Highly Universal DNA Barcoding Primers of ITS2 for Gymnosperms*

doi:10.7677/ynzwyj201313190

Abstract

Abstract:

DNA barcoding is an approach for rapid and accurate species identification using a standard DNA region. Ideal barcodes should have high level of universality in PCR and sequencing. Although the nuclear ribosomal DNA (nrDNA) internal transcribed spacers 2 (ITS2) was proposed as potential DNA barcode for seed plants, including gymnosperms, no suitable ITS2 primer pair for gymnosperms available shows high universality in either aspect. To obtain a highly universal ITS2 primer pair, we newly designed three forward primers for ITS2 based on sequences in the 5.8S gene greatly conserved among 55 genera of gymnosperms. Combined with previously published reverse primers of ITS, seven candidate primer combinations for ITS2 were assessed for their universality. A total of 56 species, representing 40 genera from all the 12 families of the eight orders of gymnosperms, were sampled in this study. Three ITS2 primer combinations, 5.8SR/ITS4, 5.8SRa/ITS4, and 5.8SF2/S3R, were excluded due to their low universality or because of yielding double bands in the PCR in the familylevel assessment. The remaining four primer combinations, GYM_5.8SF1/ITS4, GYM_5.8SF1/S3R, GYM_5.8SF2/ITS4, and S2F/S3R, showed high PCR universality (100%) on all 56 sampled species. Based on our overall assessment of PCR and sequencing universality, brightness of PCR bands, and sequence quality and coverage, we propose the primer combination GYM_5.8SF2/ITS4 as ITS2 barcode for gymnosperms because it was shown to be the most “universal”.

Key words: DNA barcoding, Gymnosperms, ITS2, Universal primer, Species identification

CLC Number:

Q 781

LI Yan-, Wu Ding-, GAO Lian-Ming. Highly Universal DNA Barcoding Primers of ITS2 for Gymnosperms*[J]. Plant Diversity, 2013, 35(6): 751-760.

References

lvarez I, Wendel JF, 2003. Ribosomal ITS sequences and plant phylogenetic inference［J］. Molecular Phylogenetics and Evolution, 29: 417—434
CBOL Plant Working Group, 2009. A DNA barcode for land plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 106: 12794—12797
Chase MW, Cowan RS, Hollingsworth PM et al., 2007. A proposal for a standardised protocol to barcode all land plants［J］. Taxon, 56: 295—299
Chen SL, Yao H, Han JP et al., 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species［J］. PLoS ONE, 5: e8613.
Chiou SJ, Yen JH, Fang CL et al., 2007. Authentication of medicinal herbs using PCRamplified ITS2 with specific primers［J］. Planta Medica, 73: 1421—1426
Christenhusz MJM, Gardner MF, Mill RR et al., 2011. A new classification and linear sequence of extant gymnosperms［J］. Phytotaxa, 19: 55—70
Coleman AW, 2003. ITS2 is a doubleedged tool for eukaryote evolutionary comparisons［J］. Trends in Genetics, 19: 370—375
Doyle JJ, Doyle JL, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue［J］. Phytochemical Bulletin, 19: 11—15
Fazekas AJ, Burgess KS, Kesanakurti PR et al., 2008. Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well［J］. PLoS ONE, 3: e2802
Ford CS, Ayres KL, Toomey N et al., 2009. Selection of candidate coding DNA barcoding regions for use on land plants［J］. Botanical Journal of the Linnean Society, 159: 1—11
Fu LG, Li N, Mill R, 1999. Taxaceae［A］. In: Wu ZY, Raven PH (eds.), Flora of China［M］. Beijing: Science Press; St. Louis, Missouri: Missouri Botanical Garden, 4: 89—96
Gao LM (高连明), Liu J (刘杰), Cai J (蔡杰) et al., 2012. A synopsis of technical notes on the standards for plant DNA Barcoding［J］. Plant Diversity and Resources (植物分类与资源学报), 34: 592—606
Gao T, Yao H, Song J et al., 2010. Evaluating the feasibility of using candidate DNA barcodes in discriminating species of the large Asteraceae family［J］. BMC Evolutionary Biology, 10: 324
GarcíaRobledo C, Erickson DL, Staines CL et al., 2013. Tropical plantherbivore networks: reconstructing species interactions using DNA barcodes［J］. PLoS ONE, 8: e52967
Gernandt DS, Liston A, 1999. Internal transcribed spacer region evolution in Larix and Pseudotsuga (Pinaceae) ［J］. American Journal of Botany, 86: 711—723
Hebert PDN, Cywinska A, Ball SL, 2003. Biological identifications through DNA barcodes［J］. Proceedings of the Royal Society of London. Series B: Biological Sciences, 270: 313—321
Hebert PDN, Penton EH, Burns JM et al., 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator［J］. Proceedings of the National Academy of Sciences of the United States of America, 101: 14812—14817
Hollingsworth ML, Clark A, Forrest LL et al., 2009. Selecting barcoding loci for plants: evaluation of seven candidate loci with specieslevel sampling in three divergent groups of land plants［J］. Molecular Ecology Resources, 9: 439—457
Hollingsworth PM, Graham SW, Little DP, 2011. Choosing and using a plant DNA barcode［J］. PLoS ONE, 6: e19254
Hollingsworth PM, 2011. Refining the DNA barcode for land plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 108: 19451—19452
Holmes FA, Walters RS, Theriault RL et al., 1991. Phase II trial of taxol, an active drug in the treatment of metastatic breast cancer［J］. Journal of the National Cancer Institute, 83: 1797—1805
Kress WJ, Wurdack KJ, Zimmer EA et al., 2005. Use of DNA barcodes to identify flowering plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 102: 8369—8374
Kress WJ, Erickson DL, 2007. A twolocus global DNA barcode for land plants: the coding rbcL gene complements the noncoding trnHpsbA spacer region［J］. PLoS ONE, 2: e508
Lahaye R, Van Der Bank M, Bogarin D et al., 2008. DNA barcoding the floras of biodiversity hotspots［J］. Proceedings of the National Academy of Sciences of the United States of America, 105: 2923—2928
Larkin MA, Blackshields G, Brown NP et al., 2007. Clustal W and clustal X version 2.0［J］. Bioinformatics, 23: 2947—2948
Li DZ, Liu JQ, Chen ZD et al., 2011a. Plant DNA barcoding in China［J］. Journal of Systematics and Evolution, 49: 165—168
Li DZ, Gao LM, Li HT et al., 2011b. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 108:19641—19646
Li Y, Gao LM, Poudel RC et al., 2011. High universality of matK primers for barcoding gymnosperms［J］. Journal of Systematics and Evolution, 49: 169—175
Linares MC, SotoCalderon ID, Lees DC et al., 2009. High mitochondrial diversity in geographically widespread butterflies of Madagascar: A test of the DNA barcoding approach［J］. Molecular Phylogenetics and Evolution, 50: 485—495
Liston A, Robinson WA, Oliphant JM et al., 1996. Length variation in the nuclear ribosomal DNA internal transcribed spacer region of nonflowering seed plants［J］. Systematic Botany, 21: 109—120
Liston A, Robinson WA, Pinero D et al., 1999. Phylogenetics of Pinus (Pinaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences［J］. Molecular Phylogenetics and Evolution, 11: 95—109
Liu J, Mller M, Gao LM et al., 2011. DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species［J］. Molecular Ecology Resources, 11: 89—100
Mller M, 2000. How universal are universal rDNA primers? A cautionary note for plant systematists and phylogeneticists［J］. Edinburgh Journal of Botany, 57 (2): 151—156
Muellner AN, Schaefer H, Lahaye R, 2011. Evaluation of candidate DNA barcoding loci for economically important timber species of the mahogany family (Meliaceae) ［J］. Molecular Ecology Resources, 11: 450—460
Nieto Feliner G, Rosselló JA, 2007. Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in specieslevel evolutionary studies in plants［J］. Molecular Phylogenetics and Evolution, 44: 911—919
Offerman J, Rychlik W, 2003. Oligo primer analysis software［A］. In: Krawetz S, Womble D (eds.), Introduction to Bioinformatics: A Theoretical and Practical Approach［M］. Totowa: Humana Press, New Jersey, USA, 345—361
Pang XH, Luo H, Sun C et al., 2012. Assessing the potential of candidate DNA barcodes for identifying nonflowering seed plants［J］. Plant Biology, 14: 839—844
Pang XH, Song JY, Zhu YJ et al., 2010. Applying plant DNA barcodes for Rosaceae species identification［J］. Cladistics, 27: 165—170
Roy S, Tyagi A, Shukla V et al., 2010. Universal plant DNA barcode loci may not work in complex groups: A case study with Indian Berberis species［J］. PLoS ONE, 5: e13674
Sass C, Little DP, Stevenson DW et al., 2011. DNA barcoding in the cycadales: testing the potential of proposed barcoding markers for species identification of cycads［J］. PLoS ONE, 2: e1154
Seberg O, Petersen G, 2009. How many loci does it take to DNA barcode a crocus?［J］. PLoS ONE, 4 (2): e4598
Simon UK, Trajanoski S, Kroneis T et al., 2012. Accessionspecific haplotypes of the internal transcribed spacer region in Arabidopsis thaliana—a means for barcoding populations［J］. Molecular Biology and Evolution, 29: 2231—2239
Valentini A, Pompanon F, Taberlet P, 2009. DNA barcoding for ecologists［J］. Trends in Ecology and Evolution, 24: 110—117
White TJ, Bruns T, Lee S et al., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics［A］. In: Innis MA, Gelfand DH, Sninsky JJ et al. (eds.), PCR Protocols: A Guide to Methods and Applications［M］. New York: Academic Press, Inc
Yao H, Song JY, Liu C et al., 2010. Use of ITS2 region as the universal DNA barcode for plants and animals［J］. PLoS ONE, 5: 370—375

[1]	Hai-Su Hu, Jiu-Yang Mao, Xue Wang, Yu-Ze Liang, Bei Jiang, De-Quan Zhang. Plastid phylogenomics and species discrimination in the “Chinese” clade of Roscoea (Zingiberaceae) [J]. Plant Diversity, 2023, 45(05): 523-534.
[2]	Shi-Yu Lv, Xia-Ying Ye, Zhong-Hu Li, Peng-Fei Ma, De-Zhu Li. Testing complete plastomes and nuclear ribosomal DNA sequences for species identification in a taxonomically difficult bamboo genus Fargesia [J]. Plant Diversity, 2023, 45(02): 147-155.
[3]	Yong Yang, David Kay Ferguson, Bing Liu, Kang-Shan Mao, Lian-Ming Gao, Shou-Zhou Zhang, Tao Wan, Keith Rushforth, Zhi-Xiang Zhang. Recent advances on phylogenomics of gymnosperms and a new classification [J]. Plant Diversity, 2022, 44(04): 340-350.
[4]	Kai-Wen Jiang, Rong Zhang, Zhong-Fu Zhang, Bo Pan, Bin Tian. DNA barcoding and molecular phylogeny of Dumasia (Fabaceae: Phaseoleae) reveals a cryptic lineage [J]. Plant Diversity, 2020, 42(05): 376-385.
[5]	HE Wei-Ying-, PAN Yue-Zhi. Study on the DNA Barcoding of Genus Ligularia Cass. (Asteraceae) [J]. Plant Diversity, 2015, 37(06): 693-703.
[6]	JIAO Li-Juan-, SHUI Yu-Min. Evaluating Candidate DNA Barcodes among Chinese Begonia (Begoniaceae) Species [J]. Plant Diversity, 2013, 35(6): 715-724.
[7]	LIU Min-Lu-, YU Wen-Bin-, Wang-Hong. Rapid Identification of Plant Species and iFlora: Application of DNA Barcoding in a Large Temperate Genus Pedicularis (Orobanchaceae) [J]. Plant Diversity, 2013, 35(6): 707-714.
[8]	ZENG Chun-Xia, HE Zheng-Shan, YANG Jun-Bei. DNA Barcoding for Plant Specimen Identification* [J]. Plant Diversity, 2013, 35(6): 693-700.
[9]	FANG Tao, ZENG Chun-Xia, YANG Jun-Bo-**, LI De-Zhu. Herbarium Collections and iFlora [J]. Plant Diversity, 2013, 35(6): 687-692.
[10]	CHEN Zhi-Duan-**, LI De-Zhu. On Barcode of Life and Tree of Life [J]. Plant Diversity, 2013, 35(6): 675-681.
[11]	LI Yan-Chun, WU Gang, YANG Zhu-Liang. DNA Barcoding of Edible Boletes (Boletaceae) from Yunnan, China [J]. Plant Diversity, 2013, 35(6): 725-732.
[12]	Lu Meng-Meng-, CI Xiu-Qin-, YANG Guo-Ping-, LI Jie. DNA Barcoding of Subtropical Forest Trees——A Study from Ailao Mountains Nature Reserve, Yunnan, China [J]. Plant Diversity, 2013, 35(6): 733-741.
[13]	ZHANG Yu-Xiao-, HU Yu-Xing-, MA Peng-Fei-, ZHANG Li-Na-, LI De-Zhu. Selection of Potential Plastid DNA Barcodes for Bambusoideae (Poaceae) [J]. Plant Diversity, 2013, 35(6): 743-750.
[14]	LUO Ya-Huang-, LIU Jie-, GAO Lian-Ming-**, LI De-Zhu. Applications and Advances of DNA Barcoding in Ecological Studies [J]. Plant Diversity, 2013, 35(6): 761-768.
[15]	TANG Min-, YI Ting-Shuang-, WANG Xin-, TAN Mei-Hua-, ZHOU Xin. The Application of Metabarcoding Technology in Identification of Plant Species Diversity [J]. Plant Diversity, 2013, 35(6): 769-773.