Exploring Regulatory Genes Of Pomegranate (Punica Granatum) Female Sterility Based On Transcriptome Analysis

Pomegranate has two kinds of flowers on a same plant: functional male flowers(FMF)and bisexual flowers(BF).BFs have well-formed pistils and can set fruits,thus they are known as female fertile(vase-shaped).FMFs,which are referred as female sterility(bell-shaped),fail to set fruit and drop finally.The ratio of FMFs is in inverse ratio to the yield of fruit.High ratio of FMFs is one of the main factor that impact the yield of pomegranate.The key reason and stage of pomegranate female sterility were studied using ‘Tunisiruanzi’ in this study,the main contents and conclusions are as follows:1.Growth characteristics and morphology differences of BFs and FMFs were studied.The results indicated that(1)the ratio of bisexual flowers existed variances in different branch types.In long fruit branch,the ratio of BFs was significantly lower than that in fruit spur,middle fruit branch and perennial branch(P < 0.01).However,there were no significant difference among fruit spur,middle fruit branch and perennial branch.(2)The ratio of BFs was different among different inflorescence types,flowers in cluster was noticeably fewer than in single and pair flowers(P < 0.01).The number of BFs in terminal was more than lateral flowers in cluster flowers(P < 0.05).(3)The ratio of BFs in fascicled phyllotaxis was higher than that in opposite phyllotaxis(P < 0.01).(4)Ovules of FMFs and BFs were observed differentially when their flower buds grow into 10.1-13.0 mm.2.Candidate genes of pomegranate female sterility were improved by transcriptome analysis.INNER OUTER(INO/YABBY4)(Gglean016270)and AINTEGUMENATA(ANT)homolog genes(Gglean003340,Gglean011480)which regulated the development of integument showed down-regulation in FMF than BF at the key stage of ovule development termination.Their upstream regulator genes like AGAMOUS-like(AG-like)(Gglean028014,Gglean026618,Gglean028632)and SPOROCYTELESS(SPL)homolog genes(Gglean005812)also showed different expressed between BF and FMF at the key stage of ovule development termination.Moreover,the differential expression of ethylene response signal ETR(ethylene-resistant)(Gglean022853)and ERF1/2(ethylene-responsive factor)(Gglean022880)between FMF and BF indicated that ethylene signaling may also involved in the formation of pomegranate female sterile.The increase of BF with the spraying of ethephon also supported this results.3.We select six candidate genes for clone and bioinformatic analysis,including ANT(Gglean003340)、 INO(Gglean016270)、 SPL(Gglean005812)、 AG(Gglean028632)、 ETR(Gglean022853),and ERF1/2(Gglean022880).The protein sequences of GgANT,GgSPL,Gg AG,GgINO,Gg ETR,and GgERF1/2 showed high similarity with ANT,SPL5,FUL,INO,ETR,and ERF1 in Arabidopsis,respectively.We also construct expression vectors of these six genes for further transgenic studies.