QTL Mapping Of Active Ingredient Contents Of Salvia Miltiorrhiza And Creation Of New Material For Male Sterility

Salvia miltiorrhiza Bunge is not only a traditional Chinese herbal medicine,but also an important raw material source for health care products and beauty industry.It has important economic value and extensive market demand.The active ingredients of S.miltiorrhiza are the basis for its efficacy in treatment,health care,and beauty maintenance.The content of active ingredient is also one of the important indicators to evaluate the quality of S.miltiorrhiza.The analysis of the biosynthesis pathway and regulatory mechanism of active ingredients of S.miltiorrhiza,and the discovery and verification of related functional genes are the important prerequisite for improving the contents of active ingredients and synthesizing active ingredients.In this study,a high-density map of S.miltiorrhiza was constructed using SNP markers developed based on GBS technology from an F₂ mapping population.Furthermore,QTL mapping was carried out based on the contents of main active components and the candidate genes involved in the biosynthesis of active components were explored.The results are not only helpful for the map-based cloning and functional verification of target genes,but also lay a theoretical foundation for further analyzing the biosynthesis pathway and regulatory mechanism of active components of S.miltiorrhiza and provide a reference for molecular marker-assisted breeding in the future.In the process of constructing the mapping population,a new material of cytoplasmic male sterility（CMS）was created and identified,and its sterility mechanism was initially explored.The main conclusions are shown as follows:1.Hybridization was performed with S.miltiorrhiza f.alba（white flower,BH）and S.miltiorrhiza（dark purple flower,ZH）as the parents.A F₂population with 214 individuals obtained from BH×ZH F₁ was constructed as the mapping population of genetic map construction.The parental lines and F₂population were sequenced using GBS and yielded about 177.37 Gb valid data.Among them,the valid data for BH and ZH were 20.84 Gb and18.68 Gb,respectively.With regard to BH and ZH,94.62%and 94.41%reads were mapped to the reference genome,respectively.The valid data of individuals in the F₂population ranged from 0.21 Gb to 2.43 Gb,and the average was 0.59 Gb.Between 89.00%and 95.34%of the reads in the F₂ individuals were mapped to the reference genome,with an average mapping rate of 93.58%.Based on the GBS results,the homozygous SNPs（aa×bb）were screened for further analysis.2,750 candidate SNPs were obtained,which were then used for genetic linkage map construction.After excluding SNP markers with similar loci,605bin markers out of the 2,750 SNPs were mapped to eight linkage groups（LG）spanning a cumulative length of 738.2 c M.The average distance between two markers was 1.22 c M.2.Through flower color statistics and cluster analysis of the parents,F₁ and F₂ progeny,it is indicated that the inheritance of S.miltiorrhiza flower color is a quantitative trait controlled by two loci of major genes.Based on the constructed genetic map and the R,G,B and R+G+B values,two QTLs were detected for flower color,located on LG4 and LG5,respectively.Two candidate genes regulating the anthocyanin biosynthesis process were identified from the QTL mapping intervals on LG4 and LG5,namely,the transcription factor TCP15 and the chalcone isomerase 1 gene（CHI1）.These two genes may play a key role in the formation of white flower of S.miltiorrhiza.Both QTL mapping results and classical genetic analysis results showed that the flower traits of S.miltiorrhiza were controlled by two loci of major genes.This also indirectly confirms that the genetic map constructed in this study was accurate and reliable.3.The contents of rosminic acid（RA）,salvianolic acid B（SAB）,cryptotanshinone（CTS）,tanshinone I（Tan I）,and tanshinone IIA（Tan IIA）of parents and F₂ population were determined using high-performance liquid chromatography.Under three different planting environments,the content of each component showed obvious super-parent phenomenon in the F₂population,and the distribution frequency basically conformed to the normal distribution.In three environments,the contents of RA and SAB showed extremely significant positive correlations,and the contents of three tanshinones（CTS,Tan I and Tan IIA）also showed extremely significant positive correlations.Composite interval mapping method was used to perform QTL analysis for the active component contents of S.miltiorrhiza in three environments,and a total of 48 QTLs were detected.These QTLs were distributed on eight LG.The likelihood of odds value of the identified QTLs ranged from3.98 to 33.37,and the phenotypic variance explained by individual QTL was between 3.11%and 44.69%.Of the 48 QTLs,13,8,10,7,and 10 QTLs were associated with RA,SAB,CTS,Tan I,and Tan IIA,respectively.Every active ingredient had at least one stable QTL loci that were simultaneously detected in three environments.4.Candidate genes were predicted according to the overlapping or partially overlapping QTL localization intervals of each active component detected in three environments,and their regulatory mechanisms were analyzed.77 candidate genes were identified.It is speculated that 20 candidate genes are involved in the regulation of the biosynthesis of phenolic acids and tanshinones.These genes involved the primary metabolic pathway（PK,PK1,PDβ,DLD,RPI4,SCo AL,and SCo ALβ）and the branching pathways of phenolic acid biosynthesis pathway（TDCOSY,POD7,CHI3,TAT3,and HST）and terpenoid biosynthesis pathway（GPPS SSU（contained two genes）,DSR24,ERG24,BSS1,LCYB,GA2ox11,and CPS）.Because the expression level differences of related enzyme genes in primary metabolic pathways may result in unbalanced distribution of glucose metabolites,this may regulate the substrate concentrations of secondary metabolic pathways and further affect the contents of secondary metabolites.The relevant enzyme genes in the phenylpropane and terpenoid metabolic pathways may affect the contents of phenolic acids and tanshinones through participating in the redistribution competition of secondary metabolic flows by regulating the biosynthetic process of the corresponding branching pathways.5.A new material of male sterility was created through forward and reverse hybridization with BH and ZH as the parents.It was verified as CMS by backcrossing.Because the traits of male sterile plants are stable,a male sterile line of nuclear-cytoplasmic interaction had been constructed.The paraffin sections showed that sterility mainly occurred at the tetrad stage of microspore development.Anthers at the tetrad stage of SW-S and fertile control SW-F were selected for comparative transcriptome analysis.In total,2,571differentially expressed genes（DEGs）were obtained.Gene Ontology enrichment analysis indicated that 18 DEGs involved in microgametogenesis and developmental maturation were downregulated in SW-S.Among these genes,the callose synthase 5 gene（Cals5）is the most closely related to the male sterility.It is speculated that downregulation of Cals5 resulted in a decrease in callose around the tetrad,which is not conducive for separation and exine formation of the developing microspores,and further leading to pollen sterility.