Analysis Of The Molecular Mechanism Of Photoperiodic Flowering Induction For Lilium × Formolongi

Lily is an important cut flower in international flower trade market.Lilium x formolongi is a lily hybrid that flowers within a year after sowing.This precious characteristic is very important for lily flowering improved breeding.The previous study indicated that photoperiod is important for the flowering transition of L.x formolongi.Due the huge genome and complex genetic background of lily,the molecular mechanism of flowering is largely unknown.Analysis the molecular mechanism of photoperiodic flowering in L.× formolongi is of great importance to such fileds.It is valuable for breeding lilies with short vegetative phase by exploration the molecular mechanism of photoperiodic flowering.And the putative gene regulatory network of flowering induction will be helpful to understand the molecular mechanism of flowering induction in lily.Furthermore,this study provides precious gene dataset for the study of molecular mechanism involved in flowering of Lilium.This study confirmed the critical stage nodes of flowering induction through the investigation of phenotype,anatomy and genes expression.Based on the transcriptome sequencing,the putative gene regulatory network of flowering induction was constructed.And we obtained some important promotive genes in photoperiodic flowering pathway.The functional and evolutionary characterizations of the 8 CONSTANS-LIKE family members of L.x formolongi were analyzed.And the critical LfCOLs members in flowering induction were confirmed.Some main results as follow:(1)The seedling of L.x formolongi was a long-day plant.Long day condition largely triggered the flowering transition of L.x formolongi.Under long day condition,the flower time of L.x formolongi was nearly four months earlier than short day,and the flower percentage was 60-70%more than short day.Moreover,the character of flowering within a year after sowing was inherited from L.formosanum.The other parent L.longiflorum could not flower within year after sowing.Futhermore,the character of L.x formolongi which was sensitive to photoperiodic flowering could be inherited.We had obtained the flowering segregation populations in selfing line.The early flowering population flowered one month earlier than normal flowering population,and two months earlier than later flowering population.These flowering segregation populations would be good population material for the study of molecular mechanism of photoperiodic flowering in L.× formolongi(2)Through investigation of the morphological characters,anatomical structure of the shoot apex meristem(SAM)and the circadian rhythm expression patterns of CONSTANSCO/FLOWERING LOCUS T(CO/FT)in long days.The critical stages of juvenile,flowering induction,adult and floral differentiation were confirmed,as well as the phenotypic traits of each stage.When the shoot apical meristem was in vegetative status,the circadian rhythm patterns of CO/FT could be used as molecular markers to identify the flowering sensitive status and flowering induction stage(3)The transcriptome sequencing of leaves and stem apexes from the stages of juvenile,flowering induction,adult and flowering differentiation germinated 52,824 Unigenes,of which 33,652(63.71%)Unigenes obtained functional annotation.The gene expression libraries of four stages generated 37,031 differentially expressed genes,most of them were up-regulated in the flowering induction stage,down-regulated in the adult stage,and up-regulated again in the floral differentiation stage.Furthermore,in this study,87 flowering homologs and 52 transcript factors were identified.The transcript factors belong to the families of bHLH,bZIP,RAV,MYB,Dof,CO-like,MADS-box and so on.And the flowering homologs distribute in six flowering regulation pathways of higher plant,of which 41(48.8%)homologs locate in photoperiodic flowering pathway,including GIGANTEA(GI),CO,FT and so on Based aboved experiment results,the putative gene regulatory network of flowering induction was constructed(4)The full length of 8 LfCOL gene family members were identified according to the RACE technology based on the RNA-seq data.And the 8 LfCOLs genes could be divided into three types according to the conserved protein domains.The first type showed two B-box motifs,only including LfCOL5.The second type exhibited one B-box motif,consisting of LfCOL6,LfCOL7,LfCOL13,LfCOL14,LfCOL15,and LfCOL16.The last type contained a normal B-box motif and a second diverged B-box motif,only including LfCOL9.Among the 8 LfCOLs,LfCOL5 showed the closest phylogenetic relationship with Arabidopsis CO,as well as the structure of conserved protein domains.(5)The expression patterns of 8 LfCOLs members and LfFT1 were investigated under different photoperiod conditions and development stages.The results indicate that LfCOL5,LfCOL6,LfCOL9,and LfFT1 all showed the highest expression in the flowering induction stage,but showed different stage-specific expression patterns under short day condition.Moreover,LfCOL5,LfCOL6,LfCOL9 exhibited circadian rhythm patterns for three days under both long day and short day conditions.Under long day,the circadian rhythm patterns of LfCOL5,LfCOL6,LfCOL9 were the same with Arabidopsis CO.Thus,LJCOL5,LfCOL6,LfCOL9 may be important promotive factors in photoperiodic flowering.(6)The plant expression vectors of LfCOL5,LfCOL6,LJCOL9 were constructed seperately.And the transgenic experiment suggested that LJCOL5,LfCOL6,LfCOL9 could trigger the Arabidopsis co-1 mutants flowering earlier.The transgenic plants with LfCOLs flowered 12-13 days earlier than co-1 mutants,6-7 days earlier than wild plants.The average leaf of transgenic plants with LfCOLs was 12-14 less than co-1 mutants,and 6-7 less than wild plants.Moreover,the expression level of AtFT in the transgenic plants with LfCOLs were all higher than the co-1 mutants when the plants just bolting.Thus,LfCOL5,LfCOL6,LfCOL9 were critical to trigger flowering induction under long day condition in L.×formolongi.Specially,LfCOL6,LfCOL9 were the distinctive genes to promote flowering in L.×formolongi.And L.x formolongi LfCOL family may be distinctive in gene phylogenetics and function.This study will be valuable for the exploration of the molecular mechanisms of flowering initiation and the short vegetative stage of L.x formolongi.The obtained critical flowering induction genes will be helpful to the breeding and cultivation of lilies with short vegetative stage.