A Comparative Analysis And Codon Usage Bias Of Complete Plastid Genomes From Sium

Objectives:The genus Sium,belongs to the tribe Oenantheae of Apiaceae subfamily Apioideae,with currently only 10 species recognized.However,duo to its wide distribution and important phylogenetic status,it has aroused widespread concern.Pimpinella crispulifolia was established by H.de Boissieu in 1909,its position is dubious as the type has no ripe fruits.In this study,the phylogenetic position of Pimpinella crispulifolia was determined by morphological and molecular analysis.Then,the complete chloroplast genomes of five Sium species were sequenced,characterized and compared,to analyze the global features and structural patterns of Sium chloroplast genomes,screen sequence divergence hotspot regions,analyze the codon usage bias and evaluate the efficacy of the whole chloroplast genome in resolving the relationships within the genus.This is the first comprehensive analysis on chloroplast genomes for the genus Sium,the results of which will provide genetic resources and molecular markers for future studies of this genus.Methods:(1)The phylogenetic position of Pimpinella crispulifolia:the new material was compared against the type specimen for morphological analysis,its ITS sequences were added into the large matrix of Zhou et al.(2020)to construct the ML tree.(2)The comparative analysis of complete plastid genomes from Sium:①assembling and annotation:raw sequence reads were quality trimmed using FastQC with default settings,with the remaining high-quality reads assembled in GetOrganelle.The chloroplast genomes were annotated using Geneious,and simultaneously counted the length,GC content,and gene composition of the whole genome and the IR,LSC and SSC regions.The circular plastid genome maps were drawn using the Organellar GenomeDRAW,and the IR/SC junction was illustrated by IRSCOPE.②Genome comparison and analysis:the chloroplast genomes were aligned by MAFFT,and mVISTA program was used to evaluate the variability of the complete chloroplast genome sequences under Shuffle-LAGAN mode,using S.suave 1 as a reference.The sliding window analysis was conducted using DnaSP to evaluate the nucleotide diversity(Pi),with a step size of 200 bp and a window length of 600 bp.③Phylogenomic analysis:Five chloroplast genomes of Oenantheae were downloaded from Genbank and added into the Sium matrix to construct the ML and BI trees,with Chamaesium paraxodum as the outgroup.④ Codon usage bias analysis:the proteincoding genes were extracted using Geneious from the annotated chloroplast genomes of Sium.Various codon indices were analyzed using DAMBE and CodonW,as well as the GC content of each gene,dinucleotide frequency and RSCU values.Relevant graphs were plotted with R ggplot,and correlation analysis metrics for gene expression and synonymous codon pairs were also calculated using the cor.test derived in R.Results:(1)The phylogenetic position of Pimpinella crispulifolia:Morphological similarity is reflected in its fascicled roots,simple pinnate leaves,long and reflexed styles,and ovoid,laterally compressed fruits with prominent and corky-thickened ribs;the phylogeny estimated showed that it allied strongly with Sium ventricosum of Oenantheae.(2)The comparative analysis of complete plastid genomes from Sium:①assembling and annotation:The Sium chloroplast genomes displayed a typical quadripartite structure including a pair of IRs(IRa and IRb),a LSC region and a SSC region,ranged in length from 153024bp(S.ventricosum)to 154929bp(S.tenue).They harbored 129 genes with the same arrangement order,including 84 protein-coding genes,37 tRNA genes,and 8 rRNA genes,while 16 genes were duplicated in the IR regions.②Genome comparison and analysis:The aligned entire chloroplast genomes are highly conserved.Except for S.ventricosum,the IR boundary regions of the remaining taxa showed no significant expansion or contraction.The sliding window analyses showed that a total of nine regions,including two gene spacers(ndhF-ndhD interval and rps15-ycf1 interval)and seven gene spacers(trnG-UCC,trnE-UUC,trnLUAA,pasl,rpl16,ndhA,ndhH).③Phylogenomic analysis:the phylogenies estimated using ML and BI analyses are well-resolved and highly consistent with one another.In all analyses,the monophyly of Sium was recovered with strong support.Clade Ⅰincluded S.suave,S.medium and S.tenue.The remaining species of the genus constituted the Clade Ⅱ,in which the two species endemic to China(S.ventricosum and S.crispulifolium)comprised a highly supported sister group relationship.Furthermore,the two accessions of S.suave from Taiwan and Xinjiang Provinces of China allied strongly as sister group.④Codon usage bias analysis:The nucleotide content showed no significant difference in the chloroplast genome of six Sium accessions,and the AU content of the third site of codons was significantly higher than the GC content.Dinucleotide frequency analysis suggested that differences in base content at different sites of codons may be related to the adjacent bases.Codons with RSCU>1 were all ended with A or U,while codons with RSCU<1 are mostly ended with G or C,with a few ended as A.Sium medium and S.tenue have four preferred codons,which were not found in S.ventricosum,and the remaining taxa have three preferred codons.The analysis of various gene parameters of the chloroplast genome(GC3,gene expression,protein length,amino acid hydrophobic and aromatic)on the ENC and RSCU values of codons showed that GC3 content is the main factor affecting its codon usage pattern,and the mutation pressure is the main evolutionary driving force of its codon preference.Conclusion:(1)The poorly known species Pimpinella crispulifolia should be transferred out of Pimpinella,and referred into the genus Sium as S.crispulifolium(H.de Boissieu)J.Zhou.(2)The plastomes of Sium are highly conserved in their structural organization,gene arrangement,gene content and GC content,but can provide sufficient information sites to resolve phylogenetic relationships and identify species.Furthermore,codon usage pattern was mainly affected by mutation pressure,while other factors such as selection and drift were also involved in this process.