plant dna barcoding

PLANT DNA BARCODING:
PROBLEMS AND APPLICATIONS
R
Massimo Labra
massimo.labra@unimib.it
ZooPlantLab

PLANT BIODIVERSITY AND
AGROBIODIVERSITY
RELATIONSHIP BETWEEN PLANT AND
ENVIRONMENT
DEVELOPMENTAL BIOLOGY AND
REGULATION MECHANISMS

TOOL FOR PLANT
BIODIVERSITY ANALYSIS

RFLP
RAPD
AFLP
SSR
cpSSR
SNP
MOLECULAR MARKERS
= Restriction Fragment length polymorphism
= Random Amplification of Polymorphic DNA
= Amplified Fragment Length Polymorphism
= Simple Sequence Repeat
= Chlorplast Simple Sequence Repeat
= Single Nucleotide Polymorphisms

1
2
3
THE MOLECULAR MARKERS
ANALYSIS
PLANT SELECTION
IDENTIFICATION MARKER(S)
SETTING TOOL(S)
ANALYSIS
DATA ANALYSIS

THE MOST FREQUENT QUESTIONS
> PLANT IDENTIFICATIONS
> RELATIONSHIPS AMONG
CLOSE RELATED TAXA
(SPECIES, CULTIVAR, ECC)

ANSWER
ATCGGATCAATGA
PLANT DNA BARCODING

Cytochrome c Oxidase 1
NOT WORK IN PLANTS !
The plant mitochondrial genome is structurally complex.
Plant mitochondrial genomes frequently undergoes
rearrangement.
Wolfe et al., 1987 PNAS, 84: 9054; Mower et al., 2007 BMC Evol. Biol 7: 135.

IN FLOWERING PLANTS, THE MITOCHONDRIAL GENES EVOLVE:
- A FEW TIMES MORE SLOWLY THAN CHLOROPLAST GENES;
- ABOUT TEN TIMES MORE SLOWLY THAN PLANT AND MAMMAL
NUCLEAR GENES;
- 50100 TIMES MORE SLOWLY THAN MAMMALIAN
MITOCHONDRIAL GENES.

must be universal for all plants.
IN PLANTS, LOW RATE OF DNA CHANGES WERE DETECTED IN
MITOCHONDRIAL GENES.
HOWEVER SOME GROUPS (I.E. PLANTAGO, PELARGONIUM,)
SHOWED A DRAMATIC INCREASE IN THE MITOCHONDRIAL
RATE OF SYNONYMOUS SUBSTITUTION.
SOME OF THESE RATE INCREASES WERE TEMPORARY, WITH
RATES APPROACHING OR RETURNING TO NORMALLY LOW
LEVELS IN CERTAIN DESCENDENT LINEAGES
SOME PLANTS CONTAIN A MIXTURE OF BOTH QUICKLY AND
SLOWLY EVOLVING MITOCHONDRIAL GENES.
Cho Y et al. 2004. Mitochondrial substitution rates are extraordinarily elevated and variable in a
genus of flowering plants. PNAS 101:17741-46.
Parkinson CL et al., 2005. Multiple major increases and decreases in mitochondrial substitution
rates in the plant family Geraniaceae. BMC Evol Biol 5:73.

Second International Barcode of
Life Conference
16 to 21 September 2007
Taipei, Taiwan.
ELIZABETH PENNISI SCIENCE 2007

Characteristic of DNA barcoding
region(s)
- POLYMORPHIC DNA REGION
- CONSERVED REGIONS FOR DESIGNING UNIVERSAL PRIMERS
- EASY AMPLIFICATION
- SEQUENCE LENGTH SUITABLE FOR SEQUENCING
- LOW INTRASPECIFIC VARIABILITY
- HIGH INTERSPECIFIC DIVERSITY
From Meyer and Paulay, PLoS Biology, 2005

Additional characteristics for plants
> the gene sequences used for barcoding should be short
enough to be PCR amplified easily also with degraded DNA!

Additional characteristics for plants
> Plants hybridize frequently! The selected
markers should be able to distinguish hybrids
from parental species!
NUCLEAR OR PLASTIDIAL
MARKERS ?
R. hirsutum L.
R. ferrugineum L.
R. x intermedium Tausch

Additional characteristics for plants
> THE PLANTS ARE OFTEN POLYPLOIDS
(ALLOPOLYPLOIDS AUTOPOLYPLOIDS)
NUCLEAR MARKERS ?

SEVERAL COMPLETE NUCLEAR
GENOME OF PLANTS
NUCLEAR DNA BARCODING
ITS > 40.000 SEQUENCES IN
GENBANK.
MORE THAN 90 PLASTIDIAL
GENOMES
(RAVI ET AL., 2008. PL SYST EVOL 271: 101)
PLASTIDIAL DNA BARCODING
- GENES (matK, rpoB, rbcL)
- Spacer region (trnH-psbA )

FIRST HYPOTHESIS:
NUCLEAR AND PLASTIDIAL REGION
ITS
trnH-psbA
THE PROBLEM OF ITS AND
NUCLEAR MARKERS!

NUCLEAR GENOME SEQUENCE
ITS
CONSIDERATIONS:
NUCLEAR DNA SEGMENT PROVIDE MORE INFORMATION ON
SPECIES IDENTITY, INCLUDING HYBRIDIZATION EVENTS,
IN DNA DATABASE THERE ARE AL LOT OF ITS SEQUENCES
PROBLEMS:
PROBLEMS ARISING FROM PARALOGOUS SEQUENCES,
PSEUDOGENES
THE LOW SPECIES DISCRIMINATORY POWER AT THE
SPECIES LEVEL IN SOME ANGIOSPERM GROUPS
THECNICAL PROBLEMS:
DIFFICULT TO OBTAIN A UNIVERSAL PCR AMPLIFICATION
ESPECIALLY FROM DEGRADED AND LOW-QUALITY DNA
DNA CONTAMINATION (i.e. PLANTS CONTAIN FUNGAL
ENDOPHYTES; HERBARIUM AND FUNGAL).

THE CBOL-PLANT WORKING GROUP HAS NOT REGARDED nrITS
SUITABLE FOR A UNIVERSAL PLANT DNA BARCODE
MAY BE A SUPPLEMENTARY LOCUS FOR TAXONOMIC GROUPS
WHICH HAVE LESS RESOLUTION WITH cpDNA AND WHERE
DIRECT SEQUENCING OF ITS IS POSSIBLE.

Different plastidial markers
- Universal (easy amplification, sequencing)
- Size
- Polymorphisms levels at the specie-genus-family..
- Sequence structure
rbcL
atpB
rpoC1
ndhJ
trnH-psbA
matK

70%
LIMITS OF DNA BARCODING ?
LIMITS OF (MORPHO)SPECIES ?

Nature (2006) 440, 524-527

907 SAMPLES:
445 ANGIOSPERM,
38 GYMNOSPERM,
67 CRYPTOGAM

ATCGGATCAATGA
rbcL
matK
CBOL Plant Working Group
rbcL + matk
-It is the best characterized gene
-There are universal primers not only for angiosperms
-It produce a high-quality bidirectional sequences
-It is one of the most rapidly evolving plastid
coding regions
-It shows high levels of discrimination among
angiosperm species
list of supplementary loci including the noncoding plastid
regions (trnHpsbA, atpFatpH), intron (trnL), ITS could be
used in the cases of degraded tissue or in the analysis of
critical taxonomic groups.

May 2011 | Volume 6 | Issue 5 | e19254

cL: discrimination ability
Kress (PNAS 2005) By comparison, ITS had a much higher
divergence value (13.6%) than any of the plastid regions, and
rbcL was by far the lowest in divergence (0.83%).

THE rbcL IS EASY TO AMPLIFY, SEQUENCE, AND ALIGN IN MOST LAND
PLANTS AND PROVIDES A USEFUL BACKBONE TO THE BARCODE DATASET,
DESPITE IT HAVING ONLY MODEST DISCRIMINATORY POWER (PLANT
WORKING GROUP CBLOD).

matK: Universal amplification
From: Dunning and Savolainen 2010

LIMITS: - SMALL NUMBER OF SPECIES FOR FAMILY
- NOT SUITABLE FOR BARCODING IDEA

- IDENTIFICATION OF CLADE -SPECIFIC PRIMERS
-MODIFICATIONS THE PRIMERS AND REACTION CONDITIONS
TO OBTAIN TO INCREASE THE AMPLIFICATION SUCCESS
DEFINITION OF A PRIMER COCKTAILS AROUND EXISTING
MATK BARCODE PRIMING SITES.
WORK IN PROGRESS!

trH-psbA
- GOOD AMPLIFICATION ACROSS LAND PLANTS WITH A
SINGLE PAIR OF PRIMERS (>90 % FOR ANGIOSPERMS)
- HIGH LEVELS OF SPECIES DISCRIMINATION (More than rbcL
+ matK in Ficus, Alnus and complex groups of Saliz and
Quercus)
PROBLEMS
-PROBLEMS TO OBTAIN HIGH QUALITY BIDIRECTIONAL
SEQUENCES.
-MEDIAN LENGTH OF trnH-psbA IS ABOUT 420 BP IN
EUDICOTS, BUT UPPER LENGTH OF >1,000 BP IN SOME
MONOCOT AND CONIFER SPECIES! TOO MUCH
-DIFFERENCES IN THE trnH-psbA LENGTH OF DIFFERENT TAXA
RESULTED IN ALIGNMENT PROBLEMS FOR ESTIMATE GENETIC
DISTANCES

Hollingswort et al., 2001

STATE OF ART
PLANT DNA BARCODING IS BASED ON TWO
CODING REGIONS: matK and rbcL
HOWEVER
THE MATK PRIMERS NEEDING IMPROVING, AND THE ABSOLUTE
LEVELS OF DISCRIMINATORY POWER OF RBCL+MATK IS
UNCERTAINTY
THUS
THE STANDARD CORE-BARCODE FOR LAND PLANTS BY CBOL IS
SUBJECT TO A REVIEW
DURING THIS REVIEW PHASE, CONTINUED SEQUENCING AND
EXPLORATION OF THE PROPERTIES OF OTHER NON-CODING
MARKERS IS RECOMMENDED (PARTICULARLY trnH-psbA AND
THE INTERNAL TRANSCRIBED SPACERS OF NUCLEAR
RIBOSOMAL DNA nrITS/nrITS2).

FACTORS INFLUENCING THE DISCRIMINATION SUCCESS
OF PLANT BARCODES
Hollingsworth 2011, Plos One 6: e19254
The DNA barcoding approach doesn work in:
- Clades where speciation has been very recent.
- Woody species with long generation times and/or slow
mutation rates
- Polyploid speciation can lead to incongruence between
barcode sequences and taxon concepts;
- In taxonomically complex groups (TCGs)

Species dispersal: in species where dispersal is poor and it is
constituted from isolated populations the neutral mutational
variants can be slow to spread throughout a range.
Thus the DNA barcoding is not universal for the same species!
A secondary consequence of poor dispersal is that the
permeability of a species to inter-specific gene flow may be
increased. No DNA barcoding gap!
Hollingsworth et al., 2011.

PLANT DNA BARCODE APPLICATIONS
A- SPECIES-LEVEL TAXONOMY
B- IDENTIFYING UNKNOWN SPECIMENS TO
KNOWN SPECIES

A- SPECIES-LEVEL TAXONOMY

TAXONOMICALLY COMPLEX GROUPS (TCGs)
Thymus (215 species)
TAXA N
Thymus brevicalyx Strobl 5
Thymus catharinae Camarda 1
Thymus oenipontanus Heinr.Braun 4
Thymus paronychioides Celak. 2
Thymus praecox subsp. polytrichus (A.Kern ex Borbàs) Jalas 3
Thymus pulegioides L. 5
Thymus spinulosus Ten. 3
Thymus striatus Vahl 8
Thymus vulgaris L. 3
Thymus dolomiticus H.J. Coste 1
Thymbra capitata (L.) Cav (=Thymus capitatus (L.)) 3
11 species; 38 samples;
Different geographical locations: IT; FR,
SV; SPA

% variation
between sp.
(S.E.%)
Range %
between sp.
Mean %
variation within
sp. (S.E.%)
Range %
within sp.
matK 0.66 (0.20) 0.29-1.84 0.48 (0.10) 0.00-0.75
trnH-psbA 2.39 (0.50) 1.33-3.53 2.18 (0.50) 0.74-3.60
rbcL 0.17 (0.10) 0.00-0.42 0.07 (0.05) 0.00-0.16

High intraspecific variability!
19 haplotypes with matK
TAXA N
Thymus brevicalyx Strobl 5
Thymus oenipontanus Heinr.Braun 4
Thymus pulegioides L. 5
Thymus striatus Vahl 8
Thymbra capitata (L.) Cav 3

B- IDENTIFYING UNKNOWN SPECIMENS TO KNOWN SPECIES

DNA BARCODING WORK WELL IN
A- PLANT IDENTIFICATION STARTING FROM A FRAGMENTS
OF PLANT MATERIALS (LEAVES, FRUITS)
B - PLANT IDENTIFICATION (INCLUDING CULTIVARS)
STARTING FROM A SET OF TARGET SPECIES.
C- IDENTIFICATION PROBLEM RELATES TO UNFAMILIARITY
WITH A GIVEN SPECIES (I.E. EXOTIC SPECIES).
D- IDENTIFICATION OF PLANT SPECIES OF A SPECIFIC
GEOGRAPHIC AREA WHERE THE SPECIES ARE NOT
NECESSARILY CLOSELY RELATED.

A- PLANT IDENTIFICATION STARTING FROM PLANT FRAGMENTS

GRUPPO I
ORMAMENTAL PLANTS WITH TOXIC METABOLITES
Nandina domestica Thunb.
Ilex aquifolium L.
Aucuba japonica Thunb.
Arum italicum Mill.
Arum maculatum L.
Convallaria majalis L.
Euphorbia pulcherrima Willd. ex Klotzsch
Spathiphyllum wallisii Regel
Sansevieria trifasciata Prain
Anthurium andreanum Linden

GRUPPO II
CONGENERIC TAXA WITH/WITHOUT TOXIC METABOLITES
Aconitum lycoctonum L.
Aconitum napellus L.
Aconitum degenii Gàyer subsp. paniculatum
(Arcang.) Mucher
Aconitum anthora L.
Gruppo IIa
Gruppo IIb
Sambucus ebulus L.
Sambucus racemosa L.
Sambucus nigra L.

Gruppo IIIa
GRUPPO III
EDIBLE AND INEDIBLE PLANTS
Prunus laurocerasus L. TOX
Prunus armeniaca L. (albicocco)
Prunus avium L. (ciliegio)
Prunus persica (L.) Batsch (pesca)
Prunus cerasus L. (amarena)
Prunus domestica L. (susino)
Gruppo IIIb
Solanum dulcamara L. TOX
Solanum nigrum L. TOX
Solanum lycopersicum L.
Solanum tuberosum L.

PLASTIDIAL rpoB; matK,
trnH-psbA
NUCLEAR Sqd1; At103
trnH-psbA and matK

B - PLANT IDENTIFICATION (INCLUDING CULTIVARS)
STARTING FROM A SET OF TARGET SPECIES.
DNA BARCODING AND FOOD TRACEABILITY

Gruppo 1
Mentha piperita L.
Mentha aquatica L.
Mentha spicata L.
Gruppo 4
Salvia officinalis L.
Salvia rutilans
Salvia sclarea
Salvia uliginosa
Gruppo 2
Ocimum gratissimum L.
Ocimum tenuiflorum L.
Ocimum basilicum L (cultivars)
Gruppo 3
Origanum majorana L.
Origanum vulgare L.
Origanum pseudodictamnius Sieber
Origanum heracleoticum
Gruppo 5
Thymus vulgaris L.
Gruppo 6
Rosmarinus officinalis L.

Markers PLASTIDIA matK; trnH-psbA; rbcL
NUCLEAR: ITS; At103; Agt1
ms oc ts mns ro mnc bs
PCR
Specific band
Nonspecific band

The non-coding trnH-psbA intergenic spacer and matK are the
most suitable marker for molecular spices identification.
In a context of food traceability the two markers are useful to
identify commercial processed spice species (sold as dried
material).
Basil: sequence divergences of
marker trnH-psbA, matK and rbcL
clearly distinguish O. gratissimum L.,
and O. tenuiflorum L. from common
basil (O. basilicum L.) while only
trnH-psbA and matK showed
appreciable differences among the
basil cultivars

Origanum samples (Group III) did not show any
sequence polymorphism !
M. piperita L. is a sterile hybrid of M. aquatica L. × M. spicata L.
The chloroplast uniparental markers used in this study, confirm
that M. spicata L. is the maternal parental of M. piperita L.
because both species showed the same plastidial DNA profile.

C- IDENTIFICATION PROBLEM RELATES TO UNFAMILIARITY
WITH A GIVEN SPECIES (I.E. EXOTIC SPECIES).
SMART DRUGS

MORPHOLOGICAL
ANALYSIS
CHEMICAL
ANALYSIS
DNA BAROCIDNG
ANALYSIS
ATCGGATCAATGA

DNA BARCODING ANALYSIS: rbcL, matK and trnH-psbA
SOME COMMERCIAL PLANT MIX SHOWED FRAGMENTS
OF MARIJUANA LEAVES
SOME COMMERCIAL PLANT
MIX SHOWED FRAGMENTS OF
PLANT RICH OF ALKALOIDS
SUCH AS Turnera diffusa L
IN SOME COMMERCIAL MIX THE
PLANT ARE ONLY A SHUTTLE FOR
TRANSPOR SYNTHETIC DRUGS

D- IDENTIFICATION OF PLANT SPECIES OF A SPECIFIC
GEOGRAPHIC AREA WHERE THE SPECIES ARE NOT
NECESSARILY CLOSELY RELATED.
INTEGRATED TAXONOMIC APPROACH: FLORA OF MONTE
VALERIO (TRIESTE).
R
ZooPlantLab
PLANT SAMPLING - DNA ANALYSIS AND
DIGITAL IDENTIFICATION KEY
STATISTICAL ANALYSIS (in progress)

Calluna vulgaris (L.) Hull
Famiglia: ERICACEAE
Nomi italiani: Brentoli, Brughiera, Brugo,
Calluna, Erica falsa, Grecchia, Scopetti,
Sorcelli.

343 PLANT COLLECTED
300 WERE AMPLIFIED WITH ALL MARKERS
trnH-psbA: 316 MOTU on 322 amplified samples
matK: 304 MOTU on 323 amplified samples
rbcL: 293 MOTU on 337 amplified samples
trnH-psba matK rbcL
Gr1- Acer (4) 4 1 1
Gr2- Euphorbia (6) 6 6 4
Gr3-Geranium (4) 4 4 2
Gr4-Medicago (4) 4 3 2
Gr5-Prunus (4) 3 4 2
Gr6-Senecio (3) 3 2 2
Gr7-Solanum (3) 3 3 3
Gr8-Trifolium (5) 4 5 3

MOLECULAR TAXONOMY
MORPHOLOGICAL TAXONOMY

Interactive keys
DNA barcoder

Fabrizio De Mattia Ilaria Bruni Alessia Losa
R
ZooPlantLab
www.zooplantlab.btbs.unimib.it
Maurizio Casiraghi Massimo Labra
Michela Barbuto
Andrea Galimberti
Emanuele Ferri
Sara Baccei
Anna
Sandionigi
Silvia Federici