Genetic Architecture of Heterophylly: Single and Multi-Leaf Genome-Wide Association Mapping in Populus euphratica

Abstract

Heterophylly is an adaptive strategy used by some plants in response to environmental changes. Due to the lack of representative plants with typical heteromorphic leaves, little is known about the genetic architecture of heterophylly in plants and the genes underlying its control. Here, we investigated the genetic characteristics underlying changes in leaf shape based on the model species, Populus euphratica, which exhibits typical heterophylly. A set of 401,571 single-nucleotide polymorphisms (SNPs) derived from whole-genome sequencing of 860 genotypes were associated with nine leaf traits, which were related to descriptive and shape data using single- and multi-leaf genome-wide association studies (GWAS). Multi-leaf GWAS allows for a more comprehensive understanding of the genetic architecture of heterophylly by considering multiple leaves simultaneously. The single-leaf GWAS detected 140 significant SNPs, whereas the multi-leaf GWAS detected 200 SNP-trait associations. Markers were found across 19 chromosomes, and 21 unique genes were implicated in traits and serve as potential targets for selection. Our results provide novel insights into the genomic architecture of heterophylly, and provide candidate genes for breeding or engineering P. euphratica. Our observations also improve understanding of the intrinsic mechanisms of plant growth, evolution, and adaptation in response to climate change.

Keywords: Populus euphratica; genome-wide association study (GWAS); geometric morphometrics; heterophylly; leaf shape.

FIGURE 1

Types and spatial distribution pattern of heterophylly in Populus euphratica. (A) An example of the four types of heterophylly in P. euphratica. (B) The spatial distribution of four types of heterophylly in the canopy of mature in P. euphratica.

FIGURE 2

Descriptive data and shape distribution and MANOVA test for heterophylly, the letters (a, b, c, and d) indicate multiple comparison results at the significant level 0.05. (A) Descriptive data distribution and MANOVA for the descriptive data of heterophylly. (B) Shape distribution and MANOVA for the shape of heterophylly. The light pink thin line denotes the shape of heterophylly for each genotype, red thick line denotes the mean shape of all genotypes for four types of heterophylly.

FIGURE 3

Principal component analysis (PCA) analysis calculated from the landmark data of 860 genotypes for heterophylly. (A) The barplot of PC contributions. (B) Biplot of the first and second principal components. The square, circle, triangle, and diamond symbols represent the linear, lanceolate, ovoid, and broad-ovate leaves, respectively. (C) The mean shape of four heterophyllous leaves on the first five PCs.

FIGURE 4

Genome-wide distribution of P. euphratica SNP loci detected based on single-leaf GWAS. (A) Distribution of the number of significant SNPs across each chromosome for all traits. (B) Manhattan plot displaying the GWAS result of the trait ‘Ovoid: Length width’ based on the LM model. The red triangle indicates the significantly associated SNPs. Redline indicates the threshold with permutation tests at 0.05.

FIGURE 5

Genome-wide association mapping of the leaf width in P. euphratica. (A) Manhattan plot displaying the GWAS result of the trait ‘leaf width’ based on the single-leaf GWAS. The significantly associated SNP markers are labeled. (B) Histograms of leaf width of different leaf types plotted as a function of genotypes at SNP255877. (C) Histograms of allelic effects of SNP255877 for the heterophylly.

FIGURE 6

Multi-leaf GWAS for the PC1 of leaf structure in P. euphratica. (A) Manhattan plot displaying the GWAS result of the PC1 based on the multi-leaf GWAS. The significantly associated SNP markers are labeled. (B) Leaf outline of PC1 of heterophylly plotted as a function of genotypes at SNP64867. (C) Visualized variation in the shape of four heterophyllous leaves in P. euphratica at a significant SNP (SNP64867 on scaffold NW_011500277.1).