Multiple FLC haplotypes defined by independent cis-regulatory variation underpin life history diversity in Arabidopsis thaliana

Abstract

Relating molecular variation to phenotypic diversity is a central goal in evolutionary biology. In Arabidopsis thaliana, FLOWERING LOCUS C (FLC) is a major determinant of variation in vernalization--the acceleration of flowering by prolonged cold. Here, through analysis of 1307 A. thaliana accessions, we identify five predominant FLC haplotypes defined by noncoding sequence variation. Genetic and transgenic experiments show that they are functionally distinct, varying in FLC expression level and rate of epigenetic silencing. Allelic heterogeneity at this single locus accounts for a large proportion of natural variation in vernalization that contributes to adaptation of A. thaliana.

Keywords: FLOWERING LOCUS C; adaptation; allelic heterogeneity; noncoding polymorphism; vernalization.

Figure 1.

Different FLC haplotypes in 1307 worldwide A. thaliana accessions. (A) Color-coded groups are indicated with numbers. The rare Hap14 and Hap16–18 are not marked. (B) Haplotype structure of the ±50-kb FLC region in 1307 global A. thaliana accessions. Each accession is represented in a column, and each SNP in the FLC ±50-kb window is represented in a row. At each SNP position, colors indicate the most likely haplotype membership for each accession as determined by fastPHASE analysis. Accessions are ordered by haplotype at the FLC locus itself, which is delineated by solid black horizontal lines. The prominent vertically oriented color blocks indicate that haplotype sharing among accessions often extends beyond the FLC locus itself. (C) Latitude and longitude of the collection site corresponding to the accessions above (Horton et al. 2012). (D–I) Geographical distribution of collection sites of A. thaliana accessions carrying the five predominant and one intermediate frequency FLC haplotypes. (J) Centroid distribution of the FLC haplotypes.

Figure 2.

Functional differentiation of FLC haplotypes. (A) Distribution of genetic polymorphism (the mean [±SEM] proportion of polymorphic sites) across the 10-kb FLC region. (B) Haplotype analysis of 10-kb FLC genomic sequences from 47 A. thaliana accessions using FLUXUS network analysis. Circle size illustrates the frequency of the corresponding FLC haplotype. The number along the branch shows the number of nucleotide differences. Numbers in brackets indicate the corresponding haplotype numbers. (C) FLC expression of haplotypes containing FLC alleles with a slow vernalization (SV, black symbols) or rapid vernalization (RV, white symbols) response. Expression values shown are mean (±SEM, n = 2–13) from plants given 4 wk of cold followed by 10 d (T10) or 30 d (T30) of warm. Significant differences in vernalization response exist between haplotypes at 10 d (T10) and 30 d (T30) (Kruskal-Wallis test: H ≥ 18.42, d.f. = 5, P ≤ 0.002 in all tests). (D) QTL analysis on populations generated from crosses between accessions containing different FLC haplotypes. Dashed horizontal lines show the significance thresholds. The FLC region is indicated by the vertical dashed lines. (E) Summary of the presence of a QTL in the FLC region from D and Supplemental Table S3. The “✓” symbol indicates that there is a QTL in the FLC genomic region in the F2 population; the “×” indicates that there is no QTL.

Figure 3.

Transgenic analysis shows that noncoding FLC polymorphism plays a central role in vernalization response of natural Arabidopsis accessions. (A) Prevernalization FLC expression (nonvernalizaion [NV]) in transgenic lines correlates weakly with natural accessions. (B) Relative FLC expression [T30/(T30 + T10)] in transgenic lines correlates significantly with expression in natural accessions. R² shows the explained amount of variation; the corresponding P-values are 0.201 and 0.025 in A and B. (C) Schematic illustration of FLC polymorphisms and the constructs of R2 chimeras. The SNPs defining the different FLC haplotypes are shown by the vertical bars. The bar transparency indicates the diversity of each SNP within each haplotype. The bars shared between haplotypes signify shared SNPs. The FLC gene structure is shown above: Filled black squares are exons, and horizontal lines are introns and untranslated regions. RSR and SRS chimeras show the exchange of R2 regions between Edi-0 and Lov-1 FLC alleles. (D) FLC expression analysis of transgenic FRI flc-2 plants containing chimeric FLC alleles. Plants were given either no cold (NV) or 4 wk of cold followed by 10 d (T10) or 30 d (T30) of warm. Values are means ±standard error from three biological repeats.

Figure 4.

Comparison of total seed production between plants with different vernalization responses. (A,B) Comparison of seed yield from F2 plants homozygous or heterozygous for the different FLC alleles after 4 wk (A; ANOVA, R² = 0.41, F₂₂₂₇ = 80.43, P < 0.001) and 12 wk (B; ANOVA, R² = 0.028, F₂₂₇₆ = 4.97, P = 0.008) of vernalization. Crosses indicate statistical outliers that fall more than three standard errors away from the mean. (C–E) Seed proxy values as a measure of fitness of field-grown accessions. The data set included only accessions with active FRIGIDA alleles (16 accessions from haplotypes RV1 and RV2 and 23 accessions from haplotype SV1-4). FLC haplotypes with rapid and slower vernalization response phenotypes were analyzed. Differences in yield were measured in field experiments in Norwich as in Fournier-Level et al. (2011). P-values are calculated from population structure-corrected ANOVA (see the Materials and Methods) (C; P = 0.0023), summer sowing (D; P = 0.00017), and fall sowing (E; P = 0.99).