High-resolution genetic mapping of allelic variants associated with cell wall chemistry in Populus

Abstract

Background: QTL cloning for the discovery of genes underlying polygenic traits has historically been cumbersome in long-lived perennial plants like Populus. Linkage disequilibrium-based association mapping has been proposed as a cloning tool, and recent advances in high-throughput genotyping and whole-genome resequencing enable marker saturation to levels sufficient for association mapping with no a priori candidate gene selection. Here, multiyear and multienvironment evaluation of cell wall phenotypes was conducted in an interspecific P. trichocarpa x P. deltoides pseudo-backcross mapping pedigree and two partially overlapping populations of unrelated P. trichocarpa genotypes using pyrolysis molecular beam mass spectrometry, saccharification, and/ or traditional wet chemistry. QTL mapping was conducted using a high-density genetic map with 3,568 SNP markers. As a fine-mapping approach, chromosome-wide association mapping targeting a QTL hot-spot on linkage group XIV was performed in the two P. trichocarpa populations. Both populations were genotyped using the 34 K Populus Infinium SNP array and whole-genome resequencing of one of the populations facilitated marker-saturation of candidate intervals for gene identification.

Results: Five QTLs ranging in size from 0.6 to 1.8 Mb were mapped on linkage group XIV for lignin content, syringyl to guaiacyl (S/G) ratio, 5- and 6-carbon sugars using the mapping pedigree. Six candidate loci exhibiting significant associations with phenotypes were identified within QTL intervals. These associations were reproducible across multiple environments, two independent genotyping platforms, and different plant growth stages. cDNA sequencing for allelic variants of three of the six loci identified polymorphisms leading to variable length poly glutamine (PolyQ) stretch in a transcription factor annotated as an ANGUSTIFOLIA C-terminus Binding Protein (CtBP) and premature stop codons in a KANADI transcription factor as well as a protein kinase. Results from protoplast transient expression assays suggested that each of the polymorphisms conferred allelic differences in the activation of cellulose, hemicelluloses, and lignin pathway marker genes.

Conclusion: This study illustrates the utility of complementary QTL and association mapping as tools for gene discovery with no a priori candidate gene selection. This proof of concept in a perennial organism opens up opportunities for discovery of novel genetic determinants of economically important but complex traits in plants.

Figure 1

LOD score profiles (solid lines) showing QTLs on Populus chromosome XIV for lignin content, S/G ratio, 5-carbon (C5) and 6-carbon (C6) sugars and co-location of QTL peaks with SNP-trait associations (closed circles) for the (A) amino acid transporter, (B) copper transport ATOX1-like, and (C) Ca ²⁺ transporting ATPase genes.

Figure 2

Partial protein and cDNA alignments of alternate alleles showing positions and effects of polymorphisms in the (A) KANADI transcription factor, Potri.014G037200; (B) Angustifolia CtBP transcription factor, Potri.014G089400; and (C) protein kinase, Potri.014G142700.

Figure 3

Differences in activation of reporter genes CesA8 (cellulose), GT43B (hemicellulose) and CCoAOMT1 (lignin) by allelic variants of the (A) KANADI transcription factor, (B) Angustifolia CtBP transcription factor, and (C) protein kinase. Error bars indicate standard deviations based on three replicates.