Genetic Improvement of Grain Quality Promoted by High and New Technology in Rice

Rice (Oryza sativa L.) is considered as one of the most important food crops all over the world. Because of the improvement of the actual living standards, there is more and more demand for improved grain quality. Although, the grain quality is a very complex quantitative trait in rice, it has great scientific significance and practical application value for the improvement of the quality of rice grain using high and up to date techniques. Here, we focus on the new advances in the application of the genetic improvement of grain quality by high throughput sequencing, genomic editing, near infrared spectroscopy, scanning electron microscopy, and other new techniques. Simultaneously, the application prospects of these high and up to date techniques have also been expected in the genetic improvement of rice quality. Thus, this will provide important information for genetic improvement of grain quality in rice breeding.


Introduction
Rice (Oryza sativa L.) is, a staple food for more than 3 billion people in more than 100 countries and regions and considered as one of the most important food crops worldwide, and it provides about 40% of the total calorie intake for Chinese people (Kusano et al., 2015;Peng et al., 2016a).The continuous growth of the world's population and the improvement of people's living standards, make the genetic improvement of grain quality and popularization of the high-quality rice to become one of the key contents (Peng et al., 2014a).Therefore, increasing rice yield and improving grain quality are two important goals of plant basic science and applied scientific research (Zhou et al., 2017), and they are also the focus of concern for plant geneticists and plant breeders in China.grain quality is generally considered the grain appearance and milling, eating, cooking, and nutritional qualities (Tsukaguchi et al., 2016;Pandey et al., 2012;Bhullar et al., 2013;Peng et al., 2014b).However, people in different countries and regions have different concerns about various grain quality traits in rice.For example, the developed countries require the good cooking and eating quality, while the people of India prefer the non-sticky rice and the people from Far East prefer the sticky and soft rice (Chen et al., 2012).For many people in the developing countries and regions, the rice is often the most important energy source (Peng et al., 2016b).Thus, it is of great scientific significance and application prospects to improve grain quality of rice.
Over the past decade, a series of new progress has been made in genetic improvement and related new technology research for rice grain quality.A large number of genes or quantitative trait loci related to grain quality traits were isolated and cloned in rice (Zhang et al., 2016;Peng et al., 2014b), important advances have also been made in the study of the functions of some important quality-related genes and their regulation.At the same time, some high and new technologies (such as the high-throughput sequencing technology and genome editing technology) have been widely used in the fields of agriculture, food industry, pharmaceuticals and medicine, especially in the process of improving and breeding the grain quality of major food crops.Detection and related analytical techniques are playing an increasingly important role in genetic improvement of rice grain quality.Here, we focus on the recent new advances in genome editing technology, high-throughput sequencing technology, near-infrared spectroscopy, scanning electron microscopy, and other high and new technologies in the application of grain quality improvement in rice.The application prospects of modern detection and analysis techniques in rice quality improvement also discussed, this will provide an important reference for the genetic improvement of the grain quality of major grain crops and the cultivation of new varieties.

Application of High-Throughput Sequencing Technology in Genetic Improvement of Rice Grain Quality
High-throughput sequencing technology, also known as next generation sequencing technology, is characterized by low sequencing cost and large amount of data output.It is a revolutionary technological innovation in the process of nucleic acid research (Ansorge, 2009;Liu et al., 2016).A large number of single nucleotide polymorphisms, insertion/deletion and structural variability sites in rice genome can be obtained by high throughput sequencing technology using the natural population, genetic mapping population or mutant individuals in rice.On the basis of these genetic variations, a large number of molecular markers can be obtained by linkage analysis, association analysis, comparative study and related bioinformatics analysis (Subbaiyan et al., 2012;Araikichise et al., 2011).At present, high-density genetic maps of rice has been constructed by high-throughput sequencing technology (Wang et al., 2011;Yu et al., 2011;Gao et al., 2016), and it contains a large number of molecular markers, which have been applied to the location quantitative trait loci (QTL) of rice quality and yield or the mining of candidate genes (Xu et al., 2015).This will promote the genetic improvement of existing cultivated rice, and then accelerate the cultivation of new varieties with high quality in rice (Figure 1).
At the moment, high-throughput sequencing technology plays an important role in the location and exploration of rice grain quality related QTLs or genes (Huang et al., 2012a;Xu et al., 2015).It has been showed by various studies that it is possible to construct genetic maps (Huang et al., 2012a;Gao et al., 2015), establish its database of single nucleotide polymorphism (SNP) and Insertions or Deletions (InDel) mutations (Li et al., 2014a;Tatarinova et al., 2016), through high-throughput genome re-sequencing in rice.These findings provide a large number of polymorphic molecular markers for the genetic improvement of grain quality and promote the molecular breeding for rice quality.For example, through a large-scale sequencing of 517 local rice varieties and 950 cultivated rice varieties, the genome wide association analysis (GWAS) was applied to study the grain quality and yield of these rice varieties.Similarly, a high density haplotype map of rice has been constructed to develop more than one million SNPs markers, and lots of new QTLs related to rice grain quality was also discovered (Huang et al., 2010(Huang et al., , 2012b;;Xu et al., 2015).Moreover, using 150 recombinant inbred populations, a genetic map containing 2334 SNPs markers was constructed by high throughput sequencing technology.Dozens of QTLs related to the important agronomic traits were identified in rice, and multiple QTLs controlling grain length and grain width were located in smaller intervals witch make it easier to analyze directly the candidate genes (Wang et al., 2011).Likewise, high throughput sequencing technology was used to detect a number of QTLs that was closely related to rice grain quality in 241 recombinant inbred lines from zhensan 97 and Minghui 63, and verified the 3 main QTLs (GS3, GW5 and OsC1) controlling rice grain quality traits (Yu et al., 2011).Therefore, high throughput sequencing technology, especially the application of re-sequencing technology to construct the rice genetic map, GWAS analysis, and even the genetic research of mutant plants in rice, will promote the discovery of QTLs or functional genes related to the important traits of rice grain quality, and then accelerate the research and extensive application of molecular breeding for rice grain quality.(Shan et al., 2015;Samanta et al., 2016;Li et al., 2017).Therefore, genome editing technology is a powerful tool in the field of crop genetic improvement and breeding.More and more genome editing technologies, such as TALENs, ZFNs and CRISPR/Cas9, will be applied to the genetic improvement of rice quality in the future so as to speed up the breeding process of new rice varieties with high quality.

Application of Scanning Electron Microscope Technology in Genetic Improvement of Rice Grain Quality
Compared with ordinary optical microscopes, scanning electron microscopy (SEM) has higher resolution rate and can analyze images of the microstructure of the surface and section of grain samples in rice.SEM is an intuitive and effective tool for detecting and analyzing the rice quality in recent years.Different types of japonica rice and fragrant rice and their corresponding rice quality have their specific starch grain shape and arrangement, and their quality could be identified by SEM (Peng et al., 2016b;Kang et al., 2007).Currently, SEM has been widely used in studies of physiological characteristics, morphological structure, and grain quality improvement in rice (Li et al., 2014b;Zhou et al., 2010;Peng et al., 2016c).
It was found that the chalky endosperm has starch grains loosely arranged and filled with a large number of bubbles in its endosperm.However, the starch granules were compact and fairly regular observed by SEM in the control rice endosperm (Li et al., 2014).The results of our previous research showed, there were mainly four types of cell arrangement on the endosperm cross sections of different japonica rice varieties in southern Henan, corresponding to the varieties with white core or white belly in rice grain, the varieties without chalkiness and the varieties with white core and white belly observed by SEM in japonica grain (Peng et al., 2016a(Peng et al., , 2016b)).Further research by SEM showed that starch granules in chalkiness endosperm were not evenly distributed in japonica rice varieties from South Henan province, and the distribution of starch granules in the central part of the endosperm and in the dorsal-ventral direction is relatively concentrated (Figure 3).However, the starch granules of the endosperm in japonica rice varieties from South Henan province with no chalky character were evenly distributed throughout the endosperm, and the appearance of starch granules was relatively regular and starch granules were arranged closely.Therefore, the density of starch granules was larger.In different fragrant rice varieties (Peng et al., 2016b), it was also observed by SEM that the starch granules of fragrant rice grain were mostly ellipsoidal or spherical in the site of chalky endosperm; the starch granules were relatively small in diameter and inconsistent.It could also be observed that the gap between the starch granules inside the chalky endosperm of the fragrant rice varieties was relatively large, and the starch granules were mostly present as a single starch grain, so the arrangement of the starch granules was loose and filled with more bubble.Based on our previous research results (Li et al., 2014b;Peng et al., 2016aPeng et al., , 2016b)), there is a clear correlation between the pattern of cell arrangement and starch granules, the development of endosperm cells and chalky endosperm observed by SEM in rice, which will provide important information for revealing the mechanism of rice chalky traits and the improvement of rice grain quality.jas.ccsenet.At present, NIRS technology has been relatively mature for the detection and analysis of the composition and content of the main components in rice grain, and the detection and analysis of micro-substance content in rice has also been successfully reported.The use of NIRS technology allows rapid and simultaneous detection of multiple quality traits and their associated traits in rice, including moisture, protein content, amylose content, total starch content, ash content, cadmium content and taste value, etc. (Bagchi et al., 2016;Wang et al., 2014;Peng et al., 2014;Zhu et al., 2015;Liu et al., 2016).Spectral data of different rice varieties with large differences in resistant starch content were collected and the mathematical model for predicting resistant starch content was constructed using the partial least squares method with the collected spectral data and the determined chemical value data (Luo et al., 2016), and then verified the mathematical model (including internal verification and external verification) to detect the content of resistant starch in rice, and the effect was better than traditional detection method.Near-infrared reflectance spectroscopy could be also used to predict the total amino acid content in brown rice and the contents of 13 amino acids in rice grain (Zhang et al., 2011).The use of NIRS analysis technology could predict the amino acid composition and total nitrogen content of rice flour, and there was a high correlation coefficient between amino acid content prediction results and traditional detection values (Wu et al., 2002;Lu et al., 2007).Using NIRS technology to predict total contents of phenol, flavonoid and antioxidant capacity in brown rice, established a calibration model to better predict the total phenol content and the antioxidant capacity of brown rice (Zhang et al., 2008;Kim et al., 2013).Rice oil was mixed with soybean oil, corn oil, rapeseed oil, restaurant waste oil, etc., respectively, and adulterated oil samples were prepared according to different proportions, then spectral collection of the mixed oil samples was performed by laser near-infrared spectroscopy (Tu et al., 2015), qualitative models and quantitative models were established simultaneously, and the accuracy of predicted samples was relatively high.Thus, modern NIRS technology can not only quickly and qualitatively detect rice oil adulteration (Tyburczy et al., 2012), but also can be used as an important technology for the detection and analysis of other oils adulterants.

Applica
The detection and analysis of rice quality in storage, processing and distribution have attracted much attention.NIRS analysis technology can quickly and accurately detect and analyze the indicators such as germination rate of rice seeds, storage time of aged rice and fresh rice.Grains of different hybrid rice varieties were aged for different lengths of time through artificial accelerated aging methods, and their spectral data were collected by near-infrared spectroscopy, then a corresponding mathematical model was established and verified, and the results showed that the error between the predicted value and the actual value was very small by NIRS analysis technology (Cossel et al., 2010), indicating that the NIRS analysis technology could rapidly and nondestructively detect the germination rate of rice seeds.Using NIRS analysis combined with nuclear partial least squares to establish a coupled mathematical model between rice spectral characteristics and storage time, which could be used for rapid detection and analysis of different rice storage periods (Ye et al., 2014;Guo et al., 2013).The two different rice samples were blended according to different proportions, then the spectral images were collected by NIRS analysis technology, and finally, a mathematical model was established for quantitative analysis of rice with different grades of rice grain quality (Luo et al., 2016;Zhou et al., 2016;Huang et al., 2012a;Feng et al., 2013).This will provide a rapid and accurate scientific detection method for quantitative detection and discrimination of adulterated rice grains.Therefore, high and new technology has been widely used in the field of rice quality detection and analysis, and has become a modern detection and analysis technology which is developing faster and more and more noticeable.

Application of Other High-Tech Technologies in Genetic Improvement of Rice Grain Quality
Traditional methods for the determination of physical and chemical indicators of rice grain quality have many deficiencies such as the complicated grain sample pretreatment, low efficiency, high cost, rigorous test procedures, and they often cause irreversible damage to rice seeds (Cossel et al., 2010).With the rapid development of modern science and technology, various high and new technologies are continuously applied to the detection and improvement of rice grain quality to make up for the inadequacies of traditional detection and analysis methods.In addition to the above mentioned high-tech technologies (such as high-throughput sequencing, genome editing technology, NIRS technology and SEM technology), there are also some high-tech technologies have been used for genetic improvement of rice grain quality, such as electronic nose detection technology, computer vision technology, texture analysis technology, differential scanning calorimetry technology and so on (Jin et al., 2015;Xu et al., 2014;Park et al., 2012;Parnsakhorn et al., 2012;Zheng et al., 2009;Roberto et al., 2006;Zhou et al., 2010;Yu et al., 2009;Thorpe et al., 2010).Electronic nose detection technology could rapidly detect and analyze odor molecules in rice grains based on the contact of odor molecules with metal oxides or biofilms (Zheng et al., 2009).At the same time, electronic nose detection technology colud also be used to predict fungal infections in grains and even the extent of contamination (Jin et al., 2015;Roberto et al., 2006).Differential scanning calorimetry technology has also been reported to detect the gelatinization temperature of starch in rice.For example, the degree of starch retrogradation in rice could be expressed by the change of the enthalpy value, if the amylose content in rice was higher, the corresponding change of enthalpy value would be greater too (Zhou et al., 2010;Yu et al., 2009).It is noteworthy that computer vision technology has been widely used in the detection and analysis of rice appearance quality traits.For example, computer vision technology could be used to quickly determine the chalky grain rate and chalkiness area in rice, and it could also be used to distinguish pure rice, whole rice, and even the different rice varieties (Ola et al., 2013;Zhou et al., 2010;Ren et al., 2014), which had greatly facilitated the improvement of rice appearance quality.Therefore, with the rapid development of detection technologies, various high-tech technologies are continuously applied to the detection and analysis of rice grain quality, which will certainly bring great convenience to the genetic improvement of rice grain quality.

Prospect of the Application of High and New Technology in Genetic Improvement of Rice Grain Quality
Rice grain quality is a complex quantitative trait, consisting mainly of processing quality, appearance quality, nutritional quality, cooking and eating quality, etc. (Tsukaguchi et al., 2016;Peng et al., 2016a;Bhullar et al., 2013;Peng et al., 2014b).The detection, analysis, and evaluation of grain quality is crucial in rice genetic breeding program.Various high and new technologies have been used in the detection and analysis of rice grain quality, such as high-throughput sequencing technology, genome editing technology, scanning electron microscopy technology, NIRS technology, electronic nose detection technology, computer vision technology, and texture analysis technology, differential scanning calorimetry technology and the other high-tech technologies.These mordern technologies are the further complement and perfection of the traditional technologies for the detection and analysis in rice grain quality, but they all have their own advantages and disadvantages in the practical application of rice quality improvement.With the continuous emergence of new instruments and equipments and the gradual improvement of rice grain quality detection and analysis methods, a variety of non-destructive technologies have gradually been used in the detection and analysis of rice grain quality, and they are rapidly developed and have a wide range of applications.At present, the high and new technologies have covered the quality detection and analysis of rice grain in production, storage, processing, circulation and other links, such as the modern near infrared spectroscopy technology and computer vision technology, etc. Especially in the rice circulation process, it should be able to achieve rapid quality detection and quality control of rice and trace it back.Therefore, for the detection and improvement of rice grain quality, the future development trend is necessary to develop the universal and simple-type equipment in terms of testing equipment.Simultaneously, in the detection and analysis field, it able to achieve a unified set of detection and analysis methods, which should have the characteristics of good accuracy, short time, fast detection speed, stable performance and simple operation.

Conclusion
Rice is considered as one of the most important nourishment crops worldwide, and it has great scientific significance and practical application value for the improvement of the quality of rice grain using high and new technologies.At present, the use of high and new technologies in the genetic improvement of grain quality have made a series of important progress; for instance, the techniques of high throughput sequencing, genomic editing, near infrared spectroscopy, scanning electron microscopy, and other new ones have been widely apllied in the aim of the genetic improvement of grain quality in rice.Moreover, we can expect that, the high and new technologies are the further complement and perfection of the traditional technologies for the detection and analysis in rice grain quality.Indeed, the new technologies have covered the quality detection and analysis of rice grain in production, storage, processing, circulation and other links.Though, the future development trend is necessary to develop the universal and simple-type equipment in terms of testing equipment, and it able to achieve a unified set of detection and analysis methods applied to genetic improvement of grain quality in rice.