| Melilotus spp is an important and widely used leguminous forage with high yield and resistance to abiotic stress.However,the coumarin in Melilotus plants will be converted into dicoumarin which is toxic to livestock under the condition of high humidity and moldy.Therefore,it is particularly important to study the genetic diversity of the genus Melilotus and to explore the coumarin synthetic pathways and related key enzyme genes.In this paper,the genetic diversity of 18 species of genus Melilotus was studied by using ITS and matK sequences.The key enzyme genes in coumarin biosynthesis pathway were identified by combining Bulk Segregant Analysis(BSA)and untargeted metabolomics analysis,as well as RNA-seq data.This study will provide the foundation for breeding of new varieties with high-yield and low-coumarin content.The main results are as follows:1.Using the nuclear gene sequence ITS and the chloroplast sequence matK,the genetic diversity was analyzed for 621 accessions of 18 Melilotus species(mainly from the National Gene Bank of Forage Germplasm,NGBFG).The phylogenetic tree constructed by Bayesian method showed that ITS is more suitable for phylogeny relationships in the low classification level of Melilotus,compared with mat K.2.The key enzyme genes in coumarin biosynthesis pathway were identified by BSA method using the near-isogenic germplasms Ma46 and Ma49 with different coumarin content of Melilotus albus.The homemade whole genome sequence of Melilotus was used as the reference genome.Five hundred and forty genes were mapped in the candidate region,and 535 genes were annotated in the candidate region.Among them,124 non-synonymous mutant genes and 16 frameshift mutant genes were annotated.Eighteen genes that may be involved in the coumarin biosynthesis pathway were selected for qRT-PCR validation.The results showed that most of the genes were expressed higher in Ma49 than Ma46.3.Using Ma46 and Ma49,LC-QE-MC detection platform was used to identify the metabolites and metabolic pathways of the test materials,and five differential metabolites were identified.The hierarchical cluster analysis(HCA)of these differential metabolites showed that the expression level of coumarin was significantly higher in Ma49 than that of Ma46.Combined with the analysis of metabolome and transcriptome data,it was found that in the phenylalanine synthesis pathway,the differential metabolite coumarin was up-regulated and four peroxidase genes were up-regulated. |
