Phenotypic And Genetic Effects In Brassica Rapa And Brassica Oleracea Autotetraploids And Trisomics Of B.rapa

Genome polyploidy is an important driving force for speciation and evolution.It is estimated that more than 75% of angiosperms are polyploids,including many important crops such as rapeseed,wheat,cotton,potato and so on.Synthetic polyploids and their derived aneuploids are important materials for genetic and breeding research.Brassica rapa(AA,2n=20)and B.oleracea(CC,2n=18)are crucial vegetables and oil-producing crops in China and worldwide.To date,development,cytology and gene expression studies of autotetraploid of B.rapa and B.oleracea are not conducted systematically.In this study,B.rapa and B.oleracea autotetraploids were developed in vitro and their phenotypic,cytogenetic,and transcriptomic changes were investigated and compared.Moreover,we developed several primary trisomics of B.rapa by continuous back-crossing between triploid and diploid and observed their phenotypic and cytogenetic expressions compared to diploid.1.Different phenotypic,cytogenetic,and transcriptomic changes induced by autopolyploidization in B.rapa and B.oleraceaThe seeds of B.rapa(cv.S9088)and B.oleracea(cv.Bo-1)were germinated in MS medium and young seedlings were treated with colchicine with plant hormone for in vitro development of tetraploid.We observed the phenotypic change between two tetraploids and found more significant morphologic and phenotypic changes in B.rapa tetraploid than B.oleracea compared to respective diploids.Both vegetative and reproductive organs of enlarged size were observed for the autotetraploid of B.rapa.In sharp contrast,the B.oleracea autotetraploid showed reduced organ size except for the seeds.We have successfully found B.rapa tetraploid which chromosome number is40,where diploid is 20;and B.oleracea tetraploid which chromosome number is 36,where diploid is 18.We noticed remarkable chromosome behavioural changes during meiosis of tetraploid.We found an average of 11.77±2.85;range 17-5 bivalent(II)pairing during meiosis in B.rapa tetraploid whereas only 5.36±2.291;range 12-2bivalent pairing(II)in tetraploid of B.oleracea.Chromosome segregation of B.rapa tetraploid follows three patterns: 20:20;19:21 and 18:22 but a high percentage(38.09%)of cells follow 20:20 segregation.On the other hand,B.oleracea tetraploid also follows three patterns: 18:18;19:17 and 20:16 but a high percentage(45.09%)of cells follow 17:19 segregation.We found 2085 differentially expressed genes(DEGs)in B.rapa tetraploid and 241 DEGs in B.oleracea tetraploid by silique wall(SW)RNA-seq analysis.Several cell wall biosynthesis-related genes were significantly upregulated in tetraploid B.rapa which is consistent with the enlarged siliques.On the other hand,two FLOWERING LOCUS T(FT)genes were strongly downregulated in the silique wall of B.oleracea tetraploid which is associated with the late flowering of this tetraploid.In conclusion,these findings indicated autopolyploidization consequence on organ size and gene expression in Brassica was species-specific and organ-specific which might be under genetic control related to the genome structure and composition.Genome re-sequencing of the parental lines revealed that the heterozygosity of B.rapa and B.oleracea genomes were 0.32% and 0.74% respectively.The corresponding SNPs of 1.45% and 2.28% were identified in the two genomes indicating that the heterozygosity of the B.oleracea genome was about twice that of B.rapa.The distribution analysis of DEGs and SNPs on chromosomes showed that there was no significant association between DEGs and SNPs changes.From this result,we could conclude that genome heterozygosity may not be the only reason for the different phenotypic and genetic effects of these two genomes after autopolyploidization.2.Phenotypic and cytogenetic expressions in primary trisomics of B.rapaPrimary trisomics of B.rapa has one additional chromosome(2n=21)in the diploid parent which has significant genetic and phenotypic effects.During continuous backcrosses with male×female or vice versa,a group of aneuploids produced which allow transmission of one additional chromosome with genes and genetic linkage groups.It will be helpful to locate specific genes or conventional linkage groups in a particular chromosome of an additional line.This study enables the development of four different primary trisomics of B.rapa(A4,A5,A7,and A9) by the continuous backcrossing of triploid as female and diploid as a male parent or vice versa.We observed the transmission rate of n+1 gamete was higher when diploid used as a pollen donor(male parent)and triploid as an accepter(female parent).The number of seeds per silique of trisomics plants was only 5-10% of that of normal diploid plants,and only 41.67-50% of mature seeds could germinate.The phenotypic,morphologic,and cytogenetic expressions of trisomics were significantly different from each other because of the effects of additional chromosomes and also chromosomal dosages balance.Additional chromosomes remained unpaired in most of the trisomics cells(>90%)and sometimes formed trivalent(<10%)at the metaphase stage.In conclusion,this study revealed the different phenotype and gene expression changes between B.rapa and B.oleracea after autotetraploidization which are species-specific and organ-specific could conclude that the heterozygosity of two genomes was not the only cause of these different changes.Meanwhile,four primary trisomics of Brassica rapa were developed and confirmed by phenotypic,cytogenetic,and genome re-sequencing analysis which laid a foundation for the genetic and epigenetic studies of Brassica trisomics in the future.