中国甜柿‘鄂柿1号’偶开雄花现象及其杂交后代早期鉴定

Occasional staminate flowering in Chinese PCNA persimmon ‘Eshi 1’ and identification of early de-astringency and sexuality in F_1 progenies by RO2 and Dl Sx-AF4S

【目的】绝大多数柿品种仅开雌花,但中国甜柿‘鄂柿1号’可偶尔开雄花,探讨该现象与DNA变异有无关系及其育种学意义。【方法】利用荧光AFLP技术探讨‘鄂柿1号’雄花枝梢与其母株间的基因组是否存在变异;通过检测其花粉生物学特性以及F1是否存在与中国甜柿自然脱涩连锁的RO2标记和与雄性性别紧密连锁的Dl Sx-AF4S标记,探讨其作为中国甜柿育种亲本的可能性。【结果】(1)AFLP分析表明已知遗传关系的‘前川次郎’和‘次郎’(前者为后者的早熟芽变类型)的相似系数0.88,而‘鄂柿1号’雄枝与母株的相似系数为1.00,说明‘鄂柿1号’偶开雄花性状可能与DNA序列变异无关;(2)‘鄂柿1号’雄枝花粉离体萌发率较低,但其与‘太秋’和‘早秋’柱头亲和性较强;(3)‘鄂柿1号’作父本与‘太秋’和‘早秋’杂交,以及作母本与‘太秋’杂交,均可获得携带RO2标记的F1代单株,且在其作父本的部分F1代单株中检测不到Dl Sx-AF4S标记。【结论】‘鄂柿1号’偶开雄花现象可能不涉及遗传物质DNA序列的改变,其变异原因有待进一步探讨;其作杂交父本时F1单株中均携带与中国甜柿自然脱涩性状连锁的RO2标记,且部分单株不含与雄性性状连锁的Dl Sx-AF4S标记,说明其在选育仅开雌花的完全甜柿新品种中具应用潜力。

【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 flow-ers）, 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 oc-casional 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 amplifica-tion 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 in vivo 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 90 i fluorescence microscope. Male-linked marker, Dl Sx-AF4 S, 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 se-lected 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. 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 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＇.‘Maekawa-jirou＇（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 F_1 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 Dl Sx-AF4 S and RO2 markers. Dl Sx-AF4 S profiles were present in some F_1 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 F_1individuals（only 8） of‘Taishuu＇בEshi 1＇. In the large-scale population of‘Eshi 1＇בTaishuu＇, both RO2 marker and Dl Sx-AF4 S marker were present in someF1 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 F_1 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.