| Citrus is an important horticultural fruit with extensive cultivation area.Using 2n gamete for sexual polyploidy can reduce breeding time,transfer heterozygosity,facilitate the generation of new mutations,improve the probability of good progenies,it has considerable application in citrus breeding.In this study,the 2n pollen frequency of 40 citrus varieties,2 hybrid combinations for two consecutive years.Expecting to analyze the occurrence regularity of 2n pollen in citrus natural population.The process of citrus meiosis was observed.And using high temperature treatment and colchicine treatment to increase 2n pollen frequency of Fortunella margarita,Fortunella hindsii and kiwi grapefruit,which is expected to explore the technical methods of increasing the 2n pollen frequency in citrus.In addition,SNP markers were used to identify the genetic sources of tetraploid progenies from five 2X × 4Xcrosses,which could provide germplasm resources for triploid seedless breeding of citrus.The main research results are as follows:1.The 2n pollen frequency in citrus natural population was between 0.00% and1.79%,which was generally not high.Based on the diameter difference between 2n pollen and normal pollen,the diameter of 2n pollen is more than 1.3 times that of normal pollen.The results showed that 2n pollen frequency of citrus was generally low.From highest to lowest was pummelo > lemon > mandarin > grapefruit > trifoliate orange > sweet orange.The 2n pollen frequency of progenies from the two hybrid combinations showed that 2n pollen frequency was increased on the whole,but some progenies were decreased.Fortunella margarita can flower continuously from April to October,the pollen was collected to investigate the 2n pollen frequency.The results showed that 2n pollen frequency was related to temperature.When the monthly mean temperature was higher than 28℃,the 2n pollen frequency was positively correlated with the temperature.When the monthly mean temperature was lower than 18℃,the frequency of 2n pollen was negatively correlated with the temperature.2.Explored the methods of inducing 2n pollen frequency of citrus.1)Determined the relationship between meiosis and flower bud morphology.According to the degree of petal exposure,the transverse and longitudinal diameter of flower bud,the buds of Fortunella margarita and kiwi grapefruit were divided into four developmental stages.During S1,meiosis has not yet begun;During S2,meiosis was from prophase I to metaphase I;During S2,meiosis was between anaphase I and metaphase II;During S4,meiosis was end.2)Determined the best stage for inducing 2n pollen in citrus.Two temperature gradients were set at 38℃ and 42℃,with no treatment as the control.Four stages of Fortunella margarita were treated for 2 h,4 h and 6 h.The results showed that in S1 and S4 the 2n pollen frequency was not significantly increased compared with the control;in S2,in 5 of the 6 treatments,the 2n pollen frequency was significantly higher than control;in S3,in 2 of the 6 treatments,the 2n pollen frequency was significantly higher than control.In conclusion,the best treatment result was in S2,the best stage for inducing 2n pollen was prophase I.3)Determined the best treatment combination for induce 2n pollen by high temperature treatment.The S2 of Fortunella margarita was treated at 38℃ and 42℃ for2 h,4 h and 6 h.The results showed that the 2n pollen frequency treated at 42℃ for 2 h and 6 h,38℃ for 6 h was significantly higher than control.The best treatment effect was42℃ for 2 h,the 2n pollen frequency was 6.59%.In 42℃ treatments,the pollen viability was negative correlation with the time,and 42℃ for 2h pollen viability was highest.In conclusion,in prophase treated at 42℃ for 2 h is the best treatment combination for induce 2n pollen.Fortunella hindsii was treated at 38℃,42℃ and 45℃ for 2 h and 4 h.The results showed that after treatment,the 2n pollen frequency increased first and then decreased,and the best treatment effect was at 42℃.4)Used colchicine to induce 2n pollen in kiwi grapefruit.Five concentration gradients(0.2%,0.4%,0.6%,0.8% and 1.0%)were set and injected 2,4,6 and 8 times at S2.Injected distilled water(0%)as controls.The results showed that the 2n pollen frequency was significantly higher than control after 8 injections at 0.4% and 1%.3.Genetic identification of tetraploid progenies from five hybrid combinations.Using SNP markers to identify the genetic origin of the 77 tetraploid progenies which crossed between diploid female parents(’Huannonghongyou’ pummelo,’Orri’,’Nules clementine’and ’Murcott’ tangor)and tetraploid male parents.The results showed that all the 77 tetraploid progenies were the sexual hybrids,and it was speculated that all tetraploid was obtained by 2n female gamete crossing with normal male gamete. |
