Artemia authentication by RFLP of mitochondrial rDNA and HSP26cDNA

1
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

CLO- Sea Fisheries Department 1
Lab Aquaculture and ARC2
INVE Technologies3
Artemia INCO workshop, Beijing September 2002
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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.

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Aim

• Develop a database(s) of DNA fingerprinting
  patterns that could serve as a tool for
  authentication commercial Artemia samples

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Methodology on mt-rDNA

• DNA extraction from a batch of cysts
• PCR amplification of rDNA
• RFLP analysis
• Data processing

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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)

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Methodology on mt-rDNA (cont)

• DNA extraction from a batch of cysts
• PCR amplification of rDNA
• RFLP analysis
• Data processing

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Methodology (cont)PCR amplification of rDNA

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Methodology (cont)PCR amplification of rDNA
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Methodology on mt- rDNA (cont)PCR amplification
of rDNA
16S rRNA
12S rRNA
1500 bp
UNIVERSAL PRIMER 12S rDNA
AAACTGGGATTAGATACCCCACTAT
CTAGGATTAGATACCCTA Artemia 12S-SP primer
UNIVERSAL PRIMER 16S rDNA
CCGGTCTGAACTCAGATCACGTAG CCGGTCTGAACTCAGATCA
Artemia 16S SP primer
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Methodology on mt-rDNA (cont)PCR amplification
of rDNA

• PCR amplification conditions
• 94C, 2 min
• (94C 75s, 52C 45sec, 72C 2 min) x 34
• 72C 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

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Methodology on mt-rDNA

• DNA extraction from a batch of cysts
• PCR amplification of rDNA
• RFLP analysis
• Data processing

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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

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Methodology on mt-rDNA (cont)

• DNA extraction from a batch of cysts
• PCR amplification of rDNA
• RFLP analysis
• Data processing

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Methodology mt-rDNA (cont)Data processing

• Gelcompar software
• Band assignment
• Matrix of Dice index of similarity 2nAB/(nA
  nB)
• UPGMA dendrogram

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UPGMA dendrogram
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Detail on the UPGMA dendrogram
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Detail on the UPGMA dendrogram
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Detail on the UPGMA dendrogram
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First analysis

• There are seven major clusters
• A. tibetiana
• Parthenogenetic Artemia
• A. franciscana
• A. sinica
• A. salina
• A. persimilis
• A. urmiana

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Techical problemsRFLP analysis on single cysts
1500
1000
500
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RFLP analysis of mitochondrial rDNA on 1 cyst
with Nde II on strain ARC1154

• more than one haplotype in a batch of cysts
• In a single cyst heteroplasmy or pseudo-genes

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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.

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Robustness of the mt-rDNA technique
These samples, all containing double restriction
patterns with one or more enzymes, were
identified against the database
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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.

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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.

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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

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UPGMA dendrogram Using 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.

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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.