This DATSETNAMEreadme.txt file was generated on YYYY-MM-DD by NAME GENERAL INFORMATION 1. Title of Dataset: Genetic differences in the temporal and environmental stability of transgenerational environmental effects 2. Author Information A. Principal Investigator Contact Information Name: Kathleen Donohue Institution: Duke University Address: Box 90338, Durham, NC 27708 USA Email: k.donohue@duke.edu B. Associate or Co-investigator Contact Information Name: Mariano Alvarez Institution: Avalo Address: 701 W Main St, Durham, NC 27701 Email: m.alvarez@avalo.ai C. Alternate Contact Information Name: Institution: Address: Email: 3. Date of data collection (single date, range, approximate date) : 2016-2019 4. Geographic location of data collection : Durham, NC, USA 5. Information about funding sources that supported the collection of the data: NSF DEB-1556855 SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on the data: N/A 2. Links to publications that cite or use the data: Forthcoming 3. Links to other publicly accessible locations of the data: N/A 4. Links/relationships to ancillary data sets: N/A 5. Was data derived from another source? No A. If yes, list source(s): 6. Recommended citation for this dataset: N/A DATA & FILE OVERVIEW 1. File List: 2. Relationship between files, if important: 3. Additional related data collected that was not included in the current data package: 4. Are there multiple versions of the dataset? yes/no A. If yes, name of file(s) that was updated: i. Why was the file updated? ii. When was the file updated? METHODOLOGICAL INFORMATION We selected six accessions ("genotypes") of A. thaliana (Supplemental Table S1) that have exhibited variation in phenological (germination and flowering) responses to environmental conditions, and temperature in particular. These genotypes have been used as founding parents for mapped recombinant inbred lines and have been sequenced in the “1001 Genomes” project (Reiter et al. 1992; Schiff et al. 2001; O’Niel et al. 2008; Bentsink et al. 2010). The genotypes used are Ag0, Col0, Cvi1, Ler2, Wa1, and Ws1. See Table S1 for accession numbers and additional information on their location of collection. Replicates of each genotype were grown in common conditions, with 12h light/12h dark at 22⁰C in replicate growth chambers (Environmental Growth Chambers, Model E7-2, Ohio, USA), in Metromix 360 (Sun Gro, Agawam, MA, USA) in 2-inch plastic pots after three days of cold (4⁰C) stratification to break dormancy. Mature seeds were harvested and stored in cryo-boxes in a desiccator cabinet at room temperature until use. Equal numbers of seeds from replicates within the same genotype and treatments were pooled to form the seed pool for the subsequent generation. Experimental design and plant growth conditions: We employed a factorial manipulation of diurnal temperature regime across three generations (Fig. 1A). This design allowed us to compare the magnitude of effects of diurnal temperature regime in three generations: present, parental, and grandparental (Fig. 1B-1). It also allowed us to test whether the response to environments experienced in past generations depends on present environmental conditions (Fig. 1B-2), that is, whether ancestral environmental effects are stably expressed over environments experienced subsequently. In addition, the design allows us to test whether responding to present environmental conditions depends on ancestral environmental conditions (Fig. 1B-3) and specifically whether ancestral environments reinforce (magnify) or antagonize responses to present environments (Fig. 1B-4). We used two diurnal 12h light/12h dark thermal regimes, resembling thermal conditions experienced during the growing season of A. thaliana at different locations in its native range, and also resembling thermal conditions in temperate climates that are likely to be experienced during spring and autumn, when seeds are known to germinate. While other aspects of the environment also vary seasonally (day length, precipitation, canopy structure, presence of competitors and herbivores, and many other factors), we have focused on diurnal thermal regime since it is known to strongly influence seasonal developmental timing in A. thaliana. The "High-temperature" thermal regime cycled between 24⁰C day and16⁰C night; the "Low-temperature" thermal regime cycled between18⁰C day and 10⁰C night. For each planting, twelve replicates from each genotype were planted in 2-inch plastic pots, as described above, with three replicates distributed over four chamber compartments (blocks), using the same chambers as described above. Plants were grown until senescence, defined as the maturation of >90% of siliques, and their seeds were then harvested and stored in a desiccator cabinet. Each generation, seeds from each genotype were pooled across blocks to seed the next generation. In the final (third) generation, 12 replicates of each of the eight environmental sequences were planted, with three replicates of each genotype from each ancestral treatment in each of the four growth chambers used, for a total of 576 plants (12 replicates x 8 treatments x 6 genotypes). Phenotypes measured: In the third generation, the following phenological traits were measured in daily censuses: the timing of seed germination, the timing of bolting (the initiation of reproduction), the timing of flowering, and the timing of fruit maturation. From these, the time intervals between germination and bolting, bolting and flowering, and flowering and fruit maturation were calculated. Two metrics of size at reproduction were also measured: the number of leaves at the time of bolting ("number of leaves"), and the length of the largest leaf at the time of bolting ("leaf length"). As a measure of fitness, all seed were collected and weighed, and the total seed biomass was measured with a microbalance. Germination assays: To estimate the proportion of seed germination in the third generation, fresh seeds (fewer than 10 days after harvest) were assayed on 0.7% agar on 35mm plastic petri dishes. Twelve replicate plates were seeded from each genotype and each ancestral environmental combination for each germination temperature regime (same as that used to grow the plants). Twenty seeds were arranged in a grid and checked for germination every 4 days for 24 days, after which point germination plateaued (or earlier), for a total of 240 seeds per genotype per treatment. Germination was recorded as radicle protrusion from the seed coat, and seeds were checked for viability at the end of 24 days by assessing firmness to touch. The proportion of viable seeds in each plate that germinated by the end of the experiment was used for statistical analyses. DATA-SPECIFIC INFORMATION FOR: EpiStabilityGen3_V5_forUpload.csv 1. Number of variables: 23 2. Number of cases/rows: 672 3. Variable List: UniqueID - Experimental unique ID Pool - Seed pool corresponding to a unique treatment combination Replicate - replicate number within seed pool Genotype - common genotype name Temp - temperature in offspring environment (warm = 24/16C, cool = 16/8C) MaternalTemp - temperature in parent environment(warm = 24/16C, cool = 16/8C) GrandmaternalTemp - temperature in grandparent environment (warm = 24/16C, cool = 16/8C) Use - Column to distinguish nucleic acid samples (not shown) from samples to be phenotyped Rand - random number for randomization purposes Chamber - Growth chamber ID Tray_ideal - tray number TraySort_ideal - temporary sorting variable Position_ideal - position within tray PickColor - toothpick color used to assess emergence time TransferredGermData - column to align data from paper recording to electronic recordings GermDate - emergence data BoltDate - bolting date LeafCount - number of leaves at bolting LL - length of longest leaf at bolting Flower - date of first flowering FirstFruit - date of first fruit production MatureFruit - date of first mature fruit SeedWeight - total reproductive biomass (g) 4. Missing data codes: N/A DATA-SPECIFIC INFORMATION FOR: 2020Epistability_GermAssay1_PoolCohort.csv 1. Number of variables: 29 2. Number of cases/rows: 4 3. Variable List: Variable "Cohort" refers to the time-batched cohort during the germination assays. All other variables refer to a specific seed pool (I.e, treatment combination). Values of these variables represent the number of replicates in a particular time cohort. 4. Missing data codes: n