The seasonal timing of seed germination determines a plant's realized environmental niche, and is important for adaptation to climate.  The timing of seasonal germination depends on patterns of seed dormancy release or induction by cold and interacts with flowering time variation to construct different seasonal life histories. To characterize  the genetic basis and climatic associations of natural variation in seed chilling responses and associated life history syndromes, we selected 559 fully-sequenced accessions of the model annual species Arabidopsis thalianafrom across a wide climate range and scored each for seed germination across a range of 13 cold stratification treatments as well as the timing of flowering and senescence. Germination strategies varied continuously along two major axes: 1) overall germination fraction and 2) induction vs release of dormancy by cold. Natural variation in seed responses to chilling was correlated with flowering time and senescence to create a range of seasonal life history syndromes. Genome-wide association (GWA) identified several loci associated with natural variation in seed chilling responses, including a known functional polymorphism in the self-binding domain of the candidate gene DOG1. A phylogeny of DOG1haplotypes revealed ancient divergence of these functional variants associated with periods of Pleistocene climate change, and Gradient Forest analysis showed that allele turnover of candidate SNPs was significantly associated with climate gradients. These results provide evidence that Arabidopsis thaliana’s germination niche and correlated life history syndromes are shaped by past climate cycles as well as local adaptation to contemporary climate.

Seed bulking

Seeds from 559 A. thalianafully-sequenced accessions from the 1001 Genomes Project (ABRC stocks) were stratified in 0.15% agar at 4°C for 7 days. Seeds were sown in soil and allowed to germinate for ten days; seedlings were vernalized for 6 weeks at 4°C with a 12/12h photoperiod and were grown at 14°C with a 12/12h photoperiod in a walk-in growth chamber (Conviron E7/2 Controlled Environment Chamber). Two individuals of each accession were grown in the same chamber (a total of 1,118 plants), planted two weeks apart in two temporal replicate blocks. Plants were watered twice a week with nutrient water until they showed 50-60% ripe siliques and seeds were individually harvested from each plant when 70-80% of the siliques were ripe. Fresh harvested seeds from each plant were immediately used for two different experiments: a) cold stratification germination experiments and b) dry seed storage germination experiments to test for seed after-ripening times. Phenology variables were recorded on the maternal plants including days to flowering (DTF) and days to senescence (DTS). DTF varied from 70 – 180 days while DTS ranged from 151 – 448 days.

Control and dark cold stratification germination experiments

Fresh seeds were stored in a drying box with desiccant in the lab for 11 days, then assigned to thirteen experimental treatments. Base germination of imbibed fresh seeds (control) was assessed under germination-inducing conditions after seven days at 22°C and 12/12 h photoperiod (80 μmols m s^-1light). We also exposed seeds to twelve stratification treatments. Seeds for these treatments were imbibed and stratified in boxes in dark chambers for 4, 8, 11, 15, 22 and 32 days at 4°C (simulating winter temperature cues) or 10°C (simulating early spring and autumn temperature cues). After these different cold treatments, dark-imbibed stratified seeds were put under the same germination inducing conditions as the control seeds. Total germination was manually scored from photographs (i.e. radicle protrusion) after seven days. Each experimental replicate consisted of 25-30 fresh seedsscattered on a Petri plate (60 ×15 mm) with blue germination paper (Blue Seed Germination Blotter, Anchor Paper) and 35 ml of a 2% PPM solution (Plant Preservative Mixture, Caisson Laboratories). PPM was added to prevent fungal growth during the 4 - 32 day stratification period. There were two replicates for each accession x treatment combination, for a total of 14,534 Petri plates scored.  Germination experiments were carried out continuously for over a year, as fresh seed from individual plants were harvested when seeds matured across over 500 days, and not during a single month period. 

Dry seed storage germination experiments

Additional germination experiments were done to test for seed after-ripening. Fresh seeds were stored under dry lab conditions and were tested for total germination every 6 weeks until 75% germination was recorded in two consecutive tests, which we considered as after-ripened seeds. Some accessions were after-ripened after 6 weeks while some others showed little evidence of after-ripening even after dry storage in the lab for up to 788 days. Germination induction conditions were the same as for the cold stratification experiment. Seed after-ripening was assessed by calculating the number of days to 50% germination by fitting a polynomial regression to the dry-stored seed germination data for each ecotype (DSDS50).

arabidopsis_germ_phen_chilling_DATA.xls - Descriptions of each phenotype column are found in the Excel sheet titled variables. Values are means from two replicates.

dog1.may22.uniq.txt - DOG1 haplotypes