Functional variants of DOG1 control seed chilling responses and variation in seasonal life-history strategies in Arabidopsis thaliana

Natural Variation in Seed Chilling Responses Creates Diverse Seasonal Life Histories.

We grew 559 Arabidopsis accessions to reproductive maturity at 14 °C with a 12/12-h photoperiod following 6 wk of vernalization at 4 °C. Fresh seeds were harvested from each plant when siliques matured, and were tested for germination at 22 °C immediately (i.e., base germination), or after 4, 8, 11, 15, 22, or 32 d of dark stratification at 4 °C and 10 °C. We performed a principal component analysis (PCA) on all germination phenotypes. The first 2 principal components (PCs) of germination timing and dormancy level explained 91% of the germination variation (PC1_germ = 83%, PC2_germ = 7%) (Fig. 1 and SI Appendix, Fig. S2). We also measured days to flowering (DTF), and days to senescence (DTS) of the maternal plants. Additionally, we assayed germination of dry-stored seeds at 6-wk intervals to quantify after-ripening requirements (days of seed dry storage required to reach 50% germination, DSDS₅₀) (Table 1).

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Fig. 1.

(A) PC scores of germination variables (base, and cold treatments: 4, 8, 11, 15, 22, and 32 d at 4 and 10 °C). Percent variance explained by each axis: PC1_germ = 83%, PC2_germ = 7%. (B) Mean percent germination in each treatment for the lines with the 10% highest and lowest scores on PC1_germ and PC2_germ. Colors indicate the amino acid sequence at the DOG1 self-binding domain (53), which we use to define haplotypes, and grey color indicates accessions for which we were not able to assign a haplotype.

All germination variables measured were positively associated to PC1_germ, (SI Appendix, Table S1), and ecotypes with a positive score along this axis had overall high germination. Accessions with high overall germination (PC1_germ) were late-flowering and late-senescing and had short after-ripening times, whereas accessions with low germination were early-flowering and early-senescing and had long after-ripening times (Table 1). Variation in overall germination fraction reflects variation in primary dormancy, consistent with previous observations of natural variation in dormancy measured as after-ripening requirement (11, 12, 50). Accessions with high germination were distributed in central and northern Europe (SI Appendix, Fig. S3A). Low dormancy allows immediate germination after dispersal, which is favored by selection in northern climates (13) and may result in multiple generations per year in midlatitudes with wet summers (7, 65). In contrast, primary dormancy that cannot be broken by chilling exposure, correlated with strong after-ripening requirements, would maintain a large population in the seed bank (7), a potential bet-hedging strategy (66). Accessions with low germination were distributed in south and central Spain and southern Europe (SI Appendix, Fig. S3A).

PC2_germ represents the germination response to cold. Base germination and germination percent of all stratification times at 10 °C were positively associated with PC2_germ, while germination percent at 11 to 32 d at 4 °C were negatively associated to this axis (SI Appendix, Table S1). Accessions with a positive score on PC2_germ had high base germination and secondary dormancy induced by prolonged cold exposure at 4 °C (Fig. 1 and SI Appendix, Fig. S2 and Table S1), flowered and senesced later, and had longer after-ripening periods (Fig. 2 and Table 1). These accessions would behave as winter annuals, with germination restricted to late summer or fall, and flowering and seed dispersal the following summer. Many of these accessions occur in Scandinavia and the Iberian Peninsula (SI Appendix, Fig. S3B). In contrast, accessions with a negative score on PC2_germ that break primary dormancy with brief chilling exposure and do not enter secondary dormancy with prolonged chilling also displayed fast phenological transitions and had long after-ripening times, preventing summer germination. Dormancy release by cold would allow germination in both fall and spring, and coupled with rapid reproduction, would facilitate rapid cycling or spring annual life histories. Seeds that do not germinate in fall would overwinter in the soil under cold temperatures and would be ready to germinate the next spring (26). This seasonally flexible germination may be favored in disturbed, ruderal landscapes. These accessions were common at midlatitudes in Europe and England (SI Appendix, Fig. S3B).

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Fig. 2.

Correlations between PC2_germ and DTF (A) and DTS (B). Colors indicate the amino acid sequence at the self-binding domain for different DOG1 haplotypes.

The timing of germination defines the temporal environment an annual plant experiences, and interacts with later life-cycle traits to generate different life histories. Our results are consistent with previous observations of covariation in seed dormancy (measured as after-ripening requirements) and flowering-time traits across climatic gradients in A. thaliana (12, 19, 20, 37). Trait syndromes allow local adaptation, as covariation in life-history traits, growth rate, and stress responses influence fitness (37). However, our observations of germination responses to chilling add a new dimension to this picture of multivariate life histories, showing that cold-induced secondary dormancy is correlated with flowering and senescence to construct a wide range of seasonal life-history strategies.

Genetic Architecture of Seed Responses to Chilling: A Major Role for DOG1.

To understand the genetic architecture of the germination niche, we performed GWA analyses on the germination PCs using 498 fully sequenced A. thaliana accessions and 3,483,598 single nucleotide polymorphisms (SNPs; 1001 Genomes Consortium). A region on chromosome (Chr.) 5 was the most highly associated to both the first and second PCs of germination. This region included 1 SNP significantly associated with PC1_germ and 42 SNPs significantly associated with PC2_germ. Several of these SNPs likely tag the same functional variant, as they are organized into only 4 linkage disequilibrium (LD) blocks spanning multiple candidate genes. Different SNPs were most highly associated to PC1_germ and PC2_germ, although the closest gene to both was DOG1 (Fig. 3 and SI Appendix, Tables S2 and S3). The SNP significantly associated to PC1_germ and PC2_germ (Chr. 5: 18,592,365) (SI Appendix, Table S2) had previously been shown to tag the promoter region of DOG1 and to be associated with after-ripening time variation (50) (SI Appendix, Fig. S4). However, in our experiments, DSDS₅₀ was associated with a different set of SNPs in several LD blocks, which did not include DOG1 (SI Appendix, Fig. S5 and Table S2). Using more permissive thresholds, we found that the top 1,000 SNPs associated to each trait were enriched for seed-specific gene ontologies (SI Appendix, Tables S4 and S5).

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Fig. 3.

Chromosome location (Mb) and association P value from GWAS for the SNPs on PC1_germ, PC2_germ, and DTF (permutation-based significance thresholds are shown by dotted horizontal lines). The vertical dashed grey line indicates the location of DOG1 gene.

As SNPs near DOG1 were associated in both analyses, we further characterized genetic variation at DOG1. Known indel polymorphism within the self-binding domain of DOG1 (amino acid positions 13 to 16) (53) is not fully captured by published SNP data for A. thaliana (43, 45, 46). We therefore locally reassembled exon 1 of DOG1 using raw sequence data from the 1001 Genomes Consortium (43), as well as African (45) and Chinese (46) A. thaliana accessions. This indel appears to be functional, as it likely affects dimerization of DOG1 proteins and alters their incorporation into larger protein complexes (53). Haplotypes in the self-binding domain have been defined by the amino acid variants present. We identified the ancestral self-binding DOG1 E-haplotype (ECCY) and 2 loss-of-function DOG1 D-haplotypes (D-RY, D-SY) previously reported (53), as well as 2 additional rare derived haplotypes without an amino acid deletion in the self-binding domain (ECSY and EFSY) found in less than 5% of individuals and restricted to Iberia (SI Appendix, Figs. S6 and S7). DOG1 haplotype identities are significantly associated with PC2_germ scores (SI Appendix, Table S6A). The loss-of-function D-RY and D-SY DOG1 haplotypes share the reference allele of 3 of the most highly associated SNPs for PC2_germ in the same LD block (Chr. 5: 18,590,327, 18,590,741, 18,590,743), while the alternative allele is associated to the self-binding E-haplotypes (SI Appendix, Fig. S8 and Table S2). Although these SNPs tag the indel and nonsynonymous variant that differentiates D- and E-containing haplotypes, our haplotype genotyping distinguishes further fine-scale genetic differences between accessions. We expect this is particularly important in refining the self-binding ability of the DOG1 protein, which is known to play an important role in the germination response to chilling (53, 59). Accessions carrying the self-binding DOG1 E-haplotypes were characterized by strong secondary dormancy induced by prolonged chilling (high PC2_germ) (Fig. 2); in contrast, the loss-of-function DOG1 D-haplotypes, had strong primary dormancy release and no secondary dormancy induced by cold (low PC2_germ) (Fig. 2).

We tested for epistatic effects of DOG1 on germination PCs by dividing the data into 2 subsets of DOG1 haplotypes (D-haplotypes, n = 158, and E-haplotypes, n = 161) and performing a GWA analysis within each haplotype. We found a peak on Chr. 5 for PC2_germ for the E-haplotype annotated to AT5G65100, an ethylene-insensitive 3 family protein (SI Appendix, Fig. S9A and Table S7). A SNP associated to PC2_germ in accessions carrying the E-haplotype falls within a region on Chr. 5 linked to a fitness quantitative trait loci related to seed coat alterations (67) and seed coat mucilage production (68). Despite not being significantly associated to PC2_germ in the D-haplotype accessions, this SNP has a similar allele frequency among the D- and E-haplotypes (SI Appendix, Table S8). Thus, this association arises not because the causative variant is absent in 1 haplotypic background, but rather because different SNPs have different effect sizes in different backgrounds. The epistatic interaction between the DOG1 E-haplotype and the associated allele in this region of Chr. 5 give additional insight into the complex gene interactions involved in the regulation of germination responses to chilling. Although not measured in our study, it is likely that DOG1 expression levels are involved in the chilling response as well (12, 53, 57, 59, 62, 63, 69⇓⇓⇓–73). Other mechanisms regulating DOG1 expression might also be involved in the chilling germination responses: For example, the antisense transcript asDOG1, which negatively regulates the expression of DOG1 (74, 75).

Life-History Syndromes Resulted from Correlated Selection on Multiple Loci.

Accessions with loss-of-function DOG1 D-haplotypes showed strong primary dormancy release and no secondary dormancy induced by cold, and also flowered and senesced early. In contrast, individuals with self-binding DOG1 E-haplotypes showed secondary dormancy induced by prolonged cold, and flowered and senesced later on average (Fig. 2). Besides PC2_germ, DOG1 haplotypes explained variation in DTF and DTS (SI Appendix, Fig. S10 and Table S6A). Similar D- and E-haplotype germination patterns were observed for geographically restricted samples within Spain and Sweden (SI Appendix, Figs. S11 and S12 and Tables S6 B and C). Pleiotropic effects of DOG1 on flowering and germination mediated by miR156 and miR172 may be a mechanism behind these trait correlations (60, 76).

Our results also showed that GWAs for DTF were polygenic (39 SNPs organized in 11 LD blocks) (Fig. 3 and SI Appendix, Table S2) and SNPs tagging DOG1 were not significantly associated with DTF. DTS showed no associated SNPs above a permutation threshold for significance (SI Appendix, Fig. S5 and Table S2). However, the 5 SNPs associated to PC2_germ tagging DOG1 have been previously associated to different flowering-time phenotypes under various conditions (Fig. 3 and SI Appendix, Fig. S13 and Table S2) (42, 43, 64, 77). The lack of association of DTF with DOG1 in our study could partly be due to the accessions sampled, as we performed an additional GWA analysis using only our accessions with subsampled 1001 Genomes Consortium flowering phenotypes (43) and did not find evidence of association of DTF with SNPs tagging DOG1 (SI Appendix, Fig. S14). Our sampling includes mostly European accessions while the 1001 Genomes phenotypes also include Asian accessions (78). Moreover, the lack of association could also be due to the different vernalization and growth conditions used in each experiment.

We also tested for epistatic effects of DOG1 haplotypes on flowering and senescence and found that SNPs significantly associated with DTF for the E-haplotype were located at different positions from those identified with the whole dataset, and the most significantly associated SNP on Chr. 1 was found closest to AT1G33440, a major facilitator superfamily protein (Fig. 3 and SI Appendix, Fig. S9B and Tables S2, S7, and S8).

Taken together, our results suggest that correlations between seed chilling responses and reproductive timing may result in winter and summer annual life histories. DOG1 variation is critical for cold induction of secondary dormancy that determines seasonal germination timing, and may also have pleiotropic effects on flowering time. However, a number of other loci are important to flowering-time variation, suggesting that the multivariate life-history syndromes we observe may be shaped by correlated selection at multiple loci in addition to DOG1.

Arabidopsis Germination Niche Today Is Explained by Current and Past Climate.

To understand the evolution of DOG1, we constructed a gene tree of the first exon of DOG1, which includes the self-binding domain (Fig. 4A). The earliest DOG1 diversification represented in extant individuals is that of the ancestral and widely distributed ECCY haplotype, associated with traits that promote a winter annual life history. The tree topology showed a deep divergence within the ancestral ECCY clade, especially for relict, African, and Swedish accessions and a few anciently diverged haplotypes in China (Fig. 4A and SI Appendix, Figs. S6 and S7, and Table S9).

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Fig. 4.

(A) Bayesian phylogeny of DOG1 haplotypes. Haplotype names represent amino acid sequence at the self-binding domain of DOG1. Triangle clade heights represent the number of individuals carrying a redundant sequence. The A. lyrata outgroup used to root the phylogeny is not shown. (B) Ages estimated from pairwise nucleotide divergence of DOG1 haplotype groups with Pleistocene deuterium isotope records (103). Confidence intervals represent a range of generation times from 1.3 (±0.3) y per generation. Pairwise divergence of D-RY appears older in B as branch lengths are not weighted by number of individuals during tree-building (Materials and Methods). In both plots, points reflect divergence of haplotypes or groups of haplotypes. Temperatures are presented as deviations from the last 1,000-y average.

The rare ECSY haplotype arose within the ECCY clade, followed by EFSY, during periods of cooler climate after the last interglacial period (Fig. 4A and SI Appendix, Table S9); both of these derived haplotypes are geographically restricted to regions in Iberia with hot, dry summers and are associated with relict and Spanish accessions (Fig. 4 and SI Appendix, Figs. S6, S7, and S15). ECSY and EFSY likely originated in the Iberian refugium (46, 79⇓–81), and may remain endemic in that region because they require hot, dry Mediterranean summers to persist.

D-SY and D-RY haplotypes are sister to one another, arising from an ECCY ancestor (Fig. 4A). Pairwise diversity within all D-clade individuals suggests this split occurred about 365 Kya (ranging from 281 to 450 Kya using differing assumptions about generation time) (Materials and Methods) during a period of climate cooling following an interglacial period (Fig. 4B and SI Appendix, Table S9), possibly during contraction into different refugia. However, we do not observe further diversification within the extant D-haplotypes until much later, potentially due to cycles of expansion and contraction out of refugia followed by local extinction. The extant, predominately Asian and east Eurasia D-SY clade diversified 115 Kya (88 to 141 Kya with varying generation times) during a cool period of the last interglacial period (Fig. 4B and SI Appendix, Table S9). Pairwise diversity among extant D-RY haplotypes suggests this west Eurasian and North American clade expanded recently 16 Kya (ranging 12 to 19 Kya), during rapid warming after the last glacial maximum (Fig. 4B and SI Appendix, Table S9), but the gene tree suggests an earlier origin (Fig. 4A). The extant distribution of D-RY in Iberia and Western Europe (SI Appendix, Figs. S6 and S7) suggests that this haplotype may have arisen and spread from the Iberian refugium.

These observations suggest that changing Pleistocene climate may have favored the rise and spread of the D-haplotypes, possibly through selection for associated traits promoting spring annual or rapid cycling life histories. The loss of cold-induced secondary dormancy in D-haplotypes, correlated with shorter life cycles, may have provided life-history flexibility facilitating persistence in changing climates and the invasion of new habitats. The ability to germinate in spring is advantageous in montane regions (17), such as Central Asia, and rapid cycling life histories may be favored in disturbed habitats with ample summer rainfall. Both D-SY and D-RY currently occupy climates with wetter summers than the E-haplotypes (SI Appendix, Fig. S15B). The wide Eurasian distribution of D-SY haplotypes suggests that their expanded germination niche may have facilitated postglacial expansion and spread of “nonrelict” genotypes out of central Asia into Europe and China (47, 82). The recent expansion of the D-RY clade is consistent with invasion of new ruderal habitats made available by the spread of agriculture through Europe (43, 82, 83). Our results show that derived loss-of-function D-haplotypes are more common in midlatitudes and England, and could have resulted from the nonrelict east–west expansion following human-mediated habitat disturbance. Relict populations in the Iberian Peninsula and Scandinavia might have preserved the ancestral DOG1 E-haplotype that promotes a winter annual cycle, advantageous in more undisturbed habitats (81, 82). Moreover, the D-RY haplotype of DOG1 is enriched in the invaded range of North America (SI Appendix, Fig. S6), where A. thaliana has arrived as a human commensal in the last 300 y (84). Although this could be due to a founder effect, phenological traits associated to this haplotype might also have played a role in invading disturbed habitats suitable for rapid cycling.

We caution that selection, geographic sampling, and demography could affect these estimates of age. But, with the exception of a more recent D-RY origin, these clade ages match the relative branching pattern on the gene tree. The origin of DOG1 haplotype variants suggests that the germination niche of A. thaliana has been shaped by climate cycling throughout the Pleistocene, as the species range repeatedly expanded out of and contracted into glacial refugia (46, 79, 80) and the changing climate and physical environment filtered which individuals were able to persist (85). Novel germination strategies, measured via the origin of DOG1 haplotypes, arose at key timepoints in the changing climate of the Quaternary. Thus, life-history strategies associated with DOG1 haplotypes may in fact shape the population structure we see today on the landscape.

SNPs Associated with Chilling Responses and DOG1 Functional Haplotypes Show a Signature of Climate Adaptation.

To understand the climatic associations between genetic variants associated to various phenotypes, we used Gradient Forest, a tree-based machine-learning regression approach, to describe nonlinear turnover functions of allele frequencies along environmental gradients (40, 86, 87). We performed Gradient Forest regression analyses between environmental gradients and LD-pruned index SNPs from the 1,000 most highly associated SNPs from our GWA results for PC1_germ (23 SNPs), PC2_germ (9 SNPs), and the DOG1 haplotypes. After accounting for spatial autocorrelation by including Moran’s eigenvector map (MEM) variables, mean temperature of the wettest quarter (Bio8) explained the highest amount of turnover in allele frequency of SNPs associated to PC2_germ and was among the top 3 predictors for PC1_germ as well (Fig. 5 and SI Appendix, Table S10). Additionally, altitude, isothermality (Bio3), and temperature mean diurnal range (Bio2) predicted allele frequencies of both germination PCs (SI Appendix, Fig. S16 and Table S10). These environmental gradients structure turnover in index SNPs more than a set of 150 random SNPs (Fig. 5 and SI Appendix, Fig. S16), evidence of local adaptation to climate at these loci. Given that the E- and D-haplotypes are very old, our data suggest that ecotypes carrying both of these haplotypes could have arrived at locations throughout the range, but that environmental filtering and selection likely give rise to the geographic patterns that we see today. As a predominately selfing annual plant, A. thaliana populations are structured across the landscape, giving rise to geographic differences in ecologically relevant alleles (88); however, our results support local adaptation to climate, as allele turnover from index SNPs is more strongly structured by temperature and precipitation gradients than alleles from random SNPs.

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Fig. 5.

Average cumulative importance functions of mean temperature of the wettest quarter for the index SNPs of the top 100 SNPs with the highest association with the first 2 PCs of germination for 752 accessions. Genomic controls are shown in grey, representing the 100 cumulative functions of 150 random SNPs with MAF > 0.01.

The response-to-cold PC2_germ cumulative importance function for mean temperature of the wettest quarter (Bio8) showed a threshold turnover in allele frequencies around 14 to 15 °C (Fig. 5), and alleles from SNPs associated to DOG1 were among the ones with the highest importance values for this function (SI Appendix, Table S11). Accessions with the reference allele, with high dormancy release under chilling and no secondary dormancy, are found in these southern regions, while accessions with the alternative DOG1 allele have strong secondary dormancy. This temperature threshold divides the winter growing season in southern regions (<14 to 15 °C) from a warm wet summer (>14 to 15 °C) growing season in northern latitudes, thus the shape of the allele turnover function suggests that germination traits might have been selected by these climatic gradients. Environmental predictors’ importance varied at explaining the distribution of different DOG1 haplotypes (SI Appendix, Fig. S17 and Tables S12 and S13). Allele distributions of DOG1 might have resulted from its effect on germination responses to chilling and on other life-history traits as well. For example, 14 °C acts both as a temperature threshold for the induction of strong maternal effects on seed dormancy (11, 22) and for vernalization disruption in flowering time (89).