The Research Of Sex Determination In Papaya Based On High-Throughput Sequencing

Sexual reproductive systems in flowering plants vazy.Most angiosperm species have perfect hermaphroditic flowers harboring both functional male(androecium)and female(gynoecium)reproductive organs,while a small proportion of angiosperms is unisexual plants,which evolve from their hermaphrodite ancestors.It is the sex determination genes of unisexual plants that accurately regulate developmental processes of floral organs to insure the stability of sex differentiation in plants.However,the mechanisms of sex determination and sex-determining genes involved in different unisexual plants might be distinct.Trioecious papaya is an excellent system to explore genetic and environmental regulatory mechanisms for sex determination,because it has three sex forms,and exhibits frequent sex-reversal in male and hermaphrodite flowers.In this dissertation,we analyzed the methylome and transcriptome of different sexual flowers and gynoecium samples to explore the sex-determining mechanisms and the associated genes.The main research findings are:1、Through profiling the methylome of female and male papaya flowers in three seasons,we verified the existence of methylation variance----consistent sex-associated differentially methylated cytocines(Cs DMCs).However,combined genome-scale transcriptomic evidences revealed that Cs DMCs did not have a significant influence on the expression profiles of neighboring genes.Differentially expressed genes(DEGs)profile showed that pytohormone signal transduction was most significantly overrepresented in DEGs,which supported the difference in pytohormone,rather than methylation variance,contributed to sex differentiation.Although there was no direct molecule evidence supporting that methylation variance has influenced sex characteristics,genome wide methylation variance uncovered distinctive change in different sexual flowers when exposed to stress conditions.Greater methylation pattern similarity in female flowers than male flower under stress was observed.It implied that stress-responsive methylome of female flowers was relatively stable while that of male was labile.Additionally,we found that early flowering of male papaya might be associated with the transcript expression alteration of SVP and APETALA1 coincides with gene-specific intergenic hypo-methylation.2、Gynoecium suppression was the phenotype of male flowers in papaya.We compared differential expression of genes in rudimentary and functional gynoecium from hermaphrodite papaya,in parallel with their dioecious papaya,to verify the genetic and environmental factors in corresponding to gynoecium development.Gene ontology annotation of key DEGs suggested corresponding DEGs were involved in 3 main biological processes,including(i)response to stimuli which may induce abnormalities,unbalanced phytohormone distribution,ectopic transcription factor modulation,and aberrant cell proliferation growth(ii)response to growth and development,which may be associated with female gametophyte and fertilization-independent development and(iii)response to phytohormones.Further enrichment analysis of metabolic pathways proved that the phytohormones signaling transduction was the most significant pathway when compared functional and rudimentary gynoecium.More importantly,auxin-related DEGs were all highlighted in the known signal transmission process,which may suggest that auxin had a much greater influence than other hormone did on the regulation of gynoecium morphogenesis and development.Our results proved the enhanced auxin signal transduction in rudimentary gynoecium and weakening of polar auxin flow by NPA treatment resulted in female fertility restorations in male flowers.The occurrence of rudimentary gynoecium patterning might be related to the alteration of auxin homeostasis.Therefore,auxin is a crucial factor that affects gynoecium development.3、The recovery of female fertility in male flowers also occur in natural conditions.Hermaphrodite papaya was diverged from its male wild papaya ancestors and was considered to effectively remove gynoecium suppression.Besides,low temperature often induces functional gynoecium in the male papaya.we compared the functional gynoecium of hermaphrodite flowers and male-to-hermaphrodite sex reversal flowers against the rudimentary gynoecium of male flowers and found that the reduction of gibberellin biosynthesis and diminution of auxin signaling were related to the release of the repression of gynoecium development.Gene expression detection indicated that the gibberellin synthesized by gynoecium could not act on itself,and weakening of auxin signaling did not result from the decline of auxin production.Multiple splice variants of CpXY14 were detected by systematic analysis of genome-scale alternative splicing.We thought CpXY14,a gene located in sex-determining region of sex chromosome,might cause the changes of auxin metabolism.CpXY14,encoding monodehydroascorbate reductase(MDAR),have a potential ability to affect the transportation and degradation of active auxin.4、The sequencing results of CpXY14 c DNA cloning from male and hermaphrodite flowers confirmed the emergence of CpXY14 splice variants.There were some amino acid variations in important residues of MDAR protein.We respectively constructed the overexpression vectors of CpXY14 splice variants,aiming for functional analysis of CpXY14 in Arabidopsis atmdar mutant and papaya via agrobacterium-mediated genetic transformation.And the transgenic lines have been obtained.The phenotypic change of Arabidopsis mutant transgenic plants might be caused by the auxin homeostasis alteration.