Identification And Functional Analysis Of BnaC08.AGP2,as A Candidate Gene Of The Main Root Morphology Locus Rt.C08-1 In Rapeseed

Root system is an essential plant organ,which not only anchors the plant and stores carbohydrates,but also absorbs and transports water,mineral nutrients,and hormones into the plant.A well-designed root system is the basis of high biomass and high yield of crops.To reveal the genetic mechanism underlying root morphology and its relationship with biomass formation,our previous study precisely identified the root traits of 280germplasms from the natural populations of Brassica napus during five vegetative developmental stages under hydroponic conditions,followed by genome-wide association study（GWAS）.Several major quality trait loci（QTL）regulating root development were identified by GWAS,and two persistent and stage-specific root development regulation patterns were proposed.The persistent main root morphology locus Rt.C08-1 obtained by GWAS was detected in nine root-related traits（root fresh weight（RFW）,root dry weight（RDW）,total root surface area（TSA）,total root volume（TRV）,primary root length（PRL）,total root length（TRL）,total root number（TNR）,shoot fresh weight（SFW）and shoot dry weight（SDW））in vegetative growth stage.The phenotypic contribution rate of Rt.C08-1 was 7.55%-16.9%.On this basis,this study investigated the regulatory pathways of root dynamic growth patterns by transcriptome analysis of root extreme materials.Further,the target gene of a persistent major effect root QTL Rt.C08-1 obtained by GWAS was mined,and the molecular mechanism of its regulation on root development was initially deciphered.The main research findings are as follows:1.Mechanism underlying rapeseed dynamic root growth.In terms of the root growth rate at five developmental stages,four types of materials of sustained slow root growth rate,sustained fast,first slow then fast,and first fast then slow were selected from the natural population.Five representative materials were selected for each type.Transcriptome analysis of root tissues of the four types was performed at ten days after transplant,three leaf stage,five leaf stage,and seven leaf stage.A total of 367differentially expressed genes（DEGs）were identified by comparative transcriptome analysis of two groups of materials with sustained slow and rapid root growth rates at four developmental stages.The DEGs were significantly enriched in plant cell wall synthesis,energy metabolism processes,and response to biotic/abiotic stress pathways.A total of485 stage-specific DEGs were obtained by comparative transcriptome analysis of two groups of materials with first slow then fast and first fast then slow root growth rate at four developmental stages.These DEGs were mainly enriched in nitrogen metabolism biological processes.By combining GWAS and transcriptomic analysis,this study preliminarily elucidated the main regulatory pathways of the two root dynamic mechanisms of persistent and stage-specific development in rapeseed,laying a foundation for further exploration of the genetic factors of persistent and stage-specific root development in rapeseed.2.Excavation and fine mapping of the persistent major root morphology locus Rt.C08-1.The GWAS result suggested that the SNP marker（Bn-Scaff＿161971-P1015150）with the highest phenotypic contribution rate in Rt.C08-1 had CC and TT genotypes in the natural population,and the SFW,RFW,PRL,TSA,and TRV of CC genotype were significantly higher than those of TT genotype.In addition,three KASP markers were developed according to the peak SNP as well as SNPs Bn-scaff＿16197＿1-p1120033 and Bn-scaff＿16197＿1-p974284 along the left and right border of the Rt.C08-1.Through the foreground selection of KASP markers in Rt.C08-1,the BC₃F₂and F_2:7near-isogenic lines（NIL）were constructed according to Rt.C08-1 using two groups of materials（8S209（large root system）and 8S158（small root system）,8S326（large root system）and 8S325（small root system））with significant differences in RFW and TRL in natural population and inconsistent genotype of Rt.C08-1,respectively.The results of phenotypic investigation in hydroponics and field environment showed that the root trait values RFW,TSA,SFW,yield per plant,number of branches,and total number of siliques of the AA genotypes（NIL-8S209 and NIL-8S326）were significantly higher than those of the BB genotypes（NIL-8S158 and NIL-8S325）.Based on the results of genotypic and phenotypic identification of the NILs,Rt.C08-1 was localized between marker C08-1-1and marker C08-1-3 whose physical distance was 145 Kb.3.Candidate gene mining of the Rt.C08-1.According to the genome annotation information of B.napus and the sequence analysis of ZS11 BAC,a total of 18 annotated genes were identified from Rt.C08-1,6 of which were highly expressed in roots with sequence difference in promoter,exon,or 3’UTR regions between NILs.Among Arabidopsis lines over-expressing the 6 candidate genes,only the lines overexpressing BnaC08.AGP2（Arabinogalactan protein 2）exhibited significantly higher RFW,PRL,and TNR than wild type.Consistently,the Arabidopsis mutant Atagp2 showed shorter PRL,less TNR,and lower RFW than wild type.4.Genetic variation of BnaC08.AGP2.BnaC08.AGP2 mainly had two genotypes in the NILs,of which AA genotype displayed 6 bp sequence deletion at-1008 bp upstream of ATG,and 38 bp and 17 bp insertion at 350 bp and 493 bp downstream of TGA,respectively.A KASP marker was developed according to the 3’UTR sequence difference and used for single gene association analysis in the natural population.The result showed that the traits values of SFW,RFW,TSA,and TRV of AA genotype were significantly higher than those of BB genotype.Dual luciferase experiment results indicated no significant difference in promoter activity.However,the 3’UTR of BnaC08.AGP2inhibited the translation of firefly luciferase,and the inhibitory effect of the 3’UTR sequence of BB genotype was stronger than that of the AA genotype.Based on this,we speculated that the sequence variation in the 3’UTR of BnaC08.AGP2 might lead to the difference in protein translation,thus resulting in differences in root development in near-isogenic lines and the natural population.Now,T₁generation plants have been obtained by transforming gene editing,overexpression,and genetic complementation vectors of BnaC08.AGP2 into rapeseed NILs,and their phenotype could be further identified.5.Functional analysis of BnaC08.AGP2.Genetic complementation test of Arabidopsis mutant Atagp2 showed that the insertion of full-length sequences of two genotypes of BnaC08.AGP2 restored the root phenotype of Atagp2.The DEGs in comparison of Atagp2 mutant line vs.wild-type were mainly enriched in cell wall synthesis and hormone response pathways.The subcellular localization showed that both BnaC08.AGP2 and AtAGP2 were localized to the cell membrane.A total of 38 proteins potentially interacting with AGP2 were screened from the root yeast library of Arabidopsis,20 of which were verified by point-to-point yeast two-hybrid,and these 20proteins included nucleolar regulator NUC-L2 and AGP family proteins such as AGP1,AGP14,and AGP22.Similar to the dynamic regulation mechanism of persistent root development in rapeseed,functional annotation of interacting proteins and comparative transcriptome analysis suggested that AGP2 might regulate root development by modulating the synthesis of root cell wall development and hormone response.