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Tephritid Barcoding Initiative
and barcoding of agricultural pest
presented by
M. Virgilio
Royal Museum for Central Africa, Tervuren, Belgium
Royal Belgian Institute of Natural Sciences, Brussels, Belgium
[email protected]
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Why DNA Barcode Fruit Flies?
• large number of species
• many economically relevant species
• difficult identification of larvae.
• need for standardized diagnostic methods
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
The Tephritid Barcode Initiative (TBI)
CBOL obtained funding from the Sloan Foundation
to support a “Demonstrator System”
Steering Committee formed in April, 2006, in Belgium
TBI Chair:
Bruce McPheron, Penn State
TBI Coordinators:
Allen Norrbom, USDA, USA
Marc De Meyer, RMCA, Belgium
Steering Committee Members:
Karen Armstrong, New Zealand
Norman Barr, USA
Amnon Freidberg, Israel
Ho-Yeon Han, South Korea
George Roderick, USA
Ian White, UK
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
TBI proposal: Goals
Generate barcode database for 2,000 species
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5 individuals/species (10,000 specimens)
100% of economically important (EI) species
>75% of EI congeners
1 species per genus in subtribes containing EI species
1 species per genus in other higher taxa
representative tephritoid families
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
TBI proposal: Beneficial Outcomes
1) Establish a globally-available DNA database of barcodes
2) Establish a globally-available DNA repository
3) Generate a collection of identified vouchers for future
systematic work
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Who is generating fruit fly barcodes?
1. Penn State University, USA: Bruce McPheron, Md. Sajedul Islam
2. Lincoln University, New Zealand: Karen Armstrong
3. Royal Museum Central Africa, BE: Marc De Meyer, Massi Virgilio
4. Yonsei University, Korea: Ho-Yeon Han
5. California Department of Agriculture, USA: Peter Kerr
6. Smithsonian National Museum of Natural History, USA: Allen
Norrbom
7. APHIS-PPQ Mission lab, USA: Norman Barr
8. University of Guelph
9. Biodiversity Institute of Ontario
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Financial support
Penn State University, USA: technical support
APHIS: collecting & sequencing
Belgian Federal Government: sequencing, pilot study mini-barcodes
California Citrus Board: general expenditure
Korean Government: sequencing
(Dutch Government: collecting)
Various contributions through staff time and general core funding of
institutions or other projects
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
What needs to be provided in BOLD for TBI
1. identification of specimen by an expert
taxonomist
2. voucher specimen
3. collection information (collection date
and location)
Euleia fratria (Trypetinae)TEPH101
(from BOLD)
4. other infos (GPS, elevation,
photodocumentation) not mandatory
but strongly encouraged
5. barcode: at least 500bp with less than
1% missing data.
Other COI records (e.g.,
Genbank submissions) are
integrated into the BOLD
database but kept separate.
6. trace files stored in BOLD.
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
http://www.barcodinglife.org
Tephritidae {family}
Search performed on October 20, 2008
Lineage: Arthropoda: Insecta:
Diptera
Specimen Record: 1047
Specimens with barcodes: 828
Public Sequences: 240
Subfamilies (5)
Dacinae [572]
Phytalmiinae [7]
Tachiniscinae [1]
Tephritinae [219]
Trypetinae [243]
Deposited in :
Barcodes :
Species :
Five Genera of Highest Economic Importance
all species
EI species
number of species
600
% of species barcoded
100
533
80
400
60
240
Dacus
0
Ceratitis
20
Bactrocera
Ceratitis
19
37
Dacus
34
Bactrocera
73
119 95
Rhagoletis
0
Anastrepha
44
40
Rhagoletis
200
Anastrepha
213
insights into
DNA barcoding
of tephritid fruitflies
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
intra- and inter-specific genetic differentiation
in 3 genera of tephritid fruitflies
ratio inter/intra specific p-dist
12.6
6.3
6.6
20%
16%
p-distances
12%
between species
8%
within species
4%
0%
Bactrocera
Ceratitis
Dacus
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
DNA barcoding of tephritid fruitflies:
where it works nicely....
44 barcodes of Bactrocera cucurbitae
from 11 countries:
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•
•
•
•
•
•
•
•
•
•
Bangladesh
Cambodia
China
Hawaii
India
Kenya
La Reunion
Malaysia
Philippines
Sudan
Tanzania
average genetic distance
within species
K2p = 0.02%
(S.E.= 0.01%)
... and where not:
the FAR complex
Ceratitis fasciventris
C. anonae
C. rosa
average genetic distances
K2P
0.02
0.01
within
species
between
species
0.00
ratio = 1.2
other potentially “problematic”
tephritid species groups and complexes:
B. dorsalis complex
C. cosyra complex
C. capitata / C. caetrata
T. occipitale / T. quadrimaculatum
...
Armstrong and Ball (2005) Phil. Trans. R. Soc. B 360: 1813-1823. (B. dorsalis complex)
Virgilio et al. (2008) Molecular Phylogenetic and Evolution 48: 270-280 (FAR complex, Ceratitis)
Dacus:
from identification issues
to classification issues
as expected low genetic differentiation
in taxonomically closer species...
D. chiwira - D. famona, p-dist.= 0.3% (subgenus Dacus)
D. apostata - D. triater, p-dist.= 0.0% (subgenus Psilodacus)
...but also discrepancies between
morphological and molecular taxonomy
subgeneric classification
DACUS
DIDACUS
LEPTOXYDA
LOPHODACUS
NEODACUS
PSILODACUS
COI
NJT-K2P
DNA barcoding may provide clues
for an improved morphological classification
Apocynaceae
Cucurbitaceae
Passifloraceae
unknown
host plant choice
COI+16S+per
Bayesian
DNA barcoding may reveal cryptic speciation:
Virgilio M., De Meyer M, White I.M., Backeljau T. (submitted) Phylogenetic relationships
among African Dacus species (Diptera: Tephritidae) as inferred from mitochondrial and
nuclear DNA.
methodological problems
in the barcoding of tephritid fruitflies
from museum collections
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
methodological problems
in the barcoding of museum specimens
DNA Extraction Procedures:
Several pilot studies performed to assess the success of an invasive,
nondestructive DNA extraction method for museum specimens.
Study 1: L. Weigt and A. Driskell (Laboratory of Analytical Biology, National
Museum of Natural History, Smithsonian Institution) -16S and COI
Study 2: N. Barr (USDA) and R. Ruiz (Penn State) -16S
Study 3. M. Virgilio (Royal Museum Central Africa, BE) – 16S, COI
Study 4. Md. Sajedul Islam (Penn State) - COI (ongoing study)
Qiagen DNeasy kit:
ok for DNA from recent dry and alcohol specimens not crushed during the
extraction process.
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
methodological problems
in the barcoding of museum specimens
age of specimens vs barcoding success
100
80
60
% specimens amplified
40
% specimens sequenced
20
< 1980
80s
90s
2000
2001
2002
2003
2004
2005
2006
0
2007
(n=394)
methodological problems
in the barcoding of museum specimens
pinned vs EtOH preserved specimens
% of succesfully sequenced specimens (n=394)
100
from EtOH specimens
80
from pinned specimens
60
40
80s
90s
2000
2001
2002
2003
2004
2005
2006
2007
0
< 1980
20
methodological problems
in the barcoding of museum specimens
<1940
32%
Ceratitis, Bactrocera and Dacus
in the collections of the RMCA:
>2000
41%
(n=1804)
40s
4%
50s
13%
80s 90s
70s
3% 4%
1%
60s
2%
methodological problems
in the barcoding of museum specimens
DNA extraction protocols tested:
• Qiagen DNeasy
• Qiagen Biosprint
• Bio101 Ancient DNA kit
• Autogen
• Machery-Nagel Filterservice kit
• Phenol-Chloroform
• DNAzol
• E.Z.N.A. kits for forensics and insects
• chargeswitch magnetic beads
• etc.
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
methodological problems
in the barcoding of museum specimens
problem:
• the quality of DNA rapidly decreases in time (shearing)
• the amount of barcodes obtained from older museum specimens
is not significantly affected by
• the extraction method used
• the use of genus- and species-specific primers
objective:
develop internal primers to improve the collection of barcodes from
sheared DNA
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
methodological problems
in the barcoding of museum specimens
the BARFLY project
short term project funded by the Belgian Science Policy (BELSPO)
Joint Experimental Molecular Unit
of RMCA and RBINS (www.jemu.be)
Jeroen Van Houdt, Floris Breman
• development of internal primers for the barcoding of Tephritids
• collection of new barcodes from museum specimens
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
a new set of internal primers
for the barcoding of tephritids
LCO 1490
full barcode - c. 670 bp
frag. 1 - 343bp
frag. 2 - 269bp
frag. 3 - 227bp
VanHoudt J., Breman F. C., Virgillio M., De Meyer M. (in prep.)
A protocol for DNA barcoding of African tephritid fruitflies from museum
collections using mini barcodes.
HCO 2198
100
% of pcr products
(n=229)
80
obtained
60
fragment 1
40
fragment 2
20
fragment 3
0
>2000
90s
80s
70s
60s
50s
40s
<1940
100
.
(n=229)
80
% of barcode sequences
60
obtained (>500bp)
40
20
0
>2000
90s
80s
70s
60s
50s
40s
<1940
a new set of internal primers
for the barcoding of tephritids
higher performances
compared to the standard primers
100
80
% of barcodes obtained (>500bp)
+7%
+32%
+6%
+7%
60
standard primers
40
internal primers
20
(n=229)
0
>2000
90s
80s
<1980
a new set of internal primers
for the barcoding of tephritids
RMCA:
internal primers as a standard protocol
for the barcoding of museum
specimens collected before 2000
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
work in progress:
could ‘minibarcodes’ represent a
temporary alternative to
‘full’ barcodes?
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Dacus armatus
400 bp
400 bp
500 bp
600 bp
400 bp
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Bactrocera oleae
500 bp
300 bp
400 bp
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
TBI current challenges
– Coordination / tracking specimens: BOLD
– Older specimens: mini barcodes / internal primers
– Fresh material: collecting activities
– Taxon coverage: non EI genera
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
RMCA current challenges
RMCA is collecting barcodes as a “complementary activity” to the currently
ongoing research lines:
- phylogeny and population genetics of African Dacus
- molecular taxonomy of species complexes in the genus Ceratitis
- phylogeny of African and Australasian Bactrocera
- population genetics and phylogeography of B. cucurbitae
contact:
Marc De Meyer: [email protected]
Massimiliano Virgilio: [email protected]
RMCA current challenges
RMCA is opened to collaborations with African Institutions aiming to:
- identify tephritid samples through morphological and
molecular characters
- prepare species inventories from African countries
- collect / rear tephritid fruit flies
- investigate population genetics and host races evolution of
tephritid species
contact:
Marc De Meyer: [email protected]
Massimiliano Virgilio: [email protected]
Acknowledgments
Karen Armstrong
Abdelaziz Babikir
Thierry Backeljau
Norman Barr
Marc DeMeyer
Sajedul Islam
Bruce McPheron
researches at the RMCA are currently funded by the
Belgian Science Policy Action 1 (project MO/37/017)
and benefit from practical support by the
Joint Experimental Molecular Unit (JEMU)
Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences