High-resolution association mapping with libraries of immortalized lines from ancestral landraces

Abstract

Association mapping with immortalized lines of landraces offers several advantages including a high mapping resolution, as demonstrated here in maize by identifying the causal variants underlying QTL for oil content and the metabolite allantoin. Landraces are traditional varieties of crops that present a valuable yet largely untapped reservoir of genetic variation to meet future challenges of agriculture. Here, we performed association mapping in a panel comprising 358 immortalized maize lines from six European Flint landraces. Linkage disequilibrium decayed much faster in the landraces than in the elite lines included for comparison, permitting a high mapping resolution. We demonstrate this by fine-mapping a quantitative trait locus (QTL) for oil content down to the phenylalanine insertion F469 in DGAT1-2 as the causal variant. For the metabolite allantoin, related to abiotic stress response, we identified promoter polymorphisms and differential expression of an allantoinase as putative cause of variation. Our results demonstrate the power of this approach to dissect QTL potentially down to the causal variants, toward the utilization of natural or engineered alleles in breeding. Moreover, we provide guidelines for studies using ancestral landraces for crop genetic research and breeding.

Fig. 1

Molecular diversity and phenotypic variation of doubled haploid libraries of six maize ancestral landraces. a Principal coordinate analysis and neighbor-joining tree based on genome-wide marker data for doubled haploid lines from six ancestral landraces and elite Flint lines (EF). The number of lines per group is given in parentheses; percentages in parentheses refer to the proportion of genotypic variance explained by the first and second principal coordinate. b Phenotypic variation of four agronomic traits (EV early vigor; KRN kernel row number; FUS Fusarium resistance; OC oil content) and four metabolites shown as normalized trait values for the 358 ancestral landrace (AL) doubled haploid lines and 51 elite Flint lines

Fig. 2

Rapid decay of linkage disequilibrium in ancestral landraces. Linkage disequilibrium (LD) decay with physical distance shown for the ancestral landraces (AL), the elite Flint (EF) lines, and for each landrace separately. The distance after which LD decays below the threshold of 0.2 (dashed horizontal line) is indicated by the dotted vertical lines

Fig. 3

Genome-wide results of association mapping in the landrace panel. Manhattan plots for the results from association mapping in the 358 doubled haploid lines from landraces shown for four agronomic traits (OC oil content; FUS Fusarium resistance; KRN kernel row number; EV early vigor) and four metabolites (Alla, allantoin; Stig, stigmastan; Gala, galacturonic acid; UN, UN_322_870). The red lines indicate the significance threshold (Bonferroni corrected P < 0.05), significant associations are shown in red. Chromosomes used for further fine-mapping are highlighted red

Fig. 4

Identification of candidate genes and potential causal variants. a Fine-mapping of the oil content QTL on chromosome 6 and identification of DGAT1-2 (Zm00001d036982, highlighted in green) with the causal phenylalanine insertion at position 469. The dashed horizontal line indicates the significance threshold (Bonferroni corrected P < 0.05), significant associations are shown in red. The boxplots show the effect of this insertion/deletion (Ins/Del) in the ancestral landraces (AL), each of the landraces, and elite Flint lines (EF). + , *, **, significant at P < 0.1, 0.05, 0.01, respectively. b Fine-mapping of the QTL for allantoin content on chromosome 10 and identification of potentially causal polymorphisms in the allantoinase gene (Zm00001d026635) and promoter. The boxplots show the effect of the TCA insertion/deletion in the promoter region at position -350

Fig. 5

Differential expression of the Allantoinase. Allantoinase mRNA expression in shoots and roots of 20 field-grown lines from the landrace ‘Walliser’ (WA) with either the insertion or deletion at the promoter position − 350. Ins and Del show the relative expression of all lines carrying the insertion or deletion, respectively

Fig. 6

Schematic roadmap for association mapping in landraces. Numbers are given exemplarily for maize. Figure adapted from Mayer et al. (2020)