2014, Li, Multiple FLC Haplotypes Genes Dev.-Li-1635-40
2014, Li, Multiple FLC Haplotypes Genes Dev.-Li-1635-40
2014, Li, Multiple FLC Haplotypes Genes Dev.-Li-1635-40
org on December 11, 2014 - Published by Cold Spring Harbor Laboratory Press
RESEARCH COMMUNICATION
GENES & DEVELOPMENT 28:1635–1640 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/14; www.genesdev.org 1635
Downloaded from genesdev.cshlp.org on December 11, 2014 - Published by Cold Spring Harbor Laboratory Press
Li et al.
with 4 wk, suggesting that the functional difference be- conclude that noncoding cis variation within FLC is the
tween haplotypes is cold exposure-dependent (Supple- major factor determining differential vernalization re-
mental Fig. S2C,D). sponse in the characterized A. thaliana population.
To conclusively demonstrate that FLC variation is the Analysis of chimeric FLC fusions previously showed
major contributor to the QTL, we generated transgenic that cis polymorphism quantitatively modulates a chro-
lines differing only in their FLC allele. Twelve-kilobase matin silencing mechanism underpinning the epigenetic
FLC genomic clones from the A. thaliana accessions Edi-0, regulation of FLC in one Northern Swedish accession
Col-0, Lov-1, Ull2-5, and Var2-6, representing haplotypes (Coustham et al. 2012). To help define which of the many
RV1, RV2, SV1, SV2, and SV4, respectively, were trans- nucleotide differences between haplotypes determined
formed individually into FRI flc-2 (Michaels and Amasino variation in vernalization response, we first looked for
2001). FLC expression in the transgenic lines was com- natural recombination events across FLC genomic re-
pared with that from the natural accessions both with gions in accessions (Martin et al. 2010) and identified one
and without vernalization. The differential response to that we called R2 (Supplemental Fig. S3A). Polymorphism
vernalization shown by the FLC haplotypes was reca- in the R2 region showed the strongest association with
pitulated in the transgenic plants (R 2 = 0.80), whereas the maintenance of FLC silencing after vernalization
the correlation of prevernalization FLC expression between (Supplemental Fig. S3B). This region includes the nucle-
accessions and transgenic lines was weaker (R2 = 0.29) ation region important for FLC silencing; the VRE (ver-
(Fig. 3A,B). The transgenic analysis therefore shows that nalization response element), defined as important for
the SNPs defining the FLC alleles confer the vernaliza- maintenance of silencing (Sheldon et al. 2002; Finnegan
tion response characteristic of the parent A. thaliana and Dennis 2007; Angel et al. 2011); and a large number of
accession, with trans-regulation playing a greater role in the SNPs defining the haplotype RV1 (Fig. 3C). We isolated
prevernalization FLC expression. These data enable us to and swapped the FLC R2 region reciprocally between Lov-1
(SV1) and Edi-0 (RV1) FLC alleles, which show slow and
rapid silencing, respectively (Fig. 3C). The vernalization
response of multiple transgenic lines was then compared.
The SV1/RV1/SV1 (SRS) chimeric FLC shows stable FLC
silencing after 4 wk of cold, which results in a rapid ver-
nalization response compared with its SV1 FLC parent
(Fig. 3D). Thus, the SRS vernalization response matches
that of the RV1 accession rather than the SV1 accession.
Similarly, the RV1/SV1/RV1 (RSR) chimera shows a ver-
nalization response similar to the SV1 accession despite
containing only a small part of the SV1 FLC allele (Fig. 3D).
These results demonstrate that polymorphisms within
R2 from RV1 and SV1 contribute to the differences in FLC
silencing after 4 wk of vernalization, respectively. This
region also contains the four single-nucleotide changes
that cause the differential silencing of Lov-1 (SV1) and
Col-0 (RV2) FLC alleles (Coustham et al. 2012). Remark-
ably, the Edi-0 (RV1) and Col-0 (RV2) FLC alleles do not
share any polymorphisms that are not also found in other
FLC haplotypes (Figs. 2B, 3C), demonstrating that the
causative nucleotide changes that distinguish slow and
rapid vernalization have evolved multiple times. The re-
combination event characterizing the RV1 haplotypes, the
relative lack of variability of SNPs in the functional R2
region, and their relatively broad geographical distribution
(Hap3/RV1) (Fig. 1E) suggest a relatively recent spread of
Figure 3. Transgenic analysis shows that noncoding FLC poly- RV1 accessions, potentially indicating a strong advantage
morphism plays a central role in vernalization response of natural of rapid vernalization in some locations.
Arabidopsis accessions. (A) Prevernalization FLC expression (non- Consistent with this, faster vernalization has been
vernalizaion [NV]) in transgenic lines correlates weakly with natural
accessions. (B) Relative FLC expression [T30/(T30 + T10)] in trans-
predicted to confer a selective advantage to plants where
genic lines correlates significantly with expression in natural acces- avoidance of summer drought or other high risk is
sions. R2 shows the explained amount of variation; the corresponding beneficial (Caicedo et al. 2004; Stinchcombe et al. 2004,
P-values are 0.201 and 0.025 in A and B. (C) Schematic illustration of 2005; Scarcelli et al. 2007; Satake 2010). We analyzed
FLC polymorphisms and the constructs of R2 chimeras. The SNPs differences in fitness-related traits conferred by different
defining the different FLC haplotypes are shown by the vertical bars.
FLC alleles in F2 populations (Fig. 4; Supplemental Fig.
The bar transparency indicates the diversity of each SNP within
each haplotype. The bars shared between haplotypes signify shared S4). After a relatively short vernalization (4 wk), the slow
SNPs. The FLC gene structure is shown above: Filled black squares vernalization (Kulturen-1) FLC allele associated with lower
are exons, and horizontal lines are introns and untranslated regions. seed yield than the rapid vernalization (Rev-1) FLC allele.
RSR and SRS chimeras show the exchange of R2 regions between After longer vernalization (12 wk), both alleles produced
Edi-0 and Lov-1 FLC alleles. (D) FLC expression analysis of trans- a similar seed yield (Fig. 4A,B; Supplemental Fig. S4A).
genic FRI flc-2 plants containing chimeric FLC alleles. Plants were
given either no cold (NV) or 4 wk of cold followed by 10 d (T10) or Total seed weight represented an increased production of
30 d (T30) of warm. Values are means 6standard error from three seed number, as no significant difference was found in the
biological repeats. weight of each seed (Supplemental Fig. S4B). High fecundity
Li et al.
Genotyping and QTL analysis of F2 populations Bandelt HJ, Forster P, Rohl A. 1999. Median-joining networks for inferring
intraspecific phylogenies. Mol Biol Evol 16: 37–48.
F2 populations were genotyped using a 384-SNP Illumina GoldenGate Box MS, Coustham V, Dean C, Mylne JS. 2011. Protocol: a simple phenol-
genotyping assay (http://www.illumina.com/technology/goldengate_ based method for 96-well extraction of high quality RNA from
genotyping_assay.ilmn). Genome Studio software (http://www.illumina.com/ Arabidopsis. Plant Methods 7: 7.
software/genomestudio_software.ilmn) and custom-made scripts in R software Broman KW, Wu H, Sen S, Churchill GA. 2003. R/qtl: QTL mapping in
(http://www.R-project.org) were used for SNP calling. experimental crosses. Bioinformatics 19: 889–890.
Quantitative trait loci mapping was performed on raw phenotypic Caicedo A, Stinchcombe JR, Olsen KM, Schmitt J, Purugganan MD. 2004.
values without any transformation. QTL analyses were carried out using Epistatic interaction between Arabidopsis FRI and FLC flowering
the default parameters of the scanone function implemented in the R/qtl time genes generates a latitudinal cline in a life history trait. Proc
package (Broman et al. 2003). The LOD significance threshold was Natl Acad Sci 101: 15670–15675.
determined by permutation test (1000 permutations, significance level Coustham V, Li P, Strange A, Lister C, Song J, Dean C. 2012. Quantitative
of 5%) for each F2 population.
modulation of polycomb silencing underlies natural variation in
vernalization. Science 337: 584–587.
M&M test of the correlation between FLC polymorphism Finnegan EJ, Dennis ES. 2007. Vernalization-induced trimethylation of
histone H3 lysine 27 at FLC is not maintained in mitotically
and gene expression
quiescent cells. Curr Biol 17: 1978–1983.
A general linear model (GLM) with T30/(T30 + T10) as response variable Fournier-Level A, Korte A, Cooper MD, Nordborg M, Schmitt J, Wilczek
and SNP as the predictor was analyzed for each SNP separately. The AM. 2011. A map of local adaptation in Arabidopsis thaliana. Science
F coefficient (adjusted MS/error variance) was calculated and plotted 334: 86–89.
against the SNP position. The strength of the association of each SNP and Hoffmann MH. 2005. Evolution of the realized climatic niche in the
the response variable was expressed by calculating the effect size genus Arabidopsis (Brassicaceae). Evolution 59: 1425–1436.
estimator h2. This statistic expresses the ratio of variance explained in Horton MW, Hancock AM, Huang YS, Toomajian C, Atwell S, Auton A,
the dependent variable by a SNP. This analysis shows that SNPs in Muliyati NW, Platt A, Sperone FG, Vilhjalmsson BJ, et al. 2012.
the R2 region (Supplemental Fig. S5B) explain ;22.1% of the variation Genome-wide patterns of genetic variation in worldwide Arabidop-
inT30/(T10 +T30) (h2 = 0.221), which is considered a large effect size. sis thaliana accessions from the RegMap panel. Nat Genet 44: 212–
216.
Kang HM, Sul JH, Service SK, Zaitlen NA, Kong SY, Freimer NB,
fastPHASE haplotype analysis Sabatti C, Eskin E. 2010. Variance component model to account for
SNPs in a window 650 kb around FLC were extracted from a preimpu- sample structure in genome-wide association studies. Nat Genet
tation version of the Regional Mapping Project SNP panel described in 42: 348–354.
Horton et al. (2012). These SNPs were used as the input into fastPHASE Kemen E, Gardiner A, Schultz-Larsen T, Kemen AC, Balmuth AL,
version 1.4.0 (Scheet and Stephens 2006), which was run using the Robert-Seilaniantz A, Bailey K, Holub E, Studholme DJ, Maclean
default settings, with the exception of invoking the Pzp option to D, et al. 2011. Gene gain and loss during evolution of obligate
output raw likelihood values. For each SNP in the analysis, the cluster parasitism in the white rust pathogen of Arabidopsis thaliana. PLoS
membership with the highest likelihood was designated as the haplotype Biol 9: e1001094.
for that SNP. Li Y, Huang Y, Bergelson J, Nordborg M, Borevitz JO. 2010. Association
mapping of local climate-sensitive quantitative trait loci in Arabidopsis
thaliana. Proc Natl Acad Sci 107: 21199–21204.
Population structure-corrected ANOVAs for seed proxy Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P. 2010.
analysis in accessions RDP3: a flexible and fast computer program for analyzing recombi-
nation. Bioinformatics 26: 2462–2463.
A standard approach for accounting for such genomic background effects Michaels SD. 2009. Flowering time regulation produces much fruit. Curr
is linear mixed models (Vilhjalmsson and Nordborg 2013). We assessed the Opin Plant Biol 12: 75–80.
significance of phenotypic differences between haplotypes by carrying out Michaels SD, Amasino RM. 2001. Loss of FLOWERING LOCUS C
an ANOVA on the residuals after subtracting the best linear unbiased activity eliminates the late-flowering phenotype of FRIGIDA and
predictors (BLUPs) of the effect explained by the genomic background. autonomous pathway mutations but not responsiveness to vernali-
The BLUPs were estimated using EMMAX (Kang et al. 2010) with zation. Plant Cell 13: 935–941.
a kinship matrix estimated from the Regional Mapping Project data Mitchell-Olds T, Schmitt J. 2006. Genetic mechanisms and evolution-
(Horton et al. 2012). ary significance of natural variation in Arabidopsis. Nature 441:
947–952.
Nordborg M, Bergelson J. 1999. The effect of seed and rosette cold
Acknowledgments treatment on germination and flowering time in some Arabidopsis
thaliana (Brassicaceae) ecotypes. Am J Bot 86: 470–475.
We thank all members of the Dean and Howard groups for discussions.
O’Neill CM, Morgan C, Kirby J, Tschoep H, Deng PX, Brennan M, Rosas U,
This research was supported by UK Biotechnology and Biological
Fraser F, Hall C, Gill S, et al. 2008. Six new recombinant inbred
Sciences Research Council (BBSRC) grant BB/I007857/1, Institute Stra-
populations for the study of quantitative traits in Arabidopsis thaliana.
tegic Programme grant BB/J004588/1, European Research Council Ad-
Theor Appl Genet 116: 623–634.
vanced Investigator grant 233039 ENVGENE, and National Science
Parachnowitsch AL, Lajeunesse MJ. 2012. Adapting with the enemy:
Foundation Frontiers in Integrative Biological Research grant NSF
local adaptation in plant-herbivore interactions. New Phytol 193:
EF-0425759.
294–296.
Redei GP. 1962. Supervital mutants of Arabidopsis. Genetics 47: 443–
460.
References
Ripatti S, Becker T, Bickeboller H, Dominicus A, Fischer C, Humphreys
Alonso-Blanco C, Mendez-Vigo B. 2014. Genetic architecture of naturally K, Jonasdottir G, Moreau Y, Olsson M, Ploner A, et al. 2009.
occurring quantitative traits in plants: an updated synthesis. Curr GENESTAT: an information portal for design and analysis of genetic
Opin Plant Biol 18C: 37–43. association studies. Eur J Hum Genet 17: 533–536.
Alonso-Blanco C, El-Assal SE, Coupland G, Koornneef M. 1998. Analysis Salome PA, Bomblies K, Laitinen RA, Yant L, Mott R, Weigel D. 2011.
of natural allelic variation at flowering time loci in the Landsberg Genetic architecture of flowering-time variation in Arabidopsis
erecta and Cape Verde Islands ecotypes of Arabidopsis thaliana. thaliana. Genetics 188: 421–433.
Genetics 149: 749–764. Satake A. 2010. Diversity of plant life cycles is generated by dynamic
Angel A, Song J, Dean C, Howard M. 2011. A Polycomb-based switch epigenetic regulation in response to vernalization. J Theor Biol 266:
underlying quantitative epigenetic memory. Nature 476: 105–108. 595–605.
Li et al.
Genes Dev. 2014 28: 1635-1640 originally published online July 17, 2014
Access the most recent version at doi:10.1101/gad.245993.114
Supplemental http://genesdev.cshlp.org/content/suppl/2014/07/11/gad.245993.114.DC1.html
Material
References This article cites 35 articles, 19 of which can be accessed free at:
http://genesdev.cshlp.org/content/28/15/1635.full.html#ref-list-1
Open Access Freely available online through the Genes & Development Open Access option.
Creative This article, published in Genes & Development, is available under a Creative Commons
Commons License (Attribution 4.0 International), as described at
License http://creativecommons.org/licenses/by/4.0.
Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the top
Service right corner of the article or click here.