Study On Phylogeny And Variation Of Characteristic Secondary Metabolites Of Camellia Linn.

Camellia Linn.,comprising more than 200 species,is an economically and phylogenetically important genus in the family Theaceae.C.sinensis,C.oleifera,C.reticulata are the representative species which has the longest history of cultivation and utilization in China due to their commercial and ornamental values.However,the continually hybridization and multipulplification,coupled with the lack of suitable DNA fragments for evolutionary analysis,resulted in a lack of understanding of the phylogenetic analysis,which to some extent hindered the efficient development and utilization of Camellia plants.To this end,on the basis of extensive collection of Camellia species,we attempts to systematically clarify the phylogeny of genus Camellia by comparative transcriptomics and metabonomics,combining with the reference genome of tea plant(C.sinensis),we make an attempt to reveal the distribution pattern and molecular mechanism of the principal secondary metabolites closely in Camellia plants,with the goal of efficiently exploring and utilizing the precious and abundant germplasm resources of Camellia and accelerating the genetic breeding of tea cultivar.The conclusions of this study are as follows:(1)RNA-seq of 121 species of the 15 section and 2 species from close related genus were sequenced with Illumina novaseq 6000,consisted of 928 Gb bases and 16,778,018 transcripts were assembled with an average N50 length of 1084 bp.With the assembly integrity of about 94.44%,7,240,353 transcripts were annotated by six well-known protein databases,accounting for 55.13% of all.220,894 transcript were identified as transcription factors,of which MYB and MYB-related are the most abundant transcription factor families in Camellia.5,568,311 EST-SSRs were identified,of which 786 were polymorphism SSR among Camellia plants.These molecular markers can provide information sites for scientific conservation and genetic improvement of Camellia.(2)A total of 89,394 paralog/ortholog homologous gene families were identified in 116 Camellia plants by conducting comparative genomics and transcriptomic analysis.Of all homologous gene families,5,793 were core orthologous gene groups,9,623 are unique for every species.Compared to species-specific genes,there are higher sequence and functional conservativeness among core OGs.C.sinensis,C.furfuracea,C.reticulata,C.Oleifera,C.cuspidata,C.tuberculata,C.impressinervis which are the most representative species of genus Camellia,derived from a most recent common ancestor(MRCA),had experienced a genome-wide duplication(WGD)event 28 million years ago(MYA).Almost 32.32% of transcripts still retain the WGD imprint,which related to the lipid metabolism,pigment metabolism and tea quality.A Camellia transcriptomic database was constructed based on an integrated information of transcripts of all Camellia plant composed of a variety of bioinformatics tools(such as functional enrichment analysis,correlation analysis,primer design,sequence comparison,etc.)to help researchers quickly retrieve and deeply mine abundant transcriptome data of Camellia plants.(3)Based on the construction of Camellia pan-transcriptome,405 high-quality lowcopy orthologous nuclear genes were identified.Taking Polyspora speciosa as an outgroup,the maximum likelihood tree and Bayesian tree of Camellia were constructed respectively.It was found that the phylogenetic relationships of Camellia constructed by the two strategies were similar,and the bootstrap value of most evolutionary branches was 100%,and the morphological characteristics of 36 leaves were highly supported.It was found that Camellia can be divided into seven clades.The wellresolved phylogenetic relationship supports the combination of Sect.Thea and Sect.Glaberrimatea,Sect.Oleifera and Sect.Paracamellia,Sect.Tuberculata and Sect.Pseudocamelliatea,Sect.Eriandria and Sect.Theopsis.It was found that Sect.Thea was monophyletic.Sect.Camellia was close to Sect.Oleifera.Sect.Chrysantha was the base group of genus Camellia.Supporting the latest revision of C.kwangtungensis and C.danzaiensis from Chinese Flora.The phylogeny relationship of 31 representative species of Camellia based on chloroplast genome was divided into five clades.It was conflict with nuclear genes tree that Sect.Thea,Sect.Camellia,Sect.Chrysantha and Sect.Oleifera were polyphyletic.Further estimation of the differentiation events of Camellia plants showed that the differentiation of genus Camellia and P.speciosa took place about 14.30 MYA,and Sect.Thea might originate about 6.67 MYA.The differentiation time of C.sinensis var.sinensis and C.sinensis var.assamica was about 1.49 – 2.16 MYA.(4)The contents of catechins,theanine and caffeine in a total of 82 species from 16 groups of Camellia plants were determined,and it was found that the characteristic secondary metabolites of Camellia plants were rich in content and diverse in types.Compared with non-Thea,Thea plants tend to accumulate high content of catechins,caffeine,theanine and other characteristic secondary metabolites closely related to the formation of tea quality.EGCG and EGC were the dominant catechins in Thea and non-Thea plants.With the differentiation of Thea plants,the content of EGC decreased gradually,while the content of EGCG increased gradually.It was also found that the accumulation of characteristic secondary metabolites in Camellia was highly correlated.The correlation between catechins and caffeine was 0.91(p-value = 1.15E-33),and the correlation between catechins and theanine was 0.71(p-value = 5.43E-14).The distribution patterns of metabolites in Sect.Thea and Sect.Glaberrimatea Thea,Sect.Tuberculata and Sect.Pseudocamellia were similar,which were highly consistent with their phylogenetic relationships,and further provided phytochemical evidence for their taxonomic combination.(5)To study the catechins metabolism pathway genes,The correlation between the xpression of genes which were crucial in the catechins metabolism pathway and the metabolite content in different tea cultivars and their wild relatives was compared.It was found that the expression patterns of genes coding flavonoid 3’,5’-hydroxylase(F3’5’H),flavonol synthase(FLS),anthocyanin reductase(ANR)and serine carboxypeptidase protein(SCPL1A)were significantly correlated with the accumulation pattern of catechins in Thea.The coding region and promoter sequence of F3’5’H were preliminarily selected and cloned.It was found that the coding region of F3’5’H was highly conserved in tea plants,but the promoter region of F3’5’H gene had 180 bp insertion mutation containing two MYB transcription factor binding sites in tea plants with high catechin content.The prediction of cis-regulatory elements showed that the 180 bp insertion mutation might enhance the activation activity of MYB transcription factor on F3’5’H,thus promoting the high accumulation of catechins and resulting in the diverse distribution of catechins in Thea.In summary,through the study of pan-transcriptome and metabonomics of Camellia plants,this study systematically clarifies the phylogenetic relationship of Camellia plants,and comprehensively reveals the distribution patterns and genetic rules of quality-related secondary metabolites in Camellia plants.It is undoubtedly helpful to speed up the efficient exploitation and utilization of precious and rich tea resources in China,and promote the rapid development of tea,tea-seed oil and Camellia planting industry and cultivation and breeding in China.