Genomic Regions Involved in Differences in Eating and Cooking Quality Other than Wx and Alk Genes between indica and japonica Rice Cultivars

In temperate rice cultivation regions, japonica rice cultivars are grown preferentially because consumers deem them to have good eating quality, whereas indica rice cultivars have high grain yields and strong heat tolerance but are considered to have poor eating quality. To mitigate the effects of global warming on rice production, it is important to develop novel rice cultivars with both desirable eating quality and resilience to high temperatures. Eating quality and agronomic traits were evaluated in a reciprocal set of chromosome segment substitution lines derived from crosses between a japonica rice cultivar ‘Koshihikari’ and an indica rice cultivar ‘Takanari’. We detected 112 QTLs for amylose and protein contents, whiteness, stickiness, hardness and eating quality of cooked rice grains. Almost of ‘Koshihikari’ chromosome segments consistently improved eating quality. Among detected QTLs, six QTLs on chromosomes 1–5 and 11 were detected that increased whiteness and stickiness of cooked grains or decreased their hardness for 3 years. The QTLs on chromosomes 2–4 were not associated with differences in amylose or protein contents. QTLs on chromosomes 1–5 did not coincide with QTLs for agronomic traits such as heading date, culm length, panicle length, spikelet fertility and grain yield. Genetic effects of the detected QTLs were confirmed in substitution lines carrying chromosome segments from five other indica cultivars in the ‘Koshihikari’ genetic background. The detected QTLs were associated with differences in eating quality between indica and japonica rice cultivars. These QTLs appear to be widely distributed among indica cultivars and to be novel genetic factors for eating quality traits because their chromosome regions differed from those of the GBSSI (Wx) and SSIIa (Alk) genes. The detected QTLs would be very useful for improvement of eating quality of indica rice cultivars in breeding programs.


Background
Rice (Oryza sativa L.) is a staple food for nearly half of the world population (GriSP (Global Rice Science Partnership), 2013). This species is divided into two main subspecies, indica and japonica, which differ in their morphological and physiological characteristics (Khush 1997). Indica is grown mainly in tropical regions, whereas japonica is grown in temperate regions such as China, Korea and Japan. The two subspecies have been suggested to have different domestication origins because, for example, seed shattering is reduced by the loss of function of different genes (Konishi et al. 2006;Li et al. 2006) and there are multiple sequence polymorphisms throughout their genomes (Garris et al. 2005;Wang et al. 2018), indicating considerable intraspecific differentiation. Generally, F 1 plants from crosses between the two subspecies show low fertility, and segregating populations such as F 2 often show sterility and hybrid weakness (Matsubara et al. 2007;Yamamoto et al. 2007). Therefore, in modern rice breeding, programs for indica-background breeding and for japonicabackground breeding are separate, although breeders use common agronomically important genes for disease resistance, plant height and stress tolerance in these programs.
The International Rice Research Institute (IRRI) developed an indica cultivar 'IR8' by introducing the semidwarf gene sd1 derived from 'Dee-geo-woo-gen'; 'IR8' has played the main role in the Green Revolution in rice (Khush 1999). Since then, IRRI has developed many high-yielding indica cultivars (e.g., 'IR64'), which are widely cultivated, mainly in tropical regions (Mackill and Khush 2018). Japanese indica cultivars such as 'Takanari' and 'Hokuriku 193' are derived from crosses with the IRRI cultivars and have significantly higher yields than Japanese japonica cultivars such as 'Koshihikari', even in temperate climates (Imbe et al. 2004;Goto et al. 2009). Global warming is likely to reach 1.5°C between 2030 and 2052 if it continues at the current rate (IPCC (Intergovernmental Panel on Climate Change), 2018). In general, indica cultivars are adapted to higher temperatures in low latitudes better than japonica cultivars. Therefore, the genetic backgrounds of indica rice will be more suitable for cultivation in temperate regions, which are likely to experience an increase in temperature during rice growth seasons in the future (IPCC (Intergovernmental Panel on Climate Change), 2018).
Indica rice cultivars are preferred by consumers in most rice cultivation areas, but not in Northeast Asia, where people are accustomed to eating japonica rice (Juliano et al. 1993;Calingacion et al. 2014). Many consumers in such countries as China, Korea and Japan tend to prefer the softness and strong stickiness of cooked grains of japonica rice cultivars. Almost all indica cultivars have low stickiness and high hardness of cooked grains (Calingacion et al. 2014;Hori and Yano 2013). One of main genetic factors controlling stickiness and hardness of cooked rice grains are allelic differences in the Waxy (Wx, GBSSI) gene involved in amylose synthesis in rice endosperm (Tan et al. 1999;Wan et al. 2004;Tian et al. 2005;Takeuchi et al. 2007;Su et al. 2011;Yang et al. 2018;Park et al. 2019;Yang et al. 2020). In general, indica cultivars have the Wx a allele, which results in high amylose content, whereas japonica cultivars have the Wx b allele, which results in moderate amylose content (Juliano et al. 1993). Several other Wx alleles have also been reported in indica cultivars such as Wx lv and Wx in with high amylose content, and in japonica cultivars such as Wx la and Wx mq with low amylose content (Sato et al. 2002;Zhou et al. 2015;Zhang et al. 2019;Zhou et al. 2020). Chromosome region at the Wx gene showed high recombination rate and various gene alleles were generated by intragenic recombination. The Alkali degradation (Alk, SSIIa) gene involved in amylopectin chain elongation is also controlling eating quality of cooked rice grains by altering starch characteristics such as amylose content, gelatinization temperature and gel consistency (Umemoto et al. 2002;Umemoto 2018). Haplotype analysis revealed that these phenotypic differences were significantly correlated with allelic differences of the Alk gene between indica and japonica rice cultivars (Umemoto et al. 2004). In general, indica cultivars have the Alk allele, which is strong functional allele and results in high gelatinization temperature, whereas japonica cultivars have the alk allele, which is weak functional allele and results in low gelatinization temperature. Another Alk allele of Alk b is reported as weak functional allele with low gelatinization temperature and gel consistency both in indica and japonica cultivars (Chen et al. 2020). Allelic difference of the Alk gene largely changed on eating and cooking qualities in near-isogenic lines (NILs) introducing the Wx b and wx gene alleles as compared with in NILs introducing the Wx a gene allele (Umemoto 2018).
Amylose and protein contents of a Japanese indica cultivar 'Takanari' are not much different from those of the japonica cultivar 'Koshihikari', which is a leading cultivar in Japan (Hori et al. 2016;Iijima et al. 2019). However, 'Takanari' has the Wx b allele introduced from japonica cultivars . 'Takanari' has harder cooked grains and significantly inferior taste in comparison with typical Japanese japonica rice cultivars including 'Koshihikari'. Therefore, the difference in eating quality between indica and japonica rice cultivars cannot be explained only by amylose and protein contents, but other major genetic factors related to eating quality are hardly known. To improve eating quality of indica rice cultivars, it is necessary to detect novel genetic factors associated with eating quality.
indica rice cultivars are considered unsuitable for consumers in Northeast Asia because of their eating and cooking characteristics. Development of novel rice cultivars with the indica genetic background, good eating quality and high yield in Northeast Asia would be an effective solution to the possible food-supply crisis caused by global warming in the future. In this study, we attempted to detect QTLs for eating quality in chromosome segment substitution lines (CSSLs) derived from crosses between 'Koshihikari' and 'Takanari' (Takai et al. 2014). We found multiple QTLs related to differences in eating quality between the two cultivars, and some of these QTLs were not associated with amylose or protein contents. These CSSLs can be promising materials to introduce novel genetic factors for eating quality into indica rice cultivars.

Plant Materials
To detect QTLs involved in the control of eating-quality traits, we used a reciprocal set of CSSLs derived from crosses between a japonica rice cultivar 'Koshihikari' and an indica rice cultivar 'Takanari' (Takai et al. 2014). Forty-one CSSLs covered most of the 'Takanari' genome in the 'Koshihikari' genetic background and 39 CSSLs covered the 'Koshihikari' genome in the 'Takanari' genetic background. Genotype information of individual CSSLs is available in Takai et al. (2014).

Evaluation of Eating Quality Traits
All plants of CSSLs and parental cultivars 'Koshihikari' and 'Takanari' were grown in an experimental field at the Institute of Crop Science, NARO, Tsukubamirai, Japan (36.01°N, 140.02°E). CSSLs of the 'Koshihikari' genetic background were grown in 2016 and 2017, and CSSLs of the 'Takanari' genetic background in 2018. Japanese leading cultivars and recently developed cultivars were grown in 2018. One-month-old seedlings of all CSSLs and cultivars were transplanted in mid-May at one plant per hill in plots with a double row for each line; there was 15 cm between plants and 30 cm between rows. Cultivation management followed the standard procedures used at the institute.
Eating quality traits were evaluated by instrumental methods according to Hori et al. (2016) and Iijima et al. (2019). Apparent amylose content was determined by using an Auto Analyzer II (Bran+Luebbe Co. Ltd., Norderstedt, Germany). Crude protein content was determined by the combustion method with an induction furnace at 900°C (American Association of Cereal Chemists International, Approved Method 46-30.01). Whiteness and grain qualities were evaluated with a Rice Grain Analyzer RGQI20B (Satake Co., Ltd., Hiroshima, Japan). Eating quality score was measured in a Cooked Rice Taste Analyzer STA1A (Satake Co., Ltd.). This instrument has been used to estimate eating quality scores by measuring transmitted light volume and reflection light volume of cooked rice grains under three wavelengths (Mikami 2009). Physical properties of cooked grains were measured by the high-compression/lowcompression method with a Tensipresser MyBoy texture analyzer (Takemoto Electric Co., Tokyo, Japan). These instrumental methods showed significant correlations with the eating quality scores by the sensory tests (Okadome 2005;Mikami 2009;Kwon et al. 2011;Hori et al. 2016).

Scoring of Agronomic Traits
For each CSSL and parental cultivar, days to heading was defined as the number of days from sowing to heading of half of the plants. Culm length, panicle length, panicle number, spikelet fertility (ratio of the number of sterile and fertile grains per panicle) and unhulled grain weight per plant were measured for five plants per CSSL and parental cultivar at maturity stage.

Statistical and Genetic Analyses
Eating quality traits and agronomic traits of CSSLs were compared with those of each recurrent parent, 'Koshihikari' or 'Takanari', by using the Dunnett's multiple comparison procedure provided by the JMP 11 software (SAS Institute Inc., NC, USA). In the Dunnett's tests, 'Koshihikari' was used as a control for 41 CSSLs of 'Takanari' and 8 CSSLs of other indica cultivars in the 'Koshihikari' genetic background, and 'Takanari' was used as a control for 39 CSSLs in the 'Takanari' genetic background. QTLs were declared present when individual trait scores were significantly different between the line and the recurrent parent.

QTLs for Eating Quality Traits in CSSLs of the 'Koshihikari' Genetic Background
The phenotypes of the CSSLs of the 'Koshihikari' genetic background varied widely in eating quality traits (amylose and protein contents, eating quality score, stickiness and hardness of cooked rice grains, and grain whiteness; Table 1, Supplementary Tables S2 and S3, Supplementary Figure 1). In comparison with 'Koshihikari', 44 QTLs in 2016 and 32 QTLs in 2017 were associated with significant differences in all six eating quality traits analyzed (Table 2). Genomic regions of almost all QTLs were consistent between 2016 and 2017.
We focused on six of the detected QTLs ( Fig. 1): qWH1 for grain whiteness on the long arm of chromosome 1, qST2 for stickiness of cooked grains on the long arm of chromosome 2, qST3-1 for stickiness of cooked grains on the short arm of chromosome 3, qHA4-2 for hardness of cooked grains on the long arm of chromosome 4, qST5 for stickiness of cooked grains on the long arm of chromosome 5 and qHA11 for hardness of cooked grains on the long arm of chromosome 11. We selected these six QTLs because of their large genetic effects and detection in both years.
In comparison with 'Koshihikari', SL1204, carrying qWH1, had significantly higher whiteness, amylose content and hardness of cooked grains, but lower stickiness of cooked grains in 2016 and 2017. SL1208, carrying qST2, had significantly lower eating quality score, stickiness of cooked grains and grain whiteness. SL1210, carrying qST3-1, had significantly lower eating quality score and stickiness of cooked grains. SL1217, carrying qHA4-2, had significantly lower eating quality score and higher hardness of cooked grains and grain whiteness. SL1219, carrying qST5, had significantly lower eating quality score and stickiness of cooked grains but higher amylose content and grain whiteness. SL1237, carrying qHA11, had significantly lower eating quality score, stickiness of cooked grains and amylose content, hardness of cooked grains and grain whiteness. The 'Takanari' allele of qWH1 increased grain whiteness. The 'Koshihikari' alleles of the remaining five QTLs resulted in high eating quality score and in strong stickiness and softness of cooked grains. qST2, qST3-1 and qHA4-2 were not associated with differences in amylose or protein contents in both years.
Eating quality traits are easily affected by many agronomic traits including heading date, grain size and weight, grain number per panicle and spikelet fertility (Juliano et al. 1993;Iijima et al. 2019). We evaluated agronomic traits of the CSSLs, in particular those of the six CSSLs each carrying a QTL on chromosomes 1-5 and 11. In comparison with 'Koshihikari', five lines showed no significant differences in days to heading (flowering time), culm length, panicle length, number of panicles, unhulled grain weight or spikelet fertility (Supplementary Tables S2 and S3). However, one line, SL1208, showed weak vigor, including few panicles and low unhulled grain weight and spikelet fertility.

QTLs for Eating Quality Traits in CSSLs of the 'Takanari' Genetic Background
Among the CSSLs of the 'Takanari' genetic background, there was also a wide range of phenotypic differences in eating quality traits (Table 3, Supplementary Table S4, Supplementary Figure 1). In comparison with 'Takanari', 36 QTLs in 2018 showed significant differences in the six eating quality traits (Table 4). The six QTLs detected in the 'Koshihikari'-background CSSLs on chromosomes 1-5 and 11 coincided well with those detected in the 'Takanari' CSSLs ( Fig. 1).
In comparison with 'Takanari', SL1303, carrying qWH1, had significantly higher grain whiteness. Thus, this chromosome region increased whiteness both in the 'Koshihikari' and 'Takanari' genetic backgrounds. SL1306, carrying qST2, had significantly higher eating quality score, stickiness of cooked grains and grain whiteness. SL1308, carrying qST3-1, had significantly higher eating quality score and grain stickiness. SL1315, carrying qHA4-2, had significantly higher eating quality  score and lower hardness of cooked grains. SL1317, carrying qST5, had significantly higher eating quality score, stickiness of cooked grains and protein content, and lower hardness of cooked grains. SL1336, carrying qHA11, had significantly higher eating quality score, amylose content, protein content and grain stickiness, and lower hardness of cooked grains. The 'Koshihikari' alleles of all of the six QTLs resulted in high eating quality score, strong stickiness and softness of cooked grains, and high grain whiteness in the 'Takanari' genetic background. qST2, qST3-1 and qHA4-2 were not associated with significant differences in amylose or protein contents.
We also evaluated agronomic traits in the CSSLs of the 'Takanari' genetic background. In comparison with 'Takanari', SL1303, SL1306, SL1308, SL1315 and SL1317 showed no significant differences in days to heading, culm length, panicle length, number of panicles or unhulled grain weight (Supplementary Table S4). However, SL1336 showed weak vigor including late flowering, short culms and panicles, few panicles and low unhulled grain weight.

Confirmation of the Effects of QTLs in CSSLs of Other indica Cultivars
To investigate whether the six detected QTLs would be commonly detected in segregating populations derived from crosses between japonica and other indica rice cultivars, we developed additional CSSLs carrying chromosome segments introduced from indica cultivars 'Naba', 'Bleiyo', 'Bei Khe', 'Tupa 121-3' and 'Basilanon' in the 'Koshihikari' genetic background. Many of the eating quality traits of these CSSLs were significantly different from those of 'Koshihikari' (Fig. 2, Supplementary Table S5). SL3202 and SL3302 each had a single segment on the long arm of chromosome 1 derived from 'Naba' and 'Bleiyo', respectively. In both lines, grain whiteness was significantly higher than in 'Koshihikari'. High whiteness in SL3202 and SL3302 was the same phenotype as that of SL1204 carrying qWH1 from 'Takanari' (Fig. 1). Eating quality scores of SL3202 and SL3302 were not significantly different from that of 'Koshihikari'. In comparison with 'Koshihikari', hardness of cooked grains was higher in SL3202 and SL3302, as it was in SL1204 in 2017. SL3205 had a single segment on the long arm of chromosome 2 derived from 'Naba'. The eating quality score and stickiness of cooked grains were significantly lower in SL3205 than in 'Koshihikari'. These phenotypes were the same as that of SL1208 carrying qST2 from 'Takanari'. SL3315, SL2412 and SL2520 each had chromosome segments on the long arm of chromosome 4 derived from 'Bleiyo', 'Bei Khe' and 'Tupa 121-3', respectively. SL3315 showed significantly lower eating quality score and higher hardness of cooked grains than those of 'Koshihikari'. SL2412 had a significantly lower eating quality score, but higher stickiness and hardness of cooked rice grains. SL2520 showed significantly higher grain whiteness and stickiness and hardness of cooked grains, but its eating quality score was not significantly different from that of 'Koshihikari'. The phenotypes of SL3315, SL2412 and SL2520 (low eating quality score and stickiness, and high hardness of cooked grains) were similar to those of SL1217, which had qHA4-2 from 'Takanari'. SL2544 and SL3042 each had a single segment on the long arm of chromosome 11 derived from 'Tupa 121-3' and 'Basilanon', respectively. SL2544 had lower eating quality score, amylose content and stickiness of cooked grains, and higher hardness of cooked grains. SL3042 had lower eating quality score and higher hardness of cooked grains. These phenotypes were similar to those of SL1237, which had qHA11 from 'Takanari'.
The QTLs in the CSSLs derived from crosses with other indica rice cultivars were detected in the same Fig. 1 Eating quality score, stickiness of the surface of cooked rice grains, hardness of whole cooked rice grains and whiteness of rice grains in 12 chromosome segment substitution lines carrying single introduced segments on chromosomes 1-5 or 11 in the 'Koshihikari' (Kos) genetic background in 2016 and 2017, and in the 'Takanari' (Tak) genetic background in 2018. White bars, homozygous for the 'Koshihikari' allele; black bars, homozygous for the 'Takanari' allele. Data for eating quality traits are presented as means ± SD (n = 6). Asterisks indicate significant differences in each eating quality traits between CSSLs and the recurrent cultivars 'Koshihikari' or 'Takanari' at P < 0.05 by the Dunnet's test Bold and underlined numbers indcate significant diffferences to 'Koshihikari' at the 5% level by the Dunnett's multiple comparison tes chromosome regions as qWH1, qST2, qHA4-2 and qHA11, confirming the genetic effects of these QTLs not only in 'Takanari' but also in several other indica cultivars. All of the CSSLs carrying qWH1, qST2, qHA4-2 and qHA11 except SL2544 showed no significant differences from 'Koshihikari' in amylose contents.

QTLs for Improving Eating Quality in indica Rice Cultivars
There is a wide range of phenotypic variations in eating quality traits among rice cultivars, and consumer preferences differ considerably worldwide (Juliano et al. 1993;Calingacion et al. 2014). Eating quality traits are very different even within indica and japonica rice (Calingacion Hardness H2 (N/m 2 × 10 4 ) qHA4-1 SL1312 Chr4 RM16260-RM5633 1.9 Positive additive effect means 'Takanari' allele increasing the trait values et al. 2014; Hori et al. 2016;Iijima et al. 2019). Generally, cooked grains of japonica cultivars are more sticky and softer than those of indica cultivars (Hori and Yano 2013). In the cultivation areas of japonica cultivars such as China, Korea and Japan, indica cultivars are often evaluated to have lower eating quality than typical japonica cultivars. In this study, we detected six QTLs for eating quality traits involved in differences between indica and japonica rice cultivars using a reciprocal set of CSSLs derived from a cross between a japonica cultivar 'Koshihikari' and an indica cultivar 'Takanari'. One eating quality QTL, qOE3, has been commonly detected on the short arm of chromosome 3 by using mapping populations derived from crosses between Japanese japonica cultivars (Kobayashi and Tomita 2008;Takeuchi et al. 2008;Wada et al. 2008;Hori and Yano 2013). In these studies, the qOE3 showed the largest genetic effect among the detected QTLs for eating quality and stickiness of cooked rice grains in the previous studies and the 'Koshihikari' allele of qOE3 was associated with good eating quality and strong stickiness of cooked grains. Here, we detected one eating quality QTL, qST3-1, also on the short arm of chromosome 3, and the 'Koshihikari' allele of this QTL was also associated with high eating quality score and strong stickiness of cooked grains. The 'Takanari' allele of qOE3 would be the same as the 'Nipponbare' allele because of consistent haplotypes between these cultivars according to the RAP-DB and TASUKE databases (Sakai et al. 2013;Kawahara et al. 2013;Kumagai et al. 2019). Therefore, qST3-1 is likely identical to qOE3.
We detected five other QTLs with large genetic effects on chromosomes 1, 2, 4, 5 and 11. Both 'Takanari' and 'Koshihikari' segments of the long arm of chromosome 1 containing qWH1 resulted in high whiteness and high eating quality score in the 'Koshihikari' and 'Takanari' genetic backgrounds, respectively. We confirmed the genetic effects of qWH1 in two additional CSSLs carrying chromosome segments derived from indica cultivars 'Naba' and 'Bleiyo'. These data suggest the presence of at least two distinct QTLs for increasing grain whiteness on the long arm of chromosome 1 in japonica and indica rice cultivars. To investigate importance of qWH1 in Japanese rice breeding programs, we investigated genotypes of the qWH1 region in Japanese leading rice cultivars and recently developed rice cultivars (Fig. 3). In the six recently released cultivars, a genome sequence between 37.0 and 39.3 Mbp of the qWH1 region containing the sd1 gene is replaced with indica-type chromosome segments, while the same region in the five leading Japanese cultivars is of japonica type. This difference may be caused not only by selection of semi-dwarf phenotypes caused by the sd1 gene during breeding, but also by selection of grain whiteness caused by qWH1. We cannot be certain whether the QTLs detected in multiple indica cultivars are the same or correspond to different genes. Further genetic analysis, including fine mapping of qWH1, is needed.
The 'Koshihikari' alleles of qST2, qHA4-2, qST5 and qHA11 on the long arms of chromosomes 2, 4, 5 and 11, respectively, improved eating quality traits by increasing stickiness and softening cooked rice grains. There were Fig. 2 Eating quality traits and genotypes of eight CSSLs derived from crosses between the recurrent parent 'Koshihikari' and donor indica rice cultivars. The short arm is on the left and the long arm on the right. White bars, homozygous for the 'Koshihikari' allele; black bars, homozygous for the donor cultivar allele. Data for eating quality traits are presented as means ± SD (n = 6). Red numbers and asterisks indicate significant differences in eating quality traits between CSSLs and 'Koshihikari' at P < 0.05 by the Dunnet's test several reports of QTL detection in mapping populations with the same Wx b allele derived from crosses between japonica cultivars. Kwon et al. (2011) found one QTL for eating quality and glossiness of cooked grains on the long arm of chromosome 4. Park et al. (2019) also detected one QTL for eating quality and glossiness of cooked grains on the long arm of chromosome 4, and they fine-mapped another eating quality QTL on the long arm of chromosome 9. Hsu et al. (2014) and Xu et al. (2016) reported QTLs for palatability of cooked grains detected by QTL analysis and genome-wide association study, respectively, in japonica rice cultivars. They reported coexistence of several QTLs and starch biosynthesis genes in the same chromosome regions. Kinoshita et al. (2017) detected three amylose content QTLs and eight protein content QTLs on chromosomes 1-4, 6, 8, 9 and 12. Takemoto-Kuno et al. (2015) found one amylose content QTL near the centromeric region on the long arm of chromosome 2. These QTLs might not be the same as those detected in our present study, because of difference in chromosome locations.
The QTLs qST2, qST3-1 and qHA4-2 on chromosomes 2, 3 and 4 were not associated with differences in amylose or protein contents. Eating quality traits are quantitative and complex, and are associated with various factors including grain composition and stickiness, hardness and whiteness of cooked grains. In this study, we found QTLs responsible for each of these factors. And, these QTLs might be eating quality genes with different molecular functions from those of previously isolated genes such as Wx, Alk and other starch biosynthesis genes, or storage protein genes. Generally, differences in eating quality between japonica and indica rice cultivars seem to be primarily due to different amylose contents caused by allelic differences of the Wx and Alk genes. Many studies have reported QTLs for eating quality and starch characteristics corresponding to the Wx and Alk genes in mapping populations derived from crosses between indica and japonica rice cultivars (Tan et al. 1999;Wan et al. 2004;Tian et al. 2005;Takeuchi et al. 2007;Su et al. 2011;Yang et al. 2018;Yang et al. 2020). The 'Takanari' allele of the Wx gene is identical with the japonica-type Wx b allele, and the amylose content is not much different from that of 'Koshihikari' Hori et al. 2016;Iijima et al. 2019). In this study, we did not also detect any QTLs in the chromosome region of the Alk gene, which also greatly affects starch properties. 'Takanari' has A-type in the G / A polymorphism and TT-type in the GC / TT polymorphism of the Alk gene based on genotyping by the DNA marker of Bao et al. (2006) and Hiratsuka et al. (2010). It was different with other weak functional alleles of the japonica-type alk (Alk a ) in 'Koshihikari', Alk (Alk c ) in typical indica type and Alk b in the previous study Fig. 3 Genotypes near the sd1 gene on the long arm of chromosome 1 in 'Dee-Geo-Woo-Gen', 'Takanari', SL1205, five Japanese leading cultivars and six high-yielding cultivars released since 2011. The centromere side is on the left, and the distal end of the long arm is on the right. White bars, homozygous for the 'Koshihikari'-type allele; black bars, homozygous for the 'Takanari'-type allele (Chen et al. 2020), but the same weak functional allele as other japonica rice cultivars such as 'Asahi' and 'Akebono' according to the RAP-DB and Rice-TASUKE database (https://ricegenomes.dna.affrc.go.jp/, Sakai et al. 2013;Kawahara et al. 2013;Kumagai et al. 2019). Therefore, 'Koshihikari' and 'Takanari' have the two weak functional alleles of both the Wx and Alk genes. This study found novel eating quality QTLs other than the Wx and Alk genes. However, we also consider other reasons for no detecting QTLs near the Wx and Alk genes. For an example, small genetic effect QTLs in this chromosome region might be concealed by other large genetic effect QTLs. Further studies are required to assess which genes or QTLs are responsible for varietal differences in eating-quality traits.

Common Location of Detected QTLs in indica Rice Cultivars
The 'Koshihikari' alleles of all QTLs except qWH1 improved eating quality traits. Therefore, the QTLs detected in this study could be used to improve eating quality of many indica cultivars. Because the effect of each single QTL did not improve eating quality to the level of typical japonica cultivars, it would be necessary to accumulate multiple QTL alleles to develop novel indica cultivars with both good eating quality and high grain yield.
Confirmation of qST2, qHA4-2 and qHA11 in CSSLs carrying chromosome segments from various indica cultivars suggests common allelic differences in these QTLs between indica and japonica subspecies. The difference in eating quality between japonica and indica cultivars might be due mainly to accumulation of genetic effects of QTLs detected in this study and Wx and Alk genes.

Perspectives for Application to Future Rice Breeding
Although indica cultivars are considered by consumers in Northeast Asian countries such as China, Korea and Japan to have lower eating quality than typical japonica cultivars, many indica cultivars and hybrid rice cultivars have high grain yield (Cheng et al. 2007;Mackill and Khush 2018) and therefore must have genes that increase grain yield. To develop novel rice cultivars that would combine high grain yield and good (japonica-like) eating quality, it is necessary to combine genetic loci that improve eating quality in the background of indica cultivars. Global warming is expected to increase temperatures by 2°C by the end of this century (IPCC (Intergovernmental Panel on Climate Change), 2018). Rice cultivars with good eating quality in the indica genetic background would be a good solution to mitigate the effects of climate warming on rice production while preserving the eating quality preferred in Northeast Asia.
However, it is often difficult to select progenies of crosses between indica and japonica rice cultivars. In modern breeding programs, the standard method includes crossing cultivars, fixing genotypes by selfpollination and selecting appropriate lines based on their phenotypes. However, many progenies derived from indica and japonica cultivars have hybrid sterility or hybrid breakdown because of differentiation between the genomes of the two subspecies and incompatibilities in many gene alleles (Matsubara et al. 2007;Yamamoto et al. 2007). If the precise chromosome positions and genetic effects of individual QTLs and genes were revealed, DNA marker technologies could allow us to improve selection efficiency in breeding when using populations derived from crosses between the subspecies. Fine-mapping of individual QTLs will be indispensable in the future. It may also be possible to reproduce favorable alleles by genome editing technology after the responsible genes are identified. Recently, we developed high-speed advanced generation technologies that use a biotron breeding system (Tanaka et al. 2016). These methods would facilitate accumulation of agronomically important genes, such as those for eating quality, grain yield, disease resistance and stress tolerance, across hybridization barriers between subspecies.

Conclusion
We detected QTLs involved in the control of eating quality traits in CSSLs derived from a cross between a japonica rice cultivar 'Koshihikari' and an indica rice cultivar 'Takanari'. Four of these QTLs, on chromosomes 1, 2, 4 and 11, were common in CSSLs derived from several other indica cultivars. These QTLs could be useful for improving eating quality of high-yielding indica cultivars to the level of typical japonica cultivars.
Additional file 1: Figure S1. Eating quality score, amylose content, protein content, stickiness of the surface of cooked rice grains, hardness of whole cooked rice grains and whiteness of rice grains in all chromosome segment substitution lines in the 'Koshihikari' genetic background in 2016 (upper) and 2017 (middle), and in the 'Takanari' genetic background in 2018 (lower). Data for eating quality traits are presented as means ± SD (n = 6).
Additional file 2: Table S1. Graphical genotypes of eight CSSLs in the 'Koshihikari' genetic background. Table S2. Eating quality traits and agronomic traits of 41 CSSLs in the 'Koshihikari' genetic background in 2016. Table S3. Eating quality traits and agronomic traits of 41 CSSLs in the 'Koshihikari' genetic background in 2017. Table S4. Eating quality traits and agronomic traits of 39 CSSLs in the 'Takanari' genetic background in 2018. Table S5. Eating quality traits and agronomic traits of eight CSSLs carrying chromosome segments from five indica rice cultivars in the 'Koshihikari' genetic background in 2018.