditions, respectively, while the other two were detected by the trait differ-
ences between the stress and normal conditions (Table 3 and Fig.). Based on their differential behaviors, these QTLs were classified into two types. The first type includes qSF5 and qSF11 which expressed only in the normal but not under stress, and the IR2061 alleles at the two loci decreased SF. The second type of QTL was qSF4 and qSF6, which were detectable only under heat stress, suggesting it was apparently induced by stress. The Xiushui 09 allele at the qSF4 and the IR2061 allele at the qSF6 caused increased SF. In addition, the two QTLs (qSF4 and qSF6) also contributed to the SF
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Table 2 QTLs affecting SRS identified at the seedling stage in the advanced backcross population of Xiushui 09/IR2061 BC2F6 QTL
Chromosome
Marker interval1)
LOD value
Additive effect (A)2)
Percentage of variance explained (%)
1 7 11 11
RM200-RM212 RM432-RM11 RM552-RM202 RM224-RM254
2.64 2.89 5.80 6.31
-0.12 10.19 -15.43 -16.21
5.69 6.50 9.58 12.22
qSRS1 qSRS7 qSRS11a qSRS11b 1) 2)
The underlined markers are those closer to the putative QTL. The same as below. The additive effect results from the effect of substitution of the Xiushui 09 allele by the IR2061 allele. The same as below.
Table 3 QTLs affecting SF identified at anthesis in the advanced backcross population of Xiushui 09/IR2061 BC2F6 QTL
Chromosome
Marker interval
Parameters1)
qSF4
4
RM518-RM401
qSF5
5
RM430-RM440
qSF6
6
RM589-RM588
qSF11
11
RM287-RM229
LOD A R2 (%) LOD A R2 (%) LOD A R2 (%) LOD A R2 (%)
1)
Normal
Stress
Difference
4.92 -11.92 10.33
3.71 10.69 8.54
4.92 10.82 14.58
6.01 -11.67 13.77
4.23 -4.16 13.55
2.93 -2.28 5.51
R2, percentage of variance explained in the population.
differences of the ILs between stress and normal conditions, and the Xiushui 09 allele at the qSF4 and the IR2061 allele at the qSF6 reduced SF difference, also known as increased stability of SF between the normal and stress conditions.
DISCUSSION CT and HT of rice are complicated quantitative traits. The SRS and SF are widely used to evaluate CT at the seedling stage and HT at anthesis, respectively, but their phenotypic values are easily influenced by environmental factors (Dilday 1990; Mackill and Lei 1997). In the present study, SRS at the seedling stage was investigated in the phytotron under normal condition. In order to avoid the influence of segregation in flowering on HT evaluation, lines having the same heading date as the recurrent parent, Xiushui 09 were selected for QTL mapping of HT at anthesis. Furthermore, environmental effects on phenotyping were eliminated to the maximum extent by setting replications and planting border lines. These measures results in accurate and reliable evaluation of SRS for CT at seedling stage and SF for HT at anthesis in this study. Cold stress causes various seedling injuries, such as
delayed heading and yield reduction due to spikelet sterility (Andaya and Mackill 2003). QTL mapping of CT at different developing stages has been conducted in many studies (Oh et al. 2004; Jiang et al. 2008; Suh et al. 2010). In the current study, most (3/4) alleles increasing SRS came from the japonica Xiushui 09, which is in agreement with the practical observation that japonica varieties usually show stronger CT than indica varieties at the seedling stage (Mackill and Lei 1997; Jeong et al. 2000). Among the four QTLs identified for SRS, the qSRS1 was co-localized in the region close to qCTs-1-c for seedling survival percentage reported by Lou et al. (2007); and the qSRS7 was located on the same region of qPSST-7 affecting percent seed set under cold water stress treatment reported by Suh et al. (2010) and also close to the region of qSCT-7 for seedling survival percentage reported by Zhang et al. (2005). Moreover, qSRS11a detected in the region of RM552-RM202 on chromosome 11 was found to be the same QTLs (fer11 and dc11) affecting spikelet fertility and leaf discoloration respectively, with the same significant marker RM552 in the study by Oh et al. (2004) and the same QTL (qSLT11-2 and qSCT-11) affecting seed set rate and seedling survival percentage with the same significant marker RM202 in the study by Liu et al. (2003) and Zhang et al. (2005). The qSRS11b was mapped in
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Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)
the same region with the qCTS11-2 for cold-induced necrosis tolerance with the same significant marker RM254 demonstrated by Andaya and Mackill (2003) in their study. These signify the three QTLs (qSRS1, qSRS7, and qSRS11a) might be the stable for CT at the seedling stage due to their detection in different genetic backgrounds. The three QTLs (qSRS7, qSRS11a, and qSRS11b) also convey partial genetic overlap underlying CT at both seedling and booting stages. These results imply that is beneficial to enhance the CT level of indica variety IR2061 through introgression of the favorable alleles from japonica variety Xiushui 09 at above mentioned stable and overlapping loci by MAS. Hybrid sterility in rice has been a subject of extensive investigation and low hybrid sterility is generally ascribed to incompatibility between indica and japonica (Oka 1988). This phenomenon was also observed in F1 hybrid plants derived from Xiushui 09/IR2061 with semi-sterility (data not shown). Recently, it was indicated that the SF of many indica-japonica hybrids was also very sensitive to temperature fluctuations and the hybrid progenies showed abnormal fertility or low seed set rate under low or high temperature conditions (Li et al. 1996; Lu et al. 2002; Zhao et al. 2006). In the present study, qSF6 detected under stress but not in normal condition was found to be located in the same region with the wide compatible gene, Sn5 on chromosome 6 (Ikehashi and Araki 1986). Obviously, the gene effect of qSF6 was induced by heat stress and had really contributed to HT due to its reduction of the trait difference between stress and non-stress conditions. Whether the Sn5 is responsive to heat stress will be depended on its allelic test to qSF6. On the other hand, a detailed comparison of the linkage map constructed in this study with the consensus map developed at Cornell University (Temnykh et al. 2001) revealed that marker order in the region of RM307-RM564 on chromosome 4, which was supported by at least 3 LOD and further confirmed by the map constructed using recombinant inbred line population derived from the same parents (data not shown), was contrary to that of the consensus map. The qSF4 was associated with SF under stress and difference of stress to normal conditions and contributed to HT. The gene effect of qSF4 probably resulted from chromosomal aberration or cryptic genomic variation in this region as indicated in the pre-
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vious studies (Henderson et al. 1958; Li et al. 1997). Unlike the qSF6, the IR2061 allele at the qSF4 had very negative effect on SF and reduced HT. Considering too low fertility might bias the detection of heat tolerant QTLs, nine lines with fertility less than 70% were excluded in our data analysis. The average SF of the nine lines was 43.8%, of which all of them had the same introgressed alleles from IR2061 at the qSF4. So we speculated that low fertility of the nine lines in normal condition was associated with chromosomal aberration resulting from introgression of IR2061. The qSF4 could not be detected under normal condition due to the deletion of the nine lines with low SF from the population. However, the gene expression of qSF4 on SF was detected under stress condition, indicating that its magnitude of gene effect was probably enhanced by heat stress. Therefore, HT based on the evaluation of SF may have different mechanisms as revealed in this study. Most HT QTLs in this study exhibited pronounced differential expressions in response to heat stress. This is substantiated by two observations. Firstly, of the four QTLs identified (Table 3), two (qSF5 and qSF11) were observed only in the normal but not in stress conditions. Secondly, the other two (qSF4 and qSF6) were detected under heat stress but not in normal conditions. Although QTLs induced only by heat stress may be associated with mechanism(s) of rice heat response, they may not necessarily contribute to HT. We believe that those QTLs that can reduce trait difference between stress and non-stress conditions should have contributed to HT because of their obvious contribution to trait stability. The qSF4 and qSF6 for SF identified in this study belonged to this kind of QTLs, affirmed by their associations with the trait differences of the ILs between the stress and non-stress conditions. CT and HT belong to the two extreme responses of plants to temperature, involving many complicated physiological and biochemical processes (Vergara 1976; Nishida and Murata 1996; Blum et al. 2001). There was no common QTL identified for CT at seedling stage and HT at anthesis in the present study. However, qSF4 detected in this study was most probably the same as the qCTB-4-1 affecting SF at the booting stage with the same significant marker RM518 as previously reported by Xu et al. (2008), suggesting there is probably partial genetic overlap between CT and HT at boot-
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ing and flowering stages. This kind of partial genetic overlap of CT and HT needs to be dissected using the present mapping population at the same developmental stage. Because most CT QTLs at seedling stage and HT QTLs at anthesis were genetically independent, it is possible to integrate CT and HT together in the progeny of Xiushui 09/IR2061 by pyramiding the favorable alleles at the three CT QTLs (qSRS1, qSRS11a, and qSRS11b) from japonica variety, Xiushui 09 and those at the two HT QTLs (qSF4 and qSF6) from indica variety IR2061 through MAS.
CONCLUSION Using survival rate of seedlings (SRS) and spikelet fertility (SF) as the index traits of cold tolerance (CT) and heat tolerance (HT), four QTLs (qSRS1, qSRS7, qSRS11a, and qSRS11b) for SRS were identified at seedling stage under cold stress, and the Xiushui 09 alleles at all loci except qSRS7 increased SRS. Four QTLs for SF were identified on chromosomes 4, 5, 6, and 11 at anthesis under heat stress. Among them, two QTLs (qSF4 and qSF6) contributed to HT because they reduced the trait difference between heat stress and normal conditions, and the IR2061 allele at the qSF6 and the Xiushui 09 allele at the qSF4 improved HT, respectively. No genetic overlap was detected between CT at seedling stage and HT at anthesis. It is possible to breed a new variety with CT and HT by pyramiding the favorable CT- and HT-improved alleles from Xiushui 09 and IR2061 by MAS.
MATERIALS AND METHODS Plant materials A set of introgression lines (ILs) population was developed using a high-yielding japonica rice cultivar, Xiushui 09 from China as a recurrent parent to cross with a drought tolerant indica breeding line-IR2061-520-6-9 (herein abbreviated as IR2061) from IRRI as a donor parent. The F1 plants were backcrossed to the recurrent parent to develop BC1F1 population with 110 plants. The BC1F1 plants were used as the male parent to backcross with the Xiushui 09, producing 253 BC2F1 plants. These plants were allowed to self 5 times without any selection via, single seed descent. Finally, a set of random ILs consisting of 240 BC2F6 lines
was developed.
Evaluation of CT at the seedling stage The experiment was conducted in a randomized complete block design with three replications. In 2009, the parents, Xiushui 09 and IR2061, and the set of 240 ILs was evaluated for CT in a phytotron in the Chinese Academy of Agricultural Sciences, Beijing, China. The seeds of the plant materials were placed in the oven at 50°C for 5 d to break any possible dormancy, and then germinated at 35°C for 48 h after surface-sterilizing with 1% sodium hypochlorite solution for 10 min and rinsing well with distilled water. The most uniform 30 germinated seeds for each line were sown in germinated trays (64 cm×33 cm×10 cm). Afterwards, the seedlings were cultured in growth cabinet, where the temperature was maintained at 25°C and relative humidity of 70%. At one-leaf stage seedling growth, 20 uniform seedlings were kept per line by thinning. When the selected seedlings grew into three-leaf stage, the seedlings were subjected to cold stress at 4°C with relative humidity of 70%. After 7 d of low temperature treatment, the temperature was adjusted gradually back to 25°C. The number of dead seedlings for each of the ILs or parental varieties was recorded from the fifth day of recovery when the seedlings started to wilt and the survival rate of seedlings (SRS) was estimated as below and used as the index of overall cold tolerance. SRS (%)=No. of surviving seedlings/No. of total seedlings×100
Evaluation of HT at anthesis The same set of ILs was used for evaluation of the HT. The experiment was carried out in the screen house in Zhejiang Academy of Agricultural Sciences, Hangzhou, China, from May to September, 2009. The seedlings of each line and the two parents were cultured under field condition. Five-leaf stage seedlings were divided into two parts for transplanting, one part was transplanted to the field under normal temperature condition as a control, while the other was transplanted into the greenhouse and subjected to heat treatment. For the control and the treatment, a two-row plot consisting of 10 plants per row per line were planted with 25 cm between row spacing and 17 cm between plant spacing with three replications. The recurrent parent, Xiushui 09 was inserted consecutively after every 20 plots in each replication. Heading date was recorded once in 2 d. The high temperature stress was imposed from the beginning of flowering of Xiushsui 09 and released the stress by the end of flowering of Xiushsui 09, which lasted around 10 d. High temperature treatment was generated by closing the windowpane. The atmosphere inside the green house was circulated by electric fans which were
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Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)
evenly fixed on the windowpane of the greenhouse and automatically controlled by temperature monitors. The temperature monitors linked to the fans were set up in central sites with the upright distance 10 cm apart from the top of the plants. As soon as the temperature at the central area rose to 38°C, the monitor was automatically switched on so that the temperature in the green house remains constant. Thus, the temperature inside the greenhouse was almost maintained at a range of 36-38°C during flowering time whereas the outdoor temperature was around 2932°C. Based on panicle size and tiller height, three consistent larger panicles per plant from the 8 plants in the middle of each plot were sampled. Only the lines having the same heading dates with the check Xiushui 09 were incorporated for data collection. Finally, 186 lines were selected for mapping analysis of HT. Spikelet fertility (SF) was investigated as the index of HT.
Genotyping, linkage map construction and QTL analysis Twenty-four individual plants per line were planted in the greenhouse in the Chinese Academy of Agricultural Sciences, Beijing, China, and 10 g of fresh leaf tissues were bulk harvested for DNA extraction using the CTAB method (Tai and Tanksley 1990) with minor modifications. The IL population was assayed with a set of 142 well-distributed SSR markers, which were used to construct the linkage map using MapManager QTX15 software (Manly and Olson 1999). Phenotypic data of the ILs, obtained from stress and non-stress conditions, were used as input data to identify QTLs affecting SRS and SF by QTLMapper 1.0 (Wang et al. 1999). In addition, trait differences (stress, non-stress) of the ILs between the stress and non-stress conditions for SF were used to identify QTLs showing differential expression between the temperature conditions. The permutation method (Churchill and Doerge 1994) was also used to obtain empirical thresholds for claiming QTLs of the experiment based on 1 000 runs of randomly shuffling the trait values, which ranged from 2.58 for SF under heat stress to 2.75 for SF in the normal condition.
Acknowledgements We thank Dr. Manly K F from Roswell Park Cancer Institute, USA, and Dr. Wang Daolong from Zhejiang University, China, for providing us the softwares MapManager QTX15 and QTLMapper 1.0. The work was funded by the Project of the 863 Program (2010AA101803) and the 948 Program of China (2006-G51 and 2010-G2B).
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Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)
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March 2012
2012, 11(3): 359-367
RESEARCH ARTICLE
Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.) CHENG Li-rui1*, WANG Jun-min2*, Veronica Uzokwe1, MENG Li-jun1, WANG Yun1, SUN Yong1, ZHU Linghua1, XU Jian-long1 and LI Zhi-kang1, 3 National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 2 Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, P.R.China 3 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines 1
Abstract A set of 240 introgression lines derived from the advanced backcross population of a cross between a japonica cultivar, Xiushui 09, and an indica breeding line, IR2061, was developed to dissect QTLs affecting cold tolerance (CT) at seedling stage and heat tolerance (HT) at anthesis. Survival rate of seedlings (SRS) and spikelet fertility (SF), the index traits of CT and HT, showed significant differences between the two parents under stresses. A total of four QTLs (qSRS1, qSRS7, qSRS11a and qSRS11b) for CT were identified on chromosomes 1, 7, 11, and the Xiushui 09 alleles increased SRS at all loci except qSRS7. Four QTLs for SF were identified on chromosomes 4, 5, 6, and 11. These QTLs could be classified into two major types based on their behaviors under normal and stress conditions. The first was QTL expressed only under normal condition; and the second QTL was apparently stress induced and only expressed under stress. Among them, two QTLs (qSF4 and qSF6) which reduced the trait difference between heat stress and normal conditions must have contributed to HT because of their obvious contribution to trait stability, and the IR2061 allele at the qSF6 and the Xiushui 09 allele at the qSF4 improved HT, respectively. No similar QTL was found between CT at seedling stage and HT at anthesis. Therefore, it is possible to breed a new variety with CT and HT by pyramiding the favorable CT- and HT-improved alleles at above loci from Xiushui 09 and IR2061, respectively, through marker-assisted selection (MAS). Key words: cold tolerance, heat tolerance, advanced backcross population, QTL mapping, rice
INTRODUCTION Rice is by far the world’s most important food crop for the poor, it supplies 27% of the calories in the lowand lower-middle-income countries, more than any other crop (Dawe et al. 2010; FAO 2010). With global climate changing, most rice growing regions are experiencing more extreme environmental fluctuations (Solomon et al. 2007). Rice is susceptible to a variety
Received 10 February, 2011
of abiotic stresses including cold and heat stresses. The chilling stress below 15°C often happen in early season rice growing regions in the lower Yangtze River and single season rice-growing regions in the Northeast, Northwest and Yungui altiplano of China. Cold damage below 15°C at seedling stage usually results in poor seedling establishment or increase of seedling mortality (Nakagahra et al. 1997; Jacobs and Pearson 1999; Ji et al. 2008), thus greatly reducing the rice yield. On the other hand, high temperature (>35°C) occurrence at flower-
Accepted 23 March, 2011
Correspondence XU Jian-long, Tel: +86-10-82105854, Fax: +86-10-82108559, E-mail: [email protected] * These authors contributed equally to this work. © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
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ing stage leads to spikelet sterility (Satake and Yoshida 1978; Matsui and Omasa 2002; Nakagawa et al. 2003; Jagadish et al. 2007; Rang et al. 2011). In 2003, large areas across Hubei, Anhui, Zhejiang, Hunan and Guangdong provinces in China, suffered high temperature stress above 38°C for a week, causing spikelet fertility lower than 50%, thus resulting in great yield deduction (Yang and Liu 2009). Consequently, in these areas, improvement of tolerance to stresses of low temperature at the seedling stage and high temperature at anthesis is important in rice breeding programs. However, identifying QTLs associated with cold tolerance (CT) and heat tolerance (HT) and elucidating their genetic relationship are the prerequisite for developing rice varieties with CT and HT. CT and HT are complex traits that are controlled by quantitative trait loci (QTL). Many QTLs related to CT at different stages such as germination, seedling, vegetative, reproductive and grain maturity have been identified by different researchers using recombinant inbred lines (RILs) (Andava and Tai 2006; Jiang et al. 2008; Suh et al. 2010), doubled haploid (DH) (Chen et al. 2006; Lou et al. 2007), F2:3 lines (Han et al. 2006), backcross (Li et al. 1997), and introgression lines (Liu et al. 2003; Xu et al. 2008; Zeng et al. 2009; Zhou et al. 2010). With regard to different stages, the genetic mechanism of CT during the early growth stage is highly essential because the evaluation of CT at seedling stage can be easily manipulated, which is also vital for the seedling establishment, seedling mortality and subsequent vigorous vegetative growth (Zhang et al. 2005; Lou et al. 2007; Jiang et al. 2008). However, the most sensitive growth stage for HT in rice is at anthesis. Recently, QTL mapping studies involving HT at flowering or grain-filling stages with seed set rate or grain quality traits as an evaluation index, revealed that HT are controlled by polygenes which were distributed on 12 chromosomes (Cao et al. 2002; Zhu et al. 2005; Zhao et al. 2006; Chen et al. 2008; Zhang et al. 2009; Jagadish et al. 2010). Both cold and heat stresses belong to the two extreme temperature stresses. Athough some CT or HT QTLs have been identified, our understanding of the genetic relationships between CT and HT is unknown. In the present study, we developed a set of BC2F6 introgression lines population derived from a cross between
japonica cultivar Xiushui 09, as a recurrent parent and indica cultivar IR2061, as a donor parent to dissect QTLs affecting CT at the seedling stage and HT at anthesis. The objective was to identify QTLs affecting the two stresses and analyze the genetic relationships between the two tolerances, provide some useful information for rice breeding of stress tolerance by MAS.
RESULTS Linkage map Among 550 SSR markers surveyed, 201 showed polymorphism between the two parents, and 142 polymorphic markers evenly distributed on 12 rice chromosomes were selected to construct the linkage map (Fig.). The linkage map spanned 1 591.1 cM with an average distance of 11.20 cM between adjacent markers. Most genome of the ILs was recovered to Xiushui 09 with an average of 91.4% of Xiushui 09 genome and a range of 73.2-98.8%. The average numbers of homozygous introgressed segments per line was 6.1.
Phenotypic variation Significant differences of SRS under cold stress treatment were observed between the two parents (Table 1), indicating that the japonica cultivar Xiushui 09 was more tolerant to cold stress than indica cultivar IR2061. The ILs expressed transgressive segregation and continuous variation which were suitable for QTL mapping. With regard to HT, significant difference was not observed for the SF between the parents under normal temperature. However, under high temperature, SF reduced to 26.3 and 47.9% for Xiushui 09 and IR2061, respectively, bringing about a significant difference between the two parents. This implied that IR2061 was more tolerant to heat stress at the anthesis than Xiushui 09 (Table 1). The ILs showed transgressive segregation for SF under high temperature.
QTLs detection of CT at the seedling stage Four QTLs (qSRS1, qSRS7, qSRS11a and qSRS11b) associated with SRS under cold treatment at seedling
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Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)
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Fig. The molecular linkage map and QTLs affecting survival rate of seedlings (SRS) under cold stress at the seedling stage and spikelet fertility (SF) under heat stress at anthesis identified in the advanced backcross population of Xiushui 09/ IR2061. , QTLs affecting SRS under cold stress at the seedling stage. , , , QTLs affecting SF in normal, heat stress and SF difference of stress to normal conditions, respectively.
Table 1 Phenotypic performance for the traits associated with cold tolerance and heat tolerance of the Xiushui 09/IR2061 BC2 F 6 introgression lines and their parents Traits 1)
Treatment2)
SRS (%) SF (%)
Cold stress Control Heat stress Difference
Xiushui 09 (P1 ) 42.8±6.4 82.1±0.8 26.3±6.5 -55.8±6.2
Parents IR2061 (P2 )
P 1 -P 2
22.1±3.0 80.6±0.3 47.9±8.0 -32.7±2.4
20.7 ** 1.5 -21.6 ** -23.1 **
Introgression lines Mean±SD Range 36.45±26.4 87.6±7.1 30.5±17.0 -69.6±20.2
0-100 70.6-97.2 1.0-77.6 12.7-93.6
1)
SRS, survival rate of seedlings at seedling stage; SF, spikelet fertility at anthesis. Difference, (Stress-Control) of the ILs between the stress and control conditions. ** , significance level at P<0.01. 2)
stage were detected on chromosomes 1, 7, and 11, accounting for a total of 33.99% of the phenotypic variance presented in the population. The IR2061 alleles at all loci except qSF7 decreased SRS (Table 2 and Fig.).
QTLs for HT-related traits at anthesis Four QTLs were identified and mapped for SF on chromosomes 4, 5, 6, and 11. Two of these QTLs were detected under normal and stress conditions, respectively, while the other two were detected by the trait differ-
ences between the stress and normal conditions (Table 3 and Fig.). Based on their differential behaviors, these QTLs were classified into two types. The first type includes qSF5 and qSF11 which expressed only in the normal but not under stress, and the IR2061 alleles at the two loci decreased SF. The second type of QTL was qSF4 and qSF6, which were detectable only under heat stress, suggesting it was apparently induced by stress. The Xiushui 09 allele at the qSF4 and the IR2061 allele at the qSF6 caused increased SF. In addition, the two QTLs (qSF4 and qSF6) also contributed to the SF
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Table 2 QTLs affecting SRS identified at the seedling stage in the advanced backcross population of Xiushui 09/IR2061 BC2F6 QTL
Chromosome
Marker interval1)
LOD value
Additive effect (A)2)
Percentage of variance explained (%)
1 7 11 11
RM200-RM212 RM432-RM11 RM552-RM202 RM224-RM254
2.64 2.89 5.80 6.31
-0.12 10.19 -15.43 -16.21
5.69 6.50 9.58 12.22
qSRS1 qSRS7 qSRS11a qSRS11b 1) 2)
The underlined markers are those closer to the putative QTL. The same as below. The additive effect results from the effect of substitution of the Xiushui 09 allele by the IR2061 allele. The same as below.
Table 3 QTLs affecting SF identified at anthesis in the advanced backcross population of Xiushui 09/IR2061 BC2F6 QTL
Chromosome
Marker interval
Parameters1)
qSF4
4
RM518-RM401
qSF5
5
RM430-RM440
qSF6
6
RM589-RM588
qSF11
11
RM287-RM229
LOD A R2 (%) LOD A R2 (%) LOD A R2 (%) LOD A R2 (%)
1)
Normal
Stress
Difference
4.92 -11.92 10.33
3.71 10.69 8.54
4.92 10.82 14.58
6.01 -11.67 13.77
4.23 -4.16 13.55
2.93 -2.28 5.51
R2, percentage of variance explained in the population.
differences of the ILs between stress and normal conditions, and the Xiushui 09 allele at the qSF4 and the IR2061 allele at the qSF6 reduced SF difference, also known as increased stability of SF between the normal and stress conditions.
DISCUSSION CT and HT of rice are complicated quantitative traits. The SRS and SF are widely used to evaluate CT at the seedling stage and HT at anthesis, respectively, but their phenotypic values are easily influenced by environmental factors (Dilday 1990; Mackill and Lei 1997). In the present study, SRS at the seedling stage was investigated in the phytotron under normal condition. In order to avoid the influence of segregation in flowering on HT evaluation, lines having the same heading date as the recurrent parent, Xiushui 09 were selected for QTL mapping of HT at anthesis. Furthermore, environmental effects on phenotyping were eliminated to the maximum extent by setting replications and planting border lines. These measures results in accurate and reliable evaluation of SRS for CT at seedling stage and SF for HT at anthesis in this study. Cold stress causes various seedling injuries, such as
delayed heading and yield reduction due to spikelet sterility (Andaya and Mackill 2003). QTL mapping of CT at different developing stages has been conducted in many studies (Oh et al. 2004; Jiang et al. 2008; Suh et al. 2010). In the current study, most (3/4) alleles increasing SRS came from the japonica Xiushui 09, which is in agreement with the practical observation that japonica varieties usually show stronger CT than indica varieties at the seedling stage (Mackill and Lei 1997; Jeong et al. 2000). Among the four QTLs identified for SRS, the qSRS1 was co-localized in the region close to qCTs-1-c for seedling survival percentage reported by Lou et al. (2007); and the qSRS7 was located on the same region of qPSST-7 affecting percent seed set under cold water stress treatment reported by Suh et al. (2010) and also close to the region of qSCT-7 for seedling survival percentage reported by Zhang et al. (2005). Moreover, qSRS11a detected in the region of RM552-RM202 on chromosome 11 was found to be the same QTLs (fer11 and dc11) affecting spikelet fertility and leaf discoloration respectively, with the same significant marker RM552 in the study by Oh et al. (2004) and the same QTL (qSLT11-2 and qSCT-11) affecting seed set rate and seedling survival percentage with the same significant marker RM202 in the study by Liu et al. (2003) and Zhang et al. (2005). The qSRS11b was mapped in
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Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)
the same region with the qCTS11-2 for cold-induced necrosis tolerance with the same significant marker RM254 demonstrated by Andaya and Mackill (2003) in their study. These signify the three QTLs (qSRS1, qSRS7, and qSRS11a) might be the stable for CT at the seedling stage due to their detection in different genetic backgrounds. The three QTLs (qSRS7, qSRS11a, and qSRS11b) also convey partial genetic overlap underlying CT at both seedling and booting stages. These results imply that is beneficial to enhance the CT level of indica variety IR2061 through introgression of the favorable alleles from japonica variety Xiushui 09 at above mentioned stable and overlapping loci by MAS. Hybrid sterility in rice has been a subject of extensive investigation and low hybrid sterility is generally ascribed to incompatibility between indica and japonica (Oka 1988). This phenomenon was also observed in F1 hybrid plants derived from Xiushui 09/IR2061 with semi-sterility (data not shown). Recently, it was indicated that the SF of many indica-japonica hybrids was also very sensitive to temperature fluctuations and the hybrid progenies showed abnormal fertility or low seed set rate under low or high temperature conditions (Li et al. 1996; Lu et al. 2002; Zhao et al. 2006). In the present study, qSF6 detected under stress but not in normal condition was found to be located in the same region with the wide compatible gene, Sn5 on chromosome 6 (Ikehashi and Araki 1986). Obviously, the gene effect of qSF6 was induced by heat stress and had really contributed to HT due to its reduction of the trait difference between stress and non-stress conditions. Whether the Sn5 is responsive to heat stress will be depended on its allelic test to qSF6. On the other hand, a detailed comparison of the linkage map constructed in this study with the consensus map developed at Cornell University (Temnykh et al. 2001) revealed that marker order in the region of RM307-RM564 on chromosome 4, which was supported by at least 3 LOD and further confirmed by the map constructed using recombinant inbred line population derived from the same parents (data not shown), was contrary to that of the consensus map. The qSF4 was associated with SF under stress and difference of stress to normal conditions and contributed to HT. The gene effect of qSF4 probably resulted from chromosomal aberration or cryptic genomic variation in this region as indicated in the pre-
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vious studies (Henderson et al. 1958; Li et al. 1997). Unlike the qSF6, the IR2061 allele at the qSF4 had very negative effect on SF and reduced HT. Considering too low fertility might bias the detection of heat tolerant QTLs, nine lines with fertility less than 70% were excluded in our data analysis. The average SF of the nine lines was 43.8%, of which all of them had the same introgressed alleles from IR2061 at the qSF4. So we speculated that low fertility of the nine lines in normal condition was associated with chromosomal aberration resulting from introgression of IR2061. The qSF4 could not be detected under normal condition due to the deletion of the nine lines with low SF from the population. However, the gene expression of qSF4 on SF was detected under stress condition, indicating that its magnitude of gene effect was probably enhanced by heat stress. Therefore, HT based on the evaluation of SF may have different mechanisms as revealed in this study. Most HT QTLs in this study exhibited pronounced differential expressions in response to heat stress. This is substantiated by two observations. Firstly, of the four QTLs identified (Table 3), two (qSF5 and qSF11) were observed only in the normal but not in stress conditions. Secondly, the other two (qSF4 and qSF6) were detected under heat stress but not in normal conditions. Although QTLs induced only by heat stress may be associated with mechanism(s) of rice heat response, they may not necessarily contribute to HT. We believe that those QTLs that can reduce trait difference between stress and non-stress conditions should have contributed to HT because of their obvious contribution to trait stability. The qSF4 and qSF6 for SF identified in this study belonged to this kind of QTLs, affirmed by their associations with the trait differences of the ILs between the stress and non-stress conditions. CT and HT belong to the two extreme responses of plants to temperature, involving many complicated physiological and biochemical processes (Vergara 1976; Nishida and Murata 1996; Blum et al. 2001). There was no common QTL identified for CT at seedling stage and HT at anthesis in the present study. However, qSF4 detected in this study was most probably the same as the qCTB-4-1 affecting SF at the booting stage with the same significant marker RM518 as previously reported by Xu et al. (2008), suggesting there is probably partial genetic overlap between CT and HT at boot-
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ing and flowering stages. This kind of partial genetic overlap of CT and HT needs to be dissected using the present mapping population at the same developmental stage. Because most CT QTLs at seedling stage and HT QTLs at anthesis were genetically independent, it is possible to integrate CT and HT together in the progeny of Xiushui 09/IR2061 by pyramiding the favorable alleles at the three CT QTLs (qSRS1, qSRS11a, and qSRS11b) from japonica variety, Xiushui 09 and those at the two HT QTLs (qSF4 and qSF6) from indica variety IR2061 through MAS.
CONCLUSION Using survival rate of seedlings (SRS) and spikelet fertility (SF) as the index traits of cold tolerance (CT) and heat tolerance (HT), four QTLs (qSRS1, qSRS7, qSRS11a, and qSRS11b) for SRS were identified at seedling stage under cold stress, and the Xiushui 09 alleles at all loci except qSRS7 increased SRS. Four QTLs for SF were identified on chromosomes 4, 5, 6, and 11 at anthesis under heat stress. Among them, two QTLs (qSF4 and qSF6) contributed to HT because they reduced the trait difference between heat stress and normal conditions, and the IR2061 allele at the qSF6 and the Xiushui 09 allele at the qSF4 improved HT, respectively. No genetic overlap was detected between CT at seedling stage and HT at anthesis. It is possible to breed a new variety with CT and HT by pyramiding the favorable CT- and HT-improved alleles from Xiushui 09 and IR2061 by MAS.
MATERIALS AND METHODS Plant materials A set of introgression lines (ILs) population was developed using a high-yielding japonica rice cultivar, Xiushui 09 from China as a recurrent parent to cross with a drought tolerant indica breeding line-IR2061-520-6-9 (herein abbreviated as IR2061) from IRRI as a donor parent. The F1 plants were backcrossed to the recurrent parent to develop BC1F1 population with 110 plants. The BC1F1 plants were used as the male parent to backcross with the Xiushui 09, producing 253 BC2F1 plants. These plants were allowed to self 5 times without any selection via, single seed descent. Finally, a set of random ILs consisting of 240 BC2F6 lines
was developed.
Evaluation of CT at the seedling stage The experiment was conducted in a randomized complete block design with three replications. In 2009, the parents, Xiushui 09 and IR2061, and the set of 240 ILs was evaluated for CT in a phytotron in the Chinese Academy of Agricultural Sciences, Beijing, China. The seeds of the plant materials were placed in the oven at 50°C for 5 d to break any possible dormancy, and then germinated at 35°C for 48 h after surface-sterilizing with 1% sodium hypochlorite solution for 10 min and rinsing well with distilled water. The most uniform 30 germinated seeds for each line were sown in germinated trays (64 cm×33 cm×10 cm). Afterwards, the seedlings were cultured in growth cabinet, where the temperature was maintained at 25°C and relative humidity of 70%. At one-leaf stage seedling growth, 20 uniform seedlings were kept per line by thinning. When the selected seedlings grew into three-leaf stage, the seedlings were subjected to cold stress at 4°C with relative humidity of 70%. After 7 d of low temperature treatment, the temperature was adjusted gradually back to 25°C. The number of dead seedlings for each of the ILs or parental varieties was recorded from the fifth day of recovery when the seedlings started to wilt and the survival rate of seedlings (SRS) was estimated as below and used as the index of overall cold tolerance. SRS (%)=No. of surviving seedlings/No. of total seedlings×100
Evaluation of HT at anthesis The same set of ILs was used for evaluation of the HT. The experiment was carried out in the screen house in Zhejiang Academy of Agricultural Sciences, Hangzhou, China, from May to September, 2009. The seedlings of each line and the two parents were cultured under field condition. Five-leaf stage seedlings were divided into two parts for transplanting, one part was transplanted to the field under normal temperature condition as a control, while the other was transplanted into the greenhouse and subjected to heat treatment. For the control and the treatment, a two-row plot consisting of 10 plants per row per line were planted with 25 cm between row spacing and 17 cm between plant spacing with three replications. The recurrent parent, Xiushui 09 was inserted consecutively after every 20 plots in each replication. Heading date was recorded once in 2 d. The high temperature stress was imposed from the beginning of flowering of Xiushsui 09 and released the stress by the end of flowering of Xiushsui 09, which lasted around 10 d. High temperature treatment was generated by closing the windowpane. The atmosphere inside the green house was circulated by electric fans which were
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Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)
evenly fixed on the windowpane of the greenhouse and automatically controlled by temperature monitors. The temperature monitors linked to the fans were set up in central sites with the upright distance 10 cm apart from the top of the plants. As soon as the temperature at the central area rose to 38°C, the monitor was automatically switched on so that the temperature in the green house remains constant. Thus, the temperature inside the greenhouse was almost maintained at a range of 36-38°C during flowering time whereas the outdoor temperature was around 2932°C. Based on panicle size and tiller height, three consistent larger panicles per plant from the 8 plants in the middle of each plot were sampled. Only the lines having the same heading dates with the check Xiushui 09 were incorporated for data collection. Finally, 186 lines were selected for mapping analysis of HT. Spikelet fertility (SF) was investigated as the index of HT.
Genotyping, linkage map construction and QTL analysis Twenty-four individual plants per line were planted in the greenhouse in the Chinese Academy of Agricultural Sciences, Beijing, China, and 10 g of fresh leaf tissues were bulk harvested for DNA extraction using the CTAB method (Tai and Tanksley 1990) with minor modifications. The IL population was assayed with a set of 142 well-distributed SSR markers, which were used to construct the linkage map using MapManager QTX15 software (Manly and Olson 1999). Phenotypic data of the ILs, obtained from stress and non-stress conditions, were used as input data to identify QTLs affecting SRS and SF by QTLMapper 1.0 (Wang et al. 1999). In addition, trait differences (stress, non-stress) of the ILs between the stress and non-stress conditions for SF were used to identify QTLs showing differential expression between the temperature conditions. The permutation method (Churchill and Doerge 1994) was also used to obtain empirical thresholds for claiming QTLs of the experiment based on 1 000 runs of randomly shuffling the trait values, which ranged from 2.58 for SF under heat stress to 2.75 for SF in the normal condition.
Acknowledgements We thank Dr. Manly K F from Roswell Park Cancer Institute, USA, and Dr. Wang Daolong from Zhejiang University, China, for providing us the softwares MapManager QTX15 and QTLMapper 1.0. The work was funded by the Project of the 863 Program (2010AA101803) and the 948 Program of China (2006-G51 and 2010-G2B).
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