Genome-Wide Association Studies Of Heterophylly In Populus Euphratica

As one of the most important organs in plants,leaves display great variability in size and shape.To better adapt to specific ecological environments,such as drought,strong light,and high salinity,plants often produce unique physiological mechanisms that drive leaf morphology to vary between different positions or different stages of development for the same individual.This phenomenon,known as heterophylly,has long been a research focus in plant biology.There has been a considerable body of literature on the formation mechanism of heterophylly and whether and how it reflects the adaptability of plants to ecological environments.However,few has been reported on the genetic architecture of heteromorphic traits,which has limited our understanding of how heterophylly is formed.This study uses Populus euphratica as the study material to deeply explore the genome-wide pattern of genetic control over heterophylly.P.euphratica is the only woody species that can grow in the northwest desert area of China.This plant generates the heteromorphic leaves as its long-term adaptation to salinity and drought The main feature of heterophylly is that leaf shape vary from broadly ovate and ovate at the upper part of canopy to lanceolate and striate at the lower part of canopy.This work is the first of its kind to investigate the detailed genetic architecture of heterophylly in P.euphratica based on a GWAS approach.A total of 29 significant SNPs are identified for heterophylly variation,many of which reside in the genomic region of candidate genes.These discoveries provide a foundation for further studies on stress resistance and could be of great use for molecular marker-assisted selection breeding programs of P.euphratica.Main results of this study are described as follows(1)In this study,the epicormic branch of 741 samples of P.euphratica were collected for hydroponication We gathered the leaves from the hydroponic branches,extracted DNA from the leaves,and resequenced the whole genome.Finally,185,740 SNPs(Single Nucleotide Polymorphism)were obtained through quality inspection and filtration.We used fastStructure software to analyze the population genetic structure,and 741 samples could be divided into a subpopulation(2)We collected and photographed the leaves from the upper,middle-upper,middle-lower and lower locations of the plants.The leaf phenotypes were quantified by the image processing techniques and we obtained the informations about shape,length,width,length-width ratio and area of leaves from the photographs.All plants were found to have heterophylly characteristics.The Elliptic Fourier method was used to fit all the leaf shapes.The inter-group anova analysis of the four location leaves showed the significant differences in leaf shapes between the four locations(P<0.001)and any two locations(P<0.001).The fitted mean shapes of the four location leaf shapes are presented as heterophylly,and the shapes were from broadly ovate to lanceolate.The results of intra-group anova analysis showed the significant differences in the middle-upper,middle-lower,and lower location leaves(P<0.01).The results of intra-group anova and cluster analysis showed that the upper location leaves were divided into one group and the shapes were broadly ovate.The middle-upper location leaves were divided into two group and the shapes were broadly ovate and ovate.The middle-lower location leaves were divided into two groups and the shapes were ovate and lanceolate.The lower location leaves were divided into three groups and the shapes were ovate,lanceolate and striate.(3)We performed GWAS to analyze the leaf length,width,length-width ratio and area of the four location leaves separately and jointly.There were 306 significant SNPs detected for the four traits affecting the four location leaves and heterophylly,and the number of SNPs affecting the traits of heterophylly was 45.24 important candidate genes were screened by functional annotation.Gene SEC6 influenced the length-width ratio of leaves by promoting cell division and cell polarity growth.Gene LOGL3 participated in the biosynthesis of cytokinins and promoted cell division,and this gene might play an important role in the formation of heterophylly of P.euphratica.(4)The contours of the leaf blades were extracted based on the Elliptic Fourier method.And the contours were the phenotypes to perform GWAS analysis.There were 168 significant SNPs detected,and the number of SNPs affecting heterophylly were 22.29 important candidate genes were screened by functional annotation.Gene ACA13 regulatde cell death and LOGL8 promoted cell division.By comparing the leaf shapes of different genotypes of the significant QTLs,and the results showed that there were obvious difference between the leaf shapes in the length and width dirctions.(5)168 significant SNPs based the GWAS analysis using the Elliptic Fourier method were detected 36-83 SNP-SNP interactions,which included 24-56 significant SNPs.The results of the epistatic effect showed,in the length and width dirctions of leaves,the proportions of the epigenetic effects and heritability in the total genetic effects and heritability were larger.(6)We used ordinary differential equation(ODE)to construct the gene regulatory networks for the significant SNP affecting the four location leaves and heterophylly.The results showed that,the number of hub SNPs in gene regulatory networks were 1-4.There were significant differences in the topologies of gene regulation networks constructed by heterophylous SNPs at different locations.We performed GWAS to study low-dimensional phenotypic data(leaf length,width,area and so on)and high dimensional phenotypic data(the contours)reflecting leaf morphology in the four locations separately and jointly.We found a larger number of SNPs affecting leaf shapes and heterophylly,and the epistatic effect played an important role.Through the reconstruction of gene regulation network,the network topologies at different locations were quite different,but each network had hub QTLs,which might be important candidate genes.Throught the gene annotation,we obtained some important candidate genes,such as LOGL3,LOGL8,ACA13 and so on.Gene ACA13 influenced the leaf shapes of middle-lower location,lower location and heterophylly of P.euphratica natural population.This study provided an important direction for the further study in heterophylly of P.euphratica and the molecular mechanism of its stress tolerance.