Identification Of Segregating Proginies With Molecular Markers Of Disease-resistance And Multi-target Gene Editing Breeding In Tomato

Applying the achievement of molecular biology to traditional breeding can improve the breeding efficiency,shorten the breeding period,accelerate the germplasm resource innovation,and realize the purposeful design breeding.Molecular marker assisted breeding and gene editing breeding are the two main aspects of molecular breeding.In this study,molecular markers were used to assist in the identification and selection of tomato breeding materials.The molecular markers involved multiple disease resistance genes,and the breeding materials involved included individual plant populations of separate generations from each hybrid combination.Gene editing adopts CRISPR/Cas9 technology,developed and improved into multi-target gene editing;and carried out gene editing of specific genes on some cherry tomato breeding materials according to breeding goals,with a view to quickly improve a few key traits of varieties and obtain ideal new varieties.1.Molecular marker assisted breeding:using the disease-resistant molecular markers developed by the tomato research group in recent years to conduct molecular identification of 558 individual individuals of isolated groups of different generations in the research group.The selected resistance gene markers are Ty1,Ty2,Sw-5,Mi-1,Mi-3,Bwr12,I-2,cf-9,etc.,fruit weight gene marker Fw11.3.The results of marker detection and identification showed that Ty-1,Mi-3,Tm-2^a,I-2,Cf-9 and other disease-resistant genes were more common in this batch of isolated materials.At the same time,there are 80 materials containing the yellow leaf curl resistance genes Ty-1 and Ty-2.Varieties containing only a single Ty gene resistance have deteriorated year by year in the field,which also drives breeders to aggregate multiple Ty genes..In this study,the isolated material group contains 125 single-plant materials with 4 or more homozygous disease-resistant genetic materials.The tomato fruit weight Fw11.3 gene has a regulatory effect on fruit size.Our marker is used to determine whether the cherry tomato disease-resistant material we retained through field screening will have large fruit isolates in the subsequent planting process.Whether it is homozygous,guide whether to use it for pairing hybrid combinations or to continue self-segregation screening.2.Gene-editing breeding of cherry tomatoes:Using the p TX editing vector in the laboratory（one or two sites can be edited）,the recessive disease resistance gene-tomato yellow leaf curl virus ty5 gene was edited,and the construction vector was transferred into the susceptible variety AC,which has been obtained 11 T₀ plants were successfully edited.The T₁plants were inoculated indoors.The homozygous edited plants showed no obvious symptoms and grew normally.The remaining plants were seriously infected with tomato yellow leaf curl virus disease.The fruit pink was formed by deletion mutation.We edited the pink fruit tomato gene yellow and constructed the vector into red fruit cherry tomato Guangdong red cherry,and obtained 8 pink fruit tomato plants successfully edited.The genes related to the regulation of the fruit pedicel node J2 were edited to obtain tomato plants with no pedicel node and multiple inflorescences.Using PTX editing vector（one or two sites can be edited）in the laboratory to edit the gene ty5 of Tomato yellow leaf curling virus,the construction vector was transferred into the non resistant variety AC of tomato,among which 11 plants of T₀ generation were successfully edited.The resistance identification was divided into field condition investigation and indoor disease identification.The disease index of the homozygous plants was less than 2,and they had good resistance.The other homozygous plants had no serious disease.After two weeks of indoor exposure,the growth of homozygous plants was normal.Other plants were seriously infected with yellow leaf curl disease.Using the improved p TX vector（multiple targets can be edited）,the tomato flowering genes SFT and SSP,disease resistance ty5,and fruitless stem j2 were simultaneously edited to obtain 6 plants with multiple genes edited at the same time.Aiming at the process of editing j2 gene,a new phenotype emerged,especially inflorescence branches,which were analyzed.Finally,corresponding molecular markers such as j2,ej2,and sb3 were developed to screen breeding materials suitable for gene editing.