| Rhododendron dauricum L.,a semi-evergreen shrub of Rhododendron L,which is a characteristic flower of extreme cold resistance in northeast China.It blooms in early spring with bright colors and high ornamental value.It can be used as cut flowers and has high medicinal value,making it an extremely important economic plant resource in Northeast China.While the Greater and Lesser Khingan mark the northernmost boundaries of native R.dauricum populations,R.dauricum is the dominant species in the undergrowth of coniferous forests or mixed broadleaf and coniferous forests in the cold temperate zone.R.dauricum population is a key factor for maintaining the stability of the fragile ecosystem in the region.Due to factors such as climate change and human interference,the diversity of R.dauricum is also undergoing dynamic changes.At present,little is known about diversity status of R.dauricum in the region,which leads to a lack of formulate data-based strategies for its protection and conservation in the region.Therefore,a field survey and relevant data collection was performed in this study to comprehensively profile the diversity characteristics of R.dauricum habitats in the Greater and Lesser Khingan Mountains;Specifically,the genetic diversity of 13 populations was assessed by associating phenotype traits and SSR molecular markers.Populations rich in genetic diversity were identified and recommendations were made for conservation efforts.Furthermore,R.dauricum and R.dauricum var.album were selected to dissect the molecular mechanisms underlying flower color polymorphisms via integrated metabolomics and transcriptomics analysis.Comparative analysis revealed key genes in regulating flower color,which were further validated experimentally.Major outcomes of this project were:(1)Field survey showed that R.dauricum is mostly distributed in a sheet-like manner,with a variety of habitat types,including coniferous forest,coniferous and broad-leaved mixed forest,and volcanic rocks,of which the coniferous forest area in the middle of the Greater Khingan is the most typical.Comparative analyses were performed on nine phenotypic traits including plant height,ground diameter,number of branches,flower diameter,flower quantity,flower color,leaf length,leaf width,and leaf aspect ratio in 13 natural populations.Significant variations were found in both intra-and inter-population comparisons.Traits analysis of variance dispersion that showed the most significant variations were flower color and flower number.Taking advantage of the natural variations,we also created two new cultivars: ‘Ao Xue’ and ‘Yanricai’.The largest variation ranges were found in Huzhong and Hongxing with average CV of 0.1838 and 0.1799,respectively.The UPGMA method clustered the 13 populations into three major groups and 6 subgroups.(2)Polymorphism was found for 9 pairs of primers,and 25 alleles were obtained.The genetic diversity analysis of 13 populations of R.dauricum showed that a Shannon information index(I)of 0.6359,polymorphism information index(PIC)of 0.3460,and genetic diversity index(Nei’s)of 0.3575.In addition,the observed heterozygosity(Ho)was 0.2514,which was lower than 0.3722 the expected heterozygosity(He),indicating a certain degree of inbreeding within the population.The average genetic differentiation coefficient(Fst)was 0.6556,suggesting that 65.56% of the variation can be attributed to inter-population variation and the remaining 34.44% can be explained by intra-population variation.Moreover,a high genetic diversity was observed in Huzhong,Tahe,and Hongxing populations,making them top candidates for conservation efforts.The NJ method was used for clustering analysis,the 13 populations are clustered into three major groups and 8 subgroups.Compared with morphological clustering,it has high consistency within the subgroup and is basically consistent with geographic distance;In large groups,there is an inconsistency phenomenon with morphological clustering,but both of the clustering are not entirely based on geographical distance.Analyze the association between polymorphic loci and phenotypic traits,two potential SSR markers with high correlation for flower color and leaf aspect ratio were obtained.(3)Focusing on the flower color polymorphism within the population,the flavonoid metabolome of R.dauricum and R.dauricum var.album was detected,170 metabolites were obtained,of which 25 were significantly different.The corolla of both flower color groups contains anthocyanidin,and six unique flavonoid metabolites were accumulated in R.dauricum,as well as cyanidins,delphinidins and medicinal compounds including farrerol,naringenin,and their derivatives were accumulated at high levels.Quercetin-like flavonols were highly expressed in R.dauricum var.album.After transcriptome sequencing,KEGG enrichment analysis found that the flavonoid biosynthesis pathway enriched 157 differentially expressed genes,of which 99 were up-regulated and 58 were down regulated.Among them,structural genes involved in flavonoid biosynthesis was generally up-regulated,including CHS/CHI that encode rate-limiting enzymes in the early stage of anthocyanin synthesis and the downstream F3H/F3’H/F3’5’H/DFR/ANS/UFGT,which promote the synthesis of anthocyanidin.Conversely,these genes were downregulated in R.dauricum var.album,the upregulation of FLS expression promoted the flavonol metabolism pathway of R.dauricum var.album,affects the synthesis of anthocyanidin,and is an important factor in the production of white flowers.(4)According to the analysis of transcriptome about differential transcription factors,revealed MYB has the highest frequency,83 differential MYB transcription factors(TFs)including 61 R2R3-MYBs;By functional clustering with published flower color genes of related and model plants,16 MYB TFs related to flavonoids were identified in the MYB subgroups.Conjoint analysis of transcriptome and metabolome,5 negative regulatory TFs and4 positive regulatory TFs of anthocyanidin were obtained.NCBI was used for sequence alignment and functional analysis of homologous proteins,and MYBA and MYB12 were considered to be the key transcription factors producing the difference of flower color between R.dauricum and R.dauricum var.album.(5)RdMYBA(OQ633005)and RdMYB12(OQ633006)were cloned.The results of online bioinformatics analysis show that: the ORFs of RdMYBA and RdMYB12 are 780 bp(encoding a protein of 259 amino acid residues)and 1356 bp(encoding a protein of 451 amino acid residues),respectively.While RdMYBA shares 93% sequence identity with DUH024860.1 in the Rhododendron reference Genome Database,RdMYB12 shows a sequence similarity of 79%to DUH012062.1.In addition,both proteins are localized to the nucleus and cytoplasmic proteins.Further phylogenetic and motif analysis showed that the two genes differ in motifs and belong to distinct evolutionary branches,indicating divergence in gene function.The plant overexpression vectors GV1300-RdMYBA-GFP and GV1300-RdMYB12-GFP were constructed and transformed into tobacco by leaf disc method.It was found that Anthocyanidin was significantly accumulated in transgenic tobacco plants,suggesting that RdMYBA was the key gene for anthocyanidin synthesis in R.dauricum;Transgenic tobacco of RdMYB12 resulted in lighter flower color.Real time fluorescence quantitative detection found that Nt FLS was significantly upregulated,which hindered anthocyanin synthesis and was consistent with the phenotype of R.dauricum var.album.Constructed a color regulation mode diagram for R.dauricum and R.dauricum var.album. |
