Analysis Of Genetic Relationship And Cause Of Seedlessness In The Sweet Orange Seedless Variant(Chunmi Sweet Orange)

The sweet orange seedless mutant,Chunmi Sweet Orange(tentative name)was discovered from top grafting of Chuncheng 1 Sweet Orange/fructus aurantii planted in Wuming District.In order to provide evidences for variety registration and germplasm innovation,we explored the biological characteristics,genetic relationship and seedlessness causes of Chunmi Sweet Orange,and evaluated its biological characteristics comprehensively by phenological observation,morphological identification,SCoT molecular markers,biochemical substances determination in leaf before and after inoculation of canker,pollen fertility detection,pollination affinity and embryonic development observation,and chromosome ploidy analysis.The results are as follow.(1)In Nanning,Chunmi Sweet Orange began to sprout spring shoots in mid-February while its flowers budded in late February and entered peak flowering period in late March.The fruits enlarged in early August and ripened from December to January of the following year having orange peel,with a fruit shape index of 0.96,a peel thickness of 3.97 mm,a seed count of 0.6,a soluble solid content of 15.63%,and a solid acid ratio of 1.35.(2)In the Q-type cluster described by leaf phenotypic traits,Chunmi Sweet Orange and Chuncheng 1 Sweet Orange were the closest to each other,and they clustered in one group when the Euclidean distance was 1.Meanwhile,in the SCoT molecular marker,the average polymorphism locus ratio of 42 sweet orange varieties was 65.26%;Chunmi Sweet Orange and Chuncheng 1 Sweet Orange were clustered in the same group at the genetic coefficient of 0.938.Combining the above results with the morphological analysis of flower,pollen and fruit in Chunmi Sweet Orange and Chuncheng 1 Sweet Orange,it was believed that Chunmi Sweet Orange was derived from the seedless high-quality bud mutation of Chuncheng 1 Sweet Orange.(3)The incidence rates of summer shoots in the field and leaves after pathogens inoculation of Chunmi Sweet Orange were 63.14%and 88.89%,which were significantly higher than those of Chuncheng 1 Sweet Orange.The leaf stomata density of Chunmi Sweet Orange was significantly greater than Chuncheng 1 Sweet Orange,while its sponge tissue thickness was significantly smaller than Chuncheng 1 Sweet Orange.There was no significant correlation between soluble protein and MDA content and the resistance to canker in leaves,which were positively correlated with POD,SOD and PPO activities.(4)The floral organs of Chunmi Sweet Orange were normal,and its pollen volume was large with 46,528 pollen grains in each single anther and low germination rate.The rates of pollen viability and in vitro culture germination were 61.83%and 4.29%,which were significantly lower than Chuncheng 1 Sweet Orange,while the deformity rate(25.64%)was significantly higher than the check variety.(5)After 8 hours of self-pollination,some pollen tubes of Chunmi Sweet Orange began to germinate;after 48 hours,the pollen tubes entered the base of the style,and after 120 hours,the fertilization was completed.By contrast,after 4h of cross-pollination(Chunmi Sweet Orange ?×Chuncheng 1 Sweet Orange?),some pollen began to germinate;after 48h,the pollen tube entered the ovary and some began to fertilize;after 120h,the fertilization was completed.(6)After self-pollination,the embryo began to shrink or had a cavity at one week post-pollination,and then the embryo began to abort and form fruit without seeds.After cross-pollination(Chunmi Sweet Orange ? xChuncheng 1 Sweet Orange ?),the spherical embryos could still be observed at two weeks post-pollination;while the embryo degradated and then disappeared after 3weeks,ultimately forming seedless fruits.(7)The results of flow cytometry showed that the chromosome multiple of Chunmi Sweet Orange was 1.46 times of Chuncheng 1 Sweet Orange,so Chunmi Sweet Orange was inferred to be triploid preliminarily.Based on the results,Chunmi Sweet Orange growthing robustly,having few seeds,excellent quality and poor resistance to canker was derived from the seedless high-quality buds of Chuncheng 1 Sweet Orange,preliminarily determining that triploid and early embryonic abortion was the reasons of its nuclear-free.