| Rice(Oryza sativa L.)is an important cultivated crop,providing food for more than half of the world’s population.Rice grain quality usually includes appearance quality,eating and cooking quality,milling quality,and nutritional quality.Rice endosperm is the main edible part,and the appearance and eating quality of rice are important factors that determine the commodity value of rice,and affect consumer’s choice.There are many factors that affect rice grain appearance quality,however,from the perspective of its genetic regulation,some factors such as grain shape and endosperm development process play key roles in the formation of rice grain appearance quality,and it is a complex quantitative trait.In our previous study,we have identified a series of rice germplasm resources with specific appearance quality traits such as translucency,low amylose content and floury endosperm.In this study,two types of rice endosperms with translucent endosperm and floury endosperm were used as experimental materials.Genetic mapping and gene cloning of the two traits were proceed by map-based cloning method.Based on gene fine mapping analysis,the low amylose controlling site LAC6 and floury gene FN20 were identified.The main results were summarized as follows.1.The underlying reasons for translucent endosperm of indica rice grain under lowmoisture conditionThe amylose content(AC)in the rice endosperm affects the eating and cooking quality along with the appearance of milled rice.In this study,four indica rice varieties with different ACs were used to study the factors affecting endosperm transparency along with the physical and chemical characteristics and eating quality of translucent endosperm varieties.The results showed that in addition to the translucent endosperm phenotype of indica rice variety 28 under low moisture conditions,it has a relative lower AC and better eating quality among the rice varieties.Besides,we found that the transparency of endosperm was positively correlated with grain moisture,and negatively correlated with the area of cavities within starch grains.These factors may be the physical and chemical reasons affecting the endosperm transparency of rice grain.To better understand the differences in starch physicochemical properties,the starch crystal structure and molecular structure were further analyzed.Starch from 28Z rice showed a relative higher relatively crystallinity among the non-glutinous rice varieties.Besides,the degree of amylopectin polymerization was higher in 28Z than in 9311 and HHZ,composed of more medium and short chains DP(16-23),reduced DP(25-34),and DP(42-50),which might be the main reason for the good taste and slow starch digestion in 28Z rice.In summary,considering the moderate AC and good taste value of 28Z,in particular,this variety achieves a trade-off between rice eating quality and appearance.We believe that 28Z is a promising germplasm to improve rice grain quality through breeding.2.Genetic mapping of rice appearance quality gene LAC6.In the present study,the indica rice variety 28Z with translucent endosperm was used for genetic mapping analysis.One indica rice variety Fengbazhan(FBZ),and one japonica rice variety Nipponbare(Nip)with transparent were selected to cross with 28Z to generate the F2 genetic mapping populations.Besides,a high-generation backcrossing population of 28Z/Nip was also generated by backcross.The phenotype of F2 individuals in the primary group were investigated,and it was found that the transparent endosperm phenotype(similar with Nip)and the translucent endosperm phenotype(similar with 28Z)in the population followed the law of single-factor genetic separation with 3:1 genetic separation,which indicated that the translucent endosperm was a trait controlled by a univariate recessive gene.Using map-based cloning technology and combining the linkage relationship between endosperm appearance phenotype and genotype,the regulatory factor was initially mapped to a region between the markers RD276 and LD6-284 on the chromosome 6.The target gene was then named as Low Amylose Content in Chromosome 6(LAC6).To further mapping the target gene,the highgeneration backcrossing population of 28Z/Nip(BC6F2)in Nip background was constructed,and used for gene fine mapping.A total of 1622 recessive individuals from the backcrossing population were analyzed and used for gene fine mapping analysis.Finally,the target gene was mapped to a region with 50.97 kb range between the molecualr markers f4-17 and LD6271.Based on sequence analysis,there are 11 open reading frames(ORFs)were found in this region,and bioinformatics analysis showed that there are no known genes related to rice endosperm appearance and starch synthesis were found in this segment,which suggested that the candidate gene was a novel one responding for low amylose content and grain appearance quality.Rice grain quality profiles were analyzed by using the rice lines Nip.28Z and NILs:the NIP(LAC6)-1 and NIP(LAC6)-2 with transparent grain,and NIP(lac6)-1 and NIP(lac6)-2)with translucent grain.The results showed that the LAC6 gene play key roles in regulating rice amylose content,grain transparence,starch viscosity,crystallinity,and starch fine structure profiles.Besides,the expression characteristics of starch synthesis related genes in rice endosperm at different filling stages were analyzed by real-time PCR.The results showed that the expression level of starch synthesis related genes in lac6 rice was significantly higher in the middle and later grain filling stages than that in plants with LAC6.These genes mainly include amylose and amylopectin biosynthesis related enzymes.Besides,OsAGPL3,OsSSIIa,OsSIIIa,OsPUL and other starch synthetases have opposite expression characteristics.Among them,OsSUT and OsAGPL3 were highly expressed in early filling stage of rice,but decreased slightly in the late filling stage.These results indicated that the LAC6 in translucent grain rice may function in affecting the fine structure of starch by regulating the expression level of major starch synthase genes in the starch anabolic process,which lead to differences in grain transparence,as well as grain quality traits.3.Map-based cloning of FN20 gene in rice with floury endosperm.Starch synthesis is a complex process.The molecular mechanism and regulatory network of starch synthesis in rice endosperm need further study.Here,a rice floury endosperm mutant with abnormal endosperm was screened and identified by using chemical mutagen EMS in the background of a japonica rice variety Ninggeng20.Compared with the wild type,the amylose content and total starch content of the mutant rice were significantly reduced.Besides,the starch granules are stunted,and the viscosity of rice flour is significantly reduced.What is more,the fine structure of starch changes significantly.The mutant was crossed with an indica rice variety Huanghuazhan to construct an F2 genetic mapping population.By phenotypic analysis of seeds(F2 generation)on Fi plants,it was found that normal grains and floury grains showed a single-gene separation pattern of approximately 3:1,which suggests that the floury endosperm is a trait controlled by a univariate recessive gene.Thus,the target gene was named as Floury Ninggeng20(FN20).Then,based on molecular marker linkage analysis of the F2 population,the target gene was preliminarily located to the short arm of chromosome 6.A total of 1759 recessive individuals from the F3 population were used for further gene fine mapping analysis,and finally,the target gene was mapped to a region with 101.4kb kb range.Based on bioinformatics analysis,there are 13 ORFs were found in this region,including the β subunit of pyrophosphate:D-fructose-1,6-diphosphotransferase(PFP)encoding gene OsPFPB which was found to control floury endosperm previously.The sequence of OsPFPB was compared between the mutant and wild type,and we found that the fn20 is a novel allele of OsPFPB.Analysis of basic agronomic traits showed that the grain thickness,1,000 grain weight and starch content of the fn20 mutant were significantly reduced,while the soluble sugar content in rice grain increased greatly.In addition,gene expression analysis showed significant changes in the expression profiles of starch synthesis-related genes in the fn20 mutant rice.At the same time,the expression profiles of genes related to glycolytic pathway was also affected.Moreover,the mutant of OsPFPB was further created by using gene editing method,and the results showed that the β subunits is essential for the maintenance of PFP function.We also generated the OsPFPA mutant rice by using gene editing method,and found that the mutation of OsPFPA gene have no effect on rice endosperm development,and grain quality trait,which indicated that the alpha subunits of PFPase may have functional redundancy.Besides,the fn20 mutant line was crossed with different Wx NILs,and the effects of fn20 mutation on rice quality under different Wx backgrounds were analyzed.The results showed that there were significant differences in rice quality profiles in the Wx/fn20 combination lines.These results suggest that FN20 plays a key role in starch granule formation,starch metabolic regulation,and rice endosperm development.To sum up,the identification and cloning of rice appearance regulation genes have further enriched the regulatory network of starch metabolism in rice endosperm,and at the same time,the new genes and genetic materials identified and created in this study will provide useful germplasm resources and technical routes for the genetic improvement of rice grain quality. |
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