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Possibilities and limitations of sequence
similarity and homology search tools implemented
in molecular nucleotide databases for organism
identification
Birgit Gemeinholzer
Botanic Garden and Botanical Museum
Berlin-DahlemFreie Universität Berlin Germany
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Lactuceae
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Tribe Cichorieae (Lactuceae) Family Asteraceae
widely distributed
varying base chromosome numbers
narrow endemics
apomicts and polyploids
reticulate evolution
intense intraspecific sampling
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molecular data Cichorieae (1550 species and 98
genera) 430 ITS-sequences in GeneBank
belonging to 199 species 222
ITS-sequences newly determined 269
species (17 ) 60 genera (61 )
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FASTA (Pearson Lipman 1988 Pearson 1990)
usefull for quick identification of long DNA
regions with low similarity WU-BLAST2.0 (Gish
1996-2003) (Washington University Basic
Local Alignment Search Tool Version 2.0., EBI)
to detect short, very conserved gene
regions BLASTN (Altschul et al. 1990 Altschul
et al. 1994 Anderson Brass 1998) standard
algorithm for sequence similarity detection of
DNA strands being similar but not equivalent to
the input sequence MEGABLAST (Altschul et al.
1990) for long alignments of very similar
sequences in short times batch option
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ITS region
ITS1
ITS2
ITS
5,8S rDNA
26S nuclear rDNA
18S nuclear rDNA
local and global alignment CTGCTAACTATGCTGGAA
GGGCCTAGGCCTGAG .........ATGCTGG-AG..............
CTGCTAACTATGCTGGAAGGGCCTAGGCCTGAG CT---AAC---GC
---ATG---CT-GG----AG
CTAACCC
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of 404 Cichorieae sequences published in
GenBank 199 are ITS of these 107 ITS 1 70 ITS
2 226 ITS1 2 und 5.8S RNA
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Optimization criteria are - adjustment of the
gap penalty (gp)
- gap extention penalty - gap
opening penalty - change of the word length
(word size)
- filter options, for high or low
similarity (hs/ls) - different impacts of base
similarity and dissimilarity - X-dropoff value
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Cichorieae number of analyzed sequences
107 taxa of these are sequences of the same
species published 25 sequences of the
same genus published 62 only different
genera of the same tribe are published
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correct Identification by 107 analyzed sequences
family
tribe
species
genus
Identifikationsgenauigkeit
100
98,8
98,4
84,0
100
correct identification
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analyses of the pitfalls

Picris
pauciflora Prenanthes purpurea RPNITS1B
88,7 Picris hieracioides AF528490
87,6 Leontodon tuberosum AF528487
86,5 1x Leontodon hispidus LHZ93830
85,7 Leontodon crispus AF588488 84,9 Lactuca
sativa Lactuca altaica LAA228610 100 Lactuca
dregeana LDA228609 100 Lactuca serriola
LSA228606 100 1x Lactuca sativa LSA228605
100 Lactuca serriola LSA228608 99,2 Cichorium
spinosum Cichorium intybus CIA228654
100 Cichorium intybus CIA228653 99,6 Cichorium
spinosum AF118918 99,6 4x Cichorium intybus
AF118917 98,4 Cichorium bottae AF118919
98,8
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ITS
4 Mikrosatelliten
Cichorium intybus 216
Cichorium intybus 235
Cichorium intybus 234
Cichorium intybus 168
Cichorium spinosum 163
Cichorium intybus 166
Cichorium intybus 217
Cichorium intybus 206
Cichorium intybus 203
Cichorium intybus 204
Cichorium intybus 136
Cichorium intybus 160
Cichorium intybus 214
Cichorium intybus 218
Cichorium intybus 167
Cichorium intybus 211
Cichorium intybus 93
Cichorium intybus 232
Cichorium pumilum 158
Cichorium spinosum 225
Cichorium pumilum 229
Cichorium spinosum 233
Cichorium intybus 228
Cichorium intybus 231
Cichorium intybus 75
Cichorium intybus 208
Cichorium pumilum 227
Cichorium intybus 76
Cichorium intybus 224
Cichorium endivia 236
Cichorium pumilum 54
Cichorium pumilum 237
Cichorium pumilum 213
Cichorium intybus 201
Cichorium pumilum 56
Cichorium pumilum 221
Cichorium calvum AF1184
Cichorium pumilum 212

Cichorium spinosum 226
Cichorium pumilum 207
Cichorium endivia AF11891
Cichorium intybus 92
Cichorium pumilum 27
Cichorium pumilum 32
Cichorium bottae AF1189
Cichorium intybus 311
Cichorium endivia 31
AFLP
21 C. intybus Germany
51 C. intybus Sweden
50 C. intybus Sweden
68 C. intybus Germany
AFLP
4 C. intybus France
28 C. intybus Iran
13 C. intybus Germany
39 C. intybus Poland
40 C. intybus Poland
41 C. intybus Poland
77 C. intybus Germany
305 C. intybus Greece
33 C. intybus Italy
49 C. intybus Slowakia
22 C. intybus Greece
45 C. intybus Romania
12 C. intybus Germany
D
11 C. intybus Germany
48 C. intybus Russia
9 C. intybus Germany
B
29 C. intybus Italy
35 C. intybus Italy
241 C. intybus Spain
34 C. intybus Italy
A
46 C. intybus Romania
188 C. intybus Austria
8 C. intybus Germany
16 C. intybus Germany
79 C. intybus Slowenia
80 C. intybus Slowenia
87 C. intybus Croatia
5 C. intybus France
6 C. intybus France
10 C. intybus Germany
211 C. intybus Turkey
23 C. intybus Greece
96 C. intybus Uzbekistan
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271 C. intybus Armenia
E
98 C. intybus Uzbekistan
100 C. intybus Uzbekistan
285 C. intybus Armenia
270 C. intybus Armenia
279 C. intybus Armenia
278 C. intybus Armenia
214 C. spinosum
234 C. spinosum
300 C. intybus Armenia
91
250 C. spinosum
C
225 C. spinosum
233 C. spinosum
235 C. spinosum
257 C. intybus Georgia
226 C. spinosum
31 C. endivia
66 C. endivia
32 C. pumilum
237 C. pumilum
27 C. pumilum
54 C. pumilum
F
55 C. endivia
62 C. endivia
221 C. pumilum
212 C. pumilum
0.1
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Cichorium spinosum Cichorium intybus

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The Hieraciinae a clade with reticulate evolution
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Polyploidy did not affect correct identification
Taraxacum
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• biological limitations
• amplificability of a locus throughout
• the complete plant kingdom
• evolutionary constraints on the variability
  between species
• -hybridization and reticulate evolution might
  lead to an overlap between inter- and
  intraspecific variability
• -multi gene families and multi loci (polyploids)

For the identification purposes in our examples,
the various mechanisms of recombination and
concerted evolution among rDNA sequences of
different clusters or different genomes within
one nucleus constituted no impediment, however,
this might not be true in different plant groups
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problems hampering species identification in
plants technical limitations Optimization
of the nucleotide databases - sequence
quality - sequence length - elimination of
wrong or bad sequences - differenciation between
verified and annotated vouchers and
not optimization of BLAST - Implimentation
of the IUB-Code - combination of local and
global BLAST
RAG YCT MAC KGT SCG WAT
HACT BCGT VACG DAGT NACGT
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The sequence similarity and homology is expressed
in the E-Value BLAST uses statistical theory to
produce a bit score and expect value (E-value)
for each alignment pair (query to hit). it
helps to know how strong an alignment can be
expected from chance alone. In this context,
"chance" can mean the comparison of (i) real but
non-homologous sequences (ii) real sequences
that are shuffled to preserve compositional
properties 1-3 or (iii) sequences that are
generated randomly based upon a DNA or protein
sequence model. The bit score evaluates the
significance of the homology in a distance like
framework and needs to be calibrated to a scoring
system, so that to calculate significance one
needs to know in addition only the size of the
search space.
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DNA-Barcoding on Diatoms Development of a
Monitoring system to be added to the EU water
quality assesment Regine Jahn, Wolf-Henning
Kusber, B. Gemeinholzer
http//www.algaterra.org
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Identification of hallocinogenic drugs Birgit
Gemeinholzer (BGBM) und Udo Zirpel (LKA-Berlin)

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