Genetic Networks Of QTL Affecting The Leaf Growth Of Populus Euphratica Oliv.in Response To Salt Stress

Leaf is an important organ for plants to produce organic nutrients,the shape with leaf plays pronounced role in maintaining plant function,adaptation to the environment and evolution.Populus euphratica Oliv.,one of the constructive and dominant tree species with the desert community,P.euphratica had evolved many excellent characteristics for stress tolerance,e.g.,salt tolerance,drought endurance,and had become an ideal model species with forest trees regarding stree-resistance research.Therefore,it is necessary to reveal the morphological changes and regulation mechanism in response to salt stress from the growth dynamic.It lays a theoretical foundation for gene cloning,functional identification and resistance breeding of P.euphratica.To this end,clonal seedlings that normally developing into complete plants were selected for experimental design,treated with control(CK)and salt stress.To track shape of leaf in a non-invasive way,the morphology reconstruction method using digital image containning leaf and an additional checkerboard was developed,with which,the time and position dependent shape with heterophylly can be recorded during the whole growth season.Genome-wide association study(GWAS)was used to analyze heterophylly based on functional mapping.Functional mapping was used to analyze leaf phenotypic traits of CK and salt stress environments,reveals the potential relevance of heterophylly and leaf dynamic growth with saline tolerance in P.euphratica,providing a new vision to study the saline resistance-related genetic mechanisms.The main results are as follows:(1)Images were used to fully track all morphological changes in the leaves,a set of camera internal parameters and external parameters were extracted.Based on the binarized shape contours converted from the RGB images,contour coordinates of leaf shape on the card or checkerboard plane were calculated to represent its authentic size and morphology,with the aid of three-dimensional reconstruction.The development of shape reconstruction technique had resulted a total of 3677 and 1780 shapes for CK and salt stress,respectively.Phenotype of leaf shape were further extracted such as leaf length,leaf width,and leaf area.(2)We develop a shape tracking method to monitor leaf shape and analyze shape using principal component analysis.PC1 explained 42.18% and 65.41% of variation in control and stress,suggesting shape variation is mainly determined by leaf length.By treating leaf length along shoot axes as a dynamic trait,we implement a functional-mapping assisted GWAS for heterophylly.We identify 171 and 134 significant loci(called QTLs)under control and stress respectively,which can be annotated to candidate genes of stress-resistance,auxin,shape,and disease-resistance related classes.Stress resistance genes of ABSCISIC ACIS-INSENSITIVE 5-like(ABI5),WRKY72 and MAPK3 are functional in linking stress sensing to many tolerance responses.The detection of AUXIN RESPONSE FACTOR17-LIKE(ARF17)suggests a balance between auxin-regulated leaf growth and the stress resistance background within the genome evolved to develop heterophylly.We do not find the same significant genes in both condition,but find genes with similar functions affecting the stress-mediated heterophylly;for example,stress-related genes ABC transporter C family member 2(ABCC2)and ABC transporter F family member(ABCF),and stomata-regulating and ROS signaling gene RESPIRATORY BURST OXIDASE HOMOLOG(RBOH).(3)Function mapping of leaf phenotypic traits showed that 371 and 347 significant QTLs were mapped,and 87 and 79 genes were annotated under CK and salt stress conditions.Genes identified in both environments are mainly involved in leaf cleavage,salt stress response,stomatal regulation,detoxification of stomatal guard cells,and wound-induced cellular reprogramming.Under CK conditions,CHX24,GA,LOG7 and transcription factor TGA1 play important roles in plant stress,maintenance of Arabidopsis shoot apex meristem and normal primary root growth,and improvement of plant disease resistance.(4)QTLs for dynamic phenotypic differences under the two conditions were mapped to 110,145 and89 significant QTLs for leaf length,width and area,respectively.A total of 37,50 and 36 genes were identified.New significant QTLs play an important role in promoting plant growth,leaf senescence,defense regulation,salt stress response,abscisic acid response,signal transduction,programmed cell death,etc.Such as unc-13,SERK2,SPIRAL1,PPRP,ATXR7 and JMJ25.(5)Leaf length,width and area were clustered into 420,408 and 300 functional modules,respectively,and the genetic effects were different among different modules.After the network reconstruction of the clustered modules,the significant QTLs were located in different modules,suggesting that these QTLs basically play a unique role in governing leaf growth.In addition,we found that the distribution of both outgoing and incoming links differs dramatically in the three networks.For example,in leaf length,the module where the significant QTLs were closely related to M396 and M212.In leaf width,the modules of significant QTLs were mainly scattered at the edge of the network,and are related to M128,M215,M296 and M44.In the leaf area,the modules of significant QTLs are mainly scattered at the edge of M250,where they are directly related to M81,M88,M128,M185 and M291,while they are indirectly related to M178,and M132 plays a correlation role in the middle.The salt-tolerant related genes obtained in this study will lay an important theoretical foundation for elucidating the internal mechanism of salt-tolerance from the aspect of molecular function and creating new transgenic germplasm with improved salt tolerance of P.euphratica.