Quantitative trait locus mapping combined with variant and transcriptome analyses identifies a cluster of gene candidates underlying the variation in leaf wax between upland and lowland switchgrass ecotypes

Abstract

Mapping combined with expression and variant analyses in switchgrass, a crop with complex genetics, identified a cluster of candidate genes for leaf wax in a fast-evolving region of chromosome 7K. Switchgrass (Panicum virgatum L.) is a promising warm-season candidate energy crop. It occurs in two ecotypes, upland and lowland, which vary in a number of phenotypic traits, including leaf glaucousness. To initiate trait mapping, two F₂ mapping populations were developed by crossing two different F₁ sibs derived from a cross between the tetraploid lowland genotype AP13 and the tetraploid upland genotype VS16, and high-density linkage maps were generated. Quantitative trait locus (QTL) analyses of visually scored leaf glaucousness and of hydrophobicity of the abaxial leaf surface measured using a drop shape analyzer identified highly significant colocalizing QTL on chromosome 7K (Chr07K). Using a multipronged approach, we identified a cluster of genes including Pavir.7KG077009, which encodes a Type III polyketide synthase-like protein, and Pavir.7KG013754 and Pavir.7KG030500, two highly similar genes that encode putative acyl-acyl carrier protein (ACP) thioesterases, as strong candidates underlying the QTL. The lack of homoeologs for any of the three genes on Chr07N, the relatively low level of identity with other switchgrass KCS proteins and thioesterases, as well as the organization of the surrounding region suggest that Pavir.7KG077009 and Pavir.7KG013754/Pavir.7KG030500 were duplicated into a fast-evolving chromosome region, which led to their neofunctionalization. Furthermore, sequence analyses showed all three genes to be absent in the two upland compared to the two lowland accessions analyzed. This study provides an example of and practical guide for trait mapping and candidate gene identification in a complex genetic system by combining QTL mapping, transcriptomics and variant analysis.

Fig. 1

Distribution of markers (per Mb, height min = 0, height max = 30) in, from outer ring to inner ring, the HH, maternal (HA) and paternal (AH) maps generated in Pop1 (a) and Pop2 (b) relative to the switchgrass AP13 reference genome (colored blocks) (color figure online)

Fig. 2

Circos diagrams showing the relationship of switchgrass AP13 genome assembly v4.0 (a, c) and assembly v5.1 (b, d) with the HH linkage map generated in Pop1 (a, b) and the HH linkage map generated in Pop2 (c, d)

Fig. 3

a Drop shape image at 5 cm from the base of the leaf blade on the abaxial leaf surface of AP13, VS16, and PV304; b Drop contact angle measurements at 5 cm from the base of the leaf blade on the abaxial leaf surface of AP13, VS16, PV304 and PV346; c QTL identified on Chromosome 7K for drop contact angles on the abaxial leaf surface and for visual scores of abaxial leaf waxiness

Fig. 4

Graphical view of the BLASTN results on Chr07K using (a) Pavir.7KG077009 genomic sequence as query; (b) Pavir.7KG030500 genomic sequence as query; (c) an ~ 70 Kb region comprising Pavir.7KG077009 and the paralog of Pavir.7KG013754/Pavir.7KG030500 [indicated with ‘2’ in (b)] located most closely to Pavir.7KG077009; the region corresponding to Pavir.7KG077009 on the ~ 70 Kb query sequence and corresponding blast hits are highlighted in blue; the region corresponding to the paralog of Pavir.7KG013754/Pavir.7KG030500 on the ~ 70 Kb query sequence and the corresponding blast hits are highlighted in yellow. Blue lines indicate homology on the same DNA strand, and red lines indicate homology on the opposite DNA strand (color figure online)

Fig. 5

Circos diagrams showing a From outer to inner ring, the gene distribution on Chr07K (number of genes per 200 Kb, height min = 0, height max = 40), percentage of Chr07K genes with a homoeolog on switchgrass Chr07N (per 200 Kb, height min = 0, height max = 100), and percentage of Chr07K genes with a paralog in the switchgrass genome (excludes homoeologs) (per 200 Kb, height min = 0, height max = 100); b From outer to inner ring, the percentage of NPs that are missing in ≥ 50% of lowland accessions (per 10 Kb, height min = 0, height max = 100), percentage of NPs that are missing in ≥ 50% of upland accessions (per 10 Kb, height min = 0, height max = 100), the gene distribution (number of genes per 10 Kb, height min = 0, height max = 25), and the repeat distribution (number of repeats per 10 Kb, height min = 0, height max = 40). The wax region is highlighted in yellow (color figure online)