Multiple Omics Analysis Unraveled Phylogenetic Relationships Among Rehmannia Species

Rehmannia is a unique distribution type in East Asia,mainly distributed in China,and the genus contains six species.The interspecies relationships previously obtained based on analysis of different gene fragments are contradictory,and there is no unified conclusion.Starting from the group level,we first comprehensively analyzed the chloroplast genome characteristics and mutation mechanism of Rehmannia,focusing on the analysis of codon usage preference patterns and RNA editing.Then comparatively analyze the chloroplast genome,mitochondrial genome,nuclear gene ITS sequence,single copy nuclear gene and simplified genomic data,provide a variety of computational analysis methods,and comprehensively explore the phylogenetic relationship of Rehmannia.The main results are as follows:(1)The chloroplast genome of Rehmannia is highly conserved,with a typical tetrad structure(LSC,SSC,IRa,IRb),with a total length of 153,477-154,407 bp,a GC content of 37.9-38%,and genome containing 133 genes.Although the chloroplast genome of Rehmannia plants is very conservative in structure and size,we have found different degrees of contraction and expansion at the border of the IR region.This expansion and contraction phenomenon exists between species within species.In the analysis of simple sequence repeat(SSR),we found that the most abundant is mononucleotide SSR(58.32%),the least is hexanucleotide SSR(0.21%).In the analysis of SSR type,we did not detect pentanucleotide SSR in R.chingii,and only detected hexanucleotide SSR in R.piasezkii.In the analysis of repetitive sequences,palindrome repeats(45.43%)are the most common type among all types of repeats,and the least common type of repeats are complementary repeats(0.53%),which only exist in tetraploid Rehmannia(R.solanifolia and R.glutinosa).Two coding regions with high variation(rps19 and rpl2)and four non-coding regions(psb Z-trn G-GCC,psa A-ycf3,cem A-pet A and psb E-pet L)can be used as potential DNA barcodes of Rehmannia in study.In the analysis of codon usage bias(CUB),there are many factors that affect codon usage bias, among which are mainly affected by base mutation and selection pressure.We preliminarily concluded that among photosynthesis-related genes,CUB is mainly affected by base mutations,and pyrimidine has a higher priority than purine in use;and for transcription-related genes,selection pressure plays a role,purine is more important than pyrimidine in use.There are about 20-22 RNA editing sites in the chloroplast genome of Rehmannia,which mainly occurs at the second position of the codon.ndh B gene with the most editing sites.Most RNA editing is the transition from hydrophilic amino acids to hydrophobic amino acids,which will greatly increase the hydrophobicity of the protein and make the structure more stable.In addition,we have successfully obtained the complete chloroplast genome from RNA-seq,and initially confirmed that the chloroplast seems to be fully transcribed in the organ and development stage of angiosperms.(2)Based on multi-omics data,we conducted a study on the developmental relationship of Rehmannia,and described its evolutionary history more completely.Among them,the development relationship based on organelle genome data considers that R.chingii is the original group of Rehmannia,and then R.henryi differentiated.The development relationship based on the nuclear genome data shows that R.chingii and R.henryi gather together.We speculate that the conflict between the results of organelle genome and nuclear genome data is due to the strong gene permeability of R.chingii to R.henry.This study not only provides a large amount of molecular data for the evolutionary relationship among the Rehmannia,but also can provide a reference for the phylogenetic analysis of Orobanchaceae.