Longer epidermal cells underlie a quantitative source of variation in wheat flag leaf size

Summary The wheat flag leaf is the main contributor of photosynthetic assimilates to developing grains. Understanding how canopy architecture strategies affect source strength and yield will aid improved crop design. We used an eight‐founder population to investigate the genetic architecture of flag leaf area, length, width and angle in European wheat. For the strongest genetic locus identified, we subsequently created a near‐isogenic line (NIL) pair for more detailed investigation across seven test environments. Genetic control of traits investigated was highly polygenic, with colocalisation of replicated quantitative trait loci (QTL) for one or more traits identifying 24 loci. For QTL QFll.niab‐5A.1 (FLL5A), development of a NIL pair found the FLL5A+ allele commonly conferred a c. 7% increase in flag and second leaf length and a more erect leaf angle, resulting in higher flag and/or second leaf area. Increased FLL5A‐mediated flag leaf length was associated with: (1) longer pavement cells and (2) larger stomata at lower density, with a trend for decreased maximum stomatal conductance (G smax) per unit leaf area. For FLL5A, cell size rather than number predominantly determined leaf length. The observed trade‐offs between leaf size and stomatal morphology highlight the need for future studies to consider these traits at the whole‐leaf level.


Fig. S1
Example stems of the eight MAGIC founders at anthesis.

Table S5
Flag leaf phenotypic data. BLUPs for the four field trials (columns C to S) and for the meta-analysis (columns).         Tables S1-S11 Tables S1-S11 provided in separate Excel file.
Methods S1 Supplementary text for the Materials and Methods and Results sections.

NIAB Elite MAGIC population trial design methods
The 2016

Genetic analysis methods
For IBS and IBD, multiple-test correction was carried out using the 'p.adjust' function in R, with

KASP genetic marker validation methods
KASP marker BS00062996_51 was validated using DNA from each of the eight founders extracted from leaf material using a Plant Mini Kit (Qiagen). KASP genotyping was undertaken as described by Downie et al. (2018) using three technical replicates per founder. Additionally, a no-template negative control was assessed, as well as a 50:50 mix of DNA from two founders known to contrast for allelic state to create an artificial heterozygote at the target SNP.

Scanning electron microscopy methods
Leaf sections were plunge-frozen in liquid nitrogen-cooled ethane and freeze-dried using a liquid-nitrogen cooled turbo freeze drier (Quorum K775X). Samples were mounted on aluminium SEM stubs using double-sided sticky tape and silver paint and were sputter coated with 35 nm gold using a Quorum K575X sputter coater. Samples were viewed using a Verios 460 scanning electron microscope (FEI/Thermo Fisher Scientific) run at 30 keV and 3.2 nA using the concentric backscatter detector in field-free mode.

NIL germplasm development methods
Comparison of the patterns homozygous and heterozygous genotypic calls for SNPs spanning the QTL with the corresponding allele calls in the eight founders allowed identification of the founders contributing to the observed heterozygous chromosomal regions. The donor founders for each of these RILs were cross-referenced with the predicted founder allelic affects at the 5A QTL, as determined by CIM analysis. Accordingly, RILs heterozygous across the target QTL were prioritised based on: (i) presence of founder alleles predicted to maximise phenotypic contrast, and (ii) minimising the genetic/physical interval heterozygous across the target QTL.

NIL field growth and phenotyping methods
NIL trials followed standard local agronomic practices, including full fungicide and pesticide treatment.

NIL glasshouse experiment growth methods
In the 2018 glasshouse experiment (GH 2018), twelve replicates for each of the four NIL germplasm stocks, as well as twelve replicates for each MAGIC founder, were randomised into 2 main blocks and 19 sub-blocks. In the 2021 glasshouse experiment (GH 2021), 22 replicates of each of the two lines were sown across two main blocks, with four sub-blocks per main block.

A priori candidate gene results
Wheat homologue of rice leaf angle gene SMOS1 was located within flag leaf angle QTL pericentromeric multi-trait genetic locus M8 controlling wheat flag leaf length, width, area and length. Some aspects of gene expression profiles as determined in the wheat gene expression atlas were broadly similar for all four genes, with strong expression in the shoot apical meristem (which included the leaf primordia) at the first leaf stage as well as in the spike at full boot stage (Fig. S3). Of note, three of the genes (TaSMOS1-D, TaCslD4-A and TaNRL1-A) also showed increased expression in the shoot apical meristem (which included the leaf primordia) at the tillering sage, in the shoot axis at the flag leaf stage, and in the ovary at anthesis.

TaSMOS1-D and
TaCslD4-A also showed increased expression in the roots and root meristem at the tillering stage. Remaining wheat homologues of cloned cereal leaf size/angle genes were located within QTL identified in a single year only, and are not discussed.