Identification Of Genetic Elements Related To Tanshinone Biosynthesis Based On Genome-wide Association,Natural Antisense Transcripts And Circular RNAs Analysis

Salvia miltiorrhiza Bunge is one of the most important traditional medicines from the Lamiaceae family,Salvia genus.Tanshinones is the diterpene quinone compounds extracted from the root.It is one of the major active compounds in S.miltiorrhiza and used to treat coronary disease and cerebrovascular disease.Due to the important medicinal and economic value of tanshinones,its biosynthesis and regulation mechanism has always been a focus of research.In this study,we identified the candidate genes of the biosynthetic pathway of tanshinones by metabolite-based genome-wide association study(mGWAS)and identified the candidate natural antisense transcripts(NATs)and circular RNAs(circRNAs)by RNA-seq data.The main results are as follows:(1)The whole genome resequencing of S.miltiorrhiza population.In this study,a total of 383 S.miltiorrhiza samples were collected and subjected to whole genome resequencing.A total of 7,179,232 high-quality single-nucleotide polymorphisms(SNPs)and 881,892 small insertions or deletions(indels)were found by mapping the reads to the reference genome.Based on the population structure,phylogenetic and PCA analysis,the S.miltiorrhiza population was divided into three subgroups.The kinship analysis showed the distant relationship of most of the samples.The linkage disequilibrium(LD)analysis showed the high genetic diversity of S.miltiorrhiza.(2)The metabolome of S.miltiorrhiza population.In this study,the metabolome of 383 S.miltiorrhiza sample were analyzed.A total of 120 metabolites were detected,30 of which were tanshinones.Tanshinones have varying degrees of content differences in S.miltiorrhiza population with a coefficient of variation ranging from 0.47 to 2.86.(3)The genome-wide association study of tanshinones.The significantly associated loci related to 8 tanshinones were identified by the mGWAS.The 8 tanshinones were salvinone,tanshinone I,1,2-dihydrotanshiquinone,dehydromiltirone,neocryptotanshinone,neocryptotanshinone II,tanshinone ⅡB,and tanshinone ⅡA.Based on reference genome,LD analysis of significantly associated SNPs,homologous gene analysis and transcriptome,we systematically analyzed the cytochrome P450(CYP450)genes and transcription factors(TF)that may be involved in the synthesis of diterpenoids.As a result,two CYP450 genes and two TFs related to the biosynthesis of tanshinone IIA,two TFs related to the biosynthesis of tanshinone ⅡB,two CYP450 genes and one TF related to the biosynthesis of salvinone,one CYP450 gene and one TF related to the biosynthesis of 1,2-dihydrotanshiquinone,two TFs related to the biosynthesis of neocryptotanshinone were identified.(4)The identification,validation and function analysis of NATs in S.miltiorrhiza.In this study,a bioinformatic pipeline was developed to identify the NATs from RNA-seq data.We identified 812 NATs,including 168 cis-NATs and 644 trans-NATs from root,flower,and leaf samples of S.miltiorrhiza using ssRNA-seq.The expression profiles for 82%of 25 pair NATs and their sense transcripts(STs)obtained from ssRNA-Seq were validated by qRT-PCR.The expression profiles of 17 NATs positively correlated with those of STs.Based on the differential expression of NATs,the expressed correlation of NATs and STs and the functional annotation of STs,NAT0001 that may have a regulatory effect on the SmKSL1 gene was identified.(5)The identification,validation and function analysis of circRNAs in S.miltiorrhiza.In this study,we identified a total of 2,476 circRNAs with at least two junction reads using software tools circRNA_finder and CIRI,of which 85%,6%,and 9%were exonic,intronic and intergenic circRNAs,respectively.A total of 31 predicted circRNAs were validated successfully by PCR and Sanger sequencing using convergent and divergent primer pairs.Among the validated circRNA,20 circRNAs and their parental genes have significantly positive correlations.Based on the differential expression of NATs,the expressed correlation of NATs and STs and the functional annotation of STs,we identified four circRNAs originated from the gene related to the growth and development of S.miltiorrhiza,and three circRNAs originated from the gene involved in the biosynthetic pathway of tanshinones.In summary,this study genome-widly identified the genetic elements related to tanshinone biosynthesis based on mGWAS,NATs and cirRNAs analysis,which will provide useful information for the study of tanshinone biosynthesis and regulation mechanism,and will be used for the selective breeding of excellent varieties.