Interaction between genetic regions responsible for the starch properties in non-glutinous rice varieties in Hokkaido, Japan (2024)

Abstract

Starch properties are the major determinants of grain quality and food characteristics in rice (Oryza sativa L.). Understanding the interactions between genetic regions responsible for starch properties will lead to the development of rice cultivars with desirable characteristics. This study investigated the genetic effect and interaction between qAC9.3, a low-amylose quantitative trait locus (QTL), and the genetic region around Starch branching enzyme IIb (SbeIIb). Both these factors are responsible for the starch properties of the Hokkaido breeding population. The amylose content, pasting temperature, and amylopectin chain-length distribution were compared using F₅ lines derived from the cross between the lower amylose content and lower pasting temperature strain ‘Hokkai332 (qAC9.3, SbeIIb)’ and the higher amylose content and higher pasting temperature variety ‘Kitagenki (-, SbeIIb^sr)’. The qAC9.3 genotype exhibited low amylose content and reduced the hardness of boiled rice but increased the ratio of amylopectin long chains and did not alter the pasting temperature. In contrast, the SbeIIb genotype was associated with pasting temperature but did not affect the amylose content and hardness of boiled rice. It was suggested that appropriately selecting genotypes of these genetic regions and QTL would allow the fine-tuning of starch properties of cooked rice suitable for future demand.

Introduction

Starch properties are the major determinants of grain quality and food characteristics in rice (Oryza sativa L.). Amylopectin, which has a highly branched structure, is the major component of starch, whereas the remainder comprises amylose, a linear polymer. In rice, the amylose content (AC) and chain-length distribution of amylopectin branches determine the physicochemical properties of starch and the cooking properties of grains through changes in gelatinisation and starch retrogradation. The rice Waxy (Wx) gene (LOC_Os06g04200) encodes granule-bound starch synthase I (GBSSI), which determines the AC of the endosperm by controlling amylose synthesis (Sano 1984). Natural variations in genotypes such as Wx-a, Wx-b, and wx have been identified as Wx alleles. The AC greatly affects the hardness of cooked rice. A higher AC increases the hardness, whereas a lower AC reduces the hardness. In Japan, a soft texture is considered to indicate good eating quality. In Japan, the taste of rice has been improved by reducing AC through breeding programs (Inatsu 1988). Wx-mq, Wx1-1, and Wx-y alleles that exhibit low AC have been generated and selected through breeding programs (Ando et al. 2010, Chuba et al. 2006, Sato et al. 2002). In addition to the Wx allele, dull (du1-5), qAC2, and qAC9.3 are genes/quantitative trait loci (QTLs) for controlling the AC in rice (Ando et al. 2010, Satoh and Omura 1981, 1986, Takemoto-Kuno et al. 2015, Yano et al. 1988). Some dull mutants induced by N-methyl-N-nitrosourea solution have already been analysed, and the causal genes were identified as Du1 (Zeng et al. 2007) and Du3 (Isshiki et al. 2008). qAC2 and qAC9.3 are considered natural mutations selected in rice breeding programs. The AC of a near-isogenic line carrying qAC2^Kuiku, the Kuiku162 allele of qAC2, was 1.1% points lower than that of the wild type in the genetic background of a japonica cultivar. In addition, qAC2^Kuiku has an epistatic interaction with the Wx allele and du1-3 (Takemoto-Kuno et al. 2015). The qAC9.3, which causes a 2.6% decrease in AC, could be useful for fine-tuning AC in rice breeding programs (Ando et al. 2010). However, it was unclear whether there was any differentiation in the pasting temperature and amylopectin chain-length distribution related to starch properties and how qAC9.3 interacts with other genes responsible for starch properties.

Starch-branching enzymes (BEs) generate amylopectin branches. Plants have two types of BE, BEI and BEII. Mutant BEI, BEIIa, and BEIIb lines have been isolated from rice (Nakamura 2002, Nishi et al. 2001, Satoh et al. 2003a, 2003b). Previously, starch branching enzyme IIb (SbeIIb) was identified on chromosome 2 as a candidate gene for the differences in starch properties between glutinous rice varieties in Hokkaido (Ikegaya and Ashida 2021). SbeIIb is known as an amylose extender (AE) gene. The sbeIIb mutant showed a decreased proportion of the amylopectin short chain (degree of polymerisation [DP] < 17) and an increased proportion of the mid-length chain (DP 18–35) (Nishi et al. 2001), resulting in a higher gelatinisation temperature (Jane et al. 1999). The glutinous variety/line with SbeIIb^sr, the genotype originating from the American variety ‘Cody’, showed a higher pasting temperature (PT) and higher hardness of rice flour paste under the condition of storage at 4°C for 24 h (Ikegaya and Ashida 2021). The chromosomal region, including the SbeIIb^sr genotype, causes changes in the chain-length distribution of amylopectin; the proportions of short (DP 6–24) and long chains (DP 25–60) decreased and increased, respectively. The SbeIIb^sr genotype has been introduced into almost all elite non-glutinous rice cultivars in Hokkaido through a breeding program. However, the effect of the SbeIIb^sr genotype on non-glutinous rice varieties/lines is unknown.

qAC9.3 and SbeIIb^sr are unique genotypes that characterise starch properties in the Hokkaido breeding strain; however, no genetic interactions between these genotypes have been reported. In this study, the non-glutinous rice cultivar ‘Kitagenki’ and breeding strain ‘Hokkai332’ developed in Hokkaido were selected. The cultivar ‘Kitagenki’, which has a SbeIIb^sr genotype, showed extremely high yield potential; however, the eating quality of cooked rice was reduced because of its high AC without qAC9.3. (Ikegaya et al. 2017, Yagioka et al. 2021). Breeding strain ‘Hokkai332’, which contains qAC9.3 and SbeIIb, showed a low AC and good eating quality. The effect of the SbeIIb^sr genotype in non-glutinous rice varieties was characterized, and the effect of qAC9.3 on the amylopectin chain-length distribution ratio was investigated by analysing F₅ progenies derived from the cross between ‘Kitagenki’ and ‘Hokkai332’. Furthermore, the genetic interaction between the SbeIIb genotype and qAC9.3 was evaluated for starch properties. This knowledge may contribute to meeting future demands for breeding varieties with finely tuned starch characteristics.

Materials and Methods

Plant material

The high-yield cultivar ‘Kitagenki’ and breeding strain ‘Hokkai332’ were used as the parental lines to detect the interaction between qAC9.3 and the genetic regions around SbeIIb responsible for starch properties. F₅ lines (n = 94), derived from a cross between ‘Kitagenki’ and ‘Hokkai332’, were investigated in 2020. Sixteen plants from each F₅ line were grown and harvested.

Growth conditions

Cultivation management followed the standard procedures used at the Hokkaido Agricultural Research Center as described by Ikegaya and Ashida (2021). Sowing and transplanting were performed on 20 April and 20 May 2020, respectively. The young leaves were sampled for DNA extraction. Seeds were harvested at the full maturity stage.

Re-sequencing and DNA analysis

Isolation of total DNA and paired-end sequencing using Illumina HiSeq 4000 was performed according to Ikegaya and Ashida (2021). Raw sequence data of ‘Kitagenki’ and ‘Hokkai332’ were deposited in the DDBJ Sequence Read Archive under the accession number DRA017497. Single-nucleotide polymorphism (SNP) markers showing polymorphisms between the parental lines were selected by comparing whole-genome sequences, and genotypes were detected using the Fluidigm EP1 System (https://dnatech.genomecenter.ucdavis.edu/fluidigm-ep1/). A total of 189 SNP markers used at the Advanced Genomics Breeding Section of the Institute of Crop Science, NARO (NICS), covering all 12 chromosomes, were tested, and 166 SNP markers were selected. The single-sequence repeat marker RM23804 was used for genotyping qAC9.3, according to Ando et al. (2010). The CAPS marker SbeIIb Ex3-1 and restriction enzyme BspT107I (HgiC I) were used according to Ikegaya and Ashida (2021) to determine the genotype of the chromosomal region around SbeIIb.

Detection of QTLs

QTL analysis was performed as described in Ikegaya and Ashida (2021).

Analysis of starch properties

AC was analysed according to the method described by Ando et al. (2010). Starch purification, analysis of chain-length distribution, and measurement of pasting properties were performed using a Rapid Visco Analyser (RVA3D+, Newport Scientific Pty Ltd., NSW, Australia), as described in Ikegaya and Ashida (2021). In this paper, apparent AC is referred to as AC.

Hardness of boiled rice: The rice was cooked using a Rapid Visco Analyser (RVA), according to Ashida-Yoshida et al. (2020). Boiled rice was stored with a cover at two different conditions, 30°C for 1 h and 4°C for 24 h. The hardness of boiled rice was measured using a digital force gauge and measurement stand (DST-2N and MX-500N, Imada, Toyohashi, Japan) equipped with a 20 N load cell. A single compression force-versus time program was used to compress boiled rice along the thickness at a test speed of 0.5 mm/s and then returned to its original position. The original clearance between the probe and the bottom of the RVA can in the load cell of the instrument was fixed at 8 mm; therefore, when the probe moved down, the test sample was compressed horizontally on the base to a distance of 6.0 mm. A stainless steel probe (P/5) 10 mm in diameter was used to compress the boiled rice. The test was repeated three times for the same sample, and samples were prepared three times for the parents and all F₅ lines. The peak force was considered as the maximum compressive force/hardness.

Results

AC and PT

The range of days to heading for the F₅ lines, including both parents, was 12 days from July 18 to July 29. Average temperatures for the 20 days after heading were 20.75°C at the lowest on July 21 and 21.88°C at the highest on July 29. The AC and PT of ‘Kitagenki’ and ‘Hokkai332’ were 22.9% and 72.2°C, and 16.5% and 71.2°C, respectively (Table 1). The phenotypic distributions of AC and PT in the F₅ lines were determined (Supplemental Fig. 1). No relationship was found between days to heading and AC (Supplemental Fig. 2). Owing to a lack of materials, the AC of the seven lines could not be measured in the F₅ lines.

QTLs for AC and PT

In total, 189 SNP markers were used to genotype the 12 chromosomes of the F₅ lines. Twenty-seven markers showed statistically significant segregation ratios in the chi-squared test (Supplemental Table 1). These 27 markers were excluded from subsequent analyses. QTL analysis assessed AC and PT (Table 2, Supplemental Table 1). The QTL for AC was mapped only on chromosome 9 between the SNP markers FA6423 and FA0535 in the 7.6 Mbp region, and the nearest marker FA3224 was located 96 kbp from RM23804, an SSR marker for qAC9.3. The QTL for PT was mapped only on chromosome 2 between the SNP markers FA1962 and FA2444 in the 5.6 Mbp region, and the nearest marker FA0803 was located 136 kbp from SbeIIb Ex3-1, a CAPS marker that determines the genotype of the chromosomal region around SbeIIb. The logarithm of the odds scores of the QTLs were 23.2 and 14.1 for AC and PT, respectively. The percentages of phenotypic variation explained were 74.3% and 57.7% for AC and PT, respectively. The additive effects of the ‘Hokkai332’ allele were –1.87 (%) and –0.47 (°C) for AC and PT, respectively. The ‘Hokkai332’ genotype lowered AC and PT (Table 2, Supplemental Fig. 1).

Amylopectin chain-length distribution patterns

The chain-length distribution was analysed to clarify the amylopectin structure of ‘Kitagenki’ and ‘Hokkai332’. The side chains were classified into four groups, according to Hanashiro et al. (1996). In the amylopectin chain-length distribution patterns of ‘Hokkai332’ compared to ‘Kitagenki’, the proportion of side chains with DP 6–12 was significantly increased, and the proportion of side chains with DP 25–36 and DP 37- were significantly decreased (Fig. 1, Table 3). The chain-length distribution of F₅ lines was measured and classified according to the genotype of qAC9.3 and SbeIIb (Fig. 2, Table 4). Strains that were heterozygous for the genotype of qAC9.3 and/or SbeIIb were excluded from subsequent analysis. No significant difference was detected in the side chain proportions of DP 6–12 and DP 13–24 between the lines with and without qAC9.3. The proportions of side chains with DP 25–36 and DP 37- were significantly increased in lines with qAC9.3 (Fig. 2a, Table 4). In lines with the genotype SbeIIb, the proportion of side chains with DP 6–12 was significantly higher than that in the SbeIIb^sr lines. However, the proportion of side chains with DP 13–24 was significantly decreased. No significant differences were detected in the side chain proportions of DP 25–36 and DP 37- (Fig. 2b, Table 4). The F₅ lines with the same genotype as ‘Kitagenki’ (-, SbeIIb^sr) or ‘Hokkai332’ (qAC9.3, SbeIIb) showed AC and PT according to genotype, although with different patterns of parental lines in comparison of amylopectin chain-length distribution (Supplemental Fig. 2, Table 4). AC and PT also corresponded to the genotypes when comparing the genotypes (qAC9.3, SbeIIb^sr) and genotypes (-, SbeIIb) that differed from those of the parental varieties.

Table 3.

HPAEC-PAD fractions of debranched amylopectin from ‘Hokkai 332’ and ‘Kitagenki’

Variety/Strain	DP 6–12 (%)		DP 13–24 (%)	DP 25–36 (%)		DP 37–60 (%)
Hokkai 332	32.9	*	54.4	9.8	*	3.0	***
Kitagenki	32.0		54.5	10.2		3.4

* indicates significant difference (Student’s t-test, * P < 0.05, *** P < 0.001).

Table 4.

Chain length distribution in the F₅ lines

Genotype	Number	AC		PT		Amylopectin chain length distribution
						DP 6–12 (%)		DP 13–24 (%)		DP 25–36 (%)		DP 37–60 (%)
qAC9.3	34	18.2	***	71.6		32.2		54.5		10.1	**	3.2	***
–	45	21.8		71.8		32.4		54.5		10.0		3.1
SbeIIb	45	20.4		71.4	***	32.4	**	54.4	**	10.1		3.1
SbeIIbsr	34	20.4		72.1		32.2		54.6		10.1		3.1
qAC9.3, SbeIIb	23	18.2	***	71.5	***	32.3		54.3	*	10.2	**	3.3	***
-, SbeIIbsr	23	21.8		72.2		32.3		54.5		10.1		3.1
qAC9.3, SbeIIbsr	11	18.3	***	72.1	***	32.1	**	54.6		10.1	**	3.2	**
-, SbeIIb	22	21.8		71.3		32.5		54.4		10.0		3.1

* indicates significant difference (Student’s t-test, * P < 0.05, ** P < 0.01,*** P < 0.001).

‘–’ indicates genotype without qAC9.3.

Hardness of boiled rice

Hardness of boiled rice was markedly higher in ‘Kitagenki’ than in ‘Hokkai332’ at 30°C for 1 h and 4°C for 24 h after cooking (Table 5, Supplemental Table 1). The average hardness of F₅ lines with qAC9.3 (30°C: 4.7N, SD ± 0.4 and 4°C: 12.8N, SD ± 1.5) were significantly lower than lines without qAC9.3 (30°C: 5.5N, SD ± 0.4 and 4°C: 15.0N, SD ± 1.7). Conversely, the average hardness of F₅ lines with SbeIIb^sr (30°C: 5.3N and 4°C: 14.3N) was not significantly higher than that of lines with SbeIIb (30°C: 5.1N and 4°C: 14.1N) (Fig. 3).

Table 5.

Hardness of boiled rice of ‘Hokkai 332’ and ‘Kitagenki’

Variety/Strain	30°C 1 h (N)		4°C 24 h (N)
Hokkai 332	4.2	***	11.3	***
Kitagenki	6.2		15.3

* indicates significant difference (Student’s t-test, *** P < 0.001).

Discussion

The breeding strain ‘Hokkai332’ shows lower AC, PT, and hardness of boiled rice than those of the high-yield cultivar ‘Kitagenki’ (Tables 1, 4). QTL analysis of F₅ lines derived from the cross between ‘Hokkai332’ and ‘Kitagenki’ revealed that differences in AC and PT between ‘Hokkai332’ and ‘Kitagenki’ were caused by qAC9.3 and the genetic region around SbeIIb, respectively. However, phenotypic values in the F₅ population showed continuous variation (Supplemental Figs. 1, 2). The cause could be other minor genetic factors other than on the candidate region. These factors would be able to be detected more accurately by performing a multiple-year QTL analysis and phenotypic evaluation. The interaction between qAC9.3 and the genetic region around SbeIIb, both of which are responsible for starch properties in rice, was investigated (Ando et al. 2010, Ikegaya and Ashida 2021). The average AC of the F₅ lines with qAC9.3 (18.2%) was lower than that of lines without qAC9.3 (21.8%) (Table 4). In contrast, there were no differences in AC caused by the genotype of SbeIIb in this study (Table 4). SbeIIb is known as the AE gene. The AC of the ae mutant was higher than that of the wild type (Nishi et al. 2001). SbeIIb^sr, the ‘Kitagenki’ genotype, decreased the short-chain ratio and increased the long-chain ratio in glutinous rice varieties as well as the ae mutant in the amylopectin chain-length distribution ratio analysis (Ikegaya and Ashida 2021). However, there was no significant effect on the AC in the glutinous rice varieties.

The average PT of F₅ lines with SbeIIb (71.4°C) was lower than that of lines with SbeIIb^sr (72.1°C) (Table 4). Although introducing low-amylose genotypes decreases PT (Satou et al. 2016, Yoshii et al. 1997), there was no significant difference in PT caused by the qAC9.3 genotype (Table 4). The F₅ lines showed different amylopectin chain-length distribution characteristics according to the genotypes of qAC9.3 and SbeIIb. Significant differences were found in DP 25–36 and 37–60 between lines with and without qAC9.3. Although Okuno et al. (1983) reported no significant differences in amylopectin chain-length distribution between glutinous, low-amylose, and non-glutinous rice, differences caused by qAC9.3 were observed. The increase in the amylopectin long-chain ratio by qAC9.3 may mask the decrease in PT due to the low AC. In contrast, in lines with SbeIIb, the proportion of side chains with DP 6–12 was significantly increased compared to that in lines with SbeIIb^sr. These results are the same as those of a previous report using glutinous varieties (Ikegaya and Ashida 2021). However, the proportion of side chains with DP 13–24 was significantly decreased. This chain-length distribution pattern was similar to that of SSIIa (Miura et al. 2018), Sbe1 (Okamoto et al. 2013), and Pho1 (Okamoto et al. 2020).

AC is known to affect the hardness of cooked rice (Yu et al. 2009). In this study, whether the hardness of cooked rice is affected by the qAC9.3 genotype and the genotype of the chromosomal region around SbeIIb was investigated. The results showed that only the qAC9.3 genotype and AC affected the hardness of boiled rice, and the SbeIIb genotype showed a slight but not significant difference in hardness. Significant differences were observed in the change in hardness of glutinous rice flour paste from lines with different SbeIIb genotypes when stored at 4°C for 24 h (Ikegaya and Ashida 2021); however, no differences were observed in the change in hardness of non-glutinous boiled rice. Itayagoshi et al. (2021) reported that AC is related to hardness but not stickiness, whereas PT is related to stickiness. This relationship between PT and stickiness is consistent with the results of previous reports (Li et al. 2016).

Wx gene expression and AC increase with the intensity of low temperatures during ripening (Hirano and Sano 1998). Under climatic conditions of low temperatures during the rice ripening period in Hokkaido, the AC tends to be high. In the Hokkaido rice breeding program, Wx1-1 and qAC9.3 were introduced to reduce AC and improve the taste of cooked rice (Ando et al. 2010). The amylopectin chain-length distribution ratio is known to increase the short-chain ratio at low temperatures during ripening (Igarashi et al. 2008). The reason for the introduction of SbeIIb^sr into Hokkaido rice varieties remains unclear. This was possibly introduced unintentionally because selection for PT and stickiness has not been conducted in rice breeding. The distribution ratio of amylopectin short chains (DP6–12) in the lines with genotypes qAC9.3 and SbeIIb^sr was the lowest among the four genotype groups (Table 4). Both genotypes additively affected the amylopectin chain-length distribution ratio.

The AC and fine structure of amylopectin greatly affect the rice quality after cooking. Here, it was revealed that qAC9.3 decreased AC while increasing the proportion of amylopectin long chains (DP25-) and did not change PT. This finding indicates that it was possible to fine-tune the AC of rice without changing its stickiness, which correlates with the PT. In contrast, SbeIIb^sr increased PT by changing the amylopectin chain-length distribution and did not change AC. It was possible to fine-tune the PT and stickiness of rice without changing its hardness, which correlated with the AC. The qAC9.3 and SbeIIb^sr were detected in Hokkaido rice breeding lines and are expected to be used as breeding materials in different cultivation areas. The association of genotypes with useful phenotypes may be facilitated using local breeding varieties because of their close genetic backgrounds and low incidence of poor traits in the evaluations. Historical studies on the unique traits that characterise elite cultivars in each local region may contribute to developing new cultivars with novel combinations of genes and more desirable agronomic traits.

Author Contribution Statement

Conceived and designed the experiments and wrote the manuscript: TI. Performed the experiments: TI. Analysed the data: TI.

Acknowledgments

We thank C. Ohga and T. Mikuni for their technical assistance and maintenance of paddy fields. This study was supported in part by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and the Food Industry). QTL mapping and sequence analysis was supported by the Advanced Genomics Breeding Section of the Institute of Crop Science, NARO (NICS) (Project ID: 20B03).

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