Fortunella (Swingle,1915) Hybrid Technology Based On SSR Molecular Marker And Pollen Fertility

Fortunella(Swingle,1915)is a citrus plant that can be eaten with its skin.It is increasingly popular with fruit consumers.The breeding of new Fortunella varieties uses field bud mutation combined with morphological identification as the main method,which has problems such as long cycle,heavy workload,inaccurate identification results and unstable mutation characteristics.Therefore,this study took 38 germplasms of Fortunella and its related genera as the research object,carried out specific molecular marker screening of germplasm resources,comparison of pollen fertility and artificial cross-pollination experiments,providing a preliminary theory for the establishment of Fortunella hybrid technology in accordance,theoretical basis for obtaining Fortunella hybrids.The main results are as follows:1.Using 23 pairs of SSR primers to amplify 38 Fortunella and its related species germplasm,a total of 265 effective bands were amplified,of which 250 were polymorphic bands,and the polymorphism ratio was approximately 92.99%.The length of the band fragments obtained after amplification is from 100 bp to 1200bp;The germplasm was screened for specific markers,and a total of 47 specific markers were obtained,of which 13 specific markers were found for 6 citrus materials,4 specific markers were found for 4trifoliate materials,and a total of 2 Fortunella hybrid materials were found.There are 6specific molecular markers,and a total of 24 specific molecular markers were found for the remaining 19 Fortunella materials.The results show that the number of specific markers between different germplasm is quite different;When the genetic similarity coefficient is0.720,the materials can be divided into three categories.The first category is Liuyangsuanju,the second category is Yuenanju and Gongsunju,and the third category is Fortunella,Hovenia and some Citrus.Liuyang jindan and Lanshan jingan clustered together at 0.98,and there may be different names of the same thing.2.The 0.5% TTC method was used to determine the pollen vigor of different species of Fortunella.The results showed that the pollen vigor of different species was different in the four stages of small bud,big bud,early opening and full bloom.All the pollen vigor of all varieties showed first growth and then growth.The weakened single peak trend and the strongest vigor in the initial blooming period.The first flowering powder vigor of Lanshan jingan is the highest among all varieties,and its pollen fertility percentage is 61.73%;the benzidine-hydrogen peroxide method is used to measure different Fortunellas The stigma activity of the cultivars,the results showed that the stigma activity of different cultivars at the four stages of small bud,big bud,first opening and full blooming is different.The stigma activity of all varieties showed a single peak trend of increasing first and then weakening.It has the strongest vitality at time,consistent with the change trend of pollen vitality.3.Some Fortunella varieties were treated with emasculation and non-pollination,and the parthenocarpy rate of different Fortunella varieties was different,and the fruiting ability was low.Among them,the parthenocarpy rate of Lanshan jingan was the highest at 8.57%and there were full seeds.It shows that Fortunella has a certain parthenocarpy and the nacreous embryo cells can develop into seeds.4.Observation of the pollen tubes found that all selfed and outcrossed pollen tubes began to germinate after 2h of pollination,and there was no significant difference;after 48 h of pollination,all outcrossed pollen tubes and 4 selfed pollen tubes could reach the ovary,while 3 of them were 12h～24h after pollination.The selfed pollen tube of the variety stops growing at 2/3 of the style.The fruit setting rate and the fruit setting rate of outcrossing are significantly higher than that of selfing.Among all self-pollination,Lanshan jingant has the highest fruit setting rate of 17.20%.Among all outcross pollinations,Liuyang jingan CQ(♀)× Lanshan jingan(♂)has the highest fruit setting rate as high as 50% all self-pollination;The fruit harvest rate of cross and outcross pollination is significantly lower than the fruit setting rate.Among the self-pollination,the fruit harvest rate of Lanshan jingan is the highest at 4.30%;among all outcross pollinations,Liuyang jingan CQ(♀)× Lanshan jingan(♂)is harvested.The fruit rate is 13.00%,which is the highest.5.A comparison between artificial outcross-pollinated fruits and naturally pollinated fruits,it is found that except for the artificial outcross-pollinated fruits of Niunaijingan(♀)× Lanshanjingan(♂),the fruit shape is different from the naturally pollinated parent fruit,other artificially different fruits.There is no significant difference between the cross-pollinated fruit shape and the parent fruit under natural pollination conditions;From the perspective of fruit size,the artificial outcross-pollinated fruit is smaller than the naturally pollinated parent fruit;The artificial outcross-pollinated seed is different from the natural.There was no significant difference in the shape of seeds of pollinated parents.A total of 146 artificial outcross-pollinated seeds were obtained,of which 20 seeds germinated into seedlings.The seedlings were identified using parent-specific markers,and a total of 2hybrid seedlings were obtained.