Genome-wide Association Mapping Study Underlying The Key Metabolites Of Chlorogenic Acid Biosynthesis Pathway In Populus Tomentosa

As the main component of phenols,chlorogenic acid(CGA)plays a crucial role in defense response,immune regulation,and the response to abiotic stress in plants.In forest trees,the enrichment CGA can significantly enhance the stress resistance and adaptability of plants,thereby improving the yield and quality of the wood.However,the biosynthesis of the CGA is controlled by a variety of enzymes and genes,and the genetic regulation network remains unclear.Therefore,the system analyzes the molecular genetic regulatory mechanism of CGA accumulation,which will greatly promote the process of molecular marker-assisted breeding and forest trees genetic improvement.Herein,this study quantitatively analyzed the 11 key metabolites in the biosynthesis pathway of CGA in 300 accessions of Populus tomentosa by high-throughput LC-MS/MS based on metabolic spectroscopy and explored their phenotypic variation patterns.Further we identified the key variation loci that affected the biosynthesis of CGA underlying the method of association genetics,and the gene regulatory network for the biosynthesis of CGA was also constructed.The main research results in this study as follows:(1)11 metabolites involved in CGA biosynthesis were determined by LC-MS/MS technology,and they have abundant phenotypic variation in the association population of P.tomentosa.Meanwhile,significant phenotypic differentiation was detected for the 11 biosynthesis of CGA related metabolic traits among the different geographic subpopulations.In addition,correlation analysis showed that CGA displayed an opposite correlation pattern with quinic acid(QA),and feruloylquinic acid(FQA).(2)The system genetic strategy,cooperating metabolite-based genome-wide association studies(mGWAS)and expression quantitative trait nucleotide(eQTN)mapping,to decipher the genetic architecture of CGA and analyze the additive and dominant genetic effects of candidate genetic marker loci.The results of the analysis showed that a total of 204 SNPs was significantly associated with 11 metabolic traits,explaining 13.22% of the phenotypic variation on average,corresponding to 206 genes of P.tomentosa,and were mainly involved in secondary metabolism and cell growth processes of P.tomentosa.Meanwhile,15 pleiotropic SNPs showed different additive and dominant effects on different metabolic traits.In the eQTN mapping,874 eQTNs were significantly associated with 109 expressed genes,representing 1066 P.tomentosa genes,and 17 eQTNs hotspot were also identified.The expression patterns and interaction mechanisms of these genes affected the accumulation of CGA biosynthesis related to metabolites.The 34 overlapping genes were detected in the results of mGWAS and eQTN,which could not only directly contribute to phenotypic variation at the genomic level,but also affect crucial metabolites of CGA biosynthesis by regulating the expression of ontology and other genes through the eQTN mapping.(3)Using the selective sweep analysis,16 GWAS signals and 78 eQTN loci were found in the selective sweep region of the P.tomentosa genome,showing significant allele frequency differences(≥ 20% allele frequency difference)in three geographical populations,which provided insights into the adaptation of CGA biosynthesis to the local environment.(4)Based on the multi-omics strategy,the two major genes RPL3 B and ERF109 that affect the biosynthesis of CGA were identified,and thus constructed an allele regulatory network underlying CGA biosynthesis.Finally,the transcriptional regulation mechanism in promoting the CGA accumulation and responding to the abiotic stress was verified in the drought-treatment population of P.tomentosa.This study provides an integrative strategy for investigating the genetic architecture of CGA metabolites in Populus,and lays a foundation for the breeding of high-resistance and excellent varieties of Populus.