Identification And Functional Analysis Of Genes Involved In Seed Dormancy In Rice(Oryza Sativa L.)

Seed dormancy pervasively exists in the plant kingdom,which is of significant importance for plants to resist unfavorable environmental conditions and maintain their prolongation of the generation.On the one hand,loss of seed dormancy is a typical feature during the domestication process of many crops which leads to pre-harvest sprouting(PHS)and reduced cereal yields and quality.On the other hand,deep dormancy causes low and un-uniform germination when seed sowing,which is difficult to meet the demands for crop production.Rice(Oryza sativa L.)is one of the most important cereal crops in China,unraveling complex genetic control of seed dormancy will contribute to breeding elite cultivars with moderate dormancy,which possessed of great significance for rice production of high yield and top quality.In this study,a genome-wide association study(GWAS)was used to explore the genetic basis of seed dormancy variation,and the physiology mechanism of seed dormancy release by cold stratification was also analyzed.The major research contents were summarized as follows:1.A diverse panel of 226 sequenced rice accessions was used to evaluate PHS over a period of two years.A total of 15 elite varieties with resistance to PHS in both 2016 and 2017 were identified,such as Carolina Gold Sel,Dhala Shaitta and DV85.By contrast,Indica group presents deeper dormancy level than Japonica group.By implementing genome-wide association studies with EMM AX model,a total of 81 loci associated with natural PHS variations were identified in both years.Among them,26,64 and 4 loci were detected in the whole panel,Indica and Japonica groups,respectively.By comparison,57%(46/81)of loci detected in this study were co-localized with previously reported QTLs for seed dormancy.In particular,12 loci were repeatedly detected in both years.Among them,8 loci overlapped with reported QTL for seed dormancy and 4 additional loci were novel.Localization analysis of significant SNPs in the candidate regions of 12 loci resulted in prediction of 72 candidate genes.The key candidate gene for qPHS9.2 was LOC_Os09g36500(an annotated zinc finger protein),which has been identified through transcriptome analysis to be differentially expressed in seeds between dormant cultivar and non-dormant cultivar.The key candidate gene for qPHS12.6 was LOC_Os12g26940(an annotated CHASE domain containing protein),the closest homologs of Arabidopsis thaliana AHK2 and AHK3 were known to participate in seed germination.2.To characterize the variation of seed dormancy in rice,a set of 216 natural accessions was analyzed through the germination test.The germination rate after 3,5 and 7 days imbibition and the germination index were measured both in the freshly harvested seeds and the after-ripened seeds.Genome-wide association analyses were implemented with EMM AX model using the four dormancy-related traits.For the freshly harvested seeds,a total of 86 distinct loci were significantly associated with seed dormancy.Of these significant loci,27,68 and 5 loci were detected in the whole panel,Indica and Japonica groups,respectively.For the after-ripened seeds,a total of 94 distinct loci were significantly associated with seed dormancy.Of these significant loci,58,51 and 17 loci were detected in the whole panel,Indica and Japonica groups,respectively.By comparison,57%(49/86)of loci detected in the freshly harvested seeds and 64%(60/94)loci detected in the after-ripened seeds were co-localized with previously identified QTLs for seed dormancy,respectively.Meanwhile,11 loci were associated simultaneously by four dormancy-related traits in the freshly harvested seeds and the after-ripened seeds,respectively,and three of which(qSD12.3,qAR2.5 and qAR11.4)were the most significant loci(-log10 PWhole>6.9,-log10 PIND>6.8 and-log10 PJAP>6.6).qSD12.3 was co-localized with qPHS12.6 that associated with PHS variation in chapter one of this thesis.Localization analysis of significant SNPs in the candidate regions of qAR2.5 and qAR11.4 resulted in prediction of 25 candidate genes.Subsequently,the causal gene of OsPK3(LOC_Os11g10980)for qAR11.4 were identified through linkage disequilibrium analysis,haplotype analysis and enzyme activity determination.OsPK3 encodes a cytosolic pyruvate kinase,which catalyses the final and irreversible step of glycolysis pathway.The CRISPR/Cas9 gene-editing system was applied to generate four mutants,including pk3-1,pk3-2,pk3-3 and pk3-4.Further phenotype evaluation showed that the disruption of OsPK3 resulted in low germination rate,seedling potential and germination index in the after-ripened seeds compared to those of the wild-type.The physiological analysis showed that the disruption of OsPK3 resulted in a significant lower pyruvate kinase activity and pyruvate/phosphoenolpyruvate(Pyr/PEP)ratio,and a significant higher PEP,Pyr and sugar contents in imbibed seeds compared to those of the wild-type.These results indicated that mutation of OsPK3 leads to disorders in the glycolysis pathway.Besides,the levels of ABA were significantly increased in imbibed seeds compared to those of the wild-type,whereas the contents of GA4 were significantly decreased in mutant lines.A significant higher of ABA/GA1 and ABA/GA4 ratios were observed in mutant lines compared to those of the wild-type.These results indicated that OsPK3 was involved in the regulation of glycometabolism and ABA/GA balance during seed germination.3.Cold stratification is commonly used for alleviating seed dormancy in plants.In this study,dormancy release by different periods(0,6,12,18 and 24 days)of cold stratification(4?)was conducted using japonica Jiucaiqing(Oryza sativa L.)Germination index,germination and seedling percentage were significantly increased with cold stratification.Seed dormancy of Jiucaiqing was completely released by cold stratification for 18 and 24 days.The expressions of the hormones,including ABA,GA and IAA,metabolism-related genes OsCYP707A5,OsCYP707A7,OsGA20ox1,OsGA20ox2,OsGH3-2,OsGH3-7 and OsGH3-8 were significantly increased while OsNCED3 and OsZEP significantly decreased in imbibed seeds by cold stratification,which resulted in the rapid decline of ABA and the increases of GA3 and IAA.The activities of antioxidant enzymes,including SOD,CAT and APX,were significantly increased in imbibed seeds by cold stratification,which resulted in a slight increase of H2O2 level.The ratios of GA/ABA,GA/IAA and IAA/ABA and H2O2 level were gradually enhanced in imbibed seeds with cold stratification,which might contribute to the increase of ?-amylase activity to promote dormancy release in rice.In this study,we explored the complex genetic architecture of seed dormancy at two different stages of storage and clarified important insights into the role of OsPK3 in seed dormancy,these results will provide a theoretical basis for the genetic improvement of seed dormancy in rice.Besides,the physiological characteristics of cold stratification on dormancy release in rice were also clarified in this study,these results will provide a basis for improving seed sowing quality in the future.