Genome-wide Identification And Characterization Of Novel LncRNAs In Ginkgo Biloba Leaves

Ginkgo biloba is an important landscape tree for fan-shaped leaves and golden leafcolor in autumn.In addition,G.biloba leaves contain a variety of active ingredients for medicine.Long non-coding RNAs(lncRNAs)are longer than 200 nucleotides in length,which play important roles in growth and development,male sterility,response to biotic and abiotic stress and other biological processes.As regulators,lncRNAs mediate gene expression through multiple mechanisms,such as target mimicry,transcription interference and methylation,and become a new research hotspot in plants.However,lncRNAs in G.biloba leaves remain unkown.In this study,we combined next-generation highthroughput sequencing technology and bioinformatics analysis to identify IncRNAs and their target genes in the leaves of G.biloba.The main results are as follows.1.To obtain comprehensive IncRNA transcripts,we used the mixed total RNA extracted from leaves of seven growth stages(from April to October)to construct the ssRNA-seq library.After paired-end sequencing and quality trimming,we obtained approximately 23.97 GB clean data(87.35%)of raw reads.By using RNA-seq,a large number of clean reads with high qualiy were obtained,87%of them can be mapped to the reference genome which means that RNA-seq results are high quality and may reflect the transcriptome features of leaves.To obtain novel lncRNA candidates,the unknown transcripts with lengths<200 nt and FPKM(Fragments Per Kilobase of transcript per million fragments mapped)<0.1 were filtered out.To remove potential transcriptional noise and DNA fragments in the library,the transcripts with exon number<2 were also excluded.Then the transcripts with coding potential were further filtered by the CPC(Coding Potential Calculator,CPC score<0),CNCI(Coding-Non-Coding-Index,CNCI score<0),CPAT(Coding-Potential Assessment Tool,default setting)and Pfam(protein family database,E-value<0.001).The remaining non-coding transcripts with lengths,200 nt,exons number ? 2,FPKM ? 0.1 in the sample were selected as high-confidence novel lncRNAs.Finally,we identified 1,323 lncRNAs expressed in the leaves of G.biloba.2.LncRNAs are categorized into lincRNAs,intronic lncRNAs,sense and anti-sense lncRNAs.Most IncRNAs in leaves are lincRNAs.Identifided lncRNAs were distributed in 947 scaffolds in G.biloba genome.Compared with protein genes,lncRNAs have the fewer in number,shorter in length,lower expression levels and lower sequence conservation.3.According to the enrichment analysis,we identified nine photosynthesis-related genes that were targeted by nine lncRNAs,17 related genes involved in secondary metabolic pathways were predicted to be targets of 15 lncRNAs.We also focused on the transcription factors(TFs)targeted by IncRNAs.A total of 31 related genes from 20 TF families identified by KEGG annotation analysis may be located within 100 kb upstream of 34 lncRNAs(cis-acting regulation).4.We predicted 12 mimic targets of lncRNAs,for example,miR165 targets MSTRG.42711.1.We constructed the miRNA,IncRNA and mRNA interaction regulatory networks in G.biloba leaves.5.Further,through subcloning technology,we validated these new IncRNA sequences,and found 3 lncRNAs which matched Illumina sequencing results exactly and 2 lncRNAs had less than three mismatches,indicating the accurance of sequencing and assembly.6.By the quantitative PCR analysis,we analyzed the IncRNA expression in different development stages of leaves and different tissues and organs,and the results revealed that lncRNAs have strong tissue and spatiotemporal specificity.7.In order to explore whether lncRNAs play a regulatory role in the development of leaf coloration,we further utilized leaf transcriptome data from the green leaves,color-changing leaves,and yellow leaves for IncRNA identification and differential expression analysis.By removing transcripts with a transcript length of ?200 bp and the expression level ?0.5,and the analysis of coding potentials,such as CPC and pfam protein domain,the intersection of the two software analysis results was taken.Finally 2,124 lncRNAs were obtained.Using green leaves as a control,we found that there were 161 differential lncRNAs between green and color-changing leaves,of which 38 lncRNAs were down-regulated and 123 lncRNAs were up-regulated;and there were 416 differential lncRNAs between green and yellow leaves,of which 134 lncRNAs down-regulated,282 lncRNAs up-regulated.8.Annotation of target genes of differentially expressed IncRNAs showed the differential gene GO enriched in cellular processes,metabolic processes and single organism.In addition,by the KEGG enrichment,the top two enriched pathways between the color transition phase and yellow leaves were metabolic pathways and secondary metabolic synthesis.In summary,the results of lncRNAs in leaves revealed that G.biloba contains a large number of lncRNAs,which participate in the growth and development,morphogenesis and signal transduction.It also suggests the complicated IncRNA regulation mechanisms involved in leaf development.This study provides abundant data and information about lncRNAs of G.biloba leaves,providing a reference for further study of the molecular mechanism of leaf development.