Rice (Oryza sativa L.) is an important food crop in China and even in the world. More than two-thirds of the population in China rely on rice as the staple food (Tian et al., 2009; Kim et al., 2013). With the continuous improvement of living standards, there is an increasing demand for high-quality rice (Rao et al., 2014; Peng et al., 2014; Zhou et al., 2015). However, the overall low performance of rice quality in China has affected its market competitiveness to some extent (Zhang et al., 2016). Therefore, breeding high quality new rice varieties has become one of the most important goals for breeders.

Rice quality is a complex comprehensive character, among which rice appearance quality, processing quality, cooking taste and nutritional quality directly affect the commercial value of rice (Huang et al., 1998). Most rice quality traits are quantitative traits with complex genetic basis. At present, a large number of rice quality-related regulatory genes have been cloned, but only a few genes have been really applied in breeding (Rao et al., 2014).

The flower organ of rice is not only the reproductive organ, but also the basis of grain formation, and its development directly affects rice yield and rice quality. During the formation and development of flower organs, in addition to being coregulated by 5 types of genes A, B, C, D and E, FZP, DL, EG1, MFS1, FON1, FON2/FON4, FON3, fon5, fon6, fon (t), OsIG1 and JMJ706 genes are also involved in the process of rice caryopsis development (Nagasawa et al., 2003; Suzaki et al., 2004, 2006; Jiang et al., 2005; Li et al., 2007; Sun et al., 2008; Zhang et al., 2008; Li et al., 2009; Zhao et al., 2011; Ren et al., 2013; Zhang et al., 2015; Bai et al., 2016). In previous studies, a japonica twin-grain mutant Zhejing22-tg1 was obtained by irradiated Zhejing22, and the key gene tg1 controlling the twin-grain trait was cloned (Ye et al., 2017). This gene is an allele of FON2/FON4, and related gene functions have been reported (Chu et al., 2006). However, its effect on rice quality remains unclear. In this study, a double-grain mutant of Zhejing22, Zhejing22-tg1, was used as the donor, and the double-grain gene tg1 was introduced into the large-grain variety SLG to analyze the effects of the double-grain gene tg1 on rice quality, so as to provide reference for breeding high-quality new rice varieties.

1 Results and Analysis

1.1 Construction of a new SLG-tg1 strain

Selection process of new SLG-tg1 strain: The large grain variety SLG was used as the female parent and crossed with Zhejing22-tg1 to obtain F₁. The recurrent parent SLG was used as the male parent and then self-crossed with F₁. The separated progenies with double phenotype were backcrossed BC₁F₂ and continued backcrossed with SLG as the male parent to obtain BC₃F₂. A new SLG-tg1 strain was obtained by combining the selection of double-grain phenotypes with agronomic traits, and the transparency of brown rice of SLG-tg1 was significantly improved compared with that of SLG (Figure 1; Figure 2).

Figure 1 Breeding process for the new line SLG-tg1

Figure 2 Appearance of rice in the husk and brown rice Note: SLG was showed in the row above, and SLG-tg1 was showed in the row below

1.2 Grouting rate analysis

The comparison of filling rates of different materials shows that the filling rates of SLG and SLG-tg1 are significantly higher than those of Zhejing22 and Zhejing22-tg1 (Table 1). The grain-filling rate of SLG-tg1 at 20 days after flowering was significantly higher than that of SLG, reaching 2.46 mg/p·d, which was 1.5 times of that of SLG at 20 days after flowering. The grain-filling rate of SLG was significantly higher than that of SLG-tg1 at 30 days after flowering, but not as much as that of SLG-tg1 at 20 days after flowering. There was no significant difference at other time points. However, there is no significant difference in filling rate between Zhejing22 and Zhejing22-tg1.

Table 1 Grain filling rate of the lines at different time points after flowering Note: * and ** indicate significant levels at p<0.05, and 0.01, respectively

1.3 Difference analysis of rice quality

After transferring tg1 gene, the appearance quality of rice was changed. Compared with SLG, the grain length of SLG-tg1 was significantly shorter, and the corresponding length to width ratio was significantly smaller, while the chalkiness grain percentage and chalkiness degree were significantly or extremely decreased, and the transparency was also increased from grade 4 to grade 1. In terms of processing quality, the brown rice rate and the head rice rate of SLG-tg1 were significantly improved. In cooking taste and nutritional quality, SLG-tg1 had significantly increased gel consistency, significantly decreased amylose content and significantly decreased protein content. Therefore, compared with SLG, SLG-tg1 has significantly improved in appearance quality, processing quality, cooking taste and nutritional quality. Compared with Zhejing22, the length-width ratio of the mutant Zhejing22-tg1 is significantly increased, chalkiness is significantly increased, transparency is reduced from grade 1 to grade 3, brown rice percentage, milled rice percentage and head rice percentage are significantly or extremely significantly decreased, and protein content is increased. In general, rice quality of the mutant Zhejing22-tg1 is significantly lower than that of Zhejing22 (Table 2).

Table 2 Comparison of grain quality for the four lines Note:* and ** indicate significant levels at p<0.05, and 0.01, respectively

1.4 Tissue and cytology observation of starch granules 

The morphological structure of starch grains in isolated offspring can be directly observed by scanning electron microscopy to roughly identify the advantages and disadvantages of rice varieties (Liang, 1996; Chang et al., 2006; Kang and Chang, 2007). Figure 3 shows the scanning electron microscope (SEM) observation of starch particles in cross section of four rice grains. Compared with Zhejing22, the starch grains of Zhejing22-tg1 are more closely arranged, but the edges and angles are blunt. Under 5.0k times field view, it can be clearly seen that there are cracks on the grain surface. Compared with SLG, the starch grains of SLG-tg1 were polyhedral and compact. Therefore, judging from the morphological characteristics of starch grains, the rice quality of the mutant Zhejing22-tg1 is lower than that of Zhejing22, while the rice quality of SLG-tg1 is significantly higher than that of SLG.

Figure 3 SEM observation of starch granules Note: a: Zhejing22- tg1, b: Zhejing22, c: SLG-tg1,d: SLG

2 Discussion

In this study, the japonica twin-grain mutant Zhejing22, Zhejing22-tg1, was used as the donor, and the twin-grain gene tg1 was introduced into the large-grain variety SLG. Since tg1 is a recessive gene, homozygous tg1 gene needs to be self-crossbred in the backcross off spring, and the single plant with twin-grain traits can be determined by naked eyes at flowering, which is beneficial for simple and rapid screening of twin-grain traits. Continuous backcross in the breeding process can eliminate the introduction of adverse genes in the mutant to the maximum extent, and accelerate the rapid stability of the new SLG-tg1 line.

In this study, the introduction of tg1 gene into large grain variety SLG not only resulted in the appearance of double grain phenotype, but also caused changes in non-target traits such as appearance quality, processing quality and nutritional quality. By comparing the differences in filling rate, rice quality indexes and starch grain morphology of each material, it is found that tg1 gene greatly improves the rice quality of large-grain varieties. However, the rice quality of Zhejing22-tg1 May be lower than that of the wild-type Zhejing22 due to the irradiated mutant. Because it is by ⁶⁰Co-γ ray irradiation mutation, the use of high-energy electromagnetic waves, will cause chromosomal aberrations and lead to gene rearrangement or mutation, gene fragments will be broken, deletion and other mutations, M₀ can show a significant decline in germination rate, M₁ can cause adverse gene expression, May have a negative regulation effect on rice quality (Pang and Wan, 1998; Wei et al., 2006; Zhang  et al., 2008).

Rice grain-filling characteristics are mainly related to processing quality and appearance quality. According to the grain length data, Zhejing22 and Zhejing22-tg1 are medium grain varieties, while SLG and 22-tg1 are large grain varieties. Chen et al. (2017) found that the filling rate of small-grain varieties was low, and the filling rate accelerated with the increase of grains. SLG is a typical large-grain variety, so the grain-filling rate of SLG is significantly higher than that of Zhejing22 and Zhejing22-tg1. Increasing grain filling rate can improve rice quality (Cai et al., 2002; 2004). Compared with SLG, the appearance quality, processing quality, cooking taste and nutrition quality of SLG-tg1 were significantly improved, which might be related to the significantly increased grain filling rate at 20 days after flowering.

The grain filling process of rice is mainly a process of starch synthesis and accumulation, which plays a crucial role in rice quality (Liu et al., 2012). Therefore, the morphological characteristics of starch grains can be used as one of the important indexes to identify the quality of rice. In this study, the surface structure of starch grains of different materials was observed by scanning electron microscopy. It was found that the starch grains of SLG-tg1 were closer than those of SLG, which was polyhedral shape of high-quality rice, which might be related to the fullness of grouting. With the increase of fullness of grouting, the starch grains were more densely arranged and the surface of starch tended to be smooth. In addition, chalkiness is closely related to the distribution of starch grains. The chalkiness of SLG-tg1 decreased and transparency increased to grade 1, which was caused by the change of starch grain structure.

3 Materials and Methods

3.1 Test materials

The experimental materials for this study are the high-quality conventional late japonica variety Zhejing22, the twin-grain mutant Zhejing22-tg1, the large-grain variety SLG, and the new strain SLG-tg1 with tg1 gene transferred. Among them, the new strain SLG-tg1 was obtained through hybrid Zhejing22-tg1 with large-grain rice variety SLG, and continuous backcross with SLG as recurrent parent, and two-grain traits were selected from separated progenies combined with rice quality identification.

3.2 Determination of grouting rate

In 2017, Zhejing22, Zhejing22-tg1, SLG and SLG-tg1 were planted in Hangzhou base of Zhejiang Academy of Agricultural Sciences, and 500 plants were planted in each plot with 3 replicates. After heading, in the morning of full flowering stage, the flowers that have blossomed from the above four materials are cut off, and the flowers that have not blossomed are removed in the afternoon of the same day, and the grains that bloom on the same day are retained for Zhejing22, Zhejing22-tg1, SLG and SLG-tg1. 1000 indica Zhejiang, Zhejing22-tg1, SLG and SLG-tg1 were collected five times every 10 days after flowering. They were baked to constant weight at 40℃, and the grain weight was determined by removing the shell, denoted by W; N is the number of grains, N₁ and N₂ are 1 000 grains. d for the day. The grain filling rate (GFR) was calculated according to the following formula:

GFR=(W2/N2-W1/N1)/10 d

3.3 Determination of rice quality-related traits 

After harvesting, the rice is threshed and stored for 3 months after drying. The physical and chemical properties are stable, weigh 6 portions of 130 g rice for each treatment. Grain length, length-width ratio, brown rice percentage, milled rice percentage, head rice percentage, chalky grain percentage, chalkiness, glue consistency, amylose and protein contents were determined according to the national standard of The People's Republic of China-High Quality Rice (GB/T 17891-1999).

3.4 Histocytological observation

Mature and dried seeds of Zhejing22, SLG, Zhejing22-tg1 and SLG-tg1 are respectively taken and shelled. Representative brown rice is selected and gently knocked near the middle of the rice to make it transverse fracture in natural state. The fracture part is cut with a knife to make a transverse section of about 2 mm. The arrangement and structure of internal starch particles were observed under a scanning electron microscope (FEI, QUANTA 450, USA) and photographed.

3.5 Data processing and analysis

Excel 2010 and SPSS 19.0 data processing system were used for data analysis.

Authors’ Contributions

YSH is the executor of the experimental design and research of this study, completed data processing and analysis and the written of the first draft of the paper. ZRR, YJ, ZGF and LYT participated in the experimental design and revision of the paper; ZXM is the architect and principal of the project, directing experimental design, data analysis, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the Zhejiang Provincial Agricultural (Grain) New Variety Breeding Major Science and Technology Special Project "Zhejiang Province Conventional Late Japonica Rice New Variety Breeding" (2016C02050-5) and Zhejiang Provincial Basic Public Welfare Research Program (LGN18C130004 and LGN20C130005).

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