Editing Of Non-heading Chinese Cabbage Haploid Induction Gene CENH3 And Preliminary Study On The Induction Line

Non-heading Chinese cabbage(Brassica campestris ssp.chinensis)is a cross-pollinated crop of the cruciferous family.It has significant heterosis.To adapt to market demand and create better non-heading cabbage varieties,it is necessary to maximize the hybridization advantage of the plant.The important content is Obtain homozygous inbred lines with homozygous genotypes and consistent traits.However,the process of using traditional breeding methods is slow.Obtaining homozygous lines requires continuous selfing for 7-8generations.If the haploid plant can be obtained by combining the haploid seed technology,and then after the artificial chromosome doubling,the pure DH plant can be obtained in only one generation,which can speed up the breeding process of non-heading Chinese cabbage." haploid induction line" refers to the use of the plant as a parent to cross with the same kind of plant,which can induce the same kind of plant to produce the corresponding maternal haploid or male haploid.CENH3,which encodes a centromere-specific protein,is a key gene related to haploid induction in dicotyledonous plants.A new haploid induction line based on CENH3 mediation has been developed in the model plant Arabidopsis.In rape,there is a rape double haploid induction line with parthenogenesis inducing ability.After crossing with genotype heterozygous rape or cruciferous female parent,the offspring can obtain only double haploid formed by doubling of the maternal chromosome Somatic plant without artificial chromosome doubling.The development of induced lines has important research value in haplotypes.In this study,for the CENH3 gene of non-heading Chinese cabbage,multiple sgRNA editing sites were designed,CRISPR/Cas9 vector was constructed,combined with pollen magnetic transformation technology,the plasmid vector was delivered into pollen,and then introduced into non-heading Chinese cabbage through fertilization.The CENH3gene-edited plants were obtained through plant resistance screening,PCR detection,and gene fragment sequencing,which laid the foundation for the use of CENH3-mediated chromosome elimination mechanism in non-heading Chinese cabbage double haploid species.It is a preliminary study of editing the CENH3 gene to establish a haploid induction system in non-heading Chinese cabbage.1.Obtained cenh3 mutant non-heading Chinese cabbage plants.This study based on the CENH3 gene of non-heading Chinese cabbage,an editing vector Brcenh3-sgRNA3 and a complementary editing vector Brcenh3-sgRNA with herbicide resistance genes as plant selection markers were constructed.After pollen nanomagnetic transformation method.,one Brcenh3-sgRNA3 vector Cas9 positive transgenic plant and three Brcenh3-sgRNA vector cas9 positive transgenic plants were obtained.The CENH3 corresponding editing site of the positive single strain was sequenced and analyzed,and the 115-1 single strain transformed by the Brcenh3-sgRNA3 vector was a repetitive sequence insertion.Sequencing of 20 monoclonals of single strain 110-1 transformed with Brcenh3-sgRNA vector revealed that base substitutions and insertions occurred in both the PAM sites and the sgRNA sequence of the two monoclonals.2.A few plants with the phenotype of the inducible line appeared in the offspring of the double haploid induction line of rapeseed and the diploid of non-heading Chinese cabbage variety.Two tetraploid offspring LCX002-4 and LCX003-2 were obtained by the stoma guard cell chloroplast counting method and flow cytometry ploidy detection,the rest of the offspring are diploid.The double haploid induction rate of rapeseed double haploid inducing line to 6 kinds of caixin varieties was 85.71%.The diploid induction rate of Aijiaohuang,Purple Tsai-tai,LCX004,LCX0034,LCX0038,and LCX0041 reached100%.The distribution of chloroplasts of stomatal guard cells in different varieties of non-heading Chinese cabbage of the same ploidy is different.The ploidy identification results of stomatal guard cell chloroplast counting method are consistent with the results of flow cytometry ploidy analysis.It proves that the chloroplast counting method of stoma guard cells can be used to initially identify the ploidy of non-heading Chinese cabbage.