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Establishing a DNA Barcode for Land plants

Establishing a DNA Barcode for Land plants. Santiago Madriñ án Restrepo Universidad de los Andes Bogotá, Colombia samadrin@uniandes.edu.co. Project Partners. COI or cox1 in Plants. Low sequence divergence Other mitochondrial genes Exhibit incorporation of foreign genes

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Establishing a DNA Barcode for Land plants

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  1. Establishing a DNA Barcode for Land plants Santiago Madriñán Restrepo Universidad de los Andes Bogotá, Colombia samadrin@uniandes.edu.co

  2. Project Partners

  3. COI or cox1 in Plants • Low sequence divergence • Other mitochondrial genes • Exhibit incorporation of foreign genes • Frequent transfer of some genes to the nuclear genome

  4. Plant Barcode Proposals

  5. Other Regions • Internal transcribed spacer regions of nuclear ribosomal DNA (ITS) • often highly variable in angiosperms at the generic and species level • divergent copies are often present within single individuals • Non-coding plastid regions • Highly length variable • rbcL • Not variable enough at species level for many plant groups

  6. Desirable Barcode attributes • Variable enough to allow identification of species but with a comparatively low level of intraspecific variation • Universally amplified/sequenced with standardized primers • Technically simple to sequence

  7. Desirable Barcode attributes • Short • Easily alignable • Readily recoverable from herbarium samples and other degraded DNA samples (e.g., forensic material)

  8. Plastid DNA • Monomorphic • High copy number • Highly diagnostic

  9. Phase 1 Mike Wilkinson University of Reading, UK • Identify suitable loci (>100) on the basis of in silico screens using Nicotiana plastid sequence • Design universal primers (sets of 4 primers/locus) • Perform initial screen for universality • Screen twice for sequence variation using diverse species-pairs • Improve universality (e.g., use all primer combinations) • Use statistical modeling approaches to identify optimal primer sets

  10. Regions and Primers

  11. PCR Mix Buffer X 1 Mg2+ 1.5mM dNTPs 0.2mM FW test primer 1µM RE test primer 1µM Taq DNA polymerase 2 units BSA 0.1mg/ml Template (variable) Water to 20 µl

  12. Thermal Profile 94ºC 1min 1 cycle 94ºC 30sec 53ºC 40sec Up to 40 cycles 72ºC 40sec 72ºC 5min 1 cycle

  13. Phase 2 Sampling Bryophytes S.L.

  14. Amplification

  15. Angiosperms

  16. Non Angiosperms

  17. Non Angiosperm Primers

  18. Variation Two Orchid genera Lauraceae

  19. Sister taxa: Cattleya and Sophronitis “Corsage orchids” Cássio van den Berg Universidade Estadual de Feira de Santana, Brasil Cattleya: 43 spp. in 2 subgenera Unifoliate species = 18 species, allopatric species “complex” Bifoliate species = 25 well-defined species, 6 species pairs Sophronitis: 63 spp. in 3 subgenera (as “sections”) Sect. Cattleyodes+Hadrolaelia – 17 well-defined species Sect. Parviflorae – 40 spp. messy complex, genetic data indicate ca. 15 spp. Sect. Sophronitis – 6 allopatric closely related species C.labiata C. aclandiae S. perrinii S. sp. nov.

  20. % species discriminated • ITS: 90.5% • psbA-trnH: 60% • matK: 33.3% • ndhJ: 37.1% • rpoB: 9.9% • rpoC1:9.9% • accD: 6.05 % Nuclear non-coding Plastid non-coding Plastid coding

  21. Orchid Results • accD, rpoB, rpoC1: variation too low for use as a single barcode • matK and ndhF: more variable but with great variation of rate among subgenera • Non-coding regions (ITS and psbA-trnH spacer) performed better, but required great manual effort for indel alignment

  22. Lauraceae • Big family • Largely unstudied • VERY difficult to id. • Economically important

  23. Species Complexes

  24. Phylogenetic Spread

  25. Lauraceae

  26. matK 974 bp

  27. matK

  28. ndhJ 428 bp

  29. ndhJ

  30. matK GenBank (134) + Samples (58) 192 sequences

  31. Cryptocarya triplinervis K-5522 Match

  32. Caryodaphnopsis cogolloy JAUM-s.n. Match

  33. Persea americana SM-s.n. Match

  34. Neolitsea aciculata K-17739 Match

  35. Laurus nobilis SM-s.n. Match

  36. Sassafras albidum K-16948

  37. Overall Results • Standardized universal primers • Different levels of variation in different groups at different taxonomic levels • Variable ID success with a single region • Score on basis of • Amplification success • Sequence variation

  38. and the winner is…

  39. Plant Barcode Proposals Option 1 matK, rpoC1 and rpoB Option 2 matK, rpoC1 and trnH-psbA

  40. Acknowledgements • Royal Botanic Gardens, Kew • Mark Chase and Robyn Cowan • Participating Institutions and Colleagues • CBOL • Funding • Alfred P. Sloan Foundation • Gordon and Betty Moore Foundation

  41. www.kew.org/barcoding

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