definitive identification of all species of the genus anguilla using the complete
DESCRIPTION
Definitive identification of all species of the genus Anguilla using the complete mitochondrial genome. Yuki Minegishi 1 , Jun Aoyama 1 , Jun G. Inoue 1 , Masaki Miya 2 , Mutsumi Nishida 1 , and Katsumi Tsukamoto 1. 1 Ocean Research Institute, The University of Tokyo - PowerPoint PPT PresentationTRANSCRIPT
Definitive identification of all speciesof the genus Anguilla using the complete
mitochondrial genome
Yuki Minegishi1, Jun Aoyama1,Jun G. Inoue1, Masaki Miya2,
Mutsumi Nishida1, and Katsumi Tsukamoto1
1 Ocean Research Institute, The University of Tokyo2 Department of Zoology, Natural History Museum and Institute
1
Genetic methods for species identification
Takagi and Taniguchi (1995)Tagliavini et al. (1995)Wakao et al. (1999)Zhang et al. (1999)Aoyama et al. (2000)Lehmann et al. (2000) Lin et al. (2002)Watanabe et al. (submitted)
RAPD
PCR-RFLP
PCR-RFLP
PCR-RFLP
PCR-RFLP
RAPD
PCR-RFLP
Real time-PCR
Reference Method
Introduction-1
2
A. reinhardtii / A. mossambica(Bastrop et al., 2000)
A. marmorata / A. nebulosa labiata(Aoyama et al., 2001)
- A. bicolor bicolor - A. malgumora (=A. borneensis)
Same sequences
Different sequences
Introduction-2
Aoyama et al., 2001 / Lin et al., 2001
3
- To obtain the exact complete mitochondrial DNA sequencesof all species of the genus Anguilla
- To collect the basic data forpopulation genetics, molecularphylogeny, DNA taxonomy, etc.
Objective
4
Materials
1
3,82,16
4,145,6,7,10
9,11,1512
13
17
1. A. anguilla2. A. australis australis3. A. australis schmidtii4. A. bicolor bicolor5. A. bicolor pacifica6. A. borneensis7. A. celebesensis8. A. dieffenbachii9. A. interioris10. A. marmorata11. A. megastoma12. A. mossambica13. A. nebulosa labiata14. A. nebulosa nebulosa15. A. obscura16. A. reinhardtii17. A. rostrata18. A. japonica (Inoue et al., 2001)Watanabe S. (2003)
Taxonomy ofthe freshwater eels, genus Anguilla, Schrank 1798
18
5
DNA extraction
Long PCR
Nested PCR
Direct sequencing
154 fish-versatile primers4 Anguilla-versatile primers78 species-specific primers
Methods
12S
16S
ND1
ND2
CO1
CO2ATP
8
ATP
6CO3ND3
ND4LND4
ND5
ND6
cybCR
6
12S ribosomal RNA
16S ribosomal RNA
tRNA-Phe
tRNA-Val
tRNA-Lue(UUR)
tRNA-IletRNA-Gln tRNA-Met
tRNA-Trp tRNA-AlatRNA-AsntRNA-Cys tRNA-Tyr
NADH dehydrogenase subunit I
Cytochrome c oxidase I
Results-1The complete mitochondrial DNA sequences of the genus Anguilla
(A part of the sequence of A. anguilla)
NADH dehydrogenase subunit II
7
- Total length; 16,550~16,833 bp
- Gene composition ; 2 rRNA genes, 22 tRNA genes, 13 protein-coding genes, 1 control region
- Base composition ; A>C>T>G
- Gene order
General characters of mitochondrial DNA of Anguilla
Identical to the mitochondrial DNAfound in other vertebrates
Results-2
Q
AN
S
EP
T
HSL
R
G
K D
W
IM
L
VF
CY
12S
16S
ND1
ND2
CO1
CO2
ATP
8
ATP
6CO3ND3
ND4L
ND4
ND5
ND6
cybCR
mtDNA
8
Why have specimens of two
different species been found
to have the same sequence?
Discussion-1
A. reinhardtii / A. mossambica(Bastrop et al., 2000)
A. marmorata / A. nebulosa labiata(Aoyama et al., 2001)
9
A. reinhardtii / A. mossambica (Bastrop et al., 2000)
Discussion-2
Bold line: distribution of Anguilla (Ege, 1939)
A. mossambica
A. reinhardtiiDistinguishable
10
Discussion-3Number of substitutions of 16S
bicangrosmarbordiemosauscelintmegnebobsrei
mosrei
-2217182623232124192417252216
1717
-1820
92420202216
72110
516
1517
-15162016211810
1011
-4
151713
9121218111916
6
67
-231924212820292416
1617
-2920182117
821
96
18
1719
-17181513151622132317
8
89
-9
19
1820
-1511202219232011
1112
-1715201514
1415
-221911
1112
-23
98
18
1719
-202217232211
1112
-16
1517
- -
jap bic mosang bormarros nebdie cel megintaus obs rei mos
01
-
1
jap
Simple technical errors
11
Discussion-4
Why have specimens of
the same species been found to
have greater sequence differences
than expected?
Aoyama et al. (2001) / Lin et al. (2001)
- A. bicolor bicolor- A. borneensis
12
12S
16S
Cytochrome b
A. bicolor bicolor
0/860 site
11/860 sites
Discussion-5
Amplification ofthe mitochondrialpseudogene
40/1140 sites
GCGTTAGCTT AAAACCCAAA GGACTTGGCG GTGCCTCAAA CCCACCTAGAGCGTTAGCTT AAAACCCAAA GGACTTGGCG GTGCCTCAAA CCCACCTAGA
AGCCCCTTG AAACAGAACA CAACCTTATT CAGGAGGACA AGGATCATATAGCCCCTTG AAACAGAACA CAACCTTACT CAGGAGGACA AGGATCATAT
CCATATTTGT CGAGACGTTA ACTACGGATG ACTAATCCGC AACCTACATGCCACATCTGC CGAGATGTTA ACTATGGATG ATTAATCCGT AACCTACATG
Sequencedifferences
13
Discussion-6
Number of substitutions of cytochrome b
Differences of 12S:38/967 sites
bicangrosmarbordiemosauscelintmegnebobsrei
bor*
-98
105101769681
10098758484799166
84
jap
-10710073
10385
100102816895767282
79
bic
-85
10899
10410410691
103
mos
-48999579
10191
113101117105101101
97
ang
-7994889887
109989491
bor
-90849691744982616758
56
mar
-9699789791
1041021181009899
100
ros
-6871
57
neb
-8165707989798776
81
die
-7570758674
84
cel
-879786
98
meg
-85576471
41
int
-95939798
10786
99
aus
-79
65
obs
-
74
rei
92* A. borneensis (=A. malgumora)
jap
- Amplification of pseudogenes- Introgression- Cryptic species
14
Conclusion
- We obtained the exact completemitochondrial DNA sequencesof all species of the genus Anguilla.
- Wrong sequences have appearedin published data.ex.) A. mossambica, A. bicolor bicolor
- Definitive identification forall anguillid species will be possible.
15
Most parsimonious tree in Lin et al. (2001)(12S, cytochrome b)
Maximum-likelihood tree from the present data(total 15,243 sites, GTR+I+G))