The Hybridization Breeding And Functional Characterization Of Flowering-related Genes In Reblooming Bearded Iris

Bearded iris（Iris spp.）could bloom in April and May,while the reblooming bearded iris could bloom not only in spring,but also in autumn.The ornamental period of reblooming bearded iris is increased,which makes it an important ornamental perennial in spring and autumn,and brings it broad application prospects.However,the breeding of reblooming bearded iris is extensively experience-oriented and labor-intensive.Therefore,it is particularly important to analyze the hybridization genetic law of important ornamental traits and the molecular regulation mechanism of reblooming to accelerate its breeding process.Through hybridization and phenotypic character analysis,transcriptome sequencing,transgene,protein interaction analysis and genome editing,this research used bearded iris as materials to analyze the genetic law of ornamental characters in reblooming populations,to screen the key genes of reblooming and to characterize the functions of flowering-related genes,thereby laying the theoretical foundation for cross breeding and molecular breeding of reblooming bearded iris.1.In all of the ten F₁ generations derived from reblooming cultivars‘Cornhusker’,‘Delta Lady’,‘English Charm’,‘Lest We Forget’and‘Many Mahalos’as female parents and‘DH’and‘HD’as male parent,the leaves on the non-blooming stems were longer and the flowers on a scape were fewer than both parents,which ensured the sufficient nutrient supply of more than once flowering.The best female parent was‘Cornhusker’,and the best hybrid combination was‘Delta Lady’בHD’,with the reblooming rate being 16.67%.The broad-sense heritability of fall length was the largest（88.03%）,and the narrow-sense heritability of scape height was the largest（66.63%）.In the six-generation populations derived from‘B-50’and‘Autumn wine’,the broad-sense heritability of height of individual flower and leaf width of non-blooming stems was the largest（83.53%and 82.81%,respectively）.The three backcross populations could have 20.31-33.33%of reblooming offsprings.2.The reblooming bearded iris（RB）and once-blooming bearded iris（OB）from the F₁ generation of‘D’and‘H’were selected for transcriptome sequencing at six developmental stages,and a total of100,391 unigenes were obtained.Combined with the characteristics of short vegetative growth period and insensitive flower bud differentiation to low temperature in RB,the differentially expressed genes（DEGs）between the two floral initiation stages and two adjacent development stages were analyzed,from the perspective of photoperiod,vernalization and growth-related pathways.The circadian clock genes PHYTOCHROME A（PHYA）and GIGANTEA（GI）,growth-related gene AUXIN RESPONSE FACTOR（ARF）and flowering inhibitory gene SHORT VEGETATIVE PHASE（SVP）had significant different expression in the two floral initiation stages,and transferred the floral initiation signal to the downstream flowering-related genes FLOWERING LOCUS T（FT）and APETALA1（AP1）in the second floral initiation stage.The increment of Ig FT and Ig AP1 in the second floral initiation stage would directly enable the second floral initiation.3.Based on the key candidate genes screened by transcriptome,the flowering-related genes SVP,TERMINAL FLOWER 1（TFL1）and FT were cloned,the total length of coding regions being 678,522and 528 bp,respectively.Both Ig SVP and Ig TFL1 Arabidopsis transgenic lines showed late flowering phenotype,while Ig FT lines showed early flowering phenotype,indicating that Ig SVP and Ig TFL1could inhibit flowering and Ig FT could promote flowering in Arabidopsis.The floral organ morphology and inflorescence branching structure of Ig SVP transgenic lines changed.Ig TFL1 transgenic lines showed variations such as the increment of trichomes on opposite leaves or branches.The basal inflorescences of Ig FT transgenic lines were increased,and the flowering time of each basal inflorescence differed by 2-3 days.The temporal and spatial expression patterns showed that the expression of flowering inhibitory gene Ig SVP in the second floral initiation stage of RB was lower than that in the first floral initiation stage of OB and RB.The expression of Ig TFL1 in the second floral initiation stage of RB was higher than that in the first floral initiation stage of RB,and the expression of flowering promoting gene Ig FT in the second floral initiation stage of RB was higher than that in the first floral initiation stage of OB and RB.These suggested that Ig SVP and Ig FT may be involved in the second floral initiation of reblooming bearded iris.4.Based on the results of functional characterization of Ig SVP,Ig TFL1 and Ig FT,the protein-protein interactions between Ig SVP,Ig TFL1,Ig FT,Ig SVPb,Ig TFL1b,Ig FTb,Ig AP1 and Ig SOC1（SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1）,which are also DEGs in the transcriptome profiling,were detected by yeast two hybrid and bimolecular fluorescent complimentary assays.The results showed that Ig AP1 and Ig SVPb could interact,and there was no interaction between other proteins,indicating that Ig SVPb may participate in the regulation of floral initiation through the interaction with Ig AP1 in bearded iris.5.In order to further analyze the effect of overexpression and knockout of the above genes in iris,we tried to establish a tissue-culture-independent genetic transformation method of iris.The naked eye visual marker genes RUBY and PRODUCTION OF ANTHOCYANIN PIGMENTS 1（PAP1）were inserted into Ig SVP overexpression vectors and Ig TFL1 genome editing vectors,respectively.The modified vectors could make the transient-expressed tobacco and stable-expressed Arabidopsis red or purple.GUS staining results showed that the effect of injecting ovary at the full exposure stage of petals was the best.Influenced by the Agrobacterium tumefaciens carrying the modified overexpression or genome editing vectors with naked eye visual maker genes,the seed setting rate of selfing in bearded iris was 3.79%,and the seed germination rate was 29.85%.There were no plants showing the color of the marker genes and no PCR positive plants among the germinated seedlings.In conclusion,in terms of cross breeding,the genetic law analysis of ornamental characters in reblooming bearded iris populations reduced the blindness of breeding.In terms of molecular breeding,based on the preliminary screening of reblooming key genes by transcriptome,the functions of key candidate genes Ig SVP,Ig TFL1 and Ig FT in reblooming were analyzed by transgene and protein-protein interaction analysis.Moreover,by injecting ovary at the full exposure stage of petals,we tried to establish a tissue-culture-independent genetic transformation system of iris,in order to lay a foundation for genome editing and transgenic research of iris.