Germplasm Discovery And Gene Mapping Of Rice Seed Germination Under Anaerobic Conditions

Due to the shortage of labour,rice planting mode,such as direct-seeding has been received great popularity.However,direct-seeding has its disadvantages that plants are typically exposed to anaerobic stress,which lead to poor germination and low seedling rate,resulting in unstable and low yield.Recently,rice germplasms were screened for anaerobic tolerance,but few varieties were applied to direct-seeding.QTL mapping with the phenotype of anaerobic tolerance at the germination stage was conducted in previous studies,but favorable allele from wild rice has not been detected.In this study,GWAS was performed in a wide range population by high-throughput sequencing technology.Meanwhile,linkage mapping population with introgression lines（ILs）was also used to detect candidate genes and favorable germplasms.More importantly,differentiated expressed genes（DEGs）and metabolites（DEMs）were added in our study.This study was aimed to explore biological mechanism involved in anaerobic germination,discover new genes functionally related with anaerobic tolerance,and provide new genetic variants for breeding rice varieties with anaerobic tolerance.1.We detected 27 QTLs associated with rice anaerobic germination by GWAS.Among them,total four QTLs,such as q AG1-1,were co-located with previous studies and the other 23 QTLs were first discovery in our study.Among them,q AG4-2（p=3.10E-05）was the most significant and total 20 candidate genes were included in it.One gene,LOC＿Os04g51040,encoding wall-associated kinase might participate in anaerobic tolerance.2.Total 7 germplasm lines containing more favorable alleles were detected and showed anaerobic tolerance by GWAS analysis.Total 63 lines showed modest tolerance for anaerobic condition.Among them,the API value of 17 lines were greater than 1.0.Furthermore,W10,W40,W64,and W228 exhibited perfect performance regarding yield,seed shape,and anaerobic germination.Among them,W10 contained 12 favorable alleles.API value was 1.304,yield was 547.7kg/667m²,and the relative ration of length and width was 3.34.These germplasms served as resources for new rice materials.3.Under anaerobic condition,109 ILs showed longer coleoptile length than receptor line,W93-11,which confirmed that chromosome segment from wild rice harbor favorable alleles to enhance anaerobic germination.Furthermore,favorable alleles from wild rice in q AGP1 and q AGP3 could increase coleoptile length under anaerobic condition.These results indicated that pyramiding favorable alleles from wild rice could increase anaerobic tolerance in rice.4.Two major QTLs were detected using a set of Oryza nivara introgression lines（ILs）among two independent experiments.q AGP1 could explained 15%,and 14%phenotype variance for replicate 1 and replicate 2,respectively.q AGP3 could also explained 15%,and14%phenotype variance,respectively.Total 26 candidate genes were located in q AGP1region.Pyruvate kinase（LOC＿Os01g16960）,peroxidase precursor（LOC＿Os01g16450、LOC＿Os01g16152）,and MYB family（LOC＿Os01g16810）were reported to be involved in the process of anaerobic germination.Total 36 candidate genes were located in q AGP3region.Among them,Os VTC1-3（LOC＿Os03g16150）,Os PYL（LOC＿Os03g18600）,and AP2family（LOC＿Os03g12950）were reported to be functionally related with anaerobic germination.Meanwhile,q AGP1 has been published in previous study and q AGP3 was first detected in our study.Furthermore,an F_2:3 population was constructed by crossing the introgression line Ra71 showing wild rice genotype in q AGP3 interval with the parent w9311.The existence of q AGP3 was confirmed by QTL analysis,and the additive effect of q AGP3 was 0.31cm,the LOD value was 9.76,and the contribution rate was 22.3%。5.Genes related with chloroplast development and glucose metabolism were differentially expressed between Ra71 and W9311,which suggested that seed development was inhibited by affecting photosynthesis.Oxidoreductase gene（LOC＿Os03g13390）and glutathione transferase gene Os GST（LOC＿Os03g17470）were induced in Ra71 under anaerobic condition.More importantly,drought tolerance gene Os RCI2-5 was up-regulated in Ra71.By performing KEGG analysis,these DEGs might be functionally related with accumulation of ROS to reduce harm of Ra71 seedling under anaerobic stress.6.DEGs and DEMs between Ra71 and W93-11 were mainly functionally related with metabolic processes of sugars,amino acids,lipids,and secondary metabolites.Among them,genes and metabolites involved in oxidative phosphorylation pathway were differentially expressed.Oxidative phosphorylation genes were up-regulated in Ra71 and induced differentiated expression of related metabolites.7.Ra71 harbor chromosome segments from wild rice in the q AGP3 region and many candidate genes were differentially expressed between Ra71 and W93-11,which suggested that chromosome segments from wild rice might enhance anaerobic germination by up or down-regulating gene expression.Among them,q AGP3 containing three DEGs（LOC＿Os03g13460、LOC＿Os03g14690、LOC＿Os03g17070）were involved in oxidative phosphorylation pathway.These genes encode tubulin,vacuolar ATP synthase subunit,and ATP synthase B chain protein,respectively,which might be candidate genes for q AGP3.But gene function and underling mechanism need further explored.