Linking transcript, QTL and association mapping to understand the genetic control of leaf size and shape in Populus

Abstract

Leaf size in Populus is an adaptive trait and early indicator of biomass yield. In order to investigate the genetic variance contributing to this variation in leaf size a collection of Populus nigra L. were made from across Europe and were planted at a single site in Belgium, in a fully randomized and replicated trial, with leaf traits measured in three consecutive years and biomass estimated at one point in time. Results indicate that leaf traits vary with latitude of sample origin, with significant differences observed in leaf area, epidermal cell number and biomass, but not in leaf shape (leaf ratio), epidermal cell area, stomatal density and stomatal index. Overall a significant positive relationship between latitude of origin and leaf traits was observed with small-leaved genotypes containing fewer epidermal cells observed in the south west (Spain), and large-leaved genotypes occurring in the north and east (the Netherlands, Germany and Italy). A sequence-based genetic study was conducted to identify Single Nucleotide Polymorphisms (SNPs) associated with leaf phenotype. Given that linkage disequilibrium (LD), decays rapidly (r2 = 0.09) in P.nigra, a candidate gene approach for association is valid. Candidate genes were selected from Quantitative Trait Loci (QTL), from a microarray experiment and from bioinformatics and literature searches, identifying sixty robust genes. From this list eight candidate genes were selected for further analysis; ASYMMETRIC LEAVES 1 (AS1), ASYMMETRIC LEAVES 2 (AS2), ACC OXIDASE ( ACO), ERECTA (ER), PHABULOSA (PHAB), ANGUSTOFOLIA (AN), E2Fc and LEAFY. Genetic association was conducted using a General Linear Model (GLM) both with and without population structure. The strongest genetic association was found in AS1, a gene involved in leaf initiation that acts by repressing KNOX genes to increase cell differentiation. Gene expression of the eight candidate genes were examined across extreme leaf genotypes using real time qPCR (RT-qPCR), at three growth stages. Extreme leaf genotypes consisted of five „small? and five „big? leaf genotypes selected from the association population. Significant differences in gene expression was seen between „small? and „big? genotypes in AN, AS2 and AS1. These results suggest that AS1 is a strong candidate gene for leaf size