The Impact Of Allopolyploid On Flavonoid Biosynthetic Pathway Genes In Brassica A/C Genome

Interspecies hybridization and allopolyploidization play an important role in the formation and evolution of plants.Allopolyploid's genome fusion which brings to the change of gene sequence,expression and the formation of new character have become the hotspots in the plant reseach field.Because it's parents origin are explicit,resynthesized B.napus has become an important plant to study species evolution.Mapping and cloning the genes that control the yellow seed character,and revealing the mechanisms underlying the formation of seed color,are very important for breeding of yellow seeded B.napus.We previously performed with the ancestral parents to form the resynthesized B.napus.In this study,we selected yellow-seeded B.rapa(AA,1151,2n=20),yellow-seeded B.oleracea(CC,T9-2,2n=18)and the stable resynthesized yellow-seeded(5027)and black-seeded(5042)B.napus strains as material.Homologue-sequence method were used to clone important genes of resynthesized B.napus and it's parent's in flavonoid metabolic pathway and RNA-Seq,Real-time PCR technologies were used to analysis the change of gene expression after A/C genome fusion.Our results are as follows:1.We cloned 24 genes in parents and 41 genes(25 genes came from A genome,16 genes came from genome)in resynthesized B.napus in flavonoid biosynthetic pathway.We analysed the sequences and found that about 58%genes have no change in sequences,they were exactly the same as those of parents.Gene sequence variation in resynthesized B.napus include fargment insertion,deletion and single nucleotide mutation,take the proportion of 21%,17%,34%.Fargment insertion,deletion and single nucleotide mutation happened in both exons and introns.Gene sequence changes in flavonoid biosynthetic pathway have bias on genomes.There were 11 and 6 genes change in A and C genome respectively,resynthesized B.napus slightly tend to retaine flavonoid biosynthetic pathway genes form C genome.2.The differences in flavonoid accumulation led to the formation of different colored seeds in resynthesized B.napus.3.RNA-seq analysis showed that the level of alternative splicing in resynthesized B.napus was lower than in the sum of two parent(reduced about 10%-15%).Participate gene number was similar in two parents,but in resynthesized B.napus,the participate gene number rise up 9%of A genome from B.rapa,declined 69%of C genome from B.oleracea.We analysised 57 genes in flavonoid biosynthetic pathway,36 genes expressed in both parents and resynthesized B.napus;1 gene expressed only in parents;6 genes expressed only in resynthesized B.napus;6 genes expressed only in black-seed resynthesized B.napus;whereas 5 genes only expressed in yellow-seed resynthesized B.napus;2 genes only expressed in black-seed resynthesized B.napus;2 genes neither expressed in parents nor resynthesized B.napus;The result singified that most genes expression decreased,rather than close one of them to replay heterologous genome impacts.4.When used B.napus genome as the reference genome,6830 DEGs were identified in yellow-seed and black-seed resynthesized B.napus.There were 3777 up-regulated genes and 3053 down-regulated genes.When used B.rapa and B.oleracea genome as the reference genome,resynthesized B.napus,4990 DEGs were identified.There were 2708 up-regulated genes and 2282 down-regulated genes.GO classification for DEGs showed that the genes were associated with cell,cell part and organelles according to cell components.In molecular biology functions,these genes were primarily related to enrichment,catalytic activity and transcription regulator.In biological processes,these genes were involved in cell processes,metabolic process and respons to stimulus.COG annotation and functional classification for these genes showed that these genes were associated with general function only,transcription,post-translational modifications and recombination and repair.5.we used the Real-time PCR to analysis the different genes expression in yellow/black resynthesized B.napus.The result showed that,compared to yellow resynthesized B.napus,genes in flavonoid biosynthetic pathway of black resynthesized B.napus are all up-regulated.Yellow or black seed colour formation,maybe not because of the genes mutation in flavonoid biosynthetic pathway,but the upstream metabolic pathways abnormality.4 pathways maybe closely related to it,include cyano-amino acid pathway and upstream pathways such as phenylpropane pathway,phenylpropane pathway and glutathione pathway which closely related to flavonoid biosynthetic pathway.47 presumed differentially expressed genes may be related to seed coat colour formation.