Genetic Effect Analysis And Regulatory Gene Identification Of Wheat Domesticated Q Gene

Q gene is the major domestication gene of wheat.It is considered to act as a master switch that regulates many different traits,most notably naked（free-threshing）grains,a non-fragile spike rachis and non-tenacious glumes.The Q gene has a pleiotropic effect on several other domestication-related traits,including plant height,spike shape（normal vs.spelt）and spike emergence time,resulting in tougher stems and higher yields.In 2006,Q gene was cloned by map-based approach,and it was found the Q encodes an APETALA2（AP2）transcription factor.Q gene has a mi R172 recognition site at the C terminal,which can be regulated by mi R172.The domestication from q to Q is due to a mutation of C>T in one of the 21 nucleotides of mi R172 recognition site,which affects the splicing efficiency of mi R172 and thus enhances the expression level of Q gene.The q/Q genes exist in all wheat ABD subgenomes.Q is located on chromosome 5A,designated as 5AQ,and its homologous genes on 5B and 5D are designated as 5Bq and 5Dq.So far,no domestication5BQ and 5DQ has been identified,and nonsense mutation in 5BQ has occurred due to functional redundancy and long-term evolutionary selection which makes it as pseudougene.Therefore,Q combinations of all the cultivated hexaploid wheat were 5AQ+5Dq.Identification of genes regulated by Q can help to understand the mechanism of plant architecture regulated by Q,and also provide important information for high yield breeding using Q.However,we have little knowledge about the regulatory genes regulated by Q gene.In our previous study,a dwarf and dense spike mutant was found from the progeny of EMS mutagenesis of Sumai 3.It was found that 5Dq had G to A transition in its mi R172recognition site,which reduced the splicing efficiency of mi R172.We designated it as 5DQ,that is,5DQ is also an anti-mi R172 AP2 gene,thereby increasing the expression of total Q protein,and further leading to dwarfing plant and dense spike.The discovery of 5DQ mutant（Q combination 5AQ+5DQ）provides a new germplasm to further study the genetic regulation mechanism of Q gene.In order to further explore the Q transcription factors on wheat development,especially the influence of the spike development,and regulatory genes by Q,this study has performed the following aspects of research:（1）Q gene expression pattern throughout whole growth period in two genotypes of different Q gene combinations;the dosage effect and allelic variation of Q in the influence of spike morphological development and the agronomic traits;（2）Identification and evaluation of NAUH164 recovery mutant;（3）The genes regulated by Q by Ch IP-Seq method,and the speculated regulatory networks and biological processes involved in Q gene pathway.The main results are as follows:1.Effects of different Q gene combinations on plant growth and developmentThe Q gene combinations of Sumai 3 and NAUH164 were 5AQ+5Dq and 5AQ+5Dq（5Bq is pseudogene）,respectively.The gene expression pattern of the whole growth period showed that 5AQ expression was high in both Sumai 3 and NAUH164 during vegetative growth stage,and decreased in the early stage of transformation from vegetative growth to reproductive growth stage.The expression level of 5AQ in Sumai 3 and NAUH164 was consistent without significant difference.The expression of 5Dq in Sumai 3 and 5Dq in Nauh164 was also high in vegetative growth stage from the whole growth stage,but due to the difference of allelic variation,the expression of 5Dq in Sumai 3 was much higher than that in Sumai 3 during the whole development stage,especially in most stages after the four-leaf stage.In general,the expression level of Q of NAUH164 was higher than that of Sumai 3,and 5AQ+5DQ had obvious dosage effect.The spike of NAUH164 and Sumai 3 at the adult stage were observed and measured.Compared with the wild type Sumai 3,the plant height and spikelet length of NAUH164were significantly shorter.The spike composition of NAUH164 were increased to a certain extent,including spike width,glume,lemma,ovary and stamen.The development of awn also changed.The awn length on the glume of NAUH164 increased significantly,while the awn length on the lemma shortened.The grain length of NAUH164 was also significantly higher than that of Sumai 3,while the seed width was not significantly different.However,the 1000-grain weight was slightly lower than that of Sumai 3,which might be caused by insufficient grain filling.The results showed that the increase of Q dosage inhibited the"vertical"reproductive development of the plant,but promoted the"horizontal"vegetative growth.Appropriate expression level of Q has the potential to increase wheat yield.2.Effects of different point mutations in the splice site of mi R172 of 5AQ gene on agronomic traitsIn order to optimize the expression level of Q,wheat variety 02Y393 was treated with chemical mutagenesis EMS to obtain different Q alleles,which were used to evaluate the effects of different Q expression levels on agronomic traits.Through screening,two plants with mutations at different positions of mi R172 binding site of 5AQ were obtained,among which SL622 was the G>A mutation at 16^thnucleotide position of mi R172 binding site,and SL480 was the G>A mutation at 6^thnucleotide position mutation of mi R172 binding site.Both of mutation were non-synonymous mutations.Gene expression analysis showed that different nucleotide mutations at mi R172 binding sites in 5AQ resulted in increased expression levels,but at different levels,and the 6th nucleotide mutation resulted in higher gene expression levels than the 16th nucleotide mutation.The investigation of agronomic traits showed that the two mutants showed different plant dwarfness and dense spike,corresponding to the expression level.Compared with SL622,SL480 showed higher"compression"between stem and cob nodes,showing extreme dwarf plant height and short spike.The length and width of each leaf of the two mutants increased significantly,the length of leaf sheath also increased,and the stem thickened.However,the increased leaf,leaf sheath and stem thickness of SL480 was not as high as that of SL622,that is,there was no positive correlation with the expression of Q,and too high level of Q expression inhibited the growth of these vegetative organs.Therefore,appropriate Q expression level can obtain the optimal plant growth effect.3.Creation of NAUH164 recovery mutantIn order to obtain mutants with restored or improved phenotype,NAUH164 was treated with EMS to create breeding materials with improved agnomic trait.Two recovery mutants,named H303 and H300,were found in EMS mutagenesis progeny of NAUH164.The phenotypes of these two plants were between Sumai 3 and NAUH164.The mutants had an improved agronomic trait on plant height,ear length,ear width,awn length,leaf size,etc,especially for the square spike morphology and a plant height of 70-80cm which are suitable to grow in the field.Gene expression analysis showed that the expression level of mutant Q gene was decreased,which further indicated that appropriate expression level of Q gene could obtain excellent agronomic traits of the plant.4.Identification of genes regulated by 5DQ transcriptional factorsAP2 protein encoded 450 amino acids,and the molecular weight of the recombinant protein was 49.27k Da.Nine candidate monoclonal antibodies were prepared by immunizing mice with wheat 5Dq recombinant protein（AP2 transcription factor）as antigen.Western-blot hybridization was performed to test the binding of antibody to Q protein,and antibody 2K24 was finally selected for subsequent chromatin immunoprecipitation-high throughput sequencing（Ch IP-seq）test.By Ch IP-Seq assay,1136 genes of 5DQ protein（AP2）were enriched at seedling stage and 524 genes were enriched at ear development stage.KEGG enrichment showed that the genes regulated by 5DQ at seedling stage were mainly involved in Oxidative phosphorylation,Photosynthesis and Ribosome synthesis pathways.The genes regulated by 5DQ during spike development are mainly related to the metabolism and synthesis of some amino acids and sugars,which may be related to the influence of sugar accumulation in the age pathway on flowering.The 5DQ transcription factor may bind to the upstream cis-regulatory element of AGL17 gene to regulate its expression through Motif enrichment analysis,and the binding motif is CTTTTTTTTTTATA.Two distinct peaks around 2200bp upstream of AGL17 were visualized by IGV.In the Ch IP-q PCR experiment,compared with the INPUT control sample,AGL17 was enriched 1.95 times.Gel migration showed that the upstream binding motif of AGL17 could specifically bind to 5DQ protein.The expression patterns of AGL17and during the whole growth period was opposite to that of Q,indicating that 5DQ could bind to the upstream cis-acting elements of AGL17.