Identification Of Fruit Characters Of Different Seedless Grape Types And Evaluation,development And Application Of Seedless Markers

Seedless grape plays an important role in the fresh and dry grape market,and it is also an important development direction of grape breeding.Seedless markers can effectively detect seedless grape lines at seedling stage,effectively exclude seedless progenies,and accelerate seedless grape breeding process.Seedless grapes can be divided into three types:parthenocarpy,triploid and seed abortion.In this study,we compared the morphological indexes and morphological characteristics of fruit grains and ovules of different seedless types of grapes,in order to explore the seedless genes and potential molecular markers related to seedless development of grapes;meanwhile,we analyzed and evaluated the known seedless markers SCC8,SCF27,SCP18,39970524-5,39970524-6,GSLP1 probe,VMC7F2,P1＿Vv AGL11、P2＿Vv AGL11、P3＿Vv AGL11 and Vv SD10 were verified in the seedless population and natural population to comprehensively evaluate the detection efficiency of seedless markers,in order to find high efficient molecular markers.Based on the location information of known high-efficiency molecular markers and grape sequencing data,the sequence characteristics of high-efficiency markers and their linkage with seedless genes were analyzed.Combined with the screening of seedless genes in the early stage of this study,new seedless markers were developed,and then the detection efficiency of new seedless markers was verified by using known grape populations.At the same time,the seedless lines in grape hybrid populations were predicted by new seedless markers.The main results are as follows:1.Through the observation,comparison and analysis of the morphological indexes and appearance characteristics of ovules and berries of different seedless grape types,it was found that the residual core variety’Jingkejing’and the soft core variety’Jingzaojing’began to abortion at 30 and 35 days after flowering,respectively,showing the characteristics of smaller size,browning color and asymmetric development.The abnormal ovule rate of both varieties reached 90%at 44 days after flowering.The ovules of triploid’8611’and’Fujiminori’treated with GA₃began to shrink in size and brown in color at 15 days after flowering,and the integument depression deepened further,and the ovules became shriveled.The abnormal ovule rate of both varieties was more than 50%at 25 days after flowering.The ovule quality of triploid varieties’Hupei 1’,’8611’and GA_3-treated’Fujiminori’and’Rasoria bianco’fluctuated slightly at the early stages of development,and then decreased gradually;the ovule diameter of seedless varieties was significantly lower.Correlation analysis showed that the vertical and horizontal diameter and quality of seedless grape ovules were significantly negatively correlated with the rate of malformed ovules,and the formation of seedless traits in different seedless types of grapes was different,which reflected that the seedless mechanism and seedless genes of different seedless types of grapes may be different.2.Using the known molecular markers SCC8,SCF27,SCP18,39970524-5,39970524-6,GSLP1 probe,and VMC7F2,P1＿Vv AGL11、P2＿Vv AGL11、P3＿Vv AGL11and Vv SD10 to verify the detection efficiency of seedless population and natural population.The results showed that the correct genotyping rates of SCF27 and 39970524-5 in seedless population were the highest（90.91%）;for different seedless types of grapes,the accuracy of seedless classification of SCF27 and 39970524-5 in residual seeded and triploid varieties were highest,and that of 39970524-6 in soft seeded varieties was 100%,and that of SCF27and 39970524-5 in Vitis vinifera×Vitis labruscana and Vitis vinifera were the highest.The results showed that SCF27 and 39970524-5 had the highest accuracy and maintenance rate of seedless typing in the natural population;39970524-5 had the highest detection rate of seedless typing;SSR markers P1＿Vv AGL11、P2＿Vv AGL11、P3＿Vv AGL11 all amplified stable polymorphic loci in the validation population,among which P1＿Vv AGL11 was the most stable in the validation population.among which P1 was the most stable in the validation population.Among them,the minimum number of alleles of P1＿Vv AGL11 was2,and the information quantity of polymorphism（PIC）of P1＿Vv AGL11 among seedless population and natural population were 0.348 and 0.344,respectively.The results of P1＿Vv AGL11 amplification showed that the frequency of heterozygous genotype AB was the highest in different seedless types of seedless population and seedless varieties of natural population,and he frequency of BB genotype was the highest in seeded varieties of natural population.Comprehensive analysis showed that SCF27,39970524-5 and P1＿Vv AGL11 had better verification effect on seedless character in verification population.3.Based on the transcriptome datas of seed/nuclear region of’Fujimino’grape treated with water/GA₃,41 genes with similar expression trend to seedless gene Vv AGL11 were screened by K-means clustering analysis.A candidate gene VvMETK5（VIT＿07s0005g02230）related to seed embryo abortion was screened by comparing those41 genes with 1261 genes near the developed markers.In the next step,we compared the SSR sites on the promoter of VvMETK5 gene in the sequencing data of’Fujimino’treated with water/GA₃,and developed a new molecular marker named P1＿VvMETK5.Using P1＿VvMETK5 to detect seedless traits in seedless population and natural population.The results showed that the frequency of heterozygous genotype was 50%in seedless population and 57.70%in natural population.The frequency of homozygous genotype was78.95%in natural population.Using P1＿VvMETK5 and high efficiency SCAR markers identified seedless plants in new hybrid population,the results showed that the individuals numbered 1,2,3,4,5 and 17 were predicted to be seedless plants under the detection of three markers,and the individuals numbered 6,8,9,10,12,13,16 and 18 were only detected in one or two markers.The plants numbered 6,10,12 had close genetic relationship with the individuals predicted to be seedless,and it was speculated that individuals numbered 6,10,12 might also have seedless traits,which indicated that the combined use of multiple markers could expand the prediction range of seedless plants and improve the detection efficiency of seedless plants...