Characteristics And Genome-wide Association Study Of Starch Fine Structure And Digestibility In The Endosperm Of Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is the most important staple food providing calories and proteins for half of the world's population,especially in developing countries.Along with economic growth and the increased level of people's lifestyle,grain quality becomes the primary consideration of rice customers and breeding programs in recent years.Rice quality is a very complex trait.Within the rice qualities,the eating and cooking quality(ECQ)and digestibility property have been focused increasingly because they closely associated with mouthfeel and human health.Therefore,understanding the detail mechanism of rice digestibility and its relationship with the ECQ could help to achieve these goals.Rice endosperm is the main consuming part of the rice grain,and the starch comprises about 90%of the dry matter of endosperm.Thus,the rice grain quality is greatly affected by starch composition and structurec and it is very important and useful for the improvement of grain quality to reveal the molecular regulation of starch metabolism in rice endosperm.In the world,there are abundant rice germplasms,including lots of local landraces,and the grain quality variation occurred widely among these landraces.Therefore,it is a key issue to clone and explore novel QTLs/genes of grain quality-related genes for rice quality improvement.In this study,we selected hundreds of natural rice accessions to analyze their ECQ qualities,starch fine structure and digestibility.Based on these quality data,we firstly analyzed the relationships between starch fine structure and ECQs,and digestibility of rice grain,expecting to find how the starch fine structure effect on rice qualities.Secondly,we explored the relationships between three important starch biosynthesis enzymes with starch fine structure to understanding how these enzymes affect the biosynthesis of starch in rice endosperm.Thirdly,we carried out the genome-wide association analysis(GWAS)to mine the known and unknown candidate genes that could affect the digestibility,and classified the mechanism of how they could affect the digestibility.Finally,as the Waxy(Wx)gene has been proved to be significantly correlated with both ECQs and digestibility of rice grain,we carefully analyzed the evolution and domestication of all cloned Wx alleles,and reported how the Wx gene affecting the digestibility of rice.The main results are shown as follows.1.Characteristics and correlationship between ECQ,digestibility and starch fine structure in rice natural populations.A total of 562 rice natural accessions that collected from all over the world and belonging to different subpopulations were used for the analysis of ECQ and digestibility properties.The ECQ properties include apparent amylose content(AAC),RVA profile and total seed protein content.Meanwhile,87 varieties were selected by different environmental and genetic backgrounds from the above 562 samples and their chain-length distributions(CLD)of amylopectin and amylose were analyzed by using size-exclusion chromatography(SEC).The SEC data were further fitted with a biosynthesis-based model and the parameters h and ? that reflected the amount of chains with different degree polymerization(DP)were derived.The correlation analyses showed that the digestibility parameters.AAC and RVA properties were correlated with each other,and all significantly correlated with the starch fine structure.While the total seed protein content has no significant correlation with other quality parameters,except with the AAC.More detail,it was found that the amounts of amylopectin intermedium chains(DP?28?58)and long amylose chains(DP?500?1000)mainly controlled the entire process of rice digestibility.Thus,it implied that increasing the amounts of short chains of both amylopectin(DP?6?24)and amylose(DP?100?500)might decrease the digestion rate but remain the fine ECQ.These data provide useful guidelines for understanding the relationship of digestibility with starch fine structure and other qualities of the rice grain,which could help rice breeders develop integrated quality by controlling the starch fine structure of rice.2.New insights into amylose and amylopectin biosynthesis in rice endosperm.Ninety-five rice varieties were selected by different environmental and genetic backgrounds from the above 562 samples and their CLDs of amylopectin and amylose were analyzed by using SEC.These CLD data were fitted with the biosynthesis-derived models based on different iso forms of soluble starch synthase(SSS),starch branching enzyme(SBE,including SBEI and SBEII)and granule-bound starch synthase(GBSS).All these enzymes are involved in amylose and amylopectin synthesis in rice endosperm,and it is usually thought that these are synthesized by separate enzymes.Based on these data and the models,how various isoforms of rice-starch biosynthesis enzymes interacted during amylose and amylopectin synthesis are explored.The results showed that different enzyme sets of SBE and SSS in amylopectin are also involved in the synthesis of rice amylose,whilst amylopectin enzyme set i is alone responsible for the synthesis of the short chains of amylopectin(DP 6?24).These short chains can also be the initial substrates for amylose synthesis.Furthermore,not only can amylopectin enzyme set iii catalyze the synthesis of intermediate-length amylopectin chains(DP 28?58),but it can also affect the synthesis of short and long amylose chains.Additionally,it is likely that the enzyme which is responsible for the synthesis of long amylopectin chains is also involved in the synthesis of long amylose chains.As some of the longer amylopectin(Ap set v)and shorter amylose(Am set i)chains share the enzyme set,the GBSS could provide more long amylopectin chains.Taken together,these data indicated that the amount of longer amylopectin chains positively correlated with that of shorter amylose chains,indicating that GBSS,SBE and SSS can affect both amylose and amylopectin synthesis.Further,the activity of GBSS in amylose significantly correlated with that of SSS in amylopectin.This new understanding of which enzymes are suggested by the statistics to be involved in both amylose and amylopectin synthesis could help rice breeders develop cereals with targeted properties.3.Genome-wide association studies of rice digestibility and starch fine structure.Digestibility of rice is significantly correlated with human health,and,in this chapter,we performed the genome-wide association study(GWAS)and set-base study on the parameters of starch fine structure and digestibility of rice.The digestibility property was detected from a total of 562 rice accessions with full genomic sequence information,and the CLDs of amylopectin and amylose were obtained from 87 varieties that selected from the above 562 samples.After the GWAS and subsequent set-base study,more than 100 candidate QTLs/genes that might be associated with the starch fine structure and digestibility were mapped.Among them,15 important candidate genes,including the Wx and RSS1 genes,were predicted according to the mapped QTLs and available SNP information of the selected rice accessions.Particularly,the effects on rice starch structure and digestibility by the important candidate gene RSS1 were further analyzed based on the genomic data and its over-expression line.The results indicated that RSS1 could control the digestibility as the minor gene,and also could control the amount of amylopectin short-medium chains(DP?6?60)and amylose long chains In addition,the digestibility of rice grain could be negatively correlated with the amount of amylose long chains,and a large number of minor genes that could be related to the cell pathway system could also affect the specific stages of digestion of rice grain.These findings provide useful information for better understanding the pathway from genetics to phenotype for rice digestibility,and may help breeders to use molecular techniques to improve the digestibility of rice.4.Allelic variation and evolution of Wx locus in rice and their effects on digestibility.Up to now,the effects of several Wx alleles on AAC and ECQ have been elucidated.Meanwhile,as shown above,we found that the Wx locus is also significantly correlated with the digestibility of rice.However,how the Wx affecting digestibility.the relationship between artificial selection and the evolution of various Wx alleles as well as their distribution remain unclear.Here,we firstly analyzed the sequence variation of the Wx locus from a total of 967 rice accessions,including 399 Oryza sativa and 568 Oryza rufipogon accessions.We demonstrated that the Wxlv allele,which is a newly cloned Wx allele in our previous work and dramatically affects the mouthfeel of rice grains by modulating the size of amylose molecules,originated directly from wild rice,and the other Wx alleles widely used in cultivated rice(such as Wxb,Wxa,and Wxin)differentiated after the substitution of one base pair at the functional sites.It indicated that Hxlv plays an important role during artificial selection and domestication of grain quality improvement.It is found that the Wx gene is significantly correlated with starch fine structure and digestibility.The Wx gene could control the biosynthesis of amylose,especially the amylose long chains.The Wxlv and Wxa alleles control the highest amount of the amylose long chains,but Wxlx controls a high amount of amylose short chains.The null wx allele has no amylose chains bit with the lowest amount of amylopectin long chains,meanwhile,its starch fine structure is also controlled by diverse factors.In addition,for the aspect of digestibility,it is indicated that the Wxa and Wxlv alleles control the lowest digestion rate constant k1 but the highest k2,while ux has the highest k1 but lowest k2.The other Wx alleles are in the middle.All these data suggested that Wx gene is a major gene,combined with other minor factors,to affect the digestibility of rice.Furthermore,we found that more amount of amylose long chains(?500?1000)could decrease both of the k1 and k2 and it is the most important factor for the digestibility.A large amount of amylopectin intermediate chains and the small amount of amylopectin long chains could be the minor factors to increase the k1 and k2,and the amylose short chain is also as minor factor that can be positively correlated with k1.The findings also shed light on the evolution of various Wx alleles and the effects of Wx alleles on digestibility,which have greatly contributed to improving the digestibility quality of rice.To sum up,in this study,we found that the biosynthesis of starch,starch fine structure,rice ECQ and digestibility are significantly associated with each other,and all of these factors are controlled by the genetics of rice.Two important genes that significantly correlated with digestibility and starch fine structure have been classified,Wx is a major gene while RSS1 is a minor gene.In addition a large number of minor genes that could be related to the cell pathway system could also affect the specific stages of the digestion of rice grain.These findings provide novel insights into the phenotype and genotype of natural variation of rice grain quality,the emphasis of rice digestibility and starch fine structure,and are expected to contribute to the breeding of rice with fine properties for human health and mouthfeel.