| Chrysanthemum(Chrysanthemum×morifolium)is one of the top ten famous flowers in China and one of the top four cut flowers in the world with a high ornamental and economic value.It has a wide variety of flowers types and colors,but there are relatively few green varieties in the market,and the mechanism and regulation research on its green color is insufficient.In order to explore the color mechanism and regulation mechanism of green ray florets of chrysanthemum,we constructed a segregation population for its green character.The aim of this study was to identify and screen the key genes affecting the color of green ray florets of chrysanthemum through genetic analysis and transcriptome analysis.This study may provide good reference for future genetic engineering in improveing the color of chrysanthemum,and lays a basis for the study of green flowers of other plants.The main research results are as follows:1.The green color of the ray florets of chrysanthemum is related to the chlorophyll content.Through the determining the pigment content of several chrysanthemum flowers with different colors by colorimeter,it was showed that the carotenoid content in petals had little effect on the green color,but the chlorophyll content showed a very significant correlation with the a*value in petals,it indicated that the green color of the ray florets of chrysanthemum was related to the chlorophyll content in its cells.In addition,we confirmed that the a*value can indirectly reflect the content of chlorophyll in chrysanthemum petals,which provides a basis for subsequent large number of flower color measurement by color difference meter.2.The segregation population was established based on the green color of the ray florets of chrysanthemum.As mentioned above that the green color of chrysanthemum formation of petals is related to chlorophyll content,to dissect the green characters,we compared and screened the flower color,flower type,petal type and other growth characteristics of the chrysanthemum resources reserved in our lab.we selected a native species Chrysanthemum vestitum and three varieties of Chrysanthemum morifolium as hybrid parents to establish 7 genetic population by 6 crosses and 2 selfings.Among them,the population progenies of C.×morifolium'ludingdang'and Chrysanthemum vestitum showed a continuous phenotype segregation on the green color of ray florets,which was used for genetic analysis and further experiments.After analyzing the phenotypic characteristics of the progenies of these population,it was found that the flower type of C.×morifolium‘Ludingdang'was convenient for crossing,which was a good parent material for the study of flower color inheritance of Chrysanthemum.3.The green character of ray florets in chrysanthemum is controlled by two pairs of major genes.Although Florist's chrysanthemum is a famous ornamental plant in China and even in the world and has variety diversity,there are relatively few green varieties and limited studies on breeding and genetic mechanisms.In this study 453 progenies from the cross between Chrysanthemum×morifolium‘Lvdingdang'(female,flat type and green)and C.vestitum(male,flat type and white)were used to investigate the heterosis and analyse the h value and a*value by mixed major genes plus polygenes inheritance models,the number of the ray florets and flower head diameter were also investigated.The results are as follows,(1)continuous variations were observed in these traits.Among these traits,all of them had very significant mid-parent heterosis,and h value and flower head diameter had very significant super-parent heterosis.(2)The h and a*values and number of ray florets in the above 453 progenies were found to be controlled by two additive-dominant-epistatic major genes,while the diameter of flower head was found to be controlled by one additive-dominant major gene.In addition,most progenies have flat petals,which are consistent with their parents,although a few progeny individuals have tube petals and spoon petals.The above a*value was found to be significantly correlated with the h value,the number of ray florets,and the flower head diameter.The above findings show the high heritability for the a*value.These results provided strong foundations in their genetic mechanisms,gene mining,parental selection,and cultivar breeding.4.Several metabolic pathways and candidate transcription factors affecting the coloration of green flower of chrysanthemum were explored.The molecular basis for green petal color in chrysanthemum is not fully understood,though the RNA sequencing analysis of white and green ray florets collected at three stages of flower development from the F1 progeny of the cross between Chrysanthemum×morifolium‘Lvdingdang'and Chrysanthemum vistitum.We found that the chlorophyll content was higher and chloroplast degradation was slower in green pools than in white pools at each developmental stage.Transcriptome analysis revealed that genes that were differentially expressed between the two pools were enriched in pathways related to chlorophyll metabolism and photosynthesis.We identified the transcription factor Cm COL16a,Cm COL16b,Cm ERF,and Cmb HLH as regulators of green flower color in chrysanthemum by differential expression analysis and weighted gene co-expression network analysis.These findings might guide future efforts to improve the color palette of chrysanthemum flowers through genetic engineering.5.Two transcription factors among the four may affect the content of chlorophyll in green ray floret of chrysanthemum.The full-length CDS sequences of the four candidate transcription factors were amplified and compared with the homologous gene sequences and protein sequences of Arabidopsis thaliana and other species.After analyzing the expression of these genes in 6 cut chrysanthemum petals with different chlorophyll content,it was found that Cm COL16b and Cmb HLH were more closely related to the chlorophyll content in petals.In a word,we established an isolated population on the green ray flrets of Chrysanthemum,and carried out genetic and transcriptome analysis to explore the genetic control of the green character of chrysanthemum ray florets,and screened several candidate transcription factors affecting the chlorophyll content in the ray florets.It laid a foundation for further exploring the color mechanism of green ray florets of chrysanthemum. |
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