Mechanism Analysis Of Double Haploid Induction System In Rapeseed

Rape belongs to Brassica napus of Cruciferae,and its heterosis is very obvious.Genetically stable homozygous inbred lines are essential materials in the utilization of rape hybrids.The acquisition of a large number of homozygous inbred lines can broaden rape germplasm resources and promote rape breeding.A synthetic polyploid material Y3380（rape dihaploid induction line）can produce homozygous bihaploid progeny by crossing with female parent rape using its pollen as donor,but its induction mechanism is not clear.In this experiment,Po lima C ytoplasmic male sterile B.napus 0068A,Common homozygous B.juncea 3496 and B.napus hybrid F₁ 4120 were selected as female parents,and Y3380-cp4,a dihaploid induction line containing glyphosate resistance gene cp4-EPSPS,was used as male parent to induce F₁.Through morphological observation,marker gene tracking,fluorescence in situ hybridization and phenotypic observation,Flow ploidy analysis and SNP chip analysis were used to analyze the induction mechanism of dihaploid induction lines.The conc lusions are as follows:1.The morphological characteristics of the formation process of induced F₁ seeds were observed.the results showed that 12～23 days after pollination,some of the induced F₁ seeds began to appear epidermis whitening and chlorosis and glossiness decreased.The seed coat slightly sunken and other signs of death,and in the subsequent growth period,the epidermis became brown,dried and concave,and finally withered to death,and the embryo death intensified after 23 days of pollination.Finally,95.60%～96.47%of the induced F₁embryos developed abnormally after fertilization,and the average number of pods was only 2～5 seeds.2.On the 8th day after pollination,the expression of cp4-EPSPS marker gene of double haploid induction line in rape could be detected in induced F₁ seeds;during seed embryo development,the probability of inducing cp4-EPSPS gene in F₁ decreased from87.31%～97.27%to 0%～16.67%,showing a downward trend,and the decline rate in each time period had nothing to do with temperature changes and seed mortality,but related to maternal genotypes.The results of fluorescence in situ hybridization showed that the induced F₁ of B.juncea（AABB）did not contain the C genome segment of the induction line,but there wo uld be one more B genome chromosome or B genome translocation to 1～2 A genome chromosomes.The results of marker gene tracking and fluorescence in situ hybridization showed that the zygote was formed when the female egg was fertilized with the spermatozoa of the induction line.During the zygote development,the specific loss of the genome of the dihaploid induction line occurred,and the loss process would affect the replication of the maternal genome.and the loss of paternal chromosomes is related to maternal genotypes and the interaction of parental genes.3.The observation of botanical characteristics of induced F1 showed that when the female parent was hybrid F₁ 4120,the leaf shape and leaf color of induced F1 were separated,and when the female pa rent was homozygous lines 0068A and 3496,the plant shape of induced F₁ was similar to that of female parent.Hybrid polyploid plants with obvious leaf thickening and shrinkage appeared in the induced F₁ of heterozygous and homozygous female parents.The results of ploidy identification of induced F₁ by flow cytometry showed that induced F₁ had tetraploid（96.52%）and parental hybrid polyploid（3.48%）.The probability of inducing F₁ double haploid was related to the maternal genotype.4.SNP chip analysis confirmed that there were heterozygous plants and homozygous plants in induced F1,and heterozygous plants were divided into two kinds,one was parent hybrid polyploid,its genetic material was 29.88～35.49%from male parent,and the rate of homozygous locus was basically unchanged compared with the control.The second type is partial locus heterozygous tetraploid,whose genetic material is 2.78%～12.43%from the paternal source and 69.75%～88.34%from the maternal source.Compared with the control group,the homozygous locus rate is increased from 67.79%～67.86%to 84.06%～90.58%.There are three sources of genetic material for homozygous plants:（1）more than99%of the genetic material comes from the female parent.（2）96.13%～97.33%comes from the female parent,and 0.32%～0.79%comes from the induction line.（3）35.42%comes from the male parent.The results of SNP chip analysis showed that the dihaploid induction line of rapeseed could improve the homozygous rate of offspring,and according to the source of genetic material of homozygous plants,the chromosomes of the induction lines were lost first and the haploid genomes of the zygotes were doubled after the formation of zygotes,and the induction effect was related not only to the interaction of parents’genotypes,but also to the induction gene dose of the induction lines.To sum up,we speculate that the induction process of dihaploid in rapeseed may be to induce the line chromosomes to enter into the female egg cells and form zygotes through fertilization.During the process of zygote mitosis,the specific loss of paternal chromosomes occurs.During the loss process,some paternal genes may infiltrate into the maternal genome through homologous exchange or transposon jumping,and then the zygotic haploid genome doubles（EH phenomenon）.After doubling,the zygote continues to develop into a complete embryo and eventually forms bihaploid offspring.The induction rate of dihaploid was greatly affected by maternal genotype and male parent single plant induction line,while the mortality rate,effective pod rate and average effective seed setting number of induced F₁ seeds were mainly affected by maternal genotypes.