Analysis Of The Whole Genome And Tepal Flavonoid Biosynthesis Pathway In The Red Flower Wintersweet

Wintersweet(Chimonanthus praecox)is a unique traditional famous flower and special economic tree species in China.It has important ornamental value,economic value,and profound cultural connotations.The color of wintersweet is mostly yellow or light yellow.The author has bred new cultivar of red flower wintersweet after years of efforts.However,its genetic background and flavonoid biosynthesis pathway are not clear,which seriously restricts the genetic improvement and industrial development of wintersweet.In this study,the whole genome of C.praecox’Hongyun’ was sequenced,assembled,and annotated;Through comparative genomics research,the evolutionary positions of magnoliids represented by wintersweet were analyzed;Through multi-omics data integration and verification,the key genes of flavonoid biosynthesis pathway in tepals of wintersweet were mined and the regulatory mechanism was studied,which laid a foundation for the genetic improvement of wintersweet and the development and utilization of flavonoids.The main results are as follows:(1)The red flower wintersweet genome was assembled and annotated at the chromosome level.Based on PacBio Sequel Ⅱ third-generation sequencing,secondgeneration sequencing data error correction and Hi-C assisted assembly,a highquality genome at chromosome level was obtained,with a size of 737.03 Mb and a conting N50 of 8.13 Mb.According to homology-based and de novo predictions,as well as transcriptome data,25832 protein-coding gene models were identified and predicted with an average coding sequence(CDS)length of 1256 bp and an average gene length of 9965 bp.Among these,24756(95.83%)of the predicted protein-coding genes could be mapped with functional annotations using public databases.Through a combination of homology-based searches and de novo prediction,466.24 Mb repetitive sequences were identified,accounting for 63.26%of the genome size.Among the repetitive sequences,DNA transposons,long interspersed nuclear elements,short interspersed nuclear elements,and long terminal repeats accounted for 9.02,7.13,0.31,and 45.86%of the genome size,respectively.We also identified 1723 non-coding RNAs.(2)The WGD events of C.praecox and other magnoliids were identified,and the phylogenetic position of magnoliids was reconstructed.The synteny and Ks analysis showed that red flower wintersweet experienced two WGD events,in which the ancient WGD event was shared by Laurales,driving the differentiation of Laurales into Calycanthaceae and Lauraceae,etc.;The young WGD event is shared by Calycanthaceae,which drives the differentiation of Calycanthaceae into C.praecox and C.salicifolia,etc.In addition,magnoliids did not share WGD events,and their branches Laurales,Magnoliales,and Piperales had experienced an independent WGD event respectively.The WGD events of Laurales and Magnoliales were relatively close.Previous studies of the evolutionary position of magnoliids showed inconsistent results possibly due to different tree-building methods,the existence of ILS,the limitation of taxon sampling,and the rapid differentiation of magnoliids in the early stages of evolution.This study fully considered various factors that may have caused errors in the evolutionary positioning of the magnoliids.Our data suggest that a bifurcating tree might not be able to fully demonstrate the early diversification of the angiosperms.We suggest that magnoliids are most likely sister to eudicots,which was supported by the results from several phylogenetic trees.(3)The key structural genes for the formation of red wintersweet tepals were screened.Based on the targeted detection of the UPLC-MS/MS platform,a total of 82 compounds were detected in tepals of different wintersweet varieties.Besides the main flavonol branch and anthocyanin branch,the flavone branch,isoflavone branch,and procyanidin branch may also be included in the flavonoid biosynthesis pathway of wintersweet tepals.Cyanidin glycosides are the characteristic metabolites of the red tepals in wintersweet,including cyanidin 3-O-glucoside,cyanidin 3-Orutinoside,and cyanidin 3-O-galactoside.Transcriptomic heat map analysis of anthocyanin metabolic pathway structural genes in tepals of different varieties,combined with KEGG annotation of different genes and metabolites of the flavonoid pathway in red and yellow tepals of different comparison groups,showed that the red tepals were mainly caused by the high expression of structure gene CpANS1.Combined with targeted metabonomic and transcriptomic analysis further confirmed that CpANS1 was positively correlated with anthocyanin glycosides,and was the key structural gene responsible for the red color of the tepals.(4)The key transcription factors regulating the anthocyanin metabolism pathway of wintersweet were explored.We isolated and cloned the CDSs of CpANSl and the corresponding promoter fragment from three different varieties of Concolor wintersweet,Patens wintersweet,and Rubrum wintersweet.Sequence analysis showed that the difference in promoter sequences or CDSs of CpANS1 was not the cause of flower color difference among these three varieties.Phylogenetic tree and WGCNA analysis showed that R2R3-MYB CpMYB1 of the S6 subfamily may be the key transcription factor regulating CpANS1 expression.The coding sequences of the CpMYBl in tepals of Concolor wintersweet were the deletion mutation type of 4 bases(AAAG),and the early termination of coding resulted in the loss of its regulatory function and termination of the anthocyanin metabolism pathway.The expression level of CpMYB1 is directly related to the red color of wintersweet tepals.(5)CpMYBl needs to form a MBW complex to fully perform its regulatory function.Two bHLH genes and two WD40 genes were obtained by homologous search in red flower wintersweet genome.A yeast two-hybrid experiment showed that MBW complex can be formed between CpbHLHl,CpWDR2 and CpMYB1.The GUS staining results showed that the CpMYB1-driven MBW complex had a strong activating activity on the CpANS1 promoter,while Cpmyb1E201 lost the ability to activate the CpANS1 promoter.We also conducted tobacco overexpression experiments to verify the function of CpMYB1.The results showed that neither CpMYB1 nor Cpmyb1E201 could deepen the color of tobacco leaves and calli,indicating that CpMYB1 may need to form a complex(MBW)with bHLH and WD40 in wintersweet to act its regulatory function.In conclusion,the red flower wintersweet genome was assembled and annotated at the chromosome level in this study,which provides a valuable resource for genome editing and molecular marker-assisted breeding.Comparative genomics identified the WGD events of C.praecox and other magnoliids and provides new insights into the evolution of the magnoliids.By integrating multiple omics data,the flavonoid biosynthesis pathway of red flower wintersweet was analyzed.The red color of the tepals was mainly attributed to CpANS1 and transcription factor CpMYB1,and CpMYBl needs to form a MBW complex to fully perform its regulatory function.