Identification,Single Nucleotide Polymorphism,Evolution And Expression Profiling Analysis Of Oleorersin Related Genes Of Loblolly Pine(Pinus Taeda L.)

Pinus taeda L.is one of the world’s most important afforestation and industrial timber species,providing around 16%of the world’s wood use annually.In addition,the Pinus taeda are also rich in turpentine,and its main component terpenoids were widely used in medicine,cosmetics and industrial chemicals.This feature has brought huge benefits to its forestry economy.In order to further understand the molecular mechanism of oleoresin biosynthesis and metabolism,and provides a theoretical basis for the directional breeding of the high-yield Pinus taeda.In this study,the 202 clone and its partial half-siblings were used as research materials.Identification of genes related to oleoresin synthesis using RNA-Seq technology.Subsequently,the single nucleotide polymorphism,molecular evolution pattern,phylogenetic and expression profiling of some candidate genes were analyzed by using bioinformatics analysis software and biotechnical methods such as Dna SPv.5.1,MEGA7.0,PAML and q RT-PCR.The main results as follows:1)Functional annotation of unigenes obtained by sequencing transcriptome sequencing of the secondary xylem of Pinus taeda and previous studies on genes related to oleoresin synthesis.A total of 422 unigenes related to oleoresin synthesis were identified in this study.It mainly includes:123 steroid skeleton biosynthesis related enzyme genes;105ABC transporter genes;67 cytochrome P450 pedigree protein genes;63 transcription factor protein genes involved in the regulation of steroid biosynthesis and metabolism;Alcohol dehydrogenase gene;13 terpene synthase genes;10 acetaldehyde dehydrogenase genes and3 non-specific lipid transporter protein genes.In addition,the results of q RT-PCR analysis indicated that the key enzyme genes of six terpenoid biosynthetic pathways were highly expressed during the high oleoresin yield period.2)Based on the functional annotation results,24 candidate genes were selected for their nucleotide diversity in 48 Pinus taeda resources and their correlation with oleoresin production.The total length of transcriptome sequencing of 24 candidate genes was 41,066bp,and a total of 1613 SNP sites were detected,with an average SNP frequency of 81bp/SNP.The average nucleotide polymorphism parametersπ_Tandθ_wof the 24 candidate genes in 48 samples were 0.0103 and 0.0109,respectively,and the average nucleotide polymorphism of the silent site was 0.0174,and the average haplotype was 29,The average haplotype diversity was 0.881,and the minimum number of historical recombination events was 20.The d N/d S value of the HDS and Nonsp gene were greater than 1 by evolutionary pressure analysis.Neutral test results showed that the Aldh1 gene was significantly greater than 0 in Tajima’s D,and the detected values in Fu and Li’s D and F were significantly less than 0.The test values of the CMK gene in Tajima’s D and Fu and Li’s D and F were significantly less than 0.A total of 33 SNP loci which are significantly associated with oleoresin yield were detected in 24 candidate genes,14 of which resulted in amino acid changes.A total of 73 genotypes were detected in 33 SNP loci,and the frequency of 17genotypes was significantly higher in high oleoresin yield samples than in low oleoresin yield samples.3)Molecular evolution analysis of 12 genes encoding biosynthesis pathways related to terpenoids showed that there was no significant correlation between d N and d S values in the whole phylogenetic tree and dicot-clade.However,in the gymnosperm clade(r~2=0.594,P<0.05)and in the mono-clade(r~2=0.551,P<0.05)there were a significant correlation between the value of d N and d S.The results of the branch model analysis showed that the selection pressure of the 12 genes was between 0.055 and 0.190,and the gene located in the MEP pathway and the gene located in the MVA pathway were significantly different in the selection pressure(P<0.05).Except for the MVK(P=0.08)gene,the selection pressure of other 11 genes in the phylogenetic tree were significantly different.The analysis of the sites model showed that the selection pressures between the points were significantly different;The analysis of the branch-site model showed that in the branch-site model of the Pinus-clade as the foreground branch,the GGPS and HMGR genes were detected 11 and 17amino acid sites faced positive selection,respectively(P>0.95).A total of 22 amino acid sites of GGPS were detected positive selection in the branch-site model of the mono-clade as a foreground branch(P>0.95).The mean values of ENC and GC3 of the 12 genes were between 47.73-55.29 and 0.29-0.63,respectively.There was a significant negative correlation between the ENC and GC3 groups of 12 genes in the mono-clade(P<0.001).4)The phylogenetic analysis of DXR,DXS,FPS,GGPS,HMGR and IDI genes in 15species showed that the DXR gene sequence has a large degree of variation,the average genetic distance reaches 0.625,and the IDI gene sequence is highly conserved.The average genetic distance is only 0.275.The phylogenetic trees of the DXS,DXR,HMGR and IDI genes have similar topological structures,consistent with the differentiation time of their corresponding species.The expression profiles of 20 candidate genes in different tissues of Pinus taeda showed that all candidate genes were highly expressed in pine needle tissues.Interestingly,the expression levels of DXS,DXR,CMS,MCS and IDS genes in the MEP pathway were higher in the mature leaf tissue than in the young leaf tissue,while the four candidate genes in the MVA pathway were show the opposite expression pattern.