Molecular Variant Mechanism Study Of Betula Pendula ’Purple Rain’ Green Mutation

Purple Rain Birch(Betula pendula ‘Purple Rain’)is a purple-leafed variant of Weeping Birch(Betula pendula Roth.),which is widely used in horticultural ornamental field because of its unique purple leaves and good growth adaptability.The research team found a green-leafed mutant of Purple Rain Birch in the previous micropropagation process of Purple Rain Birch,which has the same genetic background as the original strain and is a good material for studying leaf color trait mutation.In this study,the mutant and ordinary Purple Rain Birch were used as materials,and through multi-omics high-throughput sequencing and bioinformatics analysis methods,the causes of leaf color variation were explored,the key genes regulating anthocyanin synthesis pathway in Purple Rain Birch were mined,and genetic transformation of white birch was carried out to explore the molecular mechanism of leaf color formation in Purple Rain Birch.The results are as follows:(1)Metabolomic analysis showed that the anthocyanins accumulated in Purple Rain Birch were mainly cyanidin,delphinidin and petunidin.The anthocyanin content of green mutant was lower than that of Purple Rain Birch,but its growth rate was faster than that of Purple Rain Birch,and its young leaf color was close to that of Purple Rain Birch,with high anthocyanin accumulation.The purple color faded in mature leaves,and the color was closer to green leaves of white birch.(2)Transcriptomic analysis showed that there were 364 differentially expressed genes between them.174 genes were up-regulated and 190 genes were down-regulated in Purple Rain Birch relative to green mutant.The gene with the largest contribution to their difference was chalcone synthase(CHS)gene,which could be enriched into flavonoid metabolic pathway.(3)Gene family analysis showed that four R2R3-MYB family genes(Bp MYB113,Bp MYB114-1,Bp MYB114-2,Bp MYB4)and one b HLH family gene(Bp TT8)were related to anthocyanin synthesis pathway regulation.(4)Whole genome methylation analysis showed that obvious demethylation occurred at whole genome level in green mutant.Among them,methylation level increased in promoter region of Bp MYB113 gene and decreased in promoter region of Bp MYB114-1 gene.(5)Bp MYB113 overexpression vector was constructed.Mature seeds of white birch were used as receptors for genetic transformation by Agrobacterium-mediated method.PCR and fluorescence quantitative PCR techniques were used to detect resistance plants obtained from genetic transformation experiments.The results showed that Bp MYB113 overexpression white birch plants had been successfully obtained by genetic transformation experiments using Agrobacterium-mediated method.Compared with control group,transgenic plantlets with overexpression Bp MYB113 showed obvious purple-red color,transplanted seedlings had green leaves,fluorescence quantitative PCR analysis showed that expression levels of anthocyanin synthesis related genes increased at different degrees in transgenic strains.In summary,there are significant differences between Purple Rain Birch and green mutant at multiple dimensions such as phenotype,metabolism,transcription,methylation etc.Among them,Bp MYB113 acts as a transcriptional activator,which can promote expression of structural genes related to anthocyanin synthesis.Transgenic plantlets with overexpression Bp MYB113 have purple leaves.