Study on the Chalkiness Character of Giant Embryo Rice and Observation on Its Microstructure in Grains

Grain chalkiness greatly affects the grain appearance and milling, eating, cooking, and nutritional qualities, thus it is one of the most important traits of grain qualities. Based on the study of chalkiness characters in endosperm of different giant embryo rice, the relationship between the differences of chalkiness characters in endosperm of different giant embryo rice and the morphological structure, arrangement and development of starch granules in endosperm was clarified, which will lay a solid foundation for further revealing the formation mechanism of chalkiness characters in giant embryo rice and cultivating excellent new varieties of giant embryo rice. In this study, the chalkiness of endosperm (including chalkiness rate, chalkiness degree and chalkiness area) of 16 different rice varieties were investigated and analyzed. The results showed that the chalkiness of endosperm was closely related to the morphological structure and arrangement of starch granules in endosperm cells of different kinds of giant embryo rice, and there were significant differences between the chalkiness of different parts of the same giant embryo rice and the morphological structure and arrangement of starch granules in endosperm cells. Therefore, the results of this study will provide important information for the improvement of quality characters and the breeding of new rice varieties.


Introduction
Rice (Oryza sativa L.) is one of the most important crops in the world. More than half of the world's population uses rice as its main food. As the largest country of rice production and consumption, rice planting and processing has made an important contribution to the healthy and sustainable development of China's economy. With the increase of the world population and the gradual improvement of people's living standards, the market demand for rice, especially for high-quality rice, is also increasing (Tian et al., 2009;Kim et al., 2013;. Therefore, the grian quality of rice determines the competitive position and price of rice in the consumer market. Rice quality is a complex quantitative character, which is generally considered to include four aspects, namely cooking and eating quality, appearance quality, grinding and processing quality and nutritional quality (Peng et al., 2016). Appearance quality of rice includes grain length, grain width, grain thickness, length width ratio and chalkiness or transparency; Milling quality of rice mainly includes brown rice rate, milled rice rate and whole milled rice rate; Cooking and eating quality mainly includes amylose content, gel consistency and gelatinization temperature; Nutrition quality mainly includes protein content, amino acid content, fatty acid content and various trace elements, etc. (Hu et al., 2003;Zhang et al., 2013). The results of previous studies show that the appearance of chalkiness will reduce the appearance quality of rice and have a great negative impact on farmers' preference for rice varieties (Feng et al., 2017). Moreover, the grains with chalkiness are easy to be broken in the process of rice processing, which will greatly reduce the rate of whole rice, reduce the yield of rice, and affect the commodity value of rice.
The formation of chalkiness always affects grain quality traits in rice, especially the appearance quality, which is the direct reflection of rice commercialization (Zhu, 2017;Feng et al., 2017). Chalkiness is a typical quantitative trait in rice. The formation of chalkiness is controlled by many genes. In addition to genetic factors, it is also affected by the external environmental conditions during the filling process, such as sunshine intensity and temperature (Peng et al., 2016;Li, 2017;Zhu et al., 2017). The chalkiness area of rice was sensitive to the change of environment, and increased with the increase of light hours, daily average light and relative humidity (Li et al., 1989). Compared without chalkiness rice, the short chain amylopectin of chalky rice increased significantly, while the amylose and long chain amylopectin decreased significantly, and the transparency and milled rice rate decreased significantly, resulting in the reduction of cooking and eating quality and yield of rice (Peng et al., 2016;Kang et al., 2007). Genetic factors are the main cause of chalkiness formation, which is regulated by the genetic effect of parents and the interaction between heredity and environment (Zhang et al., 2017). At present, a large number of genes affecting rice chalkiness have been cloned from natural populations or mutants, such as OsPK2 (Cai, 2018), Chalk5 (Zhu, et al., 2017), OsPPDKB (Kang et al., 2014), GW2 (Song et al., 2007), SSIIIa (Fujita et al., 2007;Ryoo et al., 2007), flo2 (She et al., 2010) etc. The results of microstructural observation showed that chalkiness in endosperm was closely related to starch granule structure, arrangement and starch synthesis (Shen et al., 2000;Zhai et al., 1991). The structure and arrangement of starch granules is also an important character to determine the grain quality of rice. Chalkiness is formed by the opaque material refracted by light due to the round shape of composite starch granules, loose arrangement of starch and inflation between grains.
Giant embryo rice is a kind of special functional rice, whose embryo is 2-3 times larger than that of ordinary rice. The main nutrient elements in rice are concentrated in embryo, and its protein, mineral, vitamin, fat, γ-amino acid, folic acid and other nutrients are higher than that of ordinary rice Tang et al., 2020), which makes rice breeders pay more attention to the breeding of many excellent characters Guo et al., 2011). Previous studies mainly focused on gene molecular markers, gene location, growth dynamics, nutritional components and processing technology of giant embryo rice. The formation mechanism of chalkiness in endosperm of giant embryo rice is still unclear. In this study, the chalkiness of the endosperm of 16 rice varieties were detected and analyzed, and the endosperm cells and starch granules of the rice were observed by scanning electron microscopy. Thus, our results will lay a foundation for the breeding of new giant embryo rice varieties in the later stage.

Determination of Chalkiness Character
Cretaceous rate, chalkiness area and chalkiness degree of different giant embryo rice varieties were measured and analyzed according to the national standard GB/T17891-1999: 100 full and complete rice grains were randomly selected from each variety, and chalky rice grains were counted. According to the method of measuring chalkiness rate, the abdominal and cardiac white rates were determined (no back white grains were found), that is, 100 grains of fine rice were randomly selected from each variety, and the rice grains containing abdominal white and heart white were counted from them, repeated three times, and the average value was taken jas.ccsenet. as the abd SPSS softw

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Distribution of Chalkiness and Starch Granules in Giant Embryo Rice
Chalkiness not only affects the appearance quality of rice, but also affects the cooking and eating quality, milling quality and nutritional quality Liu et al., 2011;Yoshioka et al., 2007;Shen et al., 2007). The morphological structure of starch granules can be observed by scanning electron microscope, and the quality of rice can be judged preliminarily according to its morphological structure. The chalkiness was formed by oblong cells arranged in the direction of dorsal and ventral diameter (Zhai et al., 1991). The chalkiness was not only related to oblong cells, but also related to the arrangement and morphological results of starch granules in rice (Chang et al., 2006). In this experiment, through the observation of SEM, the starch granules of the varieties with higher chalkiness rate and chalkiness degree (Juhongxuanbai, Jujingzhan-GB-12 and Jujingzhan-GB-11) were obviously uneven in the cross section; the starch granules of the varieties with lower chalkiness rate and chalkiness degree (Jujingzhan-GB-7, Jujingzhan-GB-2, Baijupeishanghai, Jujingzhan-GB-2) were evenly distributed in the center of endosperm, abdomen and back. Through comprehensive analysis, we found that if the distribution of starch granules in the cross section is more uniform, the variety is not easy to produce chalkiness, and the corresponding chalkiness rate and chalkiness degree will be low or not. However, if the starch granules are distributed unevenly in the cross section and the gap between the starch granules is large, the arrangement is loose and the starch granules are poorly developed, which leads to the chalkiness easily, and the corresponding chalkiness rate and chalkiness degree will be higher. It is concluded that the starch granules distributed on the endosperm cross section of rice are closely related to the formation of chalkiness, which is consistent with previous studies.

Development of Chalkiness and Starch Granules in the Same Giant Embryo Rice
There are many granular starch granules in every starch body of rice, most of which are in the state of starch complex. Previous studies have found that the size of starch granules in the chalkiness part of rice is relatively smaller than that in the non chalkiness part (Zhao, 2015). By scanning electron microscopy, it was found that the starch granules in the endosperm of rice had different morphological structure and arrangement in different parts of the same giant embryo rice. By scanning electron microscopy, it was found that the starch granules in the endosperm of rice grain were also different in their morphological structure and arrangement in different parts of the same giant embryo rice. Compared with the non chalky part, there were spherical starch granules with different sizes in the chalky part of rice, and most of them were single starch granules in the free state. The arrangement of starch granules was loose and the gap was large, so they could not develop into starch granule complex produces chalkiness. If the starch granules in rice are closely arranged in a complex state, it is not easy to produce chalkiness.
Previous studies have found that most of the chalkiness traits tend to grow in the middle or abdomen of rice, with little or no white back trait (Ying et al., 2019;Peng et al., 2016a;Peng et al., 2016b;Kang et al., 2003). There is a certain correlation between the formation of starch granule morphology and chalkiness phenotype. Among the endosperm of 16 giant embryo rice varieties in this study, no white back character was found, and the starch granules on the back of rice grains were regular rhombus or regular and other multilateral, angular. The starch granules had little or no gap, which indicated that the starch granules on the back of rice grain developed well. The reason may be related to the transport mode of nutrients, which were generally transported to the back first to the center and abdomen. It can be seen that the chalkiness of giant embryo rice is closely related to starch development.

The Cause of Chalkiness in Giant Embryo Rice
Chalkiness is a complex quantitative trait in rice, which is not only controlled by many genes, but also related to genetic factors, starch granule structure, arrangement and external environment (Peng et al., 2016a;Li, 2017;Zhu et al., 2017). Based on the previous research and the 16 giant embryo rice varieties in this experiment, there are obvious differences in chalkiness characters of rice endosperm, indicating that the chalkiness characters of rice are mainly controlled by genetic factors. However, the formation of chalkiness is a very complex process, and the generation of chalkiness may be related to the "source", "sink" and "flow" in rice plants. If any of them changes or they are abnormal, it may lead to the occurrence of rice chalkiness (Yan et al., 2003). Therefore, it can be inferred from the results of this study that the occurrence and development of starch granules in rice endosperm and the precipitation of starch granules in the later stage are closely related to the formation of rice chalkiness. However, the distribution of starch granules, the early generation, the development process, the later deposition mode, this series of regulatory ways, how many genes are involved in the formation of chalkiness, need further research.

Conclusion
By observing the microstructure of chalkiness characters (including chalkiness rate, chalkiness degree and chalkiness area) of the 16 giant embryo rice varieties, it was found that the chalkiness characters of different giant embryo rice endosperm were closely related to the morphological structure and arrangement of starch granules in endosperm cells. In different parts of the endosperm of the same giant embryo rice variety, there were obvious differences in chalkiness characters and starch granules in endosperm cells. Therefore, our results will provide an important reference for the improvement of chalkiness character and the breeding of new rice varieties.