Antoine Kremer

Christophe Klopp

Christophe Plomion

Céline Noirot

DJ Stekel

Elodie Prince

GA Lang

Grégoire Le Provost

L Mazzitelli

S Ueno

Saneyoshi Ueno

T Pacey-Miller

Valérie Léger

English

PubMed

Springer - Publisher Connector

POSTER PRESENTATION Open Access
Detection of genes involved in bud phenology
in sessile oak (Quercus petraea Matt. Liebl)
combining digital expression analysis and Q-PCR
Saneyoshi Ueno1*, Christophe Klopp2, Céline Noirot2, Valérie Léger3, Elodie Prince3, Antoine Kremer3,
Christophe Plomion3, Grégoire Le Provost3
From IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and Delivery
Arraial d Ajuda, Bahia, Brazil. 26 June - 2 July 2011
Background
In temperate zones, the seasonal cycling of perennials
comprises two main steps: a growing period when envir-
onmental conditions are favourable and a non-growing
period in winter. Winter dormancy consists of two
phases: endodormancy and ecodormancy [1]. The for-
mer is the deepest state of the dormancy controlled by
the meristem itself, while the latter is controlled by
environmental factors, such as accumulated tempera-
tures.This phenological cycle has been shown to be
strongly affected by climatic change: higher temperature
having a positive effect on ecodormancy phase by accel-
erating meristem cell growth and early bud break is par-
ticularly crucial to consider not only in respect to
increasing length of the growing season, but also regard-
ing the risk of frost damage. For these reasons, under-
standing the molecular mechanism controlling the shift
from endo- to ecodormancy is of main importance to
optimize the management of forest tree plantations to
their most likely environmental conditions.
The objective of this study was to characterize the
transcriptome of endo- and eco-dormant sessile oaks (a
major broadleaf species in Europe) by RNA-seq and to
identify differentially expressed genes between these two
dormancy stages. Four cDNA libraries were generated
using total RNA extracted from apical buds harvested
during endo- or ecodormancy of two contrasted popula-
tions. Transcriptome characterization was performed on
a Roche 454 FLX platform. Digital expression analysis
leads us to identify a set of 48 genes differentially regu-
lated between these two developmental phases (there
was no differences between populations). Their function
was identified by sequence homology and this result
contributes to the characterization of the molecular net-
work underlying vegetative bud dormancy, an important
life history traits of these long lived organisms.
Methods
Acorns of sessile oak (Quertus petreae (Matt) Lieb.)
were collected in 2006 on two populations from North-
ern France: Longchamp (LC) and Saint Jean (SJ). These
populations are contrasted for their bud burst date
(early vs. late flushing). We obtained 560 and 468 seed-
lings for the SJ and the LC populations, respectively. A
forcing test experiment was performed in a greenhouse
from July 30th 2007 to February 25th 2008 located at
the INRA forestry research station (southwestern
France). For each population, approximately 25 seedlings
were selected each week. Ten apical buds from 10 seed-
lings were used for RNA extraction. The 15 remaining
seedlings were transferred in a greenhouse with the fol-
lowing parameters: a 16-hours photoperiod and a day/
night temperature of 25/18 °C. For each batch of seed-
lings, bud burst date was evaluated three times a week
allowing us to determine the number of days necessary
to induce bud burst. We, thus, identified periods corre-
sponding to eco- and endodormancy.
Total RNA from apical buds harvested on September
17th, 24th and October 1st for endo-dormancy as
well as on January 14th, 28th and February 11th for
eco-dormancy were extracted independently, and mixed
equimolarly to obtain a composite cDNA library for
each population and developmental stage. The libraries
were sequenced on a Roche 454 FLX to produce
495,915 reads in total. After cleaning and removal of
* Correspondence: saueno@ffpri.affrc.go.jp
1Forestry and Forest Products Research Institute, Brazil
Full list of author information is available at the end of the article
Ueno et al. BMC Proceedings 2011, 5(Suppl 7):P20
http://www.biomedcentral.com/1753-6561/5/S7/P20
© 2011 Ueno et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
duplicated reads, reads were mapped onto oak unigenes
[2]. A digital gene expression analysis was performed
using both r statistics [3]and R BioConductor packages
(edgeR, DEGseq and goseq). Homology searches were
carried out by BlastX against SWISS-PROT and TAIR9
databases with e-value cutoff of 1e-5. Gene ontology
(GO) annotations were based on the top blast hit
against SWISS-PROT database.To validate bioinformatic
analysis, Q-PCR was performed for 13 selected genes.
One microgram of total RNA was reverse transcribed
using Improm-IITM reverse transcription system (Pro-
mega) according to the manufacturer’s instructions. Q-
PCR reaction and quantification were performed on a
Chromo4 Multicolor Real-Time PCR Detection System
(Bio-Rad)
Results and conclusions
We identified 48 common contigs that showed differen-
tial expression by three bioinformatic programs (R sta-
tistics, edgeR and DEGseq) and selected 13 genes for Q-
PCR analysis with the main goal to validate the result
obtained in silico. One gene (heat shock protein)
showed multi-banding pattern and discarded from the
analysis. Among the 12 remaining genes, only one gene
(cold shock protein) showed contradictory expression
pattern between in silico and Q-PCR analysis. The high
success rate of Q-PCR analysis (11/13 = 84.6 %) indi-
cates that the verified genes are likely to represent
endo- and ecodormancy stages. Two genes (Globulin
and XET) were confirmed as up-regulated in endodor-
mancy, while nine genes (GASA, GST, DRM1, XERO2,
PIP, AWPM, GID, LEA and ELP) were found to be up-
regulated in ecodormancy .Analysis in terms of GO
showed genes responsible to abiotic and endogenous sti-
mulus as well as stress related were enriched in the set
of 48 genes. Similar trend was observed in expression
analysis for raspberry bud dormancy, where high per-
centage of stress-response/defense/detoxification-related
genes were identified [4]. In grape also, most of genes
expressed in buds at the end of dormancy were attribu-
ted to the oxidative processes and stress responses [5].
Although the number of genes indentified is small and
further verification through time course change in their
expression will be needed, these candidates will serve as
stepping stone to dissect the molecular mechanisms
involved in bud dormancy in forest trees.
Author details
1Forestry and Forest Products Research Institute, Brazil. 2Plateforme
bioinformatique Genotoul, UR875 Biométrie et Intelligence Artificielle, INRA,
Brazil. 3INRA, UMR 1202, BIOGECO, Brazil.
Published: 13 September 2011
References
1. Lang GA, Early JD, Martin GC, Darnell RL: Endo-, Para-, and Ecodormancy:
physiological terminology and classification for dormancy research.
HortScience 1987, 22:371-377.
2. Ueno S, Le Provost G, Leger V, Klopp C, Noirot C, Frigerio JM, Salin F,
Salse J, Abrouk M, Murat F, et al: Bioinformatic analysis of ESTs collected
by Sanger and pyrosequencing methods for a keystone forest tree
species: oak. BMC Genomics 2010, 11:650.
3. Stekel DJ, Git Y, Falciani F: The comparison of gene expression from
multiple cDNA libraries. Genome Res 2000, 10:2055-2061.
4. Mazzitelli L, Hancock RD, Haupt S, Walker PG, Pont SD, McNicol J, Cardle L,
Morris J, Viola R, Brennan R, et al: Co-ordinated gene expression during
phases of dormancy release in raspberry (Rubus idaeus L.) buds. J Exp
Bot 2007, 58:1035-1045.
5. Pacey-Miller T, Scott K, Ablett E, Tingey S, Ching A, Henry R: Genes
associated with the end of dormancy in grapes. Funct Integr Genomics
2003, 3:144-152.
doi:10.1186/1753-6561-5-S7-P20
Cite this article as: Ueno et al.: Detection of genes involved in bud
phenology in sessile oak (Quercus petraea Matt. Liebl) combining digital
expression analysis and Q-PCR. BMC Proceedings 2011 5(Suppl 7):P20.
Submit your next manuscript to BioMed Central
and take full advantage of: 
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at 
www.biomedcentral.com/submit
Ueno et al. BMC Proceedings 2011, 5(Suppl 7):P20
http://www.biomedcentral.com/1753-6561/5/S7/P20
Page 2 of 2


Detection of genes involved in bud phenology in sessile oak (Quercus petraea Matt. Liebl) combining digital expression analysis and Q-PCR

Ueno Saneyoshi

Klopp Christophe

Noirot Céline

Léger Valérie

Prince Elodie

Kremer Antoine

Plomion Christophe

Le Provost Grégoire

Directory of Open Access Journals

BMC Proceedings

Detection of genes involved in bud phenology in sessile oak (<it>Quercus petraea</it> Matt. Liebl) combining digital expression analysis and Q-PCR

UENO, Saneyoshi

KLOPP, Christophe

NOIROT, Céline

LÉGER, Valérie

PRINCE, Elodie

KREMER, Antoine

PLOMION, Christophe

LE PROVOST, Grégoire

Oskar Bordeaux

Crossref

POSTER PRESENTATION Open AccessDetection of genes involved in bud phenologyin sessile oak (Quercus petraea Matt. Liebl)combining digital expression analysis and Q-PCRSaneyoshi Ueno1*, Christophe Klopp2, Céline Noirot2, Valérie Léger3, Elodie Prince3, Antoine Kremer3,Christophe Plomion3, Grégoire Le Provost3From IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and DeliveryArraial d Ajuda, Bahia, Brazil. 26 June - 2 July 2011BackgroundIn temperate zones, the seasonal cycling of perennialscomprises two main steps: a growing period when envir-onmental conditions are favourable and a non-growingperiod in winter. Winter dormancy consists of twophases: endodormancy and ecodormancy [1]. The for-mer is the deepest state of the dormancy controlled bythe meristem itself, while the latter is controlled byenvironmental factors, such as accumulated tempera-tures.This phenological cycle has been shown to bestrongly affected by climatic change: higher temperaturehaving a positive effect on ecodormancy phase by accel-erating meristem cell growth and early bud break is par-ticularly crucial to consider not only in respect toincreasing length of the growing season, but also regard-ing the risk of frost damage. For these reasons, under-standing the molecular mechanism controlling the shiftfrom endo- to ecodormancy is of main importance tooptimize the management of forest tree plantations totheir most likely environmental conditions.The objective of this study was to characterize thetranscriptome of endo- and eco-dormant sessile oaks (amajor broadleaf species in Europe) by RNA-seq and toidentify differentially expressed genes between these twodormancy stages. Four cDNA libraries were generatedusing total RNA extracted from apical buds harvestedduring endo- or ecodormancy of two contrasted popula-tions. Transcriptome characterization was performed ona Roche 454 FLX platform. Digital expression analysisleads us to identify a set of 48 genes differentially regu-lated between these two developmental phases (therewas no differences between populations). Their functionwas identified by sequence homology and this resultcontributes to the characterization of the molecular net-work underlying vegetative bud dormancy, an importantlife history traits of these long lived organisms.MethodsAcorns of sessile oak (Quertus petreae (Matt) Lieb.)were collected in 2006 on two populations from North-ern France: Longchamp (LC) and Saint Jean (SJ). Thesepopulations are contrasted for their bud burst date(early vs. late flushing). We obtained 560 and 468 seed-lings for the SJ and the LC populations, respectively. Aforcing test experiment was performed in a greenhousefrom July 30th 2007 to February 25th 2008 located atthe INRA forestry research station (southwesternFrance). For each population, approximately 25 seedlingswere selected each week. Ten apical buds from 10 seed-lings were used for RNA extraction. The 15 remainingseedlings were transferred in a greenhouse with the fol-lowing parameters: a 16-hours photoperiod and a day/night temperature of 25/18 °C. For each batch of seed-lings, bud burst date was evaluated three times a weekallowing us to determine the number of days necessaryto induce bud burst. We, thus, identified periods corre-sponding to eco- and endodormancy.Total RNA from apical buds harvested on September17th, 24th and October 1st for endo-dormancy aswell as on January 14th, 28th and February 11th foreco-dormancy were extracted independently, and mixedequimolarly to obtain a composite cDNA library foreach population and developmental stage. The librarieswere sequenced on a Roche 454 FLX to produce495,915 reads in total. After cleaning and removal of* Correspondence: saueno@ffpri.affrc.go.jp1Forestry and Forest Products Research Institute, BrazilFull list of author information is available at the end of the articleUeno et al. BMC Proceedings 2011, 5(Suppl 7):P20http://www.biomedcentral.com/1753-6561/5/S7/P20© 2011 Ueno et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.duplicated reads, reads were mapped onto oak unigenes[2]. A digital gene expression analysis was performedusing both r statistics [3]and R BioConductor packages(edgeR, DEGseq and goseq). Homology searches werecarried out by BlastX against SWISS-PROT and TAIR9databases with e-value cutoff of 1e-5. Gene ontology(GO) annotations were based on the top blast hitagainst SWISS-PROT database.To validate bioinformaticanalysis, Q-PCR was performed for 13 selected genes.One microgram of total RNA was reverse transcribedusing Improm-IITM reverse transcription system (Pro-mega) according to the manufacturer’s instructions. Q-PCR reaction and quantification were performed on aChromo4 Multicolor Real-Time PCR Detection System(Bio-Rad)Results and conclusionsWe identified 48 common contigs that showed differen-tial expression by three bioinformatic programs (R sta-tistics, edgeR and DEGseq) and selected 13 genes for Q-PCR analysis with the main goal to validate the resultobtained in silico. One gene (heat shock protein)showed multi-banding pattern and discarded from theanalysis. Among the 12 remaining genes, only one gene(cold shock protein) showed contradictory expressionpattern between in silico and Q-PCR analysis. The highsuccess rate of Q-PCR analysis (11/13 = 84.6 %) indi-cates that the verified genes are likely to representendo- and ecodormancy stages. Two genes (Globulinand XET) were confirmed as up-regulated in endodor-mancy, while nine genes (GASA, GST, DRM1, XERO2,PIP, AWPM, GID, LEA and ELP) were found to be up-regulated in ecodormancy .Analysis in terms of GOshowed genes responsible to abiotic and endogenous sti-mulus as well as stress related were enriched in the setof 48 genes. Similar trend was observed in expressionanalysis for raspberry bud dormancy, where high per-centage of stress-response/defense/detoxification-relatedgenes were identified [4]. In grape also, most of genesexpressed in buds at the end of dormancy were attribu-ted to the oxidative processes and stress responses [5].Although the number of genes indentified is small andfurther verification through time course change in theirexpression will be needed, these candidates will serve asstepping stone to dissect the molecular mechanismsinvolved in bud dormancy in forest trees.Author details1Forestry and Forest Products Research Institute, Brazil. 2Plateformebioinformatique Genotoul, UR875 Biométrie et Intelligence Artificielle, INRA,Brazil. 3INRA, UMR 1202, BIOGECO, Brazil.Published: 13 September 2011References1. Lang GA, Early JD, Martin GC, Darnell RL: Endo-, Para-, and Ecodormancy:physiological terminology and classification for dormancy research.HortScience 1987, 22:371-377.2. Ueno S, Le Provost G, Leger V, Klopp C, Noirot C, Frigerio JM, Salin F,Salse J, Abrouk M, Murat F, et al: Bioinformatic analysis of ESTs collectedby Sanger and pyrosequencing methods for a keystone forest treespecies: oak. BMC Genomics 2010, 11:650.3. Stekel DJ, Git Y, Falciani F: The comparison of gene expression frommultiple cDNA libraries. Genome Res 2000, 10:2055-2061.4. Mazzitelli L, Hancock RD, Haupt S, Walker PG, Pont SD, McNicol J, Cardle L,Morris J, Viola R, Brennan R, et al: Co-ordinated gene expression duringphases of dormancy release in raspberry (Rubus idaeus L.) buds. J ExpBot 2007, 58:1035-1045.5. Pacey-Miller T, Scott K, Ablett E, Tingey S, Ching A, Henry R: Genesassociated with the end of dormancy in grapes. Funct Integr Genomics2003, 3:144-152.doi:10.1186/1753-6561-5-S7-P20Cite this article as: Ueno et al.: Detection of genes involved in budphenology in sessile oak (Quercus petraea Matt. Liebl) combining digitalexpression analysis and Q-PCR. BMC Proceedings 2011 5(Suppl 7):P20.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitUeno et al. BMC Proceedings 2011, 5(Suppl 7):P20http://www.biomedcentral.com/1753-6561/5/S7/P20Page 2 of 2

Detection of genes involved in bud phenology in sessile oak (Quercus petraeaMatt. Liebl) combining digital expression analysis and Q-PCR

Klopp, Christophe

Noirot, Celine

Léger, Valérie

Prince, Elodie

Kremer, Antoine

Plomion, Christophe

Le Provost, Grégoire

ProdInra

and Ecodormancy: physiological terminology and classification for dormancy research. HortScience

et al: Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak.

et al: Co-ordinated gene expression during phases of dormancy release in raspberry (Rubus idaeus L.) buds. J Exp Bot

Falciani F: The comparison of gene expression from multiple cDNA libraries. Genome Res

Genes associated with the end of dormancy in grapes. Funct Integr Genomics

Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance

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Detection of genes involved in bud phenology in sessile oak (Quercus petraea Matt. Liebl) combining digital expression analysis and Q-PCR

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