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Kunming Institute of Botany Chinese Academy of Sciences

DNA BARCODING OF 6000 SPECIES OF PLANTS IN CHINA. LI De-Zhu On Behalf of China Plant BOL Group. Germplasm Bank of Wild Species, and Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences.

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Kunming Institute of Botany Chinese Academy of Sciences

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  1. DNA BARCODING OF 6000 SPECIES OF PLANTS IN CHINA LI De-Zhu On Behalf of China Plant BOL Group Germplasm Bank of Wild Species, and Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences Kunming Institute of Botany Chinese Academy of Sciences

  2. Talk Overview • Plant diversity and DNA barcoding in China • Evaluation of plant DNA barcodes • Current plant barcoding activities in GBOWS • Case studies for identify species using DNA barcoding • Developing matK primers for barcoding seed plants • Future work China Plant BOL Group Kunming Institute of Botany Chinese Academy of Sciences

  3. China is a mega-diverse country with four biodiversity hotspots in the world Myers et al. 2000. Nature, 403: 853-858 • China is home to 31,000 species of vascular plants

  4. The habitats range from marine to aquatic environs, from temperate to tropical biomes, from sea level to the highest elevations of the Tibetan Plateau, and deserts, prairies and grasslands, among others. • Consequently it is not surprising that China also contains an extremely diverse biota. Kunming Institute of Botany Chinese Academy of Sciences

  5. The uplift of Tibetan Plateau dramatically since Miocene greatly changed the topography and climate of East Asia. • The East Himalaya- Hengduan Mts region is a center of distribution, diversification and endemism for many plant genera.

  6. Flora Reipublicae Popularis Sinicae (Chinese Version of Flora of China) received a First-class Prize of Natural Science of China (2009) • Prof. WU Zheng-Yi, the last Editor-in-Chief of the Flora received the Supreme Science and Technology Award of China (2007)

  7. In the Flora — Vascular plants in China — 31142 species — 3408 genera — 301 families — ca. 52% endemic species

  8. Paul Hebert’s paper on DNA barcoding in 2003 • One delegate (D.W. Huang) to the 1st International BOL Conference in London, 7-9 February 2005 • Plant DNA papers in 2005 • Two delegates (Y. P. Zhang & J. C. Ma) to the 2nd Conference in Taipei, 18-20 September 2007 Kunming Institute of Botany Chinese Academy of Sciences

  9. China joined iBOL and became a central node in April 2008

  10. 2008年10月14日 China-Canada meeting, Oct 2008 • China National Committee for Barcode of Life established in June 2008 • China-Canada joint meeting on Barcode of Life in Beijing, October 2008 • CBOL Beijing meeting, May 2009 CBOL Beijing meeting, May 2009

  11. China plant DNA barcoding pilot meeting, 3 May 2009, Beijing China plant DNA barcoding meeting, 11 July 2009, Beijing Kunming Institute of Botany Chinese Academy of Sciences

  12. One delegate (D.W. Huang) to the 1st International BOL Conference, 2005 Two delegates (D. W. Huang & J. C. Ma) to the 2nd Conference in Taipei, 2007 — Third international BOL conference, 7-13 November 2009, Mexico City — Some 450 delegates from 30 countries or regions — Ten delegates from mainland China Kunming Institute of Botany Chinese Academy of Sciences

  13. rbcL and matK were proposed

  14. The Third International Barcode of Life Conference CBOL Plant Working Group: Official CBOL plant barcode rbcL & matK = core plant barcodeSupplemented by cpDNA locus (e.g. trnH-psbA) and nuclear locus (e.g., nrITS) To be reviewed by late 2011 Mexico City, Mexico, 9-13 November, 2009 Kunming Institute of Botany Chinese Academy of Sciences

  15. China Plant Barcode of Life Project • Supported by a three-year plant barcoding project of Chinese Academy of Sciences • Coordinated by De-Zhu Li and based on the Germplasm Bank of Wild Species (GBOWS) • Involving 60 research groups from 22 institutes • Initiated in September 2009 Kunming Institute of Botany Chinese Academy of Sciences

  16. Functions of China Plant BOL Group January 5, 2010, Kunming • Collaboration to produce a consensus recommendation • Groups/consortia with comparative data on all four barcode markers • Pooling data • Targeted for ca 100 genera of seed plants with broad ordinal coverage • At least two individuals per species • Analyses of data • PWG used to compare directly with results of CBOL Plant Working group • Four methods used for analysis • Discussion meetings

  17. Plant DNA barcoding technique training Two training workshops in January and August 2010 in Kunming

  18. Talk Overview • Plant diversity and DNA barcoding in China • Evaluation of plant DNA barcodes • Current plant barcoding activities in GBOWS • Case studies for identify species using DNA barcoding • Developing matK primers for barcoding seed plants • Future work China Plant BOL Group Kunming Institute of Botany Chinese Academy of Sciences

  19. Sampling & Data Pooling • Angiosperms Order: 40, Family: 70, Genus: 131, Species: 1675 • Gymnosperms Order: 2, Family: 5, Genus: 10, Species: 82 • Total Order: 42, Family: 75, Genus: 141, Species 1757,Individual: 6286,Barcodes: 18858 (out of 21673) For analysis, ≧2 individuals per species Four barcodes: rbcL, matK, trnH-psbA, ITS (ITS2)

  20. Most of the samples collected from China (121 samples of 38 species from outside of China), which represent 6.1% species and 4.4% genera in China. Data Analysis: Two dataset, A: 5583 individuals of 1349 species in 141 genera of 42 orders B: 3011 individuals of 765 species in 83 genera of 30 orders (all samples with the four DNA markers sequenced successfully) Universality, sequence quality and Discriminatory power (four different methods )

  21. Universality Overall PCR and sequencing success was 92.7% for rbcL, 87.1% for matK, 88.5% for trnH-psbA and 76.5% for ITS (79% in angiosperms); Kunming Institute of Botany Chinese Academy of Sciences

  22. PCR and sequencing failure for ITS Case 1: Gymnosperms (only 67%): length variation A set of primers used (for 82.9% samples ) • ITS1 (ITS5)/ ITS4 for angiosperms • ITS-Leu/ITS4 for gymnosperms • Success of sequencing 71.7% indi., 75.5% spp. Case 2: Castanopsis and Fagus Case 3: Dioscorea endophytic fungi Kunming Institute of Botany Chinese Academy of Sciences

  23. Sequence quality Percent of high quality sequence traces rbcL, matK and ITS show high sequence quality. Meanwhile trnH-psbA shows lower quality as long mononucleotide repeats may interrupt the sequence runs and assembly. Kunming Institute of Botany Chinese Academy of Sciences

  24. Discriminatory power 81.8 82.8 79.1 80.7 77.4 75.3 77.2 75 72.5 69.9 69.7 67.2 68.5 66.1 62 59.4 58.5 54.6 53.1 49.7 45.2 44.8 26.4 Based on 3011 individuals representing 765 species where at least two species were sampled per genus and all four markers were successfully sequenced; I2=ITS2). (PWG distance was adopted for comparison)

  25. Comparison of the performance of four barcoding markers (I=ITS, M=matK, P=trnH-psbA and R=rbcL). (A) Universality assessment for PCR and sequencing success. PCR success was based on 6286 samples of 1757 species (5897 angiosperm samples and 389 gymnosperm samples); (B) Assessment of species discrimination success based on 3011 individuals of 765 species with multiple samples and which were successfully sequenced for all four markers. Assessment of sequence quality with QV >=30. Kunming Institute of Botany Chinese Academy of Sciences

  26. Species discriminatory power Three genera with their centers of diversification in Himalaya-Hengduan Mts. with more than 50 species were sampled to test discriminatory power: Herbaceous plants have significant higher species discriminatory power than woody plants.

  27. Laurales provide very low species discrimination using all four markers (1.8–14.3%). ITS generally performed well for the major orders of seed plants, with lowest discrimination success in Ranunculales (6.7%) and Laurales (14.3%). trnH–psbA performed well in Saxifragales, Brassicales, Caryophyllales, Celastrales, and Sapindales, but worse in Dioscoreales, Poales, and Apiales. matK performed better in Saxifragales and Asparagales but poorly in Poales, Laurales, and Dioscoreales

  28. Comparison of species discrimination among the four methods Among these methods, Blast tended to give higher discrimination rates, without exception. The lowest rates were found when using Tree-Building except that rbcL, matK, and trnH–psbA showed slightly lower rates with Distance . It is noted that, with Blast, species discrimination ranged from 29.9% (rbcL) to 81.1% (ITS) with the proposed core barcode; matK + rbcL provided 60.8% discrimination.

  29. Incongruence between nuclear ITS and plastid DNA barcode markers There are 52 of 115 (45.2%) sampled genera incongruence between ITS and plastid DNA markers observed in multiple individuals within morphologically defined species (excluding monotypic genera and genera with only one sampled species). Form Ⅰ: 22 genera Form Ⅱ: 23 genera Form Ⅲ : 15 genera ITS ITS ITS The former two scenarios clearly suggest hybridization and introgression between closely related species or shared ancestral polymorphisms; the third scenario indicates either a lower mutation rate in plastid DNA compared with ITS or possible hybridization and introgression. cpDNA cpDNA cpDNA

  30. Summary of findings • ITS shows the highest discriminatory power of the four markers, even within the most species-rich genera; • A combination of ITS and any plastid DNA marker is able to discriminate 70-79% of species, compared with only 49.7% using matK+rbcL; • Where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in 45.2% of the sampled genera (for genera with more than one species sampled). • We therefore propose that ITS should be included as part of the core barcode for seed plants. In cases where it is difficult to amplify and directly sequence the whole region, the partial sequence ITS2 could be used as an alternative. China Plant BOL Group, 2011. PNAS 108, 19641–19646

  31. Talk Overview • Plant diversity and DNA barcoding in China • Evaluation of plant DNA barcodes • Current plant barcoding activities in GBOWS • Case studies for identify species using DNA barcoding • Developing matK primers for barcoding seed plants • Future work China Plant BOL Group Kunming Institute of Botany Chinese Academy of Sciences

  32. The Germplasm Bank of Wild Species Established in Kunming • Supported by the Government of China with 148 million CN Yuan (> $ 20 million) and officially opened in November 2009 with an annual operational budget of 10 million. • Germplasm bank – seed bank – DNA bank – tissue banking, (tissues and DNAs) • Data-bases ▲ The Germplasm Bank of Wild Species

  33. The Germplasm Bank of Wild Species • Seed collection network across China • By end of 2010, 44549 accessions of seeds representing 5732 species, 1502 genera of wild plants (about 20% of the Chinese flora) have been preserved . ▲ Seed storage facility for plant conservation Kunming Institute of Botany Chinese Academy of Sciences

  34. DNA barcoding the seed accessions in GBOWS A total of 52 orders, 180 families, 9180 genera, 3500species, 9500 individuals; Barcodes obtained ca 31 000by October 2011 2010.4.19

  35. Evaluation of DNA barcodes using seed samples • Universality test based on 1800 individuals of 491 species in 290 genera of 82 families for 38 orders • Discrimination test based on a total of 622 individuals representing 166 species in 53 genera and 36 families for 28 orders; • Five DNA barcodes used for evaluation • — rbcL, matK, trnH-psbA, psbK-psbI and ITS • At least two individuals per species used for analysis Kunming Institute of Botany Chinese Academy of Sciences

  36. Primer pairs used for the five DNA markers Universality statistics for the five DNA markers Based on 1800 individuals of 491 species in 290 genera of 82 families for 38 orders

  37. ITS also showed the highest level of species discrimination with modest level of universality among the five markers (K for psbK-psbI). • The two non-coding markers trnH-psbA and psbK-psbI showed similar levels of universality and species discrimination, and can be complementary to the core barcode. Kunming Institute of Botany Chinese Academy of Sciences

  38. Talk Overview • Plant diversity and DNA barcoding in China • Evaluation of plant DNA barcodes • Current plant barcoding activities in GBOWS • Case studies for identify species using DNA barcoding • Developing matK primers for barcoding seed plants • Future work China Plant BOL Group Kunming Institute of Botany Chinese Academy of Sciences

  39. Case studies for identify unknown species in seed bank----GBOWS349 (no any taxonomic information for the specimen) GBOWS 349 matK_Blast results Maesa tenera Assigned to Species ITS showed the same blast results for this accession

  40. Case studies for identify unknown species ----GBOWS886 GBOWS 886 matK_Blast results Litsea sp. The unidentified specimen can be correctly assigned to genus but not species

  41. Case studies for correct the misidentified specimen within genus After recheck the specimen of the accession, it is real Sambucus adnata

  42. Case studies for correct the misidentified specimen Drymaria cordata Willd Z363 Jingdong, Yunnan Z364 Mengzi, Yunnan Z365 Tengchong, Yunnan Z366 Jingdong, Yunnan Z367 Yuanyang, Yunnan Pycnospora sp.

  43. Using DNA barcoding to identify Taxus species with powder of bark ITS rbcL DNA barcoding Hengduan type Liu et al. 2011. Molecular Ecology Resources 11: 89-100

  44. Talk Overview • Plant diversity and DNA barcoding in China • Evaluation of plant DNA barcodes • Current plant barcoding activities in GBOWS • Case studies for identify species using DNA barcoding • Developing matK primers for barcoding seed plants • Future work China Plant BOL Group Kunming Institute of Botany Chinese Academy of Sciences

  45. High universality of matK primers for barcoding gymnosperms • 57 species representing 40 genera, 11 families of all orders; • One of the nine candidate matK primer (Gym_F1A/Gym_R1A) showing high level of universality except Ephedraceae; • A specific matK primer for Ephedra was newly designed in this study which worked very well. Li et al., 2011. Journal of Systematics and Evolution 49:169-175 Kunming Institute of Botany Chinese Academy of Sciences

  46. New universal matK primers for DNA barcoding angiosperms Universality test of newly designed primer pair matK472F and matK1248R with 390F+1326R as a control. N: number of samples (species) used; N1: number of samples successfully amplified; N2: number of samples successfully sequenced; N3: number of samples successfully amplified 390F and 1326R Yu et al., 2011. Journal of Systematics and Evolution 49: 176-181

  47. 13 articles of plant DNA barcoding published in this special issue; • All of them were supported by the grant from the Large-Scale Scientific Facilities of the Chinese Academy of Sciences (Grant No. 2009-LSF-GBOWS-01). Kunming Institute of Botany Chinese Academy of Sciences

  48. Talk Overview • Plant diversity and DNA barcoding in China • Evaluation of plant DNA barcodes • Current plant barcoding activities in GBOWS • Case studies for identify species using DNA barcoding • Developing matK primers for barcoding seed plants • Future work China Plant BOL Group Kunming Institute of Botany Chinese Academy of Sciences

  49. Future work • Assembly of data of some 1000 more species by 2012 • Identifying gaps at ordinal, familiar and generic levels • Build-up a reference database for ALL vascular plant genera in China based on the Flora of China project (due to be completed by 2013) • Ecological applications: plant-pollinator interactions; plant community response to climate change • Build-up a tier-strategy on some ‘difficult’ groups, such as Camellia (Theaceae) • From plant DNA barcoding to iFlora Kunming Institute of Botany, Chinese Academy of Sciences

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