Development Of A Vegetable Resynthesized Brassica Napus And Assembly Of Its Genome By De Novo

Brassica napus（AⁿAⁿCⁿCⁿ,2n=38）is an allotetraploid derived from the interspecific hybridization between diploid B.rapa（A^rA^r,2n=20）and B.oleracea（C^oC^o,2n=18）and the subsequent whole genome duplication under natural conditions,with an evolutionary history of about 7500years.Because its relative short evolutionary history and the possibility of artificial resynthesis through its diploid ancestors,B.napus has become a model species for deciphering allopolyploidization.Moreover,B.napus is not only an important oil crop worldwide,but also possesses a great vegetable value because of its stem with unique flavor.However,the special germplasm resources of B.napus used as vegetables have not been enough.Herein,a vegetable B.napus was successfully resynthesized by interspecific hybridization between B.rapa“CX1”and B.oleracea“JL1”with a short reproductive period and multiple branches,which greatly broadened the germplasm resources of vegetable B.napus.Subsequently,the chromosome-scale genome of resynthesized haploid B.napus（A^rC^o,n=19）RBH-4 was de novo assembled and annotated using a combination of Illumina platform,Pac Bio Sequel IIe platform,and the high-throughput chromosome conformation capture（Hi-C）technology.This high-quality genome provides important genomic information to reveal the immediate effect of allopolyploidization in B.napus.Additionally,based on the information of Brassica A and C genome（RBH-4,B.rapa,B.oleracea,and B.napus）,the genome-wide identification and evolutionary analysis of the HIPP（heavy metal-associated isoprenylated plant proteins）gene family,has provided a theoretical basis to dissect the transport mechanisms of metal ions and to study the evolution of genome evolution during allopolyploidization of B.napus.The main results are obtained as follows:（1）In this study,four hybrid plants were obtained by artificial hybridization and embryo rescue from the cross combination of CX1（maternal）and JL1（paternal）,which were subsequently confirmed to be resynthesized haploid B.napus（A^rC^o,n=19）by specific molecular maker of the C-genome chromosome,flow cytometry,and counting of somatic chromosome number.Then,29 regenerated plants were obtained by tissue culture,and three（12.5%）allotetraploid resynthesized plants were successfully obtained by somatic chromosome doubling of regenerated plants after treated with 1%colchicine.The morphological characteristics and SPAD values of the M1 generation were intermediate between the two parents,with short reproductive period and multiple branches.However,the contents of palmitic,linoleic,erucic and nervonic acids in the seed fatty acids of M1 generation were higher than those of their parents.（2）A high-quality chromosome-scale genome of RBH-4 was de novo assembled and annotated using Illumina short reads,Pac Bio long reads,and the Hi-C assistant technology.After filtering and quality controlling,51Gb clean data of Illumina short reads,35Gb clean data of Pac Bio long reads,and 49.5Gb clean data of Hi-C were obtained.A total size of 1.04Gb chromosome-scale genome of BRH-4（named RBH-4 v1.0）was assembled using these clean data with a scaffold N50 sizes of16.12Mb,the heterozygosity rate of 0.15%,and a repeat sequence proportion of 73.04%.A total of 97,853 coding genes were predicted,with 99.06%of the genes having predicted function.For RBH-4 v1.0were evaluated by multiple methods,and the results showed an excellent consistency,completeness,and accuracy of RBH-4 v1.0,with 97.22%of the assembled genome mapping to 19 chromosomes.（3）The HIPP（heavy metal-associated isoprenylated plant proteins）which play an important role in the maintenance of metal ion homeostasis and detoxification mechanism,have been recognized as key proteins in the safety transportation of metallic ions in plants.In addition,the HIPP genes have undergone multiple rounds of gene number expansion and can be used to elucidate the evolution history of A and C genome.In this study,the genome-wide identification of the HIPP gene family in A and C genome showed that 106,53,41,and 104 HIPP genes were detected in RBH-4,B.rapa,B.oleracea,and natural B.napus by bioinformatic methods.The physicochemical property analysis and subcellular localization prediction of these HIPP genes indicated that these genes showed the tissue specificity of gene expression while highly expressed in root.Phylogenetic relationships revealed that the HIPP genes can be divided into five clades.Compared to its diploid ancestors,the HIPP genes number of A subgenome in B.napus had lost one,moreover,some genes have been demonstrated to undergo a homologous exchange with their homoeologous partners.The copy number of HIPP genes of C subgenome in B.napus was variously altered of which have undergo a significant gene number expansion（from 41 to 51）.The gene silence and elimination of HIPP genes both observed in A and C subgenome after whole genome duplication.Moreover,during the evolution of the HIPP genes,most of the HIPP have been involved in the expansion of the HIPP gene family in the form of segmental duplications.The above results not only broaden the germplasm resources for vegetable B.napus,but also provide genomic information and theoretical references for detecting allopolyploidization of B.napus and deciphering the transport mechanisms of metal ions in B.napus.