QTLs Analysis of Cold Tolerance During Early Growth Period for Rice

Rice Science, 2004, 11(5-6): 245-250 245 http://www.ricescience.org QTLs Analysis of Cold Tolerance During Early Growth Period for Rice HAN Long-zhi 1, QIAO Yong-li 1, 2, CAO Gui-lan 1, ZHANG Yuan-yuan 1, AN Yong-ping 1, 3, YE Jong-doo 4, KOH Hee-jong 2 ( 1 Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 School of Plant Science, College of Agriculture and Life Science, Seoul National University, Seoul 151-742, Korea; 3 Crop Research Institute, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750105, China; 4 National Institute of Crop Science, Rural Development Administration, Suwon 441-857, Korea) Abstract: The quantitative trait loci (QTLs) for cold tolerance during early growth period were identified using a F 2:3 population (including 200 individuals and lines derived from a cross between indica rice and japonica rice Milyang 23/Jileng 1 ) with microsatellite markers. The cold tolerance at the seedling and tillering stages, and the growth ability of seedling under low temperature conditions were evaluated. All of the traits associated with cold tolerance at early growth stages appeared a continuous distribution near to normal in F 3 lines, these were inherited as quantitative traits controlled by polygenes. Three QTLs on chromosomes 1, 5 and 9, which associated with cold tolerance at the seedling stage were detected. Among them, qcts1 accounted for 15.5% of observed phenotypic variation; Five QTLs on chromosomes 2, 3, 7, 9 and 11, associated with cold tolerance at the tillering stage were found, which explained lower percentage of observed phenotypic variation; Four QTLs on chromosomes 1, 2, 11 and 12, which associated with the growth ability of seedling under low temperature conditions were found, among them, qgas2 and qgas12 explained 26.6 and 42.9% of observed phenotypic variation, respectively, which were major genes. Key words: rice; early growth period; cold tolerance; quantitative trait locus The growth of indica and japonica rice under temperature below 18 and 15, respectively, is inhibited to different extents at early growth stage [1 3]. The growth development at early stage such as the seedling and tillering, is influenced by low temperature stress in rice planting areas of high latitudes or high altitudes, for example, high altitude area of Japan, Korea, the northeast and southwest of China. Taking South China, for instance, low temperature stress in early spring often causes the seedlings of early indica varieties rot [4, 5]. The cold tolerance at early growth stage is an important parameter in rice developmental stage, which should be paid more attention to. Up to now, many genetic studies on cold tolerance during the early growth period of rice have been reported. Chung [4] [6, 7] and Han thought that cold tolerance at the seedling stage had a continuous distribution near to approximately normal distribution as the nucleus of mean parent value in F 2 population. Li and Rutger [8] pointed out that the seedling growth potential showed the dominance and over-dominance in F 1 and F 2 generations under low Received: 17 November 2004; Accepted: 12 December 2004 temperatures, which was controlled by 4 to 5 genes. [9, 10] Li et al found the segregation ratio of leaf discoloration was 3 1 when the seedlings were treated for three to five days at low temperatures of 5 and 10, indicating the seedling cold tolerance showed complete dominance. Chuong and Omura [11] proposed that seedling etiolation was controlled by the three genes, chs1, chs2 and chs3. However, these studies on the cold tolerance during early growth period were limited to the seedling cold tolerance, the genetic studies of cold tolerance at the tillering stage and the growth ability of seedling at low temperatures had seldom been reported, and the QTL analysis by using the molecular markers was rare. This research was undertaken to identify the QTLs conferring cold tolerance at early growth stage by using SSR molecular markers including cold tolerance at the seedling, tillering stage and the growth ability of seedling under low temperatures in rice. The detected QTLs associated with the cold tolerance in early growth period will provide the effective genetics evidences for the molecular marker assisted breeding and gene cloning.

246 Rice Science, Vol. 11, No. 5-6, 2004 MATERIALS AND METHODS Construction of mapping population F 1 hybrid population was developed by a crossing combination between indica rice Milyang 23 and japonica rice Jileng 1 in 1999. A F 2:3 population with 200 lines obtained through the acceleration of generation during 2000 to 2001 was used to construct linkage map in the experiment. Total genomic DNA was extracted at the tillering stage. Evaluation of cold tolerance at the seedling and tillering stage The seeds of 200 F 3 lines were sown in plastic seedling plates with a size of 60 cm 30 cm 5 cm, supplied with nutrient soil and managed by traditional cultivation practice. When the seedlings were at 3.5-leaf stage, the plastic seedling plates were put in a cold-water pool and treated for 10 days at low temperatures. The depth of cold-water was maintained at 5 cm. During the treatment of low temperatures, the cold water in the pool was flowing and maintained at 12. After treatment, the leaf discoloration of seedling was assessed (1 9 grades) according to the method of IRRI [12]. At the same time, 200 F 3 lines were planted in cold-water nursery and under natural irrigation field conditions at National Crop Experiment Station, Rural Development Administration of Korea, in 2002. The transplanting density of rice seedling was kept as 25 cm 15 cm, with single individual per hill. Individual plots consisted of single rows with 30 lines each row, and were arranged sequentially with two replicates. The seedling was grown in cold water(17, 5-cm depth maitained) after transplanting till the maturity stage. The leaf discoloration was investigated at the tillering stage (1 9 grades) [12]. The cold tolerance at the seedling and tillering stage was evaluated by the leaf discoloration. The leaf discoloration in a smaller degree means the stronger cold tolerance; likewise, the leaf discoloration in a larger degree means the weaker cold tolerance. The experimental design was managed according to a randomized complete block design with two replications. Individual plots consisted of single rows (3.6-m long and 0.3-m apart). Evaluation of the growth ability of seedling at low temperature Fifty seeds selected out from the F 3 lines were placed in the string net bags. The soaked seeds were incubated to accelerate germination at 30 in an incubator. The seeds were taken out from the incubator when sprouting and were filtered with free water. The seeds were planted in rice seedling pool at Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences on 5 April, 2003. The sowing density was kept as 10 cm 2 cm, with single grain per hill. Individual plots consisted of single rows with 20 grains each row. The plots were in sequential arrangement with two replications. In order to maintain the wet and the constant temperature, the plots were covered with plastic film, which was unveiled when the rice seedlings reached the 2.5-leaf stage (on 18 April) to make the rice seedling grow under natural low temperature conditions. Due to the low air temperature during the period from 20 April to 20 May, the rice seedlings were under the low temperature stress. Fifteen seedlings in every plot were surveyed for the seedling height on the 31st day after the plastic film unveiled, then mean values of the replications were used in data analysis to appraise the growth ability of seedling at low temperatures. During the time from unveiling the plastic film to surveying the seedling height (on 18 May), the average daily air temperature was 12.0 24.0, the highest and lowest daily air temperatures were 15.5 30.8 and 6.7 19.0, respectively. Extraction of genomic DNA and the detection of SSR markers F 2 population was planted in the experimental field in Beijing. The total genomic DNA for analysis was extracted from the leaves taken at the tillering stage following the phenol-chloroform method. The concentration of DNA was detected with ultraviolet spectro-photometer. One hundred and ninety-six pairs of SSR primers were selected carefully from the website of Cornell University, and synthesized by

HAN Long-zhi et al. QTLs Analysis of Cold Tolerance During Early Growth Period for Rice 247 SBS Biological Company. Among them, one hundred and twenty-nine pairs of primers with diversity between two parents were selected by diversity experiment of SSR markers. DNAs of 200 F 2 seedlings were amplified through PCR and the amplified products were electrophoresed. The PCR system was as follows (total volume 20 µl): 10 PCR buffer (containing Mg 2+ ) 2.0 µl, 2.5 mmol/l dntp 1.5 µl, Taq polymerase 0.25 U, 2 µmol/l SSR primers 2.0 µl, 20 ng/µl DNA 2.0 µl, ddh 2 O 12.0 µl. Samples were amplified through a total of 38 cycles with 5 min at 94, 30 s at 95, 30 s at 60, 1 min at 72, and 10 min at 72. Amplified products were resolved by 6% polyacrylamide gel electrophoresis and then were detected by silver staining method. QTL analysis A molecular linkage map of F 2:3 population with 97 SSR markers was constructed by using SSR markers, which covered a total length of 1357.3 cm with an average distance of 13.99 cm between adjacent markers in the genome [13]. All microsatellite markers were distributed uniformly on the chromosomes. A genetic linkage map was constructed by using the software of Mapmaker/EXP 3.0 and was painted by GENEMAPPER software developed by the Chinese Academy of Sciences. Likelihood figure was generated through connecting all LOD scores of specific chromosome, with a LOD threshold of 2.0. LOD>2.0 indicated that the highest LOD score position in the interval was a QTL for one trait. QTL nomenclature system was followed according to McCouch et al [14]. RESULTS Analysis of cold tolerance in early growth period for the F 2:3 population The results of cold tolerance in early growth period for the F 3 lines and the parents had been summarized in Fig. 1. The cold tolerance at the seedling and the tillering stage, and the growth ability of seedling (seedling height) at low temperatures for F 3 lines showed the approximately normal distributions, and we speculated the traits that were quantitative in nature controlled by polygenes. The average value for cold tolerance at the seedling stage for F 3 lines was 5.65. Their variant range was from grade 1 to 9, and the standard deviation was 2.3. The average value for cold tolerance at the tillering stage for F 3 lines was 4.02, their variant range was from grade 1 to 7 and the standard deviation was 2.02. The average value for the seedling height on the 31st day after unveiled the plastic film was 25.2 cm, their variant range was from 16.7 to 37.9 cm and the standard deviation was 3.80 cm. QTL analysis of cold tolerance in early growth period Based on the molecular linkage map (Fig. 2) [13] containing 97 SSR markers, and the identification result of cold tolerance in early growth period of F 3 lines, QTLs for cold tolerance during early growth period in rice was analyzed. The results of QTL analysis were listed in Table 1. For the cold tolerance at the seedling stage, three QTLs, qcts1, qcts5 and qcts9, were identified on chromosomes 1, 5 and 9, and the corresponding marker intervals on chromosome were RM265 Fig. 1. Distribution of cold tolerance during early growth period in F 3 lines derived from a cross of Milyang 23/Jileng 1.

248 Rice Science, Vol. 11, No. 5-6, 2004 Table 1. QTL and their genetic effects for cold tolerance in early growth period for rice. Traits QTL Chromosome Interval LOD Variation Effect value (%) Additive Dominance Resource of allele Gene action CTS CTT GAS qcts1 1 RM265 RM104 4.33 15.5 1.4306 0.3921 JL PD qcts5 5 RM173 RM305 2.37 5.6-0.6081-0.8464 MY OD qcts9 9 RM328 RM245 2.61 7.5-0.3484 1.0931 MY OD qctt2 2 RM324 RM29 3.59 7.9 0.7946 0.1403 JL A qctt3 3 RM16 RM168 2.40 6.5-0.6927-0.1037 MY A qctt7 7 RM118 RM248 2.73 8.0-0.7845 0.3021 MY PD qctt9 9 RM201 RM107 2.88 6.9-0.7809-0.2758 MY PD qctt11 11 RM287 RM229 2.68 7.4 0.7340 0.2169 JL PD qgas1 1 RM265 RM104 6.04 13.8 2.3911 1.1067 JL PD qgas2 2 RM262 RM263 9.93 26.6 2.7282 0.8285 JL PD qgas11 11 RM167 RM287 2.64 7.6 1.4986-0.0797 JL A qgas12 12 RM270 RM17 17.39 42.9 3.5974 0.4176 JL A CTS, Cold tolerance at the seedling stage; CTT, Cold tolerance at the tillering stage; GAS, Growth ability of seedling at low temperature. A, Additive (DR<0.2); PD, Partial dominance (0.2<DR<0.8); OD, Over dominance (DR>1.2); JL, Jileng 1; MY, Milyang 23. RM104, RM173 RM305, RM328 RM245, respectively. The range of variation percentage explained by QTLs was from 5.6% to 15.5%, among them, qcts1 explained 15.5% of observed phenotypic variance. The genetic distance of markers interval was 22.9 cm. qcts1 was contributed by the japonica rice alleles from Jileng 1, and qcts5 and qcts9 were contributed by the indica rice alleles from Milyang 23. The effects of these QTLs were dominance or partial dominance. For the cold tolerance at the tillering stage, five QTLs, qctt2, qctt3, qctt7, qctt9, qctt11 were detected on chromosomes 2, 3, 7, 9 and 11, and the corresponding markers interval on chromosomes were RM324 RM29, RM16 RM168, RM118 RM248, RM201 RM107 and RM287 RM229, respectively. The range of variation percentage explained by QTLs was from 6.5% to 7.9%, and the variation explained by every QTL was small. Among them, qctt2 and qctt11 were contributed by japonica rice alleles from Jileng 1, qctt3, qctt7 and qctt9 were contributed by the indica rice alleles from Milyang 23. The effects of these QTLs were partial dominance or additive in nature. For the growth ability of seedling under low temperature, four QTLs, qgas1, qgas2, qgas11 and Fig. 2. Intervals distribution for QTLs of cold tolerance at early growth stage. Note: Kosambi value(cm) are indicated on the left of the chromosome, and markers are indicated on the right of the chromosome.

HAN Long-zhi et al. QTLs Analysis of Cold Tolerance During Early Growth Period for Rice 249 qgas12, were found on chromosomes 1, 2, 11 and 12, and the corresponding markers interval were RM265 RM104, RM262 RM263, RM167 RM287 and RM270 RM17, respectively. The range of variation percentage explained by QTLs was from 7.4% to 42.9%, and the variation explained of qgas2 and qgas12 was 26.6% and 42.9%, respectively. The genetic distance of markers intervals was 22.7 and 18.1 cm, respectively, presumed that they were major genes. All of the four QTLs were contributed by the japonica rice alleles from Jileng 1. The effect of these QTLs was partial dominance or additive. DISCUSSTION In the recent decade, some studies related to QTLs of the cold tolerance in early growth period had been reported with the development of molecular biology. Qian et al [15] detected four QTLs for cold tolerance at the seedling stage on chromosomes 1, 2, 3 and 4 at 6 10. Redona and Mackill [16] identified four QTLs for the seedling vigor at 18 on chromosomes 1, 3, 5 and 9, and QTLs for the seedling vigor at 25 were mapped on chromosomes 1 and 3. Miura et al found that two QTLs for the seedling growth under low temperature on chromosomes 1 and 3 at 18 [17]. Kim et al [18] detected one QTL for the seedling growth at low temperatures on chromosome 5. However, up to now the analysis of QTLs for cold tolerance during early growth period mainly focused on the seedling vigor, while the studies on cold tolerance (leaf discoloration) at the seedling and the tillering stages were rare. The results of this research for cold tolerance (leaf discoloration) at the seedling and tillering stages, and the growth ability of seedling at low temperatures showed that all of these three traits of cold tolerance had an approximately normal distribution in F 3 lines. These traits were the quantitative in nature governed by many genes. This was similar to previous studies [4,5]. The three QTLs for cold tolerance at the seedling stage, qcts1, qcts5 and qcts9, were identified on chromosomes 1, 5 and 9. Among them, qcts1 has the largest variation, could explain 15.5% of the observed phenotypic variance. The genetic distance of markers interval was 22.9 cm. A total of five QTLs were detected on chromosomes 2, 3, 7, 9 and 11, but all of these QTLs explained lower percentage of observed phenotypic variance. A total of four QTLs were found on chromosomes 1, 2, 11 and 12, among them, qgas2 and qgas12 explained 26.6% and 42.9% of the observed phenotypic variance, respectively. The genetic distance of markers interval was 22.0 and 18.1 cm, respectively, presumed that they behaved as major genes, which were contributed by japonica rice alleles from Jileng 1. It is necessary to further study the molecular assisted selection and more detailed analysis on detected major genes. The result of correlation analysis revealed that correlation coefficients among the three traits for cold tolerance at the seedling and tillering stages, and the growth ability of seedling at low temperature, ranged from 0.1632 to 0.1828, which were significant at 0.05 level (critical value was 0.159 at 0.05 level when the error degree of freedom was 150). The correlation among the above three traits for cold tolerance was probably induced by gene multi-effect of one or several QTLs in the same locus or linked tightly. qcts1 associated with cold tolerance at the seedling stage and qgas1 related to the growth ability of seedling under low temperature were mapped in the same interval on chromosome. They might be the same QTL, or have gene multi-effect or two QTLs with close linkage, and need to be further researched. qctt9 and qcts9, qgas11 and qctt11 were linked tightly on chromosomes 9 and 11, respectively. The linkage of genes causes the existence of correlation among the traits. Although some QTLs detected in this study were located on the same chromosome compared with previous reports [15 18], they yet need to be further searched because of different molecular markers being used. The detected four QTLs for the growth ability of seedling at low temperatures were contributed by japonica rice alleles from Jileng 1, but more than half of the detected QTLs for cold tolerance at the seedling and tillering stages were contributed by indica rice alleles from Milyang 23. That is to say, genes of cold tolerance are derived from not only the cold tolerance parent but also the cold sensitive parent, only some genes of cold tolerance in cold sensitive parent stashes and does not

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