Introduction

Low temperature at the booting stage of rice causes pollen sterility and results in yield loss, severe damage of which was experienced in 1993 in Japan. The developmental stage of rice most sensitive to low temperature has been revealed to be the early microspore stage just after the tetrad stage (Satake and Hayase 1970). Abnormality of sugar metabolism in anthers has been reported to be responsible for inhibition of pollen development and a decrease of fertile pollen grains, which result in inability of anther dehiscence (Nishiyama 1984).

Rice cultivar ‘Hitomebore’ has been developed as a cultivar highly tolerant to low temperature at the booting stage (Sasaki et al. 1994) and is the second most widely grown rice cultivar following ‘Koshihikari’ in Japan. The low-temperature damage in 1993 in the Tohoku region of Japan caused replacement of the major cultivar ‘Sasanishiki’, which is relatively sensitive to low temperature, by ‘Hitomebore’. However, even ‘Hitomebore’ exhibited sterility in some areas in 1993 (Sasaki et al. 1994). Further improvement of cold tolerance of rice is thus required. In previous rice breeding efforts for further improvement of cold tolerance using genetic resources as parents, inferior traits such as long culm, late maturity, low yield accompanying cold tolerance have made it difficult to develop a commercial cultivar having high yield and good quality. Development of DNA markers linked closely with cold tolerance genes is considered to be effective for breaking linkage drag to remove undesirable alleles derived from cold-tolerant parents.

Many quantitative trait loci (QTLs) for cold tolerance at the booting stage have been reported, i.e., Ctb1, Ctb2 (Saito et al. 1995, 2001, 2010), qCT-1, 7, 11 (Takeuchi et al. 2001), qCTB1, 2a, 2b, 3, 5, 6, 7, 9, 12 (Andaya and Mackill 2003), qRCT3, 6a, 6b, 7 (Dai et al. 2004), qCTB1-1, 1-2, 8 (Kuroki et al. 2007, 2011), qCTB-1-1, 4-1, 5-2, 10-2, 11-1 (Xu et al. 2008), qPSST-3, 7, 9 (Suh et al. 2010), qLTSPKST10.1 (Ye et al. 2010), qCTB3-Silewah (Mori et al. 2011), and qLTB3 (Shirasawa et al. 2012). However, it is difficult to compare the effects of these reported QTLs, since the methods for evaluation of cold tolerance and tested cultivars are different between these studies. Mori et al. (2011) have detected a QTL for cold tolerance of ‘Silewah’ different from that of ‘Silewah’ detected by Saito et al. (1995), suggesting that effects of QTLs depend on environmental conditions for cold tolerance evaluation. Temperature, day length, and parental lines used for production of segregating populations for genetic studies may have an influence on the effect of QTLs. However, a study comparing the effects of several different cold tolerance QTLs in rice has not been reported.

There are few examples of introduction of the cold tolerance QTLs into major cultivars in rice breeding (Saito et al. 2001; Zhou et al. 2012). This is probably due to small effect of each QTL. To develop a highly cold-tolerant line, QTL pyramiding may be required. Successful QTL pyramiding in rice has been reported for improvement of grain size (Wang et al. 2012a) and yield potential (Wang et al. 2012b). Kuroki et al. (2011) have reported pyramiding effects of QTLs for cold tolerance at booting stage, i.e., qCTB8.1, qCTB1.1, and qCTB1.2, but seed fertilities of all pyramiding lines were not significantly higher than those of the lines having a single QTL. Comparing seed fertilities of different genotypes of three QTLs, Shinada et al. (2014) suggested pyramiding effect of QTLs for cold tolerance at fertilization stage in rice.

In our previous investigations, pollen dispersal from flowers of ‘Kuchum’, a cultivar in Bhutan, was observed under low temperature conditions, suggesting that this cultivar has high cold tolerance. Cold tolerance of ‘Kuchum’ cannot be examined by the cold-deep-water irrigation method (Matsunaga 2005), which is commonly used for cold tolerance evaluation of breeding lines by rice breeders, because it has a long culm length, ca. 150 cm, and a late flowering trait. Although high cold-tolerant lines, e.g., ‘Tohoku 196’, have been developed from progenies of ‘Kuchum’, development of an elite line usable as a commercial cultivar from progeny of ‘Kuchum’ has been unsuccessful because of unfavorable traits of ‘Kuchum’, i.e., long culm, late flowering, red-colored grains, and low yield. In the present study, QTLs for cold tolerance in ‘Kuchum’ were analyzed, and a major QTL was detected on chromosome 4. A chromosomal region containing this QTL, named qCT-4 (QTL for Cold Tolerance at booting stage on chromosome 4), was delimited by developing near isogenic lines (NILs) of ‘Hitomebore’ with a chromosomal segment from ‘Kuchum’. Furthermore, the effect of combining a ‘Kuchum’ allele at qCT-4 with a cold tolerance qLTB3 allele of ‘Lijiangxintuanheigu’ identified in our previous study (Shirasawa et al. 2012) was investigated. Effects of other QTLs, i.e., Ctb1 and Ctb2 derived from ‘Silewah’ and qCTB8 from ‘Hokkai PL9’, under a genetic background of ‘Hitomebore’ were also examined.

Materials and methods

Plant materials

An F1 hybrid between ‘Kuchum’, a gene source of cold tolerance, and ‘Chiyohonami’, which has relatively high cold tolerance, was backcrossed once with ‘Chiyohonami’, and selfing was repeated. A BC1F6 plant named ‘BI6-12’ was crossed with ‘Chiyohonami’ for production of an F2 population of 96 plants named CKC1 (BC2F2 between ‘Kuchum’ and ‘Chiyohonami’), which were used for QTL analysis of cold tolerance. Two populations of BC6F4 plants named EBS6 with 78 plants and EBS8 with 187 plants obtained by six backcrossings of ‘Hitomebore’ with a hybrid between ‘Kuchum’ and ‘Hitomebore’ were also used for QTL analysis. Pedigrees of these plant materials are shown in Supplementary Fig. 1. A breeding line having cold tolerance genotype in qCT-4, named ‘Tou 1380’, and one having cold tolerance genotype in qLTB3, named ‘Ouu 415’, were also used for QTL pyramiding. ‘Ouu 415’ has been developed by repeated backcrossing of a hybrid between ‘Hitomebore’ and ‘Lijiangxintuanheigu’ to ‘Hitomebore’.

Fig. 1
figure 1

Graphical genotyping of ‘BI6-12’ having chromosomal segments of ‘Kuchum’ with a ‘Chiyohonami’ genetic background. Stars without marker names indicate SSR markers showing no polymorphism between ‘Kuchum’ and ‘Chiyohonami’

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Evaluation of cold tolerance of plants

Populations for QTL analysis and developed NILs were grown in a paddy field equipped for cold tolerance evaluation (Miyagi Prefectural Furukawa Agricultural Experiment Station, Osaki, Miyagi, Japan) and were treated by the cold-deep-water irrigation method (Matsunaga 2005). In this paper, “cold tolerance” means only cold tolerance at booting stage. Seeds were sown in the middle of April and seedlings were planted with 41.6 plants/m2 (24 × 10 cm) in the field with basal fertilizer of 40 kg N/ha for cold tolerance evaluation in the middle of May. Cold-water treatment was carried out from the beginning of June to the beginning of September. The water temperature was controlled at 18.5 °C (at 19.0 °C only for CKC1) and the water depth was 15 or 25 cm depending on plant heights. Seed fertility, which is the percentage of the number of fertile seeds in the number of florets, of 15 panicles from three plants was investigated. Three blocks were set for each line every year for 6 years. Dunnett’s test or Tukey–Kramer’s test was performed for multiple comparison, and the difference between two lines was analyzed by the Student’s t test.

Genotyping of plants and QTL analyses

According to Thomson and Henry (1995), genomic DNA was extracted from fresh leaves by TPS solution (100 mM Tris–HCl pH 8.0, 10 mM ethylenediaminetetraacetic acid (EDTA), 1 M KCl), precipitated with isopropanol, washed with cold ethanol, and dissolved in 1/10 TE solution (1 mM Tris–HCl pH 8.0, 0.1 mM EDTA). Using the extracted genomic DNA as a template, DNA fragments were amplified by polymerase chain reaction (PCR) under the thermal cycling condition of 7 min denaturation at 94 °C, followed by 35 cycles of 1 min denaturation at 94 °C, 1 min annealing at 55 °C, and 2 min extension at 72 °C, and a final 10 min extension at 72 °C. PCR products were separated by electrophoresis on 3 % (w/v) agarose (Sigma, Type I-A) gel and detected by staining with ethidium bromide.

DNA polymorphism between ‘Kuchum’ and ‘Chiyohonami’ was analyzed using 125 SSR markers, and graphical genotyping of ‘BI6-12’ and QTL analysis using CKC1 were carried out with 87 markers, which showed polymorphism between ‘Kuchum’ and ‘Chiyohonami’. Confirmation and delimitation of a detected QTL were performed using EBS6 and EBS8 with three markers, i.e., RM5687-2, A20I0241-4, and S38P21-1, and seven markers, i.e., RM2811, RM3658, RM7472, S72D08-2, S96E05-1, RM5687-2, and A20I0241-4 (Supplementary Table 1), respectively. A linkage map was constructed using Mapmaker/Exp Ver3.0 (Lincoln et al. 1992), and QTL was analyzed with the composite interval mapping method using Windows QTL Cartographer ver2.5 (Wang et al. 2006). The LOD threshold for QTL significance was determined by a permutation test (1000 replications) with a significance level P = 0.05.

Development of NILs of ‘Hitomebore’ with QTL regions of ‘Kuchum’, ‘Silewah’, and ‘Hokkai PL9’

From the BC6F4 population between ‘Kuchum’ and ‘Hitomebore’, 20 lines of seven types named C1 to C7 having different lengths of a chromosome segment around a QTL, i.e., qCT-4, of ‘Kuchum’ were developed. Simultaneously, 14 lines of four types that are segregants having alleles of ‘Hitomebore’ type of qCT-4 derived from the same parental plants as those of C1, C4, C5, and C7, which were named C1(-), C4(-), C5(-), and C7(-), respectively, were also developed for evaluation of the effect of qCT-4 on cold tolerance. Three lines of BC6F4, BC6F6, and BC6F7 were used for evaluation of other traits important for rice production. Among them, ‘Tou 1380’ and ‘Tou 1381’ belonging to group C1 have ‘Kuchum’ genotype in the region between RM2811 and S38P21-1 and ‘Tou 1489’ has ‘Kuchum’ genotype in the region between 9_1 and S38P21-1. ‘Tou 1380’ was used as a parent for QTL pyramiding.

An F1 hybrid between ‘Silewah’, a cold-tolerant cultivar in Indonesia, and ‘Hitomebore’ was backcrossed four times with ‘Hitomebore’, and 16 NILs of six types named S1 to S6 having ‘Silewah’ alleles at Ctb1, Ctb2, or both were selected (Supplementary Fig. 2). Three lines of S3 having ‘Hitomebore’ alleles in the Ctb1–Ctb2 region derived from the same parental plants, which was named S3(-), were also developed. Eight NILs of two types named H1 and H2 having a ‘Hokkai PL9’ allele at qCTB8 with a ‘Hitomebore’ background were developed by four backcrossings of ‘Hitomebore’ to an F1 hybrid with ‘Hokkai PL9’. Three lines named H2(-) that have lost the ‘Hokkai PL9’ allele at qCTB8 by segregation were also selected.

QTL pyramiding

For combining the cold tolerance qCT-4 allele and the cold tolerance qLTB3 allele, ‘Tou 1380’ and ‘Ouu 415’ were crossed and F2 plants were obtained. Using DNA markers Indel_2, RM5687-2, and S38P21-1 for qCT-4 and RM6970, RM7000, and RM7389 for qLTB3, 18 F2 plants of four genotypes homozygous for ‘Kuchum’, ‘Lijiangxintuanheigu’, and ‘Hitomebore’ alleles at qCT-4 and qLTB3 were selected. There were six lines of group L1 with the ‘Hitomebore’ alleles at both qCT-4 and qLTB3 and four lines of group L2 with the ‘Kuchum’ allele at qCT-4 and the ‘Hitomebore’ allele at qLTB3. There were three lines of group L3 with the ‘Hitomebore’ allele at qCT-4 and the ‘Lijiangxintuanheigu’ allele at qLTB3, and five lines of group L4 with the ‘Kuchum’ allele at qCT-4 and the ‘Lijiangxintuanheigu’ allele at qLTB3.

Investigation of agronomical traits

In a rice paddy field at the Miyagi Prefectural Furukawa Agricultural Experiment Station (Osaki, Miyagi, Japan), heading date, maturity date, culm length, panicle length, number of panicles per plant, grain yield, grain weight of 1000 seeds, grain quality, and frequency of lodging plants of the developed lines were investigated. The grain quality was rated from 1 (excellent) to 5 (poor) based on percentage of normal semi-transparent grains, and the frequency of lodging plants was rated from 0 (no lodging) to 4 (severe lodging) according to angle from upright. Resistance to leaf blast was evaluated by the upland nursery test with natural infection boosted by spreading infected leaves. Resistance was rated twice from 0 (no symptom) to 10 (dying of the whole plant) according to Asaga (1981) at the end of June and the beginning of August.

Results

QTL analysis of cold tolerance of ‘Kuchum’

DNA polymorphism between ‘BI6-12’ and ‘Chiyohonami’ was detected in 17 SSR markers among the 87 markers showing polymosphism between ‘Kuchum’ and ‘Chiyohonami’, and graphical genotyping indicated that ‘BI6-12’ has segments of chromosomes 1, 4, 5, 6, 7, 8, 9, and 11 from ‘Kuchum’ (Fig. 1). Frequency distribution of seed fertility of cold-water treated plants in CKC1, which is F2 between ‘BI6-12’ and ‘Chiyohonami’, is shown in Supplementary Fig. 3. QTL analysis of seed fertility of CKC1 detected significant LOD scores at loci of RM8146 on chromosome 1 and S72D21-1 on chromosome 4. In the QTL of S72D21-1 on chromosome 4, ‘Kuchum’ alleles showed a beneficial effect of increasing seed fertility under cold treatment, i.e., cold tolerance. The additive effect, dominance effect, and explained variance of this QTL were 37.0, 9.87, and 35.2 %, respectively (Table 1). Although LOD score and explained variance of the QTL near RM8146 on chromosome 1 were larger than those of the QTL near S72D21-1 on chromosome 4, the QTL on chromosome 4 was further investigated because the QTL on chromosome 1 had negative additive effect.

Table 1 QTL analysis of cold tolerance of‘Kuchum’
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The QTL on chromosome 4 was again analyzed using two BC6F4 populations, EBS6 and EBS8 (Supplementary Fig. 1), developed by backcrossing of ‘Kuchum’ with a recurrent parent ‘Hitomebore’ and marker-assisted selection using S72D21-1. EBS6 and EBS8 were derived from two BC6F1 plants, and independently developed by selfing, possibly having different lengths of a QTL region and genetic background. Frequency distribution of seed fertility in these backcross populations are shown in Supplementary Fig. 3. Analysis using EBS6 detected a significant LOD peak at RM5687-2 on chromosome 4, which has an additive effect of 6.51, a dominance effect of 3.72, and an explained variance of 28.1 % (Table 1). In EBS8, significant LOD peaks were detected at loci of RM3658 and S96E05-1 on chromosome 4 having additive effects of 5.42 and 4.99 and explained variances of 15.1 and 8.0 %, respectively. No QTLs for culm length and heading date were detected in EBS6 and EBS8, indicating that the detected QTL for cold tolerance is not due to high plant height, which sometimes causes inadequate cold-water treatment of developing flowers, nor to difference of the time most sensitive to low temperature.

In these QTL analyses using backcross progenies with recurrent parents ‘Chiyohonami’ and ‘Hitomebore’, a significant QTL having a high additive effect was detected in a region of ca. 14.3 Mb from RM3658 to S72D21-1. This QTL was named qCT-4 (QTL for Cold Tolerance at booting stage on chromosome 4). Replacement of homozygous ‘Hitomebore’ alleles at this QTL with homozygous ‘Kuchum’ alleles is expected to contribute to an increase in seed fertility of ca. 10 % under cold treatment.

Cold tolerance of NILs having a ‘Kuchum’ allele at qCT-4 with a ‘Hitomebore’ genetic background

Twenty NILs having a QTL region of qCT-4 from ‘Kuchum’ with a ‘Hitomebore’ genetic background were classified into seven groups according to the positions of recombination break points (Fig. 2; Table 2). Group C1 lines having ‘Kuchum’ alleles in a region from RM2811 to S38P21-1, which was developed first, showed significantly higher seed fertilities under cold stress than those of ‘Hitomebore’ and lines of C1(-) having ‘Hitomebore’ alleles in this QTL region, which was obtained by segregation in a population of group C1, in repeated cold tolerance evaluations for 5 years, indicating that qCT-4 has a significant effect on cold tolerance. Groups C2 lines having ‘Kuchum’ alleles in a region from RM2811 to S72D08-2 showed lower seed fertilities under the cold stress than those of ‘Hitomebore’ in the repeated cold tolerance evaluations for 4 years. On the other hand, lines of groups C3 and C4 having ‘Kuchum’ alleles in regions from S96E05-1 to S38P21-1 and from S72D08-2 to S38P21-1, respectively, showed significantly higher seed fertilities than those of ‘Hitomebore’ in three of the four tested years. These results suggest that the cold tolerance gene in qCT-4 is located in an 8.8 Mb region between S72D08-2 and RM5586.

Fig. 2
figure 2

Graphical genotyping of NILs developed for delimitation of the QTL region of qCT-4

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Table 2 Cold tolerance evaluation of NILs for delimitation of the QTL region of qCT-4
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Using progenies of NILs in groups from C1 to C4, recombinants within the 8.8 Mb region between S72D08-2 and RM5586 were further selected, and eight lines of three groups, i.e., group C5 to group C7 having shorter genomic regions derived from ‘Kuchum’ were developed (Fig. 2). Lines of groups C5 and C7 grown under the cold stress showed significantly higher seed fertilities than that of ‘Hitomebore’ in 2013. Although the lines of C5 and C7 did not show significantly higher seed fertilities than that of ‘Hitomebore’ in 2014, C5(-) lines and C7(-) lines having ‘Hitomebore’ alleles in the QTL region showed significantly lower seed fertilities than that of ‘Hitomebore’, suggesting that only the seed fertility of ‘Hitomebore’ was abnormally high in 2014. On the other hand, seed fertilities of lines of group C6 were comparable to those of ‘Hitomebore’ in the evaluations for 2 years of the three tested years. From these results, qCT-4 can be inferred to be in a region of ca. 1.36 Mb between DNA markers 9_1 and 10_13. Supporting data for significant effect of this region on cold tolerance were obtained using other NILs, C8 to C10 (Supplementary Fig. 4, Supplementary Table 2), although number of plants used for cold tolerance evaluation of these lines were small.

Cold tolerance of NILs having Ctb1, Ctb2, or qCTB8 with a ‘Hitomebore’ background

Ctb1 is linked with Ctb2 on chromosome 4, and NILs having each or both cold tolerance alleles at Ctb1 and Ctb2 derived from ‘Silewah’ with a ‘Hitomebore’ genetic background were developed. Lines of group S1 having only the ‘Silewah’ Ctb1 allele and group S2 having only the ‘Silewah’ Ctb2 allele showed slightly lower seed fertilities under cold stress than that of ‘Hitomebore’ in 2008, but the differences were not significant (Table 3). NILs having ‘Silewah’ alleles in chromosomal regions of different lengths covering both Ctb1 and Ctb2, i.e., groups S3 to S6, were obtained (Supplementary Fig, 2, Supplementary Table 3). Lines of S3 showed significantly lower seed fertilities than those of ‘Hitomebore’ in the evaluations for 3 years, and lines of groups S4, S5, and S6 showed slightly lower seed fertilities in 2008. Eight NILs of two groups, H1 and H2, having different lengths of a chromosomal region containing qCTB8 from ‘Hokkai PL9’ with a ‘Hitomebore’ genetic background were also developed (Supplementary Fig, 2, Supplementary Table 3). The lines of group H1 having ‘Hokkai PL9’ alleles in a 3.2 Mb region did not show higher seed fertility than that of ‘Hitomebore’ in 2008, and neither did the lines of group H2 having ‘Hokkai PL9’ alleles in a region of 1.3 Mb in 2009 and 2011 (Table 4). Furthermore, the lines of S3 and those of H2 showed seed fertilities comparable to those of S3(−) lines and H2(−) lines, respectively, having ‘Hitomebore’ alleles in the QTL region. These results indicate that Ctb1, Ctb2, and qCTB8 are ineffective for enhancing the cold tolerance of ‘Hitomebore’.

Table 3 Cold tolerance of NILs having Ctb1, Ctb2 with the ‘Hitomebore’ background
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Table 4 Cold tolerance of NILs having qCTB8 with the ‘Hitomebore’ background
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Effect of QTL pyramiding

The NIL having the ‘Kuchum’ qCT-4 allele was crossed with the line having the ‘Lijiangxintuanheigu’ qLTB3 allele for pyramiding of QTLs and cold tolerance of 18 lines at F3 and F4 generations was evaluated in 2012 and 2013 (Table 5). In both years, lines of group L4 having the cold tolerance alleles at both qCT-4 and qLTB3 showed significantly higher seed fertility than lines of group L1 having ‘Hitomebore’ alleles in these QTL regions under the cold-stress condition. Seed fertilities of the L4 lines were significantly higher than those of lines of group L2 having the ‘Kuchum’ allele at qCT-4. Although the L4 lines showed higher seed fertilities than those of lines of group L3 having the ‘Lijiangxintuanheigu’ allele at qLTB3, the differences were not significant in either 2012 or 2013.

Table 5 Cold tolerance of NILs having tolerance alleles of qCT-4 and qLTB3
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Agronomic traits of NILs having the ‘Kuchum’ qCT-4 allele with the ‘Hitomebore’ genetic background

Agronomic traits of cold-tolerant lines having the ‘Kuchum’ qCT-4 allele, which were named ‘Tou 1380’, ‘Tou 1381’, and ‘Tou 1489’, were investigated in the field under normal conditions. Days to heading of ‘Tou 1380’ and ‘Tou 1381’ were one to 2 days shorter than that of ‘Hitomebore’, and all the three NILs had slightly shorter culm lengths than ‘Hitomebore’ (Supplementary Table 4). The other traits, i.e., panicle length, grain yield, grain weight, lodging degree, grain quality, and eating quality, were comparable between the NILs and ‘Hitomebore’. ‘Tou 1489’ was especially similar to ‘Hitomebore’.

Discussion

Evaluation of cold tolerance of the many NILs having chromosomal segments from ‘Kuchum’ delimited the QTL for cold tolerance of ‘Kuchum’, i.e., qCT-4, into a region of ca. 1.36 Mb between 9_1 and 10_13, which are located at 14.9 and 16.3 Mb, respectively, on the short arm of chromosome 4. As other QTLs on chromosome 4, Ctb1, Ctb2 (Saito et al. 1995), qCTB4-1, and qCTB4-2 (Xu et al. 2008) have been reported. Comparison with the ‘Nipponbare’ genome sequence (IRGSP build5: http://rgp.dna.affrc.go.jp/E/IRGSP/Build5/) indicates that Ctb1 and Ctb2 are present between the sites of RM17341 at 28.8 Mb and RM17645 at 35.1 Mb from the end of the short arm, qCTB4-1 being between RM518 at 2.0 Mb and RM6700 at 2.8 Mb, and qCTB4-2 being between RM7200 at 4.0 Mb and RM8213 at 4.4 Mb, suggesting that qCT-4 is different from the four previously reported QTLs and is a novel QTL for cold tolerance at the booting stage.

The additive effect of qCT-4 was from 4.99 to 6.51 in the analysis using BC6F4. Corresponding to this, the NILs having the ‘Kuchum’ allele at qCT-4 as homozygotes showed an increase in seed fertility of ca. 10 % under cold stress. QTLs for cold tolerance at the booting stage so far reported have shown relatively small additive effects, generally less than 10 % of seed fertility (Takeuchi et al. 2001; Xu et al. 2008; Kuroki et al. 2011). The small additive effects of these QTLs and qCT-4 suggest the importance of pyramiding of several QTLs for improvement of cold tolerance. qLTB3 has been identified as a QTL for cold tolerance at the booting stage in the terminal region of the long arm of chromosome 3 in Chinese cultivar ‘Lijiangxintuanheigu’ by Shirasawa et al. (2012). The lines having the cold tolerance alleles at both qCT-4 and qLTB3 showed significantly higher seed fertilities than the lines having the qCT-4 allele, but the difference of seed fertilities between the lines having both QTL alleles and the lines having only the qLTB3 allele was not significant. Although the cause of this non-significant difference is unknown, it may be necessary to evaluate cold tolerance of these lines under a more severe condition lower than 18.5 °C to exhibit enhanced cold tolerance by QTL pyramiding.

Ctb1 and Ctb2 have been identified on chromosome 4 using a BC1F1 population between ‘Norin-PL8’ having high cold tolerance derived from a cross between ‘Silewah’ and ‘Kirara 397’, which has relatively high cold tolerance. Replacement of the linked DNA marker XNpb267 from ‘Kirara 397’ type to ‘Silewah’ type has increased seed fertility by 8.4 % (Saito et al. 1995, 2001). qCTB8 has been identified on the short arm of chromosome 8 by QTL analysis using F2 progeny of a hybrid between ‘Hokkai PL9’ having high cold tolerance and ‘Hokkai 287’ (Kuroki et al. 2007). Cold-tolerant lines, e.g., ‘Hokkai IL1’ having cold tolerance alleles at Ctb1 and Ctb2, ‘Hokkai IL2’ having cold tolerance alleles at qCTB8, and ‘Hokkai IL3’ having such alleles at both Ctb1 and qCTB8, have been developed in Hokkaido, Japan. However, seed fertilities of NILs having Ctb1, Ctb2, or qCTB8 with ‘Hitomebore’ background were not higher than that of ‘Hitomebore’ under the cold-stress condition. The effect of these QTLs may be dependent on genetic backgrounds of the developed NILs or environmental factors of cold tolerance evaluation. ‘Hitomebore’ has high cold tolerance, which is derived from ‘Koshihikari’. Three QTLs for cold tolerance at the booting stage in ‘Koshihikari’, i.e., qCT-1, qCT-7, and qCT-11, have been detected on chromosomes 1, 7, and 11 (Takeuchi et al. 2001). ‘Hitomebore’ has been reported to have ‘Koshihikari’ alleles at all these QTLs (Shirasawa et al. 2007). It may be inferred that Ctb1, Ctb2, and qCTB8 do not exhibit a pyramiding effect with qCT-1, qCT-7, and qCT-11.

The QTL for cold tolerance of ‘Kuchum’ was delimited to a region of ca. 1.36 Mb between 9_1 and 10_13. In the published ‘Nipponbare’ genome sequence between these DNA markers, 57 genes have been annotated. Among them, 32 genes have been reported to be expressed in inflorescence and anthers (Sato et al. 2011). Since the functions of these genes are unknown and a genomic sequence of ‘Kuchum’ in this 1.36 Mb region has not been determined, it is difficult to speculate as to which candidate gene is responsible for cold tolerance. Further narrowing of the QTL region is required for identification of the cold tolerance gene in ‘Kuchum’.

In the breeding to introduce QTLs from various genetic resources into a leading cultivar, care should be taken to avoid accompanying undesirable traits. Heading date of ‘Norin-PL8’ has been reported to be slightly late (Abe et al. 1989). In the NILs having the ‘Kuchum’ allele at qCT-4 with the ‘Hitomebore’ genetic background developed in the present study, such a delay of flowering was not observed. Grain yield and quality were not influenced by incorporation of the cold tolerance qCT-4 allele into ‘Hitomebore’. In the delimited QTL region of ca. 1.36 Mb, genes for evidently undesirable traits were not found, and DNA markers were developed in this short region. These markers will be useful for developing a cold-tolerant cultivar using ‘Kuchum’ as a gene source of cold tolerance, and the NILs having the 1.36 Mb region from ‘Kuchum’ with the ‘Hitomebore’ genetic background developed in the present study are considered to be useful lines for developing a highly cold-tolerant leading cultivar. One of the NILs having the shortest genomic region of ‘Kuchum’, i.e., group C7, will be released as a candidate of a new cultivar or a new breeding material.

Author contribution statement

TE designed the research, developed the NILs of ‘Hitomebore’ having qCT-4, and performed evaluation of cold tolerance and BC developed ‘BI6-12’ and performed QTL analysis. KW and KS controlled the deep water irrigation system for evaluation of cold tolerance. TY and TU developed NILs of ‘Hitomebore’ having Ctb1, Ctb2 and qCTB8. TA, AS, TM, and TY developed DNA markers near qCT-4 and performed DNA marker-assisted selection of NILs. TN wrote the manuscript. All authors read and approved the manuscript.