A QTL controlling low temperature induced spikelet sterility at booting stage in rice

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1 Euphytica (21) 176: DOI 1.17/s A QTL controlling low temperature induced spikelet sterility at booting stage in rice C. Ye S. Fukai I. D. Godwin H. Koh R. Reinke Y. Zhou C. Lambrides W. Jiang P. Snell E. Redoña Received: 19 October 29 / Accepted: 16 April 21 / Published online: 28 July 21 Ó Springer Science+Business Media B.V. 21 Abstract Low temperature is a major abiotic stress for rice cultivation, causing serious yield loss in many countries. To identify QTL controlling low temperature induced spikelet sterility in rice, the progeny of F2, BC1F1 and BC2F1 populations derived from a Reiziq 9 Lijiangheigu cross were exposed to 21/15 C for 15 days at the booting stage, and spikelet sterility was assessed. For genotyping, 92 polymorphic markers from 373 SSR and 325 STS primer pairs were used. A major QTL was initially indentified on the short arm of chromosome 1 by selective genotyping using highly tolerant and susceptible progeny from F2 and BC1F1 populations. The QTL (qltspkst1.1) was validated and mapped by genotyping the entire F2 (282 progeny) and BC1F1 (84 progeny) populations. The results from the F2 population showed that qltspkst1.1 could explain 2.5% of the variation in spikelet sterility caused by low temperature treatment with additive (a = 14.4) and dominant effect (d =-7.5). From the analysis of 98 selected BC2F1 progeny, the QTL located in the 3.5 cm interval between S11.9 and S114.4 was further confirmed. Based on the studies of 3 generations in 2 years, it was clear that the QTL on chromosome 1 is a major determinant of the control of low temperature induced spikelet sterility at booting stage. Keywords Cold tolerance Booting stage Spikelet sterility QTL Rice C. Ye (&) S. Fukai I. D. Godwin Y. Zhou C. Lambrides School of Land, Crop and Food Sciences, University of Queensland, Brisbane, QLD 472, Australia c.ye@irri.org H. Koh W. Jiang Department of Plant Science, Seoul National University, Seoul , South Korea R. Reinke P. Snell Department of Industry and Investment NSW, Yanco Agricultural Institute, Orange, NSW 273, Australia C. Ye E. Redoña International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines Introduction Rice is widely cultivated as a staple food for more than half of the world population. Rice productivity is limited by environmental factors, such as drought, chilling, salinity, and flood (submergence). In many high elevation and/or high latitude rice growing regions, such as China, Japan, Korea and Australia, low temperature is often a problem for stable rice production. It can cause serious yield losses, most significantly when the low temperature occurs during reproductive stages. Although the sowing time is usually adjusted to minimize the potential for low temperature damage at reproductive stages, low

2 292 Euphytica (21) 176: temperature still causes serious yield losses in these countries (Farrell et al. 26). Low temperature may affect the growth and development of rice from germination to grain filling (Ye et al. 29). Critical stages for cold injury include germination, seedling establishment, reproductive and grain filling stages. However, the most sensitive stage for cold injury in rice is the booting (early reproductive) stage, especially the early pollen microspore stage, which occurs approximately 1 12 days prior to heading. Low temperature (15 19 C) at booting stage causes sterile pollen grains which lead directly to spikelet sterility (Satake 1976). Rice genetic resources tolerant to low temperature have been identified and crossed to elite cultivars to develop cold tolerant varieties (Abe et al. 1989). QTL mapping studies for cold tolerance at booting stage have also been conducted on various rice populations (Andaya and Mackill 23; Dai et al. 24; Li et al. 1997, 23; Saito et al. 24, 21, 1995; Takeuchi et al. 21; Xu et al. 28). Saito et al. (1995) mapped two QTLs for cold tolerance at the booting stage in Norin-PL8 on chromosomes 3 and 4. Further fine mapping using a NIL (BC2F7) population revealed that the QTL for cold tolerance on chromosome 4 of Norin-PL8 was associated with two closely linked genes, Ctb1 and Ctb2. Ctb1 was further located to seven open reading frames (ORFs) in a 56-kb region of chromosome 4 (Saito et al. 21). Dai et al. (24) mapped nine QTLS for cold tolerance at reproductive stage using a F2 population derived from a cold susceptible Japanese rice variety Towada and a tolerant Chinese variety Kunmingxiaobaigu; the QTLs were distributed on chromosomes 1, 3, 4, 6, 7, 1 and 12. However, only 2 of the QTLs on chromosomes 1 and 1 were detected using BC5F3 NILs developed from the same parents, other 7 QTLs were newly identified in the NIL population (Xu et al. 28). QTL for cold tolerance at the booting stage have also been mapped on chromosome 1 and 12 using BC1F1 and F2 populations (Li et al. 1997), on chromosomes 1, 7, and 11 using doubled-haploid (DH) population (Takeuchi et al. 21), on chromosomes 1, 2, 3, 5, 6, 7, 9 and 12 using recombinant inbred lines (RIL) population (Andaya and Mackill 23), and on chromosomes 1, 6 and 7 using coldtolerant wild rice introgression lines (Liu et al. 23). Although QTLs for cold tolerance at booting stage have been mapped on all chromosomes except chromosome 8, to certain extent, the results from different research groups were not comparable, because different populations and phenotyping methods were used for QTL mapping. Furthermore, most of the reported QTL mapping studies were based on field evaluation of the cold tolerance, in which it is difficult to control environmental variation. Moreover, confirmation and fine mapping of the identified QTLs for cold tolerance were rarely done. Using mapped QTL to improve cold tolerance in rice varieties has not been achieved. Further studies on QTL mapping using accurate phenotyping technology and validations of mapped major QTL in different populations are needed to identify candidates for map based cloning of genes for cold tolerance in rice. Identifying QTL (genes) and developing markers for marker assisted breeding has high priority for rice breeding programs, especially for those traits that are difficult to screen in the field, such as cold tolerance. To this aim, high quality phenotyping is essential for any QTL mapping study, which makes the QTL identified more reliable and useful. Here we report on the identification of a QTL on chromosome 1 for low temperature induced spikelet sterility at the booting stage using three generations in experiments conducted over 2 years in a controlled-temperature glasshouse at the University of Queensland, Australia. The objectives of this study were to identify major QTL controlling low temperature induced spikelet sterility in rice, and to further confirm the QTL in different populations and years. Materials and methods Plant materials To select proper parents for QTL mapping, we have evaluated the cold tolerance of rice varieties from different countries at germination, seedling, booting and flowering stages (Ye et al. 29). Based on the performance of the varieties at low temperature, Reiziq (RZQ, female recurrent parent) and Lijiangheigu (LJHG, male donor parent) were selected and crossed. Reiziq is an Australian commercial variety with moderate cold tolerance at germination and seedling stages, but is susceptible to low temperature at booting and flowering stages. Lijiangheigu is a

3 Euphytica (21) 176: landrace from southern China which is tolerant to low temperature at all the growth stages (Ye et al. 29). The F2 (282 progeny), BC1F1 (84 progeny) and BC2F1 (819 progeny) populations were developed for mapping the QTL controlling low temperature induced sterility. Phenotyping of BC1F1 and F2 populations On 2 February 28, seeds of Reiziq, Lijiangheigu, F1, BC1F1, and the F2 population were germinated and sown, one plant per pot, in plastic pots (L 9 W 9 H = cm with draining holes) filled with sandy loam soil. The pots were randomly arranged in trays (L 9 W 9 H = cm) to ensure the plants were grown in the same water and nutrient conditions. The plants were grown in a warm glasshouse maintained at 3/19 C day/night temperatures (mean temperature 24.1 ±.7 C) under natural light. Fifteen days after sowing, 3 ml of fertilizer solution (urea 2.5 g/l, KCl 11.2 g/l, KH 2 PO g/l, CaSO g/l, MgSO g/l, ZnSO 4 7H 2 O.1 g/l, CuSO 4.1 g/l and (NH 4 ) 6 Mo 7 O 24 4H 2 O.2 g/l) was applied to each tray. Water was then applied to maintain a water depth of 1 cm. Four weeks after sowing, a young leaf (about 1 cm long) from the tiller of each plant was collected and frozen for DNA extraction. The position of the pot in the tray was changed within the tray every week to reduce any potential micro-environmental effects of light and temperature, and to avoid interweaving of the roots that protruded from the bottom of the pots. When the auricle of the flag leaf emerged (auricle distance greater than mm), the plant was moved into a new tray in a cool glasshouse maintained at 21/ 15 C day/night temperatures (mean temperature 17.7 ±.5 C) under natural light. After 15 days, the plants were moved back to the warm glasshouse and grown to maturity. The date of low temperature treatment commencement and heading date were recorded. At physiological maturity, plant height, panicle neck length (exsertion), panicle length, flag leaf length, number of fully filled spikelet and empty spikelet (include partially filled and undeveloped) were recorded. The percentage of empty spikelets was used to evaluate the cold tolerance of the plant. Other agronomic characters were used to select plants similar to recurrent parent for backcrossing. Phenotyping of BC2F1 population On 2 March 29, the seeds of Reiziq, Lijiangheigu and BC2F1 population were germinated and sown in plastic trays (L 9 W 9 H = cm, density cm) filled with sandy loam soil. The plants were grown in a glasshouse maintained at 3/19 C day/night temperatures (mean temperature 24.8 ±.8 C) under natural light. Fifteen days after sowing, a young leaf (about 1 cm long) from each plant was collected for DNA extraction and marker assisted selection (MAS) using two markers flanking the QTL. Twenty days after sowing, the selected plants were transplanted into plastic pots (L 9 W 9 H = cm with draining holes, one plant per pot) filled with sandy loam soil. The following treatment and measurements were the same as described above for BC1F1 and F2 populations. DNA extraction and PCR amplification Genomic DNA of Reiziq, Lijiangheigu, BC1F1 and F2 plants was extracted using SDS extraction buffer (1 mm Tris, 5 mm EDTA, 5 mm NaCl, 1.25% SDS and 1% v/v 2-mercaptoethanol) and chloroform/ isoamylalcohol (24:1) solution followed by ethanol precipitation. For marker-assisted selection of the BC2F1 seedlings, DNA was extracted using a modified rapid one-step extraction method (Burr et al. 21). The leaf tissue was frozen in liquid nitrogen and ground with 2 steel balls in a 2 ml microcentrifuge tube by vortexing. Then 3 ll.19 SDS extraction buffer (see above) was added to each tube. The tubes were incubated in a 65 C oven for 3 min and centrifuged at 12, rpm for 1 min. One microliter of supernatant was used for PCR amplification. PCR was performed in 1 ll reactions containing 1 ll of 1 9 PCR buffer,.5 ll of each primer (1 lm), 1 ll of dntp (2 mm each),.4 ll of MgCl 2 (5 mm),.2 unit Taq DNA polymerase, and ng of template. The PCR program was set as 94 C for 4 min, 35 cycles of 94 C for 3 s, 55 C for 3 s, 72 C for 3 s, and a final extension at 72 C for 5 min. PCR products were run on 8% (w/v) polyacrylamide gel for size separation using a MGV vertical gel electrophoresis system (CBS Scientific Co.). The gel was then stained with ethidium bromide and visualized with a UV transilluminator.

4 294 Euphytica (21) 176: Genotyping process A total of 373 SSR (McCouch et al. 22) and 325 STS (Chin et al. 27) primer pairs were used for a parental screen of Reiziq and Lijianheigu. The polymorphic markers were used for selective genotyping (Lander and Botstein 1989) using highly fertile and highly sterile progeny from BC1F1 and F2 populations exposed to low temperature at booting stage. Once a QTL was identified, the polymorphic markers flanking the QTL were used for genotyping the entire F2 and BC1F1 populations to to provide greater map resolution and to estimate the genetic effect of the QTL. Statistical analysis QTL analysis was performed using Windows QTL Cartographer V2.5 (Wang et al. 27). Other statistical analyses such as one-way ANOVA were performed using MINITAB V14. (Minitab Inc.). For selective genotyping, the mean of each marker genotype was firstly compared by using one-way ANOVA. If a significant difference was observed between different genotypes, then genotyping data of the flanking markers were used for Composite Interval Mapping (CIM) while progeny without genotyping data were treated as missing data. The genetic distance between SSR markers used for QTL analysis was estimated from maps of cv. Nipponbare available at GRAMENE ( while STS markers were based on the report of (Chin et al. 27). CIM was performed using the standard model with window size of 1 cm, and walk speed of 2 cm. The LOD thresholds were obtained from 1 permutation tests with significance level (experiment-wise type I error rate) of.1 (Churchill and Doerg 1994). The mean LOD threshold values varied among populations (F2 = 4.1, BC1F1 = 2.4, BC2F1 = 2.3). Results Cold tolerance of F2 and BC1F1 progeny Frequency distributions of F2 and BC1F1 progeny for low temperature-induced spikelet sterility (percentage of empty grains) followed normal distributions (Table 1). Spikelet sterility of Reiziq (59.4 ± 7.5%) was significantly higher than that of Lijiangheigu (17.9 ± 6.6%), while F1 progeny (4.7 ± 11.2%) were distributed between the parents. After a single backcross, the genomic component of Reiziq (susceptible) was higher in BC1F1 than in the F2 population, thus, the mean spikelet sterility of the BC1F1 population (56.1 ± 21.4%) was slightly higher that that of F2 population (44.9 ± 23.8%). Polymorphism between Reiziq and Lijiangheigu Among the screened 373 SSR and 325 STS primers, 56 SSR (15.%) and 36 STS (11.1%) loci were polymorphic between Reiziq and Lijiangheigu (Table 2). The low levels of polymorphism (13.2%) observed was because both parents were derived from the Japonica sub-species, whereas the STS primers were developed from Japonica-Indica genome sequence. The polymorphic markers were evenly distributed on each chromosome. Single marker analysis based on selective genotyping Based on the spikelet sterility of F2 and BC1F1 progeny, 11 tolerant and 11 susceptible F2 progeny were selected for genotyping using 92 polymorphic markers. Using single marker analysis (one-way ANOVA), only 3 markers, RM63 on chromosome Table 1 Spikelet sterility (%) of different populations after low temperature treatment at booting stage Year Population Count Mean SD Range Skewness Kurtosis 28 Reiziq LJHG F F BC1F Reiziq LJHG BC2F

5 Euphytica (21) 176: Table 2 Polymorphism between rice varieties Reiziq and Lijiangheigu Chromosome SSR marker STS marker Total polymorphic Screened Polymorphic Percentage Screened Polymorphic Percentage Total a a 92 The data show the number of screened markers and polymorphic markers. Percentage is the percentage of polymorphic marker a Overall percentage of polymorphic markers 5(F= 8.6, P =.2, n = 22), RM134 on chromosome 7 (F = 6.4, P =.9, n = 22) and RM216 on chromosome 1 (F = 8. 2, P =.3, n = 22) were associated with a significant difference in spikelet sterility among A (Reiziq type), H (heterozygote) and B (Lijiangheigu type) genotypes. We further genotyped 24 F2 (11 tolerant? 13 susceptible) and 22 BC1F1 (12 tolerant? 1 susceptible) progeny using markers RM63, RM134 and RM216. For RM63 with the larger F2 progeny set, the difference in spikelet sterility between A and B genotypes in the F2 population was significant (F = 3.54, P =.38, n = 46), but the difference between A and H genotypes in the BC1F1 population was not significant (F =.17, P =.688, n = 22) (Fig. 1a). QTL analysis (CIM) using RM63 and 2 other linked markers indicated that there was no QTL close to RM63 on chromosome 5. For RM134, the difference in spikelet sterility between A and B genotypes in the F2 population was significant (F = 3.7, P =.33, n = 46) when the number of progeny increased, but the difference between A and H genotypes in the BC1F1 was not significant (F = 1.71, P =.26, n = 22) (Fig. 1b). QTL analysis (CIM) using RM134 and 4 other linked markers indicated that there was no QTL close to RM134 on chromosome 7. For RM216, the difference in spikelet sterility among A (1.2 ± 4.4%), H (34.5 ± 35.9%) and B (84.6 ± 25.%) genotypes was highly significant (F = 24.9, P \.5, n = 46) after 46 F2 progeny were genotyped, and significant differences (F = 99.42, P \.5, n = 22) were also observed between A (27.4 ± 7.5%) and H (85.2 ± 17.7%) genotypes in 11 tolerant and 11 susceptible BC1F1 progeny (Fig. 1c). QTL analysis (CIM) using RM216 and 4 other linked markers identified one QTL related to spikelet sterility on the short arm of chromosome 1 in both F2 and BC1F1 populations. QTL analysis using entire F2 and BC1F1 populations To estimate the location and genetic effect of the QTL on chromosome 1, five markers (S12.7, S19.5, S114.4, CP8861 and S151.9) were used for genotyping the entire F2 and BC1F1 populations. The spikelet sterility of different genotypic classes (A and H in BC1F1 population, and A, H, B in F2 population) was significantly different (Fig. 2). The results from Composite Interval Mapping (CIM) showed a similar QTL in both F2 and BC1F1 populations (Fig. 3a, b). The QTL (peak LOD score) was located between markers S19.5 and S114.4 at 13.5 cm (from telomere) in F2 and

6 296 Euphytica (21) 176: (a) Sterility (%) RM63 Generation A(n=8) BC1F1 H(n=14) A(n=11) H(n=19) F2 B(n=16) (b) 9 8 Sterility (%) RM134 Generation A(n=13) BC1F1 H(n=9) A(n=12) H(n=19) F2 B(n=15) (c) Sterility (%) RM216 Generation A(n=11) BC1F1 H(n=11) A(n=7) H(n=18) F2 B(n=21) Fig. 1 Interval plot of spikelet sterility from selected BC1F1and F2 progeny genotyped by using markers: a RM63, b RM134 and c RM216. In BC1F1 population, 12 tolerant and 1 susceptible progeny were genotyped. In F2 population, 22 tolerant and 24 susceptible progeny were genotyped. The data above the bar shows the mean spikelet sterility (%) of each genotype. The interval bar shows the 95% confident interval of mean. Genotype A = Reiziq, H = heterozygote, B = Lijiangheigu

7 Euphytica (21) 176: Sterility (%) A(n=36) H(n=38) S114.4 Generation BC1F A(n=64) H(n=34) BC2F1 A(n=68) H(n=137) B(n=72) F2 Fig. 2 Interval plot of spikelet sterility in BC1F1, BC2F1 and F2 populations genotyped by using marker S114.4 on short arm of chromosome 1. The data above the bar shows the mean spikelet sterility of each genotype. The interval bar shows the 95% confident interval of mean. Genotype A = Reiziq, H = heterozygote, B = Lijiangheigu 12.5 cm in BC1F1 populations. In the F2 population, this QTL explained 2.5% of the variation of spikelet sterility caused by low temperature treatment. The additive effect (a = 14.4) was greater than the dominant effect (d =-7.5). The peaks at 2 cm and 28 cm may due to low recombination between S114.4 and CP8861 (over 5.7 Mb in 7.4 cm). It may also because S114.4 is a co-dominant marker and CP8861 is a dominant marker, the recombinant rate between the 2 markers could not be calculated accurately. These peaks disappeared in BC1F1 population, because there are only A and H genotypes in backcross populations. Thus co-dominant and dominant markers showed the same segregation rate. At marker S114.4 locus, the mean spikelet sterility of each genotypic class was A (Reiziq type, 34.%) \ H (heterozygote, 4.9%) \ B (Lijiangheigu type, 62.9%) in the F2 population (Fig. 2). QTL mapping using the BC2F1 population To further validate the QTL position and reduce its interval, BC1F1 plant B1-15 was selected for backcrossing to produce BC2F1 population. B1-15 was heterozygous on chromosome 1 (where the QTL was located) but was more similar to Reiziq on other chromosomes based on genotyping of the BC1F1 population. The heading date, plant height, panicle length, number of spikelets per panicle, and other agronomic characters of B1-15 were also similar to the recurrent parent Reiziq, except the spikelet sterility, which was higher than Reiziq. We first used markers S19.5 and S114.4 to screen 343 BC2F1 progeny, but only 2 recombinant plants were identified. Then, markers S15.5 and S114.4 were used to select the remaining 476 BC2F1 progeny. A total of 18 progeny exhibited recombination between these 2 markers. Another 78 randomly selected progeny (non-crossover) and the 2 selected progeny were transplanted for phenotyping and genotyping. After low temperature treatment, the spikelet sterility of BC2F1 progeny was normally distributed (Table 1). The average sterility of the BC2F1 plants (82.2%) was significantly higher than the parental varieties Reiziq (56.9%) and Lijiangheigu (13.4%). A significant difference was also observed between A and H genotypic classes in the BC2F1 population (Fig. 2). Using Composite Interval Mapping (CIM), a QTL was also mapped on chromosome 1. The QTL was in the same interval identified previously in the F2 and BC1F1 populations. The QTL (peak LOD score) was located between markers S11.9 and S114.4 with an interval of 3.5 cm (Fig. 3c). The QTL explained 29.6% of the variation in spikelet sterility caused by low temperature treatment. Discussion Cold tolerance QTL on chromosome 1 In this study, we identified a major QTL controlling low temperature induced spikelet sterility on the short arm of chromosome 1 by selective genotyping. The QTL was mapped using F2 and BC1F1 populations, and validated by using a BC2F1 population. The QTL was named as qltspkst1.1 using the nomenclature of (McCouch and CGSNL 28). The QTL qltspkst1.1 was located in a 3.5 cm interval between markers S11.9 and S This QTL explained 2.5% of the variation of spikelet sterility caused by low temperature treatment. In addition this QTL had a strong additive effect (a = 14.4) and could increase the spikelet sterility by 14% in genotypes carrying the allele from Lijiangheigu. The additive effect was greater than the dominant effect

8 298 Euphytica (21) 176: Fig. 3 Composite Interval Mapping (CIM) of spikelet sterility after low temperature treatment using a F2, b BC1F1 and c BC2F1 populations. Markers S12.7, S19.5, S114.4, CP8861 (21.8 cm) and S151.9 were used for genotyping in BC1F1 and F2 populations. Markers S15.5, S19.5, S11.9, RM216 (11.7 cm), S113.3, S114.4, RM311 (16.8 cm), CP8861 and S151.9 were used for genotyping in BC2F1 population. Genetic effect & additive effect? dominant effect (in backcross populations) (a) LOD Score (b) LOD Score Genetic distance (cm) Generation: F2 (n=282) Peak position: 13.5 cm LOD=13.6 Additive effect=14.4 Dominant effect=-7.5 R 2 =2.5% Generation: BC1F1 (n=84) Peak position: 12.5 cm LOD=5.9 Genetic effect=23.9 R 2 =29.% Genetic distance (cm) (c) 8 7 LOD Score Generation: BC2F1 (n=98) Peak position: 13.3 cm LOD=7.6 Genetic effect=18.2 R 2 =29.6% Genetic distance (cm) (d =-7.5), which will be useful in future breeding and selection. In a similar interval, a QTL for cold tolerance at booting stage (qctb-1-2) was also indentified in another rice variety Kunmingxiaobaigu (KMXBG) from Yunnan in southern China (Dai et al. 24; Xu et al. 28). Dai et al. (24) identified this QTL with peak LOD score of 1.6 and the QTL explained 37.8% of the variation in spikelet fertility in an F2 population. The results showed that the spikelet fertility of the heterozygote class was lower than that of the parental genotypic classes with a high dominant effect (-28.8) and the additive effect (5.4) of this QTL was lower than other QTL identified. Thus, it was concluded that this QTL was not important for cold tolerance. However, the same

9 Euphytica (21) 176: QTL was also identified in the following F3 and F4 populations in an interval of 15.4 cm (Dai unpublished data). Xu et al. (28) identified the same QTL (qctb-1-2) in an advanced backcross population derived from the same cross with distinct dominance effect (-18.3) which explained 14.9% of the total phenotypic variance. The heterozygote class showed higher spikelet sterility compared to the parental classes at low temperature. The interval of qctb-1-2 (about cm) was partly overlapped with that of qltspkst1.1 ( cm). In the F2 population used in the present study, for qltspkst1.1 the homozygous genotypic class of Lijiangheigu (LJHG) had greater sterility than the homozygous genotype of Reiziq, i.e. the allele from LJHG increased the spikelet sterility, and allele from RZQ decreased the spikelet sterility. It was extraordinary that the allele from KMXBG and LJHG increased the spikelet sterility at this locus, because both varieties have been considered highly coldtolerant varieties. This suggests that the expression of qltspkst1.1 may be interacting with other cold tolerance genes. It is not expressed when interacting with other cold tolerance genes exist in KMXBG and LJHG, but expressed when the cold tolerance genes do not exist, for example, in Reiziq. The QTL allele increasing the low temperature induced spikelet sterility could explain why nobody has succeeded in introducing cold tolerance from Lijiangheigu into new varieties. The failures may be partly due to the QTL identified by the present study.some other QTL have also been identified on short arm of chromosome 1, such as reverse thermo-sensitive genic male-sterile gene rtms1 (Jia et al. 21) and spikelet sterility under drought fr1.1 (Lin et al. 27). It was not clear whether qctb-1-2, rtms1 and fr1.1 are the same as or closely linked with qltspkst1.1, and whether they generally affect the spikelet sterility under abiotic stress. If the QTL in this interval controls spikelet sterility caused by various environmental stresses, it will be useful for improving the abiotic stress tolerance of new varieties in future rice breeding programs. Based on QTL mapping of F2 and BC1F1 populations, we tried to narrow the QTL interval by selecting 819 BC2F1 progeny. Although the QTL was further confirmed, we were unable to narrow the interval further. The main reason was the low frequency (4%) of crossover between markers S15.5 and S114.4 (1 cm interval). Among the 2 recombinant progeny, there were 14 crossovers occurred between markers S15.5 and S19.5 ( cm, 1.46 Mb in 4 cm), but only 6 crossovers happened within the QTL interval ( cm, 1.64 Mb in 3.5 cm). This is because the QTL interval was close to the centromere (15.7 cm), where the recombinant frequency is low (over 1 Mb/cM) (Chen et al. 22), compared to the average of 27 Kb/cM on chromosome 1 (The Rice Chromosome 1 Sequencing Consortium 23). The low frequency of recombination may also caused by linkage drag of the markers. This will make it more difficult to further fine map this region and take advantage of map-based candidate gene cloning. A solution is to continue backcrossing by using the recombinant progeny, and using marker assisted selection to enrich for recombination in the QTL interval. Cold tolerance in BC2F1 population The spikelet sterility of different genotypic classes (A \ H \ B) in the F2 population suggested that the cold tolerance (low spikelet sterility) was inherited from the susceptible parent Reiziq (A) and the allele increasing spikelet sterility was from Lijiangheigu (B). Thus, the spikelet sterility of advanced backcross populations should be higher than Reiziq when the allele of Lijiangheigu was backcrossed into the Reiziq genome. In this study, the spikelet sterility of BC2F1 population was significantly higher than that of Reiziq and Lijiangheigu. We have successfully introduced the sterile QTL qltspkst1.1 into the Reiziq background, making Reiziq more sensitive to low temperature. However, in the BC2F1 population, the spikelet sterility of the A genotypic class (75.9 ± 14.8%) was higher than that of the recurrent parent Reiziq (59.9 ± 3.9%). This suggested that qltspkst1.1 may not be the only QTL that increases spikelet sterility in the BC2F1 population; other QTL (genes) in the genome of BC2F1progeny may also increase the spikelet sterility at low temperature. It might be also because qltspkst1.1 was over expressed due to lack of interaction with other genes during the backcross. Further backcrossing combined with marker-assisted background selection will reduce this genetic noise, and increase the accuracy of fine mapping the QTL and evaluating the genetic effect of the QTL.

10 3 Euphytica (21) 176: Developing an accurate phenotyping method for evaluation of cold tolerance at booting stage The low temperature treatment system used in this study has been employed for evaluating the cold tolerance of rice varieties during the past 3 years (Ye et al. 29). It is different from the previously reported low temperature treatment methods. Although different combinations of temperature and duration have been developed for evaluation of cold tolerance in rice (Dai et al. 22; Han and Zhang 24), natural low air temperature or cool-water irrigation have been used in most QTL mapping studies for cold tolerance at booting stage (Dai et al. 24; Li et al. 1997; Liu et al. 23; Takeuchi et al. 21; Xu et al. 28). However, spikelet sterility (or fertility) is also influenced by other factors in an open field environment, such as heading time. Plants heading and flowering on different days may suffer different degrees of cold stress. Saito et al. (24) treated rice plants at the stages from the beginning of the differentiation of young panicles to the completion of heading by transferring potted plant into tanks filled with cool water maintained at 19 C. Andaya and Mackill (23) treated rice plants when auricle distance was within 2 cm by moving potted plants into a growth chamber maintained at 12 C. These methods increased the reliability of phenotyping for QTL fine mapping. To evaluate the cold tolerance of rice plants more accurately, we have developed a treatment method by moving potted plants into a cool glasshouse maintained at 21/15 C when the auricle distance was greater than cm (Ye et al. 29). The temperature regime used in this study better reflects the natural low temperature caused in rice fields than using a constant low temperature treatment. Further, the light condition was constant as plants were moved from one glasshouse to another. The results showed that the spikelet sterility from the experiments across different years was not significantly different for the parental varieties Reiziq (t =.46, P =.655, df = 8) and Lijiangheigu (t = 1.62, P =.143, df = 8). Thus, the treatment used for phenotyping in this study was repeatable. This will be useful for accurate phenotyping in fine mapping of QTL and evaluating the cold tolerance of promising breeding lines. Acknowledgments This study was supported by a postdoctoral fellowship and a travel grant from University of Queensland. The authors also appreciate the assistance from the staff and postgraduate students in Molecular Genetics Laboratory at University of Queensland and Rice Genomics & Breeding Laboratory at Seoul National University. References Abe N, Kotaka S, Toriyama K, Kobayashi M (1989) Development of the Rice Norin-PL8 with high tolerance to cool temperature at the booting stage. 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