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Abstract To provided the experimental materials for identifying and cloning the quantitative trait loci （QTLs） of cold tolerance at the seedling stage， the authors analyzed QTLs and evaluated the genetic effects of two parents and a mapping population of 213 lines （recombination inbred lines， RILs） derived from a cross between IR24 and Asominori for cold tolerance at the seedling stage with dead seedling rate by using software QTL IciMapping 4.0， based on a genetic linkage map constructed with 141 SSR molecular markers. The QTLs qCTS??6， qCTS??1 1 and qCTS??1 2 related to cold tolerance at the seedling stage were detected on chromosome 6， 11 and 12， respectively. Individual QTLs （LOD??3.194 3， LOD： 4.688 2， LOD??3.797 0） explained 5.662 7%， 8.549 6% and 12.787 7% of the observed phenotypic variance， respectively. All of the three detected QTLs alleles came from cold??tolerant parent Asominori.
Key words Rice； RIL； Cold tolerance； Mapping population； QTL
Low??temperature cold damage refers to the phenomenon that plant grows slowly or develops abnormally in the temperature range from zero degree to its suitable growth temperature [1]. Low??temperature cold damage would influence rice to different degrees at different growth and development stages. At seedling stage， cold damage causes chlorosi and withering of seedlings， and rotting and death in severe case， thereby resulting in yield decrease of rice [2]. Cold damage at seedling stage is not only a problem common in single??season rice areas in the Yunnan-Guizhou Plateau， early rice areas in the Middle and Lower Reaches of Changjiang River and rice areas in northern China， but also a problem generally existing in main rice producing areas in the world [3]. Therefore， study on rice cold??tolerance QTLs and their genetic basis attracts much attention from scholars， and is of very strong theoretical value and practical significance. In recent years， a great progress has been made in research about rice cold tolerance. Cold tolerance in rice at seedling stage is a quantitative character controlled by polygenes， which has been localized onto multiple rice cold tolerance QTLs at seedling stage by molecular markers and various genetic groups， and 12 chromosomes all have been reported to have cold tolerance??related QTLs and genes located thereon [4-9]. However， in Guizhou area where cold damage problem exists commonly， the basic research on spring cold tolerance in rice still rests on the description about cold tolerance traits in rice germplasm resources at seedling stage， and no studies have been reported on QTLs of cold tolerance at seedling stage. For this reason， with rice variety Asominori having very strong cold tolerance at seedling stage as a cold tolerance gene source， 213 IR24/Asominori recombinant inbred lines （RILs） were constructed， and according to their performance in cold tolerance at seedling stage， QTL detection and genetic effect analysis were performed for rice cold tolerance at seedling stage using 141 SSR markers， so as to find out QTLs with a large contribution rate. This study provides materials for cloning of QTLs of cold tolerance at seedling stage， and provides a theoretical basis for marker??assisted selection （MAS） of cold??tolerant varieties adaptive to Guizhou area.
Materials and Methods
Test materials
IR24/Asominori RILs were provided by doctor Zhao from Nanjing Agricultural University. These populations were obtained by single seed descent method from the F1 generation. In the summer of 2012， parents and 213 RILs were planted in experimental field of Guizhou Rice Research Institute （Guiyang） according to 20 plants per material. The seeds were harvested at maturation stage， dried and preserved for identification of cold tolerance in rice at seedling stage.
Identification of cold tolerance at seedling stage
According to the method of Nagamine [l6] and Qian et al. [11]， 213 RILs were experimented for cold tolerance at seedling stage. With two parents （IR24： not cold tolerant， Asominori： highly cold tolerant） as controls， the average value of dead seedling rates of two replicates was calculated as the phenotypic value of cold tolerance at seedling stage， to perform mapping of QTLs of cold tolerance at seedling stage. Specifically， dry seeds were soaked for 1 d， and pre??germinated for 2-4 d until the white buds were about 1 cm， and the germinated seeds were sown on 500 ml plastic cups with holes at the bottom filled with peat soil （organic substrate） （for convenience of water absorption）. Then， the seeds were cultured in a manual climatic box at room temperature （daytime： 28 ??， 14 h； night： 23 ??， 10 h） to the stage of two leaves and one heart， when diseased and weak seedlings were removed， and every material was reserved with about 12 plants in each cup， with two replicates. The seedlings were treated at 10 ?? all day long for 10 d， and then recovered at room temperature for 5 d， followed by investigation of dead seedling rate.
QTL analysis
Molecular data of SSR markers of IR24/Asominori RILs were provided by doctor Zhao from Nanjing Agricultural University. The 141 SSR markers were distributed on 12 chromosomes uniformly. Statistically analysis of experimental data was performed with software QTL IciMapping 4.0， a genetic linkage map was constructed. Recombination rate was converted to genetic distance （cM） with Kosambi function， and the genetic linkage map was drawn with software MapDraw. This map covers 1 687.1 cM of rice genome， with a mean distance of 11.96 cM between markers. QTL mapping was performed by Inclusive Composite Interval Mapping （ICIM） in BIP function of software QTL IciMapping 4.0， during which the scanning step and LOD value were set as 1.0 cM and 2.5， respectively， and the contribution rate and additive effect of each QTL to cold tolerance at seedling stage were calculated. QTLs were named according to the method put forward by McCouch et al. [12]. A positive additive effect value indicates that the increasing effective allele is from cold??tolerant parent Asominori， while a negative value means that the increasing effective allele is from non??tolerant parent IR24. Results and Analysis
Growth phenotypes of parents and RIL populations after low??temperature treatment
After low??temperature treatment， the parents and RIL populations exhibited remarkable differences. Leaves on all seedlings of parent Asominori had enough water and grew normally， all seedlings of parent IR24 showed dead seedling leaves and stems. In RIL populations， some grew the same as IR24， i.e.， all seedlings withered up， while some lines grew the same as Asominori， i.e.， all seedlings survived. Other lines were between the two， having some survived seedlings.
Cold resistance in parent and RIL populations at seedling stage
In identification of cold tolerance at seedling stage， parent Asominori was a strongly cold??tolerant variety， which had a dead seedling rate of 0%； and IR24 was a variety not tolerant to cold， which showed a dead seedling rate of 100%. The 213 IR24/Asominori RILs were subjected to low??temperature treatment， and dead seedling rates of the 213 materials were calculated. According to the distribution of dead seedling rates （Fig. 1）， the 213 RILs had the dead seedling rates in continuous distribution from 0% to 100% after the low??temperature cold treatment， with an average value of 58.6%. Cold tolerance at seedling stage exhibited the genetic characteristics of quantitative traits controlled by polygenes.
QTL analysis of cold tolerance in rice at seedling stage
QTL analysis was performed on dead seedling rate at seedling stage by ICIM in BIP function of software QTL IciMapping 4.0. Three QTLs of cold tolerance at seedling stage were detected in total， on chromosome 6， 11 and 12， respectively， and were designated as qCTS??6， qCTS??11 and qCTS??12， respectively. They were corresponding to RM6818??RM3827， RM167??RM3701 and RM6296??RM277 （Fig. 2） and had the LOD values of 3.194 3， 4.688 2 and 3.797， respectively. The three QTLs explained 5.662 7%， 8.549 6% and 12.787 7% of the observed phenotypic variance， respectively. All the three of the detected QTL alleles came from cold??tolerant parent Asominori （Table 1）.
Conclusions and Discussion
It could be seen from studies at home and abroad that cold tolerance in rice at seedling stage is a quantitative trait controlled by polygenes， with complicated genetic background and extensive genetic diversity. Different genetic populations， cold tolerance identification methods and low temperature strength all would cause great differences in QTL analysis results of cold tolerance at seedling stage. Qu et al. [7] detected three QTLs of cold tolerance at seeding stage on chromosome 3， 11， and 12 of DH population of Gui 630/02428. Zhang et al. [9] detected a major QTL of cold tolerance at seedling stage closely linked to SSR marker RM202. In this study， a molecular linkable map was constructed using 141 SSR markers based on the dead seedling rates of 213 IR24/Asominori RILs after low??temperature treatment at seedling stage， and three QTLs of cold tolerance at seedling stage were detected in total， on chromosome 6， 11 and 12 （qCTS??6， qCTS??11 and qCTS??12）， respectively， among which qCTS??12 had the highest contribution rate. Detail information of related molecular markers were researched on Gramene website （http： //archive.gramene.org/markers）， and alignment analysis showed that qCTS??l1 was different from QTLs （on chromosome 11） located by Qu [7] and Zhang et al. [9]； and qCTS??12 was different from QTL （on chromosome 12） located by Qu [7]. Furthermore， cold tolerance in rice at seedling stage is a very important agronomic trait in the life process of rice， which directly influences normal growth and development of young rice seedlings. Previous studies on cold tolerance in rice at seedling stage were mostly conducted under natural environment， and their results can be influenced by external environment easily. In this study， the influences from external environment were eliminated using a manual climatic box， and with dead seedling rate at seedling stage as the identification index of cold tolerance， the results showed that the two parents differed greatly in cold tolerance phenotype， and the cold tolerance in RIL population was in continuous distribution， exhibiting the genetic characteristics of quantitative traits controlled by polygenes， which is similar to conclusions in previous studies [3， 8，10]. The discovery and localization of these QTLs provides a fundamental basis for cloning and isolation of genes related to cold tolerance， and also fills in the blank of research on molecular mechanism of cold tolerance in rice in Guizhou Province， thereby making the basic research of spring cold tolerance in rice reach molecular level in Guizhou Province.
Agricultural Biotechnology 2018References
[1] LIU LS. Plant molecular genetics[M]. Beijing： Science Press， 2003： 11-15. （in Chinese）
[2] XIANG Y， HE HH， LIU YB， et al. Advances in research on cold tolerance in Dongxiang wild rice[J]. Acta Agriculturae Universitis Jiangxiensis， 2003， 25（4）： 482-486. （in Chinese）
[3] DAI LY， YE CR， XU FR. Study on rice cold tolerance[J]. Southwest China Journal of Agricultural Sciences， 2002， 15（1）： 41-45. （in Chinese）
[4] HAN LZ， QIAO YL， CAO GL， et al. QTL analysis on cold tolerance during early growth period in rice[J]. Chinese J Rice Sci， 2005， 19（2）： 122-126. （in Chinese）
[5] JIANG L， XUN M， WANG JK， et al. QTL analysis of cold tolerance at seedling stage in rice （Oryza sative L.） using recombination inbred lines[J]. Journal of Cereal Science， 2008， 48： 173-179.
[6] KOSEKI M， KITAZAWA N， YONEBAYASHI S， et al. Identification and fine mapping of a major quantitative trait locus originating from wild rice， controlling cold tolerance at the seedling stage[J]. Mol Genet Genomics， 2010， 28（4）： 45-54.
[7] QU TT， CHEN LY， ZHANG ZH， et al. Molecular mapping of genes conferring cold tolerance at seedling stage using doubled haploid lines from an indica??japonica cross in rice[J]. Journal of Wuhan Botanical Research， 2003， 21（5）： 385-389. （in Chinese）
[8] ZHOU Y， ZHU YB， YUAN H， et al. Characterization of cold tolerance and identification of cold tolerance qtls for rice single segment substitution lines at plumule and seedling stages[J]. Chinese Journal of Rice Science， 2013， 27（4）： 381-388. （in Chinese）
[9] ZHANG ZH， LI S， LI W， et al. A major QTL conferring cold tolerance at the early seedling stage using recombinant inbred lines of rice （Oryza sativa L.）[J]. Plant Science， 2005， 168： 527-534.
[10] NAGAMINE T， NAKAGAHRA M. Genetic variation of chilling injury at seedling stage in rice， Oryza sativa L.[J]. Japanese Journal of Breeding， 1990， 40（4）： 449-455.
[11] QIAN Q， ZENG DL， HE P， et al. QTL analysis of the rice seedling cold tolerance in a double haploid population derived from anther culture of a hybrid between indica and japonica rice [J]. Chinese Science Bulletin， 2000， 45（5）： 448-453.
[12] MACOUCH SR， CHO YG， YANG M， et al. Report on QTL nomenclature[J]. Rice Genet Newslett， 1997， 14： 11-13.
Key words Rice； RIL； Cold tolerance； Mapping population； QTL
Low??temperature cold damage refers to the phenomenon that plant grows slowly or develops abnormally in the temperature range from zero degree to its suitable growth temperature [1]. Low??temperature cold damage would influence rice to different degrees at different growth and development stages. At seedling stage， cold damage causes chlorosi and withering of seedlings， and rotting and death in severe case， thereby resulting in yield decrease of rice [2]. Cold damage at seedling stage is not only a problem common in single??season rice areas in the Yunnan-Guizhou Plateau， early rice areas in the Middle and Lower Reaches of Changjiang River and rice areas in northern China， but also a problem generally existing in main rice producing areas in the world [3]. Therefore， study on rice cold??tolerance QTLs and their genetic basis attracts much attention from scholars， and is of very strong theoretical value and practical significance. In recent years， a great progress has been made in research about rice cold tolerance. Cold tolerance in rice at seedling stage is a quantitative character controlled by polygenes， which has been localized onto multiple rice cold tolerance QTLs at seedling stage by molecular markers and various genetic groups， and 12 chromosomes all have been reported to have cold tolerance??related QTLs and genes located thereon [4-9]. However， in Guizhou area where cold damage problem exists commonly， the basic research on spring cold tolerance in rice still rests on the description about cold tolerance traits in rice germplasm resources at seedling stage， and no studies have been reported on QTLs of cold tolerance at seedling stage. For this reason， with rice variety Asominori having very strong cold tolerance at seedling stage as a cold tolerance gene source， 213 IR24/Asominori recombinant inbred lines （RILs） were constructed， and according to their performance in cold tolerance at seedling stage， QTL detection and genetic effect analysis were performed for rice cold tolerance at seedling stage using 141 SSR markers， so as to find out QTLs with a large contribution rate. This study provides materials for cloning of QTLs of cold tolerance at seedling stage， and provides a theoretical basis for marker??assisted selection （MAS） of cold??tolerant varieties adaptive to Guizhou area.
Materials and Methods
Test materials
IR24/Asominori RILs were provided by doctor Zhao from Nanjing Agricultural University. These populations were obtained by single seed descent method from the F1 generation. In the summer of 2012， parents and 213 RILs were planted in experimental field of Guizhou Rice Research Institute （Guiyang） according to 20 plants per material. The seeds were harvested at maturation stage， dried and preserved for identification of cold tolerance in rice at seedling stage.
Identification of cold tolerance at seedling stage
According to the method of Nagamine [l6] and Qian et al. [11]， 213 RILs were experimented for cold tolerance at seedling stage. With two parents （IR24： not cold tolerant， Asominori： highly cold tolerant） as controls， the average value of dead seedling rates of two replicates was calculated as the phenotypic value of cold tolerance at seedling stage， to perform mapping of QTLs of cold tolerance at seedling stage. Specifically， dry seeds were soaked for 1 d， and pre??germinated for 2-4 d until the white buds were about 1 cm， and the germinated seeds were sown on 500 ml plastic cups with holes at the bottom filled with peat soil （organic substrate） （for convenience of water absorption）. Then， the seeds were cultured in a manual climatic box at room temperature （daytime： 28 ??， 14 h； night： 23 ??， 10 h） to the stage of two leaves and one heart， when diseased and weak seedlings were removed， and every material was reserved with about 12 plants in each cup， with two replicates. The seedlings were treated at 10 ?? all day long for 10 d， and then recovered at room temperature for 5 d， followed by investigation of dead seedling rate.
QTL analysis
Molecular data of SSR markers of IR24/Asominori RILs were provided by doctor Zhao from Nanjing Agricultural University. The 141 SSR markers were distributed on 12 chromosomes uniformly. Statistically analysis of experimental data was performed with software QTL IciMapping 4.0， a genetic linkage map was constructed. Recombination rate was converted to genetic distance （cM） with Kosambi function， and the genetic linkage map was drawn with software MapDraw. This map covers 1 687.1 cM of rice genome， with a mean distance of 11.96 cM between markers. QTL mapping was performed by Inclusive Composite Interval Mapping （ICIM） in BIP function of software QTL IciMapping 4.0， during which the scanning step and LOD value were set as 1.0 cM and 2.5， respectively， and the contribution rate and additive effect of each QTL to cold tolerance at seedling stage were calculated. QTLs were named according to the method put forward by McCouch et al. [12]. A positive additive effect value indicates that the increasing effective allele is from cold??tolerant parent Asominori， while a negative value means that the increasing effective allele is from non??tolerant parent IR24. Results and Analysis
Growth phenotypes of parents and RIL populations after low??temperature treatment
After low??temperature treatment， the parents and RIL populations exhibited remarkable differences. Leaves on all seedlings of parent Asominori had enough water and grew normally， all seedlings of parent IR24 showed dead seedling leaves and stems. In RIL populations， some grew the same as IR24， i.e.， all seedlings withered up， while some lines grew the same as Asominori， i.e.， all seedlings survived. Other lines were between the two， having some survived seedlings.
Cold resistance in parent and RIL populations at seedling stage
In identification of cold tolerance at seedling stage， parent Asominori was a strongly cold??tolerant variety， which had a dead seedling rate of 0%； and IR24 was a variety not tolerant to cold， which showed a dead seedling rate of 100%. The 213 IR24/Asominori RILs were subjected to low??temperature treatment， and dead seedling rates of the 213 materials were calculated. According to the distribution of dead seedling rates （Fig. 1）， the 213 RILs had the dead seedling rates in continuous distribution from 0% to 100% after the low??temperature cold treatment， with an average value of 58.6%. Cold tolerance at seedling stage exhibited the genetic characteristics of quantitative traits controlled by polygenes.
QTL analysis of cold tolerance in rice at seedling stage
QTL analysis was performed on dead seedling rate at seedling stage by ICIM in BIP function of software QTL IciMapping 4.0. Three QTLs of cold tolerance at seedling stage were detected in total， on chromosome 6， 11 and 12， respectively， and were designated as qCTS??6， qCTS??11 and qCTS??12， respectively. They were corresponding to RM6818??RM3827， RM167??RM3701 and RM6296??RM277 （Fig. 2） and had the LOD values of 3.194 3， 4.688 2 and 3.797， respectively. The three QTLs explained 5.662 7%， 8.549 6% and 12.787 7% of the observed phenotypic variance， respectively. All the three of the detected QTL alleles came from cold??tolerant parent Asominori （Table 1）.
Conclusions and Discussion
It could be seen from studies at home and abroad that cold tolerance in rice at seedling stage is a quantitative trait controlled by polygenes， with complicated genetic background and extensive genetic diversity. Different genetic populations， cold tolerance identification methods and low temperature strength all would cause great differences in QTL analysis results of cold tolerance at seedling stage. Qu et al. [7] detected three QTLs of cold tolerance at seeding stage on chromosome 3， 11， and 12 of DH population of Gui 630/02428. Zhang et al. [9] detected a major QTL of cold tolerance at seedling stage closely linked to SSR marker RM202. In this study， a molecular linkable map was constructed using 141 SSR markers based on the dead seedling rates of 213 IR24/Asominori RILs after low??temperature treatment at seedling stage， and three QTLs of cold tolerance at seedling stage were detected in total， on chromosome 6， 11 and 12 （qCTS??6， qCTS??11 and qCTS??12）， respectively， among which qCTS??12 had the highest contribution rate. Detail information of related molecular markers were researched on Gramene website （http： //archive.gramene.org/markers）， and alignment analysis showed that qCTS??l1 was different from QTLs （on chromosome 11） located by Qu [7] and Zhang et al. [9]； and qCTS??12 was different from QTL （on chromosome 12） located by Qu [7]. Furthermore， cold tolerance in rice at seedling stage is a very important agronomic trait in the life process of rice， which directly influences normal growth and development of young rice seedlings. Previous studies on cold tolerance in rice at seedling stage were mostly conducted under natural environment， and their results can be influenced by external environment easily. In this study， the influences from external environment were eliminated using a manual climatic box， and with dead seedling rate at seedling stage as the identification index of cold tolerance， the results showed that the two parents differed greatly in cold tolerance phenotype， and the cold tolerance in RIL population was in continuous distribution， exhibiting the genetic characteristics of quantitative traits controlled by polygenes， which is similar to conclusions in previous studies [3， 8，10]. The discovery and localization of these QTLs provides a fundamental basis for cloning and isolation of genes related to cold tolerance， and also fills in the blank of research on molecular mechanism of cold tolerance in rice in Guizhou Province， thereby making the basic research of spring cold tolerance in rice reach molecular level in Guizhou Province.
Agricultural Biotechnology 2018References
[1] LIU LS. Plant molecular genetics[M]. Beijing： Science Press， 2003： 11-15. （in Chinese）
[2] XIANG Y， HE HH， LIU YB， et al. Advances in research on cold tolerance in Dongxiang wild rice[J]. Acta Agriculturae Universitis Jiangxiensis， 2003， 25（4）： 482-486. （in Chinese）
[3] DAI LY， YE CR， XU FR. Study on rice cold tolerance[J]. Southwest China Journal of Agricultural Sciences， 2002， 15（1）： 41-45. （in Chinese）
[4] HAN LZ， QIAO YL， CAO GL， et al. QTL analysis on cold tolerance during early growth period in rice[J]. Chinese J Rice Sci， 2005， 19（2）： 122-126. （in Chinese）
[5] JIANG L， XUN M， WANG JK， et al. QTL analysis of cold tolerance at seedling stage in rice （Oryza sative L.） using recombination inbred lines[J]. Journal of Cereal Science， 2008， 48： 173-179.
[6] KOSEKI M， KITAZAWA N， YONEBAYASHI S， et al. Identification and fine mapping of a major quantitative trait locus originating from wild rice， controlling cold tolerance at the seedling stage[J]. Mol Genet Genomics， 2010， 28（4）： 45-54.
[7] QU TT， CHEN LY， ZHANG ZH， et al. Molecular mapping of genes conferring cold tolerance at seedling stage using doubled haploid lines from an indica??japonica cross in rice[J]. Journal of Wuhan Botanical Research， 2003， 21（5）： 385-389. （in Chinese）
[8] ZHOU Y， ZHU YB， YUAN H， et al. Characterization of cold tolerance and identification of cold tolerance qtls for rice single segment substitution lines at plumule and seedling stages[J]. Chinese Journal of Rice Science， 2013， 27（4）： 381-388. （in Chinese）
[9] ZHANG ZH， LI S， LI W， et al. A major QTL conferring cold tolerance at the early seedling stage using recombinant inbred lines of rice （Oryza sativa L.）[J]. Plant Science， 2005， 168： 527-534.
[10] NAGAMINE T， NAKAGAHRA M. Genetic variation of chilling injury at seedling stage in rice， Oryza sativa L.[J]. Japanese Journal of Breeding， 1990， 40（4）： 449-455.
[11] QIAN Q， ZENG DL， HE P， et al. QTL analysis of the rice seedling cold tolerance in a double haploid population derived from anther culture of a hybrid between indica and japonica rice [J]. Chinese Science Bulletin， 2000， 45（5）： 448-453.
[12] MACOUCH SR， CHO YG， YANG M， et al. Report on QTL nomenclature[J]. Rice Genet Newslett， 1997， 14： 11-13.