Analysis Of Allelic Variation Of Important Wood Quality Traits In Cunninghamia Lanceolata By Co-expression Network And Association Analysis

Chinese fir(Cunninghamia lanceolata)is an important fast-growing,coniferous timber yielding species in southern China.Improvement in physical properties is one of the critical tasks of C.lanceolata breeding in the present scenario.However,like most other woody plants,C.lanceolata also has long breeding cycles and low selection efficiency problems.Therefore,rapid genetic improvements and a focus on shortening the selection cycle are the major objectives of the C.lanceolata breeding programme.In recent years,RNA-seq technology and association studies based on molecular markers have been successfully applied for the mining and molecular mechanism analysis of key candidate genes meant for important traits of non-model woody plants.In this study,we obtained a high-quality reference transcriptome of C.lanceolata.We then used RNA-Seq and association analysis to find out the key candidate genes and their excellent alleles for wood formation in C.lanceolata.The main results were as follows:(1)A high-quality C.lanceolata reference transcriptome was obtained by Single Molecule Real-time(SMRT)sequencing technology.According to BUSCO evaluation,the integrity of the transcriptome reached up to 87.4% and 88.8%.Then Unigenes were annotated in five databases,including NR and Swissprot.A total of 6,882 simple-sequence repeats(SSR)loci,6,431 long non-coding RNAs(lncRNAs)were identified by RNA coding ability prediction software.Among these identified lncRNAs,35 lncRNAs were identified as 15 microRNA(miRNA)precursors.In addition,1,566 Unigenes were identified as transcription factors(TFs)from 55 TF families.(2)RNA-seq was used to analyze the gene expression profile of the xylem of the compression wood treated at different time intervals.A total of 6,444 Unigenes were differentially expressed,including 573 lncRNAs and 283 TFs.A total number of 2,069,3,292 and 3,474 differentially expressed Unigenes were detected in 30 d,60 d and 90 d samples,respectively.Then a total number of 1,586 Unigenes were found to be involved in64 metabolic pathways,including phenylpropanoid biosynthesis pathway,amino acids biosynthesis,signal transduction of hormones etc.Weighted correlation network analysis(WGCNA)was used to mine two key modules related to wood formation,including 775 Unigenes.(3)Based on the results of transcriptome sequencing,6,646 high-quality singlenucleotide polymorphism(SNPs)were identified from 500 important Unigenes.The frequency of SNPs was 162.7 bp,and the number of transformed SNPs was more than that of transversed SNPs.A total of 57 SNP-trait associations were obtained,representing 57 SNP loci from 32 Unigenes.The phenotypic interpretation rate was 0.0571(PB.67446.1＿SNP1190)to 0.1203(PB.38620.2＿SNP608).Sixty-one haplotypes from 9Unigenes were associated with traits,and the phenotypic interpretation rate of traits ranged from 0.51%(PB.38620.2 SNP608-SNP683)to 8.08%(PB.50582.2 SNP1171-SNP1208).The most significant 200 pairs of SNP-SNP interaction sites were selected for epistasis analysis,and the phenotypic interpretation rate was between 0.0035(PB.39235.3＿SNP214& PB.50582.2＿SNP572)to 0.1509(PB.62973.3＿SNP755 & PB.66208.2＿SNP741).The interaction network of epistatic effects showed that the wood formation of these C.lanceolata species was a complex process.The above results laid the foundations for further dissection of the wood formation mechanism.Furthermore,they provided the critical candidate genes for developing molecular markers and marker-assisted selection(MAS)in C.lanceolata.