Background Brachypodium distachyon s. l. has been widely investigated across the world as a model plant for temperate cereals and biofuel grasses. However, this annual plant shows three cytotypes that have been recently recognized as three independent species, the diploids B. distachyon (2n = 10) and B. stacei (2n = 20) and their derived allotetraploid B. hybridum (2n = 30). Methodology/Principal Findings We propose a DNA barcoding approach that consists of a rapid, accurate and automatable species identification method using the standard DNA sequences of complementary plastid (trnLF) and nuclear (ITS, GI) loci. The highly homogenous but largely divergent B. distachyon and B. stacei diploids could be easily distinguished (100% identification success) using direct trnLF (2.4%), ITS (5.5%) or GI (3.8%) sequence divergence. By contrast, B. hybridum could only be unambiguously identified through the use of combined trnLF+ITS sequences (90% of identification success) or by cloned GI sequences (96.7%) that showed 5.4% (ITS) and 4% (GI) rate divergence between the two parental sequences found in the allopolyploid. Conclusion/Significance Our data provide an unbiased and effective barcode to differentiate these three closely-related species from one another. This procedure overcomes the taxonomic uncertainty generated from methods based on morphology or flow cytometry identifications that have resulted in some misclassifications of the model plant and its allies. Our study also demonstrates that the allotetraploid B. hybridum has resulted from bi-directional crosses of B. distachyon and B. stacei plants acting either as maternal or paternal parents.
References
[1]
IBI (2010) Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463: 763–768.
[2]
Mur LA, Allainguillaume J, Catalan P, Hasterok R, Jenkins G, et al. (2011) Exploiting the Brachypodium Tool Box in cereal and grass research. New Phytol 191: 334–347.
[3]
Brkljacic J, Grotewold E, Scholl R, Mockler T, Garvin DF, et al. (2011) Brachypodium as a model for the grasses: today and the future. Plant Physiol 157: 3–13.
[4]
Vain P (2011) Brachypodium as a model system for grass research. Journal of Cereal Science 54: 1–7.
[5]
Opanowicz M, Vain P, Draper J, Parker D, Doonan JH (2008) Brachypodium distachyon: making hay with a wild grass. Trends Plant Sci 13: 172–177.
[6]
Manzaneda AJ, Rey PJ, Bastida JM, Weiss-Lehman C, Raskin E, et al. (2011) Environmental aridity is associated with cytotype segregation and polyploidy occurrence in Brachypodium distachyon (Poaceae). New Phytol 193: 797–805.
[7]
Hammami R, Jouve N, Cuadrado A, Soler C, Gonzalez JM (2011) Prolamin storage proteins and alloploidy in wild populations of the small grass Brachypodium distachyon (L.) P.Beauv.. Plant Syst Evol: 99–111.
[8]
Vogel JP, Tuna M, Budak H, Huo N, Gu YQ, et al. (2009) Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium distachyon. BMC Plant Biol 9: 88.
[9]
Garvin DF, Gu YQ, Hasterok R, Hazen SP, Jenkins G, et al. (2008) Development of genetic and genomic research resources for Brachypodium distachyon, a new model system for grass crop research. Crop Sci 48: 69–84.
[10]
Filiz E, Ozdemir BS, Budak F, Vogel JP, Tuna M, et al. (2009) Molecular, morphological, and cytological analysis of diverse Brachypodium distachyon inbred lines. Genome 52: 876–890.
[11]
Catalan P, Muller J, Hasterok R, Jenkins G, Mur LA, et al. (2012) Evolution and taxonomic split of the model grass Brachypodium distachyon. Ann Bot 109: 385–405.
[12]
Robertson IH (1981) Chromosome numbers in Brachypodium Beauv. (Gramineae). Genetica 56: 55–60.
[13]
Hasterok R, Draper J, Jenkins G (2004) Laying the cytotaxonomic foundations of a new model grass, Brachypodium distachyon (L.) Beauv. Chromosome Res 12: 397–403.
[14]
Hasterok R, Marasek A, Donnison IS, Armstead I, Thomas A, et al. (2006) Alignment of the genomes of Brachypodium distachyon and temperate cereals and grasses using bacterial artificial chromosome landing with fluorescence in situ hybridization. Genetics 173: 349–362.
[15]
Idziak D, Hasterok R (2008) Cytogenetic evidence of nucleolar dominance in allotetraploid species of Brachypodium. Genome 51: 387–391.
[16]
Idziak D, Betekhtin A, Wolny E, Lesniewska K, Wright J, et al. (2011) Painting the chromosomes of Brachypodium: current status and future prospects. Chromosoma 120: 469–479.
[17]
Wolny E, Hasterok R (2009) Comparative cytogenetic analysis of the genomes of the model grass Brachypodium distachyon and its close relatives. Ann Bot 104: 873–881.
[18]
Group CPW (2009) A DNA barcode for land plants. Proc Natl Acad Sci U S A 106: 12794–12797.
[19]
Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. PLoS One 6: e19254.
[20]
Hollingsworth PM (2011) Refining the DNA barcode for land plants. 2011 108: 19451–19452.
[21]
Arca M, Hinsinger DD, Cruaud C, Tillier A, Bousquet J, et al. (2012) Deciduous trees and the application of universal DNA barcodes: a case study on the circumpolar Fraxinus. PLoS One 7: e34089.
[22]
Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C, et al. (2007) Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding. Nucleic Acids Res 35: e14.
[23]
Li DZ, Gao LM, Li HT, Wang H, Ge XJ, et al. (2011) Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proc Natl Acad Sci U S A 108: 19641–19646.
[24]
Gonzalez MA, Baraloto C, Engel J, Mori SA, Petronelli P, et al. (2009) Identification of Amazonian trees with DNA barcodes. PLoS One 4: e7483.
[25]
Valentini A, Miquel C, Nawaz MA, Bellemain E, Coissac E, et al. (2009) New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnL approach. Mol Ecol Resour 9: 51–60.
[26]
Nietto FG, Rosello JA (2007) Better the devil you know? Guidelines for insight uitilization of nrDNA ITS in species-level evolutionary studies in plants. Molecular Phylogenetics and Evolution 44: 911–919.
[27]
Chase MW, Salamin N, Wilkinson M, Dunwell JM, Kesanakurthi RP, et al. (2005) Land plants and DNA barcodes: short-term and long-term goals. Philos Trans R Soc Lond B Biol Sci 360: 1889–1895.
[28]
Wu F, Mueller LA, Crouzillat D, Petiard V, Tanksley SD (2006) Combining bioinformatics and phylogenetics to identify large sets of single-copy orthologous genes (COSII) for comparative, evolutionary and systematic studies: a test case in the euasterid plant clade. Genetics 174: 1407–1420.
[29]
Li M, Wunder J, Bissoli G, Scarponi E, Gazzani S (2008) Development of COS genes as universally amplifiable markers for phylogenetic reconstructions of closely related plant species Cladistics. 24: 727–745.
[30]
Duarte JM, Wall PK, Edger PP, Landherr LL, Ma H, et al. (2010) Identification of shared single copy nuclear genes in Arabidopsis, Populus, Vitis and Oryza and their phylogenetic utility across various taxonomic levels. BMC Evol Biol 10: 61.
Dentinger B, Didukh M, Moncalvo J-M (2011) Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina). PLoS ONE 6(9): e25081.
[33]
Casiraghi M, Labra M, Ferri E, Galimberti A, De Mattia F (2011) DNA barcoding: a six-question tour to improve users’ awareness about the method. Bioinform 4 (2): 440–453.
[34]
Fazekas AJ, Burgess KS, Kesanakurti PR, Graham SW, Newmaster SG, et al. (2008) Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PLoS One 3: e2802.
[35]
Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF, Campbell CS, et al. (1995) The ITS region of nuclear ribosomal DNA-A valuable source of evidence on Angiosperm phylogeny. Ann Mo Bot Gard 82: 247–277.
[36]
Hong SY, Lee S, Seo PJ, Yang MS, Park CM (2009) Identification and molecular characterization of a Brachypodium distachyon GIGANTEA gene: functional conservation in monocot and dicot plants. Plant Mol Biol 72: 485–497.
[37]
Wang Q, Yu Q-S, Liu J-Q (2011) Are nuclear loci ideal for barcoding plants? A case study of genetic delimination of two sister species using multiple loci and multiple intraspecific individuals. Journal of Systematics and Evolution 49: 182–188.
[38]
Hohenlohe PA, Bassham S, Etter PD, Stiffler N, Johnson EA, et al. (2010) Population genomics of parallel adaptation in threespine stickleback using sequenced RAD tags. PLoS Genet 6: e1000862.
[39]
Griffin PC, Robin C, Hoffmann AA (2011) A next-generation sequencing method for overcoming the multiple gene copy problem in polyploid phylogenetics, applied to Poa grasses. BMC Biol 9: 19.
[40]
Steele PR, Pires JC (2011) Biodiversity assessment: state-of-the-art techniques in phylogenomics and species identification. Am J Bot 98: 415–425.
[41]
Giraldo P, Rodriguez-Quijano M, Vazquez JF, Carillo JM, Benavente E (2012) Validation of microsatellite markers for cytotype descrimination in the model grass Brachypodium. Genome 55: 1–5.
[42]
Jenkins G, Hasterok R, Draper J, editors (2003) Building the molecular infrastructure of new model grass. Poznan: Institute of Plant Genetics PAS.
[43]
Roy S, Tyagi A, Shukla V, Kumar A, Singh UM, et al. (2010) Universal plant DNA barcode loci may not work in complex groups: a case study with Indian berberis species. PLoS One 5: e13674.
[44]
Vanhaecke D, Garcia de Leaniz C, Gajardo G, Young K, Sanzana J, et al. (2012) DNA barcoding and microsatellites help species delimitation and hybrid identification in endangered galaxiid fishes. PLoS One 7: e32939.
[45]
Nybom H (2004) Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants. Mol Ecol 13: 1143–1155.
[46]
Hamrick JL, Godt MJW (1996) Effects of life history traits on genetic diversity in plant species Philos Trans R Soc Lond B Biol Sci. 351: 1291–1298.
[47]
Soltis DE, Buggs RJA, Doyle JJ, Soltis PS (2010) What we still don’t know about polyploidy? Taxon 59: 1387–1403.
[48]
Soltis PS, Pires JC, Kovarik A, Tate JA, Mavrodiev E (2004) Recent and recurrent polyploidy in Tragopogon (Asteraceae): cytogenetic, genomic and genetic comparisons. Biological Journal of the Linnean Society 82: 485–501.
[49]
Stebbins GL (1950) Variation and evolution in plants. Columbia University Press, New York.
[50]
Ramsey J (2011) Polyploidy and ecological adaptation in wild yarrow. Proc Natl Acad Sci U S A 108: 7096–7101.
[51]
Paun O, Forest F, Fay MF, Chase MW (2009) Hybrid speciation in angiosperms: parental divergence drives ploidy. New Phytol 182: 507–518.
[52]
Catalan P, Olmstead RG (2000) Phylogenetic reconstruction of the genus Brachypodium P. Beauv. (Poaceae). from combined sequences of chloroplast ndhF gene nd nuclear ITS Plant Syst Evol 220: 1–19.
[53]
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure from small quantities of fresh leaf tissues. Phytochem bull 19: 11–15.
[54]
Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 17: 1105–1109.
[55]
Hsiao C, Chatterton NJ, Asay KH, Jensen KB (1995) Phylogenetic relationships of the monogenomic species of the wheat tribe, Triticeae (Poaceae), inferred from nuclear rDNA (internal transcribed spacer) sequences. Genome 38: 211–223.
[56]
Wolny E, Lesniewska K, Hasterok R, Langdon T (2010) Compact genomes and complex evolution in the genus Brachypodium. Chromosoma 120: 199–212.
[57]
Madisson WP (2000) Analysis of phylogeny and character evolution 7.0. Sunderland, MA: Sinauer Associates.
[58]
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599.
[59]
Hebert PD, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc Biol Sci 270: 313–321.
[60]
Clement M, Posada D, Crandall KA (2009) TCS: a computer program to estimate gene genealogies. Mol Ecol 9: 1657–1659.
