Development of cold-tolerant breeding lines using QTL analysis in rice

Low temperature or cold stress is one of the major abiotic stresses for rice production and productivity in temperate rice-growing regions as well as in tropical highlands worldwide. Low temperature at the reproductive stage causes high sterility and decreases production of elite rice cultivars. In this study, we used F7–8 recombinant inbred lines (RILs) which had cold-tolerance genes/quantitative trait loci (QTLs) from the donor line IR66160-121-4-2-2 in the background of a cold-sensitive japonica cultivar, Geumobyeo. The selected 15 RILs possessing QTLs for cold tolerance were phenotyped for three main agronomic traits — culm length (CL), days to heading (DTH) and spikelet fertility (SF) — which were most affected during cold stress. The RILs and cold-tolerant and cold-sensitive checks were evaluated under cold-water irrigation (18–19°C) in the field and cold-air temperature (17–18°C) in the glasshouse. The RILs showed significant differences in these traits from the cold-sensitive parent. Traits CL and DTH exhibited positive correlation with SF in the selected breeding lines. The spikelet fertility of the selected breeding lines was much higher (51–81%) than that of the coldsensitive parent Geumobyeo (7%) and the selected lines possessed at least one of the three QTLs (qPSST-3, qPSST-7 and qPSST-9) associated with cold tolerance. Our results revealed that cold tolerance is associated with spikelet fertility, but independent of the genes controlling culm length and days to heading. The cold-tolerant breeding lines developed in this study will be useful to breed cold-tolerant cultivars and increase our understanding of the mechanism of cold tolerance in rice. Introduction The cultivated rice species, Oryza sativa has two subspecies — indica and japonica. Subspecies indica is widely cultivated in the hot and humid regions of Asia, Africa and Latin America, and accounts for 80% of world rice production. Subspecies japonica is cultivated in the temperate, sub-temperate and high-altitude regions of Asia, Europe, Latin America, North America and Oceania (Mackill and Lei, 1997). In the high-latitude regions of China, Japan and Korea, japonica rice is the staple food and its productivity per hectare is comparatively higher compared to indica rice. However, low temperature or cold stress at reproductive stage is the main constraint to temperate japonica rice production and affects rice cultivars by delaying vegetative growth and heading, reducing spikelet fertility, and affecting grain quality (Suh et al., 2010). Low temperature at reproductive stage has had adverse effects on the yield of rice in Australia, China and Korea since 2000 (Lee, 2001; Xu et al., 2008). Low temperature in the range of 15–19°C during the reproductive stage impairs microspore development and leads to the production of sterile pollen grains, resulting in poor grain filling and high spikelet sterility (Satake, 1976). Analysis of mutants from the cultivar Taichung 65 treated with cold water at 19°C revealed that pollen development was inhibited, reducing spikelet fertility due to malformed embryo sac (Nagasawa et al., 1994). There are limited genetic resources for the improvement of cold tolerance of temperate japonica cultivars at different growth stages. Nonetheless, genetic sources possessing cold tolerance have been identified and crossed with cold-sensitive cultivars to develop cold-tolerant varieties. Some tropical japonica cultivars such as Silewah, Lambayque1 and Padi Lobou Alumbis, have been used in temperate japonica breeding for cold tolerance (Abe et al., 1989; Saito et al., 2001). Some genetic analyses have revealed the complexity of coldtolerance loci. Nishimura and Hamamura (1993) reported dominant digenic control of cold tolerance at reproductive stage, but Nagasawa et al. (1994) reported that cold tolerance was control by four or more genes. QTL studies of cold tolerance at reproductive stage have been conducted on several mapping populations (Saito et al., 2001; Takeuchi et al., 2001; Andaya and Mackill, 2003; Liu et al., 2003; Dai et al., 2004; Xu et al., 2008; Suh et al., 2010; Ye et al., 2010). Two QTLs for cold tolerance at booting stage derived from Norin-Pl8 were mapped on chromosomes 3 and 4 (Saito et al., 1995). Fine mapping of the QTL on chromosome 4 has identified two genes (Ctb1 and Ctb2) for cold tolerance in the 56 kb region (Saito et al., 2001). Several QTLs linked to cold tolerance at reproductive stage were mapped on different chromosomes using F2, BC5F3 and doubled-haploid (DH) populations (Dai et al., 2004; Xu et al., 2008; Li et al., 1997). Andaya and Mackill (2003) mapped QTLs for cold tolerance at reproductive stage on chromosomes 1, 2, 3, 5, 6, 7, 9 and 12 using recombinant inbred lines (RIL). Liu et al. (2003) identified QTLs for cold tolerance from wild rice introgression lines on chromosomes 1, 6 and 7. Suh et al. (2010) identified three QTLs linked to cold tolerance for seed set using an RIL population derived from a temperate-japonica × tropical-japonica cross. However, only two QTL * Corresponding author (email: [email protected]). Theme 1: Rice genetic diversity and improvement Jena et al.: Development of cold-tolerant lines using QTL analysis 1.7.2 Second Africa Rice Congress, Bamako, Mali, 22–26 March 2010: Innovation and Partnerships to Realize Africa’s Rice Potential (qCTB8; qLTSPKST10.1) were identified for cold tolerance on chromosomes 8 and 10 by using two different F2 mapping populations (Kuroki et al., 2007; Ye et al., 2010). The genetics and mechanism of cold tolerance reported by different research groups are not well understood, due to errors in phenotyping method of cold tolerance and lack of effective QTLs linked to the expression of genes at reproductive stage under cold stress. Several agronomic traits are affected during the growth stages of rice plants that eventually produce high sterility. Suh et al. (2010) have developed a reliable method of phenotyping for cold tolerance by imposing cold-water irrigation on all growth stages in the field and cold-air temperature in the glasshouse, which allowed correct measurement of the traits associated with cold tolerance. The objective of this study was to evaluate the effect of cold stress on culm length, days to heading, and spikelet fertility under field and glasshouse conditions on an F7–8 RIL population derived from a temperatejaponica × tropical-japonica cross. Here we report the agronomic performance of selected cold-tolerant breeding lines under two cold-treatment conditions, detection of correlation among the traits associated with cold tolerance, and the development of some cold-tolerant breeding lines carrying identified QTLs on chromosomes 3, 7 and 9. Materials and methods Plant materials The breeding line IR66160-121-4-4-2 was used as the donor parent for cold tolerance and cold-sensitive temperate japonica cultivar Geumobyeo was used as the recipient parent. An RIL population (F7 and F8) consisting of 153 plants was produced by single-seed descent (SSD). These RILs were used to construct a molecular genetic map and identify QTLs controlling cold tolerance. Fifteen RILs possessing QTLs for cold tolerance were developed (Fig. 1) and used for cold-tolerance analysis (Table 1). Korean japonica cultivars Jinbubyeo, Odaebyeo and Junganbyeo were used as cold-tolerant checks and Saetbyeolbyeo was used as coldsensitive check. Seeds of IR66160-121-4-4-2 were obtained from the Genetic Resources Center of IRRI, Los Baños, Philippines, and seeds of Geumobyeo, Saetbyeolbyeo, Jinbubyeo, Odaebyeo and Junganbyeo were obtained from the Rice Research Division of the National Institute of Crop Science (NICS), Rural Development Administration (RDA), Suwon, Republic of Korea. Evaluation of cold tolerance Cold-tolerance screening in cold-water irrigation plot and trait measurement Cold-tolerance evaluation of genotypes followed the method described by Suh et al. 2010. The 15 selected RILs along with the parental genotypes were planted in a cold-water irrigation plot for phenotypic evaluation during the summers of 2007 and 2008 at Chuncheon substation of NICS. Thirty-day-old seedlings were transplanted into ambient-water and cold-water irrigation plots, with 25 plants in a single row with 15-cm spacing between plants and 30 cm between rows. The field planting followed a completely randomized block design (CRBD) with two replications. Irrigation-water temperature was ambient until the tillering stage (20 days after transplanting). Cold-water at 17°C was used for irrigation to a depth of 5 cm during the entire period of rice growth from tillering to grain maturity following standard agronomic practices (NICS, 2004). Water temperature showed a gradient from 17°C at the inlet to about 21°C at the outlet in a 6-m-long screening plot. The temperature zone of 17–18°C severely affected the development of agronomic traits in most of the RILs. Therefore, we considered 18–19°C water temperature as the critical temperature zone to measure genotypes as cold tolerant or cold sensitive. Phenotypic data on culm length (CL), days to heading (DTH) and spikelet fertility (SF) were collected from five plants of each genotype at the critical temperature zone in cold-water and ambient-water irrigation plots. Culm length was measured from the soil surface to the panicle base of the main culm. Days to heading was evaluated as the number of days from seeding until 50% of the panicles had emerged. Spikelet fertility was calculated as the average percentage of fertile spikelets per panicle by counting the first three panicles of three plants of each line. Data on CL, DTH and F in normal-water irrigated plots and in cold-water treated plots were measured as the indices for cold tolerance and sensitivity at the reproductive stage, and calculated as the average CL, DTH and F in cold-water irrigated plots and ambient-water irrigated plots a. Data for CL, DTH and F were transformed to measure the effect of the cold-water treatment as follows: reductions of CL and F were calculated as the ratio of CL and seed set, respectively, in the cold-water irrigated plot to the ambient-water irrigated plot, and converted into a percentage. Heading delay (HD) was the difference in days to heading between the cold-water treated plot and the ambient-water treated plot. Theme 1: Rice genetic diversity and improvement Jena et al.: Development of cold-tolerant lines using QTL analysis Second Africa Rice Congress, Bamako, Mali, 22–26 March 2010: Innovation and Partnerships to Realize Africa’s Rice Potential 1.7.3 Figure 1. Scheme for the development of cold-tolerant breeding lines. † RIL = recombinant inbred line. Table 1. List of cultivars, breeding lines, and selected recombinant inbred lines used in the study Variety / Breeding line Selected generation Trait Geumobyeo Recipient Cold sensitive IR66160-121-4-4-2 Donor Cold tolerance Jinbubyeo Check variety Cold tolerance Odaebyeo Check variety Cold tolerance Junganbyeo Check variety Cold tolerance Satbyeolbyeo Check variety Cold sensitive IR83222-F8-11 F7–8 Cold tolerance IR83222-F8-14 F7–8 Cold tolerance IR83222-F8-18 F7–8 Cold tolerance IR83222-F8-54 F7–8 Cold tolerance IR83222-F8-66 F7–8 Cold tolerance IR83222-F8-85 F7–8 Cold tolerance IR83222-F8-134 F7–8 Cold tolerance IR83222-F8-155 F7–8 Cold tolerance IR83222-F8-156 F7–8 Cold tolerance IR83222-F8-167 F7–8 Cold tolerance IR83222-F8-173 F7–8 Cold tolerance IR83222-F8-174 F7–8 Cold tolerance IR83222-F8-201 F7–8 Cold tolerance IR83222-F8-204 F7–8 Cold tolerance IR83222-F8-206 F7–8 Cold tolerance Cold-tolerance screening in greenhouse with controlled air and water temperatures A subset of 10 cold-tolerant RILs was evaluated for cold tolerance at the booting stage in a controlledenvironment greenhouse maintained at 17°C air/water temperature under natural light following the procedure of Suh et al. (2010). Five rice seedlings each of the 10 RILs and the two parents were transplanted into a plastic pot containing pulverized dry soil with commercial fertilizer (9–4.5–5.7, N–P2O5–K2O). Extra tillers were removed from each plant in the pot, leaving a main stem per plant to avoid overcrowding and to promote better growth. The first three tillers were tagged and the plants were moved to the environment-controlled greenhouse Geumobyeo × IR66160-121-4-42 Cold sensitive Cold tolerant