| Wintersweet(Chimonanthus praecox L.)belongs to the family Calycanthaceae,a family in the order Laurales residing within Magnoliopsidae.It is a famous flowering woody tree with more than one-thousand-years history of cultivation in China.Wintersweet blooms in winter with a strong fragrance.The special traits in flowering time and strong fragrant aroma make it an ideal ornamental tree in winter.It has been widely used in garden construction,potted landscape,cut flower cultivation,and spice extraction which bring high ornamental and economical values.As a representative of the magnoliids,wintersweet possesses an important phylogenetic position.However,the relationships among magnoliids,eudicots,and monocots have not been conclusively resolved.In recent years,the studies on wintersweet were mainly focused on the molecular mechanism of quality traits such as floral fragrance,flower color,and flowering time.However,the genomic information is limited for wintersweet which hinders the process of understanding the molecular mechanism of these traits.Therefore,the genome sequencing of wintersweet was carried out in this study.Based on the genome assembly,we performed a comprehensive and systematic analysis of the genome characteristics.At the same time,the genes related to floral scents biosynthesis,regulation,and transportation were analyzed using the genome,metabolome and transcriptome data.Finally,the key gene families encoding the enzymes functioning in the formation of main component of floral scent(linalool and benzyl acetate)were discovered,among which the critical genes were functionally characterized.The main results are as follows:1.A high-quality chromosome-level reference genome of wintersweet was achieved and annotated.Based on the results of the genome survey,the genome size of wintersweet was estimated to be about 778.71 Mb,the heterozygosity was approximately 0.60% and the proportion of repetitive sequence was 56.20%.The genome was sequenced by combining three different sequencing methods that include Illumina Hi Seq,10 X Genomics,and Pac Bio SMRT sequencing.The genome was further anchored to 11 chromosomes using Hi C technology.The final assembly was 695.31?Mb in size and the N50 size of contigs,scaffolds,and super scaffolds was 2.19 Mb,4.49 Mb,and 4.49?Mb respectively.99.42% of the genome sequences were anchored to the 11 chromosomes.To assess the genome assembly quality,we performed BUSCO and CEGMA analysis and found that 95% and 92.74% complete eukaryotic conserved genes were identified in wintersweet genome respectively.Taken together,the above results indicate a high degree of continuity and completeness of the wintersweet genome.Based on de novo and homology-based predictions and transcriptome data,a total of 23,591 protein-coding genes were predicted.A total of 21,940 predicted protein-coding genes were functionally annotated.A total of 307.67 Mb repetitive sequences and 2749 non-coding RNAs(nc RNAs)were also identified.2.The phylogenetic position of wintersweet was determined and two rounds of whole genome-wide replication events were identified in wintersweet genome.Based on comparative genome analysis using the high-quality genome data from 17 flowering plant species that includes wintersweet,Calycanthus chinensis,and other 15 plant genomes,we obtained a topology with magnoliids as a sister group to eudicots.The divergence time between the magnoliids and the eudicots was estimated to be 113.0–153.1?Mya and wintersweet diverged from Calycanthus chinensis in 4.9-24.9 Mya.The collinearity and synteny analysis within or between wintersweet and other species genomes and the 4Dtv and Ks analysis of homologous genes in the collinearity blocks suggested that wintersweet experienced two genome-wide replication events,of which the ancient one occurred approximately 112.1 Mya,before the divergence of Calycantaceae and Lauraceae but after the divergence of Calycantaceae and Magnoliaceae;and the recent one occurred about 77.8 million years ago,before the differentiation of wintersweet and C.chinensis,but after the divergence of Calycantaceae and Lauraceae.During the evolution of chromosomes,49 fusions and 48 breaks have finally formed the current 11 pairs of 22 chromosomes.3.The functional genes and transcription factors related to the floral scent synthesis were identified through the conjoint analysis of the transcriptome and floral scent metabolism data.We constructed the transcriptomes using the samples collected from three developmental stages including the bud stage,full open stage,and senescence stage.The transcriptome generated 25,829 genes.The release profiles of linalool,benzyl acetate,methyl salicylate,and ocimene were determined at the three different stages.The results showed that the four kinds of volatile compounds were regulated developmentally.The emission of these volatiles increased with the flower developments,peaked at the full open stage and declined thereafter.Using the genome data,we identified 88 and 111 genes involved in the biosynthesis of floral volatile terpenes and benzenoids,respectively.48 and 63 genes were found to be expressed during flower development from these two pathways respectively.Further,the candidate genes that might be involved in the biosynthesis of floral volatiles were discovered.These genes included not only the key node genes(Cp AACTs,Cp DXSs,Cp DXRs,Cp PALs),but also the terminal reaction genes(Cp TPSs,Cp SAMTs,and Cp BAHDs).Collinearity analysis showed that most of the key node genes in floral fragrance synthesis were generated through tandem replication and whole genome-wide replication events.Besides,99 potential transcription factors that regulate the biosynthesis of floral scent in wintersweet were also identified through a systematic analysis of the floral volatile profiling and the gene expression pattern.4.The evolutionary mechanism of linalool(the characteristic aroma component of wintersweet)biosynthesis was revealed.The Cp TPS4-AS2,an allele of Cp TPS4(a linalool synthase gene),was identified as a nerolidol synthase gene.A total of 52 TPS genes were identified from wintersweet genome.Phylogenetic analysis showed that the 52 genes were assigned into 5 subfamilies,among which the TPS-b subfamily had the largest number of members.The comparative genomic analysis found that the genes from TPS-b subfamily has been enriched,and tandem replication is the main reason for the expansion of this subfamily.Transcriptome analysis showed that 21 genes expressed in the three different stages among which 4 genes in the TPS-b subfamily(Cp TPS4,Cp TPS17,Cp TPS18,and Cp TPS19)were highly expressed,and their expression pattern was consistent with the release of linalool.Cp TPS4 has three copies including Cp TPS17,Cp TPS18,and Cp TPS19.These three genes were arranged as a tandem array and highly expressed at the full open flower stage,which may have a similar function as Cp TPS4 and contribute equally to linalool production.Based on our data,we speculate that the remarkable duplication of Cp TPS4,tandem clustering of the gene,and gene selective expression may contribute to the abundant characteristic aroma linalool formation in wintersweet.Cp TPS4-AS2,the allele of Cp TPS4,was identified using genome and reference transcriptome.The analysis of the amino acid alignment of these two alleles revealed that Cp TPS4-AS2 lacks the plastid signal peptide at the N-terminal when compared with Cp TPS4.The subcellular localization results indicated that the Cp TPS4 and Cp TPS4-AS2 are located in plastids and cytoplasm respectively.The two genes have similar catalytic functions in vitro,both of which can separately use GPP and FPP as substrates to catalyze the production of linalool and nerolidol respectively.Therefore,the Cp TPS4-AS2 has the function in nerolidol biosynthesis.Real-time quantitative PCR analysis showed that Cp TPS4 and Cp TPS4-AS2 have similar expression patterns during flower development.The cis-elements in the promoter of these two alleles are quite different.5.The critical gene,which is responsible for the biosynthesis of benzyl acetate(the main component of the floral scent in wintersweet),has been identified.33 BAHD gene family members were identified from the genome and clustered into 4 different branches,most of which are distributed in the III and V clades which are related to the synthesis of volatile esters.Collinearity analysis revealed that 6 genes originated from the whole genome-wide duplication events,and the tandem duplication events produced 18 genes.Combining the genome,transcriptome,and metabolome data,four candidate genes including Cp BAHD1,Cp BAHD3,Cp BAHD4,and Cp BAHD32 were obtained.The analysis of amino acid alignment indicated that all four genes have conserved functional domains HXXXD and DFGWG.In vitro characterization of the four genes showed that Cp BAHD1,Cp BAHD3,and Cp BAHD32 were capable of generating benzyl acetate.The transient transformation experiment of tobacco showed that the benzyl acetate was separately synthesized in the Cp BAHD1,Cp BAHD3,or Cp BAHD32 transgenic leaves when supplied with benzyl alcohol respectively,confirming that these three genes have the function of benzyl acetate biosynthesis.Analysis of the expression of these three genes in different tissues showed that only the Cp BAHD1 gene was specifically and highly expressed in flowers,so Cp BAHD1 may be the dominant gene for the synthesis of benzyl acetate.In summary,we presented a chromosome-level wintersweet genome assembly,identified two whole-genome duplication events,and confirmed that Magnoliids and eudicots are sisters to monocots;uncovered the evolutionary mechanism of characteristics aroma(linalool)formation,and identified the critical genes which are responsible for the biosynthesis of benzyl acetate.These findings provide a theoretical basis for the improvement of the floral scents of wintersweet,and genetic information resources for further in-depth molecular genetic research to identify functional genes that control flowering time,floral fragrance,flower color,and many other traits. |
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