Advancing Fagaceae genomics: construction and
characterization of the first Quercus suber BAC library
Elisa Prat1, Joana Pinto2, Joelle Fourment1,Clara Graça2, Pedro Marques3, Pedro Fevereiro2, José Graça4, Jorge Paiva5 and Hélène Bergès1
(1) INRA-CNRGV, Chemin de Borde Rouge, 31326 Castanet-Tolosan, France,(2) Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal, (3) Fundação João Lopes Fernandes, Herdade do Leitões 7425-014 Montargil – Portugal, (4) Instituto Superior de
Agronomia, Departamento de Engenharia Florestal, Tapada da Ajuda, 1349 – 017 Lisboa, Portugal, (5)Instituto de Investigação Científica Tropical (IICT), Centro de Florestas e dos Produtos Florestais, Tapada da Ajuda, 1349-018 Lisboa, Portugal.
Corresponding author: [email protected]
Introduction
- Quercus suber is an evergreen oak that produces cork and has major economic and environmental importance for the
Mediterranean region. Cork is an exceptional natural material with numerous industrial applications. Portugal is the world
leading producer of cork.
- Bacterial artificial chromossomes (BAC) libraries are key genomic tools that enable positional cloning of important traits,
synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing.
Objectives:
(1) Construct and characterize the first deep-coverage Quercus suber BAC library;
(2) Library screening for the presence of several genes of interest for cork formation.
Distribution map, showing the natural distribution area
of Quercus suber. (EUFORGEN 2009)
Methods
1
Collection of Quercus suber leaves
2
Young leaves were selected and kept in obscurity for 3 days
BAC library construction and
characterization
3
Screening for genes of interest
High density BAC filters production on a 6*6 pattern.
Insert ligation in pIndigo
BA-5 vector
Hybridization with probes of genes of interest
Colony picking
gDNA
Validation of the positive clones by colony PCR
Quality tests and characterization
Results
1
Q. suber BAC library characterization
2
Qsu-B-HDL: Quercus suber BAC “Herdade dos Leitões” Library
Screening for genes of interest
Gene functional
categories
International name
Qsu-B-HDL
CNRGV name
Qsu-B-HDL
Common name
Cork oak
Host
DH10B TIR
Species
Quercus suber
Number of clones
41472
a) Fatty acid synthesis
Ecotype
Herdade dos Leitões
(Portugal)
Number of plates
108
b) Fatty acid
hydroxylation
Restriction enzyme
HindIII
Plates type
Genetix 384 wells
Vector
pIndigoBAC-5 Hind III
Average insert size
120 Kb
Selective antibiotic
Cloramphenicol
Genome size
1000 Mb
Genome equivalents
5
c) Lipid metabolism
a) Phenylpropanoid
pathway
a Average size of inserts = 120kb
b Contamination with chloroplastidial and
mitochondrial genomes
b) Phenylpropanoid
derivatives
c) Acyltransferase
d) Cross linking/oxidase
Gene name
DHPA-S-AT
CYP86A1
GDSL
PAL
CCR
Description
Acyl lipids
Dihydrolipoamide Sacetyltransferase
Fatty acid Ѡ-hydroxylase
DoLaccase
Nº of
validated
clones
GGFEK7Q01CYTRE.l.qs.1
7
GGFEK7Q01A0IF3.l.qs.1
GDSL-motif lipase/hidrolase
GGFEK7Q01A0ZV8.l.qs.1
protein like
Phenylpropanoids
Phenylalanine ammonialyase
Cinnamoyl CoA reductase
CYP84A1, F5H Ferulate-5-hydroxylase
HCBT
SUBERGENE database
(See poster 236)
3
10
qscs0011.j.05_3.1.l.qs.1
2
GGFEK7Q01A36GL.l.qs.1
3
GGFEK7Q01A0U9Z.l.qs.1
1
N-hydroxycinnamoylCoA/benzoyltransferase
GGFEK7Q01A01QL.l.qs.1
Diphenol oxidase laccase
GGFEK7Q01A0R5F.l.qs.1
11
GGFEK7Q01A0IGO.l.qs.1
3
GGFEK7Q01A65Y0.l.qs.1
4
GGFEK7Q01A0FQG.l.qs.1
2
GGFEK7Q01A2DPE.l.qs.1
3
9
Regulatory proteins
100 Kb
15
50 Kb
WRKY
b) Ethylene forming
ACC-OX
a) Monoxygenase
CYP87A2
a) Reactive oxygen
species scavenging
APX
100
100
115
100
90
120
120
110
100
vide
135
110
100
120
105
135
110
110
100
125
130
110
vide
150 Kb
110
105
110
110
80+40
100
100
70+50
120
105
140
120
120
110
125
200
a) Transcritpion factor
Choroplastidial genome
(psbA, ndhA probes)
1.16%
Mitochondrial genome
(cox3, ccb256 probes)
0,2%
Vectors
WRKY transcription factor
1- aminocyclopropane -1 carboxylic acid oxidase
Miscellaneous
Cytochrome P450 family
protein
Stress
Ascorbate peroxidase
Conclusions
Acknowledgements
We would like to thank Fundação João Lopes Fernandes for providing the plant material used in this study. This
research was partially supported by grants from the FCT (Fundação para a Ciência e Tecnologia, Portugal), within
research project (PTDC/AGR-GPL/101785/2008). Joana Pinto was supported by a BI fellowship within the
research project (PTDC/AGR-GPL/101785/2008).
• The BAC library Qsu-B-HDL constructed contains large inserts (average size 120kb) and very low extranuclear genome contamination.
• This library allowed to find single copy gene with a very high probability.
• This Q. suber BAC library represents an important milestone for the advancement of Fagaceae
genomics and forest tree research, including genome sequencing, gene isolation, functional and
comparative genomics.