Supplementary Information (SI Appendix)

Huo et al. (2016) – www.pnas.org/cgi/doi/10.1073/pnas.1600558113

DELAY OF GERMINATION1 (DOG1) Regulates both Seed Dormancy and Flowering Time

through MicroRNA Pathways

Heqiang Huo, Shouhui Wei, Kent J. Bradford

Supplementary Methods

Supplementary Text - Screening for altered flowering pathways in lettuce LsDOG1-RNAi plants

Supplementary Figures

Figure S1. Temperature sensitivity of different lettuce genotypes and characterization of lettuce DOG1

expression and function.

Figure S2. Protein amino acid sequence alignment of Arabidopsis Col DOG1 (AtDOG1) and lettuce

Salinas DOG1 (SalDOG1).

Figure S3. Gibberellin (but not fluridone, an ABA biosynthesis inhibitor) alleviates primary dormancy of

Arabidopsis seeds caused by overexpression of LsDOG1.

Figure S4. Relative mRNA levels of genes in different pathways regulating flowering.

Figure S5. Potential interactions of the DOG1-miR156-miR172 module in regulating developmental

phase transitions.

Figure S6. Overexpression of LsMIR156 resulted in delayed flowering in lettuce.

Figure S7. Relative mRNA and miRNA levels in leaves and apical meristems of Arabidopsis at different

developmental stages.

Figure S8. Primary dormancy of Col-WT, dog1-3 and dog1-5 seeds.

Supplementary Tables

Table S1. DOG1 protein amino acid sequence similarity among four lettuce and two Arabidopsis

genotypes.

Table S2. DOG1 effect on number of leaves at flowering of Arabidopsis genotypes.

Table S3. Sequences of primers used in this study.

Supplementary Methods

Generation of dog1-3 × 35S:AtMIR156 and nced 9-1× 35S:AtMIR156 Lines

Because Col-35S:AtMIR156 displayed a strong late flowering phenotype (1) we used it as a pollen

donor to cross with dog1-3 and nced9-1. The F1 seedlings from multiple crossing events were screened

based on flowering phenotype. At least three seedlings that displayed the most severe flowering

phenotypes were genotyped to confirm the T-DNA insertion in NCED9 locus and advanced to the F3

generation.

Seed Germination Assays

For lettuce seed germination tests, seeds were placed on one paper blotter disc in a 4.7-cm Petri dish

that was moistened with 3 mL of deionized water. For Arabidopsis seed germination tests, seeds were

placed in wells containing 400 µL of deionized water or solutions in 24-well plates. The plates were

sealed with Parafilm. All deionized water or solutions used for seed germination tests were supplemented

with 0.2% Plant Preservative Mixture (PPM, Caisson Labs, Smithfield, UT, USA) to protect seeds from

fungal development. Three replications of 30 lettuce seeds or 50 Arabidopsis seeds were utilized in all

tests.

Measurements of Flowering Times

To measure lettuce flowering times, at least 20 plants of each transgenic line and the segregated

control lines were grown in the greenhouse. Lettuces were irrigated on a cycle of twice with deionized

water and once with liquid nutrient solution through computer-controlled drip. The flowering times for

lettuce were measured as days from the planting date to the first open flower.

To measure Arabidopsis flowering times, two or three genotypes of Arabidopsis plants were

randomly arranged in multiple trays. Col-WT, dog1-3 and dog1-5, Col-WT and need 9-1, Col-

35S:LsMIR156, dog1-3-35S:LsMIR156, nced9-1-35S:LsMIR156 and dog1-5-35S:LsMIR156, Col-

35S:AtMIR156, dog1-3 × 35S:AtMIR156 and nced9-1× 35S:AtMIR156, Ler-WT and dog1-1, Ler-35S:

LsMIR156 and dog1-1-35S:LsMIR156 were randomly distributed within the same flat trays with multiple

replications; water and nutrition were provided as needed. The flowering times were measured as days to

flower appearance and number of rosette leaves at flowering. Data were expressed as percentages of

plants flowering or as percentages of flowering plants having similar ranges of rosette leaf numbers at

different days after trays were transferred to the growth chamber following stratification.

Isolation of LsDOG1 and Vector Construction

The Arabidopsis DOG1 (AT5G45830) coding sequence was used to BLAST search the lettuce

Transcriptome Shotgun Assembly (http://blast.ncbi.nlm.nih.gov). The best-matched EST was used for

reciprocal BLAST against the Arabidopsis genome (www.arabidopsis.org) to further confirm identity.

Primers were subsequently designed for RACE amplification of DOG1 cDNA. The lettuce DOG1 cDNA

was TA-cloned into pGEMT-easy vector (Promega, Madison, WI, USA) using the primers listed in Table

S3. For suppressing DOG1 in lettuce, the CaMV35S promoter in binary vector pGSA1165 (ABRC stock

CD3-450) was replaced by a 2.6 kb LsNCED4 promoter through the BglII and SacI restriction sites. A

350 bp fragment was amplified from the Salinas DOG1 cDNA and introduced into the previous

pGSA1165-ProLsNCED4 RNAi vector with a two-step procedure through AscI/NcoI and BamHI/SpeI

digestions and ligations. The RNAi fragment was compared to the lettuce genome

(http://compgenomics.ucdavis.edu/) by BLASTN using low stringency to confirm no potential off-targets.

For ectopic expression of LsDOG1, the CaMV35S promoter of pGSA1165 vector was replaced with the

LsDOG1 promoter through digestion and ligation of BglII/SacI, and the GUS linker of the engineered

pGSA1165 was replaced with PI-LsDOG1 through NcoI/PacI to form the ProDOG1:PIDOG1 ectopic

expression vector. For overexpression of LsDOG1 in Arabidopsis, the GUSplus fragment of

pCAMBIA1305 was replaced with LsDOG1 coding regions from Sal, PI, UC and Saligna genotypes

through NcoI/PmlI digestion/ligation to form the Pro35S:LsDOG1 overexpression vectors. All cloned

fragments were error-proofed by sequencing.

Identification of LsMIR156 and LsMIR172

The mature sequences of Arabidopsis miR156 and miR172 obtained from the miRBase database

(www.miRBase.org) were used to BLAST against the lettuce Transcriptome Shotgun Assembly

(http://blast.ncbi.nlm.nih.gov). The candidate orthologous genes to the AtMIR156 genes were first used

for BLASTX against the Arabidopsis genome and NCBI protein database (http://www.ncbi.nlm.nih.gov/)

to rule out the protein-encoding genes such as LsSPLs. The rest of the candidate sequences were further

used for BLASTN against the Arabidopsis genome for confirmation. The EST contigs of these candidates

were used to search the lettuce genome browser (http://gviewer.gc.ucdavis.edu/cgi-

bin/gbrowse/alface_version_5_1/) to obtain the 5’ and 3’ extended sequences of miRNA binding sites if

needed. CENTROIDFOLD (http://www.ncrna.org/centroidfold) was used to define the potential stem-

loop precursors within the extended sequences of these candidates, and ViennaRNA Web Services

(RNAfold web server, http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) was used for the structure

prediction.

Vector Construction for Overexpression of LsMIR156 and LsMIR172

For overexpression of LsMIR156 in lettuce, sequences comprising LsMIR156 were amplified using

the primers listed in Table S3 and cloned into the Gateway cloning vector pDONR221 through BP

reaction. The cloned LsMIR156 was subsequently transferred to the Gateway binary expression vector

pGWB402Ω via LR reaction (2). For overexpression in Arabidopsis, LsMIR156 and LsMIR172 were

amplified using the primers listed in Table S3 and cloned into the Gateway cloning vector pDONR207

through BP reaction. These were subsequently transferred to the Gateway binary expression vector

pEarleyGate100 via LR reaction (3). All cloned fragments were error-proofed by sequencing.

Plant Transformation

For lettuce, the DOG1-RNAi and ProDOG1:SalDOG1 constructs were introduced into

Agrobacterium tumefasciens LBA4404 and the LsMIR156 construct was introduced into EHA105, all of

which were then used for Salinas or PI251246 transformation at the UC Davis Ralph M. Parsons

Foundation Plant Transformation Facility. For Arabidopsis, the Pro35S:LsDOG1, Pro35S:LsMIR156 and

Pro35S:LsMIR172 overexpression vectors were introduced into Agrobacterium GV3101 and used for

transformation through the floral dip method (4). The primers in Table S3 were used to genotype

Arabidopsis T-DNA mutants prior to plant transformation and to confirm the T1 and T3 transgenic plants.

mRNA and miRNA Analyses

Three lettuce leaves of similar developmental ages were pooled from three individual plants for each

line to form one biological sample. For lettuce apical meristems, one biological sample was pooled from 8

lettuce plants for each line. For lettuce, ~100 mg seeds were used for one biological sample. For

Arabidopsis leaves and apical meristems, one biological sample was pooled from ~20 individual plants.

For Arabidopsis seeds, ~50 mg seeds were used for one biological sample. In all cases, three biological

samples were assayed for each line.

Total RNA with enriched small RNA from leaves and apical meristems of lettuce and Arabidopsis

was isolated using the RNAzol RT kit as described in the instruction manual (Molecular Research Center,

Cincinnati, OH, USA). Total RNA with enriched small RNA from seeds of lettuce and Arabidopsis was

isolated using PureLink® Plant RNA Reagent following the instruction manual (Life

Technologies/Thermo Fisher Scientific, Carlsbad, CA, USA). All total RNAs were treated with TURBO

DNAase I (Life Technologies) prior to downstream analysis. For mRNA quantitation, 1 µg of total RNA

was used for cDNA synthesis using QuantiTect Reverse Transcription Kit (Qiagen, Redwood City, CA,

USA) and real-time PCR was performed as described previously (5). miRNAs were detected with

TaqMan® MicroRNA Assay (Life Technologies) with slight modifications. In brief, 300 ng of total RNA

was used for miRNA reverse transcription and 4 µL of 400x diluted miRNA RT was used for real-time

PCR in a 10 µL of reaction buffer that contained 5 µL of TaqMan Universal PCR master Mix II and 0.5

µL of TaqMan® Small RNA Assay probe. All real-time PCR assays were performed on a StepOnePlus

Real-Time PCR system (Life Technologies).

Supplementary Text

Screening for altered flowering pathways in lettuce LsDOG1-RNAi plants

Flowering of plants is dependent on developmental programs that are mediated by both

environmental and endogenous cues and are under tight genetic control. The primary environmental cues

for regulating flowering include the photoperiod, ambient temperature and chilling (vernalization), while

endogenous factors such as phytohormones and carbohydrate status regulate the so-called autonomous,

gibberellin (GA), nutrient, and aging pathways influencing flowering (6-10). In Arabidopsis (and other

plants), these components form a complex gene regulatory network in which both types of cues converge

at a set of integrator genes including FLOWERING LOCUS C (FLC), SHORT VEGETATIVE PHASE

(SVP), FLOWERING LOCUS M (FLM), FLOWERING LOCUS T (FT), etc. Like Arabidopsis, lettuce has

a winter annual flowering habit, with flowering being promoted by long days and chilling. To understand

how suppression of LsDOG1 expression could affect flowering time in lettuce, we tested expression of

several key integrator genes involved in different pathways.

FLOWERING LOCUS T (FT) is the most important integrator for regulating flowering time. FT is

mainly expressed in leaves and is induced by the transcriptional activator CONSTANS (CO) through its

binding to the FT promoter under long-day conditions (11, 12). The CO-FT module in flowering

regulation has been demonstrated to be conserved in other plant species, including sunflowers (Helianthus

annuus) in the Asteraceae with lettuce (7, 10, 13). To test whether LsDOG1 might influence FT

expression, we measured CO and FT mRNA abundances in leaves of Salinas and DOG1-RNAi plants. FT

mRNA was almost 25-fold higher, consistent with the early flowering phenotype, while CO mRNA was

only 75% higher in the LsDOG1-suppressed plants compared to the control (Fig. S4), suggesting that

other pathways might trigger the change in FT mRNA accumulation (Fig. S5).

In addition to photoperiod, temperature is another important environmental cue that greatly influences

plant flowering (Fig. S5). Arabidopsis plants flower earlier when grown at higher temperatures (23°C)

than at lower temperatures (16°C) (14). Lettuce also responds to high temperatures (particularly night

temperatures) with early flowering (15). At lower temperature (16°C), FLM and SVP proteins form a

complex to bind to the promoters of floral activator genes like FT and SOC1 to repress their transcription

(16, 17). This repression is released by increased temperatures (16-18). In contrast, the extended low

ambient temperature during the winter season can promote rapid flowering of winter annual plants in the

following spring season. The requirement for this chilling period (also called vernalization) prevents the

improper flowering of winter annual plants until after the winter season (9, 19). In Arabidopsis, chilling

represses the transcription of FLC through epigenetic silencing that is mediated by a conserved Polycomb

(PcG) mechanism. FLC is a MADS box transcription factor that represses the floral integrators FT, TSF

and SOC1 to inhibit flowering (19-23). FLC is also a core component in the autonomous flowering

pathway (10). Autonomous pathway mutants derived from rapid-flowering accessions are characterized

by delayed flowering irrespective of day length (9, 10). Genes involved in the autonomous pathway

regulate flowering by repressing FLC through epigenetic modification and/or RNA processing (10, 24).

With respect to FLC, FLM and SVP homolog genes in lettuce, we found either no effect of silencing

LsDOG1or that mRNA amounts were increased (Fig. S4). As FLC and SVP are repressors of flowering,

this makes it unlikely that DOG1 acts on flowering through modification of the vernalization or

autonomous pathways (Fig. S5), although mRNA abundance may not always reflect protein abundance or

activity.

Gibberellin can promote both seed germination and flowering (10, 25). Because enhanced seed

germination of atdog1 and lettuce DOG1-RNAi may be attributed to alteration in GA biosynthesis and

signaling (Fig. S3) (26, 27), we tested whether there are any changes in the key enzymes involved in

biosynthesis of bioactive GA. The biosynthesis of active GA is tightly regulated by the relative activities

of anabolic (GA20ox and GA3ox) and catabolic enzymes (GA2ox) under different environmental

conditions (Fig. S5) (28). Mutations that inhibit this biosynthetic pathway or increase the degradation of

GA can delay flowering, particularly under short-day conditions (10, 29-31). It has been proposed that

GA promotes SOC1 gene expression and its target LFY in apical meristems to accelerate flowering under

short days (32-34). The GA signaling pathway also more directly regulates flowering time (Fig. S5). GA

is perceived by the GA receptors, GIBBERELLIC ACID-INSENSITIVE DWARF 1 (GID1a, GID1b, and

GID1c) (35). The lower expression of FT and TSF in a triple mutant of gid1 caused a late-flowering

phenotype in long days (36). The reduction of expression of FT and TSF is attributed to the repression by

DELLA proteins of SPL3 expression in leaves and of SPL3, SPL4 and of SPL5 expression in the shoot

apex (36, 37). We measured the transcript abundances in lettuce of GA3ox2, GA20ox1, GA2ox2, GA2ox3,

GA2ox6 homologs and of DELLA homologs RGL1 and RGA. If silencing of LsDOG1were acting to

accelerate flowering by enhancing GA levels, we would expect expression of GA3ox2 and GA20ox1 to be

elevated and expression of the GA2ox genes and of RGL1/RGA to be reduced. However, the opposite was

the case, with expression of GA3ox2 and GA20ox1 being reduced while expression of the GA2ox genes

and of RGL1/RGA were either unaffected or increased (Fig. S4). Consistent with this data, we also

observed no obvious change in internode lengths between DOG1-RNAi and its control lines. These

results indicate that the early flowering in DOG1-RNAi is unlikely to be caused by an alteration in GA-

related flowering pathways (Fig. S5).

Regulation of flowering time by the maturity (phase change) or “aging” pathway prevents flowering

until the plant transitions from the juvenile to the adult phase, even under inductive environmental

conditions like long days and high temperature (10). miR156 and miR172, and their target genes, are

major components of the aging pathway and act sequentially to regulate flowering time (Fig. S5) (8).

miR156 expression is high in the embryo and early seedling stages and decreases in older plants, resulting

in a concomitant increase in expression of SPLs with aging, especially SPL3, 4, 5, 9, 10 and 15 (38). The

up-regulation of SPL3/4/5/9/10/15 caused an early-flowering phenotype through induction of FT and

floral meristem genes by binding the promoter elements of these genes (38-41). miR156 is not regulated

by the vernalization-, photoperiod-, and GA-dependent flowering pathways (40). Inactivation or

overexpression of the flowering regulators FLC, CO, FT, or SOC1 had no obvious effects on miR156

levels, and miR156 is also not responsive to exogenous application of GA, auxin and cytokinin (41).

However, miR156 is reported to respond to ambient temperatures. At 16ºC, the miR156 level is higher

than at 23ºC (42). Higher miR156 leads to a lower level of its target gene SPL3, resulting in lower

expression of FT (40). In contrast, the level of miR172 is lower at 16°C than at 23ºC, as SPL9 and 10

(targets of miR156) directly promote miR172 expression, which has an opposite effect to miR156 on

flowering time (1). Similarly, expression of the target genes of miR172, TARGET OF EAT1 (TOE1),

TOE2 and SCHLAFMüTZE (SMZ) were observed to increase at 16ºC and act as floral repressors (8, 42).

miR172 positively regulates flowering by silencing floral repressor genes: AP2, TOE1, TOE2, TOE3,

SMZ, and SCHNARCHZAPFEN (SNZ) (43). In addition, miR156 was also reported to crosstalk with the

sugar-signaling pathway in regulating flowering time. The enzyme TREHALOSE-6-PHOSPHATE

SYNTHASE 1 (encoded by TPS1) produces trehalose- 6-phosphate (T6P) which serves as a signal for

carbohydrate availability in plants; TPS1 regulates flowering time through mediating the expression of

SPL genes in the shoot apical meristem via a miR156-dependent mechanism (44). Two additional studies

provide supporting evidence that miR156 abundance is regulated by sugars such as glucose and sucrose to

control the vegetative to flowering phase transition (45, 46).

In the early flowering DOG1-RNAi line in lettuce, we found that expression of at least three SPL

homolog genes was up-regulated several fold compared to control plants (Fig. S4). These SPLs could be

regulated by either miR156 or GA. As discussed above, the up-regulation of SPL genes is less likely to be

caused by alterations in GA biosynthesis or degradation. Thus, gene expression assays indicated that the

early flowering phenotype in DOG1-RNAi lettuce is likely to be mainly attributed to the alteration in the

miR156-SPL pathway (Fig. S5).

Supplementary Figures

Fig. S1

Fig. S1. Temperature sensitivity of different lettuce genotypes and characterization of lettuce DOG1

expression and function. (A) Sensitivity to temperature of seed germination of four lettuce genotypes: cv.

Salinas (Sal, L. sativa), PI251246 (PI, L. sativa), US96UC23 (UC, L. serriola), and PI261653 (Saligna, L.

saligna). (B) Functional tests of the ability of LsDOG1 from four lettuce genotypes to complement the

dog1-3 mutant of Arabidopsis and restore seed thermoinhibition at 32°C. (C) Relative LsDOG1 mRNA

levels in Salinas roots, leaves and dry seeds. Root and leaf tissues were from 6-week-old plants. (D)

Relative LsDOG1 mRNA levels in dry seeds of Sal and PI that were matured at 18°C or 32°C. (E)

Overexpression of PI-DOG1 inhibited germination of nced6-1 nced9-1 (nced6/9) double mutant seeds at

32°C. (F) Germination of dog1-3, nced9-1, nced 6-1 nced9-1 (nced6/9), and dog1-3/nced9-1

(dog1/nced9) seeds at 35°C. In E and F, seeds were imbibed in the light for 5 d. In panel F, no dog1-3

seeds germinated. ** Denotes a significant difference at 0.01 level (Student’s t-test). Error bars = SE, n =

3. All Arabidopsis seeds were tested at approximately 3 weeks after harvest.

Fig. S2

Fig. S2. Protein amino acid sequence alignment of Arabidopsis Col DOG1 (AtDOG1) and lettuce Salinas

DOG1 (SalDOG1). The DOG1 domain is underlined in red.

Fig. S3

Fig. S3. Gibberellin, but not fluridone (an ABA biosynthesis inhibitor), alleviates primary dormancy of

Arabidopsis seeds caused by overexpression of LsDOG1. Seeds (3 weeks after harvest) of Col plants

overexpressing LsDOG1 were imbibed in water (H2O), 300 µM fluridone (FLU) or 300 µM gibberellin

4+7 (GA) for 120 h in the light at 20˚C. No seeds germinated in water.

Fig. S4

Fig. S4. Relative mRNA levels of genes in different pathways regulating flowering. The mRNA levels in

leaves of Sal (CTL) and DOG1-RNAi line at 6 weeks after planting were normalized by the geometric

mean of three reference genes: AP2M (Clathrin adaptor complexes medium subunit family), PDF1 and

ef1α; they are shown relative to the levels in CTL leaves for each gene. The left y-axis shows values for

all genes except FT, whose values are on the right y-axis. Error bars = SE, n = 3.

See Supplementary Text for further discussion of these data.

Fig. S5. Potential interactions of the DOG1-miR156-miR172 module in regulating developmental phase

transitions. Diagramed here are potential interactions of genes and pathways described in this work. The

central focus is on our hypothesis that at least one action of DOG1 is to promote the conversion of

MIR156 and MIR172 transcripts to miR156 and miR172. This is shown as acting through DOG1

promotion of expression of DICER complex components (see Fig. 4E-G), but additional mechanisms are

possible. miR156 targets SPL transcripts for degradation, which delays or prevents flowering (6, 8). When

DOG1 function is compromised, miR156 levels are reduced, SPL expression is enhanced, and early

flowering would result, especially when MIR156 is overexpressed (Figs. 2, 3). SPL3/4/5/9/15 can directly

promote expression of FT in leaves and of LEAFY (LFY), FRUITFULL (FUL), APETALA1 (AP1),

SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and AGAMOUS LIKE 24 (AGL24)

in apical meristems (6, 8). In addition, miR156 can repress SPL10 and 11 to prevent precocious

expression of genes during seed maturation phase (47), while FUSCA3 (FUS3) can promote MIR156

expression during seed development (48), enabling generational resetting of miR156 levels. SPL9/10/15

can upregulate miR172 through promoting MIR172 expression (38, 41); since miR156 inhibits SPL

expression, when miR156 levels go down, miR172 levels increase (Figs. 2D, 3B). The conversion of

primary MIR172 to miR172 is also proposed to be affected by DOG1’s influence on the DICER complex

(Fig. 3C, 4E-G). miR172 represses AP2-like genes including SCHNARCHZAPFEN (SNZ), which can

affect seedling development (49) and juvenile to adult transition (1, 38, 41). miR172 also can promote

seed germination (Fig. 4D), although the specific mechanism remains unknown. GA promotes both seed

germination and flowering (see Supplementary Text). Other flowering regulatory pathways in the

diagram are discussed in the Supplementary Text. ABA and DOG1 can act in parallel to regulate seed

dormancy (thermoinhibition) (Fig. 4C; S1E-F; S3) through regulating GA biosynthesis or signal

transduction or possibly through additional mechanisms (27, 50-52).

SPL10,11

SPL3,4,5

SPL9,10,15

FT

miR156

DOG1

Ju

ven

ile to

ad

ult

Germination

Em

bry

o to

seed

ling

Flowering

phase

Em

bry

og

en

esis

an

d s

eed

develo

pm

en

t tr

an

sit

ion

Dormancy to germination transition

miR172

AP2-like

LFY, FUL, AP1,

SOC1, AGL24

Autonomous

pathway

FLC

Vernalization

pathway

Ambient low

temperature

SVP/FLMFT

Photoperiod

CO

SNZ

Dormant

seeds

Juvenile

phase

Adult phase

High

temperature

ABA

GA

MIR156

DICER complex

(DCL1, HYL1, TGH, SE, CDC5)

GA20ox

GA pathway

GA3ox

RGL1,

RGAGA2ox

GA

MIR172FUS3

Adult to flowering transition

Fig. S6

Fig. S6. Overexpression of LsMIR156 resulted in delayed flowering in lettuce. Representative lettuce

plants of Sal-WT (left) and Sal overexpressing LsMIR156 (Sal-LsMIR56OX) (right) are shown at 97 days

after germination.

Fig. S7

Fig. S7. Relative mRNA and miRNA levels in leaves and apical meristems of Arabidopsis at different

developmental stages. Relative levels of (A) LsMIR156, (B) miR156, (C) miR172, (D) SPL3 and (E)

SPL9 in Col-35S:LsMIR156 (Col-LsMIR156) and dog1-3-35S:LsMIR156-I (dog1-LsMIR156) leaves at

15, 25 and 35 days after germination (F). Relative mRNA levels of AtMIR156, miR156 and miR172 in

apical meristems of 25-day-old Col-35S:AtMIR156 (Col-AtMIR156) plants and in apical meristems of

dog1-3 plants of the same age into which Col-35S:AtMIR156 had been crossed (dog1×AtMIR156).

Fig. S8. Primary dormancy of Col-WT, dog1-3 and dog1-5 seeds. Freshly-harvested seeds (5 days after

harvesting) were imbibed at 25°C for 5 days.

Supplemental Tables

Table S1. DOG1 protein amino acid sequence similarity (%) among four lettuce and two Arabidopsis

genotypes.

Salinas Saligna PI251246 US96UC23 At-Col At-Cvi

Salinas 100

Saligna 96.0 100

PI251246 99.3 95.2 100

US96UC23 96.7 98.2 95.2 100

At-Col 49.5 49.8 49.1 50.2 100

At-Cvi 50.4 49.7 50.4 50.3 90.4 100

Table S2. DOG1 effect on number of leaves at flowering of Arabidopsis genotypes.

Columns are categories of the number of rosette leaves present at the time of flowering. Values for

different genotypes or growth conditions indicate the percentage of the total number of plants that have

the indicated number of rosette leaves at flowering.

Experiment 1

(LD, 22°C,135 µmol m-2s-1) Hm/Hta PNb Percentage of Flowering Plants

Number of Rosette leaves 5-10 11-15 16-20 21-25 26-30 31-35 36-40 41-50 >51

Col Hm 72 79.2 20.8

Col-35S:LsMIR156-G Hm 65 4.6 6.2 43.1 46.1

Col-35S:LsMIR156-C Hm 53 9.4 24.5 62.3 3.8

Col-35S: AtMIR156 Hm 70 1.4 4.3 11.4 42.9 40.0

dog1-3 Hm 61 67.2 32.8

dog1-3-LsMIR156-I Hm 69 23.2 52.2 24.6

dog1-3-35S:LsMIR156-G Hm 68 1.5 58.8 39.7

dog1-3 × 35S:AtMIR156-A Hm 63 3.3 39.7 39.7 17.5

dog1-3 × 35S:AtMIR156-G Hm 65 4.6 32.3 33.8 18.5 7.7 3.1

nced9-1 Hm 48 62.5 37.5

nced9-1×AtMIR156#7 Hm 40 7.5 15.0 40.0 37.5

Experiment 2

(LD, 21°C,100 µmol m-2s-1) Hm/Hta PNb Percentage of Flowering Plants

Number of Rosette leaves 5-10 11-15 16-20 21-25 26-30 31-35 36-40 41-50 >51

Col Hm 48 83.3 16.7

Col-35S:LsMIR156 Ht 72 1.4 1.4 5.6 8.3 4.2 9.7 4.2 65.2

dog1-3 Hm 36 77.2 22.8

dog1-3-35S:LsMIR156 Ht 48 1.1 28.1 27.1 14.5 25.0 2.1 2.1

dog1-5 Hm 20 80 20

dog1-5-35S:LsMIR156 Ht 28 7.1 3.6 3.6 14.3 71.4

nced9-1 Hm 47 85.1 14.9

nced9-1-35S:LsMIR156 Ht 37 2.7 5.4 2.7 8.1 5.4 13.5 62.2

Experiment 3

(LD, 22°C,135 µmol m-2s-1) Hm/Hta PNb Percentage of Flowering Plants

Number of Rosette leaves 5-10 11-15 16-20 21-25 26-30 31-35 36-40 41-50 >51

Ler Hm 48 62.5 37.5

Ler-35S:LsMIR156 Ht 63 3.2 9.6 23.8 60.3 3.1

dog1-1 Hm 57 64.9 35.1

dog1-1-35S:LsMIR156 Ht 52 34.6 44.2 21.2

Experiment 4

(SD, 22°C,135 µmol m-2s-1) Hm/Hta PNb Percentage of Flowering Plants

Number of Rosette leaves 11-20 21-30 31-40 41-50 51-60 61-70 >71

Col Hm 37 29.7 64.9 5.4

Col-35S:LsMIR172 Ht 77 18.2 45.5 22.1 11.7 2.5

dog1-3 Hm 64 3.1 17.2 67.2 12.5

dog1-3-35S:LsMIR172 Ht 100 12 27 30 25 3 3 a Hm: Homozygous; Ht: Heterozygous

b PN: number of plants when homozygous; number of independent transgenic individuals when heterozygous.

Table S3. Sequences of primers used in this study.

PRIMER NAME SEQUENCE SEQUENCE

ID

PURPOSE

AtSPL4-qF TCAAGGGTAGAGATGACACTTCCTATGC AT1G53160 RT-PCR

AtSPL4-qR TCTCTCATCATAGCAAGTGATGGACCCTG

AtSPL5-qF CCAGACTCAAGAAAGAAACAGGGTAGACAG AT3G15270 RT-PCR

AtSPL5-qF TCCGTGTAGGATTTAATACCATGACC

AtSPL9-qF CAAGGTTCAGTTGGTGGAGGA AT2G42200 RT-PCR

AtSPL9-qR TGAAGAAGCTCGCCATGTATTG

AtSPL10-qF TCAGGAGGCCTCCATGAATCTCA AT1G27370 RT-PCR

AtSPL10-qR GGCCACGGGAGTGTGTTTGAT

LBa1 TGGTTCACGTAGTGGGCCATCG PROK2 GENOTYPING T-DNA

MUTANT

AtDOG1-3LP TTCCAGGAACGTTGTCGTATC AT5G45830 GENOTYPING OF

SALK_000867

AtDOG1-3RP AGTTTGTGACCCACACAAAGC GENOTYPING OF

SALK_000867

AtDOG1-5LP AAGTTGATCATGTTCATGGGG AT5G45830 GENOTYPING OF

SALK_022748C

AtDOG1-5RP TATGGTAGCAAGGTGCAATGC GENOTYPING OF

SALK_022748C

AtNCED9-LP ATTCCGCTTGATCAACCAAC AT1G78390 GENOTYPING OF

ATNCED9-1

AtNCED9-RP CACAGTTGGATCATTGGACACT GENOTYPING OF

ATNCED9-1

AtMIR159A-qF TCAGGAGCTTTAACTTGCCCTTT AT1G73687 RT-PCR

AtMIR159A-qR CACGCTAAACATTGCTTCGGAAT

AtMIR319B-qF AGCTTTCTTCGGTCCACTCATGG AT5G41663 RT-PCR

AtMIR319B-qR GAGCTCCCTTCAGTCCAAGCATA

AtMIR156-qF TGAGCACACAAAGGCAATTT AT2G25095 RT-PCR

AtMIR156-qR CAGTGAGCACGCAAGAGAAG

AtMIR172-qF ATCTGTTGATGGACGGTGGT AT2G28056 RT-PCR

AtMIR172-qR AATAGTCGTTGATTGCCGATG

AtACT8-qF CTCAGGTATTGCAGACCGTATGAG AT1G49240 RT-PCR

AtACT8-qR CTGGACCTGCTTCATCATACTCTG

AtACT2-qF CTTGCACCAAGCAGCATGAA AT3G18780 RT-PCR

AtACT2-qR CCGATCCAGACACTGTACTTCCTT

LsDOG1-RACE-F ATGGCCAAACAAATGAAACACCA KT337314 LsDOG1 CDNA

CLONING

GENERACER-

ADAPTER

GCTGTCAACGATACGCTACGTAACGGCATGAC

AGTGTTTTTTTTTTTTTTTTTTTTTTTT

LsDOG1 CDNA

CLONING

GENERACER 3’R GCTGTCAACGATACGCTACGTAACG LsDOG1 CDNA

CLONING

GENERACER

NESTED 3’R

CGCTACGTAACGGCATGACAGTG LsDOG1 CDNA

CLONING

LsDOG1-RACE-R1 TCTCTAAAATACCCTTCAGTGTACTCATCCTC LsDOG1 CDNA

CLONING

LsDOG1-RACE-

NESTEDR1

TCTGCTTCCATCAAAACATTATACATATCA LsDOG1 CDNA

CLONING

LsDOG1-Saligna-F CACCATGGCCAAACAAATGAAACACC KT337316 SALIGNA DOG1

CONSTRUCT

LsDOG1-Saligana-R TTCAGTGTCACGTCTCCGCTG GTGGTG

LsNCED4Pro-F TTAAAGATCTAACAGACAAAAGTCAACGGAGT

TAG

KC676791 SALIGNA DOG1

CONSTRUCT

LsNCED4Pro-R TTGAGCTCTGGAGGCGGTGGTAGTGATG

LsDOG1-UC-F CACCATGGCCAAAAAAATGAAACACC JO034198 UCDOG1

CONSTRUCT

LsDOG1-UC-R TTCAGTGTCACGTCTCCGGTGGTGGTG

LsDOG1-PI/SAL-F CACCATGGCCAAACAAATGAAACACC KT337314/

KT337315

FOR SALINAS AND PI

DOG1 CONSTRUCT

LsDOG1-PI/SAL-R TTCAGTGTCACGTCTCCGGTGGTGGGT

PRODOG1-F AGATCTTATGGGTCGAAGGGACCAAT KT290282 PI DOG1 ECTOPIC

EXPRESSION

CONSTRUCT

PRODOG1-R GAGCTCTGGTTTTTTTTGTAAGGGGTGACTC

LsMIR156-qF TGATGCTGCATGTCAACAGA JI599382 RT-PCR

LsMIR156-qR CTCTATCGCCCCCACAAGTA

LsMIR156-BPF GGGGACAAGTTTGTACAAAAAAGCAGGCTTCC

GGTTCTGTTCCGATATCC

JI599382 LsMIR156

CONSTRUCT

LsMIR156-BPR GGGGACCACTTTGTACAAGAAAGCTGGGTCTC

TAAATTGGGATTCAACAAATTC

LsMIR172-BPF GGGGACAAGTTTGTACAAAAAAGCAGGCTTCT

ACTCCTCATCTCTATCCTCTCCTTG

JI585366 LsMIR172

CONSTRUCT

LsMIR172-BPR GGGGACCACTTTGTACAAGAAAGCTGGGTCAG

TTTTTGTTATCTCAAGTTGTCTAATCC

SalDOGRNAi-F GCACTAGTCCATGGCAACAGCTCGATCTGGAC

GAATTA

KT337314 LsDOG1-RNAi

CONSTRUCT

SalDOGRNAi-R CGGGATCCGGCGCGCCGCATGAAGTTCATCTA

TTCTTTTGAGC

LsDOG1-qF CCAAAAAAATCGTCTCCCACTT KT337314 RT-PCR

LsDOG1-qR CAAAAAGGAAGGCCCATCGT RT-PCR

LsSPL3-qF TAGCCGGTTTCATGAGCTTTC JI599986 RT-PCR

LsSPL3-qR TCATTGTGCCCTGCTAAACG

LsSPL4-qF GTAGGCGTTTAGCTGGGCATA JI598022 RT-PCR

LsSPL4-qR CCGTCTCCATAAGTTTCAAAGGA

LsSPL9-qF TGCTCATTCGAAAACGGCTAA JI582339 RT-PCR

LsSPL9-qR TGGAACCTGCTGCACTGTTG

LsFT-qF GGACTCTCATAGCACACAATTTCTTG JI597116 RT-PCR

LsFT-qR CATTGGTTGGTGACCGATATACC

LsSVP-qF GGCATAGCCTGCATTCAAAAA JI583990 RT-PCR

LsSVP-qR GGCATAGTTGGCGTCTTCAAC

LsFLC-qF GCTCAGAATCTTGCTCATGCTTT JI587615 RT-PCR

LsFLC-qR CGTCGCTCTTTTCATCTTTTCC

LsFLM-qF TCCGCCCCATTGATATTGA JI603388 RT-PCR

LsFLM-qR ACAAAGAAAGTTAGGTCTAGCCACAAG

LsCO-qF GTGGCAACGCCGATAGTGT JI585578 RT-PCR

LsCO-qR CATATTCCATCCCCAACTGAAAC

LsCLARI-qF CTGCTTCCGCTATCTACTTCCTAAA JI580445 RT-PCR

LsCLARI-qR TTCCCCCGACGTCATCAC

LsPDF1-qF AAGCTTGGTGCTCTCTGCAT JI579444 RT-PCR

LsPDF1-qR GGGACCAAATTCCTCTGCGA

LsEF4α-qF TCGTCATCGGCCATGTTGAC JI582565 RT-PCR

LsEF4α-qR TCGTCATCGGCCATGTTGAC

LsACT7-qF GATCACGATTGGAGCTGAAAGA JI581679 RT-PCR

LsACT7-qR GCAGCTTCCATTCCAATCAAA

LsUBQ10-qF TGGTCGTACGCTTGCTGATT JI587827 RT-PCR

LsUBQ10-qR AAAAACACCCACCACGAAGC

AtDCL1-qF CGTTGTTATGCGTTTCGACCTTGC AT1G01040 RT-PCR

AtDCL1-qR AACGCTGCGTGAGATACATTTCCTC

AtHYL1-qF TTGCCTGGATTCTTCAATCGTAAGG AT1G09700 RT-PCR

AtHYL1-qR TAGGTTCTTGCATAATCCCGTTTCG

AtTGH-qF AGATCTCGCATGTCTGCCAA AT5G23080 RT-PCR

AtTGH-qR ACCCGGAGCAATCTTTCCTG

AtSE-qF CCACCGCCTCGTAGGGATTACA AT2G27100 RT-PCR

AtSE-qR CCACCATGGTCATACCCAAATCTTC

AtCDC5-qF GTTTCCGAGCACAGGCATTG AT1G09770 RT-PCR

AtCDC5-qR TCCTCTCCAGTGGCTAGCTT

LsTGH-qF AGCGGTTTGATTTTGTCACC JI574976 RT-PCR

LsTGH-qR CGAGGGAGTTGCGACTTTAG

LsSE-qF TGGATACCAAGGTGGTCCAT JI576033 RT-PCR

LsSE-qR ACCTGCGTTCAGCTTCTGAT

LsCDC5-qF GATTATGATGGAGGCCGAGA JI575030

RT-PCR

LsCDC5-qR GAAAAATCCGAAGGATGCAA

LsDCL1-qF TGGTTGCAACAGAGGTTGGA JI573836 RT-PCR

LsDCL1-qR TTGCTGGTGCCATCTTCTTG

LsHYL1-qF GCGATAAACCTCCCCAATTTT JI587263 RT-PCR

LsHYL1-qR GGAAACGGAGTCAGCGAACA

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