development of cold-tolerant breeding lines - africa rice center

Second Africa Rice Congress, Bamako, Mali, 22–26 March 2010: Innovation and Partnerships to Realize Africa’s Rice Potential 1.7.1

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

K.K. Jena,1* S.M. Kim,1 J.P. Suh2 and Y.G. Kim2 1 Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila,

Philippines, c/o IRRI-Korea Office, National Institute of Crop Science, RDA, Suwon 441-857, Republic of Korea; 2 Rice Research Division, National Institute of Crop Science, Rural development Administration, Suwon 441-857, Republic of Korea.

Abstract 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 cold-sensitive 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 cold-tolerance 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 temperate-japonica × 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 cold-sensitive 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.



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 controlled-environment 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 × IR66160-121-4-4-2 Cold sensitive Cold tolerant

F1 (Selfing)

F2

F7 (RIL† Selfing)

F8 (RIL Selfing)

153 progenies (Selfing)

Cold-water irrigation stress phenotyping

Single-seed descent

Selection of cold-tolerant breeding lines



maintained at 17°C when the auricle of the flag leaf was approximately 4 cm inside the penultimate leaf of the tillers. After 10 days of cold treatment, the plants were taken back to the normal greenhouse and grown until maturity. The tagged tillers were harvested at maturity and the average number of fertile spikelets (as a percentage) was measured. QTL and statistical analysis QTL analysis of the 15 selected breeding lines and the parents was done following the method of Suh et al. (2010). Genomic DNA was extracted from freshly frozen leaves at seedling stage following the modified CTAB method of Murray and Thompson (1980). Simple sequence repeat (SSR) markers (RM569, RM231, RM3767, RM1377, RM24427 and RM24545) associated with identified QTLs on chromosomes 3, 7 and 9 were used to survey the presence or absence of the QTLs (Table 2). Polymerase chain reaction (PCR) was performed in a 10 µL reaction mixture containing 1 µL of 10× PCR buffer, 0.5 µL of each primer (10 µM), 1 µL of dNTP (2 mM each), 0.4 µL of MgCl2 (50 mM), and 0.2 unit of Taq polymerase (Solgent Co. Ltd., Korea). The PCR conditions and PCR product detection were as described by Suh et al. (2010).

Table 2. Informative QTLs and linked markers used to detect cold-tolerant phenotypes QTL† Chr Marker Band size (bp) Sequence‡ qPSST-3 3 RM231 186 F: 5'-CCAGATTATTTCCTGAGGTC-3' R: 5'-CACTTGCATAGTTCTGCATTG-3' qPSST-7 7 RM1377 145 F: 5'-ATTAGATACATCAGCGGGGG-3' R: 5'-GCTGCTGTACGATGTGATCC-3' qPSST-9 9 RM24545 152 F: 5'-ACAGCACAGCACCCGGAAGG-3' R: 5'-CGAGCAACAGGAAGGCGATAAGC-3' † qPSST QTLs for percent seed set in cold-water treated plot. ‡ F, forward; R, reverse.

The difference between the means of traits under control and cold-water treated conditions was analyzed at the significance level of P < 0.05 using the t-test. Percent reduction of individual traits under control and cold-water treated conditions was also calculated. The graphs were plotted with calculated means of the traits to determine the degree of cold tolerance among the breeding lines using worksheet program (Excel 2008, Microsoft). Correlation coefficient analysis between the traits was performed using the procedure of SAS version 9.2 (SAS Institute, 2000). Results and discussion QTL validation In a previous study, we identified three QTLs for cold tolerance at the reproductive stage which were located on the short arm of chromosome 3, the short arm of chromosome 7, and the long arm of chromosome 9 in the marker intervals RM569–RM231, RM3767–RM1377, and RM24427–RM24545, respectively (Suh et al., 2010). Of the 153 RILs evaluated under cold-water irrigation stress, we selected 15 RILs on the basis of their high spikelet fertility. QTL analysis of these RILs showed the presence of at least one of the three QTLs (qPSST-3, qPSST-7, qPSST-9) associated with cold tolerance (Table 3). The RILs were identical in cold tolerance to the cold-tolerant donor, IR66160-121-4-2-2, suggesting the efficiency of QTLs for selection of cold-tolerant genotypes at reproductive stage. A number of cold-tolerance QTLs have been reported, with QTLs located on all chromosomes of rice for reproductive-stage cold tolerance (Saito et al., 2001; Takeuchi et al., 2001; Andaya and Mackill, 2003; Dai et al., 2004; Kuroki et al., 2007; Xu et al., 2008; Ye et al., 2010). Most of the QTLs could not be validated due to repeatability problems with the screening methods and the complexities of trait expression under cold stress. However, the QTLs identified in our previous study for high spikelet fertility were validated in the selected RILs (Suh et al., 2010). This validation of QTLs is helpful for the selection of cold-tolerant breeding lines and development of improved cold-tolerant breeding materials. Cold tolerance phenotyping and performance of agronomic traits in the field Reproductive stage is critical for spikelet fertility and ultimately yield of rice cultivars. However, during abiotic stress conditions like drought, salinity and low temperature, the major plant traits are affected and eventually the spikelets become infertile. Lack of perfect phenotyping for cold tolerance at reproductive stage makes it difficult to correctly identify cold-tolerant genotypes. In this study, we applied cold stress to selected breeding lines by cold-water irrigation in the field during the growth stages until the end of grain filling and cold-air temperature at panicle-initiation stage in the greenhouse.



Table 3. Performance of agronomic traits of cold-tolerant lines under cold-stress conditions Selected line Linked QTLs CL (cm) DTH (days) SF (%)

Control Treat Reduction (%)

Control Treat HD Control Treat Reduction (%)

Geumobyeo – 69.08 51.90** 17.18 95 110** 15 91.7 7.0** 84.7 IR66160-121-4-4-2

qPSST-3, -7, -9 76.94 51.06** 25.88 110 126** 16 96.6 73.0* 23.6

IR83222-F8-11 qPSST-3, -7, -9 91.20 66.66** 24.54 109 126** 17 90.7 55.0** 35.7 IR83222-F8-14 qPSST-7, -9 90.32 77.94 ns 12.38 115 135** 20 95.4 74.2** 21.2 IR83222-F8-18 qPSST-3, -7, -9 78.94 55.88* 23.06 107 123** 16 94.7 73.2* 21.5 IR83222-F8-54 qPSST-3, -9 71.56 48.34** 23.22 101 121** 20 91.6 55.2** 36.4 IR83222-F8-66 qPSST-3, -7, -9 97.30 72.56* 24.74 106 116* 10 86.8 57.8** 29 IR83222-F8-85 qPSST-3 89.70 64.32** 25.38 100 119** 19 89.5 50.6** 38.9 IR83222-F8-136 qPSST-7, -9 77.58 56.02** 21.56 110 127* 17 84.8 80.6 ns 4.2 IR83222-F8-155 qPSST-3, -7, -9 82.08 62.84** 19.24 103 120** 17 88.3 59.8** 28.5 IR83222-F8-156 qPSST-3 86.22 71.94* 14.28 99 117** 18 90.1 79.8 ns 10.3 IR83222-F8-167 qPSST-3, -9 79.50 50.70** 28.8 102 118* 16 89.2 74.8 ns 14.4 IR83222-F8-173 qPSST-3, -7, -9 94.68 64.50** 30.18 111 125** 14 77.8 70.2 ns 7.6 IR83222-F8-174 qPSST-7, -9 79.18 64.66ns 14.52 106 119* 13 93.9 66.6* 27.3 IR83222-F8-201 qPSST-3, -7, -9 74.78 52.86** 21.92 109 125** 16 94.9 65.1** 29.8 IR83222-F8-204 qPSST-3, -7, -9 69.44 48.12** 21.32 107 122** 15 90.2 71.7 ns 18.5 IR83222-F8-206 qPSST-3, -7, -9 82.44 64.26 ns 18.18 106 121** 15 89.4 60.5* 28.9 CL, culm length; DTH, days to heading; SF, Spikelet fertility; Treat, cold treatment condition; HD, heading delay. *, ** significance: P < 0.05 and P < 0.01, respectively; ns, no significance.



Our phenotyping for cold tolerance was conducted for two consecutive seasons (2007 and 2008) and the breeding lines showed significant differences in three main agronomic traits compared with the cold-sensitive parent, Geumobyeo (Table 3). Spikelet fertility of the selected breeding lines under cold-water treated plot was in the range of 51–81%, in contrast to very low spikelet fertility of the cold-sensitive parent Geumobyeo (Table 3). The percentage reduction in fertility and culm length, and the delay in heading showed significant differences from the lines under normal-water treatment (Table 3). In our previous study, we identified three QTLs and validated those QTLs to correctly identify cold-tolerant breeding lines (Suh et al., 2010). This study provided correct, reproducible phenotyping and the use of QTLs for identification of cold-tolerant breeding lines. Fine mapping of these QTLs would provide clues to the mechanism of cold tolerance in reproductive stage. Performance of selected lines in two cold-treated conditions for the three traits In this study, we compared the overall cold tolerance of the 153 RILs evaluated under cold stress and classified them into five phenotypic classes. Our results showed 31% RILs were ‘tolerant’, 35% ‘moderately tolerant’ and 34% ‘sensitive’ to cold-water stress (Fig. 2). Correlations among the traits under normal- and cold-water treatment conditions were calculated and the significant correlation coefficients (P < 0.05, P < 0.01) are presented (Table 4). Under control conditions, significant correlation occurred between CL and DTH. In the cold-water treatment, significant positive correlations occurred between SF and CL, and SF and DTH. Significant positive correlation between fertility and culm length in cold-water treated plot was consistent with previous studies (Satake et al., 1988). We observed that the cold-tolerant RILs exhibited higher spikelet fertility in spite of reduction in CL and heading delay compared to the normal-water treated plot. This suggests that the genes controlling cold tolerance are independent of the genes controlling other agronomic traits.

Figure 2. Agronomic performance of 153 RILs (F7) derived from the cross Geumobyeo × IR66160-121-4-4-2 in cold-water treated plot (19°C) Vsen = very sensitive, Sen = sensitive, Nor = moderately tolerant, Tol = tolerant, Vtol = very tolerant. In this study, we selected 10 cold-tolerant lines possessing the three QTLs that showed spikelet fertility in the range of 51–81% compared to 7% in the cold-sensitive parent (Geumobyeo) and 73% in the tolerant donor, IR66160-121-4-2-2 (Fig. 3). The gain of higher spikelet fertility in some of the breeding lines may be attributed to additive effects of QTL alleles inherited from the donor parent (Fig. 4). Comparison of three agronomic traits (CL, DTH and F) of the selected 10 cold-tolerant RILs showed significant differences from the cold-sensitive parent. Our results indicate that the cold-tolerance characteristic of the traits in the selected breeding lines are attributable to the marker-assisted selection efficacy of cold-tolerant genotype.

0

10

20

30

40

50

60

Vsen Sen Nor Tol Vtol

IR66160-121-4-4-2

Geumo

No.

line

s

22

30

54

36

11



Table 4. Correlation coefficients among six traits of the RILs under two treatment conditions Trait FN FT CLN CLT DTHN

FT 0.122

CLN –0.122 0.292**

CLT –0.094 0.251** 0.831**

DTHN 0.002 0.666** 0.500** 0.307**

DTHT 0.089 0.644** 0.410** 0.213** 0.932** FN, % of spikelet fertility in normal conditions; FT, % of spikelet fertility in cold-treatment conditions; DTHN, days to heading in normal conditions; DTHT, days to heading in cold-treatment conditions; CLN, culm length in normal conditions; CLT, culm length in cold-treatment conditions. ** significance: P < 0.01.

Figure 3. Percent seed set of selected RILs with cold tolerance under different cold stress conditions The phenotyping method used in this study by using cold water in the field and cold air in the greenhouse has increased the reliability of identifying cold-tolerant genotypes compared to other studies (Saito et al., 2001; Xu et al., 2008). The QTLs identified by using these methods of phenotyping could help in the isolation of cold-tolerant breeding lines by marker-assisted selection (MAS). These QTLs might be effective in large-scale and precise screening of genotypes for cold tolerance by MAS (Jena and Mackill, 2008). The breeding lines isolated in this study have inherited the QTLs for cold tolerance from a new plant type line, IR66160-121-4-2-2 that possesses cold-tolerance genes from an Indonesian tropical japonica cultivar, Jimbrug and a Chinese japonica cultivar Shen-Nung89-386 (Suh et al., 2010). We are developing near-isogenic lines (NILs) possessing the QTLs for cold tolerance, which should be useful genetic materials for cold-tolerance breeding in the future. Conclusions Reproductive-stage cold stress severely affects spikelet fertility of temperate rice cultivars, reduces culm length and delays heading. These traits are highly correlated and are required for the development of cold-tolerant rice cultivars for temperate as well as high-altitude regions around the world. The precise screening method developed in this study by subjecting RIL genotypes to cold-water irrigation stress in the field and cold-air temperature stress in the greenhouse was comparable with the performance of the traits under nonstress conditions. The breeding materials developed in this study by using QTL information associated with high spikelet fertility are valuable and these lines could be crossed with cold-insensitive elite cultivars to develop promising recombinants for cultivation in temperate regions.

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120

Control Cold treatment controlled green house Cold water plot

Seed

set

(%)



Figure 4. Selected cold-tolerant (1–4) and cold-sensitive (5–6) breeding lines, and cold-sensitive parent, Geumobyeo (7) in a cold-water (18–19oC) treated plot. Note highly fertile lines in lanes 1–4 and highly sterile lines in lanes 5–6, expressed under cold stress. Acknowledgments We thank Rural Development Administration for providing financial support for this study. References Abe N, Kotaka S, Toriyama K and Kobayashi M. 1989. Development of the ‘rice Norin-PL8’ with high

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development of cold-tolerant breeding lines - africa rice center

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