Selection of chromosome segment substitution lines with reduced grain chalkiness without yield penalty in rice

Abstract

Grain chalkiness decreases the appearance quality (APQ) of rice (Oryza sativa L.) grains and reduces consumer satisfaction. Improving APQ is a crucial issue for both marketing and breeding. Here, we screened chromosome segment substitution lines (CSSLs) with higher APQ to find promising genetic resources. These CSSLs harbor chromosome segments derived from multiple donors in the genetic background of ‘Koshihikari’, a leading japonica rice cultivar in Japan. Three CSSLs had an increased percentage of perfect grains without panicle weight loss under field conditions across 3 years in Tsukuba city, Ibaraki prefecture, Japan. The positions of reduced chalkiness in grains differed among CSSLs, suggesting the different contribution of the harbored chromosome segments to APQ improvement. There were no significant differences in days to heading, culm length, panicle length, or panicle number in all three CSSLs, but 1000-grain weight was reduced in one. These results identify two promising genetic resources for further improvement of APQ in current japonica cultivars with reduced chalkiness but unaltered heading date and yield traits.

Introduction

The appearance quality (APQ) of rice grains is a highly complex trait which can be strongly affected by heading date, and storage compounds in grains (Chen et al. 2013, Gong et al. 2020, Ishimaru et al. 2020, Yamakawa et al. 2008). APQ is assessed by four major properties, namely grain shape, chalkiness, transparency, and grain color. Their degree of importance to APQ depends on consumer preferences. Among them, chalkiness commonly decreases APQ, especially in temperate countries. Chalkiness is indicated by opaque parts in the milled rice grain caused by the abnormal deposition of starch and storage proteins in the endosperm (Wang et al. 2008, Yoshioka et al. 2007, reviewed by Zhao et al. 2022). It is categorized by the position of the chalkiness as white basal, white belly, white back, white core, or white milky. Several quantitative trait loci (QTLs) control chalkiness (Kobayashi et al. 2013, Takehara et al. 2018, Yamakawa et al. 2008) with changes of days to heading (DTH), grain length-to-width ratio, or 1000-grain weight (TGW) (i.e. grain yield). Further investigation of QTLs that improve APQ without yield penalty is still required.

Most QTL mapping studies in rice generally rely on primary mapping populations such as recombinant inbred lines (RILs) but can fail to identify QTLs with subtle effects, owing to diminished sensitivity of detection due to the use of early generations (Kobayashi et al. 2013, Yamamoto et al. 2009). Conversely, advanced mapping populations can break through this limitation since their genetic backgrounds are more genetically fixed, resulting in higher sensitivity of detection (Nagata et al. 2015, Tanksley and Nelson 1996, Yamamoto et al. 2009). For example, chromosome segment substitution lines (CSSLs) were used to detect novel QTLs for preharvest sprouting resistance, DTH, culm length (CL), yield traits, toxic metal concentrations, and APQ components have been uncovered (Abe et al. 2013, Bian et al. 2010, Hao et al. 2009, Hori et al. 2010, Ishikawa et al. 2005, Nagata et al. 2023, Takai et al. 2014, Yamakawa et al. 2008).

Significant progress has been made in developing CSSLs with the genetic background of ‘Koshihikari’, a leading japonica cultivar in Japan, which carry chromosome segments from the indica cultivar ‘Habataki’ (Murata et al. 2014). However, CSSLs carrying chromosome segments from other indica (indica and aus) in addition to tropical japonica donors were not well screened on the purpose of increasing APQ in the ‘Koshihikari’ genetic background. Thus, evaluating the chalkiness or percentage of perfect grains (PPG) among such CSSLs may highlight the promising genetic resources with a high APQ under the present cultivation system for ‘Koshihikari’.

To evaluate chalkiness, we selected CSSLs containing chromosome segments from indica, aus, and tropical japonica cultivars in the genetic ‘Koshihikari’ background (Ishikawa et al. 2005, Nagata et al. 2015, 2023). Many agronomic traits of these materials vary widely from those of ‘Koshihikari’, but chalkiness and PPG have not yet been examined. We screened CSSLs with a similar DTH to ‘Koshihikari’ to minimize the effects of environmental factors on grain traits and found three CSSLs with reduced chalkiness and higher PPG without loss of panicle weight (PW) under field conditions across 3 years.

Materials and Methods

Plant materials and growth conditions

The 342 CSSLs, developed in previous studies (Ishikawa et al. 2005, Nagata et al. 2015, 2023), possess chromosome segments of indica (indica: ‘Bei Khe’, ‘Bleiyo’, ‘IR64’, ‘Naba’, ‘Tupa 121-3’), aus (‘Kasalath’, ‘Muha’) and tropical japonica (‘Basilanon’) cultivars in the ‘Koshihikari’ genetic background. All plants were grown under field conditions in Kannondai, Tsukuba city, Ibaraki prefecture, Japan, in 2019–2021 (36°01ʹ29.4ʺN, 140°06ʹ28.4ʺE, 21 m a.s.l.) and 2023 (36°01ʹ28.7ʺN, 140°06ʹ37.3ʺE, 23 m a.s.l.). The average temperature and relative humidity in our field were measured at 2 m above the ground (Supplemental Fig. 1). More details of the climatic conditions are available from https://www.naro.affrc.go.jp/org/niaes/aws/weatherdata.html (in Japanese). Seeds were soaked in water at 30°C for 2 d, sown in trays filled with soil and incubated at 30°C in the dark for 2 d; seeds were sown on 22 April 2019, 20 April 2020, 19 April 2021, and 26 April 2023. Seedlings were grown in a paddy field for a month and were then transplanted (9 or 11 plants, 18 cm apart × 3 rows 30 cm apart, no replicates) into a nearby paddy field, and grown for 5 months from May to September. Planting density was 18.5 hills/m². Fertilizer was applied as basal dressing at a rate of 56:16.4:56:72:120 kg/ha for N:PO₄:K:Mg:SiO₂ in 2019, 2020, and 2021; basal dressing at a rate of 60:60:45:15:60 kg/ha for (NH₄)₂SO₄:P₂O₅:K₂O:Mg in 2023. Other cultivation conditions in different cities of Ibaraki prefecture, Toyama prefecture, and Kagoshima prefecture are described in Supplemental Table 1.

Assessment of agronomic traits of rice plants

Agronomic traits were measured as previous studies (Ogawa et al. 2021a, 2021b). DTH was scored as the number of days from sowing to the appearance of the first panicle in more than half of the plants in each line. CL and panicle length (PL) of the longest culm on each plant were measured with a ruler, and panicle number (PN) was counted, from 10 days to a month after heading. For measurement of aboveground dry weight (ADW), PW, and stem and leaf weight (SLW), shoots of mature plants were dried for over a month and cut 3 cm below the panicle base to separate the parts. Five plants were evaluated per line for CL, PL and PN. Grain weight (GW) was measured after threshing. TGW was measured after hulling in only Kannondai.

To evaluate APQ, we used a 96-dpi scanned image of >400 seeds per individual plant, and 4 plants per line as biological replicates in Kannondai in 2023. Seeds were obtained from panicle bulks, and calculated PPG with a grain discriminator (RGQI90A or RGQI100B, Satake). Perfect grains were defined as non-chalky, with a normal shape (Ebitani et al. 2008, Takehara et al. 2018). Grain length (GL), width (GW), and thickness (GT) were measured by the latter grain discriminator. Each trait value of ‘Koshihikari’ was calculated as the mean of all ‘Koshihikari’ plants grown at the same time under the same conditions.

Results

Characterization of target CSSLs with similar DTH to ‘Koshihikari’

The target CSSLs (148 lines) were defined as having DTH within the range of ‘Koshihikari’ maximum +1 to minimum –1 in 2019, 2020, and 2021. DTH in ‘Koshihikari’ were 101–104 in 2019, 98–101 in 2020, and 104–106 in 2021 (Supplemental Table 2). In 2020 and 2021, we investigated the AGW, SLW, CL, PW, PL, and PN of the target CSSLs (Supplemental Fig. 2, Supplemental Table 2). The CSSLs had biomass and yield trait values ranging from –37% to +16% of ‘Koshihikari’ in AGW, –24% to +22% in SLW, –24% to +16% in CL, –36% to +18% in PW, –13% to +18% in PL, and –34% to +19% in PN. PPG ranged from –98% to +96% of ‘Koshihikari’. This motivated us to search for promising CSSLs having higher APQ without yield penalty.

Selection of promising CSSLs with higher PPG in 2020 and 2021

To screen lines with higher PPG but without yield penalty from the 148 target CSSLs, we selected the top 10% (15 CSSLs) by PPG in 2020 and 2021 (Fig. 1a, 1b) and compared PW between each CSSL and ‘Koshihikari’ in the same years. We found three promising CSSLs (SL2033, SL2037, and SL3215) with no decreased PW in both 2020 and 2021 (Fig. 1c).

Fig. 1.

Selection of promising lines with higher percentage of perfect grains without panicle weight loss. (a) Selection of three promising lines with a higher percentage of perfect grains (PPG) without panicle weight loss from 342 chromosome segment substitution lines (CSSLs) with the ‘Koshihikari’ (KO) genetic background under field conditions in 2020 and 2021. (b) PPG was assessed in 148 CSSLs with days to heading within the range of ‘Koshihikari’ ± 1 day in Tsukuba in 2020 and 2021. Dashed line marks the 90^th percentile of PPG among CSSLs, which was exceeded by 15 lines in each year. (c) Panicle weights (PW) of the 15 top CSSLs in b. ▽ Three promising lines with a higher PPG without PW loss under the field conditions in 2020 and 2021.

By comparing the locations of the introgressed chromosome segments between the three promising CSSLs (Nagata et al. 2015, 2023) and ‘Koshihikari’, we found introgressed chromosome segments between 1 × 10^–6 and 13.45 Mb on chromosome (Chr.) 10 in SL2033 (carrying ‘IR64’ chromosome segment(s)); between 1 × 10^–6 and 4.06 Mb on Chr. 11 in SL2037 (carrying ‘IR64’ chromosome segment(s)); and between 1 × 10^–6 and 19.26 Mb, and between 28.42 and 29.75 Mb on Chr. 5 in the SL3215 (carrying ‘Naba’ chromosome segments) (Table 1).

Table 1.Positions of chromosome segment substitutions in the selected CSSLs

CSSL	Donor			Chr.	Range of target chromosome per line (Mb)
	Variety name	Origin	Subspecies
SL2033	IR64	Philippines	indica	10	1 × 10–6–13.45
SL2037	IR64	Philippines	indica	11	1 × 10–6–4.06
SL3215	Naba	India	indica	5	1 × 10–6–19.26, 28.42–29.75

Changes in appearance quality of rice grains in promising CSSLs

To understand more about the chalkiness phenotypes, we analyzed and compared the ratios of different kinds of kernels among SL2033, SL2037, SL3215, and ‘Koshihikari’. All three CSSLs had lower proportions of white milky, white belly, and white back grains than ‘Koshihikari’, increasing PPG (Fig. 2). SL2033 and SL2037 also had lower proportions of white basal grains (Fig. 2b). These results implicate the different effects on grain chalkiness among the introgressed chromosome segments harbored in each CSSL.

Fig. 2.

Appearance quality of brown rice in promising CSSLs harvested in 2023. (a) Grains of a rice plant cultivated under experimental filed conditions in 2023. Three promising CSSLs (SL2033, SL2037, SL3215) exhibited higher PPG compared to ‘Koshihikari’ in 2023. Grains of ‘Koshihikari’ exhibit more chalkiness in a wide range and less transparency. Scale bar, 10 mm. (b) Percentage of different kinds of kernel in the three lines and ‘Koshihikari’. (c) Percentage of perfect grains, average of grain volume-related traits (1000-grain weight, grain length, grain width, and grain thickness) in the three lines (SL2033, SL2037, SL3215) and ‘Koshihikari’. Asterisks indicate significant differences between each CSSL and the genetic ‘Koshihikari’ background (n = 4, P < 0.05, Dunnett’s Test).

Next, we investigated the differences in TGW, GL, GW, and GT between each promising CSSL and ‘Koshihikari’ in 2023. SL2033 had significantly lower GT (P < 0.05); SL2037 had significantly longer GL and lower GW; and SL3215 had significantly lower TGW, longer GL, lower GW, and lower GT (Fig. 2c). The other grain phenotypes not described for each CSSL were not significantly different from those of ‘Koshihikari’. These results indicate that SL2033 produced flatter grains; SL2037 produced slenderer grains; and SL3215 produced lighter, slenderer, and flatter grains.

Investigation of the growth phenotypes of culms and panicles in promising CSSLs

There were no significant differences in CL, PL, PN, or PW between each CSSL and ‘Koshihikari’ in both 2020 and 2021 (Supplemental Fig. 3).

Percentage of perfect grains and yield in promising CSSLs under field conditions in different Japanese cities

To investigate the characteristics of SL2033, SL2037, and SL3215 in terms of PPG and yield in other Japanese sites, we cultivated them under field conditions in Tsukubamirai (Ibaraki prefecture), Toyama (Toyama prefecture), and Minamisatsuma cities (Kagoshima prefecture) in 2023 (Supplemental Tables 3, 4). In Tsukubamirai city, all the three CSSLs exhibited a higher PPG and yield (kg/ha) compared to ‘Koshihikari’ as same as in Tsukuba city. In Toyama city, SL2033 and SL2037 exhibited higher PPG, but all the three CSSLs exhibited lower yield compared to ‘Koshihikari’. In Minamisatsuma city, SL2037 and SL3215 exhibited higher PPG, but only SL2037 exhibited higher yield compared to ‘Koshihikari’. These results implicate that performance of the three CSSLs are different depending on the environment conditions despite the qualitatively similar performance in/near Tsukuba city.

Discussion

We screened genetic resources for reduced grain chalkiness. To minimize the impact of environmental factors, we selected the 148 target CSSLs with comparable DTH to that of ‘Koshihikari’ from 342 CSSLs in which chromosome segments of indica, aus, or tropical japonica rice donors are introgressed into the ‘Koshihikari’ genetic background. From the 148 CSSLs, we isolated SL2033 and SL2037, with reduced grain chalkiness without large effects on yield traits; and SL3215, with reduced grain chalkiness, and yield loss due to reduced grain size, under field conditions in Ibaraki prefecture. Although the performance of the three CSSLs were not qualitatively consistent in different environmental conditions, each of them still exhibited higher PPG than ‘Koshihikari’ in different sites across two or three prefectures.

Here, we will discuss the candidate QTLs related to APQs in finally selected CSSLs in this study. SL3215 has chromosome segments at 1 × 10^–6–19.26 Mb and 28.42–29.75 Mb on Chr. 5 introgressed from ‘Naba’; SL2033 has a segment at 1 × 10^–6–13.45 Mb on Chr. 10 from ‘IR64’; and SL2037 has a segment at 1 × 10^–6–4.06 Mb on Chr. 11 from ‘IR64’ (Nagata et al. 2015, 2023). Several QTLs enhancing APQ have been reported on two of these same chromosomes. Ebitani et al. (2008) detected ‘Kasalath’ alleles of QTLs on Chrs. 2, 9, 11, and 12. Although we used ‘Kasalath’ as a donor in our CSSLs, CSSLs with introgressed chromosome segments from ‘Kasalath’ were not selected here because of their lower PWs than ‘Koshihikari’ and different DTH. As the introgressed region in SL3215 has a well-known chalkiness-related QTL, CHALKINESS 5 (LOC_Os05g06480) (Li et al. 2014), it is highly likely that Chalk5 is the causal gene for the improved APQ in SL3215. Two well-known QTLs related to grain weight and size, GRAIN SIZE AND NUMBER 1 (LOC_Os05g02500) and SMALL AND ROUND SEED 3 (LOC_Os05g06280) (Guo et al. 2018, Ngangkham et al. 2018), in the same region are candidates for loci conferring smaller grain size in SL3215. To the best of our knowledge, SL2033 and SL2037 have no known QTLs enhancing APQ in the introgressed chromosome segments.

It may be possible to consider the other candidate genes from the viewpoint of mechanisms affecting chalkiness. For example, preventing sucrose from being degraded and from being used for cellulose biosynthesis in stems enhances APQ of rice grains, because the transport of more sucrose to the endosperm facilitates the synthesis of storage components and results in reduced chalkiness (Shi et al. 2024). The ‘Naba’ segment on Chr. 5 in SL3215 harbors POLYGALACTURONASE-INHIBITING PROTEINs 1 to 4 (Os05g0104200 / 4150 / 4600 / 4700) and RICE STARCH REGULATOR 1 (Os05g0121600). The ‘IR64’ segment on Chr. 10 in SL2033 harbors CELLULOSE SYNTHASE LIKE H1 and F7 (Os10g0341401 / 3400). That on Chr. 11 in SL2037 harbors PECTIN METHYLESTERASE INHIBITORs 37 to 39 (Os11g0132100 / 2300 / 2550), CHALCONE ISOMERASE-LIKE 1 (Os11g0116300) (Lam et al. 2022), and ARABINOFURANOSIDASE 3 (Os11g0131900) (Sumiyoshi et al. 2013). These genes, related to cellulose metabolism or starch accumulation, might be potential candidates for reduced chalkiness in our selected CSSLs.

This study revealed several promising genetic resources available to enhance APQ of rice grains under the field conditions in Japanese sites. Further study revealing how the APQ is enhanced in the finally selected CSSLs will be required to maximize the benefit of introgressed chromosome segments / undetected QTLs when performing gene pyramiding in the future.

Author Contribution Statement

DO conceptualized the research. HF, AF, YN, DG, NK, KM, SK, YK, MT, ST, RI, TS, JY and DO performed the investigations. SF provided the resources. HF and DO performed data curation. JY and DO helped with funding acquisition. HF wrote the manuscript. SF and DO edited the manuscript. All authors have reviewed drafts of the paper and approved the final draft.

 Acknowledgments

We gratefully acknowledge the field support of Yuko Aono and Makiko Suzuki. We are grateful to the technical staff of the Institute of Crop Science for management of the rice fields at NARO. We thank editors from ELSS, Inc. (https://elss.co.jp/en/) for editing our manuscript before submission. This study was supported by JSPS KAKENHI Grant Number JP20H0296 and by the research program on development of innovative technology grants (JPJ007097) from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN).

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