| Persimmon(Diospyros kaki Thunb.)can bear bisexual(hermaphrodite)and unisexual(male and female)flowers and mainly be classified into four types at the plant individual level,ie.pistillate plant(bearing female flowers only),staminate plant(bearing male flowers only),monoecy(bearing both male and female flowers),trimonoecy or intersexuality(bearing both bisexual and unisexual flowers),showing polygamous sexuality as opposed to other Diospyros species.Most cultivars only bears female flowers and few are monoecy.Pollination constant non-astringent(PCNA)persimmon is more popular with consumers,but most cultivated PCNA type persimmons originates from Japanese and there is still lack of independent intelligent PCNA varieties in China.Thus,the main goals of persimmon breeding is still to breed PCNA cultivars which can bear only female flowers and are strongly parthenocarpic in recent years.However,due to its relatively longer juvenile phase(it takes 5-8 years for flowering and fruiting after seeding),early elimination of male progeny has been regarded as an important strategy for enhancing breeding efficiency.In this study,we diagnosed two male sex-linked markers,DlSx-AF4S and OGI locus,in a relatively large-scale of Diospyros germplasms,and then we discussed their potential for PCNA breeding parents of ‘Eshi 1’ with male flower generation and investigated some Diospyros genotypes which displayed early flowering.The study might offer scientific benefit for Chinese PCNA persimmon breeding.The main results were as follows:Phenotype observation and DlSx-AF4S genotyping were carried out by 268 accessions,including D.kaki,F1 progenies and some related species.All of the D.kaki genotypes,with the exception of ‘Luotian Tianshi’,‘Xingyi Shuishi’,‘Amahyakume’ and ‘Shangcheng Dongshi’,displayed consistent results between DlSx-AF4S amplification and sex phenotype.A total of 118 F1 progenies were produced from 11 crosses,among which 51 individuals had flowered in 2014.In the flowering plants,DlSx-AF4S profiles of 46(90.2%)plants were identical to the field observation of flower gender.The segregation of DlSx-AF4S in ‘Huashi 1’ × ‘Luotian Tianshi’ F1 population fitted to 1:1.High transferability of DlSx-AF4S was observed in other species related to D.kaki and D.lotus.Taken together,these results indicated that the DlSx-AF4S marker could be used to distinguish male plants from female ones of persimmon at an early stage.Phenotype characterization and genotyping of the male-linked OGI marker were carried out by 205 accessions,including persimmon cultivars,F1 progenies and 9 related Diospyros species.All persimmon cultivars displayed consistent results concerning to OGI amplification and sex phenotype.A total of 143 F1 progenies were derived from 11 crosses,among which 95 individuals had flowered in 2015.In the flowering full-sib families,the amplification of the OGI marker in agreement with the sex phenotype was obtained in 85 plants(89.5%).The segregation of OGI in ‘Huashi 1’ × ‘Luotian Tianshi’ and ‘Huashi 1’ × Male 3 F1 populations fit a 1:1 ratio.Furthermore,high OGI transferability was observed in 9 related species.Overall,the results indicated that the OGI locus could be used to distinguish male from female persimmon plants at an early stage.Occasional male flowers appearance on a shoot in ‘Eshi 1’ adult tree,which was grafted on date plum(D.lotus L.)in 2009,was observed in the orchard of Persimmon Repository,Huazhong Agricultural University.The fluorescent AFLP(amplification fragment length polymorphism)marker was used to investigate genetic relationship among male shoots and female shoots of ‘Eshi 1’,and 23 D.kaki genotypes or related species.‘Maekawa-jirou’(early-ripening bud sport of ‘Jirou’)and ‘Jirou’ could be separated at the 0.88 coefficient point and there were no genomic DNA difference detection between ‘Eshi 1’ male shoot and female parts based on fluorescent AFLP markers,suggested that ‘Eshi 1’ bearing male flower occasionally couldn’t find genomic DNA difference.Persimmon and its related speices were used to investigate early flowering phenomena.Five types of early flowering presence occurred in the tested genotypes.In vitro floral morphogenesis of ‘Gongcheng Shuishi’ was observed after continuous subculture of stem-segment explant.Early flowering of Deyangshi(Diospyros sp.)seedling displayed after sown three months later.Advance flower development of adventitious shoot was observed in D.kaki cultivars(‘Unishi-wase’,‘Youhou’,‘Taishuu’,‘Zenjimaru’,‘Huashi 1’,‘Gongcheng Shuishi’),date plum(D.lotus Linn.),and some F1 progenies bred from ‘Huashi 1’ × ‘Luotian Tianshi’.Early fruit setting on ‘Gongcheng Shuishi’ secondary growth shoots was exhibited after top-grafted on adult ‘Eshi 1’ for six months.Early flowering of ‘Eshi 1’ × ‘Taishuu’ F1 plantlets via embryo rescue existed after green-grafted on adult date plum one year later.Those results suggested that there ought to be early flowering phenomena in Diospyros,as opposed to traditional concept of its juvenility.In conclusion,Dl Sx-AF4S and OGI markers exhibited high effectiveness for distinguishing male plants from female ones of persimmon at an early stage,and ‘Eshi 1’ with occasional male flower formation might not be DNA sequence mutant,and early flowering phenomenon in Diospyros were popular and might further use for transgenic systems for persimmon breeding. |
