The chloroplast PCR primer database: tools
for comprehensive phylogeographic analysis
of a whole genome
Berthold Heinze
Federal Research Centre for Forests, Department of Genetics,
Hauptstrasse 7, A-1140 Vienna, AUSTRIA
[email protected]
The data collected:
Using the database:
A data base is presented which collects published primer
information for chloroplast DNA. Additional primers were
designed in order to fill gaps were no or little primer information
could be found. Chloroplast genes evolve slowly, and many
primers have been designed to work across species ('universal
primers', e.g. Taberlet et al. 1991, Grivet et al. 2001). Amplicons
are either the genes themselves, typically in studies of sequence
variation in higher-order phylogeny, or spacers, introns, and
intergenic regions (e.g. Graham and Olmstead 2000, Small et al.
1998, Shaw et al. 2005) in studies of phylogeographic patterns
within and among species. The current list of 'generic' primers
consists of more than 500 sequences.
Our experience shows that many of the primers can be
combined into pairs for PCR quite freely when 'generic' PCR
conditions ("stepdown" or "two-step PCR") are applied. With
this set of primers it becomes possible to study the whole
chloroplast genome for variation in a comprehensive way for
many taxa (table: successful amplification with 75 primer
pairs from Fraxinus excelsior; see also list below right).
The methods:
Alignments of fully sequenced chloroplast genomes (retrieved
from GenBank 1998-2005), and primer design, were done using
standard methods (software: PC/Gene and OMIGA, Accelrys,
UK). BLASTALL (NCBI, USA) was used to search for homologies
of the primers in 8 chloroplasts (from GenBank, July 2005, exept
Populus): Nicotiana tabacum, Atropa belladonna, Spinacia
oleracea, Arabidopsis thaliana, Populus trichocarpa (Heinze et al.
unpublished), Oryza sativa, Pinus thunbergii, and Marchantia
polymorpha, with a cut-off E value of 0.5.
primer F/P
primer R/M
rpl23p46
trnHM
+
1629
rpl2f
trnHM
++
551(551-1019)
trnHf
psbA Hamilton
amplification
++
approx. length (bp)
555
trnHf
trnK1r
++
2096(2096-2452)
psbAf1
trnK1r
++
psbAf2
trnK1r
trnK1f
matK7B
trnK1f
ccmp1r
trnK1f
trnK2r
matKf2
ccmp1r
matKf2
trnK2r
ccmp1f
primer F/P
primer R/M
amplification
trnD-P
trnT-M(P*)
++
trnE Doyle
trnT-M(P*)
++
851(411-851)
trnT-f
psbD Doyle
++
approx. length (bp)
1408(1408-1690)
psbC2-P
trnS-M
+
983
589(589-764)
trnS-P
ycf9-M
++
482(482-641)
+
535(535-737)
ORF62-P
trnG-M
++
547(500-637)
+-
707
ycf9-P
trnG-M
+
482(411-550)
++
1984
trnG-P
trnfM-M
+
+-
2455
trnfM-f
psaB-FOF
++
++
371
rps14-FOF
psaB-FOF
++
611
++
864
ycf3-3f
ccmp6-r
++
824(824-938)
trnK2r
+
658
ccmp6-f
ccmp6-r
+
133
trnK2f
trnQr
++
1364,3134
ccmp6-f
trnS1-M
++
2324
psbK-P1
ccmp2r
++
713
trnS1-P
rps4-5'
++
941
ccmp2f
ccmp2r
+
213(213-353)
ucp-a/trnT-f
ucp-b
+
807(552-807)
ccmp2f
trnS0r
++
267(267-404)
ucp-c
ucp-d
++
552(552-844)
trnS0f
trnG2-r
-
754 ?
ucp-e
ucp-f/trnF-r
++
482
trnG2-f
ccmp3r
++
637
trnF-f
trnV1-r
+
3670
ccmp3f
trnG1-r
+
183
ndhC-f
trnV1-r
ccmp3f
trnRr
+-
2146(1266-2146)
trnV2-f
trnM-r
++
995
trnG1f
trnRr
+
341(341-619)
trnM-f
atpE-r2
++
652(652-2691)
trnR-f2
atpA-r1
+
210(210-551)
ccmp7-f
rbcL Samuel
+
275
trnR-f2
atpA-P3MD
+
561
atpB Samuel
rbcL Samuel
++
1002
atpA-f4
ccmp4-F
++
919
rbcL Heinze f
accD M2
++
1926
atpF2-f1
ccmp4-F
+
405(405-1512)
accD-f
psaI-r
+
1089
ccmp4-R
ccmp4-F
+
254
accD-f
ycf4-r2
++
1799
ccmp4-R
atpF1-r1
+
544(414-837)
ycf4-f3
ycf10-r1
+
1002
atpF1-f1
atpHr
++
743(743-968)
ycf10-f2
petA-r0
+
543
atpH-P
atpI-M
+-
1125
ycf10-f2
petA-r
+
1338
atpI-f
ccmp5-R
++
483
petA-f(FOF)
psbE-r(FOF)
++
2188
ccmp5-F
rpoC2-r5
+
2910
rpl20 Hamilton
5'-rps12 Hamilton
+
rpoC2-f5
rpoC1-r5
+
1965
ccmp9-f
clpP1-r
+
rpoC1-f5
rpoC1-br(Liston)
++
1331
psbB Hamilton
psbF Hamilton
++
835
rpoC1-f
rpoB-r1
+
554
petB-f
petB-r
++
2011
rpoB-f3
trnC-r
++
1422(1131-1422)
rpl16-R1661
rpl16-F71
++
1128(1128-1596)
psbM-f1
trnD-M
++
802(802-1563)
rpl16-R1516
rpl16-F71
++
971(971-1371)
psbM-f2
trnD-M
++
780(780-1457)
ccmp10-f
rpl23p46
++
2231
ccmp10-f
rpl2f
+
552
1152(1037-1152)
285
850
1270
920
489
http://bfw.ac.at/200/1859.html
total number of entries
587
results of BLASTALL homology searches:
anchored in Nicotiana tabacum
anchored in Atropa belladonna
anchored in Spinacia oleracea
anchored in Arabidopsis thaliana
anchored in Populus trichocarpa
anchored in Oryza sativa
anchored in Pinus thunbergii
anchored in Marchantia polymorpha
present in all 8 chloroplast genomes
present in Nicotiana and Atropa
present in Nicotiana, Atropa, Spinacia
present in Nicotiana Atropa Arabidopsis Populus
present in Arabidopsis and Populus
present in all but Oryza, Pinus, Marchantia
present in all but Pinus and Marchantia
present in all but Marchantia
575
530
427
421
427
395
301
226
152
530
421
369
372
333
289
152
within trn genes
100
within photosystem genes (psa, psb)
83
within ribosomal proteins
and RNA polymerase (rpl, rps, rpo)
92
within ATPase genes (atp)
28
not anchored in identified genes
165
within ycf genes
22
within rbcL
35
within NADH-specific dehydrogenase (ndh) genes 18
Variation in chloroplast DNA fragments
Efficient methods for analysing polymorphisms are necessary - traditional
sequencing may not be an option in large-scale studies. Alternatives are
simple PCR-RFLP in gels, or denaturing high-performance liquid
chromatography for simultaneous detection and analysis of polymorphisms.
Agarose PCR-RFLP
Denaturing HPLC
After PCR, samples are scanned on
agarose
gels
for
successful
amplification. An aliquot of the PCR is
treated with restriction enzymes.
Restriction polymorphisms and major
insertions-deletions can be detected
in high-percentage agarose gels.
A mixture of the PCR products of
sample and a standard plant is
heated an cooled to encourage
heteroduplex DNA formation, then
applied to a silica or polymer column
(Varian HELIX system). At a certain
temperature
specific
for
each
fragment, heteroduplex molecules
start to melt and are eluted with
different patterns (additional peaks).
Example: Polymorphism in Fraxinus
excelsior –
second lane
shows
different
banding
pattern
Example: dHPLC analysis of Fraxinus
excelsior chloroplast PCR fragment
peak unchanged
References
Graham, S.W. and Olmstead, R.G. (2000) Utility of 17 chloroplast genes for inferring the
phylogeny of the basal angiosperms. American Journal of Botany, 87, 1712–1730.
Grivet, D., Heinze, B., Vendramin, G.G. and Petit, R.J. (2001) Genome walking with
consensus primers: application to the large single copy region of chloroplast DNA. Molecular
Ecology Notes, 1, 345-349.
Shaw, J., Lickey, E.B., Beck, J.T., Farmer, S.B., Liu, W., Miller, J., Siripun, K.C., Winder,
C.T., Schilling, E.E. and Small, R.L. (2005) The tortoise and the hare II: relative utility of 21
noncoding chloroplast DNA sequences for phylogenetic analysis Am. J. Bot., 92, 142-166.
Small, R.L., Ryburn, J.A., Cronn, R.C., Seelanan, T. and Wendel, J.F. (1998) The tortoise
and the hare: Choosing between noncoding plastome and nuclear ADH sequences for
phylogeny reconstruction in a recently diverged plant group American Journal of Botany, 85,
1301-1315.
Taberlet, P., Gielly, L., Pautou, G. and Bouvet, J. (1991) Universal primers for
amplification of three non-coding regions of chloroplast DNA. Plant Mol. Biol., 17, 1105-1109.
early additional
peaks
late additional
peaks