Germplasm Innovation And Molecular Basis Of Color Formation Of Bicolor Flower In Eustoma Grandiflorum

Eustoma grandiflorum belongs to the Gentianaceae in the Gentian family,and has higher ornamental value.At present,the domestic varieties of E.grandiflorum are mainly F₁ hybrids from Japan,Europe and America.There are few varieties with independent intellectual property in China,and some special color types,such as the bicolor flowers,are more scarce,which have seriously restricted the long-term development of E.grandiflorum industry in China.In this study,we created 6 excellent inbred lines of bicolor flowers with stable traits using traditional breeding methods.The expression levels of secondary metabolites and differential genes during the development of bicolor petals in the inbred lines MT1 were analyzed by UPLC-MS and high-throughput sequencing technology,which laid a foundation for elucidation of the molecular mechanism of anthocyanin accumulation of bicolor flowers in E.grandiflorum,and provided ideas and technical support for the innovation of seed industry.The results are as follows:1.Created 6 inbred lines of bicolor flowers in E.grandiflorum with independent intellectual property.24 white-petal females and 5 red-petal males were selected to configure24 cross combination,among which 15 combinations were panchromatic flowers,and the remaining 9 combinations showed different proportions of bicolor flowers types,but the proportion was lower.Further evaluation was conducted on the bicolor flowers in 9combinations,and 6 superior combinations of bicolor flowers were identified.After 6generations of continuous self-crossing,nearly homozygous inbred lines of bicolor flowers MT1,MT2,MT3,MT4,MT5 and MT6 were obtained.Among them,the upper pigmented part of petals of MT1 and MT2 were strip type,MT5 and MT6 were spotted type,and the other two inbred lines are intermediate type.By evaluating the agronomic traits,flower characters,and fruit-setting of these 6 inbred lines,it was identified that MT1 had the strongest advantage of comprehensive trait and was used as the test material for further research.2.Three kinds of anthocyanins were identified in the comparison between the pink upper petals and the white bases of the bicolor flowers in E.grandiflorum,and pigment distribution was found in different tissues in the pigmented part of petals.The pigment distribution in the petals in inbred lines MT1 of bicolor flowers was observed.It was found that pigments were distributed in different tissues of the upper petals,with more pigments in the upper epidermis and palisade tissue adjacent to the upper epidermis than in the lower epidermis.There was no pigment distribution in the unpigmented part of the tissue.Using UPLC-MS technology,secondary metabolites were detected at different developmental stages and different parts of the petals of bicolor flowers.Five kinds of anthocyanins were identified through the analysis of upper petals at different stages,namely,cyanidin 3-O-3’’,6’’-O-dimalonylglucoside,cyanidin-3-（p-coumaroyl）-rutinoside-5-glucoside,delphinidin 5-O-beta-D-glucoside3-O-beta-D-sambubioside,delphinidin 3-glucoside 5-caffoyl-glucoside,delphinidin3-O-（6-caffeoyl-beta-D-glucoside）.Three anthocyanins were identified in the comparison between the upper petals and the petal bases at stage 3,namely,pelargonidin3-O-beta-D-glucoside 5-O-（6-coumaroyl-beta-D-glucoside）,peonidin-3-（p-coumaroyl）-rutinoside-5-glucoside,delphinidin 3-O-（6-caffeoyl-beta-D-glucoside）.3.25 differentially expressed structural gene,5 MYB and 2 b HLH transcription factors were screened.We conducted RNA-Seq analysis on samples from the four developmental stages and different parts of the petals of the bicolor flowers in E.grandiflorum.In the comparison of upper petals and petal bases at stage 3,the differentially expressed genes（DEGs）enriched in flavone and flavonol biosynthesis and the flavonoid biosynthesis pathway,both of which are related to the accumulation of pigments.The structural genes related to the anthocyanin biosynthesis pathway were analyzed,and 25 differentially expressed structural genes were screened out,of which the expression level of Eg CHS1 in the upper petals was21.5 times that in the petal bases.By constructing the evolutionary tree,it was found that 5MYBs and 2 b HLHs and Arabidopsis transcription factors related to anthocyanin biosynthesis were clustered into one branch,respectively.4.Eg CHS1 gene was found to play an important role in the pigment accumulation of bicolor flowers.We conducted a combined analysis of differential metabolites and differentially expressed genes related to anthocyanin biosynthesis pathway,drew a cluster heat map and network diagram of the two,and excavated the correlation between key genes and metabolites in network regulation.It was found that the gene Eg CHS1 was positively correlated with delphinium 3-O-（6-p-caffeoyl）glucoside.Since delphinidin3-O-（6-caffeoyl-beta-D-glucoside）is the main pigment of the color formation of bicolor flowers,it is speculated that Eg CHS1 gene may play a certain role in promoting the biosynthesis of delphinidin 3-O-（6-caffeoyl-beta-D-glucoside）.Finally,a model diagram of pigment accumulation during the formation of bicolor flowers in E.grandiflorum was drawn.5.The expression level of Eg CHS1 gene in pigmented parts of petals was significantly higher than that in other tissues,which may be the key gene leading to the color formation of bicolor flowers.We cloned the Eg CHS1 gene of the bicolor flowers in E.grandiflorum,whose coding region was 1167bp and encoded 389 amino acids.By constructing the evolutionary tree,it was found that the Eg CHS1 protein of E.grandiflorum and the CHS of Ipomoea purpurea converged into one branch,indicating the closest genetic relationship.Eg CHS1 protein was found to be a stable hydrophilic protein,and located subcellular in the cytoplasm.The expression level of Eg CHS1 gene was the highest in the pigmented parts of petals,while lower in the unpigmented parts of petals,stems,leaves and roots.Eg CHS1 may be an important gene involved in color formation of bicolor flowers in E.grandiflorum,which provides new insight into the mechanism study of color formation of bicolor flowers.