Genetic Diversity Of Male Persimmon(Diospyros Kaki Thunb.)Germplasm Resources And Regulatory Mechanism Underlying The Formation Of Male Flowers

Persimmon（Diospyros kaki）is an important woody food tree species in China belonging to family Ebenaceae and genus Diospyros.More and more attention has been paid to its value and application potential in the fields of food,medicine,health care,and chemical industry.However,most domesticated persimmon cultivars are gynoecious（bearing only female flowers）;only a few are monoecious（bearing male and female flowers on the same tree）or androgynomonoecious（bearing male,female,and hermaphroditic flowers on the same tree）.Additionally,androecious types（bearing only male flowers）are occasionally found in the rare wild persimmon（D.kaki）populations.The lack of male flowers and the low utilization efficiency seriously restricted the in-depth development of persimmon hybridization breeding,meanwhile,the restricted selection of 2n pollens with high germination rates from androecious persimmons further disadvantaged to the in-depth development of polyploid breeding.Moreover,the relatively vague genetic diversity and background of the existing male germplasm resources leads to the blindness and low efficiency in utilization.Therefore,it is necessary to improve the utilization level of existing male germplasm resources and to explore the method of artificial breeding male resources,which will play an important role in cross-and polyploid breeding for germplasm innovation and cultivation of improved varieties.In the present study,we firstly investigated and collected wild persimmons from various provinces in China,to evaluate genetic diversity among the androecious persimmon resources and to understand their specific genetic background.The result of this part will provide useful information for male parent selection for the controlled pollination of persimmons.Then,molecular biology and cell biology techniques were applied for revealing the regulatory mechanism of gender determining genes as well as the regulatory mode of programmed cell death（PCD）during male flower development.Finally,we verified the effects of plant growth regulators,soil moisture and C/N on floral development,and hence,provided valuable information and new directions for regulating sex differentiation of persimmon flowers.The major results and conclusions are described as follows:（1）Fifty-five SSR primers were used to analyse the genetic diversity of 47 male persimmon resources,2 andromonoecy resources and 11 Chinese monoecious resources.Sixty-seven female resources,38 Japanese resources（including 25 monoecious resources）and28 closely related species were used as reference out-group,the results indicated that a total of144 alleles and 316 genotypes were obtained from 15 loci,with a mean value of 9.60 alleles and 21.07 genotypes per locus.ssr DK11/DQ097479 was the most informative locus.The geographical distribution of male persimmon resources indicated that they were common in wild comfortable remote area.The 193 resources were divided into 16 and 12 clusters by UPGMA and ME cluster analysis respectively,among which,the male resources originated in Mulan Mountain,Hubei Province and in Shaanxi Province showed the highest and the lowest genetic diversity,respectively.The wild male resources in Mulan Mountain,Hubei Province could be genetically divided into two clusters,one had a near relationship with Jiangxi,Jiangsu,Hunan wild persimmons as well as main astringent cultivars such as‘Huojing’and monoecious astringent cultivars such as‘Yaoxian Wuhuashi’,while the other one was closer to Yunjia Mountain male resources and Luotian non-astringent persimmons（including‘Baogai Tianshi’）.In addition,a genetic isolation existed among wild male resources from different areas,and most Japanese resources could be classified into a same cluster exclusively.（2）Real-time quantitative polymerase chain reaction（RT-q PCR）,molecular cloning and bisulfite sequencing PCR technique（BSP）were carried out using 92 samples,including 56androecious types,15 monoecious and 21 gynoecious cultivars,to investigate the mechanism of sex determination of the specific androecious types of D.kaki in China.In conclusion,the male-linked gene（OGI）and the Kali in the OGI promoter region were conserved in androecious D.kaki genotypes.Afterwards,our result convincingly provide evidence that the expression of OGI was silenced due to the presence of Kali displaying strong methlation in the OGI promoter,meanwhile,high DNA methylated level of the Me GI promoter was closely connected with the production of male flowers.These results indicated that monoecious and androecious types of D.kaki all had OGI gene,and the expression of OGI gene was very low.The generation of male persimmon flowers was related to the high methylated level in the promoter region of Me GI gene.However,the reason why androecious persimmons can produce only male flowers,and monoecious persimmons can produce two gender types of flowers is still unknown.（3）Hematoxylin and eosin（H&E）staining,terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling（TUNEL）assay,caspase-9 and cyt-c immunohistochemistry（IHC）assays,and RT-q PCR were used to clarify the occurrence and molecular regulatory mechanism of PCD in male and female floral buds collected every 2 days from April 5（carpel primordium and anther primordium at an early development stage just after their occurrence）to April 17（when inappropriate sexual primordia were arrested to form unisexual flowers）.Accordingly,a large number of apoptotic bodies accumulated in inappropriate sex organs on April 14 during the microsporocyte and macrosporocyte period of male and female floral buds,respectively.High level of a gene homologous to AMC9（similar in structure and function to the caspase protein in animals,and the gene can induce the PCD）on April 14 may have accelerated the arrest of the pistil primordium during differentiation,leading to male unisexual flowers,and high levels of genes homologous to Me GI（female growth promoter and male growth inhibitor gene in Diospyros）,BAG5（a stress inducing gene of anti-apoptosis,which can prevent the release of cyt-c and has the ability of anti-PCD）,Aif A（a gene that leads to large-scale DNA fragmentation and cell apoptosis when translocated to the nucleus）,and HSP70（a gene encoding heat shock protein,and the expression level was increased and the number of apoptotic cells was decreased in plants under stresses）in female floral buds on April 14 were positively correlated with the arrest of stamen primordium.This may explain the abortion of inappropriate sex organs,leading to unisexual flowers.（4）The feminizing agents ethrel,5-azacytidine,a mixture of exogenous ZT,ABA and IAA were injected into androecious wild persimmon trees.Different concentrations of masculinizing GA₃ were injected into gynoecious‘Jiro’cultivar trees.‘Jiro’trees were also grown in soils differing in moisture content and carbon/nitrogen（C/N）ratio,to determine their effects on floral development and sex differentiation in persimmons.Compared to the untreated control,injections of ethrel significantly decreased the diameter of pollen grains and the length of pollen tubes.Injections of ethrel,5-azacytidine,and the mixture of ZT,ABA and IAA significantly increased the expression of the feminizing Me GI gene during the period March 14–April 14,increased the expression of a gene homologous to ACO（a gene promoting ethylene biosynthesis）,and decreased the expression of a gene homologous to WRKY28（a transcription factor negatively regulating ABA signal transduction）at the critical period of PCD in the sexual organs（April 14）;this highlights the feminizing effects of these plant growth regulators on persimmon flowers.In contrast,injections of GA₃ significantly increased the length of arrested stamens in gynoecious trees,increased the expression levels of genes homologous to WRKY28,IAA32（a gene that negatively regulates IAA signal transduction）and GA20OX2（a gene promoting GA biosynthesis）,and decreased the expression of a gene homologous to ACO in April,thus demonstrating the masculinizing effect of GA₃.High levels of soil moisture（55%and 45%volumetric moisture contents）significantly increased pistil length in gynoecious trees compared to the control（20%moisture content）.High C/N ratios also significantly increased the length of pistils and arrested stamen growth in gynoecious trees.Many of the treatments also significantly changed the contents of assimilates,and the activities of antioxidant enzymes in floral buds.