Transcript Establishing a DNA Barcode for Land plants

Establishing a DNA Barcode
for Land plants
Santiago Madriñán Restrepo
Universidad de los Andes
Bogotá, Colombia
[email protected]
Project Partners
Part n er Org ani z ations
Scient ists
Ro yal Bota n ic Gar d ens, Ke w, UK
Ro b yn Cowan
Mark Cha s e
Natural His t or y Mu seum (Lon d on) , UK
Mark Car ine
To rte lla, Pty ch o mn iaceae, As p le n ium ,
Natural His t or y Mu seum , De n mark
Gi t te Pet e rs e n
Ho rdeu m , Scale s ia, Cr o cus
New Yo rk Bota n ica l Garde n , USA
Kenn e th Cam e ron
Elapho g lo s sum , Cu pres s us, Lab ordia
Ro yal Bota n ic Gar d en Edin b u rgh, UK
Pete r Hollin gs wo rt h
Pod o carpu s , Arau caria, As t er e lla,
Anastr o ph yllu m
So u th African Na tio nal Biodi ver s it y
Ins tit u te , (Cape T o wn), S o ut h Africa
Feroza h Co n rad
Encephala rt os, M imet e s
Unive rsidad d e los Andes,
Tar g et gr o ups
Con o s t yli s , Pin us, Equis e tu m ,
Dact ylo rhiza macu lata/incarn ata
com p lex
Co lombia
Santi a go Madr iñá n
Lauraceae
Ins t it u to de Bi o lo g ia UNAM, Me x ico
Gerar d o A. Salaza r
Agave
Unive rsida d e Esta d ual de Feira de
Santana, Bras il
Cássio v a n d e n Ber g
Laelia, Ca ttl e ya
Unive rs ity of Cape To wn, So u th Afric a
Ter ry Hed d er s on
Anastr o ph yllu m - Barbi lop h ozia,
Bryu m
Impe rial Co lle ge , UK (& RBG Ke w)
Tim o th y Barrac lo u gh
(Data anal ysis)
COI or cox1 in Plants
• Low sequence divergence
• Other mitochondrial genes
– Exhibit incorporation of foreign genes
– Frequent transfer of some genes to the
nuclear genome
Plant Barcode Proposals
Other Regions
• Internal transcribed spacer regions of nuclear
ribosomal DNA (ITS)
– often highly variable in angiosperms at the generic
and species level
– divergent copies are often present within single
individuals
• Non-coding plastid regions
– Highly length variable
• rbcL
– Not variable enough at species level for many
plant groups
Desirable Barcode attributes
• Variable enough to allow identification of
species but with a comparatively low
level of intraspecific variation
• Universally amplified/sequenced with
standardized primers
• Technically simple to sequence
Desirable Barcode attributes
• Short
• Easily alignable
• Readily recoverable from herbarium
samples and other degraded DNA
samples (e.g., forensic material)
Plastid DNA
• Monomorphic
• High copy number
• Highly diagnostic
Phase 1
Mike Wilkinson
University of Reading, UK
• Identify suitable loci (>100) on the basis of in silico
screens using Nicotiana plastid sequence
• Design universal primers (sets of 4 primers/locus)
• Perform initial screen for universality
• Screen twice for sequence variation using diverse
species-pairs
• Improve universality (e.g., use all primer
combinations)
• Use statistical modeling approaches to identify
optimal primer sets
Regions and Primers
Gene
matK
rpoC 1
rpoB
accD
YCF5
ndhJ
Prim e r
2.1
2.1a
5
3.2
1
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4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
Dire ction
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Seque n ce 5' - 3'
CCTATCCATCTGGAAATCTTAG
ATCCATCTGGAAATCTTAGTTC
GTTCTAGCACAAGAAAGTCG
CTTCCTCTGTAAAGAATTC
GTGGATACACTTCTTGATAATGG
GGCAAAGAGGGAAGATTTCG
TGAGAAAACATAAGTAAACGGGC
CCATAAGCATATCTTGAGTTGG
AAGTGCATTGTTGGAACTGG
ATGCAACGTCAAGCAGTTCC
CCGTATGTGAAAAGAAGTATA
GATCCCAGCATCACAATTCC
AGTATGGGATCCGTAGTAGG
GGRGCACGTATGCAAGAAGG
TTTAAAGGATTACGTGGTAC
TCTTTTACCCGCAAATGCAAT
GGATTATTAGTCACTCGTTGG
ACTTTAGAGCATATATTAACTC
ACTTACGTGCATCATTAACCA
CCCAATACCATCATACTTAC
CATAGATCTTTGGGCTTYGA
TTGGGCTTCGATTACCAAGG
ATAATCCTTACGTAAGGGCC
TCAATGAGCATCTTGTATTTC
PCR Mix
Buffer X 1
Mg2+ 1.5mM
dNTPs 0.2mM
FW test primer 1µM
RE test primer 1µM
Taq DNA polymerase 2 units
BSA 0.1mg/ml
Template (variable)
Water to 20 µl
Thermal Profile
94ºC1min
94ºC30sec
53ºC40sec
72ºC40sec
72ºC5min
1 cycle
Up to 40 cycles
1 cycle
Bryophytes S.L.
Phase 2 Sampling
Amplification
Angiosperms
Fam ily
Agavaceae
Iridaceae
Orchidaceae
Orchidaceae
Orchidaceae
Orchidaceae
Asteraceae
Haemodoraceae
Fabaceae
Loganiaceae
Lauraceae
P oaceae
P roteaceae
Grou p
Agave
Crocus
Aulosepalum
Cattleya
Dactylorhiza
Sophronitis
Scalesia
Conostylis
Inga
Labordia
Lauraceae
Hordeum
Mimetes
accD
m atK
n dh J
rpoB
rpoC 1 trn H-psbA
Non Angiosperms
Grou p
Fam ily
Asterella
Aytoniaceae
Bryum
Bryaceae
Lophoziaceae Anastrophyllum
T ortella
P ottiaceae
T riquetrella
P ottiaceae
P tychomniaceaeP tychomniales
Equisetum
Equisetaceae
Asplenium
Aspleniaceae
Dryopteridaceae Elaphoglossum
Encephalartos
Zamiaceae
Araucariaceae Araucaria
Cupressus
Cupressaceae
P inus
P inaceae
accD
m atK
n dh J
rpoB
rpoC 1 trn H-psbA
Non Angiosperm Primers
Gene
matK
rpoC 1
rpoB
accD
ndhJ
Prim e r
X
F (Equi s et u m )
R (Equ ise t um )
F (Adian t um )
LP1
LP5
LP1.1
LP3
LP4.3
LP5.2
LP1
LP3
LP4
LP1
LP4
Dire ction
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Seque n ce 5' - 3'
TAATTTACGATCAATTCATTC
ATACCCCATTTTATTCATCC
GTACTTTTATGTTTACGAGC
GATGTTGCAGTCTATTCATTC
TATGAAACCAGAATGGATGG
CAAGAAGCATATCTTGASTYGG
TCTAATATGCARCGTCAAGG
TTTACCCAAYRAAACATCHCC
ATAATACCTTTATTWCCATG
AAATAAGGCATATCTTGTCT
AGTATGGGYTCCGTHGTDG
ACCTGCRAATGCRATATATGC
ARATATTTCGCTYAAAACACC
CATAGACCTTTRGGTTTYGA
ACCAATCCAASTATCRGGC
Variation
Two Orchid genera
Lauraceae
Sister taxa: Cattleya and Sophronitis
“Corsage orchids”
Cássio van den Berg
Universidade Estadual de Feira de Santana, Brasil
Cattleya: 43 spp. in 2 subgenera
Unifoliate species = 18 species, allopatric species “complex”
Bifoliate species = 25 well-defined species, 6 species pairs
Sophronitis: 63 spp. in 3 subgenera (as “sections”)
Sect. Cattleyodes+Hadrolaelia – 17 well-defined species
Sect. Parviflorae – 40 spp. messy complex, genetic data indicate ca. 15 spp.
Sect. Sophronitis – 6 allopatric closely related species
C.labiata
C. aclandiae
S. perrinii
S. sp. nov.
% species discriminated
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ITS: 90.5%
psbA-trnH: 60%
matK: 33.3%
ndhJ: 37.1%
rpoB: 9.9%
rpoC1:9.9%
accD: 6.05 %
Nuclear non-coding
Plastid non-coding
Plastid coding
Orchid Results
• accD, rpoB, rpoC1: variation too low for
use as a single barcode
• matK and ndhF: more variable but with
great variation of rate among subgenera
• Non-coding regions (ITS and psbA-trnH
spacer) performed better, but required
great manual effort for indel alignment
Lauraceae
• Big family
• Largely
unstudied
• VERY difficult
to id.
• Economically
important
Species Complexes
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Phylogenetic Spread
accD
Lauraceae
accD
matK
ndhJ
rpoB
rpoC
1
Genera
17
17
16
17
17
Species
42
40
36
42
43
Specimens
47
58
42
48
49
Sp. w/ >1
specimen
5
11
6
6
6
A c tinodaphne glabra K- 8 2 0 2
A c tinodaphne pruinos a K- 8 2 0 3
A iouea dubia A N - 4 1 7
B eils c hmiedia pendula B C I - 0 6 5 6 8 1
B eils c hmiedia pendula B C I - 1 7 0 4 2 1
B eils c hmiedia pendula B C I - 2 5 7 5 9 6
B eils c hmiedia tawa K- 5 5 1 9
C aryodaphnops is c ogolloy J A U M - s .n.
C innamomum c amphora K-6 4 6 9
C innamomum dic tyoneuron K- 8 2 0 1
C innamomum obtus ifolium K- 8 3 0 3
C innamomum triplinerve B C I -2 0 6 7 1 1
C innamomum triplinerve B C I -2 4 0 7 6 4
C innamomum triplinerve S M -A ma- 0 0 5
C innamomum zeylanic um K-8 3 0 6
C ryptoc arya triplinervis K- 5 5 2 2
D odec adenia grandiflora K- 5 5 2 0
E ndlic heria s p. 1 S M - A ma- 0 0 6
E ndlic heria s p. 2 S M - A ma- 0 0 3
L aurac eae s p. 1 A - 1 5 3 5
L aurac eae s p. 2 N N - B C I ?
L aurus azoric a K- 2 1 9 8 9
L aurus nobilis S M -s .n.
L indera benzoin K- 1 6 9 4 7
L its ea c ubeba K- 1 5 4 7 5
N ec tandra c is s iflora B C I - 2 1 6 3 1 4
N ec tandra c is s iflora B C I - 2 3 3 9 2 2
N ec tandra c is s iflora B C I - 2 4 4 1 4 5
N ec tandra c is s iflora B C I - 2 6 9 0 0 5
N ec tandra c us pidata FC - 1 5 7 9
N ec tandra c us pidata G amboa-s .n.
N ec tandra 'fuzzy' B C I - 0 3 6 1 5 2
N ec tandra 'fuzzy' B C I - 1 0 5 7 5 0
N ec tandra lineata B C I -0 6 5 4 4 6
N ec tandra lineata B C I -2 2 0 0 6 5
N ec tandra purpurea B C I -1 5 1 0 2 2
N ec tandra purpurea B C I -2 7 7 4 1 2
N ec tandra purpurea B C I -4 1 5 1 6 3
N ec tandra s p. 1 A N - 4 1 0
N ec tandra s p. 1 A N - 4 1 1
N eolits ea ac ic ulata K- 1 7 7 3 9
O c otea c allophyla S M - s .n.
O c otea c ernua B C I -2 0 6 4 3 7
O c otea c ernua B C I -2 1 5 9 8 8
O c otea c ernua B C I -4 1 2 9 5 1
O c otea floribunda H D -1 1 6 6
O c otea guianens is A - 8 1 8
O c otea oblonga B C I - 3 0 9 0 7 8
O c otea oblonga B C I - 4 0 3 5 1 0
O c otea puberula B C I - 1 4 6 6 8 4
O c otea puberula B C I - 2 7 2 2 1 9
O c otea puberula B C I - 7 1 6 3 1 7
O c otea s p. 1 H D - 1 1 6 7
O c otea whitei B C I - 0 0 8 0 8 6
O c otea whitei B C I - 3 0 6 2 7 7
P ers ea americ ana S M - s .n.
P ers ea c aerulea S ánc hez-4 9 1 1
P ers ea rimos a K-8 2 0 4
Rhodos temonodaphne frontinoens is B rant- 1 3 8 7
Rhodos temonodaphne kunthiana H D -1 1 7 5
Rhodos temonodaphne kunthiana M adriñán-7 1 7
Rhodos temonodaphne penduliflora S M - s .n.
S as s afras albidum K- 1 6 9 4 8
mat K
ndhJ
rpoB
rpoC1
matK
974 bp
matK
500
300
200
100
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Frequency
400
NJ
Actinodaphne pruinosa K-8203
Aiouea dubia AN-417
Ocotea cernua BCI-206437
Ocotea f loribunda HD-1166
Nectandra cissif lora BCI-216314
Rhodostemonodaphne kunthiana HD-1175
Cinnamomum zeylanicum K-8306 (matK 2.1-5)
Persea rimosa K-8204 (matK 2.1-5)
Rhodostemonodaphne f rontinoensis (matK 2.1-5)
Lauraceae sp. 2 NN-BCI?
Nectandra cissif lora BCI-269005
Nectandra lineata BCI-065446
Nectandra cissif lora BCI-244145
Nectandra cissif lora BCI-233922
Nectandra lineata BCI-220065
Nectandra cuspidata Gamboa-s.n.
Nectandra cuspidata FC-1579
Beilschmiedia pendula BCI-065681
Beilschmiedia pendula BCI-257596
Beilschmiedia pendula BCI-170421
Beilschmiedia taw a K-5519
Cryptocarya triplinervis K-5522
Dodecadenia grandif lora K-5520
Caryodaphnopsis cogolloi JAUM-s.n.
Ocotea oblonga BCI-403510 (matK X)
Ocotea callophyla SM-s. n.
Ocotea guianensis A-818
Ocotea oblonga BCI-309078
Nectandra 'f uzzy' BCI-036152
Nectandra 'f uzzy' BCI-105750
Cinnamomum dictyoneuron K-8201 (matK 2.1-5)
Persea americana SM-s.n.
Persea caerulea Sánchez-4911
Lindera benzoin K-16947
Litsea cubeba K-15475
Sassaf ras albidum K-16948
Cinnamomum triplinerve BCI-240764
Cinnamomum triplinerve SM-Ama-005
Endlicheria sp. 1 SM-Ama-006
Nectandra sp. 1 AN-411 (matK 3.2)
Endlicheria sp. 2 SM-Ama-003
Laurus azorica K-21989
Laurus nobilis SM-s.n.
Neolitsea aciculata K-17739
Ocotea sp. 1 HD-1167
Cinnamomum obtusifolium K-8303
Ocotea cernua BCI-412951
Lauraceae sp. 1 A-1535
Nectandra sp. 1 AN-410
Ocotea puberula BCI-146684
Ocotea puberula BCI-716317
Ocotea puberula BCI-272219
Cinnamomum camphora K-6469
Ocotea cernua BCI-215988
Nectandra purpurea BCI-151022
Nectandra purpurea BCI-277412
Nectandra purpurea BCI-415163
Cinnamomum triplinerve BCI-206711
0.005 substitutions/site
K2P Distance
Inter
Intra
ndhJ
428 bp
ndhJ
NJ
Actinodaphne glabra K-8202
Actinodaphne pruinosa K-8203
Aiouea dubia AN-417 (ndhJ 2)
Ocotea oblonga BCI-309078
Nectandra sp. 1 AN410
Beilschmiedia pendula BCI-170421 (ndhJ 2-3)
Beilschmiedia taw a K-5519
Beilschmiedia pendula BCI-257596
Caryodaphnopsis cogolloi JAUM-s. n.
Dodecadenia grandif lora K-5520
Nectandra cissif lora BCI-216314
Rhodostemonodaphne kunthiana HD-1175 (ndhJ 2-3)
Rhodostemonodaphne penduliflora SM-s. n. (ndhJ 2-3)
Rhodostemonodaphne kunthiana Madriñán-716 (ndhJ 2-3)
Sassaf ras albidum K-16948 (ndhJ 2)
Nectandra purpurea BCI-415163 (ndhJ 2-3)
500
Nectandra sp. 1 AN-411 (ndhJ 2-3)
Ocotea oblonga BCI-403510 (ndhJ 2-3)
Ocotea w hitei BCI-306277 (ndhJ 2-3)
Ocotea callophyla SM-s. n. (ndhJ 2-3)
Litsea cubeba K-15475
Cinnamomum obtusifolium K-8303
Nectandra purpurea BCI-277412
Persea americana SM-s.n.
Rhodostemonodaphne f rontinoensis Brant-1387
Cinnamomum camphora K-6469
Endlicheria sp. 1 SM-Ama-006
Endlicheria sp. 2 SM-Ama-003
Ocotea cernua BCI-206437
Lauraceae sp. 2 NN-BCI?
300
Ocotea sp. 1 HD-1166
Ocotea guianensis A-818
Nectandra lineata BCI-065446
Laurus azorica K-21989
Cinnamomum triplinerve BCI-240764
200
Cinnamomum zeylanicum K-8306
Nectandra f uzzy BCI-105750
Ocotea w hitei BCI-008086
Persea caerulea Sánchez-4911
100
Laurus nobilis SM-s.n.
Lauraceae sp. 1 A-1535
Ocotea puberula BCI-146688
0.005 substitutions/site
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Frequency
400
K2P Distance
Inter
Intra
matK
GenBank (134)
+
Samples (58)
192 sequences
Cryptocarya triplinervis K-5522
Match
Caryodaphnopsis cogolloy JAUM-s.n.
Match
Persea americana SM-s.n.
Match
Neolitsea aciculata K-17739
Match
Laurus nobilis SM-s.n.
Match
Sassafras albidum K-16948
Overall Results
• Standardized universal primers
• Different levels of variation in different
groups at different taxonomic levels
• Variable ID success with a single region
• Score on basis of
– Amplification success
– Sequence variation
and the winner is…
Plant Barcode Proposals
Option 1
matK, rpoC1 and rpoB
Option 2
matK, rpoC1 and trnH-psbA
Acknowledgements
• Royal Botanic Gardens, Kew
– Mark Chase and Robyn Cowan
• Participating Institutions and Colleagues
• CBOL
• Funding
– Alfred P. Sloan Foundation
– Gordon and Betty Moore Foundation
www.kew.org/barcoding