Whole Genome Sequencing And Functional Identification Of Important Terpenoid Synthase Genes In Artemisia Argyi

Artemisia argyi Lévl.et Vant.,a perennial Artemisia herb in the Asteraceae family.The dried leaf of A.argyi is a famous traditional Chinese medicine,known as"Chinese mugwort",which has a long history of application in medicine,health care,dietary therapy,folk customs,and other fields.Modern pharmacological studies indicate that the abundance of volatile terpenoids in A.argyi is one of the most important pharmacological active substances,but its complex biosynthetic mechanism is still unclear.In addition,A.argyi is one of the well-known and wide-distributed species among the Artemisia genus and has a relatively unique chromosome base(n=17),while its karyotype evolution history has not been analyzed and fully verified.In this study,we assembled a high-quality chromosome-scale genome of A.argyi to reveal the characteristics and evolutionary history of A.argyi genome.Besides,we combined genomics,transcriptomics and metabolomics to underpin the diversification of biosynthetic pathways producing major terpenes in A.argyi.Finally,the key genes encoding the enzymes functioning in the formation of important terpenes were discovered and functionally characterized.The main research results are as follows:1.A high-quality chromosome-level genome of A.argyi was achieved.Using the sequencing method combining Illumina,PacBio,and Hi-C techniques,A.argyi genome containing 17 pseudochromosomes was obtained with a total length of 3.89 Gb(contig N50 of 1.50 Mb,scaffold N50 of 214 Mb).Analysis with BUSCO and CEGMA estimated the genome completeness was up to 95.38%and 94.97%.The LTR assembly index of the genome assembly was 18.65.Collectively,these results demonstrate high quality of the A.argyi genome assembly.In addition,a total of 62,844 protein-coding genes,3.16 Gb of repetitive elements and 5,567 noncoding RNA were predicted in the A.argyi genome.2.The genome evolution of A.argyi was elucidated.Based on the phylogenetic tree of A.argyi and 11 other plant species,A.argyi clustered with A.annua,and they were most closely related to C.nankingense.The divergence time between A.argyi and A.annua was estimated to be 5.4 million years ago.Collinearity analysis and the distribution of Ks revealed that A.argyi underwent a recent lineagespecific whole genome duplication(WGD)event at 3.3 Mya after divergence from A.annua.Then two ancestor chromosomes fused to Chr 10,and finally formed a distinctive karyotype of A.argyi(2n=34).3.Discuss the molecular mechanism underlying the formation of diverse volatile terpenoids in A.argyi.According to phylogenetic relationship,we explore the lineage-specific expansion and contraction of the terpenoid synthase(TPS)gene family in A.argyi.Compared with H.annuus and A.annua,the common ancestral genes of A.argyi occurred more expansion and less lost,especially for TPS-a and TPS-b subfamilies,which contributed to the largest number of TPS genes in A.argyi.Based on the analysis of gene duplication type,almost all TPS genes originated from WGD event,which maybe the most important contributor leading to the diversity of volatile terpenoids in A.argyi.4.The key genes encoding the enzymes functioning in the formation of Germanene and Borneol were characterized.A tandem duplication gene cluster of germanene D synthase was found on Chr17 of A.argyi genome.The amino acid sequence similarity of this gene cluster was as high as 83%.However,the functional assays showed that only AarTPS114 catalyzed farnesyl pyrophosphate to produce Germacrene D,while other tandem duplication genes produced a small amount of Nerolidol and Farnesol.This indicated that duplicated genes have undergone functional differentiation.A single-copy AarTPS34 gene encoding germanene A synthase was also identified in A.argyi genome.In addition,a total of 8 borneol diphosphate synthase genes were identified in the A.argyi genome based on sequence similarity and phylogenetic analyses.One AarTPS89 produced(+)-borneol as a single product from geranyl pyrophosphate in vitro.Thus,the entire biosynthetic pathway of Germanene and Borneol was characterized in A.argyi.