| Diospyros kaki Thunb.cv.Fangshan is a geographical indication agricultural product in Yongkang,Zhejiang.It is a unique persimmon germplasm resource in Yongkang.D.kaki Thunb.cv.Fangshan has no male flower and seedless persimmon,which belongs to natural parthenocarpic variety.In recent years,many Fangshan persimmon have produced hybrid seeds,which have lost the original taste and quality,reduced the market competitiveness and user reputation,and caused huge economic losses to farmers.It was found that the hybrid male parents of Fangshan seeded persimmon were D.kaki Thunb.cv.‘Taishuu’ and D.kaki Thunb.var.sylvestris Makino.Therefore,understand the characteristics of pollen of male parents of hybrid seeds of Fangshan persimmon and the development and formation of hybrid embryos,evaluate the natural hybridization frequency of Fangshan persimmon,and identify the proportion of hybrid seeds and the source of male parents in Fangshan persimmon,to explore the effect of D.kaki Thunb.cv.‘Taishuu’ pollination distance on cross pollination of D.kaki Thunb.cv.Fangshan,it has great guiding significance for the comprehensive development of Yongkang Fangshan persimmon resources and the sustainable development of related industries.The main results are as follows:(1)In this study,pollen staining method,RGB intensity value and image segmentation technology are combined to develop a pollen vitality judgment method based on RGB intensity value and image segmentation technology,which is more accurate and efficient than traditional manual recognition,Among the different staining methods,TTC staining combined with RGB intensity value has the best effect on the detection of pollen viability,When persimmon pollen grains were stained with TTC for 25 min,the R value of active pollen grains was 30-152,the g value was 32-137 and the b value was 20-107,The results showed that the pollen vitality of D.kaki Thunb.var.sylvestris Makino was stronger than that of D.kaki Thunb.cv.‘Taishuu’.(2)This study proved that the pollen viability of D.kaki Thunb.var.sylvestris Makino was higher than that of D.kaki Thunb.cv.‘Taishuu’,The pollination affinity of D.kaki Thunb.cv.‘Taishuu’ and D.kaki Thunb.var.sylvestris Makino with D.kaki Thunb.cv.Fangshan was observed through in vivo germination,In addition,the process of hybrid seed formation and embryo development of D.kaki Thunb.cv.Fangshan is like other Diospyros L.,and there are different abortion phenomena in the process of hybrid seed formation.(3)In this study,the effects of various tissue fragmentation methods and DNA purification methods on the DNA extraction quality of persimmon seeds were compared,and a set of DNA extraction methods suitable for persimmon single hybrid seeds were established,Based on 14 pairs of EST-SSR primers of persimmon,the DNA of self-bred seeds of D.kaki Thunb.cv.‘Taishuu’ and D.kaki Thunb.var.sylvestris Makino were amplified by PCR.Two pairs of specific primers for molecular markers of D.kaki Thunb.cv.‘Taishuu’ and D.kaki Thunb.var.sylvestris Makino were selected,and the optimum annealing temperature of the primer was determined to be 54 ℃.(4)The significance test was used to analyze the seed setting rate of 292 persimmon sampled in 4 plots,3 flowering periods and 2 times.The results showed that the proportion of sexual reproduction of D.kaki Thunb.cv.Fangshan in Yongkang city reached 29.45 ± 0.01%in 2020,through the positive control test,the specific primers that can be used for the identification of male parents of hybrid seeds of D.kaki Thunb.cv.Fangshan were selected,SSR molecular marker technology was used to explore the male parent source of single seed hybrid seeds of D.kaki Thunb.cv.Fangshan.The results showed that 81.61 ± 8.83% of the male parents of D.kaki Thunb.cv.Fangshan hybrid seeds in the four sample plots were D.kaki Thunb.var.sylvestris Makino.(5)The significance test was used to analyze the seed setting rate of 211 Fangshan persimmon in 7 sampling points and 3 flowering stages,The results showed that the proportion of sexual reproduction of D.kaki Thunb.cv.Fangshan in Yongkang City reached95.47 ± 5.95% in 2021,which was significantly higher than that in 2020.SSR molecular markers and GPS positioning techniques were used to explore the natural hybridization frequency of D.kaki Thunb.cv.‘Taishuu’ and D.kaki Thunb.cv.Fangshan under different pollination distances.The results showed that the natural hybridization frequency of D.kaki Thunb.cv.‘Taishuu’ and D.kaki Thunb.cv.Fangshan first decreased with the increase of pollen transmission distance,and then stabilized after 50 m.The natural hybridization frequency of D.kaki Thunb.cv.Fangshan and D.kaki Thunb.var.sylvestris Makino increased continuously with the increase of pollen transmission distance of D.kaki Thunb.cv.‘Taishuu’and reached the maximum at 80 m.Eleven function models were used to fit the effect of pollination distance on natural hybridization frequency.The results showed that the logarithmic model had the best fitting effect. |
PDF Full Text Request