Functional Studies Of C And D Class Genes In Petunia And Development Of Molecular Markers Associated With Double Flower Trait

The double flower is considered to be a mutation in flower type,the molecular mechanism underlying the trait has been a hot issue in the research of floral development.Until now,the scientists have conducted the work from three aspects,containing the floral organ identity genes,floral determinacy and molecular markers associated with the petal number.Petunia is commonly known as a kind of horticultural plant for its flowers and is also the model plant for molecular researches.In this paper,with respective to know more about the overlap and unique functions of C and D class genes in petunia,we generated the transgenic tobacco lines which ectopically expressing Ph FBP6,Ph FBP11 and both genes;we use the near-isogenic lines(NILs)of single and double petunia to conducted the molecular marker experiments,in order to obtain the marker linked to double flower trait in petunia.The major results are as follows:1.The strong phenotype in 35S::FBP6 transgenic lines exhibited style-like tissues with stigmatic apex stretching from the sepal,the smaller petals were curled up and developed green cleft like anther structure.2.The phenotype in 35S::FBP11 transgenic lines exhibited reduced size of floral organs with the absence of homeotic transformation.The pigmentation in sepal and petal was also light,and the ovary adhered pale white surface.3.The microscopic observation exhibited that there were morphologically anomalous structures which were similar to the normal ovule on the inner side of the sepal in 35S::FBP6 transformed lines,and the epidermal cells of the inserted area seemed messy in arrangement compared to the wild-type.By contrast,the ectopic ovule did not occur in 35S::FBP11 transformed lines,indicating that FBP11 might need some factors to participate in the initiation of ovule development.4.The representative 35S::FBP6 plants were crossed with 35S::FBP11 plants and the progenies were analysed.Out of our expectation,the additive effects were not observed.The double transgenic plants only exhibited the reduced size of floral organs,and normal ovary without color changes,indicating that the FBP6 have a prior role than FBP11 when they were expressed stimultaneously.5.The vegetative organs in the single and double petunia were measured to evaluate the near-isogenic level,which showed that there was no obvious difference.While the single flower showed obviously earlier flowering than the double flower,in combination with the difference in floral morphology,we concluded that the double-flower trait might happen at the bud differentiation period.6.A total of 170 SRAP primer combinations and 38 primers were used to screen polymorphism between single and double petunia.Finally a SRAP marker was found to be tightly linked to the double flower trait,then it was successfully converted into SCAR marker designed as SC176.The SC176 marker amplified a specific band in double petunia,and there was no recombination event in the NIL population of 1150 plants.7.Based on the flanking sequences at the both ends of SC176,some SSR markers and new markers were made for polymorphic analysis.Finally there were 14 polymorphic markers in the latest scaffold,which was about 2180 Kb.Two out of the 14 markers were validated by the 1150 plants,and the results showed that the detected markers were co-segregated with double flower trait without recombination event.