CM

The Plant Cell, Vol. 30: 1322–1336, June 2018, www.plantcell.org © 2018 ASPB.

Parallel Evolution of Common Allelic Variants Confers

Flowering Diversity in Capsella rubella[OPEN]

Li Yang,a,1 Hui-Na Wang,a,1 Xing-Hui Hou,a,b Yu-Pan Zou,a,b Ting-Shen Han,a,b Xiao-Min Niu,a,b Jie Zhang,a,b
Zhong Zhao,c Marco Todesco,d Sureshkumar Balasubramanian,e and Ya-Long Guoa,b,2
a
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093,
China
b
University of Chinese Academy of Sciences, Beijing 100049, China
c
School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
d
Department of Botany, University of British Columbia, Vancouver BC V6T 1Z4, Canada
e
School of Biological Sciences, Monash University, VIC 3800, Australia
ORCID IDs: 0000‑0003‑3842‑6341 (L.Y.); 0000‑0002‑5540‑7209 (H.-N.W.); 0000‑0003‑3144‑925X (X.-H.H.); 0000‑0003‑3033‑0273
(Y.-P.Z.); 0000‑0002‑8612‑6581 (T.-S.H.); 0000‑0001‑6568‑5336 (X.-M.N.); 0000‑0002‑8476‑4088 (J.Z.); 0000‑0001‑8044‑0409
(Z.Z.); 0000‑0002‑6227‑4096 (M.T.); 0000‑0002‑1057‑2606 (S.B.); 0000‑0002‑4643‑4889 (Y.-L.G.)

Flowering time is an adaptive life history trait. Capsella rubella, a close relative of Arabidopsis thaliana and a young species,
displays extensive variation for flowering time but low standing genetic variation due to an extreme bottleneck event, pro-
viding an excellent opportunity to understand how phenotypic diversity can occur with a limited initial gene pool. Here, we
demonstrate that common allelic variation and parallel evolution at the FLC locus confer variation in flowering time in C. rubella.
We show that two overlapping deletions in the 5′ untranslated region (UTR) of C. rubella FLC, which are associated with
local changes in chromatin conformation and histone modifications, reduce its expression levels and promote flowering. We
further show that these two pervasive variants originated independently in natural C. rubella populations after speciation
and spread to an intermediate frequency, suggesting a role of this parallel cis-regulatory change in adaptive evolution. Our
results provide an example of how parallel mutations in the same 5′ UTR region can shape phenotypic evolution in plants.

INTRODUCTION understood biological process at the molecular and genetic lev-

els (Koornneef et al., 2004; Hepworth and Dean, 2015; Bloomer
Parallel evolution, which refers to different species or popula- and Dean, 2017). Natural variation for flowering time within a
tions that share a common ancestor independently evolve the species has attracted increasing attention owing to its likely cor-
same trait by changing the same genes, is known to occur and relation with adaptation to local environments (Salomé et al.,
can play a role in the evolution of adaptive traits (Wood et al., 2011; Weigel, 2012). Although mutations in more than 100
2005). It has been studied in closely related Caenorhabditis spe- genes are known to affect flowering time in Arabidopsis thaliana
cies (McGrath et al., 2011) and in natural populations within the (Srikanth and Schmid, 2011; Lee et al., 2013; Posé et al., 2013;
same species in sticklebacks (Colosimo et al., 2005; Chan et al., Wahl et al., 2013; Yan et al., 2014), only a few genes play im-
2010). For example, parallel evolution at the melanocortin-1 re- portant roles in regulating flowering time variation among nat-
ceptor gene locus Mc1r confers adaptive variation in pigmenta- ural accessions. These include FLOWERING LOCUS C (FLC),
tion in reptiles, birds, and mammals (Hoekstra, 2006; Arendt and FLOWERING LOCUS M (FLM), FRIGIDA (FRI), PHYTOCHROME
Reznick, 2008; Protas and Patel, 2008; Elmer and Meyer, 2011). C, and SHORT VEGETATIVE PHASE (Johanson et al., 2000;
Mutations underlying parallel evolution have also been stud- Gazzani et al., 2003; Caicedo et al., 2004; Lempe et al., 2005;
ied to understand the predictability of evolution, i.e., whether Shindo et al., 2005; Werner et al., 2005; Balasubramanian et al.,
certain traits can be shaped by a specific gene(s) (Stern and 2006; Méndez-Vigo et al., 2013; Sureshkumar et al., 2016; Lutz
Orgogozo, 2009; Martin and Orgogozo, 2013). et al., 2017; Zou et al., 2017).
  Flowering time is a crucial adaptive trait for plants, as flowering   Epistatic interactions between FRI and FLC regulate flower-
at the wrong time can mean failure to set seed and reproduce. ing time variation in natural populations of A. thaliana (Gazzani
Flowering time can vary tremendously both within and between et al., 2003; Lempe et al., 2005; Shindo et al., 2005; Werner
species; therefore, analysis of flowering time offers an excellent et al., 2005). Expression levels of FLC can explain up to 40% of
opportunity to study parallel evolution. It is also a relatively well the variation in flowering time between accessions of A. thaliana
1
These authors contributed equally to this work. (Lempe et al., 2005; Shindo et al., 2005). Although natural vari-
2
Address correspondence to [email protected]. ants of FLC have been described, they occur relatively rarely,
The author responsible for distribution of materials integral to the findings and much of the variation in flowering time is explained by allelic
presented in this article in accordance with the policy described in the variation in FRI and its epistatic interaction with FLC. Therefore,
Instructions for Authors (www.plantcell.org) is: Ya-Long Guo (yalong.
[email protected]).
FRI appears to be the major determinant of flowering time vari-
[OPEN]
Articles can be viewed without a subscription. ation in A. thaliana (Johanson et al., 2000; Lempe et al., 2005;
www.plantcell.org/cgi/doi/10.1105/tpc.18.00124 Shindo et al., 2005).
 Parallel Evolution of Flowering Time in C. rubella  1323

  In species other than A. thaliana, our molecular understanding   In this study, we report that FLC is an important determinant
of how variation for flowering time is achieved is still rudimentary. of flowering time variation in C. rubella. We studied three ac-
Flowering time variation has been studied in some relatives of cessions, 86IT1, 762, and MTE, which represent three distinct
A. thaliana, such as Arabidopsis lyrata (Leinonen et al., 2013), points (early, intermediate, and late, respectively) on the flower-
Arabis alpina (Wang et al., 2009), Brassica napus (Raman et al., ing time gradient in C. rubella, to map the loci responsible for
2016), Cardamine flexuosa (Zhou et al., 2013), and Capsella ru- flowering time variation. Using quantitative trait locus analysis
bella (Guo et al., 2012). C. rubella is of particular interest for sev- and fine mapping, we identified two overlapping deletions in
eral reasons. During its speciation, C. rubella went through an the 5′ untranslated region (UTR) of CrFLC as causal mutations.
extreme genetic bottleneck (a sharp reduction of population size These genetic variants were associated with the open chroma-
due to environmental change or other factors) concurrent with tin state and histone modification levels at the FLC locus, and
the transition from an outcrossing to a selfing mating system, they affected FLC expression levels and flowering time. We also
resulting in its current low genetic diversity (Foxe et al., 2009; showed that allelic variation at FLC is pervasive and that the
Guo et al., 2009). Therefore, this relatively young species con- two causal 5′ UTR deletions originated independently in natural
tains only limited genetic variation on which selection can op- C. rubella populations post speciation and subsequently spread
to intermediate frequencies. Overall, our results suggest that se-
erate. Nevertheless, there is considerable variation for flowering
quence variation in the 5′ UTR of FLC has a profound effect on
time among natural accessions of C. rubella (Guo et al., 2012).
C. rubella flowering time variation. They also support that parallel
Investigation of the molecular basis of flowering time variation
evolution of early flowering was regulated via two overlapping
in C. rubella will therefore not only broaden our understanding
recurrent deletions in CrFLC.
of the genetic factors regulating this variation in nature but also
address the fundamental question of how a young species can
adapt to diverse environments, despite limited genetic variation.
RESULTS
It is also likely to provide novel opportunities to study parallel
evolution in plants with limited standing genetic variation.
  Allelic variation at FLC and associated variation in gene ex- Genetic Mapping Identifies CrFLC as the Cause of
pression has been shown to be associated with flowering time Flowering Time Variation in Two Distinct F2 Populations
in A. thaliana relatives as well, including C. rubella (Slotte et al., To identify genetic variants controlling flowering time variation in
2009; Yuan et al., 2009; Hu et al., 2011; Guo et al., 2012; Raman C. rubella, we analyzed three accessions, MTE, 762, and 86IT1,
et al., 2016). In a previous study, we reported an allele of FLC that which differ considerably in their flowering time. The reference
causes early flowering in C. rubella (Guo et al., 2012), in which a accession MTE is one of the latest flowering accessions, 762 is
single nucleotide polymorphism (SNP) deleted a splice acceptor a intermediate flowering accession, and 86IT1 is an early flow-
site, that results in a frameshift and ultimately leading to a trun- ering accession (Supplemental Table 1). To dissect the genetic
cated, nonfunctional FLC product. However, this polymorphism basis underlying the dramatic differences in flowering time
is unique to a single accession and is therefore unable to explain among these accessions, we crossed MTE with 86IT1 and 762
variation in flowering time among other C. rubella accessions. (using 86IT1 and 762 as the female parents) and generated two
1324  The Plant Cell

F2 populations. F1 progeny from both crosses showed the late downstream sequence. We detected two polymorphisms in the
flowering phenotype, suggesting that late flowering is a poten- FLC locus of 86IT1 compared with MTE; a G-to-A SNP in in-
tially dominant phenotype. tron 1 (SNP_3256) and a 32-bp deletion (Del-32) in the 5′ UTR
  The 86IT1 × MTE F2 population (600 plants) showed a distinct that was located 44 bp upstream of the start codon (Figure 1E;
bimodal distribution for flowering time (Figure 1A), suggesting Supplemental Figure 1). Four polymorphisms distinguished the
the presence of a major causal locus regulating flowering time FLC locus of 762 from the MTE allele: two T-to-A SNPs in the
variation in this cross. A bimodal distribution for flowering time promoter (SNP_-2519 and SNP_-1400), a T-to-A SNP in intron
was also observed in the 762 × MTE F2 population (320 plants), 1 (SNP_1134), and a 54-bp deletion (Del-54) located 41 bp up-
indicating the presence of a large effect locus (Figure 1B). How- stream of the start codon in the 5′ UTR (Figure 1F; Supplemen-
ever, it was less noticeable than in 86IT1 × MTE population, sug- tal Figure 1). The 5′ UTR deletions overlapped, and the 54-bp
gesting that additional loci likely contribute to the flowering time deletion in accession 762 completely encompassed the 32-bp
variation we observed in the cross between MTE and 762. deletion observed in accession 861T1 (Figures 1E and 1F).
  To identify the causal locus (or loci), we used a mapping-by- We did not find any nonsynonymous polymorphisms, suggest-
sequencing approach, which integrates traditional genetic map- ing that the phenotypic differences were caused by regulatory
ping with next-generation whole genome sequencing by analyzing polymorphisms.
allele frequencies in a pooled sample (Schneeberger et al.,   To test whether the observed polymorphisms affected gene
2009). Tissues of 115 individuals from the 86IT1 × MTE F2 pop- expression, we measured FLC expression level in 14-d-old
ulation and 46 individuals from the 762 × MTE F2 population, seedlings of three accessions. The expression levels of FLC dif-
representing the early flowering fractions, were pooled to gen- fered significantly among the accessions (P < 0.01, Student’s
erate two distinct samples for resequencing. Short reads of each t test) (Figure 2A), with levels being lowest in 861T1, followed
pooled DNA sample were aligned to the reference MTE genome by 762 and MTE. This correlated well with their flowering time
to calculate allele frequencies using SNP data. We reasoned that phenotypes. To assess whether these different FLC alleles
regions enriched for 86IT1 or 762 SNPs would be linked to genes vary in their ability to delay flowering, we performed transgenic
underlying early flowering. complementation experiments in A. thaliana. The entire FLC
  Mapping analysis identified a region on chromosome 6 (Fig- genomic fragment (∼9.5 kb) from all three different accessions
ures 1C and 1D) that is associated with early flowering in both (MTE-FLC, 86IT1-FLC, and 762-FLC; Figure 2B) was cloned
populations. This raised the possibility that both 86IT1 and 762 and transformed into A. thaliana FRIsf2 flc-3 plants, which har-
might harbor common allelic variation resulting in early flower- bor a nonfunctional FLC allele and a functional FRI allele in the
ing. We genotyped early flowering individuals from the two F2 reference Col-0 background. The flowering phenotypes of the
populations (94 early flowering plants out of 600 individuals from transgenic plants mimicked the parental strains harboring the
the 86IT1 × MTE F2 population and 188 early flowering plants respective alleles (Figure 2D; Supplemental Figure 2). Transgenic
out of 1470 individuals from the 762 × MTE F2 population) with plants expressing the MTE-FLC allele showed strongly delayed
additional genetic markers to further narrow down the mapping flowering, while transgenic plants expressing the 86IT1-FLC and
interval (Supplemental Tables 2 and 3). Using this approach, 762-FLC alleles flowered significantly earlier (P < 0.01, Student’s
we fine-mapped the causal locus to a 120-kb interval (between t test; Figure 2D; Supplemental Figure 2). Expression analysis
markers nearby 3.09 and 3.21 Mb, containing 35 open reading revealed that their flowering phenotypes correlated with the ex-
frames) in the 86IT1 × MTE population and to a 13-kb interval pression levels of the transgene (Figures 2C and 2D). Consis-
(between markers nearby 3.113 and 3.126 M, containing five tent with these findings, the expression levels of FT and SOC1,
open reading frames) for the 762 × MTE population (Figures 1E downstream targets of FLC, were inversely correlated with the
and 1F). Interestingly, the mapping interval overlapped between level of FLC expression (Supplemental Figure 3). Taken together,
the two populations, further supporting the possibility that there our results demonstrated that allelic variation at FLC is a major
might be common allelic variation. Notably, the ortholog of A. determinant of the variation in flowering time among the tested
thaliana FLC, a critical integrator of floral transition, was located populations of C. rubella.
within the overlap between the mapped region in the two popu-
lations (Figures 1E and 1F; Supplemental Tables 4 and 5). Given Deletions in the 5′ UTR of CrFLC Reduce Its Expression
both that no other flowering time-associated genes are present and Promotes Early Flowering
in this interval and that FLC is known to be important for flow-
ering time regulation, we investigated whether CrFLC could be Among the polymorphisms that differentiated the three acces-
the gene causing the phenotypic variation that we observed in sions (Figures 1E and 1F), only the deletion in the 5′ UTR was
both F2 populations. shared between the two earlier flowering accessions (86IT1 and
762) comparing to the later flowering accession MTE, although
Sequence and Expression Variation of FLC Affects the length of the deletion varied between the accessions. There-
Flowering Time in C. rubella fore, we asked whether these deletions were sufficient to explain
the observed phenotypes. Deleting 32 bp from the 5′ UTR of the
To analyze sequence variation at the FLC locus, we sequenced MTE allele MTE-FLC (MTE-Del-32) suppressed late flowering
a 9554-bp genomic DNA fragment from the three accessions in transgenic A. thaliana plants, but reintroducing the deleted
that included the ∼2.7-kb upstream region including the puta- region in the 86IT1 allele 86IT1-FLC (86IT1-Ins-32) resulted in
tive promoter, the entire coding region (∼5.8 kb), and ∼1 kb of late flowering plants (Figures 3A and 3B; Supplemental Figure
 Parallel Evolution of Flowering Time in C. rubella  1325

Figure 1.  Mapping the Genetic Basis of the Flowering Time Variation in 86IT1 × MTE and 762 × MTE.

(A) and (B) Flowering time in 600 F2 individuals from the 86IT1 × MTE cross and 320 F2 individuals from the 762 × MTE cross. Plants were grown at
20°C in long days (16 h light/8 h dark) in 2012 and 2013, respectively. The parent population means are indicated above each graph.
(C) and (D) SHOREmap interval analysis of flowering time QTL in two combinations. The homozygosity estimator is 0 for equal allele frequencies in
both parents. Progeny from the 86IT1 × MTE cross score 1 if homozygous for the late-flowering accession MTE and −1 if homozygous for the early-
flowering accession 86IT1. Progeny from the 762 × MTE cross score 1 if homozygous for the late-flowering accession MTE and −1 if homozygous for
the intermediate-flowering accession 762. Sliding windows of 150 and 200 kb with a step size of 10 kb were used for 86IT1 × MTE and 762 × MTE
plants, respectively. The eight largest scaffolds of a preliminary C. rubella genome assembly are shown, corresponding to the majority of the eight
chromosomes.
(E) and (F) Fine mapping by a genetic-linkage analysis and sequence variation of the candidate gene C. rubella FLC. The number of homozygous MTE
and heterozygous individuals at the markers is indicated above the linkage map. Red vertical lines indicate Carubv10003343m.g (C. rubella FLC) gene
sequence polymorphisms between MTE and 86IT1, or between MTE and 762, and indel variation in the 5′ UTR is shown as well.

4A). Consistent with these findings, the expression levels of FLC Del-54), with early flowering and reduced C. rubella FLC ex-
were changed, with the lines harboring the deletions showing pression (Figures 3D to 3F; Supplemental Figure 4B). Moreover,
reduced FLC expression (P < 0.01, Student’s t test; Figure 3C). consistently, in all these transgenic lines, the expression levels of
  A similar pattern was also observed in transgenic lines har- downstream target genes SOC1 and FT were inversely correlated
boring the MTE allele with the 54-bp deletion MTE-FLC (MTE- with the FLC expression level (Supplemental Figures 5 and 6).
1326  The Plant Cell

Figure 2.  Functional Analysis of C. rubella FLC Alleles in A. thaliana.

(A) RT-qPCR analyses of C. rubella FLC expression levels in the three C. rubella accessions. Expression data are standardized to the C. rubella TUBU-
LIN (Carubv10017235m) transcript levels. Values shown are the means ± sd (n = 3 biological replicates, with 15 seedlings/replicate). **P < 0.01 for the
comparison between two early-flowering accessions and MTE (Student’s t test).
(B) Schematic illustration of constructs in the MTE, 86IT1, and 762 alleles of FLC. The structure of C. rubella FLC is illustrated at the top, and the po-
sition of ATG is defined as +1. Below the structure are illustrations of C. rubella FLC sequence variation and indel variation in the 5′ UTR region among
MTE, 86IT1, and 762.
(C) RT-qPCR analyses of the expression levels of FLC using pools of 40 independent T1 transgenic lines. Data are standardized relative to the abun-
dance of A. thaliana TUBULIN (AT5G62690) transcripts. Values shown are the means ± sd (n = 3 biological replicates; collected from 40 independent
T1 transgenic lines per replicate). **P < 0.01 between two early-flowering accessions and MTE allele transgenic lines (Student’s t test).
(D) Flowering time of transgenic plants in 20°C long-day conditions (16 h light/8 h dark). Values for two control lines (Col-0 and FRISf-2 flc-3, without
hygromycin selection) are shown on the left. Values for T1 transgenic lines with the FRISf-2 flc-3 background expressing the indicated FLC allele driven
from its native promoter are shown on the right (n = 80 T1 transgenic lines). The experiments shown in (A), (C), and (D) were repeated on three separate
occasions (three experimental replicates) from screening the transgenic plants through to RNA extraction and RT-qPCR and phenotyping, with similar
results. The results shown are from one of the three experimental replicates.

Taken together, these experiments confirmed that a deletion in   FLC expression in A. thaliana is regulated by the antisense
the 5′ UTR is sufficient to explain the observed differences in transcript COOLAIR long noncoding RNA, and increases during
FLC expression and its associated phenotype. vernalization, accelerating the silencing of FLC (Csorba et al.,
2014; Marquardt et al., 2014). Given that little is known about
antisense transcripts in C. rubella, 5′ and 3′ RACE experiments
Deletions in the 5′ UTR of CrFLC Correlate with a Reduced were performed to identify putative CrCOOLAIR transcripts ex-
Response to Vernalization pressed in C. rubella during vernalization. Three CrCOOLAIR
transcripts, all of which encompassed the FLC sense transcripts,
FLC acts as a central control in the responses to prolonged cold were detected (Supplemental Figure 7B). However, in contrast
treatment in A. thaliana and related species (Whittaker and Dean, to A. thaliana (Csorba et al., 2014), we did not find any short
2017). If the 5′ UTR deletion affects vernalization, we would ex- antisense transcripts only located at the 3′end of FLC (Supple-
pect the lines MTE and 86IT1, which differed for the 5′ UTR mental Figure 7B).
deletion, to differ in their vernalization response. Consistent with   Further analysis of the sense transcript and antisense tran-
this, a vernalization treatment reduced MTE flowering time by scripts of FLC in C. rubella revealed that the 32-bp deletion of
more than 60 d (from more than 132 to 56 d). By comparison, 86IT1-FLC was located either in the 5′ UTR of the sense transcript
vernalization reduced the flowering time of 86IT1 by only 26 d of FLC or in the 3′ region of the antisense transcript CrCOOLAIR.
(from 68 to 42 d; Supplemental Figure 7A). This suggested that We monitored the expression of FLC and COOLAIR in MTE and
the 5′ UTR deletion found in the 86IT1-FLC allele results in a 86IT1 plants at different time points during an 8-week cold treat-
considerably reduced sensitivity to vernalization. ment. While FLC expression decreased during the vernalization
 Parallel Evolution of Flowering Time in C. rubella  1327

Figure 3.  Deletions in the C. rubella FLC 5′ UTR Result in Reduced FLC Expression and Early Flowering.

(A) and (D) Schematic illustration of the DNA regions introduced or removed to create the Del-32 and Del-54 constructs in the MTE-FLC, 86IT1-FLC
and 762-FLC alleles. The 32- and 54-bp deletions in the FLC 5′ UTR are indicated with orange and green boxes, respectively. FLC is illustrated at the
top, and the position of ATG is defined as +1.
(B) and (E) Flowering time for transgenic plants in the 20°C long-day condition. Values for two control lines (Col-0 and FRISf-2 flc-3, without hygromycin
selection) are shown on the left of each graph. Values for transgenic lines with the FRISf-2 flc-3 background expressing different C. rubella FLC alleles
with or without Del-32 or Del-54 are shown to the right of the control lines.
(C) and (F) RT-qPCR analyses of FLC expression levels using both a pool of independent T1 transgenic lines (inset) and 10 randomly selected indepen-
dent T1 transgenic lines. Data are standardized relative to the abundance of A. thaliana TUBULIN (AT5G62690) transcripts and shown as means ± sd
(n = 3 biological replicates; for individual lines, each replicate was collected from 3 individual plants; for insets, each replicate was collected from 30 or
25 T1 transgenic lines in [C] and [F], respectively). The experiments shown were repeated on three separate occasions (three experimental replicates),
with similar results. The results shown are from one of the three experimental replicates.

treatment in both MTE and 86IT1, the effect was much stronger one week of cold treatment in both accessions. However, at lat-
in 86IT1, with FLC levels being consistently higher in MTE plants er stages, the difference in COOLAIR expression between the
(Supplemental Figure 7C). In contrast, COOLAIR expression in accessions was minimal (Supplemental Figure 7D). Therefore,
86IT1 was ∼50% higher than in MTE, reaching its peak after while the two accessions show partially different patterns of
1328  The Plant Cell

Figure 4.  Sequence Deletion in the FLC 5′ UTR Is Correlated with Altered Chromatin Structure State and Histone Modifications.

(A) FLC splicing efficiency in three C. rubella accessions. Intron retention was calculated as the ratio of unspliced to total (spliced + unspliced) tran-
scripts for all six FLC introns. Results were normalized to C. rubella TUBULIN (Carubv10017235m) results and are shown as means ± sd (n = 3 biolog-
ical replicates; with 15 seedlings per replicate).
(B) RNA stability assay in three C. rubella accessions. After the addition of cordycepin, the rate of mRNA degradation was measured by RT-qPCR to
determine the level of remaining transcripts relative to that of an untreated sample. C. rubella EIF4A and Carubv10017847m.g were used as controls,
representing a stable and an unstable transcript, respectively.
(C) Sensitivity of three regions (regions 1 to 3) of C. rubella FLC to increasing dosages of mononuclease in three transgenic plants. Chromatin with
loose nucleosome occupation is more susceptible to mononuclease digestion and is inefficiently amplified by PCR. The A. thaliana Ta2 region in three
transgenic plants was used as an insensitive control (Shu et al., 2013). The position of each region is labeled, and the A of ATG is referred to as +1.
(D) Regions of C. rubella FLC used for ChIP-qPCR assays.
(E) ChIP-qPCR analyses of six regions of C. rubella FLC. Data are from pooled T2 transgenic plants using antibodies against H3Ac (top), H3K36me3
(middle), and H3K27me3 (bottom), respectively. ChIP values were normalized to their respective DNA inputs. Data are shown are the means ± sd (n =
3 biological replicates; each replicate was obtained from at least 3 g of tissue corresponding to roughly 500 10-d-old seedlings from 10 independent
T2 transgenic lines).
 Parallel Evolution of Flowering Time in C. rubella  1329

COOLAIR expression in response to cold treatments, it is cur- the three FLC alleles. We found that the region around the tran-
rently unclear whether they are linked to the deletion or can scriptional start site and 5′ UTR (region 2; Figure 4C) is the most
explain the differences in flowering time and responses to ver- sensitive to mononuclease digestion and that the MTE-FLC al-
nalization that are observed between MTE and 86IT1. lele was more sensitive to mononuclease digestion compared
with the 86IT1-FLC or 762-FLC alleles in all the three tested
Deletions in the 5′ UTR of CrFLC Are Associated with regions. This is consistent with higher levels of FLC expression
Chromatin Changes in MTE-FLC transgenic plants (Figure 2C).
  Our results suggested nucleosome occupancy may be altered
To elucidate the mechanism by which the 5′ UTR deletions lead at the FLC locus. Because epigenetic regulation is a critical com-
to a reduced expression, we first checked whether the position ponent of the regulation of FLC expression in A. thaliana and the
of 32-bp region that is deleted in 86IT1-FLC 5′ UTR was import- chromatin structure state is closely associated with epigenetic
ant for early flowering. A fragment containing the 32 nucleotides modifications (He et al., 2003; Zhao et al., 2005; Jiang et al.,
that are deleted in 86IT1 was reinserted at the beginning of the 2007, 2008; Liu et al., 2007, 2010; Pien et al., 2008; Xu et al.,
upstream region (as opposed to its original position in the 5′ 2008; He, 2009; Zhang et al., 2012; Lee et al., 2015), we tested
UTR) of a Pro86IT1:gMTE-FLC construct (the FLC genomic se- whether the 5′ UTR deletions are associated with changes in
quence of MTE driven by the 86IT1-FLC promoter), generating histone modifications using transgenic A. thaliana plants ex-
a modified construct hereafter referred to as Pro(32+86IT1):gM- pressing the MTE-FLC, 86IT1-FLC, and 762-FLC alleles. We ex-
TE-FLC (Supplemental Figure 8A). The Pro(32+86IT1):MTE-FLC amined histone modification levels in six regions (A–F; Figure 4D)
construct was then transformed into FRIsf2 flc-3 A. thaliana covering the whole C. rubella FLC locus. H3K36 trimethylation
plants. These transgenic plants had a similar early-flowering and acetylated H3, which marks active transcription, displayed
phenotype as the Pro86IT1:gMTE-FLC transgenic plants (Sup- significant enrichment in the MTE-FLC allele compared with the
plemental Figure 8B). This result implied that the sequence con- 86IT1-FLC and 762-FLC alleles (Figure 4E; P < 0.01, Student’s t
text or location of the 32-bp deletion in 86IT1 is important for its test). In contrast, the H3K27me3 mark, which is associated with
role in modulating FLC activity. repressive chromatin states, was significantly enriched in the
  Subsequently, we considered whether FLC could be differen- 86IT1-FLC and 762-FLC alleles compared to MTE-FLC (Figure 4E;
tially spliced because this can lead to nonsense-mediated mRNA P < 0.01, Student’s t test). The differential enrichments were more
decay, as shown for the FLM locus in A. thaliana (Sureshkumar pronounced in the 5′ end of the gene in region C, which includes
et al., 2016). To assess this, we verified intron retention in all the 32-bp/54-bp deletions found in 86IT1-FLC and 762-FLC, and
three accessions, which would result in transcripts with pre- in region D, which includes the nucleation region of FLC (Figure
mature stop codons. For all six tested FLC introns, there was 4E) (Qüesta et al., 2016; Yuan et al., 2016). This is consistent with
no significant difference of intron retention among the three the mononuclease digestion-PCR results, indicating that MTE-
accessions, and the ratio of unspliced to total transcripts was FLC transgenic plants have a more open chromatin state than that
generally low (∼2–8%; Figure 4A). Consistent with this, treat- of 86IT1-FLC or 762-FLC in these tested regions (Figures 4C to
ment with the transcription inhibitor cordycepin and subse- 4E). These results are also consistent with the dramatic upregula-
quent expression analysis revealed no significant difference tion of FLC expression in MTE-FLC transgenic plants compared
between the three accessions, which suggested that mRNA with the expression levels for 86IT1-FLC and 762-FLC (Figure
stability is unlikely to be the underlying cause for the reduc- 2C). Taken together, these results suggest that the deletions in
tion in FLC expression associated with the 5′ UTR deletion the FLC 5′ UTR is associated with changes in histone modification
(Figure 4B). and chromatin structure state, which is likely to be the underlying
  The gene expression level could also be affected by patterns mechanism for the observed transcriptional changes.
of DNA methylation (He et al., 2011). Therefore, we considered
whether the deletion could influence the methylation status of
FLC, which would likely modulate its expression levels. Bisul- Parallel Mutations in the Same Region of CrFLC Determine
phite sequencing of the locus revealed that the total methyla- Flowering Time
tion levels of 86IT1 and 762 were not significantly different from
those of MTE for all tested regions. Also, the methylation levels Having established that allelic variation at the 5′ UTR is the
of all tested regions were very low (Supplemental Figure 9). This underlying cause for variation in flowering time of the acces-
suggested that the observed reduction in FLC expression was sions we tested, we then asked how common these variants
unlikely to be due to changes in methylation. are among C. rubella accessions. To assess this, we genotyped
  Another possible mechanism through which the 5′ UTR de- 34 C. rubella accessions for these deletions and identified all
letion could affect FLC expression is by modulating openness three haplotypes (MTE-type, 86IT1-type, and 762-type) of FLC
of the chromatin. One distinct characteristic of the genomic re- based on the two deletions in the 5′ UTR (Supplemental Table
gions of open chromatin that are associated with active gene 1). The MTE type was the most abundant haplotype with 22
expression is a pronounced sensitivity to cleavage by the en- accessions (65%). Nine other accessions belonged to the 86IT1
donuclease DNase I (Wu, 1980; Zhang et al., 2012, 2017). To type (26%) and only three accessions belonged to the 762 type
test whether the deletions altered the chromatin organization (9%) (Figure 5A; Supplemental Table 1). Accession 1408 carries
at the FLC locus, we evaluated nucleosome organization by a a unique loss-of-function allele of FLC (Guo et al., 2012), which
mononuclease digestion-PCR in A. thaliana plants expressing was included in the MTE-type haplotype.
1330  The Plant Cell

Figure 5.  Parallel Mutations in the Same Region of C. rubella FLC Shape Flowering Time.

(A) and (B) Flowering time and FLC expression levels of C. rubella accessions. Accessions were classified into three different haplotypes based on the
promoter sequence at FLC (∼2.7 kb, presence/absence of the 32-bp/54-bp deletion in 5′ UTR). The details about the C. rubella accessions used to
provide data for this figure are listed in Supplemental Table 1, except accession 1408, whose early flowering was caused by the splice variation (Guo
et al., 2012). Data are plotted for n individuals per haplotype; all of the data points are presented with box plots showing the median, upper, and lower
quartiles, and minimum and maximum range values for each haplotype. **P < 0.01 and *P < 0.05 between the 86IT1 and MTE haplotypes (Student’s t
test) for flowering time and C. rubella FLC expression level, respectively.
(C) Phylogenetic tree of 19 C. rubella accessions based on whole-genome sequences. Bootstrap values (100 replicates) are indicated at branches.
(D) Phylogeny of FLC alleles of 19 C. rubella accessions based on the FLC full-length fragment (∼9.5 kb). Bootstrap values (1000 replicates) are indi-
cated at branches. Different FLC 5′ UTR haplotypes are indicated by different colors. Orange indicates MTE-type 5′ UTR accessions, green indicates
86IT1-type 5′ UTR accessions, and blue indicates 762-type 5′ UTR accessions. Note that the 86IT1-type and 762-type accessions formed a clade on
the phylogenetic tree of FLC alleles but were separated on the phylogenetic tree based on the whole-genome sequences.

  Having established the pervasive nature of allelic variation at nificant association between allelic variation at FLC’s 5′ UTR
the FLC locus, we assessed how much variation in flowering and the expression levels (P < 0.01). Allelic variation at FLC’s
time could be explained by variation in FLC expression lev- 5′ UTR could account for 32% of the variation in FLC expres-
els. Our analysis revealed that FLC expression could account sion. Given that 60% of the variation in flowering time could
for 60% of variation (P < 0.0001, linear regression analysis of be attributed FLC expression variation, and 32% of variation in
log-transformed expression levels versus days to flowering FLC expression could be explained by allelic variation at FLC’s
[DTF] 20°C long-day condition) in flowering time among ac- 5′ UTR, we asked how much of the variation in flowering time
cessions of C. rubella, confirmed that FLC expression was a key could be attributed to allelic variation at FLC’s 5′ UTR. Regres-
determinant of flowering time variation, as has been reported sion analysis revealed that 23% of the variation in flowering
in A. thaliana (Shindo et al., 2005). To test how much of this time could be attributed to allelic variation at FLC’s 5′UTR in
variation in FLC expression could be attributed to deletions C. rubella (P < 0.05).
of 5′ UTR at FLC locus, we analyzed the association between   To assess the specific contributions of the different 5′ UTR
FLC expression and allelic variation at FLC. We detected a sig- deletions that we identified, we analyzed their individual effect
 Parallel Evolution of Flowering Time in C. rubella  1331

on flowering time. We found that accessions carrying the et al., 2011; Weigel, 2012). In the model species A. thaliana,
86IT1 haplotype displayed significantly early flowering than the epistatic interaction between FRI/FLC accounts for much
MTE-type accessions (Figure 5A; Supplemental Figure 10A; of the natural variation in flowering time, with changes in FLC
P < 0.05). However, 762-type accessions did not show a sig- expression levels resulting mostly from loss/reduction of func-
nificant difference in flowering time when compared with MTE. tion polymorphisms at the FRI locus (Sanchez-Bermejo and
This could be due to the limited sample size for 762-type Balasubramanian, 2016). Interestingly, common allelic variations
accessions (only three accessions) and/or the presence of ad- at FRI in A. thaliana occur primarily through deletions involving
ditional genetic factors affecting flowering time in these acces- 5′ UTR (Johanson et al., 2000). Although allelic variation at FLC
sions, consistent with the more continuous distribution for has been reported, it is not the major determinant of flowering
flowering time we observed in the 762 × MTE F2 population. FLC time variation in A. thaliana.
expression levels were significantly (P < 0.05, Student’s t test)   FLC, however, plays a more important role in natural variation
higher in MTE-type accessions than that in 86IT1-type acces- for flowering time in other species of the Brassicaceae family
sions but lower than those in 762-type accessions (Figure 5B). (Slotte et al., 2009; Wang et al., 2009; Yuan et al., 2009; Hu et al.,
  We then tested for a correlation between flowering time 2011; Leinonen et al., 2013; Raman et al., 2016). Sequence anal-
and climate variables (19 ecological factors downloaded from ysis of the FRI locus in C. rubella did not reveal the same level of
the WorldClim database; www.worldclim.org) using 13 acces- allelic variation that is observed in A. thaliana (Guo et al., 2012).
sions with the MTE-type 5′ UTR and nine accessions with the Therefore, it appears that the C. rubella evolutionary strategy dif-
86IT1-type 5′ UTR for which we have the accurate latitude fers from that of A. thaliana. Whereas changes in FLC expression
and longitude collection information (Supplemental Figure retain a central importance in the regulation of flowering time, in
10B). Interestingly, we found that the values of the two envi- C. rubella these changes are caused by allelic variation at FLC
ronmental factors “mean temperature of driest quarter” and itself rather than at the FRI locus.
“precipitation of wettest quarter” are significantly (P < 0.05)   This is not surprising, given that, being downstream of its
different between MTE-type 5′ UTR accessions and 86IT1- activator FRI, FLC is likely to be more capable of generating
type 5′ UTR accessions, even though there was no general phenotypic variation without affecting other functions. However,
correlation between flowering time and climate variables. This it is not clear why A. thaliana exhibits a high frequency of FRI mu-
is suggestive that the deletion of 5′ UTR might be correlated to tations related to flowering time regulation. More case studies
the adaptation of C. rubella populations to the local climates. are needed to clarify the general mechanisms underlying these
  Our findings revealed that common allelic variation at the 5′ divergent evolutionary patterns.
UTR of FLC is a major determinant of flowering time variation.
By contrast, despite FLC being one of the most studied genes in
Loss/Reduction-of-Function Alleles of FLC Have Evolved
A. thaliana, only one case of a natural 50-bp deletion at 5′ UTR
Independently Multiple Times in C. rubella
affecting flowering time in the L1-0 accession was described.
This deletion does not affect prevernalization levels of FLC ex- The study of parallel evolution provides insight into the predict-
pression, but enhances the response to vernalization (Sánchez- ability of evolution; whether different species or populations that
Bermejo et al., 2012). The deletion overlapped with the 5′ end share a common ancestor could independently evolve the same
of the deletions identified in this study (Supplemental Figure trait by changing the same genes. In numerous cases in diverse
11). Therefore, we tested whether these deletions are present in organisms, different mutations in the same gene across spe-
other species and found that the deletions were not detected in cies or natural populations are responsible for parallel pheno-
Capsella grandiflora, Capsella orientalis, A. lyrata, or Arabidopsis typic evolution (Martin and Orgogozo, 2013; Stern, 2013; Storz,
halleri (Supplemental Figure 11). Unlike the genetic variation in 2016). In plants, there are several cases of parallel evolution
the first intron of FLC orthologs in the Arabis genus, which plays documented in flower pigmentation (Schwinn et al., 2006; Smith
an important role on the flowering time variation (Kiefer et al., and Rausher, 2011). However, the extent to which mutations in
2017), the 5′ UTR deletions exist only in C. rubella. To assess the the same region in independent lineages have shaped evolu-
origin of these deletions in C. rubella, we performed a phylogenet- tionary history in plants is largely unknown.
ic analysis of 19 accessions based on whole-genome sequencing   Early flowering has evolved multiple times independently in
data (Agren et al., 2014) (Figure 5C) and FLC sequences (Figure A. thaliana associated with loss or reduction in function of alleles
5D; Supplemental File 1). These analyses revealed that the two of FRI, FLC, and FLM (Michaels and Amasino, 1999; Lempe et al.,
deletions originated independently in C. rubella. 2005; Shindo et al., 2005; Salomé et al., 2011; Sánchez-Bermejo
et al., 2012; Lutz et al., 2017). Given the recent origin and very
DISCUSSION low genetic variation of C. rubella, the study of the evolution of
phenotypic differences in this species could, to some extent,
Allelic Variation at FLC Contributes to Natural Variation in be considered akin to the experimental evolution studies often
Flowering Time in C. rubella performed in microorganisms to understand how phenotypic
diversity arises. We have demonstrated that in C. rubella, FLC
We have demonstrated that allelic variation at FLC is a ma- loss of function has evolved multiple times, conferring variation
jor determinant of flowering time variation in C. rubella. Flow- in flowering time (this study; Guo et al., 2012). Here, we have
ering time is one of the adaptive traits for which variation has shown that the cause of the early-flowering 86IT1-FLC and 762-
been reported and well studied in a variety of species (Salomé FLC alleles is two overlapping deletions in the 5′ UTR of FLC that
1332  The Plant Cell

have originated independently. In addition, in a previous study, flc-3 Arabidopsis thaliana plants, which harbor a nonfunctional FLC al-
we have demonstrated the existence of an independent a loss- lele and a functional FRI allele in the reference Col-0 background, were
of-function allele at the FLC locus (Guo et al., 2012). While the previously reported by Michaels and Amasino (1999). For this study,
plants were grown on the soil mix (soil:vermiculite = 1:1) under long-day
latter case appears to be restricted to a single accession, the
conditions (16 h light/8 h dark) at 20°C. Trays of plants were moved to
deletions are pervasive in nature. Therefore, it is clear that loss/
random positions in the growth rooms every 2 d to reduce positional
reduction-of-function alleles at FLC have repeatedly originated effects. Flowering time was measured as leaf number and DTF. DTF was
in C. rubella populations, similar to the situation in the Arabis defined as the number of days from planting to the day of the first flower
genus (Albani et al., 2012; Kiefer et al., 2017). anthesis. The flowering times of 86IT1 × MTE and 762 × MTE F2 plants,
together with their parents, were measured in 2012 and 2013 (Figures
Flowering Time Variation beyond FLC in C. rubella 1A and 1B; Supplemental Figure 7). The flowering times of 34 C. rubella
accessions was measured in year 2016 (Supplemental Figure 10A and
Despite the association between flowering time variation and the Supplemental Table 1).
5′ UTR deletions in FLC, a large fraction of the variation observed
among accessions of C. rubella cannot be explained by these
polymorphisms. For example, the two accessions in the 762-type Mapping by Sequencing
group, 1482 and 75.2, are late flowering despite carrying an ear- DNA was extracted from pooled leaves of early-flowering F2 plants using
ly-flowering allele of FLC. It is likely that other loci, aside from FLC, a CTAB (cetyltrimethyl ammonium bromide) protocol (Doyle and Doyle,
are responsible for determining flowering time in these accessions. 1987). Paired-end reads (100 bp) were sequenced using the Illumina
This is further supported by the distribution for flowering time in the HiSeq 2000 with an insert size of ∼300 bp. In total, 159,506,270 and
762 × MTE F2 population (Figure 1B), suggesting the presence of 105,233,658 reads were obtained for 86IT1 × MTE and 762 × MTE F2
other loci affecting the timing of flowering in these accession (762 populations, respectively. For 86IT1 × MTE, short reads with ∼68.9-
fold coverage were mapped to the MTE genome (Phytozome; http://
and 75.2 are closely related; Figure 5). The deletion in the FLC 5′
www.phytozome.net/capsella/), and SNPs were called using SHORE
UTR in these two accessions promotes early flowering, but other
(Ossowski et al., 2008). By applying SHORE’s scoring matrix approach to
loci may have the opposite effect. Additionally, transgenic introduc- optimize heterozygous SNP detection and strict filtering (uniqueness of
tion of either the 86IT1-FLC or the 762-FLC allele into A. thaliana reads, coverage ≥10, minor allele frequency ≥0.2, and SNP quality score
results in similarly early-flowering phenotypes (Figure 2). However, ≥25), 110,091 SNPs were retained. For the 762 × MTE combination, short
significantly later flowering was observed in C. rubella 762 plants reads with ∼54.5-fold coverage were mapped to a preliminary assem-
relative to 86IT1 plants (Supplemental Figure 10A and Supplemen- bly of the MTE genome and SNPs were called using SHORE. In total,
tal Table 1). We cannot exclude the possibility that alleles of FLC 297,682 SNPs were retained after filtering according to the following pa-
behave differently when introduced into A. thaliana versus C. rubel- rameters: coverage ≥4 and minor allele frequency ≥0.15. The SNPs from
la owing to differences between the regulatory networks controlling each pooled population were used as markers to identify regions with
an excess of homozygous alleles across the genome using SHOREmap
flowering in the two species. However, it seems more plausible that
(Schneeberger et al., 2009).
loci other than FLC are responsible for the difference in flowering
time between 86IT1 and 762.
  In this study, we focused on the role of FLC as a key de- DNA Sequence Analysis
terminant of flowering time variation in C. rubella. Although we
The FLC upstream region (∼2.7 kb) from C. rubella accessions and related
have conclusively shown both that the deletions are the under- species and an ∼9.5-kb genomic DNA fragment of FLC in MTE, 86IT1,
lying cause for early flowering and that they are associated with and 762 were obtained by PCR. Purified PCR products were sequenced
altered chromatin, exactly how the deletions affect chromatin using the ABI 3730 automated sequencer (Applied Biosystems). Laser-
structure and histone modifications is currently unclear. Do the gene Seqman (DNASTAR) was used to assemble sequences. Primer
deletions have positional and (or) length effects related to the sequences are listed in Supplemental Table 6.
regulation of C. rubella FLC chromatin structure state and his-
tone modifications? In the future, it will be intriguing to further Plasmid Construction and Plant Transformation
determine the extent to which epigenetic modifications could
account for natural phenotypic variation and adaptation in a spe- To generate C. rubella FLC transgenes, an ∼9.5-kb genomic DNA frag-
cies with extremely low genetic diversity. ment of the C. rubella FLC gene from three accessions (including ∼2.7
kb upstream of the ATG start codon and ∼1.0 kb downstream of the
  In conclusion, our results provide strong evidence that parallel
stop codon) with or without SNPs was cloned into the pCAMBIA1300
evolution of independent deletions at the same 5′ UTR region
vector (Clontech) between the KpnI and SalI sites. To produce the
shapes phenotype variation in plants. It will be of great interest to Pro(32+86IT1):gMTE-FLC construct, the ∼2.7-kb upstream sequence
clarify the extent to which parallel evolution shapes variation in of FLC from two different accessions (86IT1 and MTE) and the full FLC
other traits among natural populations of this species and, more genomic fragment from ATG to TAG (∼5.8 kb) plus 1 kb of sequence from
broadly, the role of parallel evolution in diverse taxa in general. 3′ downstream amplified from MTE were ligated together and cloned into
pCAMBIA1300 first, then we inserted the 32-bp region deleted in 86IT1
plus its around sequences, beginning from −76 bp to +64 bp (the number
METHODS
was counted according to the position of ATG is defined as +1), at the
Plant Materials and Growth Conditions beginning of the promoter region by the KpnI site.
The A. thaliana mutant accession with FRIsf2 and flc-3 alleles in the
The 34 Capsella rubella accessions used in this study were described Col-0 background was transformed using Agrobacterium tumefaciens
previously (Guo et al., 2009) and are listed in Supplemental Table 1. FRIsf2 strain GV3101 with the floral dipping method. Seeds from T0 transgenic
 Parallel Evolution of Flowering Time in C. rubella  1333

plants were selected on MS medium supplemented with 50 mg/L hygro- and suspended in 10 mL of ice-cold nuclei isolation buffer (1 M hexylene
mycin 10 d; positive lines were then transferred to soil and the presence glycol, 20 mM PIPES-KOH, pH 7.6, 10 mM MgCl2, 1 mM EGTA, 15 mM
of the transgene was confirmed by PCR. NaCl, 0.5 mM spermidine, 0.15 mM spermine, 0.5% Triton X-100, 10
mM β-mercaptoethanol, and 1× protease inhibitor cocktail [Roche]) with
gentle rotation for 15 min. The suspension was filtered through 30-nm
Analysis of Gene Transcript Levels
CellTrics, and the elute was centrifuged for 10 min at 1500g at 4°C. The
RNA was extracted from whole 14-d-old seedlings grown on MS medi- pellet was resuspended as the crude nuclei extract with digestion buffer
um for C. rubella accessions and leaves for T1 transgenic plants before (40 mM Tris-HCl, pH 7.9, 0.3 M Suc, 10 mM MgSO4, 1 mM CaCl2, and
blooming. Total RNA was extracted using the Plant RNA Kit (Omega) 1× protease inhibitor cocktail [Roche]). Equal aliquots of nuclei extract
with an OnColumn DNase I digestion treatment. Spectrophotometry and were subjected to digestion with increasing amounts of mononuclease
gel electrophoresis were performed to detect RNA quality. To synthesize at 30°C for 15 min, and DNA was extracted using phenol:chloroform:iso-
cDNA, 1 µg of RNA was used in the SuperScript III First-Strand Syn- amyl alcohol (25:24:1, v/v) treatment followed by ethanol precipitation
thesis System (Invitrogen). RT-qPCR analysis (56°C, 45S and 45 cycles) for the PCR analysis.
was performed using SYBR Premix Ex TaqII mix (TaKaRa) on the ViiA 7
Real-time PCR System (Life Technologies). Each experiment was repeat-
ed with three independent biological replicates, and RT-qPCR reactions Bisulfite Sequencing
were performed with three technical replicates for each sample. The ex- Approximately 500 ng of genomic DNA was used for bisulfite conversion
pression level was calculated as 2ΔΔCT and then normalized to C. rubel- using the EZ DNA Methylation-Gold Kit (ZYMO Research) according to
la/A. thaliana TUBLIN. All primers used are listed in Supplemental Table 6. the manufacturer’s instructions. Bisulfite-treated DNA (1 µL) was then
amplified by PCR in a 25-μL reaction using ZymoTaq DNA polymerase
5′ RACE and 3′ RACE (ZYMO Research) and degenerate primers (Supplemental Table 6). PCR
products were resolved on a 1% agarose gel, and then excised, purified,
Full-length FLC sense and antisense cDNA were obtained by 5′ and 3′ and cloned into a pGEM-T vector (Promega) for sequencing. For each
RACE with the SMARTer RACE cDNA amplification kit (Clontech) follow- plant genotype, 20 independent top-strand clones were sequenced. For
ing the manufacturer’s instruction. One microgram of RNA was used to tested regions, bisulfite DNA sequences were analyzed, and the levels
synthesis RACE-Ready cDNAs. Gene-specific primers for 5′ RACE are of DNA methylation were calculated using the online Kismeth program
listed in Supplemental Table 1. After indentifying the band(s) of interest, (Gruntman et al., 2008). For each genotype, the percentage of cytosine
DNA was extracted from the gel using the StarPrep Gel Extraction Kit methylation in each context (CG, CHG, or CHH) was calculated.
(Genestar; D205-01), and the purified fragments were ligated into the
Peasy-Blunt vector (Transgene; CB111-01). At least 15 clones for each
fragment were sequenced. Chromatin Immunoprecipitation

For the chromatin immunoprecipitation (ChIP) assay, we started with

Cordycepin Treatment ∼600 μL T2 transgenic seed for each genotype (these being transgenic
lines transformed with full-length FLC alleles of MTE-FLC, 86IT1-FLC,
Plants were grown on MS plates containing 0.3% sucrose (w/v) at 20°C.
or 762-FLC transferred into the FRISf-2 flc-3 background; see Figure 2B).
Approximately 100 14-d-old seedlings were transferred to incubation
These starting pools of seed were divided randomly into three samples of
buffer (1 mM PIPES, pH 6.25, 1 mM sodium citrate, 1 mM potassium
∼200 μL seed per genotype, and germinated into three sets of 10-d-old
chloride, and 15 mM sucrose) and incubated for 30 min with shaking.
seedlings to create three biological replicates per genotype (at least 3
Cordycepin (C-3394; Sigma-Aldrich) was then added to a final concen-
g seedling tissue, equal to more than 500 seedlings, was used for each
tration of 150 µg/mL and vacuum infiltration was performed for 30 s.
replicate). Each ∼3 g replicate tissue sample was cross-linked with 1%
Seedlings representing ∼0.5 g of material were collected at various time
formaldehyde in a vacuum for 20 min and then ground to a powder in liq-
points and used for RNA extraction. RT-qPCR was processed to analyze
uid nitrogen. The chromatin complexes were isolated and sonicated, and
the expression data. Eukaryotic initiation factor-4A (eiF4A) and the homo-
then incubated with various antibodies. A total of 10 µg of anti-AcH3 (06-
log of AT3G45970 in C. rubella named Carubv10017847m.g were used
599; Millipore), anti-H3K36me3 (ab9050; Abcam), and anti-H3K27me3
as controls to measure high and low mRNA stability levels, respectively
(07-449; Millipore) antibodies was used in a 10-μL volume for immuno-
(Golisz et al., 2013). The expression level at each time point (C. rubella
precipitation. An equal amount of sample without antibody was used as
FLC/CrTUBULIN) was normalized to the expression at time 0 and is pre-
a mock control. The precipitated DNA was recovered and analyzed by
sented on a binary logarithm scale. Three biological replicates for each
RT-qPCR with SYBR Premix Ex TaqII Mix (Takara) using specific prim-
accession were used.
ers listed in Supplemental Table 6. Each ChIP value was normalized to
its respective input DNA value. All ChIP-qPCR experiments were inde-
Measurement of Splicing Efficiency pendently performed in triplicate.
Splicing efficiency was measured as described previously (Mahrez et al.,
2016). Briefly, a primer corresponding to an exon was combined with Phylogenetic Analysis of C. rubella FLC
a primer corresponding to a neighboring intron (for the unspliced tran-
script) or covering the splicing junction (for the spliced transcript). RT A neighbor-joining tree of 19 C. rubella samples based on 1,795,299
controls were always included to confirm the absence of genomic DNA SNPs matrix across the whole genome was constructed using PHYLIP
contamination. (version 3.695; Feisenstein, 1989) (Supplemental File 1) and based on
C. rubella FLC genomic sequences (Supplemental File 2) was aligned
and constructed using Mega 6 (Tamura et al., 2013). Topological robust-
Detection of Open Chromatin by Mononuclease Digestion
ness of the phylogenetic tree was assessed by bootstrapping with 100
Open chromatin was detected according to previous studies (Shu et al., replicates for whole genome data and 1000 replicates for FLC genomic
2013; Zhang et al., 2017). About 0.3 g of 10-d-old seedlings of pooled sequences data, respectively. The C. rubella resequencing data were re-
T2 transgenic plants frozen in liquid nitrogen were ground to a powder ported in a previous study (Agren et al., 2014).
1334  The Plant Cell

Statistical Analyses Supplemental Table 6. Primers Used in This Study.

All the statistical analyses were performed in R (http://www.r-project. Supplemental File 1. Text File of the Alignment Used for the Phyloge-
org/). netic Analysis Shown in Figure 5C.
Supplemental File 2. Text File of the Alignment Used for the Phyloge-
netic Analysis Shown in Figure 5D.
Accession Numbers

The genome sequence of the pooled F2 population of MTE × 86IT1 and

MTE × 762 has been deposited in the GenBank database with acces-
sion number PRJNA305780. Promoter sequences of FLC produced ACKNOWLEDGMENTS
from PCR products were submitted to GenBank with accession num-
bers KU297662, KU297663, KU297666 to KU297672, and KU356733 We thank Song Ge, Xiaofeng Cao, Jia-Wei Wang, Yong-Xiu Liu, and
to KU356752. The accession numbers of all genes identified or Rongcheng Lin for helpful comments and discussions, as well as Lian-
used in this study list as follows: CrFLC (Carubv10003343m), CrFT wei Zhu for assistance with the ChIP experiments. This work was sup-
(Carubv10021034m), CrSOC1 (Carubv10024046m), CrTUBULIN (Carub- ported by grants from the National Natural Science Foundation of China
(91731306 and 31470331 to Y.-L.G. and 31600187 to L.Y.) and by the
v10017235m), CrEIF4A (Carubv10013828m), AtFLC (AT5G10140), AtFT
CAS/SAFEA International Partnership Program for Creative Research
(AT1G65480), AtSOC1 (AT2G45660), and AtTUBULIN (AT5G62690).
Teams (Y.-L.G.).

Supplemental Data

Supplemental Figure 1. 5′ UTR Sequence of FLC Identified in the AUTHOR CONTRIBUTIONS

MTE, 86IT1, and 762 Accessions.
Supplemental Figure 2. Leaf Number of Three Transgenic Plants Ex- L.Y., H.-N.W., and Y.-L.G. designed the experiments. L.Y., H.-N.W., X.-
H.H., X.-M.N., T.-S.H., and J.Z. performed experiments. L.Y., Y.-P.Z.,
pressing Different C. rubella FLC Alleles in 20°C LD Conditions.
Z.Z., M.T., S.B., and Y.-L.G. analyzed and interpreted the experimental
Supplemental Figure 3. RT-qPCR Analyses of FT and SOC1 Expres- data. L.Y., H.-N.W., S.B., and Y.-L.G. wrote the manuscript with input
sion Level in Transgenic Plants Expressing Different C. rubella FLC from all the other authors.
Alleles.
Supplemental Figure 4. Leaf Number ofTransgenic Plants in Figure 3
Received February 9, 2018; revised May 13, 2018; accepted May 13,
under 20°C LD Conditions.
2018; published May 15, 2018.
Supplemental Figure 5. RT-qPCR Analyses of FT and SOC1 Expres-
sion Level in Transgenic Plants Expressing Different C. rubella FLC
Alleles with or without Del-32.
REFERENCES
Supplemental Figure 6. RT-qPCR Analyses of FT and SOC1 Expres-
sion Level in Transgenic Plants Expressing Different C. rubella FLC Agren, J.A., Wang, W., Koenig, D., Neuffer, B., Weigel, D., and Wright,
Alleles with or without Del-54. S.I. (2014). Mating system shifts and transposable element evolution
Supplemental Figure 7. Deletions in the 5′ UTR of FLC May Be Essen- in the plant genus Capsella. BMC Genomics 15: 602.
tial for Variation in Vernalization Response in C. rubella. Albani, M.C., Castaings, L., Wötzel, S., Mateos, J.L., Wunder, J.,
Wang, R., Reymond, M., and Coupland, G. (2012). PEP1 of Arabis
Supplemental Figure 8. The Location of 32-bp Region Deleted in
alpina is encoded by two overlapping genes that contribute to natural
86IT1-FLC 5′-UTR Is Important for Early Flowering.
genetic variation in perennial flowering. PLoS Genet. 8: e1003130.
Supplemental Figure 9. DNA Methylation Levels in the Regions Sur- Arendt, J., and Reznick, D. (2008). Convergence and parallelism re-
rounding the Transcription Start and the End of the First Intron of C. considered: what have we learned about the genetics of adaptation?
rubella FLC in MTE, 86IT1, and 762. Trends Ecol. Evol. (Amst.) 23: 26–32.
Supplemental Figure 10. Distribution of Flowering Time and Geo- Balasubramanian, S., Sureshkumar, S., Agrawal, M., Michael, T.P.,
graphic Locations of C. rubella Accessions. Wessinger, C., Maloof, J.N., Clark, R., Warthmann, N., Chory, J.,
and Weigel, D. (2006). The PHYTOCHROME C photoreceptor gene
Supplemental Figure 11. Sequence Variation of the FLC Promoter
mediates natural variation in flowering and growth responses of Ara-
Region from −528 to −1 among Six Congeners and within Three C.
bidopsis thaliana. Nat. Genet. 38: 711–715.
rubella Accessions.
Bloomer, R.H., and Dean, C. (2017). Fine-tuning timing: natural varia-
Supplemental Table 1. Capsella rubella Accessions Used in This tion informs the mechanistic basis of the switch to flowering in Arabi-
Study. dopsis thaliana. J. Exp. Bot. 68: 5439–5452.
Supplemental Table 2. Marker Analysis of Early Flowering Plants in Caicedo, A.L., Stinchcombe, J.R., Olsen, K.M., Schmitt, J., and
the F2 Generation of 86IT1 × MTE. Purugganan, M.D. (2004). Epistatic interaction between Arabidopsis
Supplemental Table 3. Marker Analysis of Early Flowering Plants in FRI and FLC flowering time genes generates a latitudinal cline in a life
the F2 Generation of 762 × MTE. history trait. Proc. Natl. Acad. Sci. USA 101: 15670–15675.
Chan, Y.F., et al. (2010). Adaptive evolution of pelvic reduction in stickle-
Supplemental Table 4. Candidate Genes Included in the 120-kb Re- backs by recurrent deletion of a Pitx1 enhancer. Science 327: 302–305.
gion Containing the Causal Locus Responsible for Variation of Flow- Colosimo, P.F., Hosemann, K.E., Balabhadra, S., Villarreal, G., Jr.,
ering Time between 86IT1 and MTE. Dickson, M., Grimwood, J., Schmutz, J., Myers, R.M., Schluter,
Supplemental Table 5. Candidate Genes Included in the 13-kb Re- D., and Kingsley, D.M. (2005). Widespread parallel evolution in stick-
gion Containing the Causal Locus Responsible for Variation of Flow- lebacks by repeated fixation of Ectodysplasin alleles. Science 307:
ering Time between 762 and MTE. 1928–1933.
 Parallel Evolution of Flowering Time in C. rubella  1335

Csorba, T., Questa, J.I., Sun, Q., and Dean, C. (2014). Antisense Lee, J.H., Ryu, H.S., Chung, K.S., Posé, D., Kim, S., Schmid, M., and
COOLAIR mediates the coordinated switching of chromatin states Ahn, J.H. (2013). Regulation of temperature-responsive flowering by
at FLC during vernalization. Proc. Natl. Acad. Sci. USA 111: 16160– MADS-box transcription factor repressors. Science 342: 628–632.
16165. Lee, J., Yun, J.Y., Zhao, W., Shen, W.H., and Amasino, R.M. (2015). A
Doyle, J.J., and Doyle, J.L. (1987). A rapid DNA isolation procedure methyltransferase required for proper timing of the vernalization re-
from small quantities of fresh leaf tissues. Phytochem. Bull. 19: 11–15. sponse in Arabidopsis. Proc. Natl. Acad. Sci. USA 112: 2269–2274.
Elmer, K.R., and Meyer, A. (2011). Adaptation in the age of ecological Leinonen, P.H., Remington, D.L., Leppälä, J., and Savolainen, O.
genomics: insights from parallelism and convergence. Trends Ecol. (2013). Genetic basis of local adaptation and flowering time variation
Evol. (Amst.) 26: 298–306. in Arabidopsis lyrata. Mol. Ecol. 22: 709–723.
Feisenstein, J. (1989). PHYLIP-phylogeny inference package (version Lempe, J., Balasubramanian, S., Sureshkumar, S., Singh, A., Schmid,
3.2). Cladistics 5: 164–166. M., and Weigel, D. (2005). Diversity of flowering responses in wild
Foxe, J.P., Slotte, T., Stahl, E.A., Neuffer, B., Hurka, H., and Wright, Arabidopsis thaliana strains. PLoS Genet. 1: 109–118.
S.I. (2009). Recent speciation associated with the evolution of selfing Liu, C., Lu, F., Cui, X., and Cao, X. (2010). Histone methylation in higher
in Capsella. Proc. Natl. Acad. Sci. USA 106: 5241–5245. plants. Annu. Rev. Plant Biol. 61: 395–420.
Gazzani, S., Gendall, A.R., Lister, C., and Dean, C. (2003). Analysis of Liu, F., Quesada, V., Crevillén, P., Bäurle, I., Swiezewski, S., and
the molecular basis of flowering time variation in Arabidopsis acces- Dean, C. (2007). The Arabidopsis RNA-binding protein FCA requires
sions. Plant Physiol. 132: 1107–1114. a lysine-specific demethylase 1 homolog to downregulate FLC. Mol.
Golisz, A., Sikorski, P.J., Kruszka, K., and Kufel, J. (2013). Arabidopsis Cell 28: 398–407.
thaliana LSM proteins function in mRNA splicing and degradation. Lutz, U., Nussbaumer, T., Spannagl, M., Diener, J., Mayer, K.F., and
Nucleic Acids Res. 41: 6232–6249. Schwechheimer, C. (2017). Natural haplotypes of FLM non-coding
Gruntman, E., Qi, Y., Slotkin, R.K., Roeder, T., Martienssen, R.A., and sequences fine-tune flowering time in ambient spring temperatures in
Sachidanandam, R. (2008). Kismeth: analyzer of plant methylation Arabidopsis. eLife 6: e22114.
states through bisulfite sequencing. BMC Bioinformatics 9: 371. Mahrez, W., Shin, J., Muñoz-Viana, R., Figueiredo, D.D., Trejo-Arellano,
Guo, Y.L., Bechsgaard, J.S., Slotte, T., Neuffer, B., Lascoux, M., Weigel, M.S., Exner, V., Siretskiy, A., Gruissem, W., Köhler, C., and Hennig,
D., and Schierup, M.H. (2009). Recent speciation of Capsella rubel- L. (2016). BRR2a affects flowering time via FLC splicing. PLoS Genet.
la from Capsella grandiflora, associated with loss of self-incompat- 12: e1005924.
ibility and an extreme bottleneck. Proc. Natl. Acad. Sci. USA 106: Marquardt, S., Raitskin, O., Wu, Z., Liu, F., Sun, Q., and Dean, C.
5246–5251. (2014). Functional consequences of splicing of the antisense tran-
Guo, Y.L., Todesco, M., Hagmann, J., Das, S., and Weigel, D. (2012). script COOLAIR on FLC transcription. Mol. Cell 54: 156–165.
Independent FLC mutations as causes of flowering-time variation in Martin, A., and Orgogozo, V. (2013). The loci of repeated evolution:
Arabidopsis thaliana and Capsella rubella. Genetics 192: 729–739. a catalog of genetic hotspots of phenotypic variation. Evolution 67:
He, Y. (2009). Control of the transition to flowering by chromatin modifi- 1235–1250.
cations. Mol. Plant 2: 554–564. McGrath, P.T., Xu, Y., Ailion, M., Garrison, J.L., Butcher, R.A., and
He, G., Elling, A.A., and Deng, X.W. (2011). The epigenome and plant Bargmann, C.I. (2011). Parallel evolution of domesticated Caenor-
development. Annu. Rev. Plant Biol. 62: 411–435. habditis species targets pheromone receptor genes. Nature 477:
He, Y., Michaels, S.D., and Amasino, R.M. (2003). Regulation of flowering 321–325.
time by histone acetylation in Arabidopsis. Science 302: 1751–1754. Méndez-Vigo, B., Martínez-Zapater, J.M., and Alonso-Blanco, C.
Hepworth, J., and Dean, C. (2015). Flowering Locus C's lessons: con- (2013). The flowering repressor SVP underlies a novel Arabidopsis
served chromatin switches underpinning developmental timing and thaliana QTL interacting with the genetic background. PLoS Genet.
adaptation. Plant Physiol. 168: 1237–1245. 9: e1003289.
Hoekstra, H.E. (2006). Genetics, development and evolution of adaptive Michaels, S.D., and Amasino, R.M. (1999). FLOWERING LOCUS C
pigmentation in vertebrates. Heredity (Edinb.) 97: 222–234. encodes a novel MADS domain protein that acts as a repressor of
Hu, G.L., Hu, Z.L., Li, Y., Gu, F., Zhao, Z.P., and Chen, G.P. (2011). A flowering. Plant Cell 11: 949–956.
splicing site mutation in BrpFLC1 and repressed expression of Brp- Ossowski, S., Schneeberger, K., Clark, R.M., Lanz, C., Warthmann,
FLC genes are associated with the early flowering of purple flowering N., and Weigel, D. (2008). Sequencing of natural strains of Arabidop-
stalk. Russ. J. Plant Physiol. 58: 431–438. sis thaliana with short reads. Genome Res. 18: 2024–2033.
Jiang, D., Yang, W., He, Y., and Amasino, R.M. (2007). Arabidopsis Pien, S., Fleury, D., Mylne, J.S., Crevillen, P., Inzé, D., Avramova, Z.,
relatives of the human lysine-specific Demethylase1 repress the ex- Dean, C., and Grossniklaus, U. (2008). ARABIDOPSIS TRITHORAX1
pression of FWA and FLOWERING LOCUS C and thus promote the dynamically regulates FLOWERING LOCUS C activation via histone 3
floral transition. Plant Cell 19: 2975–2987. lysine 4 trimethylation. Plant Cell 20: 580–588.
Jiang, D., Wang, Y., Wang, Y., and He, Y. (2008). Repression of Posé, D., Verhage, L., Ott, F., Yant, L., Mathieu, J., Angenent, G.C.,
FLOWERING LOCUS C and FLOWERING LOCUS T by the Ara- Immink, R.G., and Schmid, M. (2013). Temperature-dependent regu-
bidopsis Polycomb repressive complex 2 components. PLoS One lation of flowering by antagonistic FLM variants. Nature 503: 414–417.
3: e3404. Protas, M.E., and Patel, N.H. (2008). Evolution of coloration patterns.
Johanson, U., West, J., Lister, C., Michaels, S., Amasino, R., and Dean, Annu. Rev. Cell Dev. Biol. 24: 425–446.
C. (2000). Molecular analysis of FRIGIDA, a major determinant of natu- Qüesta, J.I., Song, J., Geraldo, N., An, H., and Dean, C. (2016). Arabi-
ral variation in Arabidopsis flowering time. Science 290: 344–347. dopsis transcriptional repressor VAL1 triggers Polycomb silencing at
Kiefer, C., Severing, E., Karl, R., Bergonzi, S., Koch, M., Tresch, A., FLC during vernalization. Science 353: 485–488.
and Coupland, G. (2017). Divergence of annual and perennial spe- Raman, H., Raman, R., Coombes, N., Song, J., Prangnell, R.,
cies in the Brassicaceae and the contribution of cis-acting variation Bandaranayake, C., Tahira, R., Sundaramoorthi, V., Killian, A., Meng,
at FLC orthologues. Mol. Ecol. 26: 3437–3457. J., Dennis, E.S., and Balasubramanian, S. (2016). Genome-wide
Koornneef, M., Alonso-Blanco, C., and Vreugdenhil, D. (2004). Nat- association analyses reveal complex genetic architecture underlying
urally occurring genetic variation in Arabidopsis thaliana. Annu. Rev. natural variation for flowering time in canola. Plant Cell Environ. 39:
Plant Biol. 55: 141–172. 1228–1239.
1336  The Plant Cell

Salomé, P.A., Bomblies, K., Laitinen, R.A., Yant, L., Mott, R., and Wahl, V., Ponnu, J., Schlereth, A., Arrivault, S., Langenecker, T., Fran-
Weigel, D. (2011). Genetic architecture of flowering-time variation in ke, A., Feil, R., Lunn, J.E., Stitt, M., and Schmid, M. (2013). Regu-
Arabidopsis thaliana. Genetics 188: 421–433. lation of flowering by trehalose-6-phosphate signaling in Arabidopsis
Sanchez-Bermejo, E., and Balasubramanian, S. (2016). Natural vari- thaliana. Science 339: 704–707.
ation involving deletion alleles of FRIGIDA modulate temperature- Wang, R., Farrona, S., Vincent, C., Joecker, A., Schoof, H., Turck, F.,
sensitive flowering responses in Arabidopsis thaliana. Plant Cell Alonso-Blanco, C., Coupland, G., and Albani, M.C. (2009). PEP1
Environ. 39: 1353–1365. regulates perennial flowering in Arabis alpina. Nature 459: 423–427.
Sánchez-Bermejo, E., Méndez-Vigo, B., Picó, F.X., Martínez-Zapater, Weigel, D. (2012). Natural variation in Arabidopsis: from molecular ge-
J.M., and Alonso-Blanco, C. (2012). Novel natural alleles at FLC and netics to ecological genomics. Plant Physiol. 158: 2–22.
LVR loci account for enhanced vernalization responses in Arabidopsis Werner, J.D., Borevitz, J.O., Uhlenhaut, N.H., Ecker, J.R., Chory, J., and
thaliana. Plant Cell Environ. 35: 1672–1684. Weigel, D. (2005). FRIGIDA-independent variation in flowering time of
Schneeberger, K., Ossowski, S., Lanz, C., Juul, T., Petersen, A.H., natural Arabidopsis thaliana accessions. Genetics 170: 1197–1207.
Nielsen, K.L., Jørgensen, J.E., Weigel, D., and Andersen, S.U. Whittaker, C., and Dean, C. (2017). The FLC locus: a platform for dis-
(2009). SHOREmap: simultaneous mapping and mutation identifica- coveries in epigenetics and adaptation. Annu. Rev. Cell Dev. Biol. 33:
tion by deep sequencing. Nat. Methods 6: 550–551. 555–575.
Schwinn, K., Venail, J., Shang, Y., Mackay, S., Alm, V., Butelli, E., Wood, T.E., Burke, J.M., and Rieseberg, L.H. (2005). Parallel genotypic
Oyama, R., Bailey, P., Davies, K., and Martin, C. (2006). A small adaptation: when evolution repeats itself. Genetica 123: 157–170.
family of MYB-regulatory genes controls floral pigmentation intensity Wu, C. (1980). The 5′ ends of Drosophila heat shock genes in chromatin
and patterning in the genus Antirrhinum. Plant Cell 18: 831–851. are hypersensitive to DNase I. Nature 286: 854–860.
Shindo, C., Aranzana, M.J., Lister, C., Baxter, C., Nicholls, C., Nordborg, Xu, L., Zhao, Z., Dong, A., Soubigou-Taconnat, L., Renou, J.P., Steinmetz,
M., and Dean, C. (2005). Role of FRIGIDA and FLOWERING LOCUS A., and Shen, W.H. (2008). Di- and tri- but not monomethylation on
C in determining variation in flowering time of Arabidopsis. Plant histone H3 lysine 36 marks active transcription of genes involved in
Physiol. 138: 1163–1173. flowering time regulation and other processes in Arabidopsis thaliana.
Shu, H., Gruissem, W., and Hennig, L. (2013). Measuring Arabidopsis Mol. Cell. Biol. 28: 1348–1360.
chromatin accessibility using DNase I-polymerase chain reaction and Yan, Y., Shen, L., Chen, Y., Bao, S., Thong, Z., and Yu, H. (2014). A
DNase I-chip assays. Plant Physiol. 162: 1794–1801. MYB-domain protein EFM mediates flowering responses to environ-
Slotte, T., Huang, H.R., Holm, K., Ceplitis, A., Onge, K.S., Chen, J., mental cues in Arabidopsis. Dev. Cell 30: 437–448.
Lagercrantz, U., and Lascoux, M. (2009). Splicing variation at a Yuan, W., Luo, X., Li, Z., Yang, W., Wang, Y., Liu, R., Du, J., and He,
FLOWERING LOCUS C homeolog is associated with flowering time Y. (2016). A cis cold memory element and a trans epigenome reader
variation in the tetraploid Capsella bursa-pastoris. Genetics 183: mediate Polycomb silencing of FLC by vernalization in Arabidopsis.
337–345. Nat. Genet. 48: 1527–1534.
Smith, S.D., and Rausher, M.D. (2011). Gene loss and parallel evolution Yuan, Y.X., Wu, J., Sun, R.F., Zhang, X.W., Xu, D.H., Bonnema, G.,
contribute to species difference in flower color. Mol. Biol. Evol. 28: and Wang, X.W. (2009). A naturally occurring splicing site mutation in
2799–2810. the Brassica rapa FLC1 gene is associated with variation in flowering
Srikanth, A., and Schmid, M. (2011). Regulation of flowering time: all time. J. Exp. Bot. 60: 1299–1308.
roads lead to Rome. Cell. Mol. Life Sci. 68: 2013–2037. Zhang, L., et al. (2017). A natural tandem array alleviates epigenetic repres-
Stern, D.L. (2013). The genetic causes of convergent evolution. Nat. sion of IPA1 and leads to superior yielding rice. Nat. Commun. 8: 14789.
Rev. Genet. 14: 751–764. Zhang, W., Wu, Y., Schnable, J.C., Zeng, Z., Freeling, M., Crawford,
Stern, D.L., and Orgogozo, V. (2009). Is Genetic Evolution Predictable? G.E., and Jiang, J. (2012). High-resolution mapping of open chroma-
Science 323: 746–751. tin in the rice genome. Genome Res. 22: 151–162.
Storz, J.F. (2016). Causes of molecular convergence and parallelism in Zhao, Z., Yu, Y., Meyer, D., Wu, C., and Shen, W.H. (2005). Prevention
protein evolution. Nat. Rev. Genet. 17: 239–250. of early flowering by expression of FLOWERING LOCUS C requires
Sureshkumar, S., Dent, C., Seleznev, A., Tasset, C., and Balasubra- methylation of histone H3 K36. Nat. Cell Biol. 7: 1256–1260.
manian, S. (2016). Nonsense-mediated mRNA decay modulates Zhou, C.M., Zhang, T.Q., Wang, X., Yu, S., Lian, H., Tang, H., Feng,
FLM-dependent thermosensory flowering response in Arabidopsis. Z.Y., Zozomova-Lihová, J., and Wang, J.W. (2013). Molecular basis
Nat. Plants 2: 16055. of age-dependent vernalization in Cardamine flexuosa. Science 340:
Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. 1097–1100.
(2013). MEGA6: Molecular Evolutionary Genetics Analysis version Zou, Y.P., et al. (2017). Adaptation of Arabidopsis thaliana to the Yangtze
6.0. Mol. Biol. Evol. 30: 2725–2729. River basin. Genome Biol. 18: 239.
 Parallel Evolution of Common Allelic Variants Confers Flowering Diversity in Capsella rubella
Li Yang, Hui-Na Wang, Xing-Hui Hou, Yu-Pan Zou, Ting-Shen Han, Xiao-Min Niu, Jie Zhang, Zhong
Zhao, Marco Todesco, Sureshkumar Balasubramanian and Ya-Long Guo
Plant Cell 2018;30;1322-1336; originally published online May 15, 2018;
DOI 10.1105/tpc.18.00124
This information is current as of May 3, 2019

Supplemental Data /content/suppl/2018/05/15/tpc.18.00124.DC1.html

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