Transcriptionally Functional Differentiation Of Flowering Time-Related Duplicated Genes In Brassica Napus

Alternative mRNA processing such as alternative splicing and alternative polyadenylation enables the ability of individual genes to generate multiple transcripts.With the advancement of genome sequencing,up to 60%of total genes in a higher eukaryote genome have been found to produce alternative transcripts(ATRs),resulting in doubling or tripling the number of transcripts predicted from gene models in a genome.Some case studies on individual genes have showed that ATRs from a single gene have different functions in biological processes or just one of them has its biological function and the others are subject to degradation.However,the scale and extent of such differentiation between ATRs of all genes is unknown.Polyploid duplicate genes are considered a genetic matter source for functional differentiation in plant evolution.It is also unknown whether duplicate genes have undertaken function differentiation by producing different ATRs.If yes,an undiscovered layer of functional differentiation of duplicated genes may exist in polyploid genome evolution.Brassica napus(Bn,AnAnCnCn)is an allotetraploid that evolved through hybridization between B.rapa and B.oleracea,an excellent model for studying the evolution of polyploid duplicate genes.The objectives of the study are to address the above questions by investigating Bn flowering time(FT)genes as an example.The following summarizes the main results:1.The identification of ATRs and alternative splicing eventsBn FT genes and Arabidopsis thaliana FT genes were retrieved from gene function annotation of the Bn Darmor-bzh reference genome and the TAIR10 database,respectively.The retrieved genes were analysed to select syntenic orthologous Bn genes of A.thaliana FT genes as Bn FT genes according to gene function conservation between A.thaliana and Bn.In total,732 Bn FT genes were obtained.Bn full-length transcripts were generated by third-generation PacBio RNA-seq from the bud,leaf and root tissues.The transcript sequences were mapped to the Darmor-bzh genome genome and then compared with the Bn FT genes to identify transcripts of Bn FT genes.The total number of transcripts is 1822,of which 1412 are ATRs produced by 322 genes,accounting for 44%of Bn FT genes.These ATR genes were almost equally distributed in both subgenomes(160 An vs 162 Cn).Furthermore,the numbers of genes producing ATRs and ATR numbers of duplicate An and Cn gene pairs showed no significant difference(pairedt-test,p>0.05)between the two subgenomes.For all FT genes producing ATRs,the greater proportion of genes presented two ATRs.For all 322 Bn genes that produce ATRs,a total of 1075 alternative splicing(AS)events were identified.Approximately 50%of these AS events were complex AS types.The remaining AS events were intron retention events>alternative acceptor splice site events>donor splicing site events>exon skipping events>mutually exclusive exon events.Intron retention was the most common type of AS events in Bn FT genes.Alternative polyadenylation(APA)analysis identified 924 poly(A)sites from 396 Bn FT genes whereas 250 FT genes had alternative polyadenylic acid sites and 69 genes had at least 4 poly(A)sites in their ATRs.Between the duplicate gene pairs,166 An and 159 Cn genes produced polyadenylation sites(p>0.05).These results showed that extensive mRNA processing occurred in Bn FT genes.2.Bn genes producing ATRs are more highly expressed than those with single transcriptQuantification of gene expression was conducted by employing second-generation Illumina RNAseq data of three tissues(bud,leaf and root),in which PacBio full length transcripts were used as reference to avoid cross-mapping of Illumina short reads owing to high similarity of duplicated genes.Our results demonstrated that total 55%of Bn FT genes(408 of 732)and 47%of total transcripts(857 out of 1822)were highly expressed which was defined as FPKM≥0.5 in any single tissue,we found out that 95%(308)of genes producing ATRs were indeed highly expressed,whereas only 24%(100)of single transcript genes showed FPKM≥0.5;88%of 857 transcripts were ATRs.Genes and transcripts with the feature were equally distributed in An and Cn subgenomes(p>0.05).These results indicated that genes producing ATRs are more actively expressed,suggesting a novel and complex regulation involved in these genes in B.napus.3.The prediction of functional differentiation by expression differentiation of flowering time gene pairs of An and CnThere were 258 pairs of duplicate FT genes in the An and Cn subgenome.We observed that only 19%alternative splicing(AS)events were conserved between An and Cn duplicate gene pairs,suggesting their functional differentiation.Quantification of the duplicated genes were conducted as described above using Illumina reads.By setting FPKM≥0.5 in any of the three tissues for a gene to be highly expressed,we found that the gene pairs were classified into the three groups:(1)68(26%)pairs of 258 pairs were in low expression levels(FPKM<0.5).(2)In 19(7%)gene pairs,only An genes were highly expressed.(3)In 24(9%)pairs,only Cn genes were highly expressed.(4)147(57%)gene pairs were highly expressed in both subgenomes.Further,we categorized these gene pairs based on their single or alternative transcripts except for 68 gene pairs due to their minor expression in both An and Cn subgenomes.The eight groups were divided.(1)10(4%)pairs of genes with single transcript showed high expression in the An members but low in their respective partners(the Cn members).(2)in 6(2%)pairs of genes with single transcript,the Cn members were high but the An members were low.(3)in 14(5%)pairs of genes producing ATRs,the An members were high,but the Cn members were low.(4)in 14(5%)pairs of genes producing ATRs,the Cn members were high but the An members were low.(5)in 17(7%)pairs of genes with single transcript,all were high.(6)in 18(7%)pairs of genes,all were high,but An members produced single transcript whereas the Cn members produced ATRs.(7)in 17(7%)pairs of genes,all were high,but the Cn members produced single transcript,whereas the An members produced ATRs.(8)95(37%)pairs of genes,both An and Cn members produced ATRs and all were high.These results indicated severe differentiation of duplicate genes although some gene pairs(95)involved more complex regulation by ATRs.Furthermore,when taking account of expression between duplicate genes in the group 6 to 8(in total 130 duplicated pairs)in which both An and Cn members were highly expressed and one gene of each pairs produced ATRs,the results showed that the expression of genes in Cn were significantly higher than that in An(t-test,***p<0.0001),confirming differentiation of duplicated genes.By analyzing the tissue-specific expression,we categorized the duplicated gene(127 duplicated pairs)into three groups;(1)only Cn subgenome gene was tissue specific(2)only An subgenome gene was tissue specific and(3)both An and Cn pair was tissue specific.The tissue specific genes were ATR producing gene as well.The results showed that all the duplicated genes used in this analysis were differentiated while in group 3 duplicated genes,the complex expression pattern was observed.Furthermore,the results showed that the expression of genes in Cn were significantly higher than that in An(t-test,**p<0.001),verifying the differentiation of duplicated genes In terms of the principle that protein functions are mainly determined by their structure domains,we further checked structure domain variation between alternative transcripts of a single gene or duplicate genes.79%of genes producing ATRs displayed minor changes between alternative transcripts,identified as a same domain or the same combinations between sister transcripts or ATRs of duplicate genes,while the remaining 21%presented major changes in structure domains between transcripts with multiple domains.In total,64 genes producing ATRs had different multiple domains in their transcripts.These sister transcripts or paralogous transcripts of duplicate genes with different domains are considered with potential functional differentiation.4.The identification of functional differentiation of flowering time gene pairs by the population association analysisWe associated the alternatively spliced transcripts of flowering time(FT)genes with flowering time(Days to flowering,DTF,25%plants flowering in a field plot)in the population comprising 324 accessions that all were conducted RNA-seq for shoot apical meristem(SAM).In the case of whole gene expression,the cumulative expression of all transcripts of a gene in the SAM tissue was used.In the identified 322 Bn FT genes producing alternative transcripts,Spearman’s correlation analysis indicated that 68 genes were significantly correlated with FT(p<0.05).92 transcripts from 68 genes that were significantly correlated with flowering time in the population.We also found that the correlations between cumulative expression quantity of a gene and flowering time did not represent the correlations between the alternative transcript abundance of the genes and flowering time.For example,in a gene pair of BnaA02g00370D and BnaC02g00490D,a region containing BnaA02g00370D was significantly associated with flowering time,but its paralogous region with BnaC02g00490D did not;when using transcripts to do association analysis,only the transcript PB.27283.3 of three transcripts from BnaC02g00490D was significantly associated with flowering time(p<0.0001)and no significant association for the two transcripts of,BnaA02g00370D.These indicated functional differentiation of transcripts of An and Cn gene pairs in control of flowering time.Taking account of structure domain changes,we found that just one gene of each pair of nine gene pairs with various domains in their transcripts were significantly correlated with flowering time,these indicated not only functional differentiation of duplicate genes,but also functional differentiation of the sister transcripts of a gene.5.Genome-wide characterization of Ariadne gene family associated with flowering timeGenes in a gene family generally originate from a common ancestor gene and then undergo sequence and functional differentiation with various divergent extents,depending on birth time and selection pressure of every member.Young members compose duplicate genes and thus are a good model to study mechanisms of duplicate gene evolution.Here,we analyzed a E3 ligase-related gene family in B.napus for their alternative transcript status and effect on flowering time.E3 ligases are involved in protein ubiquitination and degradation,regulating every aspect of eukaryotic life.The Ariadne(ARI)proteins of RBR(Ring Between Ring Fingers)protein subfamily were discovered as a group of potential E3 ubiquitin ligases.A few studies showed their role in the plant adaptation.The functions of ARI proteins are largely unknown in plants.Therefore,in this study,we performed a genome-wide analysis to identify the ARI gene family and explore functional differentiation of their members in B.napus.We identified 39 ARI genes in the B.napus genome with almost equal distribution of genes in both An and Cn subgenome.Based on phylogenetic analysis,the BnARI genes were classified into three subfamilies(A,B and C),in which the subfamily B contains the most genes(21 genes)with the conserved motifs.The expansion of BnARI gene family was mainly the result of segmental duplication events and the duplicate gene pairs were under purifying selection.The proteinprotein interaction networks and enrichment analysis indicated that BnARI genes could be involved in endoreduplication,DNA repair,proteasome assembly,ubiquitination,protein kinase activity and stress adaptation.A total of 91 alternative transcripts were detected 20 BnARI genes in Pacbio Iso-seq data including distinct AS events.There are varied number of transcripts between the duplicate gene pairs,suggesting possibility for functional differentiation at the transcriptional level.Furthermore,of eight BnARI genes that were identified as candidate genes for multiple agronomic traits through association mapping analysis in the above B.napus population,BnaA02g12100D which is orthologous to AtARI8 was significantly associated with flowering time.This study provided an angle of gene family to reveal functional differentiation of duplicate genes.