| Persimmon(Diospyros kaki)is a specie belonging to family Ebenaceae and genus Diospyros,whose fruit is rich in nutrients and taste is delicious and the plant has good appearance and colorful leaf,thus is used for fruits,leaves and even as ornamental trees.Our country has the biggest amount of persimmon germplasm resources,most proportions are astringent persimmon varieties except ‘Luotian’ nonastrigent persimmon.However,tannin is abundant in astrigent persimmon fruits,they can only be eaten after deastringency process.And those nonastrigent persimmon varieties vary greatly in quality characteristics such as deastringency,storage,flavor and active constituents.And non-astringent types did not have the special flavor as the the astringent ones.Therefore,priority will given to selecting and cultivating the varieties that possess several good traits(easy deastringency,long-time storage and high active constituents)in the improvement of varieties and breeding in persimmon.Compared with lower efficiency in introduction and selective breeding,the purpose of hybrid breeding is clear thus becoming the most effective means to cultivate new germplasms.It is especially important to cultivate more male varieties,because they are scare.Firstly,we must make it clear that the mechanism of sex differentiation so as to cultivate male resources artificially in the future.Illumina HiSeq transcriptome sequencing was conducted with the samples of male and female buds collected from the ‘Zenjimaru’ tree on April 13 th,which was just before the crucial stage when flower sex differentiation occurred morphologically.Firstly,assessment of sequencing quality and data statistics was conducted.Meanwhile,we developed SSR and SNP markers to promote the researches on varieties identification and genetic diversity in Diospyros kaki and selected the reference genes suitable for different experimental conditions to obtain reliable qRT-PCR data in this study.A selection of genes may be related to sex differentiation was performed based on the comparative analysis,after that gene expression patterns in flower buds developmental stages was further studied to elucidate the mechanism of flower sex differentiation.The major results are described as followed:(1)We obtained 54.47 Gb clean reads,which sequence were assembled and obtained 154741 unigenes with average length of 663 bp.The percentage was 38.49%(59570)that sequences were matched against seven public databases through blast.The sequences that matched with NR database were the most,with the proportion of 30.65%(47435),which was followed by SwissProt with 21.53 %(33320).KO ranked the last with 9.57%(14813).Further analysis was conducted with the Gene Ontology(GO)terms.A total of 32646 unigenes was assigned at least one GO term,the results showed that they can be classified into cellur component,molecular function,biology process,and further classified into 56 terms.16098 unigenes was assigned at KOG term,the results showed that they can be classified into 26 categories.Annotated unigenes were fed into Pathways.A total of 129 pathway were predicted.(2)MISA software was used to detect SSR and SNP loci to do polymorphism analysis.44304 SSRs occurred in 59570 unigenes were identified,including 83 SSR repeat motifs.Mononucleotide repeats(20006,47.63%)based on A/T accounted for great proportions in complete SSRs,Dinucleotide repeats(16055,38.23%)mainly on AG/CT followed.405685 candidate SNP bit points were searched and the average appearance frequency was 1/ 253 bp,and the number of the conversion types were significantly higher than the transversion types among 6 kinds of SNP types.SSR and SNP loci were abundant in persimmon,the development of molecular marker would provide the basic information for genetic map structure,analysis of genetic diversity and identificaiton of relative relationships for Diospyros kaki.(3)After comparative analysis,there were 1228 genes expressed significantly in different levels between the male and female buds.Of which,849 genes were up-regulated in male buds and 379 genes were up-regulated in female buds.Genes expressed differentially were classified into 6 GO categories and 81 metabolic pathways.A total of 27 genes were screened by the GO and KEGG analysis,which were related to the plant hormone synthesis and signal transduction or involved in reproduction organs development.Genes up-regulated in male buds included GA20OX2 related to gibberellin biosynthesis,protein phosphatase 2C(PP2C)(negative regulator in the ABA signal transduction pathway),s-adenosylmethionine synthase(SAM)in ethylene biosynthesis of gene,CKX6,CKX3,CKX5 involved in degradation of zeatin and PMADS2 belonged to C/D gene,C,AG belonged to C family.HEC1 associated with pistil,Beta-amyrin 28-oxidase-like classified into sex differentiation,tapetum transcription factor DYT1 and AMC9 related to apoptosis process in metacapase family,MMD1 indispensable in male meiosis.In female buds,up-regulated genes included auxin responsive protein AUX/IAA gene IAA3 and IAA32,ethylene biosynthesis key enzyme gene ACO and EBF1 in ethylene signal transduction pathway,AP2-like ANT ethylene response factor,ERF034,ERF115 transcription factor and female gene MeGI.(4)We also evaluated the stability of 6 reference genes in persimmon in different organs,different developmental stages and different varieties to select the genes expressed relatively stably.The results showed that there were significant differences in the stability of reference genes in different species or even in different tissues in the same plant or developmental stages.The stability of GAPDH was best when the materials were the flower buds of developmental stages or organs in one plant.The stability of EF1-α was best when the flower buds were used as materials.Therefore,GAPDH was selected as reference genes for qRT-PCR in this study.(5)5 genes with down-regulated expression and 6 with up-regulated expression in male flowers were selected to verify the RNA-Seq results,which proved to be in consistent with the results of qRT-PCR.The change of gene expression of GA20OX2,MeGI,HEC1,ACO,Beta-amyrin 28-oxidase-like and IAA32 were analysed in different developmental stages.As for auxin responsive protein AUX/IAA family gene IAA32,Beta-amyrin 28-oxidase-like and HEC1,the gene expressions were significantly higher from stage 1 from stage 4(early June to late March in the next year)in male buds than that in female buds,and that indicated that they may induce male buds formation in the early stages of the flower development process.The gene expression for GA20OX2,crucial in gibberellin biosynthesis is much higher in male buds from stage 7 to stage 11(from mid April to early May),it may signified that higher gibberellin content may induce female differentiation.While,gene expression of the sex determinant MeGI and ACO gene involved in ethylene biosynthesis rose up from stage 6 to stage 10(early April to early May)evidently,which was the key period when MeGI induced female buds formation and the high ethylene contents help female buds formation.The correlation analysis showed the gene expression of ACO was negatively related to that of IAA32 and GA20OX2 in the female buds and male buds,respectively.While in both sex buds,the gene expression of IAA32 Beta-amyrin 28-oxidase-like and HEC1 was significantly positively correlated.That indicated the metobolic pathway these genes regulated may have interaction and played important roles in persimmon flower sex differentiation. |
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