Construction Of Transcriptional Network And Functional Characterization Of Genes Controlling Silique Dehiscence In Brassica Napus L.

Brassica napus(oilseed rape or canola)has appeared as an essential oilseed crop species cultivated in temperate climates of both the southern and northern hemispheres.Among oil crops,rapeseed ranked 2nd worldwide,as a main source of vegetable oil.The main reducing factors to attain potential crop yield in China and in the world are less resistance against various traits and poor mechanization.The growth and development of crop frequently face harsh environmental conditions.An asynchronous flowering in rapeseed maneuvers pod dehiscence or shattering at maturity especially under the drastic environmental conditions,diminishing the yield and gross profit.However,studying the mechanism of pod shattering or dehiscence zone(DZ)in rapeseed,helps to identify the key genes involved in this mechanism.Firstly,we screened out pod shatter resistant and susceptible varieties by keenly observing the pod development and maturation stages,we aimed to find out highly resistant and susceptible varieties.Then we analyzed the dehiscence resistance mechanism of rapeseed at transcriptional level and identify key genes in this mechanism.Later,we functionally characterized key genes by insertion mutants(T-DNA)to find out novel trait specific relationship.Additionally,we selected polygalacturonase(PG)gene family to explore the evolutionary fruit developmental role in land plants and its interaction with pod shatter resistance.The precise results are as follows:1.Comparative transcriptome study of Brassica napus accessionsFirstly,we screened out the silique resistant and susceptible materials by phenotypic and silique shatter resistance observation of more than 500 rapeseed accessions.Then two datasets were selected for transcriptome analysis,1)resistant(P22)and susceptible(P124)accessions,2)ZS11 pod developmental stages(3 to 46 DAP).ZS11(silique pericarp;SP)transcriptome was selected to better understand the time frame transcriptional changes involved in pod dehiscence.After the selection of materials,we sampled pods at different developmental stages in both(P22 and P124)accessions.By comparative transcriptomic study we identified 7,587 differentially expressed genes(DEGs)by pairwise comparison between accession and among developmental stages(1449 days after pollination,with 7 days of interval).The presence of higher number of DEGs at 42-49 DAP in each accession suggesting that it has important role in the dehiscence zone formation.Then DEGs were subjected to K-means clustering and WGCNA analysis to subtilize the candidate genes.Through,K-means clustering we attained two related clusters in both accessions,each of which comprised of senescence associated genes closely allied to variation in dehiscence.GO enrichment analysis showed that these genes ere enriched in senescence,aging,cell wall disassembly,water deprivation and hormonal activity which was consistent with the GO analysis of DEGs among adjacent stages and between accessions,suggesting the role of senescence,hormone,and lignin deposition in pod shattering.A Weighted Gene Co-Expression Network Analysis(WGCNA)unveiled two critical co-expression modules consisting of MEyellow and MEpink correlated with dehiscence.The color modules in 49 DAP were significantly correlated as MEyellow(r2=0.82,p=9e-10)and MEpink(r2=0.62,p=6e-5).MEyellow possessed genes participated in“senescence"(GO:0010150),"leaf senescence"(GO:0010150),“stomatal closure"(GO:0090332)and "gibberellin metabolic process"(GO:0009685);while in MEpink "fruit development"(GO:0010154),"lignin metabolic process"(GO:0009808)and“cell wall modification"(GO:0042545).The functions of all the corresponding genes in these modules directly or indirectly correlated with pod dehiscence mechanism and suggested that late developmental stages have crucial role in pod dehiscence of B.napus.Then we selected two TFs as hub genes from MEyellow module,having highest K-within values,Bna.A05ABI5(BnaA05g08020D,K-within=379.15)and Bna.C03ERF/AP2-3(BnaC03g09040D,K-within=422.50).Upregulation of transcription factor Bna.C03ERF/AP2-3 decreased the transcription of upstream SHP1/2 to enhance dehiscence resistance by downregulating the transcription of upstream genes.However,the other hub gene Bna.A05ABI5 upregulates the expression of SAG2,ERF/AP2 and several LEA proteins related genes to fluctuate the mechanical force in lignified cells.Among pod-shattering genes,we found the upregulation of Bna.C07SHP1/2 and Bna.PGl/2 genes in susceptible accession associated with minimal resistance.Furthermore,we observe the upregulation of Bna.WAG2 in resistant accession reduced the polygalacturonase activity in the cell loosening process.Moreover,in lignin biosynthesis pathway CCoAOMT genes may increase the lignin deposition to promote dehiscence.We further approved the authenticity of transcriptome results by quantitative real-time PCR of 12 DEGs including BnaPG,BnaWAG2 and BnaABI5.On the other hand,ZS11 SP were sampled from 3 DAP to 46 DAP to find out the upregulation of genes involved in pod maturation.WGCNA analysis revealed that MEblue has higher correlation(r2=0.87,p=5E-05)with SP.GO analysis of MEblue module showed the important terms as "senescence"(GO:0010149),"leaf senescence"(GO:0010150),cell wall modification"(GO:0042545)"regulation of cell death"(GO:0010941)and“ethylene mediated signaling pathway"(GO:0009873).The gene expression analysis found the gradual upregulation(40 to 46 DAP)of ADPG1/2 and several other senescence-related genes.1.1 Overlapping analysis of ZS11 and silique shatter specific materials(P22 and P124)To examine the relationship between time series SP development and silique shatter(SS)transcriptome,we overlapped the MEyellow and MEblue modules.The overlapping analysis was highly significant(p=2e-105)and contained 671 common genes.The GO study of 671 genes revealed the important biological terms relevant to the pod dehiscence as senescence,reproductive structure development,ABA response,fruit development,aging etc.Interestingly "reproductive structure development"(GO:0048608)in overlapped analysis possessed Bna.A07PG1,showing the significance of PG genes in pod dehiscence.Moreover,these results showsd that time series transcriptional changes in SP development might have important role in DZ formation.Further study of Bna.A07PGl may provide new insights to improve pod dehiscence in B.napus.Eventually,we short listed 30 trait-specific genes from both transcriptome datasets to further explore the functional relevance with pod-dehiscence by T-DNA study.2.T-DNA mutant study of candidate genes and editing of Key genes by CRISPR Cas9 SystemFrom the transcriptome analysis of trait specific materials(pod shatter)and ZS11,we selected 30 candidate genes for functional characterization by T-DNA insertion mutants(Arabidopsis thaliana).After genotyping and expression verification,several mutants(naxt1,dox1,rpk1,cpy7,wakl4,sbt3,wak1,sqp1 and asmt)were excluded from further study due to the higher expression.The remaining mutants were used to analyze the trait specific parameters such as silique shatter resistance index(SSRI).By SSRI analysis two highly susceptible mutants(erd6 and tmk3)and three resistant mutants(nst1,adpgl and adpg2)were short listed and further analyzed by PG activity,lignin accumulation and lignin staining.According to the results and previous literature,PG genes are involved in separation layer formation in DZ.So.we selected ADPG1 and ADPG2 to construct the multiplex knockout lines to better understand the DZ formation in Brassica napus.Three vectors were constructed in such a way that two of them capable of targeting ADPG1(Bna.A07PG1,Bna.A09PG1、Bna.C08PGl and Bna.CnnPG1)and ADPG2(Bna.A04PG2,Bna.A05PG2,Bna.C04PG2-1 and Bna.C04PG2-2)genes individually and the third vector can target both ADPG1 and 2.The positive transgenic plants(T0)were confirmed by PCR.Further analysis may explore the individual and combined role of ADPG genes in Brassica napus and could provide new insights to improve the pod shattering resistance.3.Evolutionary analysis of PG gene family and its role in fruit developmentPG is a hydrolase that contributes to pectin disassembly,fruit softening and pod shattering.In this study,we found 2,786 PG members across fifty-four plants,that could be classified into three maj or groups.Evolutionary analysis of these plants implied that PG family originated from the charophyte green algae,and Subgroups A2-A4 evolved from the Subgroup A1 after the tracheophyte-angiosperm split.Whole-genome duplication(WGD)was the major force heading to PG gene expansion.Remarkably,plant species that underwent more WGD and whole genome triplication(WGT)events throughout their evolutionary history kept more PG family members.For instance,Cucumis sativus and Glycine max derived from the common ancestor(Fabidae),Cucumis sativus contains 61 PG members,while Glycine max have 97 PG members owing to the additional WGD event in the evolutionary history.Intriguingly,the PG proteins constantly expanded in eudicots,whereas it contracted in monocots after the eudicotmonocot split.Expression analysis showed that PG genes in Group A exhibit higher expression in floral organs,though genes present in Groups B and C are expressed at high levels in numerous tissues as in reproductive and vegetative parts.Additionally,three BnaPG15 members were noticed for their possible function in pod shattering in rapeseed.These findings indicating that expansion of PG gene family seemed to be linked with the evolution of steadily more complex organs in plants as moving from non-angiosperms to angiosperms plant species.Mosses and early vascular plants have several PG members,and they might have originated from the lycophytes after the divergence of seed-bearing plants.Gene expression patterns shows the functional divergence and preservation of the PG family proteins in higher plants.In conclusion,we analyzed two transcriptome datasets and found important genes(ABI5,SAG2,and ERF/AP2,ADPG etc.)involved in DZ formation or pod shattering.From T-DNA mutant study ADPG1 and 2 genes were chosen to construct knockout lines by CRISPR-Cas9 system.Furthermore,evolutionary analysis of PG gene family was done to comprehend the role of this family in fruit development and fruit setting process in plants.