Targeted Generation Of ZmCENH3 Mutations Using Reporter-Assisted CRISPR/Cas9 And Identification Method Of Essentially Derived Varieties

The CRISPR/Cas9 gene editing technology can achieve precise mutation of endogenous genes,and has significant application value in the genetic improvement of crops such as maize.To simplify the identification of gene editing materials,the establishment of a CRISPR/Cas reporting system based on seeds can significantly improve the efficiency of the plant gene editing system.Double haploid breeding has become an important technical strategy for maize breeding,and gene editing technology provides a new idea for creating materials with a high induction rate.This study aims to establish a Seed Fluorescence Reporter(SFR)-assisted CRISPR/Cas9(SFR/Cas9)to further improve the efficiency of the technical maize gene editing system.Based on this,a series of mutations were created by SFR/Cas9 targeting the important functional domain of maize ZmCENH3 desired sequences,as basic materials for double haploid breeding.Additionally,with the help of classical improved inbred lines,basic parameters related to the analysis of substantive derived varieties,such as Residual Heterozygosity(RH)and Genetic Drift(GD),were observed,laying a foundation for the EDV analysis method based on genome-wide molecular markers to analyze the EDV of receptor mutant lines produced by gene editing and other techniques in the future.The main research results of this experiment are as follows:1.The red fluorescent protein DsRED was driven by the embryo-specific promoter ZmESP and the endosperm-specific promoter HvASP.The Em-SFR and En-SFR expression cassettes were constructed and linked in tandem with the CRISPR/Cas9 expression cassette to establish the SFR/Cas9 system in maize.Both SFRs exhibited distinguishable fluorescent patterns in the embryo and seed endosperm,enabling the identification of seeds carrying the transgene and those that had segregated it out.2.To test the efficiency of the SFR/Cas9 systems,we transformed 14 targeted genes.We constructed 7 Em-SFR/Cas9 vectors and 7 En-SFR/Cas9 vectors,with each targeting a single gene.A total of 137 T0 transgene events were obtained through stable genetic transformation.We analyzed the fluorescence,transgenic elements,and mutation types.Both tests showed that the kernel’s fluorescence status is a reliable visible reporter for sorting kernels based on the presence or absence of the transgene.This system also successfully generated a single target mutant or mutant library.3.Based on Em-SFR and En-SFR,we have established multiple gene editing systems,namely Em-SFR/Ca9,Em-SFR/CBE,and Em-SFR/ABE.These systems were combined with En-SFR/sgRNA to edit the ZmCENH3 gene that is fundamental in haploid induction.Consequently,we have generated a set of mutant materials that exhibit a N-terminal truncation of the CENH3 protein by 25 amino acids.Furthermore,we have developed the Segregated Gene Editing System,which is composed of sgRNA transcription cassette and Cas expression cassette that are not connected.This system has allowed us to create an extensive allelic variation material of exon 1 and exon 4 of the ZmCENH3 gene.4.Using 21 classical maize inbred lines from various sources as materials,the average rate of recombination between homologous chromosomes(RH)for their whole genome was observed to be around 1.03 % using high-throughput SNP(Single Nucleotide Polymorphisms)genotyping technology.Nevertheless,the RH rate for some accessions was found to be higher than 3 %.The genetic diversity(GD)between two accessions from different years or breeding programs ranged from 0.13 % to 13.16 %.Both RH and GD data suggest that genetic purity analysis is an essential procedure before determining the estimated breeding value(EBV).By investigating the genome compositions of 11 n derivative lines,their breeding histories can be inferred.The data provides insight into underlying fundamental parameters that help define the EDV threshold.This study presents a novel technical scheme to enhance the efficiency of crop gene editing.Specifically,we generated a ZmCENH3 targeted mutation that serves as a fundamental material for double haploid breeding.Additionally,we established a basic parameter for maize EDV analysis.