artemia authentication by rflp of mitochondrial rdna and hsp26-cdna w. xiaomei 1,2, f. catania 1,2,...
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Artemia authentication by RFLP of mitochondrial
rDNA and HSP26-cDNA
W. Xiaomei1,2, F. Catania1,2, F. Dooms1,2,
G. Van Stappen2,
E. Naessens3,
P. Sorgeloos2, P. Bossier1
Artemia INCO workshop, Beijing September 2002
CLO- Sea Fisheries Department 1
Lab Aquaculture and ARC2
INVE Technologies3
Introduction• Diversification of commercial
Artemia cyst supply
• Broader spectrum of cyst samples and characteristics, e.g.– Cyst size– Nauplii size– HUFA– Vitamine C
• Hence need for the possibility to authenticate cyst samples.
Aim
Develop a database(s) of DNA fingerprinting
patterns that could serve as a tool for
authentication commercial Artemia
samples
Methodology on mt-rDNA
DNA extraction from a batch of cysts
PCR amplification of rDNA
RFLP analysis
Data processing
Methodology on mt-rDNA (cont)
• Isolation of DNA from cysts
– Pretreatment• Suspension of 10 mg cyst in 50 µl
for 2h• Crushing with sterile pestil
– DNA extraction by• Promega kit (requiring mostly extra
Ph/Chl purification)• CTAB extraction• Chelex (works also on single cysts)
Methodology on mt-rDNA (cont)
DNA extraction from a batch of cysts
PCR amplification of rDNA
RFLP analysis
Data processing
Methodology (cont)PCR amplification of rDNA
Homo sapiens Gadus morhua Penaeus monodon Artemia franciscana
cox1 cox1 cox1 cox1cox2 cox2 cox2 cox2atp8 atp8 atp8 atp8atp6 atp6 atp6 atp6cox3 cox3 cox3 cox3nad3 nad3 nad3 nad3nad4L nad4L nad5 nad5nad4 nad4L nad4 nad4nad5 nad5 nad4L nad4Lnad6 nad6 nad6 nad6cob cob cob cobUNK UNK nad1 nad112s rRNA 12s rRNA 16s rRNA 16s rRNA16s rRNA 16s rRNA 12s rRNA 12s rRNAnad1 nad1 UNK UNKnad2 nad2 nad2 nad2
Methodology on mt- rDNA (cont)
PCR amplification of rDNA
16S rRNA 12S rRNA
1500 bp
UNIVERSAL PRIMER 12S rDNA AAACTGGGATTAGATACCCCACTAT
CTAGGATTAGATACCCTAArtemia 12S-SP primer
UNIVERSAL PRIMER 16S rDNA CCGGTCTGAACTCAGATCACGTAG CCGGTCTGAACTCAGATCA Artemia 16S SP primer
Methodology on mt-rDNA (cont)
PCR amplification of rDNA
• PCR amplification conditions
• 94°C, 2 min• (94°C 75s, 52°C 45sec, 72°C 2 min) x 34• 72°C 2 min
• Mg Cl2: 2.5 mM
• dNTPs 0.2 mM
• DNA polymerase: 1.75 U
• primers: 0.5 µM
• Target DNA: 50 –100 ng DNA
Methodology on mt-rDNA
DNA extraction from a batch of cysts
PCR amplification of rDNA
RFLP analysis
Data processing
Methodology on mt-rDNA (cont)
RFLP analysis• Purification of the fragment
using Promega kit
• RFLP analysis using– MseI – Tsp509I – HpaII – NdeII – TaqI – HaeIII – HinfI– DdeI
Methodology on mt-rDNA (cont)
DNA extraction from a batch of cysts
PCR amplification of rDNA
RFLP analysis
Data processing
Methodology mt-rDNA (cont)
Data processing• Gelcompar software
• Band assignment
• Matrix of Dice index of similarity: 2nAB/(nA + nB)
• UPGMA dendrogram
RFLP HaeIII+RFLP mseI+RFLP HpaII+RFLP NdeII+RFLP TaqI+RFLP Tsp509I+RFLP HinfI+RFLP DdeI
8RFLPs
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
unidentified Artemia
parthenogenetic Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
parthenogenetic Artemia
parthenogenetic Artemia
parthenogenetic Artemia
parthenogenetic Artemia
unidentified Artemia
parthenogenetic Artemia
unidentified Artemia
parthenogenetic Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
parthenogenetic Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
A. tibetiana
A.franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
unidentified Artemia
A. franciscana
A. franciscana
A. franciscana
A.franciscana
unidentified Artemia
A. franciscana
A. monica
unidentified Artemia
A. franciscana
unidentified Artemia
A. persimilis
parthenogenetic Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
parthenogenetic Artemia
parthenogenetic Artemia
Artemia sinica
unidentified Artemia
unidentified Artemia
A. urmiana
A. salina
A. salina
A.salina
A. salina
A. salina
Karabogaz-Gol, Turkmenistan
Karabogaz-Gol, Turkmenistan
Gahai, Qinghai Province, P.R. China
Karabogaz-Gol, Turkmenistan
Sayten, Kazakhstan
Uzbeksitan
Bolshoe Yarovoe, Russia
Kazakhstan
Bolshoe Yarovoe, Russia
Pavlodar, Kazakhstan
Jingyu (Whale Lake), Xinjiang, P.R. China
Tanggu, Tianjin Province, P.R. China)
Wu Shen Qi County,Inner Mongolia, P.R.China
Bameng, Inner-Mongolia Province, P.R. China (id.)
Karabogaz-Gol (RHI), Turkmenistan
Karabogaz-Gol, Turkmenistan
Ankiembe, Madagascar
Sorkaidak, Kazakhstan
Vineta Swakopmund, Namibia
Medvezhe (Bear) Lake, Kurgan, Russia
Urmia Lake, Iran
Urmia Lake, Iran
Urmia lake, Iran
Urmia Lake, Iran
Aibi Lake, Xinjiang Province, P.R. China
Coqen Lake, Tibet, P.R.China
Tibet, P.R. China
Bozi Co, Tibet, P.R. China
Tibet, P.R. China
Laggor Co, Tibet, P.R. China
Vinh Chau, Vietnam
San Francisco Bay, USA
Vinh Chau, Vietnam
Vinh Chau, Vietnam
Vinh Chau, Vietnam
Vinh Chau, Vietnam
San Francisco Bay (location 3), USA
San Francisco Bay (location 5) USA
Great Salt Lake, USA
Blenheim, New Zealand
GSL '00 ( N.A.) DOUG 11038
GSL '99 ( N.A.) >90%
Great Salt Lake, USA
Great Salt Lake,South Arm, USA
Estuario de Virrila, Peru
Macau, Brazil
Mono Lake, USA
Bonaire Duinmeer, Neth. Antilles
Porto Arraya, Venezuela
Curacao Fuik, Neth.Antilles
Argentina
Wu Shen qi County, Inner Mongolia, P.R. China
Xiechi Lake, Yuncheng, PR China
Yuncheng, P.R. China
Tuhum lake, Mongolia
Megalon Embolon, Greece
Citros, Pieria, Greece
Yuncheng, Shanxi Province, P.R. China
Yimeng, Inner Mongolia, P.R. China
Haolebaoji, Inner Mongolia, PR China, A. sinica
Urmia Lake, Iran
Megrine, Tunisia
Wadi Natron, Egypt
Sfax, Tunisia
Larnaca, Cyprus
Larnaca, Cyprus
1374
1322
1265
1380
1367
1162
1553
1016
1552
1518
1524
1187
1242
1317
1407
1504
1314
1523
1186
1507
1505
1229
1444
1226
1236
1526
1329
1461
1346
1347
1301
1258
1456
1455
1457
1454
1470
1472
1287
1422
1509
1508
1320
1520
479
1300
1277
28
554
502
1321
1243
1434
1206
1463
1420
1280
1218
1188
1415
1230
1268
1290
1269
1011
1148
UPGMA dendrogram
RFLP HaeIII+RFLP mseI+RFLP HpaII+RFLP NdeII+RFLP TaqI+RFLP Tsp509I+RFLP HinfI+RFLP DdeI
8RFLPs
100
80
60
unidentified Artemia
parthenogenetic Art.
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
parthenogenetic Art.
parthenogenetic Art.
parthenogenetic Art.
parthenogenetic Art.
unidentified Artemia
parthenogenetic Art.
unidentified Artemia
parthenogenetic Art.
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
parthenogenetic Art.
unidentified Artemia
unidentified Artemia
unidentified Artemia
unidentified Artemia
A. tibetiana
Karabogaz-Gol, Turkmenistan
Karabogaz-Gol, Turkmenistan
Gahai, Qinghai Province, P.R. Ch.
Karabogaz-Gol, Turkmenistan
Sayten, Kazakhstan
Uzbeksitan
Bolshoe Yarovoe, Russia
Kazakhstan
Bolshoe Yarovoe, Russia
Pavlodar, Kazakhstan
Jingyu (Whale Lake), Xinjiang, P.
Tanggu, Tianjin Province, P.R. C.
Wu Shen Qi County,Inner Mongol.
Bameng, Inner-Mongolia Provinc.
Karabogaz-Gol (RHI), Turkmenis.
Karabogaz-Gol, Turkmenistan
Ankiembe, Madagascar
Sorkaidak, Kazakhstan
Vineta Swakopmund, Namibia
Medvezhe (Bear) Lake, Kurgan, .
Urmia Lake, Iran
Urmia Lake, Iran
Urmia lake, Iran
Urmia Lake, Iran
Aibi Lake, Xinjiang Province, P.R..
Coqen Lake, Tibet, P.R.China
Tibet, P.R. China
Bozi Co, Tibet, P.R. China
Tibet, P.R. China
Laggor Co, Tibet, P.R. China
1374
1322
1265
1380
1367
1162
1553
1016
1552
1518
1524
1187
1242
1317
1407
1504
1314
1523
1186
1507
1505
1229
1444
1226
1236
1526
1329
1461
1346
1347
Detail on the UPGMA dendrogram
RFLP HaeIII+RFLP mseI+RFLP HpaII+RFLP NdeII+RFLP TaqI+RFLP Tsp509I+RFLP HinfI+RFLP DdeI
8RFLPs
100
806040
A.franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
unidentified Artemia
A. franciscana
A. franciscana
A. franciscana
A.franciscana
unidentified Artemia
A. franciscana
A. monica
unidentified Artemia
A. franciscana
unidentified Artemia
A. persimilis
Vinh Chau, Vietnam
San Francisco Bay, USA
Vinh Chau, Vietnam
Vinh Chau, Vietnam
Vinh Chau, Vietnam
Vinh Chau, Vietnam
San Francisco Bay (location 3), .
San Francisco Bay (location 5) U.
Great Salt Lake, USA
Blenheim, New Zealand
GSL '00 ( N.A.) DOUG 11038
GSL '99 ( N.A.) >90%
Great Salt Lake, USA
Great Salt Lake,South Arm, USA
Estuario de Virrila, Peru
Macau, Brazil
Mono Lake, USA
Bonaire Duinmeer, Neth. Antilles
Porto Arraya, Venezuela
Curacao Fuik, Neth.Antilles
Argentina
1301
1258
1456
1455
1457
1454
1470
1472
1287
1422
1509
1508
1320
1520
479
1300
1277
28
554
502
1321
Detail on the UPGMA dendrogram
RFLP HaeIII+RFLP mseI+RFLP HpaII+RFLP NdeII+RFLP TaqI+RFLP Tsp509I+RFLP HinfI+RFLP DdeI
8RFLPs
100
80604020
parthenogenetic Art.
unidentified Artemia
unidentified Artemia
unidentified Artemia
parthenogenetic Art.
parthenogenetic Art.
Artemia sinica
unidentified Artemia
unidentified Artemia
A. urmiana
A. salina
A. salina
A.salina
A. salina
A. salina
Wu Shen qi County, Inner Mongol.
Xiechi Lake, Yuncheng, PR China
Yuncheng, P.R. China
Tuhum lake, Mongolia
Megalon Embolon, Greece
Citros, Pieria, Greece
Yuncheng, Shanxi Province, P.R..
Yimeng, Inner Mongolia, P.R. Ch.
Haolebaoji, Inner Mongolia, PR .
Urmia Lake, Iran
Megrine, Tunisia
Wadi Natron, Egypt
Sfax, Tunisia
Larnaca, Cyprus
Larnaca, Cyprus
1243
1434
1206
1463
1420
1280
1218
1188
1415
1230
1268
1290
1269
1011
1148
Detail on the UPGMA dendrogram
First analysis
There are seven major clusters
•A. tibetiana•Parthenogenetic Artemia•A. franciscana•A. sinica•A. salina•A. persimilis•A. urmiana
Techical problems:RFLP analysis on single
cysts
RFLP analysis of mitochondrial rDNA on 1 cyst with Nde IIon strain ARC1154
1500
1000
500
100
• more than one haplotype in a batch of cysts•In a single cyst: heteroplasmy or pseudo-genes
Techical problems
• In most of cyst samples these technical problems only occur with one to two enzymes
• If a real mixture of two species is present in a cyst sample, double restriction patterns are found with all enzymes.
• In the previously shown dendrogram only samples without apparent technical problems are included.
Robustness of the mt-rDNA technique
Sample Biological ID origin RFLP ID
ARC 1264 p Yimeng, China pARC 1315 p Yimeng, China pARC 1243 p Wu Shen Qi, China pARC 1154 p InnerMongolia, China pARC 1387 p Karasjuk; Karakhstan pARC 1388 p Altai, Karakhstan pARC 1389 p Kuchuckshkoe, Russia pARC 1406 f Ingebright, Canada f
These samples, all containing double restriction patterns with one or more enzymes, were identified against the database
Conclusions on the rDNA database
• The RFLP technique on a batch of cysts is able to differentiate easily between species.
• Within each species a lot a diversity can be found. Using 8 restriction enzymes only 8x4 nucleotides are probed with this RFLP experimental design, while the fragment is 1500 bp long (so only 2.1%). It is clear that the actual DNA sequence of this fragment would unvail much more variability.
Conclusions on the rDNA database (cont)• The mitochondrial rDNA is normally
used to differentiate families or even species within a genus. Here, using 8 R.E. below-species diversity is very high. This is an indication for high genetic isolation of Artemia habitats.
• Probably depending on the local geographical conditions, the 8xRFLP technique might be able to differentiate Artemia samples at a local-salt-lake complex level. This would need more validation.
Methodology for HSP26 RFLP
Total RNA extraction from a batch of cysts
RT-PCR amplification of HSP26, yielding a 650 bp fragment
RFLP analysis using 7 restriction enzymes
Data processing
RFLP Hae III+RFLP Nde II+RFLP Hinf I+RFLP Hpa II+RFLP Tru9 I(Mse I)+RFLP Taq I+RFLP Tsp509 I
RFLP (7. R.E)10
0
9080706050
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. franciscana
A. parthenogen.
A. persimilis
SFB
Vietnam
Vietnam
Vietnam
Vietnam
South Arm GSL 2001
Karabogaz-Gol
Argerntina
1258
1454
1456
1457
1455
1520
1407
1321
UPGMA dendrogramUsing HSP26 data
Conclusion: • HSP26 is suppossed to be involved in the
indogenous stress resistance of cysts. Hence, polymorphism might reflect local adaptation (non-neutral mutations). This needs further study.
• By studying only the cDNA, the putative non-neutral polymorphism can be studied in detail.
• HSP26 cDNA can be isolated from cysts. So stored samples can be used as well in such a study.
General conclusions
• RFLP of the rDNA with eight restriction enzymes reveals a lot of polymorphism
• Within a species, and probably even within a lake, different rDNA haplotypes can be found.
• Commercial samples can be identified at the species level, even if technical problems like double restriction patterns, are complicating the analysis.
• For phylogenetic and population genetic studies, RFLP analysis on single cysts or even cloned rDNA fragment are necessary.
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