Molecular Markers And Rna-seq Of Atractylodes Chinensis And Atractylodes Japonica

Objective: To investigate the kinship between Atractylodes chinensis(Bunge)Koidz and Atractylodes japonica Koidz.ex Kitam by DNA barcode identification;to find a means to better differentiate the two by using molecular marker methods;to investigate the reasons for the difference in the content of the active ingredient Atractylon by RNA-seq combined with metabolomics analysis,and to find the link between the differential genes and differential metabolites in the key pathways.Methods:(1)The ITS2 sequences of DNA barcoding technology were selected as amplification primers,and sequenced sequences were compared with ITS2 sequences of the same genus on Genbank by MEGA to analyze base variation sites,genetic distances,establish phylogenetic trees,and predict the secondary structure of ITS2 sequences at the same time.(2)Molecular markers were selected by SRAP,ISSR and DAMD methods and specific bands were recovered and purified.(3)Combined RNA-seq and metabolomics analyses was performed to explore the differential genes,differential metabolites,and co-enrichment pathways between A.chinensis and A.japonica,and to investigate the possible reasons for the differences in the content of Atractylon between the two.Results:(1)The PCR amplification,sequencing success rate and sequence acquisition rate of all experimental samples for DNA barcoding were 100%,the PCR products showed good electrophoresis results,and the data results showed significant interspecific variation between A.chinensis and A.japonica.A total of 198 pairs of primer were screened for PCR amplification between A.chinensis and A.japonica,and 7 pairs of SCAR primers were designed for stable identification of the two,including 1 pair of primers converted from SRAP,3 pairs from ISSR and 3 pairs from DAMD.(2)The RNA-seq results showed that a total of 84109 genes were detected in A.chinensis and A.japonica,with an average length of 456 bp,of which the shortest was201 bp and the longest was 15176 bp,with high fragment assembly integrity.A total of 84109 functional annotations were obtained,with 40179 annotations to the Nr database(47.77%)and 23948 annotations to the Swiss-prot database(28.47%).(3)A total of 10034 differentially expressed genes were obtained by RNA-seq,with 4903 up-regulated genes and 5131 down-regulated genes in A.japonica compared with A.chinensis.Those related to phytoalexin synthesis included AUX,IAA and ARF,and those related to Atractylon synthesis included ACS,ACC,PAL and NOS,while the number of similar differentially expressed genes was less in the comparative group of rhizome and leaf of A.chinensis.(4)The results of metabolomics principal component analysis showed that both groups were more clustered within the group,and the two groups were farther apart.There were a total of 171 differential metabolites in the A.chinensis and A.japonica rhizomes,of which 111 were up-regulated and 60 were down-regulated.The most classified were lipids and lipid-like molecules,and the least were alkaloids and their derivatives.KEGG enrichment analysis showed that the differential metabolites were involved in a total of 34 pathways,with the main enrichment pathways being metabolic pathways,amino acid biosynthesis,and 2-oxocarboxylic acid metabolism.(5)The results of the combined analysis showed that KEGG enriched a total of94 pathways,with only 128 differential genes enriched and only 37 differential metabolites enriched.The largest number of co-enrichment was in metabolic pathways,and genes and metabolites related to phytohormones were co-enriched in phytohormone signaling pathways.Among them,salicylic acid content was related to the endophytic fungal inducer-mediated ability,while differential expression of TGAL4 and DOGL3 transcription factors within the pathway affected the accumulation of Atractylon.Conclusions:(1)The interspecific variation between A.chinensis and A.japonica species is evident and ITS2 sequences can be used for the identification of A.chinensis.(2)A total of seven pairs of SCAR primers were designed and developed for efficient and stable identification of A.chinensis and A.japonica,among which SCAR-2,SCAR-3,SCAR-4,SCAR-6,SCAR-7 could identify A.chinensis,SCAR-1and SCAR-5 could identify A.japonica.(3)The results of the combined RNA-seq and metabolomics analysis reasonably suggested that the low salicylic acid content in A.chinensis and A.japonica plants would lead to their poor ability to mediate endophytic fungal inducers,resulting in the down-regulation of TGAL4 transcription factor expression and up-regulation of DOGL3 transcription factor expression in Atractylode cells,which in turn would affect the accumulation of Atractylon.