Occasional staminate flowering in Chinese PCNA persimmon ‘Eshi 1’ and identification of early deastringency and sexuality in F1 progeniesby RO2 and DlSx-AF4S

Abstract: 【Objective】Persimmon (Diospyros kaki Thunb.) can bear bisexual (hermaphrodite) and unisexu⁃al (male or female) flowers and mainly be classified into four types, that is, pistillate plant (bearing femaleflowers 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 sexu⁃ality as opposed to other related Diospyros species. Most commercial persimmon cultivars only bear femaleflowers and few cultivars are monoecious (bearing both male and female flowers). However, some pistillategenotypes (bearing female flowers only), such as Japanese PCNA (pollination constant non-astringent) per⁃simmon cultivars‘Fuyuu’and‘Jirou’, can occasionally generate male flowers under certain circum⁃stance. Recently, male flowers were found on a shoot of‘Eshi 1’adult tree (female‘Eshi 1’) in the or⁃chard of Persimmon Repository, Huazhong Agricultural University, Wuhan, Hubei, China. This tree wasgrafted 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 flow⁃ers of the mother plant and to examine its potential of being used as a parent for crossing breeding pro⁃gram 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 shootswith 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 dendrogramwas constructed by UPGMA method. The pollens of‘Eshi 1’‘Zenjimaru’and‘Taishuu’were observedby optical microscope for counting pollen grain number via three replications. Artificial pollination and invivo pollen tube growth observation were conducted among three cross combinations of‘Taishuu’בEshi1’,‘Soshuu’בEshi 1’and‘Eshi 1’בTaishuu’. Fluorescence observation of squashed pistils was per⁃formed by Nikon ECLIPSE 90i fluorescence microscope. Male-linked marker, DlSx-AF4S, and ChinesePCNA de-astringency associated marker, RO2, were used to detect the individuals of the F1 progenies ofabove 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 fluorescentAFLP markers, accounting for 96.0% polymorphisms. Based on UPGMA cluster analysis, D. lotus, D. glau⁃cifolia, 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 ChinesePCNA 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) culti⁃vars‘( Mopanshi’and‘Tongpenshi’). All the cultivars originated from Japanese were grouped into GroupII 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 withregard 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 pollengermination rates of‘Eshi 1’was lower than that of‘Zenjimaru’, and fluctuated significantly in 2015 and2016.‘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 stigmaof‘Eshi 1’. A total of 571 F1 progenies, which were obtained from‘Taishuu’בEshi 1’,‘Soshuu’בEshi 1’and‘Eshi 1’בTaishuu’via embryo rescue, were used to early diagnose their sexuality and astringencytrait using DlSx-AF4S and RO2 markers. DlSx-AF4S profiles were present in some F1 progenies of‘Tai⁃shuu’בEshi 1’and‘Soshuu’בEshi 1’, but RO2 was only present in‘Soshuu’בEshi 1’and ab⁃sent in‘Taishuu’בEshi 1’due to limited F1 individuals (only 8) of‘Taishuu’בEshi 1’. In the largescalepopulation of‘Eshi 1’בTaishuu’, 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 mutantationbut exhibit stable phenotype modification. Considering the pollen biology characteristics and the early vali⁃dation of F1 progenies by MAS (marker-assisted selection) system, the shoot occasionally bearing maleflower of Chinese PCNA persimmon‘Eshi 1’might be further used as an excellent breeding parent for ge⁃netic improvement of PCNA persimmon.