| Lily is an important commercial cut flower.It is an important direction to shorten the breeding cycle and breed early flowering cultivars.Lilium × formolongi flowers within a year after sowing,which precious characteristic is very important for study of early flowering breeding and flowering regulation mechanism of lily.In this study,the backcross 1(BC1)generation with meiotic and mitosis ployploidization origin were constructed using L.x formolongi as parents.The obstacles to interspecies cross were identified and overcome.The inheritance of flowering traits and chromosomes were analized by genomic in situ hybridization(GISH).Based on the identification of different flower induction types of lily,integrating factor FLOWERING LOCUS T(FT)gene family of L.× formolongi and other lily cultivars were studied by gene cloning and bioinformatics analyses.The expression of FT genes in different cultivars at different stages were compared and the function of these genes were studied.This study are of great theoretical and practical value for the breeding of new early flowering lily cultivars and the research on the regulation mechanism of flower formation in lily.The main research results are as follows(1)The L.x formolongi was more suitable used as female parents for interspecies cross with L.Oriental cultivars,and had a higher compatibility for hybridization with 'Sorbonne' and 'Muscadet'.In order to construct BC1 population,L.FO and L.FA were used to backcross with L.Oriental hybrids,L Asiatic hybrids and L.x formolongi.In the backcross combination with L.FO cultivars,'Huigui' and'Hongyun' showed a relatively higher compatibility with L.Oriental cultivars and were more suitable used as female parents;L.FA lily 'Jiaoyang' showed a generally lower compatibility with L.Asiatic cultivars;and the cross compatibility between L.x formolongi and L.FO/FA cultivars was low.Compared with diploid L.FO 'Huigui',more hybrid offspring were obtained using the tetraploid L.FFOO '7-4' coming from somatic doubling as parents.However,L.FFOO '7-4' had different effects when they are used as female and male parent.There was a strong recognition reaction when it was used as female parent,and the pollen hardly germinated on the stigma.This allotetraploid was more suitable for cross breeding as the male parent.(2)The bolting and flowering time of the BC1 from 'Raizan 3' × '7-4'(X4)were significantly earlier than those of other combinations,and 0.8cm tissue culture bulds could flower in 265 days after seeding.Although the BC1 from 'Sorbonne' × '7-4' failed to flower within one year,the number,length and width of basal leaves were significantly greater than those of the offspring from meiotic polyploidization.The results of chromosome composition analysis showed that the distant hybrid F1'Huigui' could produce activated euploid and aneuploid gametes between n and 2n.The BC1 were euploid and aneuploid between diploid and triploid with composition chromosomes.Therefore,BC1 from meiotic polyploidization had significant advantages in growth and development than those from meiotic polyploidization pathway,which inherited the "early flowering" characteristic of L.×formolongi.However,meiotic polyploidization resulted in chromosomes recombination,which was helpful to introgression breeding.(3)The key time points of flower bud differentiation in different lily cultivars were determined.The cultivars were divided into two types according to the time of flweing transition:pre-transplanting flower bud differentiation type and post-transplanting flower bud differentiation type.'Sorbonne','Tresor' and potted L.Asiatic cultivars 'Tiny Ghost','Tiny Todd' and 'Tiny Dino' enter the flower transformation stage at 2-6 weeks in the cold storage period,which is the pre-planting flower bud differentiation type.In contrast,the flower bud differentiation process of L.FO 'Huigui',L.FA'Jiaoyang' and L.longiflorum 'White Heaven' didn't start this process until 4-6 weeks after transplanting,which was the flower bud differentiation type after transplanting.(4)The full-length coding sequences of three members of LfFT gene family were identified in L.×formolongi by RACE technique.Homologous cloning of FT genes were performed in different lily cultivers,and the results of sequence alignment showed that these genes were highly conserved in different lily cultivars.According to the analysis of protein system evolutionary relationships and conservative sequence structure domain,these three FT genes were all belong to FT-like subfamily of PEBP protein family,FT1 gene was most related to the FT of arabidopsis thaliana which promoting flowering,and more variations were detected in the conservative protein structure domain of FT2.(5)The spatiotemporal expression of LfFT gene in the seeding-plant of L.x formolongi were analyzed.LfFTl was highly expressed in leaves and bulds during the induction period,and the expression of LfAPl and LfSOC in stem tips were up-regulated with the up-regulation of LfFT1 LfFT2 was highly expressed in bulds.Although LfFT3 showed a similar expression pattern to LfFT1 in leaves,the expression had been down-regulated before induction.Therefore,LfFT1 plays an important role in flower formation induction of L.× formolongi,LfFT2 might be related to bulb formation of L.x formolongi,and LfFT3 might participate in flower formation process of L.x formolongi.(6)The spatiotemporal expression of FT gene in different lily cultivars were analyzed.FTI were highly espressed in stem apex during transition period in L.FO 'Huuigui',L.FA 'Jiaoyang' and L.Oriental 'Sorbonne',while FT3 were highly espressed in Asiatic 'Tresor'.FT2 was up-regulated in the bulds of L.FO 'Huuigui',L.Oriental 'Sorbonne' and L.Asiatic 'Tresor' after flower formation,while it was mainly highly expressed in the undifferentiated stage,inflorescence formation stage and flowering stage in stem apex and leaves.In the process of buld differentiation and growth of BC1 Q1-16,FT2 peaked when the sacles differentiated into clumps of small bulds,and it was gradually down-regulated in the buld growth period,while FT1 was low in the scale differentiation process,and gradulated in the buld growth period.Therefore,FT genes could be directly transcribed and expressed in the stem apex of lily bulbs,and then induced flowering.FT1 and FT3 play a promoting role in flower induction in different lily cultivars,and FT1 might promotes buld growth,while FT2 might be involved in the differentiation of small bulb.(7)The function of LfFT genes were verified through transformation of arabidopsis thaliana.The mean bolting and flowering time of the transgenic lines of LfFT1 were significantly earlier than that of wild-type arabidopsis thaliana,and the number of rosette leaves was significantly lower than that of wild-type.The mean bolting and flowering time of the transgenic lines of LfFT3 were slightly earlier than that of the wild type,but the number of rosette leaves was significantly lower than that of the wild type.The mean bolting and flowering time and rosette number of LfFT1 transgenic lines were not significantly different from those of wild type.Therefore,both LfFT1 and LfFT3 genes could promote the early flowering of arabidopsis thaliana,and LfFT1 was the important promoter of lily flowering induction.LfFT2 had no significant effect on flowering of arabidopsis thaliana,and its function in flower induction and bulb formation of lily still needed to be further studied. |
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