| Waterlogging stress affects the growth,yield,and quality of barley(Hordeum vulgare L.).At present,the researches mainly focus on trait indicators of the stress and quantitative trait locus(QTL)for waterlogging tolerance studies in barley,only a few genes have been verified.Therefore,it is essential to study the physiological differences of barley genotypes in response to waterlogging stress and to identify tolerance genes for revealing the genetic mechanism of barley in response to waterlogging stress.In this study,100 barley core accessions from 41 countries were used to carry out physiology and genetic association analysis under waterlogging stress.The main results are as follows:1.Physiological differences of barley genotypes in response to waterlogging stress at germination stage.Using three tolerance genotypes(No.37,No.87,and No.88)and three sensitive genotypes(No.8,No.20,and ZD9)as materials,we compared the microstructure and antioxidant enzyme system of different barley genotypes during the germination period under waterlogging stress.In the root cells of the waterlogging-sensitive genotypes,it was found that the cell wall was destroyed,the cell structure became loose and the thickness was uneven,flocs appeared on both sides and the phenomenon of the plasmolysis generally appeared in the cells;invaginating plasmalemma was observed and the mitochondria were injured,the membrane and inner cristae in deformed mitochondria were absent;the nucleus was squeezed to one side,the structure of the nuclear membrane was not clear and nuclear chromatin condensation was observed.However,in the root cells of the waterlogging-tolerance genotypes,the phenomenon of the plasmolysis rarely appeared in the cells;the cytoplasm was slightly shrunken and collapsed,and the degree of vacuolization was less than the sensitive genotypes;the mitochondrial residual cristae could still be seen vaguely;the nuclear membrane was more complete and the nucleoplasm was more uniform than the sensitive genotypes.Besides,the results of the antioxidant enzyme assay showed that SOD enzyme activity in the waterlogging-tolerant genotypes was significantly higher than that in the sensitive genotypes after the end of the waterlogging stress.2.Genome-wide association analysis of waterlogging tolerance in barley at germination stage.Using 100 global cultivated barley core accessions as materials,the mixed linear model (MLM)was used for genome-wide association analysis(GWAS)of relative root length,relative shoot length,relative root fresh weight,and relative shoot fresh weight between under waterlogging and control condition.The results showed that the above four traits were associated with 41,10,17,and 30 significant SNP loci respectively.Among these associated SNPs,3 loci were detected for multiple traits,namely RL-36(SL-5),SL-1(SFW-1),and SL-4(SFW-12),in which RL-36(SL-5)explained the phenotypic variation of 16.21% relative root length and 18.33% relative shoot length,SL-1(SFW-1)explained the phenotypic variation of 17.25% relative shoot length and 15.68% relative shoot fresh weight,and SL-4 (SFW-12)explained the phenotypic variation of 13.08% relative shoot length and 13.25% relative shoot fresh weight.The candidate genes were screened in the 100 kb region upstream and downstream of the significant association sites,and then 77,28,33,and 76 annotated genes were identified.Here,12 annotated genes were detected in multiple traits.Among them,HORVU3Hr1G092660 and HORVU3Hr1G092680 encoded glycosyltransferase and aldehyde dehydrogenase,were associated with relative root length and relative root fresh weight,which could be considered as the candidate genes related to waterlogging tolerance in barley.3.Analysis of differentially expressed genes of barley in response to waterlogging stress at germination stage by transcriptomic methods.Using barley cultivar ZD9 as materials,the RNA-Seq technology was used to analyze the molecular responses in barley to waterlogging stress at germination stage.Compared with under normal condition,waterlogging stress caused a serious effect on the development of roots and shoots of ZD9.The result of differentially expressed genes(DEGs)analysis showed that 5,383 DEGs were identified,including 2,158 up-regulated genes and 3,225 down-regulated genes.Moreover,GO functional enrichment analysis showed that DEGs were mainly enriched in the redox process and metabolic process.Besides,pathway enrichment analysis indicated that DEGs were mainly involved in pathways such as phenylpropanoid biosynthesis,plant-pathogen interaction,and plant hormone signal transduction.According to GO function annotation,three down-regulated genes involved in RAM development were identified,encoding ethylene response factor(HORVU3Hr1G089160),tryptophan aminotransferase 2(HORVU3Hr1G016490),and receptor-like protein kinase 2(HORVU0Hr1G001770).Meanwhile,five DEGs involved in RAM development were identified.In detail,YABBY4(HORVU6Hr1G060770)was significantly up-regulated expression,while three ERFs(HORVU2Hr1G061430,HORVU2Hr1G061440,HORVU2Hr1G061450),and HOX10(HORVU4Hr1G090030)were significantly down-regulated expression.Therefore,it was speculated that the differential expression of the above 8 genes related to meristem development caused the damaged development of roots and shoot in barley during germination stage under waterlogging stress.In summary,based on the GWAS and the RNA-Seq analysis,five genes encoding glycosyltransferase(HORVU3Hr1G092660),short-chain dehydrogenase(HORVU1Hr1G005730),β-1,3-galactosyltransferase(HORVU1Hr1G005680),thaumatin-like protein(HORVU1Hr1G005760),and ROTUNDIFOLIA-like protein(HORVU1Hr1G005690)were identified as candidate genes related to waterlogging tolerance in barley.The present results not only provide the reference for revealing the molecular mechanism of waterlogging tolerance in barley but also laid the foundation for genetic improvement of barley on waterlogging tolerance. |
