Research On Genetic Characters And Molecular Mechanism Of Double Flower Trait In Calibrachoa Hybrida

Calibrachoa hybrida,also known as mini-petunia,is a herb of perennial,belonging to Solanaceae Calibrachoa.Double flowers,as one of the important traits of ornamental plants,the genetic characters and molecular mechanism have always being studied and explored.So far,there has been no reports about Calibrachoa hybrida.In this study,commercial varieties contain both double flower lines and single flower lines were used to construct segregation populations for studying the genetic behavior of double flower trait.The probable molecular mechanism were studied by isolating and analyzing the expression pattern of several genes related to flower development.The main results were as follows:1.Single flower lines had five sepals,five petals,five stamens and one pistil.The doubled-flowers come from five stamens.The segregation populations were constructed by interspecies hybridization between double and single varieties.The plants that the F1 hybrids backcross with male parent was double flower.The genetic analysis showed that the double flower trait of Calibrachoa hybrida were likely to be dominant.Through the hybridization between Calibrachoa and Petunia,the genetic materials of Calibrachoa hybrida was transferred to Petunia,which laid a foundation for the future studies of Calibrachoa hybrida.2.Twelve flower development related genes were isolated and amplified which belong to MADS-box.Two A-class genes: Ca PFG,Ca FBP26;There B-class genes:Ca TM6,Ca MADS1,Ca MADS2;Two C-class genes: Ca MADS3,Ca FBP6;Two D-class genes: Ca FBP7,Ca FBP11;There E-class genes: Ca FBP2,Ca FBP5,Ca FBP9.The coding sequences of B,C and D genes were compared between double and single varieties,the results showed that was no difference.Because of the high homology between Ca MADS3 and Ca FBP6,Ca FBP7 and Ca FBP11 coding sequences,we used3'RACE to amplify their 3'UTR sequences in order to design primers for semi-quantitative expression.But,we only obtained the 3'UTR sequences of Ca MADS3 and Ca FBP6 genes.We also compared the sequences of each between single and double varieties,the results showed that there were no differences.3.The spatial expression of Ca MADS3 and Ca FBP6 genes was studied by semi-quantitative expression analysis.The results showed that:(1)Ca MADS3 and Ca FBP6 genes were mainly expressed in stamens and pistil.(2)The expression levels were related to the degree of petalization: When the degree of petalization was strong,theexpression of Ca MADS3 and Ca FBP6 was significantly decreased in stamens,not even at all,but increased in pistil;When the degree of petalization was week,the expression of Ca MADS3 and Ca FBP6 genes in stamens and pistil were decreased,but the decrease was not obvious.4.The second intron of Ca MADS3 and Ca FBP6 genes were cloned in this study.The results showed,compared double and with single varieties,except differences of some individual bases,the second intron of Ca MADS3 was inserted in a 9bp fragment and the second intron of Ca FBP6 was inserted in a 10 bp and a 70 bp fragments in double varieties,but the second intron of Ca FBP6 did not clone all sequences now.It is speculated that the formation of the double flower may be related to the insertion of these fragments.