Comparison Of Flowering Mechanism Of Different Seasons In Asiaticus Group Of Osmanthus Fragrans

Osmanthus fragrans Lour.(Oleaceae)was cultivated as an ornamental and aromatic plant for a long history in China.The cultivars of O.fragrans was divided into Asiaticus Group and Autumn Group,the formor flowered many times in different seasons of a year,but the latter one time in a year.To study flowering mechanisms in different seasons of Asiaticus Division,3cultivars were selected invovling O.fragrans ‘Sijigui',O.fragrans ‘Rixianggui' and O.fragrans‘ZAFU-S1'.Firstly,the microdissection technique and paraffin method were employed to observe flower bud differentiation and flowering characteristics in different sensons of O.f‘Sijigui',O.f ‘Rixianggui'and O.f ‘ZAFU-S1'.Then in order to explore the flowering mechanism of Asiaticus Group in different seasons,designed FT and TFL1 primers according to transcriptome of Of ‘yanhonggui',and cloned their homologous genes from Asiaticus division O.f ‘Sijigui',O.f ‘Rixianggui',O.f ‘ZAFU-B' and Autumn Division O.f ‘yanhonggui',and further analysised their sequence differences at the mRNA and DNA level,respectively.In addition,the expression level of OfFT and OfTFL were determined at the different stage of flower bud differentiation,we aimed that to further explore the flowering mechanism of Asiaticus Group.The main results of our study were as follows:1.Comparison on flower bud differentiation and development in different seasons of Asiaticus division:O.f ‘Sijigui' and O.f ‘Rixianggui' flowered 3 times one year,the flower bud was observed in early March and continued to late April,then the second stage of flower bud begun early June and finished in the late August,the last stage begun from late October to the late November or early December.In addition,O.f ‘ZAFU-S1' flowered 2 times one year,flower bud formation begun from early March and early June and finished in late April,late august.In 3cultivars,the flower bud differentiation begun in June and flowering process were similar to Autumn Group which flowered only at autumn one year,but other seasons were different.The flower buds formatted in June and experienced 6 stages involving inflorescence primordium differentiation stage,floret primordium differentiation stage,sepal primordium differentiation stage,petal primordium differentiation stage,stamen primordium differentiation stage and carpel differentiation stage.Additionally,we found that it could not bloom after finishing flower bud differentiation unless experienced low temperature and finally formed cyme which had no inflorescence stalks,and the pollen developmented nomally.In O.f ‘Sijigui' and O.f ‘Rixianggui',the buds begun in October flowered immediately after finishing differentiation and had inflorescence stalks which were divided into short stalks and long stalks,and the lengthes were0.8±0.11 cm,1.03±0.12 cm and 3.5±0.71 cm,2.98±0.41 cm;The buds begun in March developmented with new shoots at the same time,and these two differentation formed panicle finally,the flowers had unnomal pollen.The results show that flowering mechanisms in different reasons of Asiaticus Group had significant differencesand it needed further researches.2.The cloning and sequence analysis FT and TFL1 homologous gene in Asiaticus Group and Autumn Group : we designed FT and TFL1 primers according to transcriptome of O.f‘yanhonggui' to clone those homologous from O.f ‘Sijigui',O.f ‘Rixianggui',O.f ‘ZAFU-S1'and Autumn Group O.f ‘yanhonggui'.We obtained the OfFT cDNA sequence and 2 TFL1 homologous genes OfTFL1 A and OfTFL1 B cDNA and DNA sequences.After sequence alignment,we found these genes contained 4 exons and 3 introns.OfTFL1 A and OfTFL1 B in 3cultivars were above 99.3% and 97.85% at the DNA level,above 99.86% and 97.69% at the cDNA level respectively with O.f ‘yanhonggui'.The homology of OfFT were above 90.48% at the cDNA level with O.f ‘yanhonggui'.In addition,the functional sites of three genes were simile with O.f ‘yanhonggui'.All of these indicated that OfFT,OfTFL1 A and OfTFL1 B sequences have no differences with O.f ‘yanhonggui'.These further excluded possibility that the sequences difference of OfFT,OfTFL1 A or OfTFL1 B caused flower in different seasons.3.Expression analysis of OfFT,OfTFL1 A and OfTFL1B:We analyzed the expression pattern of OfFT,OfTFL1 A and OfTFL1 B from 3 cultivars in the one year using the real-time quantitative PCR,ACT as a reference gene.The results showed that OfTFL1 A and OfTFL1 B associated with flowering transitionand OfFT can promote flower bud formation and flowering.In addition,there was no obvious difference in OfFT expression between before and after flowering transition in spring and winter,the expression were lower than autumn.So,the flowering pathway in spring and winter differed from autumn,and we can excluded Photoperiod pathway,Vernalization pathway and Ambient temperature pathway.So,we predicted GA pathway.In conclusion,floral development,inflorescence,ambient temperature and illumination conditions when flowering transition of Asiaticus Group in spring and winter differed from autumn,and the sequences and functional sites of OfFT,OfTFL1 A and OfTFL1 B were simily with O.f ‘yanhonggui',there was no obvious in expression between before and after flowering transition in spring and winter and the expression were lower than autumn.These indicated that there are different flowering mechanisms and pathway between different seasons in Asiaticus Group.