Occasional staminate flowering in Chinese PCNA persimmon ‘Eshi 1’ and identification of early de-astringency and sexuality in F1 progenies by RO2 and DlSx-AF4S
[Objective] Persimmon (Diospyros kaki Thunb.) can bear bisexual (hermaphrodite) and unisexual (male or female) flowers and mainly be classified into four types,that is,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 related Diospyros species.Most commercial persimmon cultivars only bear female flowers and few cultivars are monoecious (bearing both male and female flowers).However,some pistillate genotypes (bearing female flowers only),such as Japanese PCNA (pollination constant non-astringent) persimmon cultivars ‘Fuyuu’ and ‘Jirou’,can occasionally generate male flowers under certain circumstance.Recently,male flowers were found on a shoot of ‘Eshi 1’ adult tree (female ‘Eshi 1’) in the orchard of Persimmon Repository,Huazhong Agricultural University,Wuhan,Hubei,China.This tree was grafted on D.lotus L.in 2009.The aim of this study was to analyze the genetic status of the shoot with occasional staminate flowering of ‘ Eshi 1’ (male ‘ Eshi 1’) compared with the other fhoots with female flowers of the mother plant and to examine its potential of being used as a parent for crossing breeding program of PCNA persimmon.[Methods]The fluorescent AFLP (amplification fragment length polymorphism) markers were used to investigate genetic relationship between the shoot with male flowers and the shoots with female flowers of ‘Eshi 1’,with 23 D.kaki genotypes or related species.The AFLP PCR amplification of AFLP markers were tested by capillary electrophoresis (CE) and then the production were scored as "1/0" matrix for genetic distance analysis by the software NTSYS-pc v2.1,and the clustering dendrogram was constructed by UPGMA method.The pollens of ‘Eshi 1’ ‘Zenjimaru’ and ‘Taishuu’ were observed by optical microscope for counting pollen grain number via three replications.Artificial pollination and in vivo pollen tube growth observation were conducted among three cross combinations of ‘Taishuu’בEshi 1 ’,‘Soshuu’ × ‘Eshi 1’ and ‘Eshi l’× ‘Taishuu’.Fluorescence observation of squashed pistils was performed by Nikon ECLIPSE 90i fluorescence microscope.Male-linked marker,DlSx-AF4S,and Chinese PCNA de-astringency associated marker,RO2,were used to detect the individuals of the F1 progenies of above three crossing combinations.[Results] Totally,8 AFLP primers with robust amplification were selected to analyze these tested materials.In short,898 amplification bands were generated by fluorescent AFLP markers,accounting for 96.0％ polymorphisms.Based on UPGMA cluster analysis,D.lotus,D.glaucifolia,D.rhombifolia and D.virginiana were clustered together,which showed a far distance from 21 D.kaki genotypes of 2 groups.Group Ⅰ was comprised of fourteen Chinese persimmons,including 6 Chinese PCNA persimmons (‘Baogai Tianshi’ ‘Luotian Tianshi’ ‘Xiaoguo Tianshi’ ‘Sifang Tianshi’,female ‘Eshi 1’ and male ‘Eshi 1 ’),6 androecious resources and 2 pollination constant astringent (PCA) cultivars (‘Mopanshi’ and ‘Tongpenshi’).All the cultivars originated from Japanese were grouped into Group Ⅱ and ‘ Youhou’ was tightly clustered together with its breeding parents ‘ Fuyuu’ and ‘Jirou’.‘ Maekawajirou’ (early-ripening bud sport of ‘Jirou’) and ‘Jirou’ could be separated at the 0.88 coefficient point.However,there were no genomic DNA difference between ‘Eshi 1 ’ male shoot and female parts with regard of fluorescent AFLP markers.The pollen in vitro germination rates of ‘Eshi 1’,‘Zenjimaru’ and ‘Taishuu’ were measured in 2015 and repeated in 2016 during flowering periods.In total,in vitro pollen germination rates of ‘Eshi 1’ was lower than that of ‘Zenjimaru’,and fluctuated significantly in 2015 and 2016.‘Eshi 1’ pollen tube could develop and penetrate into the style of two Japanese PCNA cultivars ‘Taishuu’ and ‘Soshuu’,meanwhile the similar result was obtainedd for ‘Taishuu’ pollen on the stigma of‘Eshi 1’.A total of 571 F1 progenies,which were obtained from ‘Taishuu’ × ‘Eshi 1’,‘Soshuu’ × ‘Eshi 1’ and ‘Eshi 1’ × ‘Taishuu’ via embryo rescue,were used to early diagnose their sexuality and astringency trait using DlSx-AF4S and RO2 markers.DlSx-AF4S profiles were present in some F1 progenies of ‘Taishuu’ × ‘Eshi 1’ and ‘Soshuu’ × ‘Eshi 1’,but RO2 was only present in ‘Soshuu’ × ‘Eshi 1’ and absent in ‘Taishuu’ × ‘Eshi 1’ due to limited F1 individuals (only 8) of ‘Taishuu’ × ‘Eshi 1 ’.In the largescale population of ‘Eshi 1’ × ‘Taishuu’,both RO2 marker and DlSx-AF4S marker were present in some F1 individuals and were fitted to a 1∶1 ratio by Chi test.[Conclusion]The overall results suggested that ‘Eshi 1’ occasionally bearing male flower (male ‘Eshi 1 ’) could not be involved in genetic mutantation but exhibit stable phenotype modification.Considering the pollen biology characteristics and the early validation of F1 progenies by MAS (marker-assisted selection) system,the shoot occasionally bearing male flower of Chinese PCNA persimmon ‘Eshi 1’ might be further used as an excellent breeding parent for genetic improvement of PCNA persimmon.
Persimmon (Diospyros kaki Thunb.)‘Eshi 1’Staminate occasionallyGenetic variationBreeding value
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