artemia authentication by rflp of mitochondrial rdna and hsp26-cdna w. xiaomei 1,2, f. catania 1,2,...

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)




RFLP analysis

Data processing

Methodology (cont)PCR amplification of rDNA

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)


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



• 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



RFLP analysis

Data processing


RFLP analysis• Purification of the fragment

using Promega kit

• RFLP analysis using– MseI – Tsp509I – HpaII – NdeII – TaqI – HaeIII – HinfI– DdeI




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




























A. tibetiana

A.franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana


A. franciscana

A. franciscana

A. franciscana

A.franciscana


A. franciscana

A. monica


A. franciscana


A. persimilis







Artemia sinica



A. urmiana

A. salina

A. salina

A.salina

A. salina

A. salina

Karabogaz-Gol, Turkmenistan


Gahai, Qinghai Province, P.R. China


Sayten, Kazakhstan

Uzbeksitan

Bolshoe Yarovoe, Russia

Kazakhstan


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


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,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


8RFLPs

100

80

60


parthenogenetic Art.




























A. tibetiana



Gahai, Qinghai Province, P.R. Ch.


Sayten, Kazakhstan

Uzbeksitan


Kazakhstan


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.


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


8RFLPs

100

806040

A.franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana


A. franciscana

A. franciscana

A. franciscana

A.franciscana


A. franciscana

A. monica


A. franciscana


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.


Blenheim, New Zealand

GSL '00 ( N.A.) DOUG 11038

GSL '99 ( N.A.) >90%


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



8RFLPs

100

80604020







Artemia sinica



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


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.

artemia authentication by rflp of mitochondrial rdna and hsp26-cdna w. xiaomei 1,2, f. catania 1,2,...

Documents

mtrdna dna extraction

mt rdna dna extraction

urmiana slide

mt rdna cont dna extraction

ng dna slide

rflp of mitochondrial

mt rdna technique

mt rdna cont isolation