| Freesia is one of the world’s most famous flower bulbs.It is rich in flower colors and floral.It is an important economic flower in the market and is very popular among people.As an important phenotypic trait of freesia,flower color has always been an important target of freesia breeding,but the physiological and molecular mechanism of pigmentation of flower color has not been revealed.In this paper,11 cultivars were selected as materials.Based on the analysis of the result of anthocyanin components and contents in the petals,transcriptome analysis was carried out by RNA-seq technique,and the differential gene expression profiles were constructed.The key gene expression characteristics were studied to explore and make a basis for revealing the molecular mechanism of petals of freesia.The main findings are as follows:1.Anthocyanin composition profiles in petals of Freesia hybridaWe analyzed the anthocyanin profiles in 11 cultivars of freesia.The petal color was observed according to the Royal Horticultural Society Color Card（RHSCC）and color meter.Pigment type was determined by characteristic color reaction.UPLC-Q-TOF-MS was used for qualitative and quantitative analysis of anthocyanins.The results showed that the 11selected varieties covered four major colors of freesia:white,yellow,red,and blue-purple.All selected cultivars contained flavonoids,which did not contain or contain very low amounts of carotenoids;meanwhile,every cultivar,except for‘White River’,Fragrant Sunburst’,‘Gold River’,‘Tweety’,contained anthocyanins in petals.Among seven anthocyanin contained cultivars,the highest anthocyanin content was detected in petals of‘Red Passion’and the lowest was‘Lovely Lavender’.A total of ten anthocyanin components were detected in petals of eleven cultivars.‘Red Passion’and‘Shangnong Hongtaige’mainly contained Cyanidin derived anthocyanins.‘Pink Passion’,‘Castor’,‘Shangnong Danxueqing’and‘Shangnong Purple Rose’mainly contained Delphinidin and Malvidin derived anthocyanins.‘Lovely Lavender’only contained Delphinidin derived anthocyanins.The colors of petals of different freesia cultivars were related to anthocyanin components,meanwhile,the degree of petal coloration is proportional to the total content of anthocyanins in the petals.2.Transcriptome analysis of petals of Freesia hybridaIn order to analyze the molecular mechanism of the difference in the coloration of freesia petals from the genetic level,four representative varieties were selected:’Red Passion’（RP）,’Gold River’（GR）,’Castor’（CA）and’White River’（WR）petals was analyzed by transcriptome,and115.51 Gb of Clean Data was obtained.The percentage of Q30 base was not less than 93%and GC%was about 45%.A total of 103,828 Unigenes were obtained through assembly,with N50 of 1,388 and 50%of the Unigenes were more than 500 nt in length.The assembly integrity was high.By comparison with various database platforms,a total of 29,309genes obtained functional annotations.Functional analysis was performed through GO and COG databases,and it was found that the annotation genes involved in Carbohydrate metabolism and Secondary metabolic processes were more concentrated.3.Quantitative analysis of genes involved in anthocyanin biosynthesis pathway in petals of Freesia hybridaA digital gene expression profile of freesia was constructed,and the expression levels of key structural genes and transcription factors in the anthocyanin biosynthesis pathway of different Freesia varieties were studied by using q RT-PCR technology.Comparing the distribution of DEGs,it was found that the number of DEGs obtained by RP and CA with WR was 1835 and 1455,respectively,and mainly down-regulated genes.The number of differential genes obtained by RP and CA with GR was 1111 and 848,respectively,and were mainly down-regulated genes.Among them,the number of DEGs obtained by comparison between WR and GR was the smallest,with only 15,indicating there are differences between red-purple and yellow-red freesia flowers in the color formation of peals.The KEGG pathway analysis showed that there are more than 20metabolic pathways involved in the formation of folor color.The number of DEGs enriched in the flavonoid biosynthetic pathway.It is speculated that the flavonoid biosynthetic pathway（ko0094）was closely related to flower color.The main reason for the difference in color of freesia flowers is the difference in the anthocyanin species and accumulation.Therefore,we focus on the branch of the anthocyanin synthesis pathway in the flavonoid biosynthesis pathway.Based on the FPKM values of key genes,a map of the main regulatory patterns of anthocyanin metabolism in freesia petals was constructed.The difference in anthocyanin accumulation and composition in petals mainly resulted in different colors of freesia cultivars.Therefore,we further studied the expression characteristics of key DEGs in the anthocyanin biosynthesis pathway by q RT-PCR.The results showed that anthocyanin synthesis was indeed regulated by structural genes and transcription factors.The expression levels of Chalcone synthase（CHS）,Flavonol 3-hydroxylase（F3H）,Flavonoid 3’5’-hydroxylase（F3’5’H）,Flavonoid 3’-hydroxylase（F3’H）dihydroflavonol 4-reductase（DFR）,Anthocyanin synthase（ANS）,glycosyltransferase（A3GT）in petals of RP and CA were significantly higher than that of WR and GR,which was consistent with the difference of total anthocyanin content,while the expression of flavonol synthase（FLS）in petals of GR and WR was significantly higher than that of RP and CA.In petals of RP and CA,the expression of upstream structural genes such as CHS,F3H and F3’H in RP was significantly higher than that in CA,and F3’H was hardly expressed in petals of CA,while F3’5’H was highly expressed,which corresponded to the content of cyanidin and delphinidin in the four cultivars.The expression levels of MYB,TT8L,GL3L and TTG1 were positively correlated with total anthocyanin content,MYB was almost not expressed in WR and GR,and the expression level of TT8L was higher than that of other three transcription factors.Based on this,it is speculated that the expression levels of upstream structural genes such as CHS determine the total anthocyanin content in the petals of freesia,and F3’5’H and F3’H are important genes that determine the red and purple color of Freesia petals.The different expressions of MYB and TT8L in different species of petals is one of the reasons for the difference in color of Freesia. |
