Abstract
Understanding the yield attributes of the popular rice (Oryza sativa L.) hybrids can provide useful information for developing new hybrid rice varieties. Field experiments were conducted at two subtropical sites (Hengyang and Xingyi) in two years (2021 and 2022) to compare grain yield and yield attributes of three currently popular hybrid rice varieties (Jingliangyouhuazhan, Jingliangyou 534, and Longliangyouhuazhan) with three representative super hybrid rice varieties (Y-liangyou 1, Y-liangyou 2, and Y-liangyou 900). No significant differences in grain yield were observed between the group of popular hybrids and the group of super hybrids at Hengyang and Xingyi in 2021 or at Xingyi in 2022, but at Hengyang in 2022, the group of popular hybrids produced a 15% higher grain yield. The grain yield at Hengyang in 2022 was lower than that at Hengyang and Xingyi in 2021 and at Xingyi in 2022. At Hengyang in 2022, the group of popular hybrids had 9–15% higher panicles per m2, spikelet filling percentage, and harvest index but similar spikelets per panicle and total biomass production and 12% lower grain weight compared to the group of super hybrids. Correlation plot analysis showed that grain yield was significantly related to panicles per m2 but not other yield attributes across six varieties at Hengyang in 2022. These results indicated that the currently popular hybrid rice varieties had higher yield performance than the representative super hybrid rice varieties under the condition of relatively lower productivity, and the key factor for this higher yield performance in the popular hybrids was the higher panicles per m2. This finding highlights that more attention should focus on the yield performance under medium- and low-productivity conditions in hybrid rice breeding programs, and an effective breeding strategy is to select the varieties with high panicle numbers.
1. Introduction
Rice is the most important staple food in China, providing energy and nutrition for about 65% of the population [1]. To ensure national food security, a super rice breeding program was initiated by the Chinese Ministry of Agriculture in 1996 [2]. Professor Longping Yuan proposed a strategy to breed super rice varieties by combining heterosis utilization with morphological improvement [3], and his team has developed multiple super hybrid rice varieties, including three representative ones: Y-liangyou 1, Y-liangyou 2, and Y-liangyou 900 [4].
However, although the three representative super hybrid rice varieties have been cumulatively grown on more than 3 million ha (https://www.ricedata.cn/variety, accessed on 18 December 2023), they are not currently popular hybrid rice varieties in China. In contrast, Jingliangyouhuazhan, Jingliangyou 534, and Longliangyouhuazhan have been the top three hybrid rice varieties by annual planting area in China since 2018 [5]. There is, however, limited information available on why these varieties are more popular than the representative super hybrid rice varieties. Better yield performance may be a reason, but investigations are needed to confirm this possibility. In addition, a further understanding of the yield attributes of the popular hybrids can provide useful information for developing new hybrid rice varieties.
Rice grain yield is determined by four components: panicles per m2, spikelets per panicle, spikelet filling percentage, and grain weight [6]; it also can be expressed as a function of biomass production and harvest index [7]. Previous studies have demonstrated that super hybrid rice varieties produce over 10% higher grain yield than ordinary hybrid and inbred rice varieties, and the higher grain yield of super hybrid varieties is attributable to both higher spikelets per panicle and higher biomass production [8,9].
The gain yield and yield attributes of rice are also affected by the environment. Although it has been well documented that rice crops grown in high-yielding environments generally have high biomass production [9,10,11,12], no consistent conclusion is obtained regarding the key yield components for the difference in grain yield of rice between environments, and different studies have highlighted different components or combinations of components as being responsible. For example, Li et al. [10] reported that higher panicles per m2, spikelet filling percentage, and grain weight were responsible for higher grain yields of rice in the subtropical environment of Taoyuan, China compared to the subtropical environment of Nanjing, China. Katsura et al. [11] observed that the higher grain yield of rice in the subtropical environment of Yunnan, China compared to the temperature environment of Kyoto, Japan was attributable to higher panicles per m2 and spikelets per panicle. Ibrahim et al. [12] found that higher spikelets per panicle were responsible for the higher grain yield of rice in the subtropical environment of Changsha, China than in two tropical environments in Gazipur and Habiganj, Bangladesh.
In this study, grain yield, yield components, biomass production, and harvest index were compared between three currently popular hybrid rice varieties and three representative super hybrid rice varieties at two subtropical sites in two years. The objective of this study was to determine whether the yield performance is better in the popular hybrids than in the super hybrids, and if so, which yield attributes are responsible for the better yield performance in the popular hybrids.
2. Materials and Methods
2.1. Sites and Soils
Field experiments were conducted at Hengyang (26°52′31″ N, 112°30′07″ E, 73 m a.s.l.), Hunan Province and Xingyi (25°01′14″ N, 104°55′45″ E, 1165 m a.s.l.), Guizhou Province, China, in 2021 and 2022. Both experimental sites have a subtropical monsoon climate. The average daily temperature during the rice growing season (from sowing to harvesting) was 29.0 and 28.9 °C at Hengyang and 23.7 and 22.9 °C at Xingyi in 2021 and 2022, respectively. Total incident solar radiation during the rice growing season was 1905 and 1787 MJ m−2 at Hengyang and 2191 and 2527 MJ m−2 at Xingyi in 2021 and 2022, respectively. The soils of the experimental field were a purple sandy soil at Hengyang and a yellow podzolic soil at Xingyi. The soil chemical properties in the upper 20 cm layer before transplanting in 2021 are provided in Table 1.
Table 1.
Soil chemical properties of the experimental fields at two subtropical sites.
Three representative super hybrid rice varieties (Y-liangyou 1, Y-liangyou 2, and Y-liangyou 900) and three currently popular hybrid rice varieties (Jingliangyouhuazhan, Jingliangyou 534, and Longliangyouhuazhan) were used in the experiment. Information about the varieties is given in Table 2. The experiment was arranged in a randomized complete-block design with three replicates. The plot size was 20 m2 at Hengyang and 15 m2 at Xingyi.
Table 2.
Information about varieties used in the experiment.
Pre-germinated seeds were sown on the 8th of May at Hengyang and the 9th of April at Xingyi. Thirty- and 20-day-old seedlings were transplanted at Hengyang and Xingyi, respectively. Transplanting was performed with two seedlings per hill at a hill spacing of 20 cm × 20 cm. Urea (46% N), superphosphate (12% P2O5), and potassium chloride (60% K2O) were used as N, P, and K fertilizers, respectively. The N fertilizer rate was determined based on the average N application rate of China (180 kg N ha−1). The P and K fertilizer rates were determined according to a locally recommended N:P2O5:K2O ratio of 1:0.5:1. Basal fertilizer (90 kg N ha−1, 90 kg P2O5 ha−1, and 90 kg K2O ha−1) was applied one day before transplanting. The first top dressing (36 kg N ha−1) was applied seven days after transplanting. The second top dressing (54 kg N ha−1 and 90 kg K2O ha−1) was applied at the panicle initiation stage. Continuous flooding (5–10 cm water depth) was practiced in all plots from transplanting until one week before maturity, when the plots were drained for harvesting. Plant diseases, insects, and weeds were intensively controlled by pesticides.
2.2. Sampling and Measurements
Ten hills of rice plants were sampled diagonally from each plot at the maturity stage to determine yield attributes. Sampled plants were hand threshed after counting the panicle number. Filled and unfilled spikelets were separated by submerging them in tap water. Three subsamples of 30 g of filled spikelets and all unfilled spikelets were used to count the spikelet number. The number of filled spikelets was counted with a digital automatic seed counter (SLY-C, Zhejiang Top Cloud-Agri Technology Co., Ltd., Hangzhou, China). The number of unfilled spikelets was counted manually. All plant organs were oven-dried at 70 °C to a constant weight to determine biomass. Yield components (panicles per m2, spikelets per panicle, spikelet filling percentage, and grain weight), total biomass production, and harvest index were calculated. Rice plants were harvested from a 5 m2 area in each plot to determine grain yield. Harvested grains were weighed after sun-drying. A subsample of 50 g of sun-dried grains was oven-dried at 70 °C to a constant weight to determine moisture content. Grain yield was calculated by adjusting to a moisture content of 14%.
2.3. Statistical Analysis
All data were analyzed separately for each year. Analysis of variance (ANOVA) was performed after the Shapiro–Wilk normality test (Statistix 8.0, Analytical Software, Tallahassee, FL, USA). The statistical model of the ANOVA included varietal group, site, and the interaction between varietal group and site. A post hoc LSD test was performed if a significant effect was detected by the ANOVA. Correlation plot analysis was used to evaluate the relationships between grain yield and yield attributes (Origin 2024, OriginLab Corp., Northampton, MA, USA). The significant level was set at p < 0.05 and p < 0.01 in the ANOVA, at p < 0.05 in the LSD test, and at p < 0.05 and p < 0.01 in the correlation analysis.
3. Results
3.1. Grain Yield
There was no significant difference in grain yield between the currently popular hybrid rice varieties and the representative super hybrid rice varieties at Hengyang and Xingyi in 2021 (Table 3). In 2022, a significant difference in grain yield between the groups was observed at Hengyang but not at Xingyi. Mean grain yield was 15% higher in the popular hybrids compared to the super hybrids at Hengyang in 2022. The experiment at Hengyang in 2022 produced the lowest grain yield among the four experiments.
Table 3.
Grain yield (t ha−1) in three representative super hybrid rice varieties and three currently popular hybrid rice varieties grown at two subtropical sites in two years.
3.2. Yield Components
The popular hybrids had significantly higher mean panicles per m2 than the super hybrids by 21% and 17% at Hengyang and Xingyi in 2021 and by 15% and 13% at Hengyang and Xingyi in 2022, respectively (Table 4 and Table 5). The difference in mean spikelets per panicle between the popular hybrids and the super hybrids was not significant at Hengyang or Xingyi in 2021 or at Hengyang in 2022, while at Xingyi in 2022, mean spikelets per panicle were 16% lower in the popular hybrids than in the super hybrids. There was no significant difference in mean spikelet filling percentage between the popular hybrids and the super hybrids at Hengyang and Xingyi in 2021 or at Xingyi in 2022, while at Hengyang in 2022, the popular hybrids had a 10% higher mean spikelet filling percentage. Mean grain weight was significantly lower in the popular hybrids than in the super hybrids by 14% at Hengyang in 2021 and by 12% and 7% at Hengyang and Xingyi in 2022, respectively, whereas the difference was not significant at Xingyi in 2021.
Table 4.
Yield components of three representative super hybrid rice varieties and three currently popular hybrid rice varieties grown at two subtropical sites in 2021.
Table 5.
Yield components of three representative super hybrid rice varieties and three currently popular hybrid rice varieties grown at two subtropical sites in 2022.
3.3. Biomass Production and Harvest Index
The difference in mean total biomass production between the popular hybrids and the super hybrids was not significant at either site or in either year (Table 6 and Table 7). There was no significant difference in the mean harvest index between the popular hybrids and the super hybrids at Hengyang and Xingyi in 2021 or at Xingyi in 2022, while at Hengyang in 2022, the popular hybrids had a 9% higher mean harvest index than the super hybrids.
Table 6.
Total biomass production and harvest index of three representative super hybrid rice varieties and three currently popular hybrid rice varieties grown at two subtropical sites in 2021.
Table 7.
Total biomass production and harvest index of three representative super hybrid rice varieties and three currently popular hybrid rice varieties grown at two subtropical sites in 2022.
3.4. Relationships between Grain Yield and Yield Attributes
Based on the yield performance at each site in each year (Table 3), the data from four experiments were divided into two sets: a set with high productivity (Hengyang and Xingyi in 2021 and Xingyi in 2022) and a set with relatively lower productivity (Hengyang in 2022), and relationships between grain yield and yield components were separately evaluated for these two sets. Results showed that grain yield was significantly related to panicles per m2 (r = 0.570, p = 0.014), grain weight (r = 0.708, p = 0.001), and total biomass production (r = 0.835, p = 0.000) but not to spikelets per panicle, spikelet filling percentage, and harvest index across six varieties at Hengyang and Xingyi in 2021 and Xingyi in 2022 (Figure 1A). At Hengyang in 2022, grain yield was significantly related to panicles per m2 (r = 0.910, p = 0.012) but not to other yield attributes across six varieties (Figure 1B).
Figure 1.
Correlation plot analysis between grain yield and yield attributes across six varieties (A) at Hengyang and Xingyi in 2021 and Xingyi in 2022 (n = 18) and (B) at Hengyang in 2022 (n = 6). * and ** indicate significant relationships at p < 0.05 and p < 0.01, respectively. NS indicates a non-significant relationship at p < 0.05.
4. Discussion
The currently popular hybrid rice varieties produced similar mean grain yields as the representative super hybrid rice varieties in three of four experiments (Hengyang and Xingyi in 2021 and Xingyi in 2022) in this study. In the other experiment (Hengyang in 2022), a higher mean grain yield was achieved in the popular hybrids than in the super hybrids. This experiment had the lowest grain yield among the four experiments. These findings suggest that the popular hybrids have better yield performance than the super hybrids under conditions of relatively lower productivity. This could be an important determinant for the popularity of hybrid rice varieties among Chinese farmers because medium- and low-productivity land accounts for as much as 70% of the total arable land in China [13]. The results of this study suggest a need for further research comparing yield performance between the currently popular hybrid rice varieties and representative super hybrid rice varieties under medium- and low-productivity conditions.
A higher mean number of panicles per m2 was observed in the popular hybrids compared to the super hybrids in all four experiments in this study. In rice crops, strong compensation always exists between the number of panicles per m2 and the number of spikelets per panicle [14,15]. However, in this study, the higher number of panicles per m2 in the popular hybrids did not lead to lower spikelets per panicle compared to the super hybrids in three of four experiments (Hengyang and Xingyi in 2021 and Hengyang in 2022), indicating reduced compensation between the two components in the popular hybrids. In general, decoupling the compensatory relationship between the number of panicles per m2 and the number of spikelets per panicle can be accomplished by increasing biomass production during panicle formation [16]. However, it is not clear whether this is also the case for the popular hybrids in the present study. This highlights the need for a greater understanding of physiological processes governing the relationship between the number of panicles per m2 and the number of spikelets per panicle in the currently popular hybrid rice varieties.
The mean spikelet filling percentage between the popular hybrids and the super hybrids was different in only one of four experiments: Hengyang in 2022. In this experiment, the popular hybrids had a higher mean spikelet filling percentage than the super hybrids. In rice crops, a higher spikelet filling percentage can be achieved by increasing source capacity and/or by increasing the transportation of assimilates into grains under favorable conditions [17]. In this study, mean total biomass production was similar, while the mean harvest index was higher in the popular hybrids compared to the super hybrids at Hengyang in 2022. Thus, the higher mean spikelet filling percentage in the popular hybrids at Hengyang in 2022 was likely attributable to the higher transport of assimilates to grains. Further investigations are required to clarify the difference in physiological characteristics related to assimilating transportation, such as the activity of key enzymes in carbon metabolism and the content of hormones in grains [17,18], between the popular hybrids and the super hybrids, especially under medium- and low-productivity conditions.
The popular hybrids had lower grain weight than the super hybrids in three of four experiments (Hengyang in 2021 and Hengyang and Xingyi in 2022). However, the lower grain weight did not result in lower grain yields in the popular hybrids compared to the super hybrids because it was compensated for by a higher panicle number (Hengyang in 2021 and Xingyi in 2022) or both a higher panicle number and a higher spikelet filling percentage (Hengyang in 2022). Grain weight is not only a yield component but also an important trait in determining the grain quality in rice. In general, appearance quality increases with decreasing grain weight [19]. Most recently, Huang et al. [20] reported that grain weight and chalky grain rate decreased synchronously with the year of varietal release in middle-season hybrid rice cultivars released in Hunan Province of China from 2006 to 2021. Although this study did not determine grain quality, regional variety testing showed that the quality of the popular hybrids used in this study reached the second or third grade of high-quality rice standards in China (https://www.ricedata.cn/variety, accessed on 18 December 2023). This could also be a reason why farmers preferred these varieties.
Taken together, the group of currently popular hybrid rice varieties had higher panicles per m2, spikelet filling percentage, and harvest index, and, consequently, higher grain yield than the group of representative super hybrid rice varieties under the condition of relatively lower productivity (Hengyang in 2022). However, correlation plot analysis showed that relationships between grain yield and yield attributes under the condition of relatively lower productivity were different from those under conditions of high productivity (Hengyang and Xingyi in 2021 and Xingyi in 2022), and grain yield was only significantly related to panicles per m2 under the condition of relatively lower productivity. This suggests that the selection of the varieties with high panicle numbers can be effective for achieving a high grain yield of rice under conditions of relatively lower productivity. This finding is consistent with the study by Liu et al. [21], which showed that introgression of the allele associated with a high tillering into modern rice varieties increased panicle number and, consequently, boosted grain yield under conditions of medium and low productivity. The finding of this study highlights that further investigations are required to clarify the physio-biochemical and molecular mechanisms for the high yield of the popular hybrids under conditions of relatively lower productivity.
5. Conclusions
The group of three currently popular hybrid rice varieties (Jingliangyouhuazhan, Jingliangyou 534, and Longliangyouhuazhan) produced a higher mean grain yield than the group of three representative super hybrid rice varieties (Y-liangyou 1, Y-liangyou 2, and Y-liangyou 900) under conditions of relatively lower productivity. This higher yield performance in the group of popular hybrids was attributable to higher panicles per m2, spikelet filling percentage, and harvest index. However, further correlation analysis indicated that grain yield was only significantly related to panicles per m2 across six varieties under the condition of relatively lower productivity. These highlight that the yield performance under medium- and low-productivity conditions should receive more attention in hybrid rice breeding programs, and the selection of the varieties with high panicle numbers is an effective breeding strategy.
Author Contributions
Conceptualization, M.H.; conducting field experiments, S.F., H.Z. and L.L.; sampling and measurements, C.L., S.F., H.Z., J.X., X.L. and F.C.; data collection and preprocessing: J.X., X.L. and F.C.; data analysis, C.L., J.C. and M.H.; writing—original draft preparation, C.L. and M.H.; supervision: M.H., L.L. and J.C.; funding acquisition, M.H. All authors have read and agreed to the published version of the manuscript.
Funding
This study was funded by the Science and Technology Innovation Program of Hunan Province, grant number 2021RC3088, and the Earmarked Fund for China Agriculture Research System, grant number CARS-01.
Data Availability Statement
The data presented in this study are available upon request from the corresponding author.
Acknowledgments
The authors thank the other members of the Rice and Product Ecophysiology for their help with this study.
Conflicts of Interest
The authors declare no conflicts of interest.
References
- Nie, L.; Peng, S. Rice production in China. In Rice Production Worldwide; Chauhan, B.S., Jabran, K., Mahajan, G., Eds.; Springer: Cham, Switzerland, 2017; pp. 33–52. [Google Scholar]
- Cheng, S.; Liao, X.; Min, S. China’s ‘‘super’’ rice research: Background, goals and issues. China Rice 1998, 1, 3–5. [Google Scholar]
- Yuan, L. Hybrid rice breeding for super high yield. Hybrid Rice 1997, 12, 1–6. [Google Scholar]
- Yuan, L. Development of super hybrid rice for food security in China. Engineering 2015, 1, 13–14. [Google Scholar] [CrossRef]
- Zhang, Y.; Fu, J.; Wang, K.; Han, X.; Yan, T.; Su, Y.; Li, Y.; Lin, Z.; Qin, P.; Fu, C.; et al. The telomere-to-telomere gap-free genome of four rice parents reveals SV and PAV patterns in hybrid rice breeding. Plant Biotechnol. J. 2022, 20, 1642–1644. [Google Scholar] [CrossRef] [PubMed]
- Xiong, D.; Flexas, J.; Huang, J.; Cui, K.; Wang, F.; Douthe, C.; Lin, M. Why high yield QTLs failed in preventing yield stagnation in rice? Crop Environ. 2022, 1, 103–107. [Google Scholar] [CrossRef]
- Panda, B.; Mondal, S.; Mohanty, A.; Senapaty, J.; Meher, J.; Sahoo, C.E.; Samal, K.C.; Dash, M.; Chakraborty, K.; Dash, S.K. Exploring the physiological basis of yield enhancement in New Generation Rice (NGR): A comparative assessment with non-NGR rice genotypes. Plant Physiol. Rep. 2023, 28, 543–555. [Google Scholar] [CrossRef]
- Huang, M.; Zou, Y.; Jiang, P.; Xia, B.; Ibrahim, M.; Ao, H. Relationship between grain yield and yield components in super hybrid rice. Agric. Sci. China 2011, 10, 1537–1544. [Google Scholar] [CrossRef]
- Zhang, Y.; Tang, Q.; Zou, Y.; Li, D.; Qin, J.; Yang, S.; Chen, L.; Xia, B.; Peng, S. Yield potential and radiation use efficiency of super hybrid rice grown under subtropical conditions. Field Crop. Res. 2009, 114, 91–98. [Google Scholar] [CrossRef]
- Li, G.; Xue, L.; Gu, W.; Yang, C.; Wang, S.; Ling, Q.; Qin, X.; Ding, Y. Comparison of yield components and plant type characteristics of high-yield rice between Taoyuan, a ‘special eco-site’ and Nanjing, China. Field Crop. Res. 2009, 112, 214–221. [Google Scholar] [CrossRef]
- Katsura, K.; Maeda, S.; Lubis, I.; Horie, T.; Cao, W.; Shiraiwa, T. The high yield of irrigated rice in Yunnan, China: ‘A cross-location analysis’. Field Crop. Res. 2008, 107, 1–11. [Google Scholar] [CrossRef]
- Ibrahim, M.; Peng, S.; Tang, Q.; Huang, M.; Jiang, P.; Zou, Y. Comparisons of yield and growth behaviors of hybrid rice under different nitrogen management methods in tropical and subtropical environments. J. Integr. Agric. 2013, 12, 621–629. [Google Scholar] [CrossRef]
- Wang, Y. The Challenges and strategies of food security under rapid urbanization in China. Sustainability 2019, 11, 542. [Google Scholar] [CrossRef]
- Ying, J.; Peng, S.; He, Q.; Yang, H.; Yang, C.; Visperas, R.M.; Cassman, K.G. Comparison of high-yield rice in tropical and subtropical environments I. Determinants of grain and dry matter yields. Field Crop Res. 1998, 57, 71–84. [Google Scholar] [CrossRef]
- Ata-Ul-Karim, S.T.; Begum, H.; Lopena, V.; Borromeo, T.; Virk, P.; Hernandez, J.E.; Gregorio, G.B.; Collard, B.C.Y.; Kato, Y. Genotypic variation of yield-related traits in an irrigated rice breeding program for tropical Asia. Crop Environ. 2022, 1, 173–181. [Google Scholar] [CrossRef]
- Fukushima, A. Varietal differences in tiller and panicle development determining the total number of spikelets per unit area in rice. Plant Prod. Sci. 2019, 22, 192–201. [Google Scholar] [CrossRef]
- Yang, J.; Zhang, J. Grain-filling problem in ‘super’ rice. J. Exp. Bot. 2009, 61, 1–5. [Google Scholar] [CrossRef] [PubMed]
- Panigrahi, R.; Kariali, E.; Panda, B.B.; Lafarge, T.; Mohapatra, P.K. Controlling the trade-off between spikelet number and grain filling: The hierarchy of starch synthesis in spikelets of rice panicle in relation to hormone dynamics. Funct. Plant Biol. 2019, 46, 595. [Google Scholar]
- Dwiningsih, Y.; Kumar, A.; Thomas, J.; Ruiz, C.; Alkahtani, J.; Al-hashimi, A.; Pereira, A. Identification of genomic regions controlling chalkiness and grain characteristics in a recombinant inbred line rice population based on high-throughput SNP markers. Genes 2021, 12, 1690. [Google Scholar] [CrossRef] [PubMed]
- Huang, M.; Cao, J.; Chen, J.; Cao, F.; Zhou, C. Slimming the grain through breeding is a practical way to reduce the chalky grain rate of middle-season hybrid rice. Agronomy 2022, 12, 1886. [Google Scholar] [CrossRef]
- Liu, Y.; Wang, H.; Jiang, Z.; Wang, W.; Xu, R.; Wang, Q.; Zhang, Z.; Li, A.; Liang, Y.; Ou, S.; et al. Genomic basis of geographical adaptation to soil nitrogen in rice. Nature 2021, 590, 600–605. [Google Scholar] [CrossRef] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).