Identification Of MicroRNAs Involved In Pomegranate Female Sterility And Functional Analysis On The Regulation Of Pg-miR166a-3p On Pistil Development

Pomegranate?Punica granatum L.?has two kinds of flowers on a same plant:functional male flowers?FMFs?and bisexual flowers?BFs?.BFs,also named as‘vase-shaped',poss well-formed pistil and can set fruits.FMFs which are refered as‘bell-shaped'are known to be female sterile,so they fail to set fruits and eventually fall from the tree.The proportion of FMFs is negatively associated with fruit yield.Preliminary results showed that ovule development in FMFs ceased following the formation of the inner integument primordium.Many candidate genes may affect pomegranate female sterility were caved by RNA-seq,the factors regulating those candidate genes remained unknown.MiRNAs play important roles in ovule development.Thus,in this study we explored the regulatory mechanism of pomegranate female sterility based on previous studies.Moreover,ethephon has a significant effect on the proportion of BFs,but the valid ways to prevent fruits physiological dropping keep unsolved.So,we aim to find out the effective measures to improve the fruit setting rate at the same time in this study,the main contents and results are as follows:1.A final set of 103 known miRNAs and 58 novel miRNAs were identified from pomegranate BFs and FMFs'pistils.Among all identified miRNAs,57 miRNAs were differentially expressed in comparison of FMFs and BFs,39 miRNAs showed significantly different expression among different stages of BFs,38 miRNAs exhibited remarkable expression difference among different stages of FMFs.2.A total of 6 potential miRNA-mRNA regulatory network that may related to pomegranate female sterility were identified by miRNA-mRNA interaction identification.Based on the analyses of the differential expressed miRNAs in pistil between FMFs and BFs at different stages,a strong association between Pg-miR858b/Pg-miRN01 and pistil development was identified at the early stage.Pg-miR444b.1,Pg-miRN11,Pg-miR166a/165a-3p and Pg-miR952b may influence pomegranate integument development at later stages.3.Arabidopsis thaliana plants transformed with Pg-miR166a-3p driven by the Ca MV35S promoter exhibited declining in plant biomass,clip size and seeds number.The results also showed that the number of meristems of inflorescence increased in transformants,whereas the number of ovule primordia decreased significantly.4.Pg-miR166a-3p and its predicted targeted genes Gglean012177.1 and Gglean013966.1showed reciprocally expression patterns during BFs and FMFs'pistil development.The results of transient assay indicated that Pg-miR166a-3p can interact with its targets?Gglean012177.1 and Gglean013966.1?to affect the ovule development.5.Using transgenic analyses in Arabidopsis,we verified the candidate gene,Gglean016270.1,which showed lower expression level in FMFs than that in BFs.We found the curled leaves phenotype in over-expression lines.Based on the microscopic observation,the increase of cell number in the back of leaves might be the major reason for leaves curl,suggesting the cell division and abaxial-adaxial growth could be regulated by Gglean016270.1.Meanwhile,the consistent expression pattern of Gglean016270.1 and Gglean012177.1 indicated that Gglean012177.1 may regulate the pistil development via the interaction with Gglean016270.1.6.In 2017,the results showed that fruit setting rate of the single spraying of GA₃ 30.0 mg/L at the first early to full-bloom stage?May 5?harbored 48.3%higher than that of the control group,and the fruit setting rate was significantly increased by 16.0±0.32%?P<0.01?.Single spraying of GA₃?10.0mg/L and 20.0 mg/L?at May 18 increased the fruit setting rate by 20.8%and 31.5%than control,respectively,and significantly increased the fruit setting rate?P<0.05?.However,the female sterility flowers can't drop naturally in this treatment,which leads to nutrition wasting.The results of single spraying of GA₃ 30 mg/L at May 5 in 2018 showed significantly increase in fruit setting rate by 28.5%compared with the control?P<0.05?,consistent with the results in 2017.Soluble solid content of fruits with GA₃ 30.0 mg/L treatment increased significantly,which significantly improve the fruit setting rate without affecting the appearance.Taken together,we concluded that miRNAs play important roles in the development of female sterile.Pg-miR858b and Pg-miRN01 strongly associated with pistil development at the early stage.Pg-miR444b.1,Pg-miRN11,Pg-miR166a/165a-3p and Pg-miR952b may influence pomegranate integument development at later stages.Pg-miR166a-3p may affect pomegranate female development by interacting with its targets Gglean012177.1/Gglean013966.1.The development termination and failure of further elongation of ovule integument in functional male flower might be involved in the lower expression of Gglean016270.1.Foliar spraying with GA₃?30 mg/L?can effectively improve the fruit setting rate of pomegranate.