DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL؉matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.matK ͉ rbcL ͉ species identification L arge-scale standardized sequencing of the mitochondrial gene CO1 has made DNA barcoding an efficient species identification tool in many animal groups (1). In plants, however, low substitution rates of mitochondrial DNA have led to the search for alternative barcoding regions. From initial investigations of plastid regions (2-4), 7 leading candidates have emerged (5, 6). Four are portions of coding genes (matK, rbcL, rpoB, and rpoC1), and 3 are noncoding spacers (atpF-atpH, trnH-psbA, and psbK-psbI). Different research groups have proposed various combinations of these loci as their preferred plant barcodes, but no consensus has emerged (5-12). This lack of an agreed standard has impeded progress in plant barcoding.Our aim here is to identify a standard DNA barcode for land plants. To achieve this goal, we have pooled data across laboratories including sequence data from 907 samples, representing 445 angiosperm, 38 gymnosperm, and 67 cryptogam species. Using various subsets of these data, we evaluated the 7 candidate loci using criteria in the Consortium for the Barcode of Life's (CBOL) data standards and guidelines for locus selection (http:// www.barcoding.si.edu/protocols.html). Universality: Which loci can be routinely sequenced across the land plants? Sequence quality and coverage: Which loci are most amenable to the production of bidirectional sequences with few or no ambiguous base calls? Discrimination: Which loci enable most species to be distinguished? ResultsUniversality. Direct universality assessments using a single primer pair for each locus in angiosperms resulted in 90%-98% PCR and sequencing success for 6/7 regions. Success for the seventh region, psbK-psbI, was 77% (Fig. 1A). Greater problems were encountered in other land plant groups, with rpoB, matK, atpF-atpH, and psbK-psbI all showing Ͻ50% success in gymnosperms and/or cryptogams based on data compiled from several laboratories (Fig. 1 A).Sequence Quality. Evaluation of sequence quality and coverage from the candidate loci demonstrated that high quality bidirectional sequences were routinely obtained from rbcL, rpoC1, and rpoB (Fig. 1B, x axis). The remaining 4 loci required more manual editing and produced f...

Sesamum indicum is an important crop plant species for yielding oil. The complete chloroplast (cp) genome of S. indicum (GenBank acc no. JN637766) is 153,324 bp in length, and has a pair of inverted repeat (IR) regions consisting of 25,141 bp each. The lengths of the large single copy (LSC) and the small single copy (SSC) regions are 85,170 bp and 17,872 bp, respectively. Comparative cp DNA sequence analyses of S. indicum with other cp genomes reveal that the genome structure, gene order, gene and intron contents, AT contents, codon usage, and transcription units are similar to the typical angiosperm cp genomes. Nucleotide diversity of the IR region between Sesamum and three other cp genomes is much lower than that of the LSC and SSC regions in both the coding region and noncoding region. As a summary, the regional constraints strongly affect the sequence evolution of the cp genomes, while the functional constraints weakly affect the sequence evolution of cp genomes. Five short inversions associated with short palindromic sequences that form step-loop structures were observed in the chloroplast genome of S. indicum. Twenty-eight different simple sequence repeat loci have been detected in the chloroplast genome of S. indicum. Almost all of the SSR loci were composed of A or T, so this may also contribute to the A-T richness of the cp genome of S. indicum. Seven large repeated loci in the chloroplast genome of S. indicum were also identified and these loci are useful to developing S. indicum-specific cp genome vectors. The complete cp DNA sequences of S. indicum reported in this paper are prerequisite to modifying this important oilseed crop by cp genetic engineering techniques.

This study reports the complete chloroplast (cp) DNA sequence of Eleutherococcus senticosus (GenBank: JN 637765), an endangered endemic species. The genome is 156,768 bp in length, and contains a pair of inverted repeat (IR) regions of 25,930 bp each, a large single copy (LSC) region of 86,755 bp and a small single copy (SSC) region of 18,153 bp. The structural organization, gene and intron contents, gene order, AT content, codon usage, and transcription units of the E. senticosus chloroplast genome are similar to that of typical land plant cp DNA. We aligned and analyzed the sequences of 86 coding genes, 19 introns and 113 intergenic spacers (IGS) in three different taxonomic hierarchies; Eleutherococcus vs. Panax, Eleutherococcus vs. Daucus, and Eleutherococcus vs. Nicotiana. The distribution of indels, the number of polymorphic sites and nucleotide diversity indicate that positional constraint is more important than functional constraint for the evolution of cp genome sequences in Asterids. For example, the intron sequences in the LSC region exhibited base substitution rates 5-11-times higher than that of the IR regions, while the intron sequences in the SSC region evolved 7-14-times faster than those in the IR region. Furthermore, the Ka/Ks ratio of the gene coding sequences supports a stronger evolutionary constraint in the IR region than in the LSC or SSC regions. Therefore, our data suggest that selective sweeps by base collection mechanisms more frequently eliminate polymorphisms in the IR region than in other regions. Chloroplast genome regions that have high levels of base substitutions also show higher incidences of indels. Thirty-five simple sequence repeat (SSR) loci were identified in the Eleutherococcus chloroplast genome. Of these, 27 are homopolymers, while six are di-polymers and two are tri-polymers. In addition to the SSR loci, we also identified 18 medium size repeat units ranging from 22 to 79 bp, 11 of which are distributed in the IGS or intron regions. These medium size repeats may contribute to developing a cp genome-specific gene introduction vector because the region may use for specific recombination sites.

The nucleotide sequence of the chloroplast genome from Abies koreana is the first to have complete genome sequence from genus Abies of family Pinaceae. The circular double-stranded DNA, which consists of 121,373 base pairs (bp), contains a pair of very short inverted repeat regions (IRa and IRb) of 264 bp each, which are separated by a small and large single-copy regions (SSC and LSC) of 54,197 and 66,648 bp, respectively. The genome contents of 114 genes (68 peptide-encoding genes, 35 tRNA genes, four rRNA genes, six open reading frames and one pseudogene) are similar to the chloroplast DNA of other species of Abietoideae. Loss of ndh genes was also identified in the genome of A. koreana like other genomes in the family Pinaceae. Thirteen genes contain one (11 genes) or two (rps12 and ycf3 genes) introns. In phylogenetic analysis, the tree confirms that Abies, Keteleeria and Cedrus are strongly supported as monophyletic. Other inverted repeat sequences located in 42-kb inversion points (1186 bp) include trnS-psaM-ycf12- ψtrnG genes.

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