Genetic Effect On Key Agronomic,Fine Mapping And Candidate Gene Analysis Of Wheat Dwarf Gene Rht5

Wheat plant height is one of the key phenotypes determining yield.The utilization of dwarfing genes Rht1 and Rht2 significantly reduced wheat plant height and improved its lodging resistance,resulted in a significant increase in wheat yield.However,currently,high-yielding semi-dwarf varieties planted worldwide still rely mainly on Rht1 and Rht2,which severely limited the potential for exploring wheat production.Therefore,it is necessary to explore and enhance our understanding of new dwarf genes as a reference basis for breeding new wheat dwarf varieties.The wheat dwarf gene Rht5 located on chromosome 3B and had a strong dwarf effect without negatively affecting the coleoptile length and the vigor of the seedling.However,there has been relatively little research on the genetic effects and dwarfing mechanisms of Rht5.In this study,we conducted genetic effect analysis and fine mapping of Rht5 using offspring obtained by crossing the donor parents Marfed M with Jinmai47 and Ningchun45,respectively.This study was done to promote the utilization,cloning,and elucidation of the dwarfing mechanism of Rht5 in wheat breeding.We used pure homozygous tall and dwarf lines from F_2:3,F_3:4,and F_4:5 populations as experimental materials to evaluate the effects of Rht5 on important field agronomic traits and grain-related traits.We also analyzed the response of Rht5 to exogenous hormones such as gibberellin（GA₃）and brassinolide（BR）,and explored the relationship between dwarfing and hormones.In addition,we performed RNA-Seq sequencing on the F₅ progeny of the Jinmai47×Marfed M population under elongating spikes to analyze differences in transcription levels between dwarf and tall lines and identify candidate genes related to plant height through correlation analysis.Furthermore,we used two F₂ populations of Jinmai47×Marfed M and Ningchun45×Marfed M as materials,and performed fine mapping and candidate gene analysis of Rht5 using BSA+660K chip analysis and resequencing data.The main research results are as follows:（1）Genetic effects of the dwarfing gene Rht5 in wheat.Rht5 significantly reduced plant height,with a reduction effect of 27.84%（33.62 cm）and 32.67%（32.53 cm）in the Jinmai47 and Ninguang45 populations,respectively.Analysis revealed that this was related to a shortening of internode length,with the largest contribution coming from the peduncle,accounting for 23.54%（7.3 cm）and 35.45%（11.49cm）in the two populations,respectively.Further analysis showed that the shortening of the peduncle was related to a decrease in the number of stem epidermal cells,but not cell length.Rht5 significantly reduced the diameter of each stem node,but its effect on the thickness of the stem node wall was influenced by genetic background.In the Jinmai47 population,Rht5significantly increased the wall thickness of all internodes except the peduncle,while in the Ninguang45 population,there was no significant difference in the wall thickness between dwarf and tall strains.Rht5 increased the stem lodging resistance index（CLRI）by 1.87 to3.44 times,and further analysis showed that the increase in CLRI was achieved by lowering the center of gravity and stem fresh weight.In terms of yield traits,Rht5 had a significant negative effect on spikelet number and grain weight,although it significantly increased the number of fertile tillers by 25.15%to 31.81%.However,it still reduced yield by 27.99%to35.63%.Indoor seedling experiments showed that Rht5 inhibited seedling root growth,and the effect on coleoptile length was influenced by genetic background.In the Jinmai47population,Rht5 significantly shortened the embryonic sheath length by 13.5%（0.96 cm）,while in the Ninguang45 population,Rht5 had no effect on embryonic sheath length.Overall,Rht5 could improve wheat lodging resistance and effective tiller number,but it had a significant negative effect on final yield.（2）Response of wheat dwarfing gene Rht5 to exogenous hormonesResearch results showed that there were significant differences in the response of different dwarfing genes to 10^-4 M GA₃.Rht5（29.74%）had a stronger response to exogenous GA₃ than the gibberellin signaling pathway mutant Rht1（0.79%）,but weaker than the gibberellin-deficient mutant Rht12（54.28%）.After treatment with 10^-8 M BR,the change rate of Rht5（32.14%）seedling height was between Rht1（9.95%）and Rht12（54.39%）.Furthermore,during the jointing stage,dwarfing lines（Rht5Rht5）and tall lines（rht5rht5）produced by the hybridization of Jinmai47 and Marfed M were treated with GA₃and BR.The results showed that both lines had a stronger response to GA₃ than to BR,but exogenous GA₃ could not completely restore the phenotype of the dwarfing lines to that of the tall lines.The determination of gibberellin component content showed that the GA₃content in Marfed M（3.353 ng/g）was significantly lower than that in tall materials Jinmai47（9.953 ng/g）and Ningchun45（9.835 ng/g）.These results indicated that Rht5might have a negative effect on both gibberellin biosynthesis and signal transduction.（3）Transcriptome analysis identifies stem development-related genesDwarf lines（Rht5Rht5）and tall lines（rht5rht5）derived from the cross between Jinmai47 and Marfed M were used for BSR-Seq.The results of differentially expressed genes analysis showed that 131 genes and 781 genes were up-regulated and down-regulated in the dwarf pool,respectively.The enrichment results of GO,KEGG,and GSEA showed that genes related to gibberellin biosynthesis pathway and gibberellin hormone signal transduction pathway were suppressed in the dwarfing lines,indicating that Rht5 may cause dwarfing by affecting gibberellin synthesis and hormone signal transduction.In addition,high-quality SNPs were selected between the pools,and fitting analysis associated the height trait with the end of chromosome 3BS,and identified Ta GAD1＿3B and Ta PSS1＿3B,which had a similarity of 88.00%and 96.71%to the Os GAD1 and Os PSS1 genes that regulate plant growth and development in rice,respectively,and were upregulated in the dwarfing lines.In conclusion,this study provided a reference for the dwarfing mechanism of Rht5 at the transcriptome level.（4）Fine mapping and candidate gene analysis of wheat dwarfing gene Rht5Using BSA+660K chip analysis,248 and 143 SNPs on chromosome 3B were identified to be polymorphic between Marfed M and Jinmai47,and Marfed M and Ningchun45,respectively.10 KASP markers and 9 In Del markers were developed using these SNPs and In Del sites resequenced between parents.By analyzing F₂ populations of Jinmai47×Marfed M and Ningchun45×Marfed M,Rht5 was mapped to the region between In Del-23（10195566 bp）and Xbarc102（17572724 bp）,and Kasp-42（5940010 bp）and In Del-8（12176700 bp）,respectively.The two mapping intervals overlapped at 10195566 bp to 12176700 bp（IWGSC v1.1）.Based on this,4 tightly linked polymorphic markers within the interval were selected to screen recombinants in a Ningchun45×Marfed M population containing 800 individual plants.Finally,Rht5 was located between markers KASP-23 and KASP-25,with a genetic distance of 0.41 c M,corresponding to the physical interval of10659551-11501716 bp（IWGSC v1.1）.This interval contained 17 highly annotated genes,and q RT-PCR analysis revealed that Ta DEx H3＿3B,Ta FBA3＿3B,and Ta CYP709B2＿3B were significantly down-regulated in dwarfing genotypes compared to their parents.Sequence analysis showed that Ta CYP709B2＿3B had a 30 bp deletion in the first exon of the dwarf lines and their parents.In summary,this study narrowed down the candidate gene region for Rht5 to a 1 Mb interval on wheat chromosome 3B,providing a foundation for further cloning of Rht5 and exploring its dwarfing mechanism.This study clarified the genetic effect of Rht5,which resulted in a decrease in plant height by 27.84%to 32.67%.Rht5 improved wheat resistance to lodging and promoted tiller development,but had a significant negative effect on final yield.Results from gibberellin response experiments and content measurements suggested that Rht5 might have negative effects on gibberellin biosynthesis and signal transduction.Transcriptome analysis identified two genes involved in plant growth,Ta GAD1＿3B and Ta PSS1＿3B.Furthermore,the candidate gene range for Rht5 was narrowed down based on fine mapping results,annotation information,and expression analysis,which will facilitate the cloning of Rht5.