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DNA-barcoding on a different scale: challenges within Coccoidea

DNA-barcoding on a different scale: challenges within Coccoidea. Lyn Cook, The University of Queensland, Australia. SIBI Scale Insect Barcode Initiative (Stellenbosch, Feb 2007). Outline. What are scale insects? Why have they been a challenge? Prospects for a universal scale insect barcode.

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DNA-barcoding on a different scale: challenges within Coccoidea

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  1. DNA-barcoding on a different scale: challenges within Coccoidea Lyn Cook, The University of Queensland, Australia

  2. SIBI Scale Insect Barcode Initiative (Stellenbosch, Feb 2007)

  3. Outline • What are scale insects? • Why have they been a challenge? • Prospects for a universal scale insect barcode

  4. SCALE INSECTS APHIDS 210 - 160 Myr ago

  5. 1st scale insect “barcode” • 1995 - DNA extracted from new morphotype. Some ovarian tissue frozen at -70C. Cuticle slide mounted as voucher. Other specimens from same collection frozen or stored in 100% ethanol • 1999 - PCR and sequencing • 2003 - paper published including sequences and stored DNA as part of holotype material Apiomorpha gullanae

  6. A. munita

  7. A. munita 18S rDNA (nuclear SSU rRNA)

  8. 18S variation within a morphospecies

  9. PCR product for 5’ end of nuclear ssu rDNA (18S) 1160 bp 980 bp 720 bp single genus * 480 bp 360 bp

  10. 18S helix E10-1

  11. 18S NJ K2P+G

  12. Cryptococcus

  13. results of COI barcoding attempts * major pest groups

  14. Why such low success with COI?

  15. mtDNA nucleotide frequencies (%)

  16. COI barcode region • Taxa that amplify appear to have higher CG in 3rd positions than failures • not direct comparison • Amino acid divergence of up to 8% in aphid crown • Amino acid divergence of up to 55% within scale crown

  17. Alternatives

  18. Case study Florida lac scale (= lobate lac scale)

  19. Paratachardina lobata

  20. 28S conserved regions only COI - 3’ region

  21. 28S D2 region for lac scales length of the region varies from 703-1114

  22. 28S D2 Aligned using muscle -no adjustment Analysed using NJ K2P

  23. India India Florida Christmas Is. Bermuda &, as of 8 weeks ago, Cuba

  24. Conclusions • Proceed with non-COI barcode - 28S when successful • 18S might be an alternative in “decision tree” • 18S to assign specimen to group or direct ID • next step depends on grouping • (e.g. COI barcode or COI 3’, 12S etc with group-specific primers) • Attempt to find solution to COI barcode problem. Options include: • more primer design • mini COI barcode • cocktails • whole mt genome • other?

  25. Acknowledgements CBOL SAFRANET NSF NSF-PEET ARC ABRS SIBI, Stellenbosch, February, 2007Participants: BB Normark, W Pieterse, PJ Gullan, I Millar, J Giliomee, D Downie, F Haas, C Eardley, LG Cook, A Rung, D Masinga, A Venter, D Muller, DR Miller, R Hanna

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