Cross-locational experiments to reveal yield potential and yield-determining factors of the rice cultivar ‘Hokuriku 193’ and climatic factors to achieve high brown rice yield over 1.2kg m^-2 at Nagano in central inland of Japan

ABSTRACT

Understanding the yield potential and yield-determining factors of recent high-yielding cultivars is essential for further increasing rice yield. In this study, a cross-locational field experiment was conducted across 3 years using ‘Hokuriku 193ʹ (H193), a high-yielding cultivar, at four sites including one in Nagano Prefecture, which is the highest-yielding region in Japan. The highest mean yields of 3 years, 1214 g m⁻² for brown rice grains and 1586 g m⁻² for rough grains, were recorded at the Nagano site. The yields from the 17 environments were strongly correlated with spikelet number per square meter while percentage of filled grain was relatively stable, suggesting that sink capacity is the primary determining factor for grain yield of H193. The climatic factors for high spikelet number at the Nagano site can be explained by the high cumulative radiation before heading associated with longer duration until heading by low night temperature. In addition, a large increase in shoot dry weight during grain filling (ΔW) and high radiation use efficiency (ΔW/rad) at the Nagano site could satisfy large source demand by the large sink size. The high ΔW/rad at the Nagano site associated with low night temperature. This study demonstrated high yield potential of H193 and revealed an environment that achieves extra-high yields in H193, which provided insight to attain further increase in rice yield.

Graphical abstract

KEYWORDS:

• Oryza sativa
• High yield
• Non-structural carbohydrate
• Radiation
• Source-sink balance
• Temperature

Introduction

Rice (Oryza sativa L.) is one of the world’s most important crops (FAO, Godfray et al., 2010). Many high-yielding cultivars have been developed to feed the increasing global population. Understanding the yield potential and yield-determining factors of these cultivars is essential to reveal breeding targets and achieve further increases in yield. In China, a project for development of ‘super rice’ (rice with a super-high yield potential) was launched in 1996 (Wang & Peng, 2017). Many super rice cultivars were developed exploiting intersubspecific (indica and japonica) heterosis. Some of these cultivars have produced grain yields of more than 1500 g m⁻² (Amano et al., 1996; Katsura et al., 2008; Chang et al., 2016). In Japan, many high-yielding inbred cultivars have been developed from hybridization of indica and japonica cultivars, and the majority have a predominantly indica genetic background (Yonemaru et al., 2014). Among these indica-dominant cultivars, ‘Hokuriku 193ʹ (H193) and ‘Takanari’ are the highest-yielding cultivars grown in relatively warm regions in Japan, with more than 1100 g m⁻² recorded for brown rice grains (corresponding to more than 1400 g m⁻² if the ratio of rough grain yield to brown rice yield was 1.29) (Nagata et al., 2016).

The grain yield of rice is determined by the sink capacity (total number of spikelets per unit area × filled grain weight) and the filled grain ratio (percentage of grains that are filled). Yoshinaga et al. (2013) reported that the main factor for the high yield of recently developed high-yielding cultivars was large sink capacity, suggesting the importance of enlarging sink capacity as breeding target. On the other hand, the filled grain ratios of many cultivars often showed negative correlation with sink capacities (Yagioka et al., 2021; Yoshinaga et al., 2013) and some studies using near isogenic or mutant lines reported that increasing sink capacity did not substantially improve yield (Fukushima et al., 2017; Nakano et al., 2017; Ohsumi et al., 2011). In these cases, sink capacities did not determine grain yield. Therefore, understanding whether sink capacity or filled grain ratio (source abundance and/or translocation efficiency) limits yield of each cultivar under diverse conditions was important to achieve further increases in grain yield.

Nagano Prefecture, located in central inland of Japan, is the highest-yielding region for rice production in Japan. The mean farmers’ yield in the most recent 5 years (2016–2020) in Nagano Prefecture (619 g m⁻²) was the highest among Japanese prefectures, whereas the Japanese average brown rice yield was 533 g m⁻² (Ministry of Agriculture, Forestry and Fisheries, 2021). Murata (1964) analyzed the statistical nation-wide yield data published by Ministry of Agriculture, Forestry and Fisheries of Japan. He suggested that the high yield in cool regions, including Nagano Prefecture, partly depends on the high radiation use related to low temperature during grain filling stages. Horie et al. (1997) conducted cross-locational experiment at the sites including Nagano Prefecture using ‘Koshihikari’, a standard Japanese cultivar, and reported that high dry matter production at Nagano Prefecture compared with Kyoto Prefecture was attributed to higher cumulative radiation during the growing season. However, to the best of our knowledge, there are no precise studies investigating the potential yield and yield determining process associated with climatic variables in the sites including Nagano Prefecture at high-yielding level higher than 1000 g m⁻² for brown rice grains using recently developed high-yielding cultivars under high fertilized nitrogen levels.

In the present study, a cross-locational field experiment was conducted at four sites, including one in Nagano Prefecture, in 3 years under high nitrogen fertilizer application rate to reveal the yield potential of H193, selected as a representative high-yielding cultivar, under a favorable environment. Based on the relationships between yield traits related to sink and source capacity and climatic variables before and after heading stage, yield-determining factors of H193 in each environment and the climatic factors for achieving high yielding at Nagano Prefecture were discussed.

Materials and methods

Plant materials and growth conditions

The rice cultivar ‘Hokuriku 193ʹ (H193) and ‘Nipponbare’ were planted in 17 and 11 environments, respectively, at four sites (listed in Table 1) across 3 years with some management differences (Supplemental Table S1). H193 is a Japanese high-yielding indica inbred cultivar. Nipponbare is a standard japonica inbred cultivar and was used as reference. The data for yield, yield components, shoot dry weight, and stem non-structural carbohydrate (NSC) content at the Ibaraki site in 2016 were identical to those reported by Okamura et al. (2018).

Table 1. Details for the experimental sites

Site name	City	Latitude (N)	Longitude (E)	Altitude above sea level (m)
Ibaraki	Tsukubamirai, Ibaraki, Japan	36°00′	140°02′	10
Nagano	Suzaka, Nagano, Japan	36°39′	138°17′	334
Hiroshima	Fukuyama, Hiroshima, Japan	34°50′	133°39′	1
Aichi	Togo, Aichi, Japan	35°11′	137°09′	64

Seedlings (20–24 days old) were transplanted to paddy fields on the days listed in Supplemental Table S1. The plants were grown at a density of 22.2 hills m⁻² (spacing of 15 cm × 30 cm) except in the Aichi site where a density was 23.9–24.7 hills m⁻². The plot sizes were 5.3–15.0 m⁻². The plots were arranged in a randomized block design with three replicates except in the Aichi site where only single plot (>32.0 m²) was employed to accommodate three replicated samplings. Climatic variables at the four sites were measured by a weather station at each experimental station except for the Aichi site. The weather data for the Aichi site was obtained from the nearest weather station of Japan Meteorological Agency (2021), located in Nagoya city. Cumulative temperature and radiation for the period from the day X to day Y were calculated by sum of daily mean values from the day X to the 1 day before Y. The soil in the 10-cm-deep plow layer was sampled in each field of each site before fertilization in 2016. The soil chemical properties shown in Supplemental Table S2 were measured by a commercial service (Katakura and Co-op Agri Corporation, Tokyo, Japan).

Yield and yield components

At maturity, when approximately 85% of grains became yellow, 40–80 plants were air-dried for more than 2 weeks. The yield and yield components were measured in accordance with the procedure of Okamura et al. (2018). In brief, whole grains with the hull attached were weighed to determine the rough grain yield. Half of the rough grains were hulled and weighed to obtain the rough (whole) brown rice yield. The rough brown rice grains were sieved with a grain sorter with a sieve size of 1.6 mm, the retained grains were weighed to calculate the actual brown rice yield. Rough grain yield, rough brown rice yield, brown rice yield, and 1000-grain weight were adjusted to 15% (w/w) moisture content. Sink capacity was estimated as following formula based on Yoshinaga et al. (2013):

Sink capacity = (spikelets number × 1000-grain weight)/1000 (1)

Dry weight, non-structural carbohydrate content, and nitrogen content

At full heading and at maturity, 10–12 successive hills per plot were harvested. The heading date and full-heading date were defined as the dates when approximately 50% and 80%, respectively, of panicles had emerged. The sampling dates for each stage are shown in Supplemental Table S1. The samples were divided into panicles, leaf sheaths + culms (stems), and leaf blades, and then weighed in accordance with the method of Okamura et al. (2018). The divided samples were powdered in a mill for measurement of stem NSC content and shoot nitrogen content. The contents of starch, sucrose, glucose, and fructose in the powdered samples were measured in accordance with the methods of Okamura et al. (2016) using glucoamylase (Toyobo, Osaka, Japan), the F-kit #716,260 (J.K. International, Tokyo, Japan), and a microplate reader (Epoch 2, BioTek, Winooski, VT, USA). The NSC content was calculated as the sum of the contents of these carbohydrates.

The nitrogen content was measured using an elemental analyzer (Flash EA 1112, Thermo Fisher Scientific, Waltham, MA, USA or JM3000CN, J-Science Lab, Kyoto, Japan).

The increase in shoot dry weight during grain filling (ΔW) and potential source adjusted to 15% (w/w) moisture content, were calculated as following formula based on Morita and Nakano (2011):

ΔW = shoot dry weight at maturity − shoot dry weight at full heading (2)

Potential source = (stem NSC content at full heading + ΔW) × 1.15 (3)

The spikelet number per shoot dry weight at full heading (Spik/W_FH), the harvest index, the spikelet number per shoot nitrogen content at full heading (Spik/N), the brown rice yield per radiation until maturity (Y/rad), the shoot dry weight per radiation until maturity (W_M/rad), the spikelet number per radiation until heading (Spik/rad), the shoot dry weight per radiation until full heading (W_FH/rad), ΔW per radiation during grain filling (ΔW /rad), and shoot nitrogen content per radiation until full heading (N_FH/rad) were calculated as:

Spik/W_FH = spikelet number/shoot dry weight at full heading (4)

Harvest index = (brown rice yield × 0.85/shoot dry weight at maturity) × 100 (5)

Spik/N = spikelet number/shoot nitrogen content at full heading (6)

Y/rad = brown rice yield/cumulative radiation until maturity (7)

W_M/rad = shoot dry weight at maturity/cumulative radiation until maturity (8)

Spik/rad = spikelet number/cumulative radiation until heading (9)

W_FH/rad = shoot dry weight at full heading/cumulative radiation until full heading (10)

ΔW/rad = ΔW/cumulative radiation during grain filling (11)

N_FH/rad = shoot nitrogen content at full heading

/ cumulative radiation until full heading (12)

Statistical analysis

The significance of differences between sites was assessed with Tukey’s test after one-way or two-way analysis of variance (ANOVA) using R software (https://www.r-project.org). The one-way and two-way ANOVA were conducted with site or year and site as fixed factors, respectively. The correlation coefficient and regression line were calculated using Microsoft Excel.

Results

Grain yield and yield components

Yield and yield components of Hokuriku 193 (H193) in the 17 environments at four sites for 3 years with some management differences were investigated (Table 2). Rough grain yield ranged from 1020 to 1708 g m⁻², and brown rice yield ranged from 757 to 1305 g m⁻², at the Ibaraki site in 2018 and the Nagano site in 2016, respectively. The spikelet number per square meter was strongly correlated with brown rice yield (Figure 1a). Conversely, the percentage of filled grains was not correlated with brown rice yield and fell within a relatively narrow range (Figure 1b and Table 2): from 78.9% at the Nagano site in 2018 to 94.1% at Aichi in 2017. Although only 11 of 17 environments, we investigated the yield and yield component of Nipponbare, a standard japonica cultivar, and the relationships of brown rice yield with spikelet number and percentage of filled grains were shown (Figure 1c,d). Both spikelet number and percentage of filled grains were significantly correlated with brown rice yield in Nipponbare unlike H193.

Table 2. Yield and yield component

Year	Site/ planting time	Total nitrogen fertilizer (g m^−2)	Panicle number (m^−2)	Spikelets per panicle	Spikelet number (×10^3 m^−2)	Thousand-grain weight (g)	Sink capacity (g m^−2)	Percentage of filled grains (%)	Rough grain yield (g m^−2)	Rough brown rice yield (g m^−2)	Brown rice yield (g m^−2)
2016	Ibaraki	16^a	202	227	45.6	23.1	1054	85.9	1176	912	905
	Nagano	19	385	167	64.0	22.6	1449	90.0	1708	1316	1305
	Hiroshima	19	336	163	54.7	22.0	1201	93.1	1503	1121	1118
	Aichi	19	290	164	47.5	23.1	1098	93.5	1444	1033	1027
2017	Ibaraki	16	290	169	48.7	22.5	1096	85.1	1187	942	933
	Ibaraki	19	352	144	50.5	22.1	1119	83.5	1205	944	935
	Nagano	19	321	178	57.1	23.3	1328	85.6	1483	1153	1138
	Nagano	38	363	172	62.2	23.1	1434	84.6	1579	1233	1214
	Hiroshima	19	351	163	56.9	21.4	1218	86.9	1485	1069	1059
	Aichi	19	321	126	40.5	22.8	924	94.1	1163	878	869
	Ibaraki – April	19	345	172	59.2	22.7	1347	84.4	1474	1159	1136
2018	Ibaraki	16	269	171	45.9	22.1	1015	81.6	1112	852	829
	Ibaraki	19	318	136	43.2	21.9	946	80.0	1020	778	757
	Nagano	19	366	168	61.4	23.4	1435	83.6	1568	1229	1201
	Nagano	38	404	160	64.6	23.6	1527	78.9	1595	1234	1204
	Hiroshima	19	316	176	55.7	21.7	1211	91.6	1514	1115	1109
	Ibaraki – April	19	346	141	48.9	21.8	1067	89.4	1235	959	954
Mean of three years	Ibaraki	19^b	290 c	169 a	46.4 c	22.4 b	1040 c	83.2 c	1134 c	878 c	866 c
	Nagano	19	357 a	171 a	60.9 a	23.1 a	1404 a	86.4 b	1586 a	1232 a	1214 a
	Hiroshima	19	334 b	167 a	55.8 b	21.7 c	1210 b	90.5 a	1500 b	1102 b	1095 b
ANOVA	Year (Y)		**	***	n.s.	n.s.	n.s.	***	*	*	**
	Site (S)		***	n.s.	***	***	***	***	***	***	***
	Y × S		***	***	***	***	**	*	**	**	**

Sink capacity = (spikelet number × 1000-grain weight)/1000; Percentage of filled grains = filled spikelet number/total spikelet number. ^aThe data from Okamura et al. (2018). ^bThe results under the 16 g m⁻² application rate were used in 2016. Values are means (n = 3). Different lower-case letters within a column indicate a significant difference among sites (P < 0.05, Tukey’s test after two-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 1. Relationship of brown rice yield with spikelet number (a, c) and percentage of filled grains (b, d) in Hokuriku 193 (H193) (a, b) and Nipponbare (c, d). Value are means (n = 3). *P < 0.05, ***P < 0.001.

To compare yield under different management practices in H193, the panicle number under 16 g m⁻² total nitrogen fertilizer content with one plant per hill (16 N) was significantly lower than that under 19 g m⁻² with three plants per hill (19 N) at the Ibaraki site in 2018–2019 (Table 3). However, no significant differences in spikelet number per square meter and yields were observed because the spikelet number per panicle was higher under 16 N. Similarly, although panicle number and spikelet number per square meter under 38 g m⁻² with three plants per hill (38 N) were significantly higher than those under 19 N at the Nagano site in 2018–2019, no significant differences in yields were observed because the percentage of filled grains tended to be lower under 38 N. Early transplanting increased yield at the Ibaraki site in 2018–2019 owing to the higher spikelet number per panicle, heavier 1000-grain weight, and higher percentage of filled grains.

Table 3. Comparison of yield under different management practices

Year	Site/planting time	Total nitrogen fertilizer (g m^−2)	Panicle number (m^−2)	Spikelets per panicle	Spikelet number (×10^3 m^−2)	Thousand-grain weight (g)	Sink capacity (g m^−2)	Percentage of filled grains (%)	Rough grain yield (g m^−2)	Rough brown rice yield (g m^−2)	Brown rice yield (g m^−2)
2018– 2019	Ibaraki	16	280	170	47.3	22.3	1056	83.4	1150	897	881
	Ibaraki	19	335	140	46.9	22.0	1033	81.8	1113	861	846
	Year (Y)		n.s.	n.s.	*	n.s.	**	*	**	**	***
	Nitrogen fertilizer (N)		**	**	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
	Y × N		n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
2018–2019	Nagano	19	343	173	59.3	23.3	1382	84.6	1525	1191	1169
	Nagano	38	383	166	63.4	23.3	1480	81.7	1587	1234	1209
	Year (Y)		***	n.s.	*	n.s.	*	*	n.s.	n.s.	n.s.
	Nitrogen fertilizer (N)		***	n.s.	**	n.s.	*	n.s.	n.s.	n.s.	n.s.
	Y × N		n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
2018–2019	Ibaraki	19	335	140	46.9	22.0	1033	81.8	1113	861	846
	Ibaraki-Apr.	19	346	157	54.1	22.3	1207	86.9	1355	1059	1045
	Year (Y)		n.s.	**	***	***	***	n.s.	***	***	***
	Planting time (P)		n.s.	*	***	*	***	***	***	***	***
	Y × P		n.s.	n.s.	n.s.	*	n.s.	**	n.s.	n.s.	n.s.

Sink capacity = (spikelet number × thousand-grain weight)/1000; Percentage of filled grains = filled spikelet number/total spikelet number. Values are means from 2 years. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA).

To examine regional differences in yield, the yield and yield components at sites in Ibaraki, Nagano, and Hiroshima prefectures, where experiments were conducted for 3 years, were compared (Table 2). In this analysis, results under 19 N were used except for those of the Ibaraki site in 2016, for which results under 16 N were used. Given that the yield and yield components, except for panicle number and spikelet number per square meter, did not differ either under 16 N or 19 N as already mentioned (Table 3), the management practice had little or no effect on the results of analysis. Yield and all yield components except spikelet number per panicle showed significant differences among sites (Table 2). Mean rough grain and brown rice yield were highest at the Nagano site, followed by the Hiroshima site, and lowest at the Ibaraki site. Panicle number, spikelet number per square meter, and sink capacity were higher in the same order as that observed for brown rice yield. In contrast, 1000-grain weight was highest at the Hiroshima site, followed by the Nagano site, and lowest at the Ibaraki site.

Yield-related traits

Yield-related traits of H193 in the 17 environments are shown in Tables 4–6. The regional differences were compared in the same manner as the yield and yield components. The shoot dry weight at full heading and maturity were highest at the Nagano site (Table 4). The increase in shoot dry weight during grain filling (ΔW), potential source, sum of ΔW and stem NSC contents at full heading, were higher in the same order as those observed for brown rice yield: Nagano, Hiroshima, and Ibaraki. The spikelet number per shoot dry weight at full heading (Spik/W_FH) and harvest index were not highest at the Nagano site. The shoot nitrogen content at full heading and maturity were higher at the Nagano site than other two sites and there was no regional difference in spik number per nitrogen content at full heading (Spik/N) (Table 5). The leaf dry weight and leaf nitrogen concentration at the Nagano site were not remarkably higher than those at the other two sites (Table 6).

Table 4. Dry matter production-related traits

Year	Site/ planting time		Total nitrogen fertilizer (g m^−2)	Shoot dry weight (g m^−2)		ΔW (g m^−2)	Stem NSC content at full heading (g m^−2)	Potential source (g m^−2)	Spik/WFH (g^−1)	Harvest index (%)
				Full heading	Maturity
2016	Ibaraki		16^a	1511^a	2030^a	519	275^a	934	30.2	37.9
	Nagano		19	2257	3650	1393	270	1957	28.4	30.4
	Hiroshima		19	1814	2489	675	223	1057	30.2	38.2
	Aichi		19	1767	2539	772	202	1146	27.1	34.4
2017	Ibaraki		16	1743	2512	769	134	1062	28.0	31.6
	Ibaraki		19	1786	2773	987	156	1345	28.3	28.6
	Nagano		19	1666	2817	1151	163	1546	34.3	34.3
	Nagano		38	1819	3230	1412	160	1849	34.2	31.9
	Hiroshima		19	1545	2493	948	162	1306	36.9	36.1
	Aichi		19	2070	2561	490	231	849	19.7	28.9
	Ibaraki – April		19	–	2752	–	–	–	-	35.1
2018	Ibaraki		16	1946	2095	148	263	484	23.6	33.6
	Ibaraki		19	2055	2263	208	289	585	21.0	28.4
	Nagano		19	2042	3625	1583	273	2183	30.1	28.2
	Nagano		38	2246	3777	1531	309	2165	28.8	27.1
	Hiroshima		19	1569	2391	822	180	1180	35.5	39.4
	Ibaraki – April		19	1927	2649	722	227	1116	25.6	30.6
Means of three years	Ibaraki		19^b	1784 b	2355 b	572 c	240 a	955 c	26.4 c	32.7 b
	Nagano		19	1988 a	3364 a	1376 a	235 a	1895 a	30.9 b	31.0 b
	Hiroshima		19	1643 c	2458 b	815 b	188 b	1181 b	34.2 a	37.9 a
ANOVA		Year (Y)		***	n.s.	n.s.	***	n.s.	***	**
		Site (S)		***	***	***	***	***	***	***
		Y × S		***	***	***	**	***	***	***

ΔW = shoot dry weight at maturity − shoot dry weight at full heading; NSC = non-structural carbohydrate; Potential source = (ΔW + Stem NSC content at full heading) × 1.15; Spik/W_FH = spikelet number/shoot dry weight at full heading; Harvest index = (brown rice yield × 0.85/shoot dry weight at maturity) × 100; ^aThe data from Okamura et al. (2018).^bThe results under the 16 g m⁻² application rate were used in 2016. Values are means (n = 3). Different lower-case letters within a column indicate a significant difference among sites (P < 0.05, Tukey’s test after two-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

Table 5. Shoot nitrogen concentration and content

Year	Site/ planting time		Total nitrogen fertilizer (g m^−2)	Shoot nitrogen concentration (%)		Shoot nitrogen content (g m^−2)		Spik/N (g^−1)
				Full heading	Maturity	Full heading	Maturity
2016	Ibaraki		16	0.89	1.02	13.4	20.7	3399
	Nagano		19	1.10	1.02	24.8	37.1	2578
	Hiroshima		19	1.13	0.86	20.5	21.5	2670
	Aichi		19	0.94	1.01	16.7	25.7	2847
2017	Ibaraki		16	1.32	1.03	23.0	26.0	2113
	Ibaraki		19	1.21	0.93	21.6	25.9	2340
	Nagano		19	1.27	0.78	21.2	21.9	2695
	Nagano		38	1.49	0.96	27.2	31.0	2289
	Hiroshima		19	1.22	0.93	18.8	23.2	3023
	Aichi		19	1.16	0.90	24.0	23.0	1688
	Ibaraki – April		19	–	–	–	–	-
2018	Ibaraki		16	0.88	0.67	17.2	14.1	2671
	Ibaraki		19	1.03	0.75	21.2	17.1	2038
	Nagano		19	1.16	0.78	23.7	28.3	2596
	Nagano		38	1.17	0.94	26.2	35.3	2466
	Hiroshima		19	1.40	0.92	22.0	22.0	2530
	Ibaraki – April		19	1.03	0.58	19.9	15.3	2455
Means of three years	Ibaraki		19^a	1.04 b	0.90 a	18.7 b	21.2 b	2649 n.s.
	Nagano		19	1.18 ab	0.86 a	23.2 a	29.1 a	2638 n.s.
	Hiroshima		19	1.25 a	0.90 a	20.4 ab	22.2 b	2747 n.s.
ANOVA		Year (Y)		*	**	n.s.	*	*
		Site (S)		**	n.s.	*	***	n.s.
		Y × S		n.s.	*	*	***	n.s.

Spik/N = spikelet number/shoot nitrogen content at full heading. ^aThe results under the 16 g m⁻² application rate were used in 2016. Values are means (n = 3). Different lower-case letters within a column indicate a significant difference among sites (P < 0.05, Tukey’s test after two-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

Table 6. Leaf nitrogen concentration and content at full heading

Year	Site/ planting time	Total nitrogen fertilizer content (g m^−2)	Leaf dry weight (g m^−2)	Leaf nitrogen concentration (%)	Leaf nitrogen content (g m^−2)
2016	Ibaraki	16	293	2.16	6.3
	Nagano	19	466	2.27	10.6
	Hiroshima	19	446	2.40	10.7
	Aichi	19	387	2.02	8.0
2017	Ibaraki	16	437	2.58	11.3
	Ibaraki	19	474	2.36	11.3
	Nagano	19	351	2.75	9.7
	Nagano	38	385	3.04	11.7
	Hiroshima	19	406	2.41	9.8
	Aichi	19	548	2.36	13.1
	Ibaraki-Apr.	19	-	-	-
2018	Ibaraki	16	405	2.07	8.4
	Ibaraki	19	519	2.16	11.4
	Nagano	19	474	2.44	11.6
	Nagano	38	496	2.49	12.4
	Hiroshima	19	414	2.84	11.8
	Ibaraki-Apr.	19	426	2.28	9.8
Means of three years	Ibaraki	19^a	429 n.s.	2.23 b	9.7 n.s.
	Nagano	19	430 n.s.	2.49 a	10.6 n.s.
	Hiroshima	19	422 n.s.	2.55 a	10.8 n.s.
ANOVA	Year (Y)		**	*	*
	Site (S)		n.s.	**	n.s.
	Y × S		***	*	*

^aThe results in 16 g m⁻² were used in 2016. Values are means (n = 3). The difference letters indicate the significant differences among site (P < .05, Tukey’s test after two-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

The relationship between potential sources and sink capacities in H193 is shown in Figure 2. The potential sources were higher than the sink capacities at the Nagano site in all years and fertilized nitrogen levels (black and white square symbols).

Figure 2. Relationship between sink capacity and potential source. Values are means (n = 3).

Growth duration and climatic variables

The mean growth durations at the three sites in 3 years are shown in Table 7. The total growth duration was longer at the Nagano site than the other two sites by the longer days to heading and grain filling duration.

Table 7. Mean growth duration at the three sites as mean values of 3 years

Site	Total nitrogen fertilizer (g m^−2)	Total growth duration (d)	Days to heading (d)	Grain filling duration (d)
Ibaraki	19^a	141 b	87.8 b	53.6 b
Nagano	19	161 a	97.1 a	63.9 a
Hiroshima	19	135 b	83.8 b	51.6 b

Total growth duration is days from transplanting to maturity. Days to heading is days from transplanting to heading. Grain filling duration is days from heading to maturity. ^aThe results in 16 g m⁻² were used in 2016. Values are means of three years. Different lower-case letters within a column indicate a significant difference among sites (P < 0.05, Tukey’s test after one-way ANOVA).

The climatic variables at the three sites in 3 years are shown in Table 8. The daily mean temperature was the lowest at the Nagano site through growing period mainly due to lower minimum temperature. The mean temperature after heading was remarkably higher at the Hiroshima site than the other two sites. The mean radiation until heading was higher at the Nagano site, while that after heading had no significant difference from the other two site. The cumulative radiations until and after heading were both higher at the Nagano site than the other two sites mainly due to extended duration of the grain filling.

Table 8. Mean climatic variables at the three sites as mean values of 3 years

Stage	Site	Total nitrogen fertilizer (g m^−2)	Mean temperature (°C)	Mean maximum temperature (°C)	Mean minimum temperature (°C)	Cumulative temperature (°C)	Mean radiation (MJ m^−2 d^−1)	Cumulative radiation (MJ m^−2)
Transplanting to maturity	Ibaraki	19^a	23.8 b	28.3 b	20.0 a	3370 n.s.	16.2 c	2287 b
	Nagano	19	21.4 c	27.9 b	14.9 b	3441 n.s.	18.7 a	3010 a
	Hiroshima	19	25.3 a	29.9 a	21.2 a	3429 n.s.	17.4 b	2360 b
Transplanting to heading	Ibaraki	19^a	24.0 a	28.8 n.s.	20.4 a	2107 n.s.	18.1b	1585 b
	Nagano	19	22.1 b	29.0 n.s.	15.5 b	2145 n.s.	21.7 a	2104 a
	Hiroshima	19	25.0 a	29.6 n.s.	20.8 a	2093 n.s.	18.8 b	1577 b
Heading to Maturity	Ibaraki	19^a	23.6 b	27.5 b	19.4 a	1263 n.s.	13.1 b	701 b
	Nagano	19	20.3 c	26.1 b	14.1 b	1297 n.s.	14.2 ab	905 a
	Hiroshima	19	25.9 a	30.3 a	22.0 a	1336 n.s.	15.2 a	783 b

^aThe results in 16 g m⁻² were used in 2016.Values are means of three years. Different lower-case letters within a column indicate a significant difference among sites (P < 0.05, Tukey’s test after one-way ANOVA). n.s. not significant.

Relationships between climatic variables and yield-related traits

The relationships between climatic variables and spikelet number in the 17 environments are shown in Figure 3. The daily mean and minimum temperature until heading showed significantly negative correlation with spikelet number. The daily mean and cumulative radiation until heading showed significantly positive correlation with spikelet number. The days to heading also negatively correlated with minimum temperature until heading (Figure 4). During grain filling, increase of shoot dry weight (ΔW) showed negative correlation with mean and minimum temperature and positive correlation with cumulative radiation (Figure 5). There was no significant correlation between ΔW and daily mean radiation.

Figure 3. Relationship between climatic variables and spikelet number. Values are means (n = 3). **P < 0.01, ***P < 0.001.

Figure 4. Relationship between mean minimum temperature until heading and days to heading. Values are means (n = 3). ***P < 0.001.

Figure 5. Relationship between climatic variables and ΔW. ΔW = The increase of shoot dry weight during grain-filling. Values are means (n = 3). **P < 0.01, ***P < 0.001.

As indices for radiation use efficiency, the ratios of yield and yield-related traits to cumulative incident radiation and shoot nitrogen content were calculated and regional difference at the tree site was shown in Table 9. Among the three sites, the W_M/rad and ΔW/rad were highest at the Nagano site. Y/rad and Spik/rad were higher at the Hiroshima site than at the other two sites. The W_FH/rad was highest at the Ibaraki site, followed by the Hiroshima site, and lowest at the Nagano site, which was the opposite order observed for yield. No significant differences in N_FH/rad was observed. The relationships between minimum temperature during grain filing and ΔW/rad in the 17 environments are shown in Figure 6. They showed significantly negative correlation.

Table 9. Indices for radiation use efficiency at the three sites as mean values of 3 years

Site/planting time	Total nitrogen fertilizer (g m^−2)		Y/rad (g MJ^−1)			WM/rad (g MJ^−1)	Spik/rad (MJ^−1)	WFH/rad (g MJ^−1)	ΔW/rad (g MJ^−1)	NFH/rad (mg MJ^−1)
Ibaraki	19^a		0.380 c			1.03 b	29.3 b	1.08 a	0.98 b	11.3 n.s.
Nagano	19		0.404 b			1.12 a	28.9 b	0.92 c	1.79 a	10.8 n.s.
Hiroshima	19		0.465 a			1.04 ab	35.4 a	1.02 b	1.22 b	12.7 n.s.
ANOVA		Year (Y)		***	n.s.		*	***	*	**
		Site (S)		***	*		***	***	***	n.s.
		Y × S		***	***		*	***	***	***

Y/rad = brown rice yield/cumulative radiation until maturity; W_M/rad = shoot dry weight at maturity/cumulative radiation until maturity; Spik/rad = spikelet number/cumulative radiation until heading; W_FH/rad = shoot dry weight at full heading/cumulative radiation until full heading; ΔW/rad = ΔW/cumulative radiation during grain filling; N_FH/rad = shoot nitrogen content at full heading/cumulative radiation until full heading; ^aThe results under the 16 g m⁻² application rate were used in 2016. Different lower-case letters within a column indicate a significant difference among sites (P < 0.05, Tukey’s test after two-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant.

Figure 6. The relationship between mean minimum temperature during grain filing and ΔW/rad. ΔW = The increase of shoot dry weight during grain-filling; ΔW/rad = ΔW/cumulative radiation during grain filling. **P < 0.01.

Discussion

Highest yield of ‘Hokuriku 193ʹ: 1708 g m⁻² for rough grain yield

The highest yields of 1305 g m⁻² for brown rice yield and 1708 g m⁻² for rough grain yield were recorded at the Nagano site in 2016 (Table 2). In previous studies, the highest yields in Japan recorded in a field experiment were 1173 g m⁻² for ‘Takanari’ and 1131 g m⁻² for H193 for rough brown rice yield at Fukuyama city, Hiroshima Prefecture (Nagata et al., 2016), to the best of our knowledge. The present record of H193 in Nagano Prefecture was higher than all the yield records obtained in selected previous studies (Table 10). These studies used the different cultivars from the present study or applied lower nitrogen fertilizer. Therefore, it is considered that the present highest yield at Nagano in Japan in this study was achieved by using H193 partly due to high fertilizer input.

Table 10. Summary of previous studies at high-yielding regions including Nagano Prefecture

Year	Site	Grain yield^a (g m^2)	Cultivar name	Cultivar type	Total nitrogen fertilizer content (g m^−2)	Mean temperature (°C)^b	Mean Radiation (MJ m^−2 day^−1)^b	Method for Filled-grain selection	Reference
2016	Nagano, Japan	1645	H193	indica inbred	19	22	19	calculated from rough grain^c	Present study
1992	Nagano, Japan^d	968	Koshihikari	japonica inbred	20	20^e	16	specific gravity (>1.06 g m^3)	Horie et al., 1997
2002	Nagano, Japan^d	919	Takanari	indica inbred	no data	22^e	no data	calculated from rough grain^c	Yoshida & Horie, 2009
2008–2010 ^f	Nagano, Japan	1333	H193	indica inbred	10	21 ^g	19 ^g	calculated from brown rice^h	Sakai et al., 2011
2017	Nagano, Japan	1306	Oonari	indica inbred	19	21 ^g	19 ^g	calculated from brown rice^h	Arai-Sanoh et al., 2020 Arai-Sanoh, Okamura, Hosoi et al., 2020
1985	Kafl El Shak, Egypt	1571	Giza 172	Egyptian local	15	25	26	specific gravity (>1.06 g m^3)	Namba, 2003
1991	Yanco, Austraria	1341	Koshihikari	japonica inbred	13	22^i	24	specific gravity (>1.06 g m^3)	Horie et al., 1997
1994	Yunnan, China	2005	Yu-Za 29	japonica hybrid	14	23	20	wind selection	Amano et al., 1996
2003	Yunnan, China	1589	Liangyoupeijiu	indica hybrid	28	25	17	calculated from rough grain^c	Katsura et al., 2008
2003	Yunnan, China	1454	Takanari	indica inbred	28	25	17	calculated from rough grain^c	Katsura et al., 2008
2013	Ghuizhou, China	1409	Y-liangyou 1	indica hybrid	23	23 ^j	21	specific gravity (>1.00 g m^3)	Jiang et al., 2016

^a14% Moisture. ^bMean during the growing season. ^cRough grain yield was multiplied by 0.963, which is the mean ratio of wind selected filled-grain yield to rough grain obtained from the present data (n = 24). ^dThe different site from the present study, located at Minamiminowa village. ^eMean from May to September in the nearest weather station of Japan Meteorological Agency (2021), located in IIda city. ^fOnly the mean for three years was obtained. ^gMean from May to September in the nearest weather station of Japan Meteorological Agency (2021), located in Nagano city. ^hBrown rice yield was multiplied by 1.289, which is the mean ratio of wind selected filled-grain yield to brown rice obtained from the present data (n = 24). ⁱCalculated by Namba (2003). ^jMedian between maximum and minimum temperature.

Many field experiments have been conducted in various regions regarded as a high-yielding region for rice production worldwide using inbred and hybrid cultivars and the results are summarized in Table 10. The present result at the Nagano site in 2016 with H193 is the second-highest record among these studies, suggesting high yield potential of H193, an inbred cultivar, under favorable conditions.

Yield determining factor for H193

While it was reported that the main factor for the high yield of recently developed high-yielding cultivars was large sink capacity (Yoshinaga et al., 2013), some recently developed cultivars with a large sink capacity show poor grain-filing even if the source abundance is adequate as a result of low translocation efficiency of carbohydrate (Okamura et al., 2018; 2021). It is therefore important to understand the determinant mechanism of the percentage of filled grains in each cultivar. In this study with H193, the spikelet number per square meter was strongly correlated with brown rice yield but not with the percentage of filled grains (Figure 1a,b). Meanwhile, both spikelet number and the percentage of filled grains showed moderate correlation with brown rice yield in Nipponbare, a standard japonica cultivar (Figure 1c,d), suggesting the grain filling of H193 was more stable than that of Nipponbare and the sink capacity is the primary limiting factor for grain yield of H193.

To investigate the reason for the stable grain filling in H193, the relationships between sink capacity (product of spikelet number and single grain weight) and potential source for grain filling (sum of ΔW and stem NSC content at heading) are shown in Figure 2. The potential sources were adequate to sink capacity in most of the environments, suggesting the stable grain filling was supported by a high source ability of H193. High biomass production of H193 obtained in this study with the highest value 3650 g m⁻² recorded in 2016 in the Nagano site reflected a high potential of source ability of H193. High source ability with high photosynthetic activity and translocation of NSC is generally observed in indica-dominant high-yielding cultivars (Hirasawa et al., 2010; Ohsumi et al., 2007; Yoshinaga et al., 2013). Grain filling percentage was low <85% in some years and locations for unclarified reasons (Table 2). A further investigation is required to clarify the possibility of involvement of sterility caused by low assimilation around flowering (Kobata et al. 2017).

The reason for high spikelet number of H193 at Nagano prefecture

Although the previous study with Koshihikari showed higher dry matter accumulation in Nagano Prefecture was responsible for higher cumulative radiation during growing season (Horie et al., 1997), the effect of radiation and other climatic factors during growing periods on yield determining process remain ununderstood, especially in recently developed high-yielding cultivars. In the present study with H193, not only cumulative radiation until maturity, but also the shoot dry weight at maturity per radiation (W_M/rad) were the highest at the Nagano site (Tables 8 and 9). This result suggests that the reason for high yielding of H193 at the Nagano site cannot be explained only by cumulative radiation during the whole growing season. Since we showed a primary yield-determining factor for H193 is the sink capacity in the above section, we analyzed the relationships between sink production and the climatic variables until heading separately from grain-filling period.

Until heading, mean daily temperature was lowest at the Nagano site mainly due to the lowest minimum temperature (Table 8). Mean and cumulative radiation were highest at the Nagano site. These climatic variables were closely correlated with spikelet number in the 17 environments (Figure 3). The correlation coefficient was higher in cumulative radiation than mean radiation (Figure 3c,d), and the spikelet number per cumulative radiation until heading (Spik/rad) at the Nagano site were not higher than those at the other two sites (Table 9). These results indicated that a primarily important environmental determining factor for spikelet number of H193 is probably cumulative radiation until heading. Given that the latitude of the experimental sites and the transplanting days were similar among all environments, the day length during the vegetative period differed negligibly. In these environments, the days to heading was determined largely by temperature because the phenological development of a cultivar is well explained by two environmental factors: temperature and day length (Horie & Nakagawa, 1990). In support of the present results, no significant difference in cumulative temperature until heading was observed among experimental sites (Table 8) and mean minimum temperature until heading was strongly correlated with days to heading (Figure 4). Therefore, not only higher mean radiation but also the long duration to heading caused by lower minimum (night) temperature were responsible for the higher spikelet number at the Nagano site as compared with other sites. Somewhat similar trends were reported by Ying et al. (1998) who compared yield between subtropical condition at Yunnan, China and tropical conditions at IRRI, the Philippines, using high-yielding cultivars with high N rate (20 g m⁻² in total) and showed that the much higher yield in Yunnan than IRRI was mainly related to longer duration in Yunnan associated with lower minimum temperature, but not maximum temperature, which led to larger panicle number and sink capacity (spikelet number per area).

Cool night temperature alone may increase spikelet number because night-time warming is considered to cause declines in yield and biomass owing to complex reasons, such as increase in respiration loss, early leaf senescence, and other factors (Sadok & Jagadish, 2020). Estimations of yield and/or biomass loss in response to night-time warming in previous studies have varied widely: 0–10% decrease in yield for each 1°C increase in night temperature (Lyman et al., 2013; Peng et al., 2004; Peraudeau et al., 2015). Shoot dry weight at full heading was increased by 2.3% for each 1°C decrease in daily minimum temperature on average in a comparison of the Nagano and Ibaraki sites in the present study (Tables 4 and 8). While the high biomass production at heading was one of factors for high spikelet number under high cumulative radiation at Nagano, biomass production may not be only determinant factor for spikelet number since spikelet number per shoot dry weight at full heading (Spik/W_FH) varied by locations (Table 2) and correlation between biomass and spikelet number was not high (r = 0.205, p = 0.430, data not shown). High nitrogen uptake at heading is also considered to be related to the high spikelet number at Nagano (Table 5). However, nitrogen uptake could not totally explain variation in spikelet number since its correlation with spikelet number was not very high including all environments (r = 0.571, p = 0.021, data not shown). It would be interesting to further examine the direct effect of a cool night temperature on spikelet number.

The reason for high dry matter production during grain filling of H193 at Nagano prefecture

The high yields at the Nagano site were achieved by not only high spikelet number but also adequate source abundance to fill them (Figure 2). There were regional differences in potential source (sum of ΔW and stem NSC content at heading) mainly due to the differences of dry matter production after heading (ΔW) (Table 4). During grain filling, the Nagano site showed the lowest daily mean temperature due to the lowest minimum temperature and the highest cumulative radiation owing to the long grain-filling duration despite that the mean radiation was not so high at the Nagano site (Tables 7 and 8). The mean and minimum temperature and cumulative radiation were correlated with ΔW (Figure 5). It is well known that the grain-filling duration was determinant by cumulative temperature after heading (Arai-Sanoh, Okamura, Mukouyama et al., 2020; Takahashi et al., 2007) and cumulative temperature after heading had no regional differences in this study (Table 8), when the maturity was judged by grain color. Therefore, one possible reason for high ΔW at the Nagano site was as follows. The low minimum temperature prolonged the grain-filling duration. Then, the high cumulative radiation caused by prolonged grain-filling duration was one reason for high ΔW.

However, ΔW per radiation during grain filling (ΔW/rad) was also much higher at the Nagano site than at the other sites (Table 9), indicating that efficient utilization of radiation during grain filling supported high source abundance at the Nagano site as well as the high cumulative radiation. One possible reason for the high ΔW/rad at the Nagano site is the direct effect of cooler night temperature to the biomass production similar to that before heading (Table 7) because ΔW/rad was correlated with minimum temperature (Figure 6). The further study to estimate the effect of cool night temperatures to mitigate biomass loss due to respiration quantitatively is needed.

The additional factors for the high biomass production at the Nagano site should not be ignored. For example, radiation interception ratio by canopy should be considered. Although leaf dry weight and leaf nitrogen content at full heading were not higher at the Nagano site (Table 6), it is possible that the difference of plant architecture affects radiation use efficiency (San et al., 2018; Yang & Hwa, 2008) and further analysis for canopy structure was needed. The shoot nitrogen content was highest at the Nagano site, especially at maturity (Table 5), resulting highest nitrogen absorption during grain filling at the Nagano site (2.5 g m⁻², 5.9 g m⁻², 1.8 g m⁻², at the Ibaraki, Nagano and Hiroshima sites, respectively). This might lead to delayed leaf senescence followed by high ΔW/rad at late grain filling, although no significant differences in leaf nitrogen content at full heading and maturity were observed (data not shown), and a further time-course analysis of leaf nitrogen content is needed. This delayed leaf senescence might be associated with the differences in soil characteristics (Supplemental Table S2) to affect root activity such as N uptake. In addition, photosynthesis rates in diverse plants, including rice, are affected by the sink–source balance owing to feedback regulation from carbohydrate (Fabre et al., 2019; Kasai, 2008; Sugiura et al., 2019). Therefore, the possibility that the high sink capacity at the Nagano site enhanced photosynthesis, which was responsible for the high ΔW/rad, cannot be denied. If so, the increase in sink capacity may be directly linked to improvement in yield at the other sites. Further studies are needed to test whether genetic increase in sink capacity enhances dry matter production at these sites. As discussed here, high yield at the Nagano site in H193 were probably achieved by several interactions of plant characters of H193, such as phenological development, tiller and spikelet formation, dry matter productivity, N uptake and others, and climatic factors inferred from the results in this study. To quantitatively estimate the effect of these factors on yield determination quantitatively, sensitivity analysis using crop growth modeling (such as Yoshida & Horie, 2009, 2010) would be a valuable option in future study.

Yield determining factors in other sites

The relationship between spikelet number and cumulative radiation until heading was plotted (Figure 3d). Although the greater spikelet number at the Nagano site (square symbols) and the early planting at the Ibaraki site (grey-colored circle symbols) could be explained by cumulative radiation, it was not the case at the Hiroshima site (triangle symbols). At the Hiroshima site, which was the second-highest-yielding site in the present study (Table 2), the Spik/rad was highest whereas W_FH/rad was lower than that at the Ibaraki site (Table 9). These results indicated that additional environmental and/or cultural factors limit spikelet number of H193 as well as cumulative radiation. Given that the difference of shoot nitrogen content per radiation until full heading (N_FH/rad) between the Ibaraki site and the Hiroshima site tended to be larger than that of spikelet number per shoot nitrogen content at full heading (Spik/N) (Tables 5 and 9, 12.3% and 3.7% larger in the Hiroshima site, respectively), this unknown factor responsible for higher Spik/rad at the Hiroshima site (Table 9) may be associated with nitrogen absorption. Spik/W_FH was also highest in Hiroshima indicating efficient sink production in this site, which possibly resulted in high harvest index (Table 4). It is possible that the differences in timing of application or type of fertilizer (Supplemental Table S1) were related to the higher Spik/rad and Spik/W_FH. The difference in the nitrogen mineralization characteristics of soil to release available nitrogen among sites might be also involved as indicated in Table S2. There may be a research opportunity to optimize nitrogen supplying patterns and amount for attaining maximum Spik/rad in H193. In any case, the present results suggested that increasing Spik/rad was an effective strategy to increase yield of H193 where radiation is limited. On the other hand, there were limited regional difference in Spik/N among sites (Table 9), which is consistent with previous finding that the number of differentiated spikelets of a cultivar is well explained by nitrogen content of the plant at 2 weeks before heading and this efficiency in spikelet differentiation per nitrogen is largely affected by genotypic factor (Yoshida et al., 2006). Therefore, it is also useful to seek to the increase the Spik/N by breeding for further increasing yield potentials. Since the 1000-grain weight showed less variation among environments than spikelet number in the current study (Table 2), breeding to increase sink capacity by increasing grain size might be also effective.

Conclusion

The results of cross-locational experiments achieved extra high yield, over 1.2 kg m⁻² on three-year average, in brown rice yield, and suggest that sink capacity is the primary determining factor for grain yield of H193 rather than grain filling rate. The stable grain filling was supported by the high source ability of H193. Therefore, a breeding target for further increase in grain yield of H193 is improvement in grain number or grain size under favorable conditions such as Nagano. The high sink capacity at the Nagano site can be explained by the high cumulative radiation before heading. In addition, high dry matter production per incident radiation after heading stage at the Nagano site could satisfy large source demand by the large sink size. Although this high radiation use efficiency at the Nagano site associated with low night temperature, further studies are needed to clarify the factors attributed to regional differences in dry matter production. This study has revealed attainable yield of H193 and a climatic factor that enables extra-high yield production in rice, and provides valuable information to attain further increases in rice yield.

Acknowledgments

We express our gratitude to the staff of the research support section at all the sites and the technical support staff of our laboratories.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Cabinet Office, Government of Japan, Cross-ministerial Strategic Innovation Promotion Program (SIP), ‘Technologies for creating next-generation agriculture, forestry and fisheries’ (funding agency: Bio-oriented Technology Research Advancement Institution, NARO) and by JSPS KAKENHI Grant no. JP20H02965.