Analysis Of Brassicaceae DMP9 Locus And Screening Of B.rapa DMP9 Mutants

Brassica rapa is a variant of Brassica genus Brassica subspecies of Brassicaceae that can form leaf balls.The number of chromosomes is 2n=2x=20.It is a biennial herb.Brassica rapa is a typical cross-pollinated crop with significant heterosis.Double haploid(DH)line is crucial for Brassica rapa breeding.Double haploid production restores inbred lines with recombinant haploid genomes and shortens the breeding process.The traditional DH line is obtained by multi-generation self-purification,which requires 7-8 generations.In addition,it can also be obtained by techniques such as embryo induction,bud regeneration medium and microspore culture.However,these are highly dependent on variety genotype.The recent emergence of haploid induction technology has revolutionized crop breeding,which makes it possible to obtain a double haploid system in 2 generations,which greatly reduces the human,material and time cost of crop breeding.This study analyzed the evolutionary relationship of DMP9 homologous genes using 26 sequenced Brassicaceae genomes and 18 Brassica pan-genomes,and analyzed haploidy based on Brassica rapa and Brassica oleracea transcriptome data Functional evolution and differentiation of somatically induced genes after polyploidization.We created a library of Brassica rapa mutants using EMS reagents for secondary induction of germinated seed mutations.Since the high conservation of the amino acid sequence of DMP9 gene and the conservation of its function have been verified in monocotyledonous and dicotyledonous plants and diploid or polyploid crops,it can be speculated that the haploid-inducing function of DMP9 gene is also suitable for Brassica rapa.The analysis of the DMP9 locus in Brassicaceae plants and the creation of the Brassica rapa mutant library provide a theoretical basis and germplasm resources for the subsequent creation of Brassica rapa haploid inducer line.The results of the study are as follows:1.This study demonstrates the evolution of DMP9 homologous genes in Brassicaceae plants.It also means that the DMP8 gene is a unique replication event that occurs in Arabidopsis.The DMP9 homologous gene is divided into two parts according to the genomic blocks(GBs): S block and R block.In Brassica diploid,the LF and MF1 subgenomes retain one S block and S block,respectively.2.In the process of pan-genome evolution of Brassica rapa,the comparative analysis of DMP9 genes showed that the gene of Chinese cabbage B9008 was complicated,and there was a tandem duplication in LF subgenome.These multicopy phenomena are ubiquitous in the genome and have important implications for plant diversity and adaptation to the environment.3.In order to study the expression of multiple copies of DMP9 gene in Brassica plants,this study selected Brassica oleracea JZS,Chinese cabbage Chiifu-401-42 and Chinese cabbage B9008 as research materials.First,I observed the pollen of cabbage,Chinese cabbage Chiifu-401-42 and Chinese cabbage B9008 at different meiotic stages using microscopy,and stained the pollen with DAPI.Combined with the results of previous studies on the expression of DMP genes in corn,wheat,rice and other crops.The meiotic period of pollen is mainly divided into six stages: promeiosis,dyad,tetrad,one nucleus,two nucleus,and three nucleus(mature pollen).By analyzing the expression patterns of DMP9 homologus genes in Chinese cabbage,it was found that the two genes in Chiifu had highly similar expression patterns to At DMP9,while two genes of LF subgenome which were tandem duplication in B9008 were significantly differentiated,and one of them was completely similar to At DMP9.Differently,the expression of copies of MF1 subgenome in the two Chinese cabbages was significantly higher than that in LF,and it was predicted that the DMP9 homologous gene of MF1 subgenome played a major role.4.In this study,the method of secondary mutagenesis of B.rapa B9008 germinated seeds by EMS was used to create a B.rapa mutant library,which with relatively saturated mutations and high density.And 4 lines containing stop gained mutations were screened for two genes of B9008 with haploid-inducing potential,and the results laid a foundation for further obtaining Brassica rapa inducer lines.