De Novo Genome Assembly Of A Highly Embryogenic And Transformable Maize KN5585 And Its Genomics Characters

Maize is one of the most important staple crops in the world,as well as a model species in genomic research.The publication of reference genome sequences of several maize inbred lines(such as B73,Mo17,etc.)has facilitated basic research and laid foundation for breeding improvement.Massive SNPs,In Dels and structural variations have been identified between different maize lines,which contributed to various phenotypes observed among maize lines.High quality reference genomes of representative maize lines are of great importance to genetic diversity studies and germplasm improvement.With genetic transformation techniques such as plant tissue culture and genetic engineering,researchers can accurately and rapidly improve the traits of plants.Agrobacterium transformation is the most widely used method in plant genetic transformation currently.Besides,the regeneration rate of Agrobacterium-mediated transformation varies among different maize lines,which vastly limits the application of genetic transformation in different maize.KN5585 is one of the most important maize line for the research of genetic transformation in China,and its mutant library has been constructed.The reference of KN5585 would be a key resource for various genetic transformation studies.In genetic transformation,ectopic expression of transcription factors is one of the important induction methods of somatic embryogenesis,which can improve the regeneration rate of genetic transformation.However,the molecular mechanism underlying maize somatic embryogenesis induced by this method is still elusive.In this study,a highly embryogenic and transformable maize line KN5585,was sequenced through ONT platform.We constructed a high-quality reference genome for KN5585 and finely annotated the genome.We identified massive structural variations between KN5585 and B73,Mo17 and SK by whole genome sequence comparison.The immature embryos of maize KN5585 and maize Chang7-2,which cannot form embryogenic callus,were cultured for 3 days under the induction of plant hormones and ectopic expression of Zm WUS and Zm BBM.By analyzing the transcriptome sequencing data of immature embryos and immature embryos cultured under different conditions,seven candidate genes related to the transformation regeneration rate were discovered.The main results are as follows:1.The maize inbred line KN5585 genome was sequenced with ONT Prometh ION platform and MGISEQ-2000 platform.A high-quality reference genome was constructed based on the sequencing data.The final assembled genome is 2.23 Gb in size,with a contig N50 and contig N90 of 24.52 Mb and 5.05 Mb,respectively.Of these,2.13 Gb(95.5%)sequences were anchored to 10 pseudochromosomes using Hi-C data.2.We performed repeat annotation for the KN5585 genome.In total,1.95 Gb of sequences were annotated as repeat elements,accounting for 89.46% of the genome.By integrating evidence derived from ab initio prediction,homologous protein alignments,RNA-seq data,and full-length transcript sequencing data,40,765 protein-coding genes and100,254 transcripts were annotated in maize KN5585 genome.For the proteins encoded by the annotated transcripts,97.93% of them can be functionally annotated.3.We performed whole genome sequence comparison between KN5585 and maize B73,SK,and Mo17.The syntenic relationship between KN5585 and B73、Mo17、SK genomes was conservative.However,a large number of structural variations were identified between KN5585 and the other three genomes.4.To unravel the differences in gene regulation network underlying embryogenic callus between the KN5585 and Chang7-2,we sequenced the transcriptome of immature embryos and embryogenic callus of these two maize lines.In total,2,812 line-specific differential expression genes,including Zm YUC2,Zm IAA,were identified by analysing the transcriptome data,indicating that the anabolic signal pathway of endogenous auxin synthesis is the key to the induction of embryogenic callus.5.We identified 7 key candidate genes that might affect maize somatic embryogenesis,by analyzing transcriptome data of maize KN5585 and Chang7-2 immature embryos that cultured in different conditions: 3 days in the Zm BBM and Zm WUS2(induced group);the control group(Auxin induced group,negative plasmid induced group).In summary,we constructed a high-quality reference genome for maize KN5585,based on sequencing data generated by ONT platform and MGISEQ platform,and formulated a ready-to-use strategy for de novo assembly of large,repetitive genomes.By analyzing differentially expressed genes during the process of genetic transformation,we identified multiple candidate genes that involved in somatic embryogenesis process and pinpointed potential pathways that contributing to transformation efficacy differences among maize genotypes.It lays foundation for the generation of genotype-independent highly efficient maize transformation system.