Functional Identification And Mechanism Analysis Of TaCOL6 In Regulation Of Wheat Flowering Time

Wheat(Triticum aestivum L.)is the leading source of calories for the human population.Deployment of genetic resources in breeding program is crucial for improvement of wheat yield,adaptivity and resistance to biotic and abiotic stresses.The growth of wheat can be divided into several steps,which mainly include those at vegetative stage and reproductive stage.Regulation of these growth stages in wheat,especially for flowering time,has been evidenced important to achieve high production and adaptivity in diverse environments.For instance,the extensive application of photo-insensitive gene alleles of Ppd1 had played an important role in the “Green Evolution” of wheat.However,the underlying mechanisms of regulation wheat flowering remains largely unknown and only a few flowering related genes have been evidenced applicable in marker associated selection(MAS)in breeding.In our previous study,we have analyzed the population of wild emmer wheat at the“Evolution Canyon”(ECI)and its beside locations/countries.By genotyping 168 accessions of wild emmer wheat via 55 K genechip and genome resequencing,we found three independent populations(SFS1,SFS2 and NFS)had sympatrically diverged at the microsite of ECI,all of which bear different adaptation strategies.It is notated that the SFS1 and SFS2 populations have split on the same south facing slop(SFS),within a 30 meters transect.Facing the high drought and irradiance environment at SFS,the SFS2 population had evolved especially early flowering feature,probably to keep from much higher abiotic stresses at later stage.In contrast,the SFS1 population had evolved direct resistance to high irradiance but with relative late flowering time.In order to investigate the genetic and molecular mechanisms of flowering divergence of SFS1 and SFS2 populations,we performed the current study,details as followings:1.A preliminary analysis using Fst index showed paticulary high degree of genetic differentiation between NFS,SFS1,and SFS2 populations,wherein the Fst values between different populations were generally above 0.6 across genome level.To avoid the allele fixation by genetic drift,we combined the newly split SFS2 population and NFS populationas a single population and performed the selection analysis against the SFS1 population by using XPCLR,Fst and Pi diversity.By annotating the top 5 genes in selection sweeps,we found that a homolog of CONSTANS,a known gene involved in regulating flowering time,showed a different genotype and fixed in the SFS1 population.SNP analysis of the TaCOL6 gene among the three populations revealed that the 321 amino acid in the SFS1 population had changed from lysine to glutamic acid.This mutation of K321 E was only found in SFS1 population when we examined extensive wild emmer wheat accessions from ECI beside location or countries,implying this mutation has been evolutionarily occurred at ECI and fixed in the SFS1 population.2.The biological function of TaCOL6 gene was verified in brachypodium podium(bd21)by over-expression that the flowering time of the transgenic brachypodium showed significantly earlier flowering time than that of the control plants,indicating that the TaCOL6 gene did participate in and positively regulate the flowering period,advancing the flowering time.3.Through yeast one-hybrid and EMSA verification,it was confirmed that TaCOL6 protein can bind to the CORE box on the FT1 promoter and transactivate FT1 gene expression,thereby promoting flowering time.In the Electrophoretic Mobility Shift Assay(EMSA),we found that the K321 E mutated protein of TaCOL6 losed substantial activity binding at CORE element,which indicated that this mutation could lead to late flowering of SFS1 population by failed driving FT1 expression.4.To validate the flowering-related function of TaCOL6 in wheat background,we over-expressed the wild type of this gene in the cultivar of ‘Fielder' and found no significant difference in flowering time when comparing the control plants under normal conditions.However,the early flowering phenotype was observed when drought stress was applied to the transgenic plants.This result implies that TaCOL6 mediates the transcription of FT1 to promote flowering under abiotic stress conditions.5.To unfold the underlying mechanism,we carried out yeast two-hybrid assay and two-molecule fluorescence complementary assay(BIFC)to speculate the interaction partners of TaCOL6.Our results showed that TaCOL6 was able to broadly interact with NF-Y family that are recognized to be involved in the regulation of stress,which indicated that the abioticstress induced early flowering regulation of TaCOL6 might be associated with transcription complex with these NF-Y factors.6.Finally,we investigated the allele divergence of TaCOL6 in wheat germplasm using a‘mini core' collection.Significant correlation between genotypes and phenotypes was found for an allele on 7B chromosome.Beyond the correlation with flowering time,this genotype was also found to be associated with spike-layer uniformity.This result indicates the potential for application of TaCOL6 in future breeding program by marker-assisted selection.