Population genomics and haplotype analysis in spelt and bread wheat identifies a gene regulating glume color

Abstract

The cloning of agriculturally important genes is often complicated by haplotype variation across crop cultivars. Access to pan-genome information greatly facilitates the assessment of structural variations and rapid candidate gene identification. Here, we identified the red glume 1 (Rg-B1) gene using association genetics and haplotype analyses in ten reference grade wheat genomes. Glume color is an important trait to characterize wheat cultivars. Red glumes are frequent among Central European spelt, a dominant wheat subspecies in Europe before the 20^th century. We used genotyping-by-sequencing to characterize a global diversity panel of 267 spelt accessions, which provided evidence for two independent introductions of spelt into Europe. A single region at the Rg-B1 locus on chromosome 1BS was associated with glume color in the diversity panel. Haplotype comparisons across ten high-quality wheat genomes revealed a MYB transcription factor as candidate gene. We found extensive haplotype variation across the ten cultivars, with a particular group of MYB alleles that was conserved in red glume wheat cultivars. Genetic mapping and transient infiltration experiments allowed us to validate this particular MYB transcription factor variants. Our study demonstrates the value of multiple high-quality genomes to rapidly resolve copy number and haplotype variations in regions controlling agriculturally important traits.

Fig. 1. Spike morphology and spikelet disarticulation in spelt.

a Representative spikes of spelt accessions collected in Central Europe, Asia, and the Iberian Peninsula. Scale bar = 5 cm. b Spikelet disarticulation of spelt. Spikelet disarticulation in plants with brittle rachis can be classified as barrel-type (upper rachis segment pressed against the lower spikelet) and wedge type (rachis segment pointing down). Central European spelt shows a barrel-type disarticulation, whereas disarticulation in Asian and Iberian spelt is of the wedge type. Scale bar = 2 cm. Arrows point to the rachis segment after spikelet disarticulation.

Fig. 2. Whole-genome analysis of spelt and bread wheat.

a Principal component analysis (PCA) across the 339 spelt and bread wheat accessions. Samples are colored according to subspecies and geographical origin. b Maximum-likelihood tree constructed with SNPhylo. The colors used to label the accessions are identical to a and the branch size is indicated below the tree. c ADMIXTURE ancestry coefficients (K = 3 and K = 7) for the 339 accessions of spelt and bread wheat. Stacked bars represent accessions and colors represent ancestry components. Accessions are ordered according to subspecies and geographic origin.

Fig. 3. Haplotype variation at the red glume Rg-B1 locus.

a Images of a white glume spelt accession (right) and a red glume spelt accession (left). b Manhattan plot showing a significant association for glume color at the Rg-B1 locus on chromosome arm 1BS. The physical confidence interval spans positions 2.24–4.17 Mb in the RefSeq v1.0 assembly of Chinese Spring. The region contains a MYB transcription factor gene (TraesCS1B02G005200). c Copy number and allele variation for the candidate MYB transcription factor gene in ten high-quality wheat assemblies. Shown are the first 10 megabases of chromosome arm 1BS in the ten different wheat assemblies (left). Arrows indicate MYB-like transcription factor genes that are paralogous to TraesCS1B02G005200. Colors refer to one of five different allele groups (G1–G5) shown on the right. Group 3 is associated with red glumes in bread wheat and spelt. d Haplotype-specific PCR marker for the group 3 alleles. The red arrow points to the 334 bp amplicon specific for the group 3 Rg-B1 alleles. Scale bar = 1 cm. e Phylogenetic analysis of MYB transcription factors regulating the flavonoid biosynthesis pathway. SG = subgroup based on conserved amino-acid motifs. The specific flavonoids that are linked to each subgroup are indicated.

Fig. 4. Transient expression of Rg-B1 in Nicotiana benthamiana.

a Transcript levels of endogenous flavonoid biosynthetic genes in N. benthamiana infiltrated with the 35 S:GFP vector control and the Rg-B1-overexpressing constructs 35 S:Rg-B1b_h1:GFP and 35 S:Rg-B1b_h3:GFP. Error bars represent standard errors of three biological replicates. b Spectrofluorometric profile of agroinfiltrated leaves pre and post DPBA staining. The region between the two vertical dotted lines from 550 to 650 nm coincides with the previously reported peak for the flavonol quercetin. The peak from ~665 to 685 nm is autofluorescence from chlorophyll/chloroplasts.